Resistant Head And Neck Squamous Cell Carcinoma Cell Research Articles

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

ABSTRACT Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

Abstract 3251: Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1

Abstract Effective targeted therapies are lacking for head and neck squamous cell carcinoma (HNSCC) that is common and fatal. Developing targeted therapies for HNSCC is challenging as the genomic landscape is dominated by mutations in tumor suppressors, including NOTCH1. To address this need, we previously demonstrated that PI3K inhibition led to apoptosis in NOTCH1 mutant HNSCC cell lines. The underlying mechanism of this sensitivity is unknown and important to elucidate as modest responses and development of acquired resistance are the leading causes of failure for targeted therapies. To address this knowledge gap, we measured the levels of 304 proteins using reverse phase protein array in NOTCH1WT and NOTCH1MUT HNSCC cell lines upon PI3K inhibition and identified Aurora kinase B (AURKB) to be differentially regulated. We also identified AURKB levels to be maintained in the NOTCH1MUT cell lines with acquired resistance to PI3K inhibition. To determine if the maintenance of AURKB expression contributes to PI3K inhibitor resistance, we depleted AURKB in resistant NOTCH1WT HNSCC cells and overexpressed AURKB in sensitive NOTCH1MUT HNSCC cells. This manipulation of AURKB levels led to increased sensitivity and resistance to PI3K inhibition respectively. To use a pharmacologic approach, we combined the pan-Aurora kinase inhibitor danusertib (0-2µM) with PI3K/mTOR inhibitor omipalisib (0-200nM) in 56 HNSCC cell lines for 72h and observed a substantial decrease in cell viability in >80% of NOTCH1WT and >90% of NOTCH1MUT HNSCC lines. Additionally, concurrent Aurora kinase and PI3K inhibition in NOTCH1WT, NOTCH1MUT, and NOTCH1MUT acquired resistant HNSCC cells for 24h resulted in elevated cell death compared to single agents, as measured by the induction of cleaved PARP and cleaved Caspase 3; and Annexin V positive cells. Mice bearing NOTCH1MUT xenografts showed complete tumor regression when treated with a combination of pan-PI3K inhibitor Copanlisib and Aurora inhibitor Alisertib as compared to control groups. AURKB depletion in NOTCH1WT and overexpression in NOTCH1MUT HNSCC cells revealed significant changes in the total protein levels of AKT and PDK1. Similarly, AKT depletion and overexpression in NOTCH1WT and NOTCH1MUT HNSCC cells altered sensitivity to PI3K inhibition. However, manipulation of AKT levels affected PDK1 and not AURKB levels, demonstrating AURKB to be upstream to AKT and PDK1. Therefore, maintenance of AURKB levels mediates resistance to PI3K inhibition in HNSCC through AKT and PDK1. We identified AURKB as a central player governing the sensitivity to PI3K inhibitor-induced apoptosis in the context of NOTCH1 mutation status in HNSCC through its effects on AKT and PDK1. These novel findings may lead to the development of more robust therapeutic approach for NOTCH1 mutant squamous carcinoma as well as patients who develop acquired resistance to targeted therapies. Citation Format: Pooja A. Shah, Anne M. Fernandez, Vaishnavi Sambandam, Hongyun Zhao, Tuhina Mazumdar, Li Shen, Jing Wang, Faye M. Johnson. Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3251.

Targeting PI3Kα/δ and the ErbB Family of Protein-Tyrosine Kinases in Cisplatin-Resistant Head and Neck Squamous Cell Carcinomas

<h3>Purpose/Objective(s)</h3> The mortality rate for advanced head and neck squamous cell carcinoma (HNSCC) remains very high due to cancer recurrence and metastasis. Cisplatin is a commonly used anti-cancer drug for treatment of recurrent and metastatic HNSCC, but the majority of patients will eventually develop resistance. Therefore, there is an urgent, yet still unmet, need for improved therapies for cisplatin-resistant HNSCC. Increasing evidence has demonstrated that ErbB family kinase and PI3K/Akt are the most important regulators and targets in HNSCC. Our goal is to find new therapeutics to treat cisplatin resistant HNSCC without use of cisplatin. Here, we determined the efficacy of a combination of the PI3Kinase inhibitor, Copanlisib with the ErbB inhibitor, Afatinib, on suppressing cisplatin resistant HNSCC <i>in vitro</i> and <i>in vivo</i>. <h3>Materials/Methods</h3> Both cisplatin sensitive (Cal27 and FaDu) and resistant (Cal27CP and FaDu-CP) HNSCC cells were treated with copanlisib, afatinib, or a combination of copanlisib and afatinib. The effects on cell signaling pathways were tested by Western blot assay and RT-PCR. Cell proliferation was determined by MTT assay. Apoptosis was assayed by Annexin V. Migration and invasion was monitored via the xCELLigence real-time cell system tic cells. Xenograft tumor formation was observed in nude mice. <h3>Results</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals. <h3>Conclusion</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals.

Stimulator of interferon response cGAMP interactor overcomes ERBB2-mediated apatinib resistance in head and neck squamous cell carcinoma.

Purpose: Apatinib resistance is the main obstacle to the effective treatment of advanced head and neck squamous cell carcinoma (HNSCC). This study aimed to evaluate the function of Erb-B2 receptor tyrosine kinase 2 (ERBB2) and stimulator of interferon response cGAMP interactor (STING) in apatinib resistance in HNSCC.Method: The Cancer Genome Atlas database of HNSCC was used to analyze the relationship between vascular endothelial growth factor receptor 2 (VEGFR2) expression and prognosis. An apatinib resistant (AR) HNSCC cell line was constructed based on the CAL27 cell line. RNA sequencing was performed to explore the differentially expressed mRNAs. Quantitative real-time reverse transcription PCR (qRT-PCR) and western blotting were used to evaluate the expression and phosphorylation level VEGFR2, ERBB2, STING, and related proteins. Apatinib resistance was evaluated by colony formation and cell viability assays. A mouse subcutaneous tumor formation model was established to evaluate the efficiency of combination treatment and vascularization was evaluated by assessing CD31 immunofluorescence.Result: The expression of VEGFR2 was high in tumor of patients with HNSCC. Western blotting and qRT-PCR revealed that in AR cells, ERBB2 expression was high, whereas the expression of STING was low. Targeted treatment of ERBB2 using lapatinib could attenuate apatinib resistance. Further research confirmed that overexpressing STING could decrease ERBB2 expression.Conclusion: STING could sensitize AR cells to apatinib by decreasing ERBB2 expression. The combination of lapatinib or a STING agonist with apatinib ameliorated acquired apatinib resistance in a synergistic manner.

Abstract 1095: Overcoming acquired cetuximab resistance in head and neck squamous cell carcinoma: An in vitro study on the potential of PI3K inhibition

Abstract Resistance to therapies targeting the epidermal growth factor receptor (EGFR), such as cetuximab, remains a major roadblock in the search for effective therapeutic strategies in head and neck squamous cell carcinoma (HNSCC). Increasing evidence suggests that aberrant signalling of the PI3K/Akt pathway is involved in resistance to cetuximab treatment. Therefore, this study aims to investigate whether combined inhibition of EGFR and PI3K might be a novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC. Acquired cetuximab resistant HNSCC cell lines (SC263-R and SCC22b-R) were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines (SC263-PBS and SCC22b-PBS) were established by exposing these cells to the vehicle control (PBS). All cell lines were HPV-negative. Cytotoxicity of cetuximab (72h, 25 and 50 nM) plus the PI3K inhibitor buparlisib (72h, 0-1μM) was assessed using the Tecan Spark Cyto. Cells were stained with Hoechst 33342 and Cytotox Green (Sartorius) after 72h of treatment. Possible synergism between cetuximab and buparlisib was determined by calculating the combination index (CI) using the additive model. CI&amp;lt;0.8, CI = 1.0±0.2, and CI&amp;gt;1.2 indicated synergism, additivity, and antagonism, respectively. The effects of the combination treatment on cell cycle arrest and apoptosis were analysed flow cytometrically using the Vindelov and Annexin V-FITC/PI method, respectively. Simultaneous exposure to cetuximab and buparlisib revealed a synergistic interaction in the SC263-PBS and SC263-R cell lines (CI≤0.797). Furthermore, the percentage of cell death was the highest in the combination treatments, with a maximum of 30.33±4.18% for 50 nM cetuximab plus 1 μM buparlisib. Cetuximab monotherapy induced a small cell cycle arrest, while buparlisib monotherapy had little to no effect on cell cycle distribution. The addition of buparlisib to cetuximab resulted in a small increase in G0/G1 phase cells. Therapy-induced apoptosis was limited when cells were treated with cetuximab monotherapy. Buparlisib monotherapy induced a small percentage of apoptosis in a concentration-dependent manner. The addition of buparlisib to cetuximab resulted in an increase in the percentage of apoptotic cells with a maximum of 22.30±7.48%. These results are currently being validated in other acquired resistant cell lines. Preliminary results also show an additive to synergistic interaction between both drugs in the SCC22b-PBS and SCC22b-R cell lines. In conclusion, these results can serve as a preclinical rationale for a novel therapeutic strategy combining buparlisib with cetuximab in HNSCC patients who experience acquired cetuximab resistance. Further research is necessary to obtain a more profound understanding of the molecular mechanisms underlying this targeted combination therapy. Citation Format: Hannah Zaryouh, Hasan Baysal, Patrick Pauwels, Marc Peeters, Jan B. Vermorken, Filip Lardon, An Wouters, Ines De Pauw. Overcoming acquired cetuximab resistance in head and neck squamous cell carcinoma: An in vitro study on the potential of PI3K inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1095.

A chemical genetic screen identifies Aurora kinases as a therapeutic target in EGFR T790M negative, gefitinib-resistant head and neck squamous cell carcinoma (HNSCC).

BackgroundOverexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities.MethodsGenomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies.FindingsWe derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival.InterpretationOur study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors.FundingAgency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).

The Polo-like kinase 1 inhibitor onvansertib represents a relevant treatment for head and neck squamous cell carcinoma resistant to cisplatin and radiotherapy.

Rationale: Head and neck squamous cell carcinoma (HNSCC) represent the 4th most aggressive cancer. 50% of patients relapse to the current treatments combining surgery, radiotherapy and cisplatin and die two years after the diagnosis. Elevated expression of the polo-like kinase 1 (Plk1) correlated to a poor prognosis in epidermoid carcinomas.Methods: The molecular links between Plk1 and resistance to cisplatin/radiotherapy were investigated in patients and cell lines resistant to cisplatin and/or to radiotherapy. The therapeutic relevance of the Plk1 inhibitor onvansertib, alone or combined with cisplatin/radiotherapy, was evaluated on the proliferation/migration on HNSCC cell lines, in experimental HNSCC in mice, in a zebrafish metastasis model and on patient-derived 3D tumor sections.Results: Plk1 expression correlated to a bad prognosis in HNSCC and increased after relapse on cisplatin/radiotherapy. Onvansertib induced mitotic arrest, chromosomic abnormalities and polyploidy leading to apoptosis of sensitive and resistant HNSCC cells at nanomolar concentrations without any effects on normal cells. Onvansertib inhibited the growth of experimental HNSCC in mice and metastatic dissemination in zebrafishes. Moreover, onvansertib combined to cisplatin and/or radiotherapy resulted in a synergic induction of tumor cell death. The efficacy of onvansertib alone and in combination with reference treatments was confirmed on 3D viable sections of HNSCC surgical specimens.Conclusions: Targeting Plk1 by onvansertib represents a new strategy for HNSCC patients at the diagnosis in combination with reference treatments, or alone as a second line treatment for HNCSCC patients experiencing relapses.

P38 Mitogen-activated protein kinase modulates cisplatin resistance in Head and Neck Squamous Cell Carcinoma cells

ObjectiveThis study aims to investigate the role of p38 Mitogen-activated protein kinase (MAPK) in imparting cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) cells. DesignLaboratory generated cisplatin resistant HNSCC cells were treated with p38 inhibitor and were subjected to increasing dosage of cisplatin. Western blot, immunohistochemistry and RT PCR analysis were performed to investigate expression level of p-p38 and Cancer stem cell (CSC) markers in cisplatin resistant HNSCC cells with or without p38 inhibitor. Chemoresistance, wound healing capacity and Spheroids formation capacity were assessed following p38 inhibition in cisplatin resistant HNSCC cell lines. In addition, alkaline comet assay and γ-H2AX immunostaining were performed to evaluate the DNA damage response and repair abilities in cisplatin resistant HNSCC cells after p38 inhibition. ResultsIt was observed that following p38 inhibition, cisplatin resistant HNSCC cells exhibited significant reduction in expression of CSC markers, β-catenin, reduced migration potential and sphere forming ability along with increased apoptotic index demonstrating there was increased sensitivity towards Cisplatin. Molecular docking study identified several interface amino acid residues between p-p38 with CSC markers (Klf4 and CD44). p38 inhibited cisplatin resistant HNSCC cells also exhibited increased DNA damage as measured by Comet assay and γ-H2AX foci formation index. There was significant decrease in DNA repair as confirmed by reduced ERRC1 expression. ConclusionsOur study demonstrated that p38 MAPK inhibition can be a targeted approach to overcome resistance in HNSCC thereby escalating the effectiveness of chemotherapy in HNSCC.

Abstract 3371: Role of the epidermal growth factor receptor expression and internalization in cetuximab-induced antibody-dependent cellular cytotoxicity in head and neck squamous cell carcinoma

Abstract The epidermal growth factor receptor (EGFR)-targeted therapies and immunotherapies are now at the forefront for treatment of head and neck squamous cell carcinoma (HNSCC). However, resistance to cetuximab, an anti-EGFR monoclonal antibody (mAb), is a major clinical problem, limiting the clinical benefit of treatment. Besides inhibition of downstream signalling, cetuximab can also induce antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to investigate the role of baseline EGFR expression and EGFR internalization following treatment with cetuximab as possible mechanisms that may explain differences in cetuximab-induced ADCC between HNSCC cell lines. Ten HNSCC cell lines, comprising cetuximab sensitive as well as intrinsically and acquired resistant cell lines, differing in HPV-status, were used. ADCC was assessed using the xCELLigence RTCA system, by co-culturing HNSCC cells with natural killer cells from healthy volunteers (E:T; 5:1). Baseline EGFR expression was flow cytometrically determined, following staining with an EGFR PE-conjugated antibody or a PE-conjugated isotype control. Experiments were performed under normoxic and hypoxic (1% O2) conditions. To determine EGFR internalization, cetuximab and the corresponding isotype control were stained with FabFluor-pH reagent prior to incubation with HNSCC cells. Phase-contrast and red fluorescence kinetics were quantified using the IncuCyte Zoom. We demonstrate that cetuximab-induced ADCC was highest in intrinsically cetuximab resistant cell lines compared to the acquired resistant and cetuximab sensitive cell lines. Under hypoxic conditions, a reduced ADCC was observed in the cetuximab sensitive and acquired resistant HNSCC cell lines but not in the intrinsically resistant cell lines. Baseline EGFR was expressed in a cell line-dependent manner and was not correlated with cetuximab sensitivity or HPV-status. Moreover, there was no correlation between baseline EGFR expression and the amount of cetuximab-induced ADCC. Importantly, the intrinsically cetuximab resistant cells demonstrated the highest level of EGFR internalization compared to the acquired resistant and cetuximab sensitive cell lines. Correlation analysis showed that the degree of EGFR internalization positively corelated with cetuximab-induced ADCC. In conclusion, we showed that induction of ADCC is maintained, independent of EGFR expression. Furthermore, internalization of EGFR might play a role in cetuximab resistance and ADCC-induction. Future research further unravelling this mechanism will provide a better understanding of mAb-based anti-EGFR therapies in the future. Citation Format: Hasan Baysal, Ines De Pauw, Hannah Zaryouh, Marc Peeters, Jan Baptist Vermorken, Evelien Smits, Filip Lardon, Julie Jacobs, An Wouters. Role of the epidermal growth factor receptor expression and internalization in cetuximab-induced antibody-dependent cellular cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3371.

Cisplatin exposure causes c-Myc-dependent resistance to CDK4/6 inhibition in HPV-negative head and neck squamous cell carcinoma

The loss of p16 is a signature event in Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) that leads to increased Cyclin Dependent Kinase 4/6 (CDK) signaling. Palbociclib, a CDK4/6 inhibitor, is active for the treatment of a subset of HNSCC. In this study, we analyzed patient response data from a phase I clinical trial of palbociclib in HNSCC and observed an association between prior cisplatin exposure and CDK inhibitor resistance. We studied the effects of palbociclib on cisplatin-sensitive and -resistant HNSCC cell lines. We found that while palbociclib is highly effective against chemo-naive HNSCC cell lines and tumor xenografts, prior cisplatin exposure induces intrinsic resistance to palbociclib in vivo, a relationship that was not observed in vitro. Mechanistically, in the course of provoking a DNA damage-resistance phenotype, cisplatin exposure upregulates both c-Myc and cyclin E, and combination treatment with palbociclib and the c-Myc bromodomain inhibitor JQ1 exerts a synergistic anti-growth effect in cisplatin-resistant cells. These data show the benefit of exploiting the inherent resistance mechanisms of HNSCC to overcome cisplatin- and palbociclib resistance through the use of c-Myc inhibition.

Abstract 2946: Role of EphB3 receptor in mediating head and neck tumor growth, cell migration, and response to PI3K inhibitor

Abstract Introduction: Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Dysregulated expression of EphB3, a member of the Eph receptor gene family, has been reported in different cancers. However, the precise role of EphB3 in head and neck squamous cell carcinoma (HNSCC) has not been explored. In the current study, we examined the effect of EphB3 downregulation on HNSCC tumor cell growth, migration, and response to PIK3CA inhibition both in vitro and in an orthotopic model of head and neck cancer. Materials and methods: Alterations in EPHB3 across cancer types in the TCGA database were accessed via cBioportal. shRNA approach was used to knockdown the levels of EphB3 in human and murine HNSCC cell lines. The effect of EphB3 knockdown on cell viability and cell migration was determined by MTT/Trypan blue assay and by Boyden chamber assay respectively. We also generated a BKM120 resistant HNSCC cell line to elucidate the functional role of EphB3 in mediating sensitivity to PI3K inhibitor. The phospho-EphB3 levels were analyzed by an immunoprecipitation assay. For in vivo testing, we used an orthotopic model of HNSCC. Immunohistochemistry and Western blot analysis was performed on cultured HNSCC cells and on tumor tissues to determine key proteins that are altered in response to EphB3 knockdown in the presence and absence of PI3K inhibition. Results: Our TCGA data analysis showed that EphB3 is frequently co-amplified with PIK3CA in HNSCC. We therefore hypothesized that EphB3 amplification plays a pro-tumorigenic role in HNSCC and that EphB3 and PIK3CA are co-operating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported since EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is associated with an increase in pro-survival proteins, S6 and BcL-XL in the EphB3 shRNA tumors treated with BKM120 compared to controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo. To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. While total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2-Fc fusion protein resulted in decreased HNSCC migration and cell growth and enhanced response to BKM120 in vitro. Conclusions: Our data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K inhibitor. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC. Citation Format: Shilpa Bhatia, Anastacia Griego, Shelby Lennon, Ayman Oweida, Jaspreet Sharma, Christina Rohmer, Nomin Uyanga, Sanjana Bukkapatnam, Benjamin Van Court, David Raben, Christian Young, Lynn Heasley, Sana D. Karam. Role of EphB3 receptor in mediating head and neck tumor growth, cell migration, and response to PI3K inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2946.

Abstract 05: Untangling the gene-epigenome networks: Timing of epigenetic regulation of gene expression in acquired cetuximab resistance gene programs

Abstract The current study performs time course RNA-seq and DNA methylation profiling to determine the complex interactions between gene expression and epigenetic changes in acquired therapeutic resistance. The genomics era provides widespread characterization of the genomic landscape of tumors and has enabled precision treatment strategies. Currently, epidermal growth factor receptor (EGFR) inhibitors are the only FDA-approved targeted therapy for clinical use in head and neck squamous cell carcinoma (HNSCC). EGFR inhibitors are only effective in a subset of each of these tumors. Moreover, patients with de novo sensitivity to EGFR inhibitors often subsequently acquire resistance and succumb to their tumors. Numerous genetic and epigenetic alterations occur in tumors with acquired resistance. However, their timing and function remain unknown. Therefore, we develop new computational techniques to find gene programs associated with the acquisition of resistance. High-throughput transcriptional profiling enables unprecedented characterization of individual genes during cancer treatment. However, identifying and targeting mechanisms of EGFR resistance from these high-throughput data requires novel systems biology techniques that can discriminate altered cellular signaling pathways in response to cancer treatment. Therefore, we developed a new bioinformatics algorithm Coordinated Gene Activity in Pattern Sets (CoGAPS) to quantify multiplexed regulation and gene reuse in oncogenic signaling. To test this algorithm, we modified the HaCaT keratinocyte cell line model of premalignancy to simulate cancer cells with constitutive overexpression of wild-type EGFR and with activating mutations in HRAS and PIK3CA in a controlled genetic background. We apply CoGAPS to gene expression data from these models. This algorithm quantified relative changes in cellular signaling pathway activity in these data, not accessible to standard gene comparisons. Increases in CoGAPS pathway signatures from HRAS also occurred in gene expression data from the cetuximab resistant HNSCC cell line (1CC8) relative to its parental, sensitive cell line (UMSCC1). Investigation of the mechanisms of acquired resistance has previously been limited by reliance on case-control comparisons between sensitive and resistant cells, such as the UMSCC1 and 1CC8 cell lines. Since activity in cellular signaling pathways evolves during cetuximab resistance, it is essential to extend these case-control paradigms to quantify the dynamics responsible for resistance. Therefore, we developed a novel time course analysis to determine the molecular mechanisms of acquired cetuximab resistance in HNSCC. Specifically, we treated the cetuximab sensitive SCC25 HNSCC cell line over generations with both cetuximab and PBS. This long-term treatment protocol models the progression of acquired therapeutic resistance, including controls for clonal selection unrelated to treatment. Overexpression of the previous HRAS gene program also occurs in gene expression data measured during acquired cetuximab resistance in SCC25. We also measure DNA methylation during this time course to find the driver of this aberrant signaling associated with resistance. We apply CoGAPS analysis to the time course data for both DNA methylation and gene expression. This analysis distinguished early gene expression changes from cetuximab treatment from longer-term epigenetic alterations to gene expression during acquired resistance. Epigenetic regulation of FGFR1 expression emerged as the dominant mechanism of acquired therapeutic resistance in this system. Thus, our integration of time course DNA methylation and gene expression data enables unprecedented inference of the timing of targetable gene-epigenome programs responsible for acquired resistance. Citation Format: Genevieve Stein-O'Brien, Luciane T. Kagohara, Sijia Li, Manjusha Thakar, Ruchira Ranaweera, Michael Considine, Ludmila V. Danilova, Hiroyuki Ozawa, Joseph A. Califano, Daria A. Gaykalova, Michael F. Ochs, Christine H. Chung, Elana J. Fertig. Untangling the gene-epigenome networks: Timing of epigenetic regulation of gene expression in acquired cetuximab resistance gene programs [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 05.

Genome-scale Modeling of NADPH Metabolism in Radiation-resistant Head and Neck Squamous Cell Carcinoma Patients

Tumor resistance to radiation therapy remains a significant obstacle to long-term cancer patient survival, especially for head and neck squamous cell carcinoma (HNSCC), a cancer type with poor long-term outcomes. NADPH metabolism is a viable target for personalized radiation-sensitizing chemotherapies due to the reliance of tumors on NADPH for DNA repair, antioxidation, and cell survival following ionizing radiation. We have previously shown that genome-scale metabolic models that incorporate transcriptomic, kinetic, thermodynamic, and metabolite concentration data can make accurate predictions of NADPH production and sensitivity to redox-cycling chemotherapeutics such as β-lapachone in radiation-sensitive and -resistant HNSCC cell lines. Through the incorporation of transcriptomic data from the Cancer Genome Atlas, we have extended our modeling approach to develop personalized flux balance analysis-based metabolic models of HNSCC patient tumors. This collection of 132 models predicts that radiation-resistant tumors display greater conversion of NADP+ to NADPH than radiation-sensitive tumors, consistent with our prior work in matched HNSCC cell lines. Differences in NADPH production are manifested by the re-routing of metabolic flux through alternate NADPH-producing pathways in the human metabolic network. In addition, simulated gene knockdown of isocitrate dehydrogenase (IDH) isoforms has a more pronounced effect on NADPH production in radiation-resistant tumors, supporting prior evidence of IDH as a promising chemotherapeutic target for multiple cancer types. Finally, we discovered a subset of radiation-sensitive patients that displayed significantly lower NADPH-producing flux throughout the metabolic network as well as greater sensitivity to knockdown of NADPH production genes. Almost all of these TCGA patients with lower fluxes had tumors of the larynx as opposed to other head and neck cancer regions, indicating that NADPH metabolism in HNSCC tumors may depend greatly on the tumor’s anatomical location. This modeling approach can be extended to investigate the synergistic action of redox-based chemotherapeutics and radiation therapy to treat radiation-resistant HNSCC patients.

Dual Targeting of Epidermal Growth Factor Receptor and HER3 by MEHD7945A as Monotherapy or in Combination with Cisplatin Partially Overcomes Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma Cell Lines

Aberrant signaling of the epidermal growth factor receptor (EGFR) plays a crucial role in the tumorigenesis of many cancer types, including head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. After initial promising results of EGFR-targeted therapies such as cetuximab, the problem of therapeutic resistance is emerging and new treatment options are necessary. In contrast to first-generation EGFR inhibitors, MEHD7945A (duligotuzumab) is a monoclonal antibody with dual EGFR/HER3 specificity. Consequently, treatment with MEHD7945A may result in a more pronounced therapeutic benefit. In this study, sensitivity to MEHD7945A as a single agent and in combination with cisplatin was investigated in cetuximab-sensitive and -resistant HNSCC cell lines under normal and reduced oxygen conditions. The results demonstrated that sensitivity to MEHD7945A was cell line dependent and influenced by oxygen concentration. An additive, but not synergistic, interaction between MEHD7945A and cisplatin was observed. In conclusion, MEHD7945A has the potential to partially overcome cetuximab resistance. Nevertheless, further research is warranted to determine additional resistance mechanisms to cetuximab treatment besides HER3 signaling. Unraveling these mechanisms will ultimately lead to the development of new therapeutic strategies to improve the response to EGFR blockage.

Erratum to: Insulin-like growth factor 1 receptor mediated tyrosine 845 phosphorylation of epidermal growth factor receptor in the presence of monoclonal antibody cetuximab.

BACKGROUND The epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and several other human cancers. Monoclonal antibodies, such as cetuximab that block EGFR signaling, have emerged as valuable molecular targeting agents in clinical cancer therapy. Prolonged exposure to cetuximab can result in cells acquiring resistance by a process that remains incompletely understood. METHODS In this study, we analyzed the immediate early molecular response of cetuximab on physical interactions between EGFR and Insulin growth factor 1 like receptor (IGF-1R) in head and neck cancer cells that are resistant to cetuximab. Co-immunoprecipitation, small molecule inhibitors against phospho-Src and IGF-1R, quantitative western blot of EGFR and Src phosphorylation, cell proliferation assays were used to suggest the role of IGF-1R mediated phosphorylation of specific tyrosine Y845 on EGFR via increased heterodimerization of EGFR and IGF-1R in cetuximab resistant cells. RESULTS Heterodimerization of EGFR with IGF-1R was increased in cetuximab resistant HNSCC cell line UMSCC6. Basal levels of phosphorylated EGFR Y845 showed significant increase in the presence of cetuximab. Surprisingly, this activated Y845 level was not inhibited in the presence of Src inhibitor PP1. Instead, inhibition of IGF-1R by picropodophyllin (PPP) reduced the EGFR Y845 levels. Taken together, these results suggest that heterodimerization of EGFR with IGF-1R can lead to increased activity of EGFR and may be an important platform for cetuximab mediated signaling in head and neck tumors that have become resistant to anti-EGFR therapy. CONCLUSIONS EGFR-IGF-1R interaction has a functional consequence of phosphorylation of EGFR Y845 in cetuximab resistant HNSCC cells and dual targeting of EGFR and IGF-1R is a promising therapeutic strategy.

Insulin-like growth factor 1 receptor mediated tyrosine 845 phosphorylation of epidermal growth factor receptor in the presence of monoclonal antibody cetuximab.

BackgroundThe epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and several other human cancers. Monoclonal antibodies, such as cetuximab that block EGFR signaling, have emerged as valuable molecular targeting agents in clinical cancer therapy. Prolonged exposure to cetuximab can result in cells acquiring resistance by a process that remains incompletely understood.MethodsIn this study, we analyzed the immediate early molecular response of cetuximab on physical interactions between EGFR and Insulin growth factor 1 like receptor (IGF-1R) in head and neck cancer cells that are resistant to cetuximab. Co-immunoprecipitation, small molecule inhibitors against phospho-Src and IGF-1R, quantitative western blot of EGFR and Src phosphorylation, cell proliferation assays were used to suggest the role of IGF-1R mediated phosphorylation of specific tyrosine Y845 on EGFR via increased heterodimerization of EGFR and IGF-1R in cetuximab resistant cells.ResultsHeterodimerization of EGFR with IGF-1R was increased in cetuximab resistant HNSCC cell line UMSCC6. Basal levels of phosphorylated EGFR Y845 showed significant increase in the presence of cetuximab. Surprisingly, this activated Y845 level was not inhibited in the presence of Src inhibitor PP1. Instead, inhibition of IGF-1R by picropodophyllin (PPP) reduced the EGFR Y845 levels. Taken together, these results suggest that heterodimerization of EGFR with IGF-1R can lead to increased activity of EGFR and may be an important platform for cetuximab mediated signaling in head and neck tumors that have become resistant to anti-EGFR therapy.ConclusionsEGFR-IGF-1R interaction has a functional consequence of phosphorylation of EGFR Y845 in cetuximab resistant HNSCC cells and dual targeting of EGFR and IGF-1R is a promising therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2796-x) contains supplementary material, which is available to authorized users.

Abstract 1866: HER3 targeting sensitizes HNSCC to cetuximab - evidence from cell line and patient derived xenograft (PDX) models

Abstract Background and Objectives: Our previous study demonstrated that a combination of EGFR-targeted antibody cetuximab and HER3-specific antibody MM121 could significantly inhibit growth of head and neck squamous cell carcinoma (HNSCC) as compared with either of the single drugs. The current study aims to understand the role of HER3 in cetuximab resistance and the anti-tumor mechanisms of dual targeting of EGFR/HER3 in HNSCC. Materials and Methods: A cetuximab resistant HNSCC cell line SCC1-C, its parental cell line SCC1-P, and a HER3 knockdown counterpart SCC1-C/H were treated with cetuximab, MM121 and their combination. SRB and colony formation assays were performed to investigate the combined effect of MM121 and cetuximab on cancer cell growth. Activations of EGFR, HER2, HER3, AKT, and ERK signaling pathways were evaluated by Western blotting. The SCC1-C xenograft animal model and PDX models from 6 patients with HNSCC were utilized to determine the combined efficacy of MM121 and cetuximab. Results: Cetuximab induced HER3 activation and HER2/HER3 dimerization in HNSCC cell lines. The combined treatment blocked both EGFR and HER3 activation and inhibited both PI3K/AKT and ERK signaling pathways as well as HNSCC cell growth more effectively compared to either single antibody treatment in vitro. Furthermore, knock down of HER3 re-sensitized the resistant cell line to cetuximab. The anti-HER3/EGFR combination also demonstrated better efficacy than either single agent antibody in the cetuximab resistant HNSCC xenograft model and the PDX animal models. In PDX models from patients 1-4, both cetuximab and the combination significantly inhibited tumor growth in nude mice compare to the control (p&amp;lt;0.001 for both treatments) and there was no difference between single cetuximab and the combination. In 1 of the 4 PDXs, tumors treated with cetuximab grew back while no relapse could be detected in the combination group. In the PDX from patient 5, only the combination treatment significantly inhibited tumor growth (p&amp;lt;0.004) which was also more effective than cetuximab treatment (p&amp;lt;0.032). In the PDX from patient 6, both cetuximab and the combination significantly inhibited tumor growth in nude mice as compared with the control (p&amp;lt;0.001 for both treatments). However, the combination was significantly more effective than either of the single agents (p&amp;lt;0.0004 and p&amp;lt;0.003, respectively). Conclusion: Our study indicates that dual targeting of EGFR and HER3 is more effective than cetuximab alone in both cetuximab resistant HNSCC cell line and PDX models. These results pave the way for further clinical investigations using multiple targeting strategies in patients who have failed cetuximab based therapy. (The current study is supported by NIH grant R21 CA182661 to NFS and ZGC). Citation Format: Dongsheng Wang, Guoqing Qian, hongzheng zhang, kelly R. Magliocca, sreenivas nannapaneni, zhengjia chen, mihir R. patel, Mark W. El-Deiry, J.trad Wadsworth, dong M. Shin, fadlo R. khuri, Nabil F. Saba, Zhuo G. chen. HER3 targeting sensitizes HNSCC to cetuximab - evidence from cell line and patient derived xenograft (PDX) models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1866.

Abstract 3579: Identification of biomarkers of AXL-mediated drug resistance in head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer. In 2013, there were ∼53,000 newly diagnosed cases and ∼11,000 deaths related to HNSCC in the USA. Overexpression of EGFR is seen in 90% HNSCC; but, only ∼10% of patients treated with the anti-EGFR antibody cetuximab show increased response rates to cetuximab and these eventually gain resistance by poorly-characterized mechanisms. We showed an association between EMT and resistance to EGFR inhibitors in lung cancers (LC) and HNSCC using a 76-gene EMT signature. AXL was identified as a therapeutic candidate linking EMT and drug resistance, showing significantly higher expression in erlotinib resistant cell lines. Other groups have linked AXL to drug resistance in HNSCC, LC and breast cancers. Here we identify signaling pathways that are regulated by AXL, mediate drug resistance, and identify potential therapeutic targets to combine with AXL inhibition. Methods: Using 6 clinical cohorts including The Cancer Genome Atlas (TCGA N = 493) and PROSPECT (N = 142) across 3 cancer types, we identified genes whose mRNA expression was highly correlated with AXL. Protein profiling by reverse phase protein array (RPPA) to analyze total and phospho-proteins in HNSCC cell lines, pre- and post-AXL inhibitor treatment was used to identify pathways altered upon AXL inhibition. The response to AXL inhibition was assayed in HNSCC cell lines by proliferation assays and correlated to mRNA and protein expression. Results: Using gene-expression and RPPA analysis we saw the highest association of AXL with pathways involved in EMT (TGF-β, Rho GTPases), autophagy and immune response. Following treatment with an AXL inhibitor, we observed a decrease in phospho-proteins in the PI3K-AKT pathway, increased expression of markers associated with apoptosis, an epithelial phenotype, and p-EGFR. Using an AXL knockdown model system in HNSCC cell lines, we validated an increase in EGFR signaling (EGFR and p-Erk), epithelial (E-cadherin), apoptotic (cleaved PARP and caspase-7) and DNA repair proteins (RAD51, ku-80 and PARP) and a decrease in Slug, Twist and ZEB-1, indicating that AXL may be directly involved in mediating EMT. AXL knockdown reduced proliferation of HNSCC cell lines and AXL inhibition was able to re-sensitize resistant HNSCC cell lines to erlotinib, an EGFR tyrosine kinase inhibitor. Conclusions: In summary, we identified potential therapeutic targets that are upregulated with AXL expression in HNSCC and LC patient tumors and cell lines. Using AXL inhibitor and knockdown in HNSCC cell lines, we validated biomarkers involved in EMT, EGFR signaling and apoptosis that are altered upon AXL inhibition. AXL inhibition led to an epithelial phenotype in cells and re-sensitized resistant cells to erlotinib. Studies are ongoing to validate the mechanisms of AXL-mediated drug resistance and to identify potential combination treatments that can synergize with AXL-inhibition. Citation Format: Kavitha Balaji, Robert Cardnell, Lixia Diao, Pan Tong, Milena Mak, You Hong Fan, Fatemeh Masrorpour, Steven L. Warner, David J. Bearss, Ignacio Wistuba, Gordon B. Mills, John Heymach, Khandan Keyomarsi, Jing Wang, Lauren Averett Byers. Identification of biomarkers of AXL-mediated drug resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3579. doi:10.1158/1538-7445.AM2015-3579

Liposome encapsulated curcumin-difluorinated (CDF) inhibits the growth of cisplatin resistant head and neck cancer stem cells.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with 600,000 new cases every year worldwide. Although chemotherapeutics exist, five-year survival is only 50%. New strategies to overcome drug resistance are required to improve HNSCC treatment. Curcumin-difluorinated (CDF), a synthetic analog of curcumin, was packaged in liposomes and used to evaluate growth inhibition of cisplatin resistant HNSCC cell lines CCL-23R and UM-SCC-1R generated from the parental cell lines CCL-23 and UM-SCC-1 respectively. Growth inhibition in vitro and expression levels of the CD44 (cancer stem cell marker), cytokines, and growth factors were investigated after liposomal CDF treatment. The in vivo growth inhibitory effect of liposomal CDF was evaluated in the nude mice xenograft tumor model of UM-SCC-1R and the inhibition of CD44 was measured. Treatment of the resistant cell lines in vitro with liposomal CDF resulted in a statistically significant growth inhibition (p < 0.05). The nude mice xenograft study showed a statistically significant tumor growth inhibition of UM-SCC-1R cells and a reduction in the expression of CD44 (p < 0.05), indicating an inhibitory effect of liposomal CDF on CSCs. Our results demonstrate that delivery of CDF through liposomes may be an effective method for the treatment of cisplatin resistant HNSCC.