Head And Neck Squamous Cell Carcinoma Xenografts Research Articles

ERBB3/HER2 Signaling Promotes Resistance to EGFR Blockade in Head and Neck and Colorectal Cancer Models

EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.

Caspase-8 mutations in head and neck cancer confer resistance to death receptor-mediated apoptosis and enhance migration, invasion, and tumor growth

Little is known regarding molecular markers in head and neck squamous cell carcinoma (HNSCC) that predict responsiveness to different therapeutic regimens or predict HNSCC progression. Mutations in procaspase-8 occur in 9% of HNSCC primary tumors, but the functional consequences of these mutations are poorly understood. In this study, we examined the impact of four, representative, HNSCC-associated procaspase-8 mutations on activation of the extrinsic apoptosis pathway, as well as cellular migration and invasion, and in vivo tumor growth. All four mutant proteins acted to potently inhibit activation of apoptosis following treatment with TRAIL or agonistic anti-Fas. In contrast to wild-type procaspase-8, the mutant proteins were not recruited to FADD following treatment with TRAIL or anti-Fas, but may be constitutively bound by FADD. Three of the four procaspase-8 mutants promoted enhanced cellular migration and invasion through matrigel, relative to that seen with the wild-type procaspase-8 protein. Procaspase-8 mutation also stimulated the growth of HNSCC xenograft tumors. These findings indicate that HNSCC-associated procaspase-8 mutations inhibit activation of the extrinsic apoptosis pathway and are likely to represent markers for resistance to therapeutic regimens incorporating death receptor activators. Moreover, procaspase-8 mutations may serve as markers of HNSCC tumor progression, as exemplified by enhanced migration, invasion, and tumor growth.

Bitter Melon Reduces Head and Neck Squamous Cell Carcinoma Growth by Targeting c-Met Signaling

Head and neck squamous cell carcinoma (HNSCC) remains difficult to treat, and despite of advances in treatment, the overall survival rate has only modestly improved over the past several years. Thus, there is an urgent need for additional therapeutic modalities. We hypothesized that treatment of HNSCC cells with a dietary product such as bitter melon extract (BME) modulates multiple signaling pathways and regresses HNSCC tumor growth in a preclinical model. We observed a reduced cell proliferation in HNSCC cell lines. The mechanistic studies reveal that treatment of BME in HNSCC cells inhibited c-Met signaling pathway. We also observed that BME treatment in HNSCC reduced phosphoStat3, c-myc and Mcl-1 expression, downstream signaling molecules of c-Met. Furthermore, BME treatment in HNSCC cells modulated the expression of key cell cycle progression molecules leading to halted cell growth. Finally, BME feeding in mice bearing HNSCC xenograft tumor resulted in an inhibition of tumor growth and c-Met expression. Together, our results suggested that BME treatment in HNSCC cells modulates multiple signaling pathways and may have therapeutic potential for treating HNSCC.

EGFR-Inhibition Enhances Apoptosis in Irradiated Human Head and Neck Xenograft Tumors Independent of Effects on DNA Repair

Epidermal growth factor receptor (EGFR) inhibition using cetuximab improves the efficacy of radiotherapy in only a subgroup of head and neck squamous cell carcinoma (HNSCC) patients. Therefore, to improve patient selection a better understanding of tumor characteristics that affect treatment is necessary. Here, we investigated the effect of cetuximab on repair of radiation-induced DNA damage in a HNSCC xenograft model, which shows a synergistic effect to cetuximab and radiotherapy (SCCNij185) and a HNSCC model, which shows no additive effect of cetuximab to radiotherapy (SCCNij153). In both tumor models, clear increases were seen in the number of 53BP1 and Rad51 foci after irradiation. 53BP1 foci were present at comparable levels in hypoxic and normoxic tumor areas of the tumor xenografts, while the number of Rad51 foci was significantly higher in normoxic areas compared to hypoxic areas (P < 0.05). In both SCCNij185 and SCCNij153 xenografts an increased number of 53BP1 foci was observed in tumors treated with cetuximab and radiotherapy compared to radiotherapy alone. In SCCNij185 this increase was statistically significant in normoxic tumor areas (P = 0.04) and in SCCNij153 in both hypoxic and normoxic areas (P = 0.007 and P = 0.02, respectively). The number of Rad51 foci was not significantly different when cetuximab was added to radiotherapy compared to radiotherapy alone. Levels of pEGFR and pERK1/2 were decreased when cetuximab was added to radiotherapy in SCCNij185, but not in SCCNij153. Apoptosis was also only increased in SCCNij185 tumors at 4 days after cetuximab and radiotherapy treatment (P < 0.01). In conclusion, cetuximab inhibited DNA repair in both HNSCC models, but this effect was not predictive for the radiosensitizing effect of cetuximab in vivo. This lack of correlation may be related to differential effects of cetuximab and radiotherapy on ERK1/2 signaling and a decreased induction of apoptosis by cetuximab and radiotherapy in the resistant model.

111In-cetuximab-F(ab’)2 SPECT imaging for quantification of accessible epidermal growth factor receptors (EGFR) in HNSCC xenografts

Background and purposeImmunohistochemical epidermal growth factor receptor (EGFR) expression does not correlate with treatment response in head and neck squamous cell carcinomas (HNSCC). Aim was to apply the tracer 111In-cetuximab-F(ab’)2 for EGFR microSPECT imaging and to investigate if tracer uptake correlated with response to EGFR-inhibition by cetuximab in HNSCC xenografts. Usage of F(ab)2 fragments allows for shorter interval between tracer injection and imaging. Materials and methodsMice with HNSCC xenografts, SCCNij202, 153, 185 and 167 were imaged with microSPECT using 111In-cetuximab-F(ab’)2. Subsequently, tumors were analyzed by autoradiography and immunohistochemistry and tracer concentration was determined. Tumor uptake was correlated with previously assessed response to cetuximab treatment. ResultsMicroSPECT imaging showed preferential uptake in HNSCC xenografts. Tumor-to-liver ratios were 3.1±0.2 (SCCNij202), 2.8±0.4 (SCCNij153), 2.0±0.8 (SCCNij185), 2.0±0.4 (SCCNij167). Immunohistochemical EGFR fractions (fEGFR) differed significantly between xenografts; 0.77±0.07 (SCCNij202), 0.66±0.11 (SCCNij153), 0.57±0.19 (SCCNij185), 0.16±0.10 (SCCNij167) (p<0.001). Tumor fEGFR correlated with 111In-cetuximab-F(ab’)2 tumor uptake (r=0.6, p<0.01) and tracer autoradiography (r=0.7, p<0.0001). Tumor uptake of 111In-cetuximab-F(ab’)2 was proportionally associated with cetuximab treatment response in three out of four xenograft models. Conclusion111In-cetuximab-F(ab’)2 showed good tumor-to-background contrast on microSPECT imaging, allowing noninvasive assessment of EGFR expression in vivo, and possibly evaluation of treatment response to EGFR-inhibition.

A novel extracellular drug conjugate significantly inhibits head and neck squamous cell carcinoma

Despite advances in treatment modalities, head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat with poor survival and high morbidity, necessitating a therapy with greater efficacy. EDC22 is an extracellular drug conjugate of the monoclonal antibody targeting CD147 (glycoprotein highly expressed on HNSCC cells) linked with a small drug molecule inhibitor of Na, K-ATPase. In this study, EDC22's potential as a treatment modality for HNSCC was performed. HNSCC cell lines (FADU, OSC-19, Cal27, SCC-1) were cultured in vitro and proliferation and cell viability were assessed following treatment with a range of concentrations of EDC22 (0.25-5.00μg/mL). Mice bearing HNSCC xenografts (OSC-19, SCC-1) were treated with either EDC22 (3-10mg/kg), anti-CD147 monoclonal antibody, cisplatin (1mg/kg) or radiation therapy (2Gy/week) monotherapy or in combination. In vitro, treatment with minimal concentration of EDC22 (0.25μg/mL) significantly decreased cellular proliferation and cell viability (p<0.0001). In vivo, systemic treatment with EDC22 significantly decreased primary tumor growth rate in both an orthotopic mouse model (OSC-19) and a flank tumor mouse model (SCC-1) (p<0.05). In addition, EDC22 therapy resulted in a greater reduction in tumor growth in vivo compared to radiation monotherapy (p<0.05) and a similar reduction in tumor growth compared to cisplatin monotherapy. Combination therapy provided no significant further reduction in tumor growth relative to EDC22 monotherapy. EDC22 is a potent inhibitor of HNSCC cell proliferation in vitro and in vivo, warranting further investigations of its clinical potential in the treatment of HNSCC.

HSP90 Inhibitor SNX5422/ 2112 Targets the Dysregulated Signal and Transcription Factor Network and Malignant Phenotype of Head and Neck Squamous Cell Carcinoma

Heat shock protein 90 (HSP90) is a chaperone protein that stabilizes proteins involved in oncogenic and therapeutic resistance pathways of epithelial cancers, including head and neck squamous cell carcinomas (HNSCCs). Here, we characterized the molecular, cellular, and preclinical activity of HSP90 inhibitor SNX5422/2112 in HNSCC overexpressing HSP90. SNX2112 inhibited proliferation, induced G2/M block, and enhanced cytotoxicity, chemosensitivity, and radiosensitivity between 25 and 250 nM in vitro. SNX2112 showed combinatorial activity with paclitaxel in wild-type (wt) TP53-deficient and cisplatin in mutant (mt) TP53 HNSCC lines. SNX2112 decreased expression or phosphorylation of epidermal growth factor receptor (EGFR), c-MET, v-akt murine thymoma viral oncogene homolog 1 (AKT), extracellular signal-regulated kinases (ERK) 1 and 2, inhibitor êB kinase, and signal transducer and transcription factor 3 (STAT3), corresponding downstream nuclear factor êB, activator protein-1, and STAT3 reporter genes, and target oncogenes and angiogenic cytokines. Furthermore, SNX2112 enhanced re-expression of TP53 and targets p21WAF1 and PUMA, while TP53 inhibitor Pifithrin or siRNA attenuated the antiproliferative activity of SNX2112 in wtTP53 HNSCC in vitro. Prodrug SNX5422 similarly down-modulated key signal targets, enhanced TP53 expression and apoptosis, and inhibited proliferation, angiogenesis, and tumorigenesis in a wtTP53-deficient HNSCC xenograft model. Thus, HSP90 inhibitor SNX5422/2112 broadly modulates multiple key nodes within the dysregulated signaling network, with corresponding effects upon the malignant phenotype. Our data support investigation of SNX5422/2112 in combination with paclitaxel, cisplatin, and radiotherapy in HNSCC with different TP53 status.

Vascular priming enhances chemotherapeutic efficacy against head and neck cancer

The need to improve chemotherapeutic efficacy against head and neck squamous cell carcinomas (HNSCC) is well recognized. In this study, we investigated the potential of targeting the established tumor vasculature in combination with chemotherapy in head and neck cancer. Experimental studies were carried out in multiple human HNSCC xenograft models to examine the activity of the vascular disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in combination with chemotherapy. Multimodality imaging (magnetic resonance imaging, bioluminescence) in conjunction with drug delivery assessment (fluorescence microscopy), histopathology and microarray analysis was performed to characterize tumor response to therapy. Long-term treatment outcome was assessed using clinically-relevant end points of efficacy. Pretreatment of tumors with VDA prior to administration of chemotherapy increased intratumoral drug delivery and treatment efficacy. Enhancement of therapeutic efficacy was dependent on the dose and duration of VDA treatment but was independent of the chemotherapeutic agent evaluated. Combination treatment resulted in increased tumor cell kill and improvement in progression-free survival and overall survival in both ectopic and orthotopic HNSCC models. Our results show that preconditioning of the tumor microenvironment with an antivascular agent primes the tumor vasculature and results in enhancement of chemotherapeutic delivery and efficacy in vivo. Further investigation into the activity of antivascular agents in combination with chemotherapy against HNSCC is warranted.

Effect of obatoclax (GX15-070) on cell survival, apoptosis, and prosurvival autophagy in head and neck squamous cell carcinoma (HNSCC).

e13587 Background: Approximately 500,000 new cases of head and neck cancer are diagnosed annually worldwide, with 300,000 deaths per year. Patients with recurrent and metastatic disease have a dismal prognosis with a median survival of less than one year. Current standard of care involves a platinum-based regimen for patients with good performance status, but is usually difficult to tolerate. Novel therapeutic approaches are in great need for this patient population. The antiapoptotic BCL-2 family proteins, including BCL-2, BCL-XL, and MCL-1 are involved in oncogenesis and chemoresistance and are overexpressed in HNSCC. Obatoclax is a pan-BCL2 inhibitor capable of inhibiting MCL-1, in addition to BCL-2 and BCL-XL. Methods: We determined the activity of obatoclax in 4 well-characterized HNSCC cell lines (UMSCC-1, Cal-33, 1483, UMSCC-22A). Cell viability was determined by MTT assay and apoptosis by Annexin-V binding, FACS analysis, and immunoblotting. Autophagy was assessed by immunofluorescence and immunoblotting. Results: All 4 HNSCC cell lines were highly sensitive to single-agent obatoclax with IC50’s ranging from 50-200 nM. Obatoclax induced apoptosis in all cell lines as evidenced by increased Annexin-V binding at 48 hours in a dose-dependent manner from less than 10% in vehicle treated cells to 30-60% in cells treated with obatoclax. Processing/activation of caspase-3 and cleavage of PARP protein was also observed. In addition, obatoclax induced prosurvival autophagy in all cell lines and the addition of the autophagy inhibitor chloroquine enhanced obatoclax cytotoxicity. In vivo studies of obatoclax using human HNSCC xenograft tumors is currently underway. Conclusions: This preclinical study suggests that obatoclax might have therapeutic value in HNSCC, as a single agent or in combination with an autophagy inhibitor.

Interleukin-8 as a modulator of response to bevacizumab in preclinical models of head and neck squamous cell carcinoma

Bevacizumab, a monoclonal antibody to VEGF-A, is under active clinical evaluation in head and neck squamous cell carcinoma (HNSCC) and appears to be a promising therapy in at least a subset of patients. However, there are no reliable predictive biomarkers to identify those patients most likely to benefit. In this study, we assessed the efficacy of bevacizumab in HNSCC xenograft models to characterize escape mechanisms underlying intrinsic resistance and identify potential biomarkers of drug response. We evaluated the angiogenic profile of HNSCC cells from sensitive and resistant cell lines using antibody array. We further examined the role of interleukin-8 (IL-8) in contributing to resistance both in vitro and in vivo, using a loss- and gain-of-function approach. Angiogenic profiling indicated that resistant cells expressed higher levels of proangiogenic factors including IL-8, interleukin-1α (IL-1α), vascular endothelial growth factor (VEGF), fibroblast growth factor-a (FGF-a), and tumor necrosis factor-α (TNF-α). IL-8 was the most differentially expressed protein. IL-8 signaling compensated for VEGF inhibition in endothelial cells. Downregulation of IL-8 resulted in sensitization of resistant tumors to bevacizumab by disrupting angiogenesis and enhancing endothelial cell apoptosis. Overexpression of IL-8 in sensitive tumors conferred resistance to bevacizumab. Serum analysis of HNSCC patients treated with a bevacizumab-containing regime revealed high baseline IL-8 levels in a subset of patients refractory to treatment but not in responders. These results implicate IL-8 in mediating intrinsic resistance to bevacizumab in HNSCC. Hence, co-targeting of VEGF and IL-8 may help overcome resistance and enhance therapeutic efficacy.

Abstract 2263: Biliverdin inhibits head and neck cancer cell growth via activation of retinoblastoma signaling pathway.

Abstract Head and neck cancer patients often present with advanced disease and, despite advances in chemoradiation treatment protocols, continue to have a poor prognosis while experiencing severe treatment induced toxicity. There is an increased demand for natural, and non-toxic agents for treatment, which can be either used alone or in combination with our therapies. In this study, we are using biliverdin (BV), an antioxidant bile pigment, which is oxidized from bilirubin. As a potent antioxidant, accumulating data from observations in experimental and human studies indicate that the BV may be protective against certain diseases, including cancer. Based on our own observations that BV suppresses cell proliferation in head and neck squamous cell carcinoma (HNSCC) cells and clinical observations finding a lesser incidence of cancer in healthy individuals with slightly elevated serum bilirubin levels, we hypothesized that BV might suppress tumor cell proliferation. The present study looks to evaluate whether BV treatment can suppress cell proliferation in HNSCC cells in vitro and in vivo, and to elucidate the potential mechanism through which it exerts this effect. HNSCC cell lines, with varying resistance to chemoradiation, and a mouse model with human HNSCC xenografts were used in this study. As potential effectors, we analyzed key proteins that are involved in apoptosis and cell cycle progression. In vitro, we investigated the effect of BV on five HNSCC cell lines and the signaling pathways involved in BV action on tumor cell proliferation using western blots. We also analyzed the antiproliferative effects of the BV by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell cycle analysis where BV caused cell cycle arrest by hypophosphorylation (activating) of the retinoblastoma (Rb) tumor suppressor protein in HNSCC cells. Ex vivo, we found significant suppression of xenograft tumor growth in HNSCC cells pretreated with BV before intradermal injection in nude mice as compared to controls. In vivo, tumor growth was assessed in BALB/c nude mice bearing HNSCC xenografts that were treated with BV. The signaling pathway responsible for this action included dephosphorylation of epidermal growth factor receptor (EGFR), Akt, NF-κB, and hypophosphorylation of the Rb tumor suppressor protein as well as caspase activation. Our study provides a rationale for a novel therapeutic approach using exogenous BV, a nontoxic antioxidant product of heme catabolism, as an anti-cancer agent against HNSCC through its effect on the Rb/Akt/NF-κB signal transduction pathway. Our findings indicate that BV's actions on these key tumorogenic pathways may extend its therapeutic potential to head and neck cancer. Citation Format: Jun Zheng, Danish A. Nagda, Shayanne A. Lajud, Sanjeev Kumar, Anas Mouchli, Orysia Bezpalko, Bert W. O'Malley, Daqing Li. Biliverdin inhibits head and neck cancer cell growth via activation of retinoblastoma signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2263. doi:10.1158/1538-7445.AM2013-2263

Abstract 2439: Obatoclax (GX15-070) potently induces apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines and enhances the activity of EGFR inhibitors.

Abstract HNSCC represents 90% of head and neck tumors. More than half of HNSCC patients are initially treated with curative intent, but will relapse over the course of their disease and have poor prognosis with a median survival of 6-9 months. Novel therapeutic approaches are in desperate need for this patient population. The antiapoptotic BCL-2 family proteins like BCL-2, BCL-XL, and MCL-1 are involved in oncogenesis and chemoresistance and are overexpressed in HNSCC. Obatoclax is a small-molecule antagonist of the BH3-binding groove of the prosurvival (or anti-apoptotic) members of the BCL-2 family of proteins. It is the only compound among the current BCL-2 inhibitors in clinical trials that is capable of inhibiting MCL-1, in addition to BCL-2 and BCL-XL. We determined the activity of obatoclax in 4 HNSCC cell lines (UMSCC-1, Cal-33, 1483, UMSCC-22A). Cell viability was determined by MTT assay, and apoptosis by Annexin-V binding, FACS analysis and immunoblotting. All four HNSCC cell lines were highly sensitive to single agent obatoclax with IC50’s ranging from 50-300 nM. Obatoclax induced apoptosis in all four HNSCC cell lines as evidenced by increased Annexin-V binding at 48 hours from 10% in vehicle treated cells to 30-60% and through cleavage of caspase 3 and PARP. Interestingly, obatoclax enhanced the activity of the two small tyrosine kinase inhibitors of EGFR (erlotinib and gefitinib). We are the first to show the activity of obatoclax in HNSCC alone and in combination with EGFR inhibitors. In vivo study of obatoclax using human HNSCC xenograft tumors is currently underway. This preclinical study suggests that obatoclax might have a therapeutic role in HNSCC either alone or in combination with EGFR inhibitors. Citation Format: Victor Y. Yazbeck, Kathleen Dorritie, Changyou Li, Jennifer Grandis, Yan Zang, Daniel E. Johnson. Obatoclax (GX15-070) potently induces apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines and enhances the activity of EGFR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2013-2439

Antitumor Effects of EGFR Antisense Guanidine-Based Peptide Nucleic Acids in Cancer Models

Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.

Targeting TORC1/2 Enhances Sensitivity to EGFR Inhibitors in Head and Neck Cancer Preclinical Models

Head and neck squamous cell carcinoma (HNSCC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) where treatments targeting EGFR have met with limited clinical success. Elucidation of the key downstream-pathways that remain activated in the setting of EGFR blockade may reveal new therapeutic targets. The present study was undertaken to test the hypothesis that inhibition of the mammalian target of rapamycin (mTOR) complex would enhance the effects of EGFR blockade in HNSCC preclinical models. Treatment of HNSCC cell lines with the newly developed TORC1/TORC2 inhibitor OSI-027/ASP4876 resulted in dose-dependent inhibition of proliferation with abrogation of phosphorylation of known downstream targets including phospho-AKT (Ser473), phospho-4E-BP1, phospho-p70s6K, and phospho-PRAS40. Furthermore, combined treatment with OSI-027 and erlotinib resulted in enhanced biochemical effects and synergistic growth inhibition in vitro. Treatment of mice bearing HNSCC xenografts with a combination of the Food and Drug Administration (FDA)-approved EGFR inhibitor cetuximab and OSI-027 demonstrated a significant reduction of tumor volumes compared with either treatment alone. These findings suggest that TORC1/TORC2 inhibition in conjunction with EGFR blockade represents a plausible therapeutic strategy for HNSCC.

Carfilzomib and ONX 0912 Inhibit Cell Survival and Tumor Growth of Head and Neck Cancer and Their Activities Are Enhanced by Suppression of Mcl-1 or Autophagy

Carfilzomib is a selective, irreversible inhibitor of the chymotrypsin-like activity of the proteasome and is undergoing clinical evaluation in myeloma. ONX 0912 (oprozomib) is an orally bioavailable derivative. The activities of carfilzomib and ONX 0912 against solid tumor malignancies are less well understood. We investigated the impact and mechanisms of action of carfilzomib and ONX 0912 in preclinical models of head and neck squamous cell carcinoma (HNSCC). The effects of carfilzomib and ONX 0912 on HNSCC cell survival and xenograft tumor growth were evaluated. The impact and mechanisms of both agents on apoptosis and autophagy induction were also investigated. The contribution of the unfolded protein response (UPR) to autophagy induction and the role of autophagy in attenuating HNSCC cell death were determined. Carfilzomib and ONX 0912 potently induced apoptosis in HNSCC cell lines via upregulation of pro-apoptotic Bik. Upregulation of Mcl-1 by these agents served to dampen their efficacies. Carfilzomib and ONX 0912 also induced autophagy, mediated, in part, by activation of the UPR pathway involving upregulation of ATF4 transcription factor. Autophagy induction served a prosurvival role. Oral administration of ONX 0912 inhibited the growth of HNSCC xenograft tumors in a dose-dependent manner. These results show that carfilzomib and ONX 0912 are potently active against HNSCC cells, and the activities of these agents can be enhanced via suppression of Mcl-1 or inhibition of autophagy. Oral ONX 0912 exhibits in vivo activity against HNSCC tumors and may represent a useful therapeutic agent for this malignancy.

Evaluation of tyrosine receptor kinases in the interactions of head and neck squamous cell carcinoma cells and fibroblasts

Despite treatment advancements, disease-free survival of head and neck squamous cell carcinoma (HNSCC) has not significantly improved. This may be a result of tumor-fibroblasts interactions providing protective pathways for oncogenic cells to resist therapy. Further understanding of these relationships in HNSCC may improve effectiveness of targeted therapies. In this article, we investigated the role of several receptor tyrosine kinases (RTKs) in the interactions between HNSCC cells and supporting cells (fibroblasts). HNSCC cell lines and human tumor samples were evaluated for FGFR1/2/3, and PDGF-beta expression levels. Cell lines (FADU, SCC1, OSC19, Cal27, SCC22A) were treated with a range of physiological concentrations of dovitinib and assessed for proliferation, cytotoxicity, and apoptosis. Mice bearing HNSCC xenografts were treated with dovitinib (20 mg/kg). Evaluation of HNSCC tumor specimens, cell lines and fibroblasts found variable expression of multiple RTKs (fibroblasts growth factor receptor, platelet derived growth factor receptor and vascular endothelial growth factor receptor) and their ligands, supporting previous theories of paracrine and autocrine signaling within the microenvironment. In a dose-dependent fashion, RTK inhibition reduced proliferation of HNSCC cell lines and fibroblast in vitro. When HNSCC cells were cocultured with fibroblasts, RTK inhibition resulted in a smaller reduction in the proliferation relative to untreated conditions. In vivo, RTK inhibition resulted in significant tumor regression and growth inhibition (p<0.05) and reduced the incidence of regional lymph node metastasis. Effective treatment of HNSCC, therefore, may require inhibition of multiple RTKs in order to adequately inhibit the microenvironment's various signaling pathways.

Antitumor Mechanisms of Targeting the PDK1 Pathway in Head and Neck Cancer

G-protein-coupled receptors (GPCR) activate the epidermal growth factor receptor (EGFR) and mediate EGFR-independent signaling pathways to promote the growth of a variety of cancers, including head and neck squamous cell carcinoma (HNSCC). Identification of the common signaling mechanisms involved in GPCR-induced EGFR-dependent and EGFR-independent processes will facilitate the development of more therapeutic strategies. In this study, we hypothesized that phosphoinositide-dependent kinase 1 (PDK1) contributes to GPCR-EGFR cross-talk and signaling in the absence of EGFR and suggests that inhibition of the PDK1 pathway may be effective in the treatment of HNSCC. The contribution of PDK1 to the EGFR-dependent and EGFR-independent signaling in HNSCC was determined using RNA interference, a kinase-dead mutant, and pharmacologic inhibition. In vivo xenografts studies were also carried out to determine the efficacy of targeting PDK1 alone or in combination with the U.S. Food and Drug Administration-approved EGFR inhibitor cetuximab. PDK1 contributed to both GPCR-induced EGFR activation and cell growth. PDK1 also mediated activation of p70S6K in the absence of EGFR. Blockade of PDK1 with a small molecule inhibitor (AR-12) abrogated HNSCC growth, induced apoptosis, and enhanced the antiproliferative effects of EGFR tyrosine kinase inhibitors in vitro. HNSCC xenografts expressing kinase-dead PDK1 showed increased sensitivity to cetuximab compared with vector-transfected controls. Administration of AR-12 substantially decreased HNSCC tumor growth in vivo. These cumulative results show that PDK1 is a common signaling intermediate in GPCR-EGFR cross-talk and EGFR-independent signaling, and in which targeting the PDK1 pathway may represent a rational therapeutic strategy to enhance clinical responses to EGFR inhibitors in HNSCC.

Lyn Kinase Mediates Cell Motility and Tumor Growth in EGFRvIII-Expressing Head and Neck Cancer

EGF receptor variant III (EGFRvIII) has been detected in several cancers in which tumors expressing this truncated growth factor receptor show more aggressive behavior. The molecular mechanisms that contribute to EGFRvIII-mediated tumor progression that are amenable to targeted therapy are incompletely understood. The present study aimed to better define the role of Src family kinases (SFKs) in EGFRvIII-mediated cell motility and tumor growth of head and neck squamous cell carcinomas (HNSCC). HNSCC models expressing EGFRvIII were treated with dasatinib, a pharmacologic inhibitor of SFKs. SFK inhibition significantly decreased cell proliferation, migration, and invasion of EGFRvIII-expressing HNSCC cells. Administration of dasatinib to mice bearing EGFRvIII-expressing HNSCC xenografts resulted in a significant reduction of tumor volume compared with controls. Immunoprecipitation with anti-c-Src, Lyn, Fyn, and Yes antibodies followed by immunoblotting for phosphorylation of the SFK activation site (Y416) showed specific activation of Lyn kinase in EGFRvIII-expressing HNSCC cell lines and human HNSCC tumor specimens. Selective inhibition of Lyn using siRNA decreased cell migration and invasion of EGFRvIII-expressing HNSCCs compared with vector control cells. These findings show that Lyn mediates tumor progression of EGFRvIII-expressing HNSCCs in which strategies to inhibit SFK may represent an effective therapeutic strategy.

MTOR as a Molecular Target in HPV-Associated Oral and Cervical Squamous Carcinomas

The incidence of head and neck squamous cell carcinomas (HNSCC) associated with human papillomavirus (HPV) infection has increased over the past decades in the United States. We aimed at examining the global impact of HPV-associated HNSCC and whether the established key role of mTOR activation in HNSCC is also observed in HPV(+) HNSCC lesions, thereby providing novel treatment options for HPV-associated HNSCC patients. An international HNSCC tissue microarray (TMA) was used to analyze the expression of p16(INK4A), a surrogate for HPV infection, and Akt-mTOR pathway activation. Results were confirmed in a large collection of HPV(-) and HPV(+) HNSCC cases and in a cervical cancer (CCSCC) TMA. Observations were validated in HNSCC and CCSCC-derived cell lines, which were xenografted into immunodeficient mice for tumorigenesis assays. Approximately 20% of all HNSCC lesions could be classified as HPV(+), irrespective of their country of origin. mTOR pathway activation was observed in most HPV(+) HNSCC and CCSCC lesions and cell lines. The preclinical efficacy of mTOR inhibition by rapamycin and RAD001 was explored in HPV(+) HNSCC and CCSCC tumor xenografts. Both mTOR inhibitors effectively decreased mTOR activity in vivo and caused a remarkable decrease in tumor burden. These results emphasize the emerging global impact of HPV-related HNSCCs and indicate that the activation of the mTOR pathway is a widespread event in both HPV(-) and HPV-associated HNSCC and CCSCC lesions. The emerging results may provide a rationale for the clinical evaluation of mTOR inhibitors as a molecular targeted approach for the treatment of HPV-associated malignancies.

Abstract 4684: Synthetic lethal interaction between EGFR and PARP inhibition

Abstract Purpose: Dysregulation of the epidermal growth factor receptor (EGFR) family is a hallmark of many cancers. Although targeting of EGFR family has improved outcomes, many patients ultimately fail this therapy. PARP inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing homologous recombination (HR)-deficient tumors while maintaining minimal toxicity in normal tissues. We have previously shown that EGFR inhibition induces synthetic lethality with PARPi in head and neck squamous cell carcinomas (HNSCCs) in vitro by attenuating DNA repair pathways. In this study, we validated these observations in vivo in HNSCCs xenografts. Importantly, we also tested this strategy in other EGFR dysregulated cancers. Methods: Cervical, breast, and head and neck cancer cell lines were used in this study. DNA damage and repair were assessed by immunofluorescence staining of cells for γ-H2AX, Rad51, and DNA-Pk foci and/or chromosomally integrated HR and nonhomologous end-joining based GFP reporter assays. Cell viability was determined via colony formation and ATPlite assays. Tumor growth delay was assessed in mice bearing tumor xenografts. Results: Combined EGFR and PARP inhibition resulted in the greatest tumor growth delay versus either agent alone in mice bearing head and neck tumor xenografts (9 week tumor growth delay when compared to PARPi alone, 3 week tumor growth delay when compared to EGFR inhibitor alone). This correlated with persistent γ-H2AX foci indicative of unresolved DNA damage. Interestingly, this combination was also effective in other EGFR dysregulated tumors. Human cervical and breast cancer cells exhibited enhanced cytotoxicity with EGFR and PARP inhibition compared to either agent alone (80-99% cytotoxicity with combination vs 0-20% with either agent alone). Similar to head and neck cancer, susceptibility to PARPi induced by EGFR inhibition was associated with deficient NHEJ- and HR-mediated DNA repair and subsequent persistence of DNA damage. Conclusions: Thus, by generating a DNA repair deficit, EGFR inhibition can induce synthetic lethality with PARP inhibition. This can be an innovative treatment strategy for tumors with dysregulated EGFR signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4684. doi:1538-7445.AM2012-4684