Deric Wheeler, PhD

Deric Wheeler, PhD

Professor

Department of Human Oncology

I am a professor in the Department of Human Oncology. My laboratory is focused on understanding mechanisms of resistance to molecular targeting agents directed against receptor tyrosine kinases. Specifically, we center our work on cancers of the head and neck and breast. We use molecular biology, proteomics, mouse modeling, including patient derived xenografts, and human tissues to understand how tumors that are initially responsive to therapy become resistant over time. The overall goal of this research is to identify these mechanisms of resistance and to target specific pathways to increase the efficacy of molecular targeting agents. Ultimately, our goal is to take basic science discoveries in the lab and translate them to the clinic.

In addition to my leadership and research roles, my laboratory provides a rich environment for students interested in pursuing translational research. I teach undergraduate, graduate and medical students as well as postdoctoral fellows. Being within a clinical department provides ample opportunities to interact with physicians and physician-scientists. This interaction allows for meaningful understanding of cancer and the best way for my students to advance their findings to the clinic. Ultimately, we hope our work impacts patients and their families in a positive way.

Education

Postdoctoral Fellow, University of Wisconsin, Cancer Biology (2008)

Postdoctoral Fellow, Fred Hutchinson Cancer Research Center, Cancer Biology (2006)

PhD, University of Wisconsin, Cancer Biology (2004)

BS, University of Idaho, Botany and Zoology (1996)

Academic Appointments

Professor, Human Oncology (2021)

Director of Cancer Biology and Translational Medicine, Human Oncology (2015)

Associate Professor, Human Oncology (2014)

Assistant Professor, Human Oncology (2008)

Selected Honors and Awards

NCI Scholar, Molecular Targets of Cancer; Keystone Symposia (2007)

Travel Scholarship Winner, Multidisciplinary Head and Neck Cancer Symposium (2007)

Postdoctoral Fellowship, American Cancer Society (2006-2009)

Postdoctoral Fellow Travel Scholarship, Fred Hutchinson Cancer Research Center (2006)

AACR Scholar-in-Training Award, AACR Annual Meeting (2004)

International Travel Award, The Vilas Fellowship (2002)

Phi Sigma National Biological Society

Dean's list University of Idaho (1994-1996)

Research Focus

Head and Neck Cancer, Triple Negative Breast Cancer


Dr. Deric Wheeler serves as the director of Translational Medicine and Cancer Biology. His laboratory is focused on understanding mechanisms of resistance to molecular targeting agents directed against receptor tyrosine kinases in cancers of the head and neck and breast.

Resistance to drugs that target cancers cells is one of the most pressing problems in all of oncology.

In the Wheeler lab, we use state of the art molecular biology, proteomics and mouse modeling to understand how tumors become resistant to cancer drugs. Our goal is to identify unique and shared pathways that tumors use to circumvent therapy and translate these findings to the clinic to impact the lives of our patients.

The Role of the HER Family in Resistance to Cetuximab Therapy

Monoclonal antibody therapy has been developed over the past 30 years to target receptor tyrosine kinases (RTKs), which sit on the outer membrane of cancerous cells. These receptors are responsible for activating key pathways in cancer cells that lead to their proliferation and survival. Gene amplification, overexpression and hyperactivity of the epidermal growth factor receptor (EGFR) is frequently observed in non-small cell lung cancer (NSCLC), head and neck cancer (HNSCC), breast and colorectal cancer (CRC). Therefore, the EGFR has been one of the most targeted receptors in all of oncology. Targeting EGFR has been intensely pursued in the last decade as a cancer treatment strategy. One approach uses monoclonal antibodies (mAbs) to target the extracellular domain of the EGFR to block natural ligand binding. Cetuximab (IMC- C225, Erbitux) prevents receptor activation and dimerization and ultimately induces receptor internalization and. Cetuximab exhibits promising antitumor activity as monotherapy or in combination with chemotherapy and/or radiation in head and neck cancer and metastatic colorectal cancer. However, despite initial response, acquired and intrinsic resistance to this promising molecular therapeutic eventually develops. Understanding resistance to antibody-based molecular targeting agents is my labs long-term goal.

In 2008 our lab was the first to report that tumor cells that acquired resistance to cetuximab exhibited dramatic up regulation of the EGFR, secondary to loss of ubiquitination, which led to enhanced signals to other EGFR family members (HER and HER3) that promoted proliferation and survival. More in-depth characterization of cetuximab-resistant model systems led to additional works highlighting the interplay between the EGFR and Src Family Kinases (SFKs) in cetuximab resistance. Ultimately, this work has led to our understanding of targeting SFKs and HER family members simultaneously.  This work has advanced the field, along with other investigators, in combinatorial therapy directed at RTKs in several human cancers.

Further studies in the arena of cetuximab resistance, using both in vitro and in vivo modeling, established that nuclear EGFR (nEGFR) can play a direct role in cetuximab resistance. It is known that nEGFR, specifically the C-terminal of the EGFR, acts as a co-transcription factor to regulate a series of genes that promotes proliferation and survival. Nuclear EGFR leads to resistance to cetuximab because this antibody is too large to pass through the membrane of the cell and thus nEGFR can transcribe a series of genes that lead to cetuximab resistance. We further, identified nEGFR as a prognostic factor in NSCLC.

Therapeutic Targeting of Nuclear EGFR

After thorough investigation on how nEGFR leads to resistance to cetuximab, we turned our attention to successful targeting of the nEGFR signaling node.  In models of cetuximab resistance it was clear that the SFKs were dramatically overexpressed. We showed that SFK inhibition could impact nEGFR levels.  Collectively, this work highlighted a tight linkage between SFKs, EGFR translocation to the nucleus and resistance to cetuximab. However, the mechanism of how SFKs mediate EGFRs movement from the membrane to the nucleus remained unresolved.  In 2012 we published a critical paper identifying the exact SFKs, Yes and Lyn, which directly phosphorylates the EGFR on tyrosine 1101 and showed that this is an early, critical step for nuclear translocation of the EGFR.  This was the first paper to identify the function of Y1101 of the EGFR and how SFKs mediate nuclear translocation, an area for therapeutic intervention. We were the first to publish methods on targeting the nEGFR signaling pathway in NSCLC, TNBC and CRC.  This work culminated in a novel approach to target both EGFR signaling nodes, membrane and nuclear.  Investigations found that targeting both nEGFR, via abrogation of its nuclear translocation with dasatinib, and simultaneously targeting classical EGFR signaling with cetuximab, can lead to robust anti-proliferative effects in numerous cell models of cetuximab resistance.

Illustration depicting how nEGFR leads to resistance cetuximab therapy

Collectively this work addresses a central problem in the field of EGFR biology and its subsequent targeting as a cancer therapeutic. Current EGFR targeting agents engage only the membrane-bound, tyrosine kinase activity of EGFR and not the nEGFR-signaling network. The innovation of our work lies in the refining and improving paradigms in EGFR targeting and shifting toward targeting the nEGFR-signaling network. It is clear that nEGFR leads to resistance to cetuximab therapy and that by targeting nEGFR we will be able to increase the efficacy of these therapeutic modalities where nEGFR plays a central role. These studies have the potential to improve and refine current EGFR-centric therapeutic approaches in NSCLC, HSNCC and TNBC.

Identification of HER Family Transcriptional Domains

It has been readily reported that HER family members exhibit a novel function distinct from membrane signaling and that is functions of a transcription factor. HER3 is expressed in the nucleus, and therefore we performed a functional mapping of HER3 to identify the individual amino acid sequences in the HER3, and other family members (EGFR and HER2), that confer their transcriptional activity. This will help define the critical elements of the EGFR, HER2 and HER3 that impart its nuclear function so we can approach designing novel allosteric inhibitors to block its nuclear function and target nuclear HER family members in specific cancers. This was the first report identifying the amino acids, which confer transcriptional activity in an RTK.

Role of AXL in Therapeutic Resistance of NSCLC and HNSCC

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. Studies in our lab have shown that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). We showed that AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab. Further, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in models of cetuximab resistance. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in cetuximab resistant models, creating a positive feedback loop that maintained EGFR activation by AXL. Using cell line xenograft and patient-derived xenograft models, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers.

In further studies, we found AXL to be highly over-expressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases and shorter relapse-free survival in HNSCC patients. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration and invasion. Additionally, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab and radiation. Moreover, radiation-resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Collectively, this work has provided evidence for the role of AXL in HNSCC pathogenesis and supports further pre-clinical and clinical evaluation of anti-AXL therapeutics for the treatment of HNSCC patients.

Investigating Molecular Pathways that Lead to Resistance to AXL Targeting Agents 

The TAM (TYRO-3, AXL, MERTK) family receptor tyrosine kinases (RTKs) play an important role in promoting growth, survival and metastatic spread of several tumor types. AXL and MERTK are over-expressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. AXL is the most well-characterized TAM receptor and mediates resistance to both conventional and targeted cancer therapies. Since AXL is over-expressed in aggressive tumor types, cancer patients are currently being enrolled in clinical trials testing AXL inhibitors. In the current study, we analyzed the effects of AXL inhibition using a small molecule AXL inhibitor, monoclonal antibody therapy, and siRNA in HNSCC, TNBC and NSCLC preclinical models. Anti-AXL targeting strategies had limited efficacy across these different models, which our data suggests could be attributed to up-regulation of MERTK. MERTK expression was increased in cell lines and patient-derived xenografts treated with AXL inhibitors and inhibition of MERTK sensitized HNSCC, TNBC and NSCLC preclinical models to AXL inhibition. Dual targeting of AXL and MERTK led to a more potent blockade of downstream signaling, synergistic inhibition of tumor cell expansion in culture and reduced tumor growth in vivo. Furthermore, ectopic over-expression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These observations suggest that therapeutic strategies co-targeting both AXL and MERTK could be highly beneficial in a variety of tumor types where both receptors are expressed, leading to improved survival for patients with lethal malignancies.

Image of the Wheeler Lab members

CURRENT TEAM MEMBERS

  *   MARI IIDA, PHD, SCIENTIST III
  *   Kourtney Kostecki, Graduate student
  *  Bridget Mehall, Graduate student
  *  Christine Glitchev, Undergraduate researcher
  *  Jillian Adams, Undergraduate researcher
  *  Maddie Harris, Undergraduate researcher
  *  Madisen Crow, Highschool YAP student

 

ALUMNI

  *  Nellie McDaniel (Ph.D.), 2015-2020
  *   Toni Brand (Ph.D.), 2009-2015
  *   Rebecca Myer (Pharm. D), 2008-2011
  *   David Campbell (M.D.), 2009-2011
  *   Kellie Kostopoulus (M.S.), 2011-2012
  *   Megan Starr (RN), 2011-2013
  *   Matthew Wleklinski, (M.D./Ph.D), 2012-2013
  *   Evan Huppert (PA), 2013-2014
  *   Kelsey Corrigan (M.D.), 2013-2015
  *   Cara Braverman (Pharm. D), 2013-2015
  *   John Coan (Ph.D.), 2014-2015
  *   Hannah Pearson (M.D.), 2014-2017
  *   Rachel Orbuch (M.D), 2015-2017
  *   Bailey Flanigan, 2015-2016
  *   Olivia Ondracek, 2017-2018
  *   Grace Kang, 2017-2019
  *   Mahyar Abbariki, 2018-2019
  *   Meghan Gallagher, 2018-2019
  *   Alison Schultz, 2017-2020
  *   Noah Welke, 2018-2020
  *  Sophia Patton , 2019-2020
  *   Kathryn Uhl, 2019-2021
  *  Carkene Kranjac , 2019-2022
  • Immune Escape Strategies in Head and Neck Cancer: Evade, Resist, Inhibit, Recruit Cancers
    Kostecki KL, Iida M, Crossman BE, Salgia R, Harari PM, Bruce JY, Wheeler DL
    2024 Jan 11;16(2):312. doi: 10.3390/cancers16020312.
    • More

      Head and neck cancers (HNCs) arise from the mucosal lining of the aerodigestive tract and are often associated with alcohol use, tobacco use, and/or human papillomavirus (HPV) infection. Over 600,000 new cases of HNC are diagnosed each year, making it the sixth most common cancer worldwide. Historically, treatments have included surgery, radiation, and chemotherapy, and while these treatments are still the backbone of current therapy, several immunotherapies have recently been approved by the Food and Drug Administration (FDA) for use in HNC. The role of the immune system in tumorigenesis and cancer progression has been explored since the early 20th century, eventually coalescing into the current three-phase model of cancer immunoediting. During each of the three phases-elimination, equilibrium, and escape-cancer cells develop and utilize multiple strategies to either reach or remain in the final phase, escape, at which point the tumor is able to grow and metastasize with little to no detrimental interference from the immune system. In this review, we summarize the many strategies used by HNC to escape the immune system, which include ways to evade immune detection, resist immune cell attacks, inhibit immune cell functions, and recruit pro-tumor immune cells.

      PMID:38254801 | PMC:PMC10814769 | DOI:10.3390/cancers16020312


      View details for PubMedID 38254801
  • Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors iScience
    Guo L, Mohanty A, Singhal S, Srivastava S, Nam A, Warden C, Ramisetty S, Yuan Y, Cho H, Wu X, Li A, Vohra M, Saladi SV, Wheeler D, Arvanitis L, Massarelli E, Kulkarni P, Zeng Y, Salgia R
    2023 Jul 10;26(8):107302. doi: 10.1016/j.isci.2023.107302. eCollection 2023 Aug 18.
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      This study investigates the role of integrin β4 (ITGB4) and stemness-associated factor SOX2 in platinum resistance in lung squamous cell carcinoma (LUSC). The expression of SOX2 and ITGB4 is found to be high in all LUSC subtypes, but the impact of ITGB4 expression on overall patient survival varies by subtype. Cancer stem cells (CSCs) isolated from LUSC patients were found to be resistant to cisplatin, but knocking down ITGB4 or SOX2 sensitized them to cisplatin. Carfilzomib (CFZ) synergized with cisplatin and suppressed CSC growth by inhibiting ITGB4 and SOX2 expression. Additionally, CFZ was found to inhibit SOX2 expression epigenetically by inhibiting histone acetylation at the SOX2 promoter site. CFZ also suppressed the growth of SOX2-dependent small cell lung cancer cells in vitro and in vivo. The study highlights the unique function of CFZ as a transcriptional suppressor of SOX2, independent of its proteasome inhibitory function.

      PMID:37554452 | PMC:PMC10405066 | DOI:10.1016/j.isci.2023.107302


      View details for PubMedID 37554452
  • Radiation Sensitivity: The Rise of Predictive Patient-Derived Cancer Models Seminars in radiation oncology
    Berube LL, Nickel KP, Iida M, Ramisetty S, Kulkarni P, Salgia R, Wheeler DL, Kimple RJ
    2023 Jul;33(3):279-286. doi: 10.1016/j.semradonc.2023.03.005.
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      Patient-derived cancer models have been used for decades to improve our understanding of cancer and test anticancer treatments. Advances in radiation delivery have made these models more attractive for studying radiation sensitizers and understanding an individual patient's radiation sensitivity. Advances in the use of patient-derived cancer models lead to a more clinically relevant outcome, although many questions remain regarding the optimal use of patient-derived xenografts and patient-derived spheroid cultures. The use of patient-derived cancer models as personalized predictive avatars through mouse and zebrafish models is discussed, and the advantages and disadvantages of patient-derived spheroids are reviewed. In addition, the use of large repositories of patient-derived models to develop predictive algorithms to guide treatment selection is discussed. Finally, we review methods for establishing patient-derived models and identify key factors that influence their use as both avatars and models of cancer biology.

      PMID:37331782 | PMC:PMC10287034 | DOI:10.1016/j.semradonc.2023.03.005


      View details for PubMedID 37331782
  • Circulating tumor cell abundance in head and neck squamous cell carcinoma decreases with successful chemoradiation and cetuximab treatment Cancer letters
    Poellmann MJ, Bu J, Kim D, Iida M, Hong H, Wang AZ, Wheeler DL, Kimple RJ, Hong S
    2023 May 28;562:216187. doi: 10.1016/j.canlet.2023.216187. Epub 2023 Apr 15.
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      Head and neck squamous cell carcinoma (HNSCC) is a common and deadly cancer. Circulating tumor cell (CTC) abundance may a valuable, prognostic biomarker in low- and intermediate-risk patients. However, few technologies have demonstrated success in detecting CTCs in these populations. We prospectively collected longitudinal CTC counts from two cohorts of patients receiving treatments at our institution using a highly sensitive device that purifies CTCs using biomimetic cell rolling and dendrimer-conjugated antibodies. In patients with intermediate risk human papillomavirus (HPV)-positive HNSCC, elevated CTC counts were detected in 13 of 14 subjects at screening with a median of 17 CTC/ml (range 0.2-2986.5). A second cohort of non-metastatic, HPV- HNSCC subjects received cetuximab monotherapy followed by surgical resection. In this cohort, all subjects had elevated baseline CTC counts median of 73 CTC/ml (range 5.4-332.9) with statistically significant declines during treatment. Interestingly, two patients with recurrent disease had elevated CTC counts during and following treatment, which also correlated with growth of size and ki67 expression in the primary tumor. The results suggest that our device may be a valuable tool for evaluating the success of less intensive treatment regimens.

      PMID:37068555 | PMC:PMC10510654 | DOI:10.1016/j.canlet.2023.216187


      View details for PubMedID 37068555
  • Active recruitment of anti-PD-1-conjugated platelets through tumor-selective thrombosis for enhanced anticancer immunotherapy Science advances
    Wang Y, Li W, Li Z, Mo F, Chen Y, Iida M, Wheeler DL, Hu Q
    2023 Mar 29;9(13):eadf6854. doi: 10.1126/sciadv.adf6854. Epub 2023 Mar 29.
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      Immune checkpoint inhibitors (ICIs) can reinvigorate T cells to eradicate tumor cells, showing great potential in combating various types of tumors. We propose a delivery strategy to enhance tumor-selective ICI accumulation, which leverages the responsiveness of platelets and platelet-derivatives to coagulation cascade signals. A fused protein tTF-RGD targets tumor angiogenic blood vessel endothelial cells and initiates the coagulation locoregionally at the tumor site, forming a "cellular hive" to recruit anti-PD-1 antibody (aPD-1)-conjugated platelets to the tumor site and subsequently activating platelets to release aPD-1 antibody to reactivate T cells for improved immunotherapy. Moreover, on a patient-derived xenograft breast cancer model, the platelet membrane-coated nanoparticles can also respond to the coagulation signals initiated by tTF-RGD, thus enhancing the accumulation and antitumor efficacy of the loaded chemotherapeutics. Our study illustrates a versatile platform technology to enhance the local accumulation of ICIs and chemodrugs by taking advantage of the responsiveness of platelets and platelet derivatives to thrombosis.

      PMID:36989364 | PMC:PMC10058243 | DOI:10.1126/sciadv.adf6854


      View details for PubMedID 36989364
  • Dual Axl/MerTK inhibitor INCB081776 creates a proinflammatory tumor immune microenvironment and enhances anti-PDL1 efficacy in head and neck cancer Head & neck
    Kostecki KL, Iida M, Wiley AL, Kimani S, Mehall B, Tetreault K, Alexandridis R, Yu M, Hong S, Salgia R, Bruce JY, Birge RB, Harari PM, Wheeler DL
    2023 May;45(5):1255-1271. doi: 10.1002/hed.27340. Epub 2023 Mar 20.
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      BACKGROUND: The tyrosine kinase receptors Axl and MerTK are highly overexpressed in head and neck cancer (HNC) cells, where they are critical drivers of survival, proliferation, metastasis, and therapeutic resistance.

      METHODS: We investigated the role of Axl and MerTK in creating an immunologically "cold" tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Effects of INCB081776 and/or anti-PDL1 on mouse oral cancer (MOC) cell growth and on the TIME were evaluated.

      RESULTS: Targeting Axl and MerTK can reduce M2 and induce M1 macrophage polarization. In vivo, INCB081776 treatment alone or with anti-PDL1 appears to slow MOC tumor growth, increase proinflammatory immune infiltration, and decrease anti-inflammatory immune infiltration.

      CONCLUSIONS: This data indicates that simultaneous targeting of Axl and MerTK with INCB081776, either alone or in combination with anti-PDL1, slows tumor growth and creates a proinflammatory TIME in mouse models of HNC.

      PMID:36939040 | PMC:PMC10079616 | DOI:10.1002/hed.27340


      View details for PubMedID 36939040
  • Targeting HER3-dependent activation of nuclear AKT improves radiotherapy of non-small cell lung cancer Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology
    Toulany M, Iida M, Lettau K, Coan JP, Rebholz S, Khozooei S, Harari PM, Wheeler DL
    2022 Sep;174:92-100. doi: 10.1016/j.radonc.2022.07.008. Epub 2022 Jul 15.
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      BACKGROUND: AKT1 must be present and activated in the nucleus immediately after irradiation to stimulate AKT1-dependent double-strand breaks (DSB) repair through the fast non-homologous end-joining (NHEJ) repair process. We investigated the subcellular distribution of AKT1 and the role of HER family receptor members on the phosphorylation of nuclear AKT and radiation response.

      MATERIALS AND METHODS: Using genetic approaches and pharmacological inhibitors, we investigated the subcellular distribution of AKT1 and the role of HER family receptor members on the activation of nuclear AKT in non-small cell lung cancer (NSCLC) cells in vitro. ɤH2AX foci assay was applied to investigate the role of AKT activating signaling pathway on DSB repair. A mouse tumor xenograft model was used to study the impact of discovered signaling pathway activating nuclear AKT on the radiation response of tumors in vivo.

      RESULTS: Our data suggests that neither ionizing radiation (IR) nor stimulation with HER family receptor ligands induced rapid nuclear translocation of endogenous AKT1. GFP-tagged exogenous AKT1 translocated to the nucleus under un-irradiated conditions and IR did not stimulate this translocation. Nuclear translocation of GFP-AKT1 was impaired by the AKT inhibitor MK2206 as shown by its accumulation in the cytoplasmic fraction. IR-induced phosphorylation of nuclear AKT was primarily dependent on HER3 expression and tyrosine kinase activation of epidermal growth factor receptor. In line with the role of AKT1 in DSB repair, the HER3 neutralizing antibody patritumab as well as HER3-siRNA diminished DSB repair in vitro. Combination of patritumab with radiotherapy improved the effect of radiotherapy on tumor growth delay in a xenograft model.

      CONCLUSION: IR-induced activation of nuclear AKT occurs inside the nucleus that is mainly dependent on HER3 expression in NSCLC. These findings suggest that targeting HER3 in combination with radiotherapy may provide a logical treatment option for investigation in selected NSCLC patients.

      PMID:35839938 | PMC:PMC10083767 | DOI:10.1016/j.radonc.2022.07.008


      View details for PubMedID 35839938
  • AXL regulates neuregulin1 expression leading to cetuximab resistance in head and neck cancer BMC cancer
    Iida M, McDaniel NK, Kostecki KL, Welke NB, Kranjac CA, Liu P, Longhurst C, Bruce JY, Hong S, Salgia R, Wheeler DL
    2022 Apr 23;22(1):447. doi: 10.1186/s12885-022-09511-6.
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      BACKGROUND: The receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) is overexpressed and an important therapeutic target in Head and Neck cancer (HNC). Cetuximab is currently the only EGFR-targeting agent approved by the FDA for treatment of HNC; however, intrinsic and acquired resistance to cetuximab is a major problem in the clinic. Our lab previously reported that AXL leads to cetuximab resistance via activation of HER3. In this study, we investigate the connection between AXL, HER3, and neuregulin1 (NRG1) gene expression with a focus on understanding how their interdependent signaling promotes resistance to cetuximab in HNC.

      METHODS: Plasmid or siRNA transfections and cell-based assays were conducted to test cetuximab sensitivity. Quantitative PCR and immunoblot analysis were used to analyze gene and protein expression levels. Seven HNC patient-derived xenografts (PDXs) were evaluated for protein expression levels.

      RESULTS: We found that HER3 expression was necessary but not sufficient for cetuximab resistance without AXL expression. Our results demonstrated that addition of the HER3 ligand NRG1 to cetuximab-sensitive HNC cells leads to cetuximab resistance. Further, AXL-overexpressing cells regulate NRG1 at the level of transcription, thereby promoting cetuximab resistance. Immunoblot analysis revealed that NRG1 expression was relatively high in cetuximab-resistant HNC PDXs compared to cetuximab-sensitive HNC PDXs. Finally, genetic inhibition of NRG1 resensitized AXL-overexpressing cells to cetuximab.

      CONCLUSIONS: The results of this study indicate that AXL may signal through HER3 via NRG1 to promote cetuximab resistance and that targeting of NRG1 could have significant clinical implications for HNC therapeutic approaches.

      PMID:35461210 | PMC:PMC9035247 | DOI:10.1186/s12885-022-09511-6


      View details for PubMedID 35461210
  • Retraction Science signaling
    Brand TM, Iida M, Corrigan KL, Braverman CM, Coan JP, Flanigan BG, Stein AP, Salgia R, Rolff J, Kimple RJ, Wheeler DL
    2021 Nov 9;14(708):eabn0168. doi: 10.1126/scisignal.abn0168. Epub 2021 Nov 9.
  • Impact of immediate cryopreservation on the establishment of patient derived xenografts from head and neck cancer patients Journal of translational medicine
    Abel L, Durmaz A, Hu R, Longhurst C, Baschnagel AM, Wheeler D, Scott JG, Kimple RJ
    2021 Apr 28;19(1):180. doi: 10.1186/s12967-021-02850-1.
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      BACKGROUND: Patient-derived xenografts established from human cancers are important tools for investigating novel anti-cancer therapies. Establishing PDXs requires a significant investment and many PDXs may be used infrequently due to their similarity to existing models, their growth rate, or the lack of relevant mutations. We performed this study to determine whether we could efficiently establish PDXs after cryopreservation to allow molecular profiling to be completed prior to implanting the human cancer.

      METHODS: Fresh tumor was split with half used to establish a PDX immediately and half cryopreserved for later implantation. Resulting tumors were assessed histologically and tumors established from fresh or cryopreserved tissues compared as to the growth rate, extent of tumor necrosis, mitotic activity, keratinization, and grade. All PDXs were subjected to short tandem repeat testing to confirm identity and assess similarity between methods.

      RESULTS: Tumor growth was seen in 70% of implanted cases. No growth in either condition was seen in 30% of tumors. One developed a SCC from the immediate implant but a lymphoproliferative mass without SCC from the cryopreserved specimen. No difference in growth rate was seen. No difference between histologic parameters was seen between the two approaches.

      CONCLUSIONS: Fresh human cancer tissue can be immediately cryopreserved and later thawed and implanted to establish PDXs. This resource saving approach allows for tumor profiling prior to implantation into animals thus maximizing the probability that the tumor will be utilized for future research.

      PMID:33910584 | PMC:PMC8082827 | DOI:10.1186/s12967-021-02850-1


      View details for PubMedID 33910584
  • Activation of EPHA2-ROBO1 Heterodimer by SLIT2 Attenuates Non-canonical Signaling and Proliferation in Squamous Cell Carcinomas iScience
    Srivastava S, Pang KM, Iida M, Nelson MS, Liu J, Nam A, Wang J, Mambetsariev I, Pillai R, Mohanty A, McDaniel N, Behal A, Kulkarni P, Wheeler DL, Salgia R
    2020 Oct 16;23(11):101692. doi: 10.1016/j.isci.2020.101692. eCollection 2020 Nov 20.
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      The tyrosine kinase receptor ephrin receptor A2 (EPHA2) is overexpressed in lung (LSCC) and head and neck (HNSCC) squamous cell carcinomas. Although EPHA2 can inhibit tumorigenesis in a ligand-dependent fashion via phosphorylation of Y588 and Y772, it can promote tumorigenesis in a ligand-independent manner via phosphorylation of S897. Here, we show that EPHA2 and Roundabout Guidance Receptor 1 (ROBO1) interact to form a functional heterodimer. Furthermore, we show that the ROBO1 ligand Slit Guidance Ligand 2 (SLIT2) and ensartinib, an inhibitor of EPHA2, can attenuate growth of HNSCC cells and act synergistically in LSCC cells. Our results suggest that patients with LSCC and HNSCC may be stratified and treated based on their EPHA2 and ROBO1 expression patterns. Although ~73% of patients with LSCC could benefit from SLIT2+ensartinib treatment, ~41% of patients with HNSCC could be treated with either SLIT2 or ensartinib. Thus, EPHA2 and ROBO1 represent potential LSCC and HNSCC theranostics.

      PMID:33196021 | PMC:PMC7644594 | DOI:10.1016/j.isci.2020.101692


      View details for PubMedID 33196021
  • Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers Cancers
    Tiwari A, Iida M, Kosnopfel C, Abbariki M, Menegakis A, Fehrenbacher B, Maier J, Schaller M, Brucker SY, Wheeler DL, Harari PM, Rothbauer U, Schittek B, Zips D, Toulany M
    2020 Sep 29;12(10):2795. doi: 10.3390/cancers12102795.
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      The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

      PMID:33003386 | PMC:PMC7601769 | DOI:10.3390/cancers12102795


      View details for PubMedID 33003386
  • An Avidity-Based PD-L1 Antagonist Using Nanoparticle-Antibody Conjugates for Enhanced Immunotherapy Nano letters
    Bu J, Nair A, Iida M, Jeong W, Poellmann MJ, Mudd K, Kubiatowicz LJ, Liu EW, Wheeler DL, Hong S
    2020 Jul 8;20(7):4901-4909. doi: 10.1021/acs.nanolett.0c00953. Epub 2020 Jun 11.
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      Upregulation of programmed death ligand 1 (PD-L1) allows cancer cells to evade antitumor immunity. Despite tremendous efforts in developing PD-1/PD-L1 immune checkpoint inhibitors (ICIs), clinical trials using such ICIs have shown inconsistent benefits. Here, we hypothesized that the ICI efficacy would be dictated by the binding strength of the inhibitor to the target proteins. To assess this, hyperbranched, multivalent poly(amidoamine) dendrimers were employed to prepare dendrimer-ICI conjugates (G7-aPD-L1). Binding kinetics measurements using SPR, BLI, and AFM revealed that G7-aPD-L1 exhibits significantly enhanced binding strength to PD-L1 proteins, compared to free aPD-L1. The binding avidity of G7-aPD-L1 was translated into in vitro efficiency and in vivo selectivity, as the conjugates improved the PD-L1 blockade effect and enhanced accumulation in tumor sites. Our results demonstrate that the dendrimer-mediated multivalent interaction substantially increases the binding avidity of the ICIs and thereby improves the antagonist effect, providing a novel platform for cancer immunotherapy.

      PMID:32510959 | PMC:PMC7737517 | DOI:10.1021/acs.nanolett.0c00953


      View details for PubMedID 32510959
  • AXL Mediates Cetuximab and Radiation Resistance Through Tyrosine 821 and the c-ABL Kinase Pathway in Head and Neck Cancer Clinical cancer research : an official journal of the American Association for Cancer Research
    McDaniel NK, Iida M, Nickel KP, Longhurst CA, Fischbach SR, Rodems TS, Kranjac CA, Bo AY, Luo Q, Gallagher MM, Welke NB, Mitchell KR, Schulz AE, Eckers JC, Hu R, Salgia R, Hong S, Bruce JY, Kimple RJ, Wheeler DL
    2020 Aug 15;26(16):4349-4359. doi: 10.1158/1078-0432.CCR-19-3142. Epub 2020 May 21.
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      PURPOSE: Radiation and cetuximab are therapeutics used in management of head and neck squamous cell carcinoma (HNSCC). Despite clinical success with these modalities, development of both intrinsic and acquired resistance is an emerging problem in the management of this disease. The purpose of this study was to investigate signaling of the receptor tyrosine kinase AXL in resistance to radiation and cetuximab treatment.

      EXPERIMENTAL DESIGN: To study AXL signaling in the context of treatment-resistant HNSCC, we used patient-derived xenografts (PDXs) implanted into mice and evaluated the tumor response to AXL inhibition in combination with cetuximab or radiation treatment. To identify molecular mechanisms of how AXL signaling leads to resistance, three tyrosine residues of AXL (Y779, Y821, Y866) were mutated and examined for their sensitivity to cetuximab and/or radiation. Furthermore, reverse phase protein array (RPPA) was employed to analyze the proteomic architecture of signaling pathways in these genetically altered cell lines.

      RESULTS: Treatment of cetuximab- and radiation-resistant PDXs with AXL inhibitor R428 was sufficient to overcome resistance. RPPA analysis revealed that such resistance emanates from signaling of tyrosine 821 of AXL via the tyrosine kinase c-ABL. In addition, inhibition of c-ABL signaling resensitized cells and tumors to cetuximab or radiotherapy even leading to complete tumor regression without recurrence in head and neck cancer models.

      CONCLUSIONS: Collectively, the studies presented herein suggest that tyrosine 821 of AXL mediates resistance to cetuximab by activation of c-ABL kinase in HNSCC and that targeting of both EGFR and c-ABL leads to a robust antitumor response.

      PMID:32439698 | PMC:PMC7442604 | DOI:10.1158/1078-0432.CCR-19-3142


      View details for PubMedID 32439698
  • EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma Oncogenesis
    Tan YC, Srivastava S, Won BM, Kanteti R, Arif Q, Husain AN, Li H, Vigneswaran WT, Pang K, Kulkarni P, Sattler M, Vaidehi N, Mambetsariev I, Kindler HL, Wheeler DL, Salgia R
    2019 Sep 4;8(9):49. doi: 10.1038/s41389-019-0159-6.
    • More

      Squamous cell carcinoma (SCC) and malignant pleural mesothelioma (MPM) are thoracic malignancies with very poor prognosis and limited treatment options. It is an established fact that most of the solid tumors have overexpression of EPHA2 receptor tyrosine kinase. EPHA2 is known to exhibit opposing roles towards cancer progression. It functions in inhibiting cancer survival and migration via a ligand and tyrosine kinase dependent signaling (Y772). Whereas it is known to promote tumor progression and cell migration through a ligand-independent signaling (S897). We analyzed the expression profile and mutational status of the ephrin receptor A2 (EPHA2) in SCC and MPM cell lines and primary patient specimens. The EPHA2 receptor was found to be either overexpressed, mutated or amplified in SCC and MPM. In particular, the EPHA2 mutants A859D and T647M were interesting to explore, A859D Y772 dead mutant exhibited lower levels of phosphorylation at Y772 compared to T647M mutant. Molecular Dynamics simulations studies suggested that differential changes in conformation might form the structural basis for differences in the level of EPHA2 activation. Consequently, A859D mutant cells exhibited increased proliferation as well as cell migration compared to controls and T647M mutant. Kinomics analysis demonstrated that the STAT3 and PDGF pathways were upregulated whereas signaling through CBL was suppressed. Considered together, the present work has uncovered the oncogenic characteristics of EPHA2 mutations in SSC and MPM reinstating the dynamics of different roles of EPHA2 in cancer. This study also suggests that a combination of doxazosin and other EPHA2 inhibitors directed to inhibit the pertinent signaling components may be a novel therapeutic strategy for MPM and Non-small cell lung cancer patients who have either EPHA2 or CBL alterations.

      PMID:31484920 | PMC:PMC6726628 | DOI:10.1038/s41389-019-0159-6


      View details for PubMedID 31484920
  • Correction: Nuclear Epidermal Growth Factor Receptor Is a Functional Molecular Target in Triple-negative Breast Cancer Molecular cancer therapeutics
    Brand TM, Iida M, Dunn EF, Luthar N, Kostopoulos KT, Corrigan KL, Wleklinski MJ, Yang D, Wisinski KB, Salgia R, Wheeler DL
    2019 Apr;18(4):868. doi: 10.1158/1535-7163.MCT-18-1183.
  • Correction: AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma Clinical cancer research : an official journal of the American Association for Cancer Research
    Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Coan JP, Pearson HE, Bahrar H, Fowler TL, Bednarz BP, Saha S, Yang D, Gill PS, Lingen MW, Saloura V, Villaflor VM, Salgia R, Kimple RJ, Wheeler DL
    2018 Dec 1;24(23):6099. doi: 10.1158/1078-0432.CCR-18-3194.
  • Corrigendum to: "Nuclear EGFR as a molecular target in cancer" [Radiother Oncol 108 (2013) 370-77] Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology
    Brand TM, Iida M, Luthar N, Starr MM, Huppert EJ, Wheeler DL
    2019 Jan;130:195. doi: 10.1016/j.radonc.2018.10.011. Epub 2018 Nov 13.
  • MERTK Mediates Intrinsic and Adaptive Resistance to AXL-targeting Agents Molecular cancer therapeutics
    McDaniel NK, Cummings CT, Iida M, Hülse J, Pearson HE, Vasileiadi E, Parker RE, Orbuch RA, Ondracek OJ, Welke NB, Kang GH, Davies KD, Wang X, Frye SV, Earp HS, Harari PM, Kimple RJ, DeRyckere D, Graham DK, Wheeler DL
    2018 Nov;17(11):2297-2308. doi: 10.1158/1535-7163.MCT-17-1239. Epub 2018 Aug 9.
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      The TAM (TYRO3, AXL, MERTK) family receptor tyrosine kinases (RTK) play an important role in promoting growth, survival, and metastatic spread of several tumor types. AXL and MERTK are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. AXL is the most well-characterized TAM receptor and mediates resistance to both conventional and targeted cancer therapies. AXL is highly expressed in aggressive tumor types, and patients with cancer are currently being enrolled in clinical trials testing AXL inhibitors. In this study, we analyzed the effects of AXL inhibition using a small-molecule AXL inhibitor, a monoclonal antibody (mAb), and siRNA in HNSCC, TNBC, and NSCLC preclinical models. Anti-AXL-targeting strategies had limited efficacy across these different models that, our data suggest, could be attributed to upregulation of MERTK. MERTK expression was increased in cell lines and patient-derived xenografts treated with AXL inhibitors and inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibition. Dual targeting of AXL and MERTK led to a more potent blockade of downstream signaling, synergistic inhibition of tumor cell expansion in culture, and reduced tumor growth in vivo Furthermore, ectopic overexpression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These observations suggest that therapeutic strategies cotargeting both AXL and MERTK could be highly beneficial in a variety of tumor types where both receptors are expressed, leading to improved survival for patients with lethal malignancies. Mol Cancer Ther; 17(11); 2297-308. ©2018 AACR.

      PMID:30093568 | PMC:PMC6215511 | DOI:10.1158/1535-7163.MCT-17-1239


      View details for PubMedID 30093568
  • Exosomal miRNAs species in the blood of small cell and non-small cell lung cancer patients Oncotarget
    Poroyko V, Mirzapoiazova T, Nam A, Mambetsariev I, Mambetsariev B, Wu X, Husain A, Vokes EE, Wheeler DL, Salgia R
    2018 Apr 13;9(28):19793-19806. doi: 10.18632/oncotarget.24857. eCollection 2018 Apr 13.
    • More

      Lung cancer is a devastating disease with overall bleak prognosis. Current methods to diagnose lung cancer are rather invasive and are inadequate to detect the disease at an early stage when treatment is likely to be most effective. In this study, a shotgun sequencing approach was used to study the microRNA (miRNA) cargo of serum-derived exosomes of small cell lung cancer (SCLC) (n=9) and non-small cell lung cancer (NSCLC) (n=11) patients, and healthy controls (n=10). The study has identified 17 miRNA species that are differentially expressed in cancer patients and control subjects. Furthermore, within the patient groups, a set of miRNAs were differentially expressed in exosomal samples obtained before and after chemotherapy treatment. This manuscript demonstrates the potential of exosomal miRNAs for developing noninvasive tests for disease differentiation and treatment monitoring in lung cancer patients.

      PMID:29731983 | PMC:PMC5929426 | DOI:10.18632/oncotarget.24857


      View details for PubMedID 29731983
  • Anti-Trop2 blockade enhances the therapeutic efficacy of ErbB3 inhibition in head and neck squamous cell carcinoma Cell death & disease
    Redlich N, Robinson AM, Nickel KP, Stein AP, Wheeler DL, Adkins DR, Uppaluri R, Kimple RJ, Van Tine A, Michel LS
    2018 Jan 5;9(1):5. doi: 10.1038/s41419-017-0029-0.
    • More

      ErbB3 has been widely implicated in treatment resistance, but its role as a primary treatment target is less clear. Canonically ErbB3 requires EGFR or ErbB2 for activation, whereas these two established treatment targets are thought to signal independently of ErbB3. In this study, we show that ErbB3 is essential for tumor growth of treatment-naive HNSCC patient-derived xenografts. This ErbB3 dependency occurs via ErbB3-mediated control of EGFR activation and HIF1α stabilization, which require ErbB3 and its ligand neuregulin-1. Here, we show that ErbB3 antibody treatment selects for a population of ErbB3-persister cells that express high levels of the transmembrane protein Trop2 that we previously identified as an inhibitor of ErbB3. Co-treatment with anti-ErbB3 and anti-Trop2 antibodies is synergistic and produces a greater anti-tumor response than either antibody alone. Collectively, these data both compel a revision of ErbB-family signaling and delineate a strategy for its effective inhibition in HNSCC.

      PMID:29305574 | PMC:PMC5849045 | DOI:10.1038/s41419-017-0029-0


      View details for PubMedID 29305574
  • Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells Cell death discovery
    Toulany M, Maier J, Iida M, Rebholz S, Holler M, Grottke A, Jüker M, Wheeler DL, Rothbauer U, Rodemann HP
    2017 Oct 30;3:17072. doi: 10.1038/cddiscovery.2017.72. eCollection 2017.
    • More

      Akt1 through the C-terminal domain interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulates the repair of DNA double-strand breaks (DSBs) in K-RAS-mutated (K-RASmut) cells. We investigated the interactions of distinct domain(s) of DNA-PKcs in binding to full-length Akt1. Similarly, we analyzed potential interactions of DNA-PKcs with Akt2 and Akt3. Finally the effect of Akt isoforms in cell proliferation and tumor growth was tested. We demonstrated that Akt1 preferentially binds to the N-terminal domain of DNA-PKcs using pull-down studies with distinct eGFP-tagged DNA-PKcs fragments that were expressed by plasmids in combination with mCherry-tagged full-length Akt isoforms. These binding studies also indicated an interaction with the intermediate and C-terminal domains of DNA-PKcs. In contrast, Akt3 interacted with all four DNA-PKcs fragments without a marked preference for any specific domain. Notably, we could not see binding of Akt2 to any of the tested DNA-PKcs fragments. In subsequent studies, we demonstrated that Akt inhibition interferes with binding of Akt1 to the N-terminal domain of DNA-PKcs. This indicated a correlation between Akt1 activity and the Akt1/DNA-PKcs complex formation. Finally, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)-induced DNA DSBs, leading to radiosensitization. Furthermore, in a xenograft study the expression of shAkt1 or shAkt3, but not shAkt2 in K-RASmut breast cancer cell line MDA-MB-231 showed major tumor growth delay. Together, these data indicate that Akt1 and Akt3, but not Akt2, physically interact with DNA-PKcs, thus stimulating the repair of DSBs and therefore protecting K-RASmut cells against IR. Likewise, interaction of Akt isoforms with DNA-PKcs could be crucial for their role in regulating tumor growth.

      PMID:29090098 | PMC:PMC5661268 | DOI:10.1038/cddiscovery.2017.72


      View details for PubMedID 29090098
  • Overcoming Resistance to Cetuximab with Honokiol, A Small-Molecule Polyphenol Molecular cancer therapeutics
    Pearson HE, Iida M, Orbuch RA, McDaniel NK, Nickel KP, Kimple RJ, Arbiser JL, Wheeler DL
    2018 Jan;17(1):204-214. doi: 10.1158/1535-7163.MCT-17-0384. Epub 2017 Oct 20.
    • More

      Overexpression and activation of the EGFR have been linked to poor prognosis in several human cancers. Cetuximab is a mAb against EGFR that is used for the treatment in head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancer. Unfortunately, most tumors have intrinsic or will acquire resistance to cetuximab during the course of therapy. Honokiol is a natural compound found in the bark and leaves of the Chinese Magnolia tree and is established to have several anticancer properties without appreciable toxicity. In this study, we hypothesized that combining cetuximab and honokiol treatments could overcome acquired resistance to cetuximab. We previously developed a model of acquired resistance to cetuximab in non-small cell lung cancer H226 cell line. Treatment of cetuximab-resistant clones with honokiol and cetuximab resulted in a robust antiproliferative response. Immunoblot analysis revealed the HER family and their signaling pathways were downregulated after combination treatment, most notably the proliferation (MAPK) and survival (AKT) pathways. In addition, we found a decrease in phosphorylation of DRP1 and reactive oxygen species after combination treatment in cetuximab-resistant clones, which may signify a change in mitochondrial function. Furthermore, we utilized cetuximab-resistant HNSCC patient-derived xenografts (PDX) to test the benefit of combinatorial treatment in vivo There was significant growth delay in PDX tumors after combination treatment with a subsequent downregulation of active MAPK, AKT, and DRP1 signaling as seen in vitro Collectively, these data suggest that honokiol is a promising natural compound in overcoming acquired resistance to cetuximab. Mol Cancer Ther; 17(1); 204-14. ©2017 AACR.

      PMID:29054984 | PMC:PMC5752575 | DOI:10.1158/1535-7163.MCT-17-0384


      View details for PubMedID 29054984
  • Cotargeting mTORC and EGFR Signaling as a Therapeutic Strategy in HNSCC Molecular cancer therapeutics
    Swick AD, Prabakaran PJ, Miller MC, Javaid AM, Fisher MM, Sampene E, Ong IM, Hu R, Iida M, Nickel KP, Bruce JY, Wheeler DL, Kimple RJ
    2017 Jul;16(7):1257-1268. doi: 10.1158/1535-7163.MCT-17-0115. Epub 2017 Apr 26.
    • More

      Head and neck squamous cell carcinomas (HNSCC) are frequently altered along the PI3K/AKT/mTORC signaling axis. Despite excellent preclinical data, the use of compounds targeting this pathway as monotherapy has been underwhelming in initial clinical trials, and identification of predictive biomarkers remains challenging. To investigate mTORC-specific inhibition, we tested catalytic mTORC (AZD8055) and PI3K/mTORC (NVP-BEZ-235) inhibitors ± cetuximab in a panel of HNSCC cell lines and patient-derived xenografts (PDX). Cell lines were assayed for response to all agents and siRNA knockdown of targets by multiple approaches. All cell lines showed similar response to both drug and siRNA inhibition of both PI3K and mTORC pathways, with anti-EGFR combination producing modest additive effect. Five PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and AZD8055. In vivo single-agent mTORC inhibition inhibited growth of one PIK3CA-mutant cancer, but had little effect on any PIK3CAWT or a second PIK3CA-mutant model. In all models, the combination therapy showed greater growth delay than monotherapy. The uniform ability of PI3K and mTORC inhibition to suppress the growth of HNSCC cells highlights the pathway's role in driving proliferation. Although single-agent therapy was largely ineffective in vivo, improved response of combination treatment in an array of PDXs suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy. Overall, these results add to a growing body of evidence, suggesting that approaches that attempt to match biomarkers to the optimal therapy in HNSCC remain complex and challenging. Mol Cancer Ther; 16(7); 1257-68. ©2017 AACR.

      PMID:28446642 | PMC:PMC5505754 | DOI:10.1158/1535-7163.MCT-17-0115


      View details for PubMedID 28446642
  • Targeting the HER Family with Pan-HER Effectively Overcomes Resistance to Cetuximab Molecular cancer therapeutics
    Iida M, Bahrar H, Brand TM, Pearson HE, Coan JP, Orbuch RA, Flanigan BG, Swick AD, Prabakaran PJ, Lantto J, Horak ID, Kragh M, Salgia R, Kimple RJ, Wheeler DL
    2016 Sep;15(9):2175-86. doi: 10.1158/1535-7163.MCT-16-0012. Epub 2016 Jul 15.
    • More

      Cetuximab, an antibody against the EGFR, has shown efficacy in treating head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer, and non-small cell lung cancer (NSCLC). Despite the clinical success of cetuximab, many patients do not respond to cetuximab. Furthermore, virtually all patients who do initially respond become refractory, highlighting both intrinsic and acquired resistance to cetuximab as significant clinical problems. To understand mechanistically how cancerous cells acquire resistance, we previously developed models of acquired resistance using the H226 NSCLC and UM-SCC1 HNSCC cell lines. Cetuximab-resistant clones showed a robust upregulation and dependency on the HER family receptors EGFR, HER2, and HER3. Here, we examined pan-HER, a mixture of six antibodies targeting these receptors on cetuximab-resistant clones. In cells exhibiting acquired or intrinsic resistance to cetuximab, pan-HER treatment decreased all three receptors' protein levels and downstream activation of AKT and MAPK. This correlated with decreased cell proliferation in cetuximab-resistant clones. To determine whether pan-HER had a therapeutic benefit in vivo, we established de novo cetuximab-resistant mouse xenografts and treated resistant tumors with pan-HER. This regimen resulted in a superior growth delay of cetuximab-resistant xenografts compared with mice continued on cetuximab. Furthermore, intrinsically cetuximab-resistant HNSCC patient-derived xenograft tumors treated with pan-HER exhibited significant growth delay compared with vehicle/cetuximab controls. These results suggest that targeting multiple HER family receptors simultaneously with pan-HER is a promising treatment strategy for tumors displaying intrinsic or acquired resistance to cetuximab. Mol Cancer Ther; 15(9); 2175-86. ©2016 AACR.

      PMID:27422810 | PMC:PMC5010956 | DOI:10.1158/1535-7163.MCT-16-0012


      View details for PubMedID 27422810
  • Dual targeting of PI3K and MEK enhances the radiation response of K-RAS mutated non-small cell lung cancer Oncotarget
    Toulany M, Iida M, Keinath S, Iyi FF, Mueck K, Fehrenbacher B, Mansour WY, Schaller M, Wheeler DL, Rodemann HP
    2016 Jul 12;7(28):43746-43761. doi: 10.18632/oncotarget.9670.
    • More

      Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.

      PMID:27248324 | PMC:PMC5190057 | DOI:10.18632/oncotarget.9670


      View details for PubMedID 27248324
  • Feedback regulation of biological networks: Examples relevant to signalling pathways and resistance to pharmacological interceptors Seminars in cell & developmental biology
    Yarden Y, Wheeler DL
    2016 Feb;50:83-4. doi: 10.1016/j.semcdb.2016.02.016.
  • Adaptive responses to antibody based therapy Seminars in cell & developmental biology
    Rodems TS, Iida M, Brand TM, Pearson HE, Orbuch RA, Flanigan BG, Wheeler DL
    2016 Feb;50:153-63. doi: 10.1016/j.semcdb.2016.01.001. Epub 2016 Jan 22.
    • More

      Receptor tyrosine kinases (RTKs) represent a large class of protein kinases that span the cellular membrane. There are 58 human RTKs identified which are grouped into 20 distinct families based upon their ligand binding, sequence homology and structure. They are controlled by ligand binding which activates intrinsic tyrosine-kinase activity. This activity leads to the phosphorylation of distinct tyrosines on the cytoplasmic tail, leading to the activation of cell signaling cascades. These signaling cascades ultimately regulate cellular proliferation, apoptosis, migration, survival and homeostasis of the cell. The vast majority of RTKs have been directly tied to the etiology and progression of cancer. Thus, using antibodies to target RTKs as a cancer therapeutic strategy has been intensely pursued. Although antibodies against the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) have shown promise in the clinical arena, the development of both intrinsic and acquired resistance to antibody-based therapies is now well appreciated. In this review we provide an overview of the RTK family, the biology of EGFR and HER2, as well as an in-depth review of the adaptive responses undertaken by cells in response to antibody based therapies directed against these receptors. A greater understanding of these mechanisms and their relevance in human models will lead to molecular insights in overcoming and circumventing resistance to antibody based therapy.

      PMID:26808665 | DOI:10.1016/j.semcdb.2016.01.001


      View details for PubMedID 26808665
  • Genetic deletion of TNFα inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas in PKCε transgenic mice via inhibition of cell survival signals Carcinogenesis
    Singh A, Singh A, Bauer SJ, Wheeler DL, Havighurst TC, Kim K, Verma AK
    2016 Jan;37(1):72-80. doi: 10.1093/carcin/bgv162. Epub 2015 Nov 19.
    • More

      Protein kinase C epsilon (PKCε), a Ca(2+)-independent phospholipid-dependent serine/threonine kinase, is among the six PKC isoforms (α, δ, ε, η, μ, ζ) expressed in both mouse and human skin. Epidermal PKCε level dictates the susceptibility of PKCε transgenic (TG) mice to the development of cutaneous squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by using the DMBA initiation-TPA (12-O-tetradecanoylphorbol-13-acetate) tumor promotion protocol (Wheeler,D.L. et al. (2004) Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas. Cancer Res., 64, 7756-7765). Histologically, SCC in TG mice, like human SCC, is poorly differentiated and metastatic. Our earlier studies to elucidate mechanisms of PKCε-mediated development of SCC, using either DMBA-TPA or UVR, indicated elevated release of cytokine TNFα. To determine whether TNFα is essential for the development of SCC in TG mice, we generated PKCε transgenic mice/TNFα-knockout (TG/TNFαKO) by crossbreeding TNFαKO with TG mice. We now present that deletion of TNFα in TG mice inhibited the development of SCC either by repeated UVR exposures or by the DMBA-TPA protocol. TG mice deficient in TNFα elicited both increase in SCC latency and decrease in SCC incidence. Inhibition of UVR-induced SCC development in TG/TNFαKO was accompanied by inhibition of (i) the expression levels of TNFα receptors TNFRI and TNFRII and cell proliferation marker ornithine decarboxylase and metastatic markers MMP7 and MMP9, (ii) the activation of transcription factors Stat3 and NF-kB and (iii) proliferation of hair follicle stem cells and epidermal hyperplasia. The results presented here provide the first genetic evidence that TNFα is linked to PKCε-mediated sensitivity to DMBA-TPA or UVR-induced development of cutaneous SCC.

      PMID:26586792 | PMC:PMC5006113 | DOI:10.1093/carcin/bgv162


      View details for PubMedID 26586792
  • AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma Clinical cancer research : an official journal of the American Association for Cancer Research
    Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Coan JP, Pearson HE, Bahrar H, Fowler TL, Bednarz BP, Saha S, Yang D, Gill PS, Lingen MW, Saloura V, Villaflor VM, Salgia R, Kimple RJ, Wheeler DL
    2015 Jun 1;21(11):2601-12. doi: 10.1158/1078-0432.CCR-14-2648. Epub 2015 Mar 12.
    • More

      PURPOSE: Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard-of-care treatments for patients with HNSCC include surgery, radiation, and chemotherapy. In addition, the anti-EGFR monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult malignancy to treat. Thus, identification of new molecular targets is critical.

      EXPERIMENTAL DESIGN: In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient-derived xenografts (PDX), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small-molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard-of-care treatment regimens used in HNSCC.

      RESULTS: AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases, and shorter relapse-free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. In addition, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation-resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance.

      CONCLUSIONS: This study provides evidence for the role of AXL in HNSCC pathogenesis and supports further preclinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC.

      PMID:25767293 | PMC:PMC5032632 | DOI:10.1158/1078-0432.CCR-14-2648


      View details for PubMedID 25767293
  • Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy Molecular cancer
    Iida M, Brand TM, Starr MM, Huppert EJ, Luthar N, Bahrar H, Coan JP, Pearson HE, Salgia R, Wheeler DL
    2014 Oct 24;13:242. doi: 10.1186/1476-4598-13-242.
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      BACKGROUND: Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (Ctx(R)) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in Ctx(R) clones due to decreased EGFR ubiquitination and degradation and 2) Ctx(R) clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the Ctx(R) setting.

      RESULTS: Since HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in Ctx(R) models was evaluated. First, EGFR and HER3 expression were knocked down with siRNAs. Compared to the Ctx(S) parental cell line (HP), all Ctx(R) clones exhibited robust decreases in cell proliferation upon dual knockdown. Analysis of Ctx(R) clones indicated that neuregulin-1 was highly overexpressed compared to HP cells. Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects. Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo. Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy.

      CONCLUSIONS: These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.

      PMID:25344208 | PMC:PMC4283113 | DOI:10.1186/1476-4598-13-242


      View details for PubMedID 25344208
  • AXL mediates resistance to cetuximab therapy Cancer research
    Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Luthar N, Toulany M, Gill PS, Salgia R, Kimple RJ, Wheeler DL
    2014 Sep 15;74(18):5152-64. doi: 10.1158/0008-5472.CAN-14-0294. Epub 2014 Aug 18.
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      The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR.

      PMID:25136066 | PMC:PMC4167493 | DOI:10.1158/0008-5472.CAN-14-0294


      View details for PubMedID 25136066
  • Nuclear epidermal growth factor receptor is a functional molecular target in triple-negative breast cancer Molecular cancer therapeutics
    Brand TM, Iida M, Dunn EF, Luthar N, Kostopoulos KT, Corrigan KL, Wleklinski MJ, Yang D, Wisinski KB, Salgia R, Wheeler DL
    2014 May;13(5):1356-68. doi: 10.1158/1535-7163.MCT-13-1021. Epub 2014 Mar 14.
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      Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e., estrogen receptor-negative, progesterone receptor-negative, and HER2-negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBCs overexpresses the EGF receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: (i) classical membrane-bound signaling and (ii) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. On the basis of these findings, we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Furthermore, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of patients with TNBC.

      PMID:24634415 | PMC:PMC4013210 | DOI:10.1158/1535-7163.MCT-13-1021


      View details for PubMedID 24634415
  • Combined MET inhibition and topoisomerase I inhibition block cell growth of small cell lung cancer Molecular cancer therapeutics
    Rolle CE, Kanteti R, Surati M, Nandi S, Dhanasingh I, Yala S, Tretiakova M, Arif Q, Hembrough T, Brand TM, Wheeler DL, Husain AN, Vokes EE, Bharti A, Salgia R
    2014 Mar;13(3):576-84. doi: 10.1158/1535-7163.MCT-13-0109. Epub 2013 Dec 10.
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      Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)-induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC.

      PMID:24327519 | PMC:PMC4286701 | DOI:10.1158/1535-7163.MCT-13-0109


      View details for PubMedID 24327519
  • Sym004, a novel EGFR antibody mixture, can overcome acquired resistance to cetuximab Neoplasia (New York, N.Y.)
    Iida M, Brand TM, Starr MM, Li C, Huppert EJ, Luthar N, Pedersen MW, Horak ID, Kragh M, Wheeler DL
    2013 Oct;15(10):1196-206. doi: 10.1593/neo.131584.
    • More

      The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don't respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resistance using the non-small cell lung cancer line NCI-H226. These cetuximab-resistant (Ctx(R)) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for Ctx(R) tumor cells. Sym004 treatment of Ctx(R) clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo Ctx(R) NCI-H226 mouse xenografts and subsequently treated Ctx(R) tumors with Sym004. Sym004 treatment of mice harboring Ctx(R) tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly decreased in Ctx(R) tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for Ctx(R) tumors.

      PMID:24204198 | PMC:PMC3819635 | DOI:10.1593/neo.131584


      View details for PubMedID 24204198
  • Combining radiotherapy with MEK1/2, STAT5 or STAT6 inhibition reduces survival of head and neck cancer lines Molecular cancer
    Stegeman H, Kaanders AM, Verheijen MG, Peeters JM, Wheeler DL, Iida M, Grénman R, Kogel vd, Span PN, Bussink J
    2013 Nov 5;12(1):133. doi: 10.1186/1476-4598-12-133.
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      BACKGROUND: Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients.

      METHODS: An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines.

      RESULTS: The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition.

      CONCLUSIONS: Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.

      PMID:24192080 | PMC:PMC3842630 | DOI:10.1186/1476-4598-12-133


      View details for PubMedID 24192080
  • Human epidermal growth factor receptor 3 (HER3) blockade with U3-1287/AMG888 enhances the efficacy of radiation therapy in lung and head and neck carcinoma Discovery medicine
    Li C, Brand TM, Iida M, Huang S, Armstrong EA, Kogel vd, Wheeler DL
    2013 Sep;16(87):79-92.
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      HER3 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In the present study, we investigated the capacity of the HER3 blocking antibody, U3-1287/AMG888, to modulate the in vitro and in vivo radiation response of human squamous cell carcinomas of the lung and head and neck. We screened a battery of cell lines from these tumors for HER3 expression and demonstrated that all cell lines screened exhibited expression of HER3. Importantly, U3-1287/AMG888 treatment could block both basal HER3 activity and radiation induced HER3 activation. Proliferation assays indicated that HER3 blockade could decrease the proliferation of both HNSCC cell line SCC6 and NSCLC cell line H226. Further, we demonstrated that U3-1287/AMG888 can sensitize cells to radiation in clonogenic survival assays, in addition to increasing DNA damage as detected via λ-H2AX immunofluorescence. To determine if U3-1287/AMG888 could enhance radiation sensitivity in vivo we performed tumor growth delay experiments using SCC6, SCC1483, and H226 xenografts. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation could decrease tumor growth in studies using single or fractionated doses of radiation. Analysis of HER3 expression in tumor samples indicated that radiation treatment activated HER3 in vivo and that U3-1287/AMG888 could abrogate this activation. Immunohistochemistry analysis of SCC6 tumors treated with both U3-1287/AMG888 and a single dose of radiation demonstrated that various cell survival and proliferation markers could be reduced. Collectively our findings suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by increasing DNA damage and cell death. These findings suggest that HER3 may play an important role in response to radiation therapy and blocking its activity in combination with radiation may be of therapeutic benefit in human tumors.

      PMID:23998444 | PMC:PMC3901945


      View details for PubMedID 23998444
  • Mapping C-terminal transactivation domains of the nuclear HER family receptor tyrosine kinase HER3 PloS one
    Brand TM, Iida M, Luthar N, Wleklinski MJ, Starr MM, Wheeler DL
    2013 Aug 8;8(8):e71518. doi: 10.1371/journal.pone.0071518. eCollection 2013.
    • More

      Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁) and 27 (B₂) amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

      PMID:23951180 | PMC:PMC3738522 | DOI:10.1371/journal.pone.0071518


      View details for PubMedID 23951180
  • Dasatinib Inhibits DNA Repair after Radiotherapy Specifically in pSFK-Expressing Tumor Areas in Head and Neck Xenograft Tumors Translational oncology
    Stegeman H, Span PN, Rijken JW, Cockx SC, Wheeler DL, Iida M, Kogel vd, Kaanders AM, Bussink J
    2013 Aug 1;6(4):413-9. doi: 10.1593/tlo.13259. Print 2013 Aug.
    • More

      Src family kinases (SFKs) have been implicated in resistance to both radiation and epidermal growth factor receptor (EGFR) inhibition. Therefore, we investigated whether inhibition of SFK through dasatinib (DSB) can enhance the effect of radiotherapy in two in vivo human head and neck squamous cell carcinoma (HNSCC) models. Response to DSB and/or radiotherapy was assessed with tumor growth delay assays in two HNSCC xenograft models, SCCNij153 and SCCNij202. Effects on EGFR signaling were evaluated with Western blot analysis, and effects on DNA repair, hypoxia, and proliferation were investigated with immunohistochemistry. DSB and radiotherapy induced a significant growth delay in both HNSCC xenograft models, although to a lesser extent in SCCNij202. DSB did not inhibit phosphorylated protein kinase B (pAKT) or phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) but did inhibit (phosphorylated) DNA-dependent protein kinase. Moreover, DSB reduced repair of radiation-induced DNA double-strand breaks as shown by an increase of p53-binding protein 1 (53BP1) staining 24 hours after radiation. This effect on DNA repair was only observed in the cell compartment where phosphorylated SFK (pSFK) was expressed: for SCCNij153 tumors in both normoxic and hypoxic areas and for SCCNij202 tumors only in hypoxic areas. No consistent effects of DSB on hypoxia or proliferation were observed. In conclusion, DSB enhances the effect of radiotherapy in vivo by inhibition of radiation-induced DNA repair and is a promising way to improve outcome in HNSCC patients.

      PMID:23908684 | PMC:PMC3730016 | DOI:10.1593/tlo.13259


      View details for PubMedID 23908684
  • Nuclear EGFR as a molecular target in cancer Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology
    Brand TM, Iida M, Luthar N, Starr MM, Huppert EJ, Wheeler DL
    2013 Sep;108(3):370-7. doi: 10.1016/j.radonc.2013.06.010. Epub 2013 Jul 3.
    • More

      The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cell's nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future.

      PMID:23830194 | PMC:PMC3818450 | DOI:10.1016/j.radonc.2013.06.010


      View details for PubMedID 23830194
  • Mouse models of the skin: models to define mechanisms of skin carcinogenesis Journal of skin cancer
    Wheeler DL, Verma AK, Denning MF
    2013;2013:971495. doi: 10.1155/2013/971495. Epub 2013 Jun 2.
  • Paxillin mutations affect focal adhesions and lead to altered mitochondrial dynamics: relevance to lung cancer Cancer biology & therapy
    Kawada I, Hasina R, Lennon FE, Bindokas VP, Usatyuk P, Tan YC, Krishnaswamy S, Arif Q, Carey G, Hseu RD, Robinson M, Tretiakova M, Brand TM, Iida M, Ferguson MK, Wheeler DL, Husain AN, Natarajan V, Vokes EE, Singleton PA, Salgia R
    2013 Jul;14(7):679-91. doi: 10.4161/cbt.25091. Epub 2013 May 31.
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      Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.

      PMID:23792636 | PMC:PMC3742497 | DOI:10.4161/cbt.25091


      View details for PubMedID 23792636
  • Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab Cancer biology & therapy
    Iida M, Brand TM, Campbell DA, Starr MM, Luthar N, Traynor AM, Wheeler DL
    2013 Jun;14(6):481-91. doi: 10.4161/cbt.24342.
    • More

      The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.

      PMID:23760490 | PMC:PMC3813564 | DOI:10.4161/cbt.24342


      View details for PubMedID 23760490
  • Nuclear EGFR protein expression predicts poor survival in early stage non-small cell lung cancer Lung cancer (Amsterdam, Netherlands)
    Traynor AM, Weigel TL, Oettel KR, Yang DT, Zhang C, Kim K, Salgia R, Iida M, Brand TM, Hoang T, Campbell TC, Hernan HR, Wheeler DL
    2013 Jul;81(1):138-41. doi: 10.1016/j.lungcan.2013.03.020. Epub 2013 Apr 28.
    • More

      INTRODUCTION: Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC).

      METHODS: Resected stages I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive.

      RESULTS: Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p = 0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p = 0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1-10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5-17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15-3.10]; p = 0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3-22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1-29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12-2.99]; p = 0.014).

      CONCLUSIONS: Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC.

      PMID:23628526 | PMC:PMC3679338 | DOI:10.1016/j.lungcan.2013.03.020


      View details for PubMedID 23628526
  • Predictive value of hypoxia, proliferation and tyrosine kinase receptors for EGFR-inhibition and radiotherapy sensitivity in head and neck cancer models Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology
    Stegeman H, Kaanders JH, Kogel vd, Iida M, Wheeler DL, Span PN, Bussink J
    2013 Mar;106(3):383-9. doi: 10.1016/j.radonc.2013.02.001. Epub 2013 Feb 28.
    • More

      BACKGROUND AND PURPOSE: EGFR-inhibitor Cetuximab (C225) improves the efficacy of radiotherapy in only a subgroup of HNSCC patients. Identification of predictive tumor characteristics is essential to improve patient selection.

      MATERIAL AND METHODS: Response to C225 and/or radiotherapy was assessed with tumor growth delay assays in 4 HNSCC xenograft models with varying EGFR-expression levels. Hypoxia and proliferation were quantified with immunohistochemistry and the expression of proteins involved in C225-resistance with Western blot.

      RESULTS: EGFR-expression did not predict response to C225 and/or radiotherapy. Reduction of hypoxia by C225 was only observed in SCCNij202, which was highly sensitive to C225. Proliferation changes correlated with response to C225 and C225 combined with radiotherapy, as proliferation decreased after C225 treatment in C225-sensitive SCCNij202 and after combined treatment in SCCNij185, which showed a synergistic effect to combined C225-radiotherapy. Furthermore, C225-resistant SCCNij153 tumors expressed high levels of (activated) HER3 and MET.

      CONCLUSIONS: EGFR-expression is needed for C225-response, but is not sufficient to predict response to C225 with or without radiotherapy. However, basal expression of additional growth factor receptors and effects on proliferation, but not hypoxia, correlated with response to combined C225-radiotherapy treatment and are potential clinically relevant predictive biomarkers.

      PMID:23453541 | PMC:PMC3627829 | DOI:10.1016/j.radonc.2013.02.001


      View details for PubMedID 23453541
  • Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck BMC cancer
    Stegeman H, Kaanders JH, Wheeler DL, Kogel vd, Verheijen MM, Waaijer SJ, Iida M, Grénman R, Span PN, Bussink J
    2012 Oct 10;12:463. doi: 10.1186/1471-2407-12-463.
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      BACKGROUND: Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo.

      METHODS: Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O(2) and treated with MK-2206.

      RESULTS: We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs = 0.77, p = 0.10, rs = 0.89, p = 0.03) and rs = 0.93, p = 0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all r(s)<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs = 0.51, p = 0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells.

      CONCLUSIONS: These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.

      PMID:23046567 | PMC:PMC3517352 | DOI:10.1186/1471-2407-12-463


      View details for PubMedID 23046567
  • Activation of multiple ERBB family receptors mediates glioblastoma cancer stem-like cell resistance to EGFR-targeted inhibition Neoplasia (New York, N.Y.)
    Clark PA, Iida M, Treisman DM, Kalluri H, Ezhilan S, Zorniak M, Wheeler DL, Kuo JS
    2012 May;14(5):420-8. doi: 10.1596/neo.12432.
    • More

      Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.

      PMID:22745588 | PMC:PMC3384429 | DOI:10.1596/neo.12432


      View details for PubMedID 22745588
  • KRAS mutant colorectal tumors: past and present Small GTPases
    Brand TM, Wheeler DL
    2012 Jan-Mar;3(1):34-9. doi: 10.4161/sgtp.18751.
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      The treatment of metastatic colorectal cancer (mCRC) remains one of the largest hurdles in cancer therapeutics to date. The most advanced treatment option for mCRC patients are anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) that bind to and inhibit the activity of EGFR. While the use of anti-EGFR mABs has had great impact in the treatment of mCRC, it has now been widely accepted that mCRC tumors with a mutation in the small GTPase KRAS do not respond to these therapies. KRAS mutations allow for EGFR independent activation of various oncogenic signaling cascades. In attempts to inhibit KRAS mutant tumor growth, BRAF, MEK and farsenyltransferase inhibitors have been used, however, their clinical efficacy is still accruing in the setting of CRC. Recent data suggests that various other inhibitors, including inhibitors of Src family kinases (SFK) and hepatocyte growth factor receptor (MET), may have potential preclinical and clinical success in KRAS mutant tumors. Additionally, it is becoming increasingly clear that different KRAS missense mutations may have varied biological responses to cetuximab, suggesting that cetuximab may still be a potential therapeutic option in some KRAS mutant tumors. In this review, we highlight the importance for both improved multimodality approaches for treating KRAS mutant mCRC tumors and stratification of KRAS mutations in response to different treatment regimes in order to optimize the best possible care for mCRC patients.

      PMID:22714415 | PMC:PMC3398915 | DOI:10.4161/sgtp.18751


      View details for PubMedID 22714415
  • The nuclear epidermal growth factor receptor signaling network and its role in cancer Discovery medicine
    Brand TM, Iida M, Li C, Wheeler DL
    2011 Nov;12(66):419-32.
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      The epidermal growth factor receptor (EGFR) is a member of the EGFR family of receptor tyrosine kinases (RTKs). EGFR activation via ligand binding results in signaling through various pathways ultimately resulting in cellular proliferation, survival, angiogenesis, invasion, and metastasis. Aberrant expression or activity of EGFR has been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, pancreatic cancer, and brain cancer. Thus intense efforts have been made to inhibit the activity of EGFR by designing antibodies against the ligand binding domains (cetuximab and panitumumab) or small molecules against the tyrosine kinase domain (erlotinib, gefitinib, and lapatinib). Although targeting membrane-bound EGFR has shown benefit, a new and emerging role for EGFR is now being elucidated. In this review we will summarize the current knowledge of the nuclear EGFR signaling network, including how it is trafficked to the nucleus, the functions it serves in the nucleus, and how these functions impact cancer progression, survival, and response to chemotherapeutics.

      PMID:22127113 | PMC:PMC3305885


      View details for PubMedID 22127113
  • p53 modulates acquired resistance to EGFR inhibitors and radiation Cancer research
    Huang S, Benavente S, Armstrong EA, Li C, Wheeler DL, Harari PM
    2011 Nov 15;71(22):7071-9. doi: 10.1158/0008-5472.CAN-11-0128. Epub 2011 Nov 8.
    • More

      There is presently great interest in mechanisms of acquired resistance to epidermal growth factor receptor (EGFR) inhibitors that are now being used widely in the treatment of a variety of common human cancers. To investigate these mechanisms, we established EGFR inhibitor-resistant clones from non-small cell lung cancer cells. A comparative analysis revealed that acquired resistance to EGFR inhibitors was associated consistently with the loss of p53 and cross-resistance to radiation. To examine the role of p53, we first knocked down p53 in sensitive parental cells and found a reduction in sensitivity to both EGFR inhibitors and radiation. Conversely, restoration of functional p53 in EGFR inhibitor-resistant cells was sufficient to resensitize them to EGFR inhibitors or radiation in vitro and in vivo. Further studies indicate that p53 may enhance sensitivity to EGFR inhibitors and radiation via induction of cell-cycle arrest, apoptosis, and DNA damage repair. Taken together, these findings suggest a central role of p53 in the development of acquired resistance to EGFR inhibitors and prompt consideration to apply p53 restoration strategies in future clinical trials that combine EGFR inhibitors and radiation.

      PMID:22068033 | PMC:PMC3229180 | DOI:10.1158/0008-5472.CAN-11-0128


      View details for PubMedID 22068033
  • Erlotinib is a viable treatment for tumors with acquired resistance to cetuximab Cancer biology & therapy
    Brand TM, Dunn EF, Iida M, Myers RA, Kostopoulos KT, Li C, Peet CR, Wheeler DL
    2011 Sep 1;12(5):436-46. doi: 10.4161/cbt.12.5.16394. Epub 2011 Sep 1.
    • More

      The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of tumorigenesis in many human tumors. Currently there are five EGFR inhibitors used in oncology, two monoclonal antibodies (panitumumab, and cetuximab) and three tyrosine kinase inhibitors (erlotinib, gefitinib, and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical successes, however many tumors remain non-responsive or acquire resistance during therapy. To explore potential molecular mechanisms of acquired resistance to cetuximab we previously established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to escalating doses of cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in steady-state expression of EGFR, HER2, and HER3 receptors as well as increased signaling through the MAPK and AKT pathways. RNAi studies demonstrated dependence of cetuximab-resistant clones on the EGFR signaling network. These findings prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to the EGFR targeted TKI erlotinib. In vitro, erlotinib was able to decrease signaling through the EGFR axis, decrease cellular proliferation, and induce apoptosis. To determine if erlotinib could have therapeutic benefit in vivo, we established cetuximab-resistant NCI-H226 mouse xenografts, and subsequently treated them with erlotinib. Mice harboring cetuximab-resistant tumors treated with erlotinib exhibited either a tumor regression or growth delay as compared to vehicle controls. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation and increase rates of apoptosis. The work presented herein suggests that 1) cells with acquired resistance to cetuximab maintain their dependence on EGFR and 2) tumors developing resistance to cetuximab can benefit from subsequent treatment with erlotinib, providing rationale for its use in the setting of cetuximab resistance.

      PMID:21725209 | PMC:PMC3219082 | DOI:10.4161/cbt.12.5.16394


      View details for PubMedID 21725209
  • Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab Cancer biology & therapy
    Brand TM, Iida M, Wheeler DL
    2011 May 1;11(9):777-92. doi: 10.4161/cbt.11.9.15050. Epub 2011 May 1.
    • More

      The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.

      PMID:21293176 | PMC:PMC3100630 | DOI:10.4161/cbt.11.9.15050


      View details for PubMedID 21293176
  • Treating PIK3CA and EGFR overexpressing breast cancers with lithium citrate Cancer biology & therapy
    Brand TM, Wheeler DL
    2011 Feb 1;11(3):368-70. doi: 10.4161/cbt.11.3.14696. Epub 2011 Feb 1.
  • The use of single-agent dasatinib in molecularly unselected non-small-cell lung cancer patients Expert opinion on investigational drugs
    Kruser TJ, Traynor AM, Wheeler DL
    2011 Feb;20(2):305-7. doi: 10.1517/13543784.2011.550873. Epub 2011 Jan 5.
    • More

      A Phase II study of the Src family kinase (SFK) inhibitor dasatinib was recently reported in molecularly unselected patients with metastatic NSCLC. SFK inhibition has a strong rationale as a clinical strategy in NSCLC. The reviewed study unfortunately showed disappointing activity as monotherapy in this molecularly unselected patient cohort and toxicity in terms of pleural effusion was problematic. Therefore, dasatinib as monotherapy in this setting does not appear promising. Nonetheless, the reviewed study may be used in conjunction with other studies of dasatinib in NSCLC to identify patients more likely to benefit from dasatinib either as monotherapy or in combination with other agents. Future studies of dasatinib in NSCLC should examine the agent in combination with EGFR inhibitors and/or cytotoxic chemotherapies.

      PMID:21204748 | PMC:PMC3303592 | DOI:10.1517/13543784.2011.550873


      View details for PubMedID 21204748
  • Regulation of heparin-binding EGF-like growth factor by miR-212 and acquired cetuximab-resistance in head and neck squamous cell carcinoma PloS one
    Hatakeyama H, Cheng H, Wirth P, Counsell A, Marcrom SR, Wood CB, Pohlmann PR, Gilbert J, Murphy B, Yarbrough WG, Wheeler DL, Harari PM, Guo Y, Shyr Y, Slebos RJ, Chung CH
    2010 Sep 13;5(9):e12702. doi: 10.1371/journal.pone.0012702.
    • More

      BACKGROUND: We hypothesized that chronic inhibition of epidermal growth factor receptor (EGFR) by cetuximab, a monoclonal anti-EGFR antibody, induces up-regulation of its ligands resulting in resistance and that microRNAs (miRs) play an important role in the ligand regulation in head and neck squamous cell carcinoma (HNSCC).

      METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide changes in gene and miR expression were determined in cetuximab-sensitive cell line, SCC1, and its resistant derivative 1Cc8 using DNA microarrays and RT-PCR. The effects of differentially expressed EGFR ligands and miRs were examined by MTS, colony formation, ELISA, and western blot assays. Heparin-binding EGF-like growth factor (HB-EGF) and its regulator, miR-212, were differentially expressed with statistical significance when SCC1 and 1Cc8 were compared for gene and miR expression. Stimulation with HB-EGF induced cetuximab resistance in sensitive cell lines. Inhibition of HB-EGF and the addition of miR-212 mimic induced cetuximab sensitivity in resistant cell lines. MicroRNA-212 and HB-EGF expression were inversely correlated in an additional 33 HNSCC and keratinocyte cell lines. Six tumors and 46 plasma samples from HNSCC patients were examined for HB-EGF levels. HB-EGF plasma levels were lower in newly diagnosed HNSCC patients when compared to patients with recurrent disease.

      CONCLUSIONS/SIGNIFICANCE: Increased expression of HB-EGF due to down-regulation of miR-212 is a possible mechanism of cetuximab resistance. The combination of EGFR ligand inhibitors or miR modulators with cetuximab may improve the clinical outcome of cetuximab therapy in HNSCC.

      PMID:20856931 | PMC:PMC2938338 | DOI:10.1371/journal.pone.0012702


      View details for PubMedID 20856931
  • Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology
    Li C, Iida M, Dunn EF, Wheeler DL
    2010 Nov;97(2):330-7. doi: 10.1016/j.radonc.2010.06.010.
    • More

      BACKGROUND AND PURPOSE: The aberrant expression of epidermal growth factor receptor (EGFR) has been linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III trial combining cetuximab with radiation confirmed a strong survival advantage. However, both cetuximab and radiation can promote EGFR translocation to the nucleus where it enhances resistance to both of these modalities. In this report we sought to determine how to block cetuximab- and radiation-induced translocation of EGFR to the nucleus in HNSCC cell lines.

      MATERIAL AND METHODS: We utilized three established HNSCC cell lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of EGFR after treatment with cetuximab or radiation. We then utilized dasatinib (BMS-354825), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases, to determine if SFKs blockade could abrogate cetuximab- and radiation-induced nuclear EGFR translocation.

      RESULTS: Cetuximab and radiation treatment of all three HNSCC lines lead to translocation of the EGFR to the nucleus. Blockade of SFKs abrogated cetuximab- and radiation-induced EGFR translocation to the nucleus.

      CONCLUSIONS: The data presented in this report suggest that both cetuximab and radiation can promote EGFR translocation to the nucleus and dasatinib can inhibit this process. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC.

      PMID:20667610 | PMC:PMC2974772 | DOI:10.1016/j.radonc.2010.06.010


      View details for PubMedID 20667610
  • Understanding resistance to EGFR inhibitors-impact on future treatment strategies Nature reviews. Clinical oncology
    Wheeler DL, Dunn EF, Harari PM
    2010 Sep;7(9):493-507. doi: 10.1038/nrclinonc.2010.97. Epub 2010 Jun 15.
    • More

      EGFR is a tyrosine kinase that participates in the regulation of cellular homeostasis. Following ligand binding, EGFR stimulates downstream cell signaling cascades that influence cell proliferation, apoptosis, migration, survival and complex processes, including angiogenesis and tumorigenesis. EGFR has been strongly implicated in the biology of human epithelial malignancies, with therapeutic applications in cancers of the colon, head and neck, lung, and pancreas. Accordingly, targeting EGFR has been intensely pursued, with the development of a series of promising molecular inhibitors for use in clinical oncology. As is common in cancer therapy, challenges with respect to treatment resistance emerge over time. This situation is certainly true of EGFR inhibitor therapies, where intrinsic and acquired resistance is now well recognized. In this Review, we provide a brief overview regarding the biology of EGFR, preclinical and clinical development of EGFR inhibitors, and molecular mechanisms that underlie the development of treatment resistance. A greater understanding of the mechanisms that lead to EGFR resistance may provide valuable insights to help design new strategies that will enhance the impact of this promising class of inhibitors for the treatment of cancer.

      PMID:20551942 | PMC:PMC2929287 | DOI:10.1038/nrclinonc.2010.97


      View details for PubMedID 20551942
  • Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors Clinical cancer research : an official journal of the American Association for Cancer Research
    Kruser TJ, Wheeler DL, Armstrong EA, Iida M, Kozak KR, Kogel vd, Bussink J, Coxon A, Polverino A, Harari PM
    2010 Jul 15;16(14):3639-47. doi: 10.1158/1078-0432.CCR-09-3385. Epub 2010 May 27.
    • More

      BACKGROUND: Motesanib is a potent inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit receptors. In this report we examine the interaction between motesanib and radiation in vitro and in head and neck squamous cell carcinoma (HNSCC) xenograft models.

      EXPERIMENTAL DESIGN: In vitro assays were done to assess the impact of motesanib on VEGFR2 signaling pathways in human umbilical vein endothelial cells (HUVEC). HNSCC lines grown as tumor xenografts in athymic nude mice were utilized to assess the in vivo activity of motesanib alone and in combination with radiation.

      RESULTS: Motesanib inhibited VEGF-stimulated HUVEC proliferation in vitro, as well as VEGFR2 kinase activity. Additionally, motesanib and fractionated radiation showed additive inhibitory effects on HUVEC proliferation. In vivo combination therapy with motesanib and radiation showed increased response compared with drug or radiation alone in UM-SCC1 (P < 0.002) and SCC-1483 xenografts (P = 0.001); however, the combination was not significantly more efficacious than radiation alone in UM-SCC6 xenografts. Xenografts treated with motesanib showed a reduction of vessel penetration into tumor parenchyma, compared with control tumors. Furthermore, triple immunohistochemical staining for vasculature, proliferation, and hypoxia showed well-defined spatial relationships among these parameters in HNSCC xenografts. Motesanib significantly enhanced intratumoral hypoxia in the presence and absence of fractionated radiation.

      CONCLUSIONS: These studies identify a favorable interaction when combining radiation and motesanib in HNSCC models. The data presented suggest that motesanib reduces blood vessel penetration into tumors and thereby increases intratumoral hypoxia. These findings suggest that clinical investigations examining combinations of radiation and motesanib are warranted in HNSCC.

      PMID:20507929 | PMC:PMC3216115 | DOI:10.1158/1078-0432.CCR-09-3385


      View details for PubMedID 20507929
  • Mechanisms of resistance to HER family targeting antibodies Experimental cell research
    Kruser TJ, Wheeler DL
    2010 Apr 15;316(7):1083-100. doi: 10.1016/j.yexcr.2010.01.009. Epub 2010 Jan 11.
    • More

      The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.

      PMID:20064507 | DOI:10.1016/j.yexcr.2010.01.009


      View details for PubMedID 20064507
  • BB, a novel epidermal growth factor receptor inhibitor Cancer biology & therapy
    Wheeler DL, Dunn EF
    2009 Sep;8(17):1648-50. doi: 10.4161/cbt.8.17.9358. Epub 2009 Sep 29.
  • The role of Src in solid tumors The oncologist
    Wheeler DL, Iida M, Dunn EF
    2009 Jul;14(7):667-78. doi: 10.1634/theoncologist.2009-0009. Epub 2009 Jul 6.
    • More

      The proto-oncogene c-Src (Src) encodes a nonreceptor tyrosine kinase whose expression and activity are correlated with advanced malignancy and poor prognosis in a variety of human cancers. Nine additional enzymes with homology to Src have been identified and collectively are referred to as Src family kinases (SFKs). Together, SFKs represent the largest family of nonreceptor tyrosine kinases and interact directly with receptor tyrosine kinases, G-protein-coupled receptors, steroid receptors, signal transducers and activators of transcription, and molecules involved in cell adhesion and migration. These interactions lead to a diverse array of biological functions including proliferation, cell growth, differentiation, cell shape, motility, migration, angiogenesis, and survival. Studies investigating mutational activation of Src in human cancers suggest that this may be a rare event and that wild-type Src is weakly oncogenic. Thus, the role of Src in the development and progression of human cancer remains unclear. Recently, it was suggested that increased SFK protein levels and, more importantly, SFK tyrosine kinase activity are linked to cancer progression and metastatic disease by facilitating the action of other signaling proteins. This accumulating body of evidence indicates that SFKs may represent a promising therapeutic target for the treatment of solid tumors. This review discusses the role of SFKs in solid tumors and the recent therapeutic advances aimed at targeting this family of tyrosine kinases in cancer.

      PMID:19581523 | PMC:PMC3303596 | DOI:10.1634/theoncologist.2009-0009


      View details for PubMedID 19581523
  • Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab Cancer biology & therapy
    Wheeler DL, Iida M, Kruser TJ, Nechrebecki MM, Dunn EF, Armstrong EA, Huang S, Harari PM
    2009 Apr;8(8):696-703. doi: 10.4161/cbt.8.8.7903. Epub 2009 Apr 22.
    • More

      The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab when treated with dasatinib. These results indicate that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab.

      PMID:19276677 | PMC:PMC2895567 | DOI:10.4161/cbt.8.8.7903


      View details for PubMedID 19276677
  • Establishment and characterization of a model of acquired resistance to epidermal growth factor receptor targeting agents in human cancer cells Clinical cancer research : an official journal of the American Association for Cancer Research
    Benavente S, Huang S, Armstrong EA, Chi A, Hsu K, Wheeler DL, Harari PM
    2009 Mar 1;15(5):1585-92. doi: 10.1158/1078-0432.CCR-08-2068. Epub 2009 Feb 3.
    • More

      PURPOSE: The epidermal growth factor receptor (EGFR) is recognized as a key mediator of proliferation and progression in many human tumors. A series of EGFR-specific inhibitors have recently gained Food and Drug Administration approval in oncology. These strategies of EGFR inhibition have shown major tumor regressions in approximately 10% to 20% of advanced cancer patients. Many tumors, however, eventually manifest resistance to treatment. Efforts to better understand the underlying mechanisms of acquired resistance to EGFR inhibitors, and potential strategies to overcome resistance, are greatly needed.

      EXPERIMENTAL DESIGN: To develop cell lines with acquired resistance to EGFR inhibitors we utilized the human head and neck squamous cell carcinoma tumor cell line SCC-1. Cells were treated with increasing concentrations of cetuximab, gefitinib, or erlotinib, and characterized for the molecular changes in the EGFR inhibitor-resistant lines relative to the EGFR inhibitor-sensitive lines.

      RESULTS: EGFR inhibitor-resistant lines were able to maintain their resistant phenotype in both drug-free medium and in athymic nude mouse xenografts. In addition, EGFR inhibitor-resistant lines showed a markedly increased proliferation rate. EGFR inhibitor-resistant lines had elevated levels of phosphorylated EGFR, mitogen-activated protein kinase, AKT, and signal transducer and activator of transcription 3, which were associated with reduced apoptotic capacity. Subsequent in vivo experiments indicated enhanced angiogenic potential in EGFR inhibitor-resistant lines. Finally, EGFR inhibitor-resistant lines showed cross-resistance to ionizing radiation.

      CONCLUSIONS: We have developed EGFR inhibitor-resistant human head and neck squamous cell carcinoma cell lines. This model provides a valuable preclinical tool to investigate molecular mechanisms of acquired resistance to EGFR blockade.

      PMID:19190133 | PMC:PMC2903727 | DOI:10.1158/1078-0432.CCR-08-2068


      View details for PubMedID 19190133
  • Molecular target approaches in head and neck cancer: epidermal growth factor receptor and beyond Seminars in radiation oncology
    Harari PM, Wheeler DL, Grandis JR
    2009 Jan;19(1):63-8. doi: 10.1016/j.semradonc.2008.09.009.
    • More

      Approximately 50,000 new cases of head and neck squamous cell carcinoma (HNSCC) will be diagnosed in the United States in 2009. Although the gradual decline in smoking rates in the United States is a highly favorable trend, the future global HNSCC incidence will likely reflect the increased marketing and penetration of tobacco products across several of our most populous countries. Although modern surgery, radiation, and conventional chemotherapy strategies for HNSCC continue to provide gradual improvement in outcome, the first molecular targeting approach to show a survival advantage for HNSCC patients has recently emerged in the context of epidermal growth factor receptor biology. The scientific background and current challenges accompanying this recent advance are described in this article as are several additional promising molecular targets for HNSCC. There is cautious anticipation that the logical advancement of molecular targeting agents in conjunction with radiation may afford increased cure rates and diminished normal tissue toxicity profiles for HNSCC patients over the years to come.

      PMID:19028347 | PMC:PMC3216119 | DOI:10.1016/j.semradonc.2008.09.009


      View details for PubMedID 19028347
  • Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer International journal of radiation oncology, biology, physics
    Kruser TJ, Armstrong EA, Ghia AJ, Huang S, Wheeler DL, Radinsky R, Freeman DJ, Harari PM
    2008 Oct 1;72(2):534-42. doi: 10.1016/j.ijrobp.2008.06.1490.
    • More

      PURPOSE: To examine the interaction between panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, and radiation in head-and-neck squamous cell carcinoma and non-small-cell lung cancer cell lines and xenografts.

      METHODS AND MATERIALS: The head-and-neck squamous cell carcinoma lines UM-SCC1 and SCC-1483, as well as the non-small-cell lung cancer line H226, were studied. Tumor xenografts in athymic nude mice were used to assess the in vivo activity of panitumumab alone and combined with radiation. In vitro assays were performed to assess the effect of panitumumab on radiation-induced cell signaling, apoptosis, and DNA damage.

      RESULTS: Panitumumab increased the radiosensitivity as measured by the clonogenic survival assay. Radiation-induced epidermal growth factor receptor phosphorylation and downstream signaling through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was inhibited by panitumumab. Panitumumab augmented radiation-induced DNA damage by 1.2-1.6-fold in each of the cell lines studied as assessed by residual gamma-H(2)AX foci after radiation. Radiation-induced apoptosis was increased 1.4-1.9-fold by panitumumab, as evidenced by Annexin V-fluorescein isothiocyanate staining and flow cytometry. In vivo, the combination therapy of panitumumab and radiation was superior to panitumumab or radiation alone in the H226 xenografts (p = 0.01) and showed a similar trend in the SCC-1483 xenografts (p = 0.08). In vivo, immunohistochemistry demonstrated the ability of panitumumab to augment the antiproliferative and antiangiogenic effects of radiation.

      CONCLUSION: These studies have identified a favorable interaction in the combination of radiation and panitumumab in upper aerodigestive tract tumor models, both in vitro and in vivo. These data suggest that clinical investigations examining the combination of radiation and panitumumab in the treatment of epithelial tumors warrant additional pursuit.

      PMID:18793955 | PMC:PMC2927815 | DOI:10.1016/j.ijrobp.2008.06.1490


      View details for PubMedID 18793955
  • Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice Toxicologic pathology
    Li Y, Wheeler DL, Ananthaswamy HN, Verma AK, Oberley TD
    2007 Dec;35(7):942-51. doi: 10.1080/01926230701748164.
    • More

      Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

      PMID:18098040 | DOI:10.1080/01926230701748164


      View details for PubMedID 18098040
  • Protein kinase Cepsilon and development of squamous cell carcinoma, the nonmelanoma human skin cancer Molecular carcinogenesis
    Verma AK, Wheeler DL, Aziz MH, Manoharan H
    2006 Jun;45(6):381-8. doi: 10.1002/mc.20230.
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      Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)-dependent serine/threonine protein kinases. At least five PKC isoforms (alpha, delta, epsilon, eta, and zeta) are expressed in epidermal keratinocytes. PKC isoforms are differentially expressed in proliferative (basal layer) and nonproliferative compartments (spinous, granular, cornified layers), which exhibit divergence in their roles in the regulation of epidermal cell proliferation, differentiation, and apoptosis. Immunocytochemical localization of PKC isoforms indicate that PKCalpha is found in the membranes of suprabasal cells in the spinous and granular layers. PKCepsilon is mostly localized in the proliferative basal layers. PKCeta is localized exclusively in the granular layer. PKCdelta is detected throughout the epidermis. PKC isozymes exhibit specificities in their signals to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform mediates the induction of squamous cell carcinoma (SCC) elicited either by the DMBA-TPA protocol or by repeated exposures to ultraviolet radiation (UVR). PKCepsilon overexpression, which sensitizes skin to UVR-induced carcinogenesis, suppresses UVR-induced sunburn (apoptotic) cell formation, and enhances both UVR-induced levels of TNFalpha and hyperplasia. UVR-induced sunburn cell formation is mediated by Fas/Fas-L and TNFalpha NFR1 extrinsic apoptotic pathways. The death adaptor protein termed Fas-associated death domain (FADD) is a common adaptor protein for both of these apoptotic pathways. PKCepsilon inhibits UVR-induced expression of FADD leading to the inhibition of both apoptotic pathways. It appears that PKCepsilon sensitizes skin to the development of SCC by UVR by transducting signals, which inhibit apoptosis on one hand, and enhances proliferation of preneoplastic cells on the other hand.

      PMID:16683253 | DOI:10.1002/mc.20230


      View details for PubMedID 16683253
  • Protein kinase C delta overexpressing transgenic mice are resistant to chemically but not to UV radiation-induced development of squamous cell carcinomas: a possible link to specific cytokines and cyclooxygenase-2 Cancer research
    Aziz MH, Wheeler DL, Bhamb B, Verma AK
    2006 Jan 15;66(2):713-22. doi: 10.1158/0008-5472.CAN-05-2684.
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      Protein kinase C delta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the novel PKCs (delta, epsilon, and eta) expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 8-fold) PKCdelta protein in basal epidermal cells and cells of the hair follicle are resistant to the development of both skin papillomas and squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion protocol. We now present that PKCdelta overexpression in transgenic mice failed to suppress the induction of SCC developed by repeated exposures to UV radiation (UVR), the environmental carcinogen linked to the development of human SCC. Both TPA and UVR treatment of wild-type mice (a) increased the expression of proliferating cell nuclear antigen (PCNA) and apoptosis; (b) stimulated the expression of cytokines tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF); and (c) increased cyclooxygenase-2 (COX-2) expression and expression of phosphorylated Akt (p-Akt), p38, extracellular signal-regulated kinase-1 (ERK1), and ERK2. PKCdelta overexpression in transgenic mice enhanced TPA-induced but not UVR-induced apoptosis and suppressed TPA-stimulated but not UVR-stimulated levels of cell PCNA, cytokines (TNF-alpha, G-CSF, and GM-CSF), and the expression of COX-2, p-Akt, and p38. The results indicate that UVR-mediated signal transduction pathway to the induction of SCC does not seem to be sensitive to PKCdelta overexpression. The proapoptotic activity of PKCdelta coupled with its ability to suppress TPA-induced expression of proinflammatory cytokines, COX-2 expression, and the phosphorylation of Akt and p38 may play roles in the suppression of TPA-promoted development of SCC.

      PMID:16424000 | DOI:10.1158/0008-5472.CAN-05-2684


      View details for PubMedID 16424000
  • Early epidermal destruction with subsequent epidermal hyperplasia is a unique feature of the papilloma-independent squamous cell carcinoma phenotype in PKCepsilon overexpressing transgenic mice Toxicologic pathology
    Li Y, Wheeler DL, Alters W, Chaiswing L, Verma AK, Oberley TD
    2005;33(6):684-94. doi: 10.1080/01926230500323441.
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      Protein kinase C epsilon (PKCepsilon) overexpressing transgenic (PKCepsilon Tg) mice develop papilloma-independent squamous cell carcinomas (SCC) elicited by 7,12-dimethylbenz[a]anthracene (DMBA) tumor initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) tumor promotion. We examined whether epidermal cell turnover kinetics was altered during the development of SCC in PKCepsilon Tg mice. Dorsal skin samples were fixed for histological examination. A single application of TPA resulted in extensive infiltration of polymorphonuclear neutrophils (PMNs) into the epidermis at 24 h after TPA treatment in PKCepsilon Tg mice while wild-type (WT) mouse skin showed focal infiltration by PMNs. Complete epidermal necrosis was observed at 48 h in PKCepsilon Tg mice only; at 72 h, epidermal cell regeneration beginning from hair follicles was observed in PKCepsilon Tg mice. Since the first TPA treatment to DMBA-initiated PKCepsilon Tg mouse skin led to epidermal destruction analogous to skin abrasion, we propose the papilloma-independent phenotype may be explained by death of initiated interfollicular cells originally destined to become papillomas. Epidermal destruction did not occur after multiple doses of TPA, presumably reflecting adaptation of epidermis to chronic TPA treatment. Prolonged hyperplasia in the hair follicle may result in the early neoplastic lesions originally described by Jansen et al. (2001) by expanding initiated cells in the hair follicles resulting in the subsequent development of SCC.

      PMID:16243773 | DOI:10.1080/01926230500323441


      View details for PubMedID 16243773
  • Overexpression of protein kinase C-{epsilon} in the mouse epidermis leads to a spontaneous myeloproliferative-like disease The American journal of pathology
    Wheeler DL, Reddig PJ, Ness KJ, Leith CP, Oberley TD, Verma AK
    2005 Jan;166(1):117-26. doi: 10.1016/s0002-9440(10)62237-7.
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      Protein kinase C (PKC)-epsilon, a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mouse lines that overexpress (8- or 18-fold) PKC-epsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate-promotion or by repeated ultraviolet radiation exposures. The susceptibility to the development of SCC was proportional to the level of expression of the PKC-epsilon transgene. We now report that PKC-epsilon FVB/N transgenic mice (line 215) that overexpress in epidermis approximately 18-fold PKC-epsilon protein more than their wild-type littermates spontaneously develop a myeloproliferative-like disease (MPD) in 100% of PKC-epsilon transgenic mice. The MPD was characterized by an excess of neutrophils and eosinophils, resulting in invasion of almost all vital organs of the mouse by 6 months of age. On gross examination these mice present with splenomegaly, hepatomegaly, and severe lymphadenopathy. Examination of the bone marrow revealed almost complete effacement by neutrophils, eosinophils, and their precursors. Furthermore, the spleen and lymph nodes were enlarged and exhibited marked extramedullary hematopoiesis. Complete pathological analysis of the second PKC-epsilon transgenic mouse (line 224) that expresses approximately eightfold PKC-epsilon protein more than their wild-type littermates revealed no remarkable findings in any of the affected organs as seen in line 215. However, peripheral blood analyses of PKC-epsilon transgenic mice indicated significant increases of neutrophils in the circulating blood in both PKC-epsilon transgenic lines. To determine whether there was an imbalance of cytokines in PKC-epsilon transgenic mice (line 215), resulting in aberrant myelopoiesis, we analyzed 17 cytokines in the peripheral blood. This analysis indicated that interleukin-5, interleukin-6, and granulocyte-colony stimulating factor were up-regulated as a function of age. The transgene PKC-epsilon was not detected in any of the affected organs (bone marrow, liver, spleen, lung) We suggest that overexpression of PKC-epsilon in the epidermis may lead to the induction of specific cytokines that may, in a paracrine mechanism, perturb normal hematopoiesis in bone marrow resulting in a granulocytic skew toward that of neutrophils and eosinophils. The susceptibility of PKC-epsilon transgenic mice to the induction of SCC and the spontaneous development of MPD are unrelated.

      PMID:15632005 | PMC:PMC1602310 | DOI:10.1016/s0002-9440(10)62237-7


      View details for PubMedID 15632005
  • Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas Cancer research
    Wheeler DL, Martin KE, Ness KJ, Li Y, Dreckschmidt NE, Wartman M, Ananthaswamy HN, Mitchell DL, Verma AK
    2004 Nov 1;64(21):7756-65. doi: 10.1158/0008-5472.CAN-04-1881.
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      Chronic exposure to UV radiation (UVR), especially in the UVA (315-400 nm) and UVB (280-315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C epsilon (PKCepsilon), a member of the phospholipid-dependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKCepsilon transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8- and 18-fold PKCepsilon protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m(2) three times weekly) induced irreparable skin damage in high PKCepsilon-overexpressing mouse line 215. However, the PKCepsilon transgenic mouse line 224, when exposed to UVR (2 kJ/m(2) three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKCepsilon transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor alpha (TNFalpha) and other growth stimulatory cytokines, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. The role of TNFalpha in UVR-induced cutaneous damage was evaluated using PKCepsilon transgenic mice deficient in TNFalpha. UVR treatment three times weekly for 13 weeks at 2 kJ/m(2) induced severe cutaneous damage in PKCepsilon transgenic mice (line 215), which was partially prevented in PKCepsilon-transgenic TNFalpha-knockout mice. Taken together, the results indicate that PKCepsilon signals UVR-induced TNFalpha release that is linked, at least in part, to the photosensitivity of PKCepsilon transgenic mice.

      PMID:15520180 | DOI:10.1158/0008-5472.CAN-04-1881


      View details for PubMedID 15520180
  • Protein kinase Cepsilon is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-alpha ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase Cepsilon transgenic mice Cancer research
    Wheeler DL, Ness KJ, Oberley TD, Verma AK
    2003 Oct 1;63(19):6547-55.
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      Protein kinase Cepsilon (PKCepsilon), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 18-fold) PKCepsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited by 7,12-dimethylbenz(a)anthracene-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. We now present that PKCepsilon transgenic mice elicit elevated serum tumor necrosis factor (TNF)alpha levels during skin tumor promotion by TPA, and this increase may be linked to the development of mSCC. A single topical application of TPA (5 nmol) to the skin, as early as 2.5 h after treatment, resulted in a significant (P < 0.01) increase (2-fold) in epidermal TNFalpha and more than a 6-fold increase in ectodomain shedding of TNFalpha into the serum of PKCepsilon transgenic mice relative to their wild-type littermates. Furthermore, this TPA-stimulated TNFalpha shedding was proportional to the level of expression of PKCepsilon in the epidermis. Using the TNF-alpha converting enzyme (TACE) inhibitor, TAPI-1, TPA-stimulated TNFalpha shedding could be completely prevented in PKCepsilon transgenic mice and isolated keratinocytes. These results indicate that PKCepsilon signal transduction pathways to TPA-stimulated TNFalpha ectodomain shedding are mediated by TACE, a transmembrane metalloprotease. Using the superoxide dismutase mimetic CuDIPs and the glutathione reductase mimetic ebselen, TPA-stimulated TNFalpha shedding from PKCepsilon transgenic mice could be completely attenuated, implying the role of reactive oxygen species. Finally, i.p. injection of a TNFalpha synthesis inhibitor, pentoxifylline, during skin tumor promotion completely prevented the development of mSCC in PKCepsilon transgenic mice. Taken together, these results indicate that: (a) PKCepsilon activation is an initial signal in TPA-induced shedding of TNFalpha from epidermal keratinocytes; (b) PKCepsilon-mediated signals to TACE are possibly mediated through reactive oxygen species; and (c) TPA-induced TNFalpha shedding may play a role in the development of mSCC in PKCepsilon transgenic mice.

      PMID:14559850


      View details for PubMedID 14559850
  • Inhibition of the development of metastatic squamous cell carcinoma in protein kinase C epsilon transgenic mice by alpha-difluoromethylornithine accompanied by marked hair follicle degeneration and hair loss Cancer research
    Wheeler DL, Ness KJ, Oberley TD, Verma AK
    2003 Jun 15;63(12):3037-42.
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      The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.

      PMID:12810623


      View details for PubMedID 12810623
  • Protein kinase Cdelta-mediated signal to ornithine decarboxylase induction is independent of skin tumor suppression Oncogene
    Wheeler DL, Reddig PJ, Dreckschmidt NE, Leitges M, Verma AK
    2002 May 16;21(22):3620-30. doi: 10.1038/sj.onc.1205451.
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      Protein Kinase Cdelta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately eightfold) PKCdelta protein in basal epidermal cells are resistant to skin tumor formation by the 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. However, despite being resistant to skin tumor promotion by TPA, PKCdelta transgenic mice elicited a 3-4-fold increase in TPA-induced epidermal ODC activity and putrescine levels than their wild-type littermates. PKCdelta was observed to be the key component of the TPA signal transduction pathways to the induction of mouse epidermal ODC activity. To determine if TPA-induced ODC activity and associated putrescine levels in PKCdelta transgenic mice contributed to PKCdelta-mediated suppression of skin tumor promotion by TPA, the irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO), was used. PKCdelta transgenic mice and their wild-type littermates were initiated with 100 nmol DMBA and then promoted twice weekly with 5 nmol TPA. The experimental group was given 0.5% DFMO in their drinking water, while the control group was given tap water. After 25 weeks, the number of papillomas (>2 mm) per mouse was counted. The DFMO treatment did not affect the skin tumor multiplicity of PKCdelta transgenic mice. These results indicate that PKCdelta-induced ODC activity is not involved in PKCdelta-mediated tumor suppression. Thus, the signaling pathways via PKCdelta to epidermal ODC induction and skin tumor suppression appear to be independent.

      PMID:12032864 | DOI:10.1038/sj.onc.1205451


      View details for PubMedID 12032864

Contact Information

Deric Wheeler, PhD

1111 Highland Avenue,
3159 WIMR
Madison, WI 53705-2275
Email