Head And Neck Squamous Cell Cancer Cell Research Articles

Repurposing Albendazole: new potential as a chemotherapeutic agent with preferential activity against HPV-negative head and neck squamous cell cancer.

Albendazole is an anti-helminthic drug that has been shown to exhibit anti-cancer properties, however its activity in head and neck squamous cell cancer (HNSCC) was unknown. Using a series of in vitro assays, we assessed the ability of albendazole to inhibit proliferation in 20 HNSCC cell lines across a range of albendazole doses (1 nM–10 μM). Cell lines that responded to treatment were further examined for cell death, inhibition of migration and cell cycle arrest. Thirteen of fourteen human papillomavirus-negative HNSCC cell lines responded to albendazole, with an average IC50 of 152 nM. In contrast, only 3 of 6 human papillomavirus-positive HNSCC cell lines responded. Albendazole treatment resulted in apoptosis, inhibition of cell migration, cell cycle arrest in the G2/M phase and altered tubulin distribution. Normal control cells were not measurably affected by any dose tested. This study indicates that albendazole acts to inhibit the proliferation of human papillomavirus-negative HNSCC cell lines and thus warrants further study as a potential chemotherapeutic agent for patients suffering from head and neck cancer.

Induction of tumor regression by intratumoral STING agonists combined with anti-programmed death-L1 blocking antibody in a preclinical squamous cell carcinoma model.

Cyclic dinucleotides (CDNs) are bacterial intracellular messengers that have demonstrated antitumor activity in melanoma and breast tumors, although their role in immunotherapy of head and neck squamous cell cancers (HNSCCs) has not been well investigated. We measured primary tumor growth rates, mechanism of antitumor activity, and efficacy of programmed death-L1 blockade combinatorial therapy in SCCFVII tumor-bearing C3H/HeOUJ mice undergoing intratumoral injections with RR-cyclic-di-guanine (synthetic CDG), CDG (natural cyclic-di-guanine), R848 (TLR 7/8 agonist), or phosphate buffered saline (PBS, control). Intratumoral CDN treatment groups showed decreased tumor size and enhanced splenocyte Th1 response when compared to the PBS treatment control group (p < .05). The RR-CDG tumor microenvironment showed upregulated interferon (IFN)-γ+CD8+ and programmed death-L1. Combining programmed death-L1 blocking antibody with RR-CDG induced regression of established tumors. This study demonstrates the antitumor effects of CDNs in a HNSCC cell line. These preclinical data strongly support the future clinical development of intratumoral CDN in patients with HNSCC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1086-1094, 2017.

461 - Metabolic Plasticity Regulates Head and Neck Squamous Cell Cancer Growth-State Specific Oxidative Stress Response

Surgical resection followed by post-operative radiation is used as a standard treatment approach for locally advanced head and neck squamous cell cancer (HNSCC) patients. Unfortunately, even with aggressive approaches roughly 40% of patients with locoregionally advanced disease will ultimately succumb. 18F- Fluorothymidine Positron Emission Tomography (FLT PET) is a non-invasive imaging method that can be used in vivo to assess tumor proliferative status. Results showed that FLT PET successfully images HNSCC subjects with high lesion-to-background contrast. Surprisingly, our results also showed that HNSCC subjects with low FLT uptake (indicative of low proliferative index; LPI) in the primary tumor at base line have significantly poorer outcome compared to subjects with high tumor FLT uptake (indicative of high proliferative index; HPI). This intriguing clinical observation suggests that HNSCC growth-state significantly impacts therapy outcome. Consistent with this hypothesis, results from in vitro HNSCC cell culture studies showed that LPI-HNSCC cultures ( 20% S-phase cells). Radioresistance of LPI-HNSCC cultures is associated with a higher basal manganese superoxide dismutase activity, increases in radiation induced forkhead box M1 expression, suppression of late accumulation of reactive oxygen species (ROS), and absence of a radiation induced shift in cellular metabolism towards mitochondrial respiration. Interestingly, treatment with palmitate (substrate for the TCA cycle) increased oxygen consumption rate and ROS levels resulting in radiation sensitization of LPI-HNSCC cultures. These results support the hypothesis that metabolic plasticity regulates HNSCC growth-state specific radiation sensitivity.

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.

We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

Abstract 4666: Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts

Abstract Background: There is an urgent need for improved therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. The Phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in HNSCC, particularly in human papillomavirus (HPV) positive disease. PI3K pathway inhibitors are under active investigation in preclinical models and clinical trials, however it is not clear which patient population will benefit most from these agents. Methods: Thirty unique HNSCC cell lines including five HPV positive lines were characterized with whole exome sequencing and copy number arrays. All lines were treated over a 10-point dose range with a PIK3CA specific inhibitor (BYL719), a pan PI3K inhibitor (GDC-0941), a combined pan-PI3k and mTOR inhibitor (BEZ-235), mTOR inhibitor (everolimus) and a combination of BYL719 and everolimus. Five unique patient derived xenografts (PDX) were treated with BYL719 and everolimus alone and in combination. Results: The selective PIK3CA inhibitor BYL719 was preferentially active in cell lines with PIK3CA activating mutations and amplifications (&amp;gt;100% relative PIK3CA amplification), however HRAS mutant lines were resistant. These genomic markers of sensitivity and resistance did not apply to the pan-PI3K inhibitor GDC-0941 or combined PI3K-mTOR inhibitor BEZ-235, however these agents were more active in HPV-positive cell lines (P&amp;lt;0.05). BYL719 and everolimus were individually effective at decreasing tumor growth in four PDX models including a model with an activating PIK3CA mutation, two PIK3CA amplified models and a PIK3CA wild-type model. Combined PIK3CA and mTOR inhibition appeared to be synergistic and evolved resistance never developed to this treatment, however the combination caused significant weight loss in the PDX models. Interpretation: PI3K inhibitors were broadly active in HNSCC cell lines and PDXs and their application should not be solely restricted to tumors with activating PIK3CA mutations or amplifications. Agents with broader isoform activity and combined PI3K and mTOR treatment were more potent, however combined inhibitor treatment appeared toxic in the PDX models. This trade off of toxicity and efficacy needs to balanced in the clinical setting. Citation Format: Anthony C. Nichols, Laurie Ailkes, Ren Sun, Morgan Black, Alessandro Datti, Frederick Vizeacoumar, Nicole Pinto, Kara Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, Joe Mymryk, Paul C. Boutros, John W. Barrett. Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts. [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 4666.

Abstract 372: Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC 0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to taselisib, we profiled over 550 cell lines, encompassing 13 of the main tissue types, to increasing concentrations of taselisib. As expected, a small percentage of all tumor types responded to taselisib and correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 0.5uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Interestingly, the majority of HNSCC cell lines displayed an activated PI3K pathway as evidenced by phosphorylation of key proteins, such as AKT, S6 and PRAS40 implicating that downstream phospho-protein analysis of the PI3K pathway may not predict for sensitivity to taselisib. Mutational profiling identified that only 3 out of the tested 31 HNSCC cell lines harbored a mutation within PIK3CA, suggesting that additional genotypes may explain the sensitivity to taselisib. Additional molecular analysis assessing both gene expression and copy number levels of genes relevant to the PI3K pathway found that many of the taselisib sensitive cell lines had an activated ERBB signaling node through low level amplification of ERBB receptors (e.g. EGFR, FGFR1) or via a NRG1:ERBB3 autocrine mechanism. As many ERBB receptors heterodimerize with ERBB3, which contains p85 binding sites and potentially activates the PI3K pathway, we next tested whether taselisib would enhance the potency of ERBB receptor inhibitors in biomarker-defined subset of HNSCC cell lines. Combination screens were performed in cell lines with amplified EGFR, ERBB2, and NRG1:ERBB3 autocrine signaling with taselisib + tarceva or lapatinib. We found that combination effects assessed using the BLISS excess method showed a synergistic interaction. These results suggest that taselisib may have therapeutic potential for the treatment of HNSCC. Citation Format: Heidi M. Savage, Carol O’Brien, Heather Moore, Mark R. Lackner, Robert Yauch, Jeffrey Settleman, Timothy R. Wilson. Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines. [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 372.

Cancer Stem Cell Signaling during Repopulation in Head and Neck Cancer.

The aim of the study was to investigate cancer stem signaling during the repopulation response of a head and neck squamous cell cancer (HNSCC) xenograft after radiation treatment. Xenografts were generated from low passage HNSCC cells and were treated with either sham radiation or 15 Gy in one fraction. At different time points, days 0, 3, and 10 for controls and days 4, 7, 12, and 21, after irradiation, 3 tumors per group were harvested for global gene expression, pathway analysis, and immunohistochemical evaluation. 316 genes were identified that were associated with a series of stem cell-related genes and were differentially expressed (p ≤ 0.01 and 1.5-fold) at a minimum of one time point in UT-SCC-14 xenografts after radiation. The largest network of genes that showed significant changes after irradiation was associated with CD44, NOTCH1, and MET. c-MET and ALDH1A3 staining correlated with the changes in gene expression. A clear pattern emerged that was consistent with the growth inhibition data in that genes associated with stem cell pathways were most active at day 7 and day 12 after irradiation. The MET/CD44 axis seemed to be an important component of the repopulation response.

Cold Atmospheric Plasma: A Promising Complementary Therapy for Squamous Head and Neck Cancer.

Head and neck squamous cell cancer (HNSCC) is the 7th most common cancer worldwide. Despite the development of new therapeutic agents such as monoclonal antibodies, prognosis did not change for the last decades. Cold atmospheric plasma (CAP) presents the most promising new technology in cancer treatment. In this study the efficacy of a surface micro discharging (SMD) plasma device against two head and neck cancer cell lines was proved. Effects on the cell viability, DNA fragmentation and apoptosis induction were evaluated with the MTT assay, alkaline microgel electrophoresis (comet assay) and Annexin-V/PI staining. MTT assay revealed that the CAP treatment markedly decreases the cell viability for all tested treatment times (30, 60, 90, 120 and 180 s). IC 50 was reached within maximal 120 seconds of CAP treatment. Comet assay analysis showed a dose dependent high DNA fragmentation being one of the key players in anti-cancer activity of CAP. Annexin-V/PI staining revealed induction of apoptosis in CAP treated HNSCC cell lines but no significant dose dependency was seen. Thus, we confirmed that SMD Plasma technology is definitely a promising new approach on cancer treatment.

FGFR1 Expression Levels Predict BGJ398 Sensitivity of FGFR1-Dependent Head and Neck Squamous Cell Cancers.

FGFR1 copy-number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors, and patient-derived xenografts (PDX) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398. FGFR1 status, expression levels, and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n = 353) were assessed for FGFR1 CNG and mRNA levels, and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n = 39) were submitted to FGFR1 copy-number detection and mRNA assays to identify putative FGFR1-dependent tumors. Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. Thirty-one percent of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The nonamplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398. FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts tyrosine kinase inhibitor sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC.

Abstract 2152: Oncolytic virus (RT3D) administration in combination with cetuximab in head and neck squamous cell cancer (HNSCC) models harboring active EGFR/RAS/PI3K signaling

Abstract Introduction: Reovirus type 3 Dearing (RT3D) exerts selective toxicity against cells with an activated MAPK pathway. In reovirus-resistant cells, viral protein transcripts phosphorylate and activate the double-stranded RNA activated protein kinase (PKR) which subsequently phosphorylates the translation initiation factor (elF-2) which in turn inhibits viral gene translation. We sought to study the proliferation and oncolytic function of RT3D in HNSCC cells in relation to constitutively active RAS/PI3K pathway. We also sought to determine whether the combination of cetuximab(C) treatment and RT3D infection in HNSCC cells and animal models bearing constitutively active EGFR/RAS/PI3K pathway is more efficient compared to treatment only with C. Methods: We used a eEF1a1 KrasG12D transgenic mouse to test the therapeutic effect of combination of C treatment and RT3D infection in a mouse model of ras-driven oral SCC. Mice tumors were either treated with C or RT3D alone, or with a combination of RT3D and C. Tumor proliferation and apoptosis were measured by Ki67 and caspase 5,9 immunostaining before and after treatment. We also infected C resistant BB49 (HRAS mutant) and CAL33 (PIK3CA mutant) cell lines with RT3D and we compared the growth rates of RT3D infected and non-infected cells using MTT viability assay. We subsequently silenced HRAS expression using a lentivirus expressing shRNA and we compared the growth rates of parental and HRAS depleted cells that were either RT3D- infected or C treated. Protein extracts were subjected to immunobloting for elF2,p-elF2, PKR, p-PKR. Results: Combinatorial C and RT3D treatment decreased proliferation and enhanced apoptosis more efficiently compared to C or RT3D monotherapy in oral cancers that were developed in eEF1a1 KrasG12D transgenic mice. Our experiments also showed that RT3D infection suppressed the growth of CAL33 and BB49 cells more efficiently, compared to C treatment. Interestingly, RT3D infection also exerted an equipotent antiproliferative effect on HRAS silenced BB49 and Cal-33 cells. Immunoblot analysis revealed that PKR does not phosphorylate elF2 in HRAS depleted BB49 and CAL33 cells, upon RT3D infection providing evidence that reovirus proliferation is not exclusively dependent on HRAS signaling in HNSCC cells. Conclusions: RT3D infection exhibits a strong oncolytic effect in C resistant HNSCC with activated EGFR/RAS/MAPK signaling. Our findings provide evidence that RAS independent molecular mechanisms can also support the RT3D proliferation in this subset of HNSCC. Interestingly, combinatorial C and RT3D treatment was more effective compared to C or RT3D monotherapy in oral cancers developed in eEF1a1 KrasG12D transgenic mice, possibly due to the inhibitory effect of C in EGFR-dependent signaling pathways that promote resistance to RT3D infection and proliferation. Citation Format: Amanda Psyrri, Panagiota Economopoulou, Ioannis Kotsantis, Vassilios Ramfidis, Elena Vagia, George Koutsontodis, Clarence Sasaki, Theodoros Rampias. Oncolytic virus (RT3D) administration in combination with cetuximab in head and neck squamous cell cancer (HNSCC) models harboring active EGFR/RAS/PI3K signaling. [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 2152. doi:10.1158/1538-7445.AM2015-2152

Exploiting high-throughput cell line drug screening studies to identify candidate therapeutic agents in head and neck cancer.

BackgroundThere is an urgent need for better therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. Recent advances in high-throughput drug screening techniques and next-generation sequencing have identified new therapeutic targets in other cancer types, but an HNSCC-specific study has not yet been carried out. We have exploited data from two large-scale cell line projects to clearly describe the mutational and copy number status of HNSCC cell lines and identify candidate drugs with elevated efficacy in HNSCC.MethodsThe genetic landscape of 42 HNSCC cell lines including mutational and copy number data from studies by Garnett et al., and Barretina et al., were analyzed. Data from Garnett et al. was interrogated for relationships between HNSCC cells versus the entire cell line pool using one- and two-way analyses of variance (ANOVAs). As only seven HNSCC cell lines were tested with drugs by Barretina et al., a similar analysis was not carried out.ResultsRecurrent mutations in human papillomavirus (HPV)-negative patient tumors were confirmed in HNSCC cell lines, however additional, recurrent, cell line-specific mutations were identified. Four drugs, Bosutinib, Docetaxel, BIBW2992, and Gefitinib, were found via multiple-test corrected ANOVA to have lower IC50 values, suggesting higher drug sensitivity, in HNSCC lines versus non-HNSCC lines. Furthermore, the PI3K inhibitor AZD6482 demonstrated significantly higher activity (as measured by the IC50) in HNSCC cell lines harbouring PIK3CA mutations versus those that did not.ConclusionHNSCC-specific reanalysis of large-scale drug screening studies has identified candidate drugs that may be of therapeutic benefit and provided insights into strategies to target PIK3CA mutant tumors. PIK3CA mutations may represent a predictive biomarker for response to PI3K inhibitors. A large-scale study focused on HNSCC cell lines and including HPV-positive lines is necessary and has the potential to accelerate the development of improved therapeutics for patients suffering with head and neck cancer. This strategy can potentially be used as a template for drug discovery in any cancer type.Electronic supplementary materialThe online version of this article (doi:10.1186/2050-6511-15-66) contains supplementary material, which is available to authorized users.

Abstract 2631: Neuregulin-1 expression correlates with sensitivity to the PI3K inhibitor, GDC-0032, outside of PIK3CA mutations in head and neck cancer

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that GDC-0032 has increased activity against PIK3CA mutant cancer cell lines (Olivero A. et al., DDT02-01, AACR 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to GDC-0032, we profiled over 550 cell lines, encompassing 13 of the main cancer types, to increasing concentrations of GDC-0032. As expected, a small percentage of all tumor types responded to GDC-0032 and, were assessed, correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 1uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Molecular profiling identified that only 10% of the HNSCC cell lines had a mutation within PIK3CA, and no consistent copy number alterations were observed in receptor tyrosine kinases or PTEN. To investigate this observation further, we developed a ”PIK3CA activation” algorithm, by utilizing the mutant-selective property of GDC-0032, and calculated a ratio of GDC-0032 to GDC-0941, a pan PI3K inhibitor with reduced in vitro selectivity for PIK3CA mutations. Following a proof of principle experiment in breast cancer cells, a GDC-0941/GDC-0032 ratio of ≥4 enriched for breast cancer cells with mutant PIK3CA (11 out of 13 cell lines). Application of the algorithm, to our panel of HNSCC cell lines identified 10 out of 34 cell lines that had a GDC-0941/GDC-0032 ratio of ≥4, with only 1 of these cell lines being PIK3CA mutant. Further molecular characterization identified high mRNA expression of neuregulin-1, the ligand for HER3, in GDC-0032 cell lines with a GDC-0941/GDC-0032 ratio of ≥4, compared to cell lines with a ratio of &amp;lt;4. Pharmacodynamic analysis of sensitive and refractory HNSCC cell lines demonstrated that GDC-0032 inhibited the phospho-AKT pathway in both groups of cell lines, but GDC-0032 only suppressed downstream phospo-S6 signaling in the sensitive cell lines. In conclusion, high throughput cell line screening identified a “PIK3CA activated” subset of HNSCC cell lines that correlated with increased neuregulin-1 expression and corresponding sensitivity to GDC-0032, outside of PIK3CA mutations. Citation Format: Heidi M. Savage, Carol O'Brien, Bob Yauch, Jeff Settleman, Mark R. Lackner, Timothy R. Wilson. Neuregulin-1 expression correlates with sensitivity to the PI3K inhibitor, GDC-0032, outside of PIK3CA mutations in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2631. doi:10.1158/1538-7445.AM2014-2631

Loss of Trop2 causes ErbB3 activation through a neuregulin-1-dependent mechanism in the mesenchymal subtype of HNSCC.

In head and neck squamous cell cancer (HNSCC), four intrinsic subtypes (or groups) have been identified, and each one possesses a unique biology that will require specific treatment strategies. We previously reported that mesenchymal (group 2) tumors exhibit reduced levels of Trop2 expression. In this study, we investigated the functional role of Trop2 in HNSCC and find that loss results in autocrine activation of the EGFR family member ErbB3 via neuregulin-1. Trop2 localizes to both the cell surface and cytosol of HNSCC cells and forms a complex with neuregulin-1, which is predominantly cytosolic. Inactivation of Trop2 increases the concentration of neuregulin-1 at the cell surface where it is cleaved to activate ErbB3. In primary HNSCC, detection of ErbB3 activation was limited to Trop2 negative tumors. An analysis of the Cancer Genome Atlas (TCGA) HNSCC dataset confirms enrichment for ErbB3 activity in mesenchymal tumors. Notably, Trop2 loss triggers sensitivity to anti-ErbB3 antibodies, which results in reduced proliferation and tumorigenic growth of Trop2 negative HNSCC cancer cells. These results uncover a molecular mechanism by which tumor cells control the amount of cell-surface neuregulin-1 available for cleavage and ErbB3 activation. Moreover, we demonstrate that Trop2 is a potential surrogate biomarker to identify tumors with ErbB3 activation and may therefore respond to anti-ErbB3 therapeutics.

Broad Phenotypic Changes Associated with Gain of Radiation Resistance in Head and Neck Squamous Cell Cancer

The central issue of resistance to radiation remains a significant challenge in the treatment of cancer despite improvements in treatment modality and emergence of new therapies. To facilitate the identification of molecular factors that elicit protection against ionizing radiation, we developed a matched model of radiation resistance for head and neck squamous cell cancer (HNSCC) and characterized its properties using quantitative mass spectrometry and complementary assays. Functional network analysis of proteomics data identified DNA replication and base excision repair, extracellular matrix-receptor interaction, cell cycle, focal adhesion, and regulation of actin cytoskeleton as significantly up- or downregulated networks in resistant (rSCC-61) HNSCC cells. Upregulated proteins in rSCC-61 included a number of cytokeratins, fatty acid synthase, and antioxidant proteins. In addition, the rSCC-61 cells displayed two unexpected features compared with parental radiation-sensitive SCC-61 cells: (i) rSCC-61 had increased sensitivity to Erlotinib, a small-molecule inhibitor of epidermal growth factor receptor; and (ii) there was evidence of mesenchymal-to-epithelial transition in rSCC-61, confirmed by the expression of protein markers and functional assays (e.g., Vimentin, migration). The matched model of radiation resistance presented here shows that multiple signaling and metabolic pathways converge to produce the rSCC-61 phenotype, and this points to the function of the antioxidant system as a major regulator of resistance to ionizing radiation in rSCC-61, a phenomenon further confirmed by analysis of HNSCC tumor samples. The rSCC-61/SCC-61 model provides the opportunity for future investigations of the redox-regulated mechanisms of response to combined radiation and Erlotinib in a preclinical setting.

Abstract 4668: Antitumor efficacy of AZD1480 in head and neck squamous cell carcinoma.

Abstract Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been implicated in cancer cell proliferation and survival. AZD1480, a pharmacologic inhibitor of JAK2, has been tested in several cancer models including glioblastoma, lung, renal, prostate, breast, and ovarian cancer. However JAK inhibitors, including AZD1480 has not been evaluated in head and neck squamous cell cancer (HNSCC). In the present study, the in vitro and in vivo efficacy of AZD1480 was evaluated in HNSCC preclinical models. AZD1480 decreased HNSCC proliferation in cell lines with varying levels of pJAK2 and pSTAT3 with an IC50 ranging from 0.9μM - 4μM. Further evaluation demonstrated downmodulation of pJAK2 and pSTAT3 in HNSCC cell lines by AZD1480. Furthermore, in vivo antitumor efficacy of AZD1480 was tested in heterotopic xenograft tumor models derived from 2 HNSCC patient tumors. AZD1480 at a dose of 30mg/kg, was given twice daily via oral gavage and compared with vehicle (0.5% Hypromellose) control. Tumors treated with AZD1480 exhibited significant growth inhibition relative to the tumors treated with vehicle (p=0.0037). In addition, significant decrease in pSTAT3Tyr705 expression was observed in both the patient tumors treated with AZD1480 compared to vehicle alone indicating that AZD1480 targets pSTAT3 and may be considered for study in the treatment of HNSCC patients. Citation Format: Malabika Sen, John Black, Sarah Wheeler, Maria L. Freilino, Sonali Joyce, Vivian Lui, Yan Zeng, Korey Marshall, Jennifer R. Grandis. Antitumor efficacy of AZD1480 in head and neck squamous cell carcinoma. [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 4668. doi:10.1158/1538-7445.AM2013-4668

Abstract 5359: Gene expression characterization of different cancer stem cell isolation methods in head and neck cancer

Abstract Background: Head and neck squamous cell cancer (HNSCC) is a heterogeneous cancer that continues to pose major clinical problems. There is growing evidence that cancer stem cells (CSCs) may be an important factor in disease progression and resistance to current treatments. Establishing that stem cells are present in HNSCC and characterizing their global gene expression patterns provides the rationale for this study. Methods: A series of 5 HNSCC cell lines that have been maintained at low passage number such that they maintain phenotypic and morphological characteristics similar to the primary tumor were used in this study. Three different strategies were used to isolate stem cells by flow cytometry sorting: 1) Hoechst 33342 “side” population, 2) Aldefluor, and 3) CD44 bright cells. Global gene expression for each isolated CSC-enriched population and its associated CSC-depleted population was analyzed using Affymetrix Human Exon 1.0ST arrays. Gene set enrichment analysis was performed using Ariadne Pathway Studio. Results: The different methods of identifying CSCs produced widely different populations. In side population cells, there were 77 differentially expressed genes (p&amp;lt;0.05 and 1.5-fold cutoff) while only 59 and 17 genes were differentially expressed in the CD44 bright and Aldefluor bright populations, respectively. Not a single gene was commonly altered between any two of the isolation methods. Gene set enrichment analysis, however, was able to identify categories that differed between the CSC-enriched and CSC-depleted populations. Biological processes of cell cycle, cell division, mitosis, translational elongation, and spindle assembly were highly regulated. Microtubule binding/microtubule plus-end binding and structural constituent of ribosome were commonly altered molecular functions. Conclusions: This study suggests that current methods for isolating putative stem cells in HNSCC result in diverse populations with differing expression of specific genes. However, when examined for global expression patterns, several important categories of genes are commonly altered between the CSC-enriched and CSC-depleted populations. Some of the known characteristics of stem cells such as asymmetric division and unlimited growth potential are suggested by the highly regulated categories of cell division and spindle assembly. From these studies we hope to gain greater insight into the importance of stem cells in HNSCC as well as improve the capability of identifying these potentially important new targets for treatment. 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 5359. doi:1538-7445.AM2012-5359

Novel C-Terminal Hsp90 Inhibitor for Head and Neck Squamous Cell Cancer (HNSCC) with in vivo Efficacy and Improved Toxicity Profiles Compared with Standard Agents

Current therapies for HNSCC, especially platinum agents, are limited by their toxicities and drug resistance. This study evaluates a novel C-terminal Hsp90 inhibitor (CT-Hsp90-I) for efficacy and toxicity in vitro and in vivo in an orthotopic HNSCC model. Our hypothesis is that C-terminal inhibitors exhibit improved toxicity/efficacy profiles over standard therapies and may represent a novel group of anticancer agents. MDA-1986 HNSCC cells were treated with doses of 17-AAG or KU363 (a CT-Hsp90-I) and compared for antiproliferation by GLO-Titer and trypan blue exclusion and for apoptosis by PARP cleavage and caspase-3 inactivation by Western analysis. In vivo studies in Nu/Nu mice examined an orthotopic model of MDA-1986 cells followed by drug dosing intraperitoneally for a 21-day period (mg/kg/dose: cisplatin = 3.5, low-dose KU363 = 5, high-dose KU363 = 25, 17-AAG = 175). Tumor size, weight, and toxicity (body score) were measured 3×/week. The IC(50) levels for KU363 = 1.2-2 μM in MDA-1986. KU363 induces apoptosis at 1 μM with cleavage of PARP and inactivation of caspase-3 levels after 24 h. Client proteins Akt and Raf-1 were also downregulated at 1-3 μM of drug. In vivo, 100% of controls had progressive disease, while 100% of cisplatin animals showed some response, all with significant systemic toxicity. High-dose KU363 showed 88% of animals responding and low-dose KU363 showed 75% responding. KU363 animals showed significantly less toxicity (P < 0.01) than cisplatin or 17-AAG. This novel CT-Hsp90-I KU363 manifests potent anticancer activity against HNSCC, showing excellent in vivo efficacy and reduced toxicity compared with standard agents justifying future translational evaluation.

Sensitization of Head and Neck Cancer to Cisplatin Through the Use of a Novel Curcumin Analog

To determine whether a novel small molecule inhibitor derived from curcumin (FLLL32) that targets signal transducer and activator of transcription (STAT) 3 would induce cytotoxic effects in STAT3-dependent head and neck squamous cell cancer (HNSCC) cells and would sensitize tumors to cisplatin. Basic science. Two HNSCC cell lines, UM-SCC-29 and UM-SCC-74B, were characterized for cisplatin [cis-diammineplatinum(II) dichloride] sensitivity. Baseline expression of STAT3 and other apoptosis proteins was determined. The FLLL32 50% inhibitory concentration (IC(50)) dose was determined for each cell line, and the effect of FLLL32 treatment on the expression of phosphorylated STAT3 and other key proteins was elucidated. The antitumor efficacy of cisplatin, FLLL32, and combination treatment was measured. The proportion of apoptotic cells after cisplatin, FLLL32, or combination therapy was determined. The UM-SCC-29 cell line is cisplatin resistant, and the UM-SCC-74B cell line is cisplatin sensitive. Both cell lines express STAT3, phosphorylated STAT3 (pSTAT3), and key apoptotic proteins. FLLL32 downregulates the active form of STAT3, pSTAT3, in HNSCC cells and induces a potent antitumor effect. FLLL32, alone or with cisplatin, increases the proportion of apoptotic cells. FLLL32 sensitized cisplatin-resistant cancer cells, achieving an equivalent tumor kill with a 4-fold lower dose of cisplatin. FLLL32 monotherapy induces a potent antitumor effect and sensitizes cancer cells to cisplatin, permitting an equivalent or improved antitumor effect at lower doses of cisplatin. Our results suggest that FLLL32 acts by inhibiting STAT3 phosphorylation, reduced survival signaling, increased susceptibility to apoptosis, and sensitization to cisplatin.

Fas ligand gene transfer effectively induces apoptosis in head and neck cancer cells

Conclusion: Fas ligand (FasL) gene therapy may provide a new efficient therapeutic model for head and neck squamous cell cancer (HNSCC). Acid ceramidase (AC) may not play an important role in the sensitivity of HNSCC cell lines to Fas-induced apoptosis. Objectives: The aims of this study were to investigate the efficacy of FasL gene therapy for HNSCC in vitro and to determine whether the expression of AC in different kinds of HNSCC cell lines is related to the sensitivity of HNSCC cell lines to Fas-mediated apoptotic induction. Methods: Three HNSCC cell lines (Hep-2, MMSI, and SCCVII) were transfected with pEGFP-FasL, a plasmid containing a modified human FasL gene fused to enhanced green fluorescent protein (GFP). pEGFP-C1, a plasmid containing the GFP gene alone, was used as a control. Cell death was observed by fluorescence imaging and quantified using a tetrazolium-based (MTS) assay. SCCVII cells were analyzed by flow cytometry to determine the presence of apoptotic induction. Hep-2 and MMSI cells were evaluated by quantitative real-time PCR to evaluate the expression of AC. Results: Transfection of pEGFP-FasL plasmid was shown to be able to induce cell death, the sensitivity of Fas-mediated apoptosis in HNSCC was different, and the level of AC did not correlate with the sensitivity of HNSCC cells to Fas-induced apoptosis.

Abstract 125: Exogenous MiR-125b inhibits head and neck squamous cell carcinoma growth by suppressing Bcl-2 expression

Abstract PURPOSE: Head and neck squamous cell cancer (HNSCC) is one of top ten cancers in the United States. The overall 5-year survival rate of HNSCC has not improved in the past two decades. B-cell lymphoma 2 (Bcl-2), an oncogene, is a key regulator of the apoptosis to promote cell proliferation and apoptosis arrest. Therefore, targeting Bcl-2 is an attractive strategy for restoring a normal apoptotic process and inhibiting cancer cell growth in HNSCC. MicroRNAs (miRNAs) are a class of ∼22-nucleotide-long, endogenously expressed, highly conserved of noncoding RNAs with important regulatory functions in proliferation, apoptosis and metastasis. In this study, we screened eight cancer-associated miRNAs levels (mir-21, -124, -125b, -147, -200a, -200b, -200c and -429) in twenty five human tissue specimens and four cell lines, including twenty HNSCC specimens and five normal controls, and three HNSCC cell lines (JHU-13, -22 and -29) and one transformed oral keratinocytes cell line (OKF-6). Based on the screening data, we further investigated the mechanism of miRNA-125b (miR-125b) regulated Bcl-2 expression and the therapeutic potential of exogenous miR-125b in a miR-125b stable transfected HNSCC cell line. Design Methods: The miRNA expression level was determined by quantitative RT-PCR. A stable expressed exogenous miR-125b HNSCC cell line (JHU-22miR125b) was generated by using lentivirus transfection. Colony formation and MTT assayed cell growth and cell viability and Western blot was used to analyze protein expression. Results: The expression levels of mir-124 and mir-125b were significant lower in both tumor tissue and tumor cell lines than control, and mir-21 was significant higher in tumors. Thus, mir-125b was selected as a candidate for further study. The level of miR-125b in the transfected cell lines (JHU-22miR125b) was increased more than five times compared with the control cells (Transfected with vector only, JHU-22vector). The levels of Bcl-2 protein and cell viability were significantly lower in JHU-22miR125b than JHU-22 vector. CONCLUSION: Bcl-2 is a direct target in miR-125b regulated Bcl-2 expression. Exogenous miR-125b suppresses the proliferation and growth of cultured HNSCC cells. The effect of exogenous miR-125b on inhibition of HNSCC growth via suppressing Bcl-2 expression will be further validated in tumor xanografts. This work was supported in part by grants P20 CA118770 from National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 125. doi:10.1158/1538-7445.AM2011-125