Head Neck Squamous Cell Carcinoma Cells Research Articles

Abstract A028: Cornulin, An epithelial differentiation marker: A novel antitumor protein and prognosticator in patients with Head and Neck Squamous Cell Carcinoma

Abstract Background: Cornulin, a relatively unexplored protein, was reported to be downregulated in esophageal carcinoma along with the association of low tissue Cornulin level with disease stage. In our preliminary study, based on TMT-based LC-MS/MS analysis, the salivary Cornulin was also found to be downregulated ~10-fold in Head Neck Squamous Cell Carcinoma (HNSCC) patients. The current study further explored the biomarker potential of salivary Cornulin in HNSCC patients and the anti tumor effects of Cornulin in in vitro as well as in in vivo HNSCC models. Methods: Sandwich ELISA and immunohistochemistry techniques were utilized to assess the levels of Cornulin in the saliva and tissues of patients diagnosed with HNSCC, respectively. The expression of Cornulin in HNSCC was examined by analyzing the TCGA database using the GEPIA2 tool. To investigate the antitumor effects of Cornulin, its levels were increased through lentiviral transduction in the Cal27 and FaDu, HNSCC cell lines with low Cornulin expression. Subsequently, various cell assays were conducted to examine the antitumor effects of Cornulin upregulation. Additionally, the effects of increased Cornulin expression was also evaluated in an in vivo nude mice model. Results: The pre-treatment salivary Cornulin levels were significantly low (p=0.0001) in HNSCC patients (n=128,146.4±5.589pg/mL) as well as in oral premalignant disease patients (n=25, 174.6 ± 9.924 pg/mL) with respect to healthy controls (n=84,185.2±7.170pg/mL). Also, the tumor tissue expression of Cornulin (n=113, H-score=12.70±2.396) was significantly downregulated (p<0.0001) in comparison to the tumor-free margin (n=72, H-score=139.6±10.34). The patients showing complete clinical response regained normal salivary within 6 months of completion of treatment (p<0.0001). More importantly, low salivary Cornulin level at diagnosis was associated with poor overall survival (p=0.0282), indicating it to be a potential prognostic marker. On upregulation of Cornulin in low expressing HNSCC cell lines (Cal27 and FaDu), interestingly, the cellular viability, migration, and invasion showed significant decrease. These findings suggest that Cornulin plays a role in inhibiting tumor growth, indicating its potential as an anti-tumor factor. The analysis of RNAseq data of Cornulin overexpressed cell lines and further Gene set enrichment analysis, we found that G2/M checkpoint pathway to be positively enriched in the Cornulin overexpressed cell line. Furthermore, the cell cycle analysis revealed Cornulin overexpression arrests G1/S phase of cell cycle. In nude mice xenograft model, decreased tumor progression as well as tumor volume was documented after Cornulin overexpression. There was increased in differentiation in the xenograft tumors formed by Cornulin overexpressed cells. Conclusion: Here, we report for the first time intrinsic Cornulin to have a role as a potent anti-tumor and its potential as prognostic marker. However the pathways by which Cornulin shows its antitumor activity needs to be further evaluated. Citation Format: Rajandeep Kaur, Krishan Kumar Saini, Debajyoti Chatterjee, Jaimanti Bakshi, Radhika Srinivasan, Sushmita Ghoshal, Dipak Datta, Arnab Pal. Cornulin, An epithelial differentiation marker: A novel antitumor protein and prognosticator in patients with Head and Neck Squamous Cell Carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A028.

ETV5 facilitates tumor progression in head-neck squamous cell carcinoma.

E26 transformation-specific (ETS) factors have emerged as key mediators underlying human tumorigenesis. Here, we sought to characterize the expression pattern, biological roles, and clinical significance of ETS Variant Transcription Factor 5 (ETV5) in head neck squamous cell carcinoma (HNSCC). ETV5 expression pattern in HNSCC was determined by bioinformatics interrogations and immunohistochemical staining in primary samples. The associations between its abundance with clinicopathological parameters, and patient survival were evaluated. Colony formation, CCK-8, flow cytometry, wound healing, and Transwell invasion assays, as well as xenograft models, were utilized to determine the phenotypic changes after ETV5 silencing in vitro and vivo. The potential binding of ETV5 in the Slug promoter was determined by ChIP-qPCR. ETV5 was significantly overexpressed in HNSCC samples. Its overexpression is significantly associated with aggressiveness features and reduced survival. ETV5 knockdown significantly inhibited cell proliferation, migration, invasion, and induced apoptosis in vitro, and impaired tumor growth in vivo. Moreover, ETV5-activated Slug transcription by binding its promoter region in HNSCC cells. Patients with ETV5highSlughigh had the worst survival across multiple HNSCC cohorts. Our findings reveal that ETV5 serves as a novel prognostic biomarker and putative oncogene for HNSCC progression likely by activating Slug transcription.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

MiR-381-3p suppresses biological characteristics of cancer in head-neck squamous cell carcinoma cells by targeting nuclear autoantigenic sperm protein (NASP).

MiR-381-3p and nuclear autoantigenic sperm protein (NASP) have regulatory functions in tumors. Whether NASP is targeted by miR-381-3p to influence biological characteristics of cancer in head-neck squamous cell carcinoma (HNSCC) cells was investigated. StarBase (version 3.0) found that the expression of NASP was increased with the down-regulation of miR-381-3p in laryngocarcinoma tissue, AMC-HN-3，FaDu，HNE-3，and Detroit 562 cell lines. MiR-381-3p could target NASP, reduce the expression of MMP-2 and MMP-9, Vimentin, repress the cell viability, invasion, and migration, and promote the expression of E-cadherin in AMC-HN-3 cells. Overexpressed NASP could increase the viability, migration and invasion rates in AMC-HN-3 cells, which could be partially reversed by overexpressed miR-381-3p. Thus, miR-381-3p targeted and suppressed NASP gene, reduced the viability, migration, invasion, EMT of HNSCC cells, demonstrating that miR-381-3p has the potential to be a therapeutic target in inhibiting the progression of HNSCC.

Restoration of TET2 deficiency inhibits tumor growth in head neck squamous cell carcinoma.

BackgroundTet methylcytosine dioxygenase 2 (TET2) has been increasingly recognized as an important tumor suppressor involved in tumorigenesis. Here, we aimed to explore the expression pattern of TET2, its clinical significance as well as functional roles in head neck squamous cell carcinoma (HNSCC).MethodsBoth mRNA and protein levels of TET2 in primary HNSCC samples were detected via immunohistochemistry and qRT-PCR, respectively. Correlations between TET2 expression with multiple clinicopathological parameters and patient survival were determined. The biological roles of TET2 in HNSCC were assessed via a gain-of-function approach and in 4-nitroquinoline-1-oxide (4NQO)-induced HNSCC model. Restoration of TET2 by chemicals including 5-Aza-2'-deoxycytidine (5-AZA), metformin or Vitamin C (VC) to inhibit tumor growth was determined in vitro and in a xenograft animal model.ResultsReduced TET2 expression was found in a large fraction of HNSCC samples. Downregulated TET2 significantly correlated with larger tumor size, advanced clinical stage and inferior prognosis. Reduced TET2 and 5-hydroxymethylcytosine (5hmC) were observed along with disease progression in the 4NQO-induced HNSCC model. Enforced TET2 overexpression significantly inhibited cell proliferation, migration and enhanced the chemosensitivity of cisplatin in HNSCC cells. Restoration of TET2 following 5-AZA, metformin or VC exposure impaired cell proliferation and migration in vitro. Moreover, VC alone or in synergistic with cisplatin potently inhibited tumor growth in vivo.ConclusionsOur data reveal that reduced TET2 associates with tumor aggressiveness and reduced survival in HNSCC. Genetic or pharmacological restoration of TET2 might be a viable therapeutic strategy for HNSCC patients with TET2 deficiency.

The Hippo effector TAZ promotes cancer stemness by transcriptional activation of SOX2 in head neck squamous cell carcinoma

The Hippo-TAZ signaling has emerged as a fundamental regulator underlying cancer stem cells (CSCs) stemness which intricately associates with local recurrence and metastatic spreading in head neck squamous cell carcinoma (HNSCC). However, the precise downstream targets of TAZ responsible for HNSCC CSCs maintenance remain largely underexplored. Here, we identified Sex determining region Y box 2 (SOX2) as a putative downstream target of TAZ to promote CSCs maintenance and tumorigenicity in HNSCC. Both TAZ and SOX2 were significantly enriched in CSCs subpopulation (CD44+CD133+) isolated from Cal27 and Fadu cells via fluorescence-activated cell sorting. TAZ knockdown significantly reduced expression of SOX2 at both mRNA and protein levels, whereas its ectopic overexpression markedly increased its abundance in HNSCC cells. Moreover, reintroduction of ectopic SOX2 abolished, at least in part, the reduced tumorsphere formation and tumorigenicity in vivo induced by TAZ knockdown. Mechanistically, transcriptional complex formed by TAZ and TEAD4 was recruited to two binding sites in SOX2 promoter, which in turn facilitated transcription of SOX2 in HNSCC cells. In addition, the abundance of TAZ and SOX2 was positively correlated in HNSCC clinical samples, and both upregulations of TAZ and SOX2 associated with the worst survival. Taken together, our data reveal a previously unknown mechanistic linkage between TAZ and SOX2 and identify SOX2 as a direct downstream target of TAZ in modulating CSCs self-renewal and maintenance in HNSCC. These findings suggest that targeting TAZ-SOX2 axis might be a promising therapeutic strategy for HNSCC.

Overexpression of lncRNA WWTR1-AS1 associates with tumor aggressiveness and unfavorable survival in head-neck squamous cell carcinoma.

Long noncoding RNAs (lncRNAs) have been recognized as novel biomarkers and therapeutic targets in human cancer including head-neck squamous cell carcinoma (HNSCC). Here, we aimed to investigate the expression of a novel lncRNA WW domain containing transcription regulator 1 antisense RNA 1 (WWTR1-AS1) as well as its clinical significance and biological roles in HNSCC. The expression level of endogenous WWTR1-AS1 in primary HNSCC and paired adjacent nontumor mucosa was evaluated by quantitative reverse-transcription polymerase chain reaction. The correlations were assessed between WWTR1-AS1 expression and clinical data such as clinicopathological parameters and patient survival. Further, the molecular mechanisms of WWTR1-AS1 underlying HNSCC progression were detected by loss-of-function assay in vitro and bioinformatics analysis. Aberrant overexpression of WWTR1-AS1 in HNSCC samples showed a significant correlation between its higher expression levels and larger tumor size, cervical node metastasis, and poor prognosis. WWTR1-AS1 knockdown mediated by antisense oligonucleotide markedly inhibited cell proliferation, migration, invasion, tumorsphere formation as well as apoptosis resistance probably by modulating WWTR1 messenger RNA (mRNA) stability in HNSCC cells. Bioinformatics analysis revealed that WWTR1-AS1 expression positively correlated with WWTR1 expression in TCGA-HNSCC data sets. Together, our data provide evidence that aberrant upregulation of WWTR1-AS1 associates with malignant features and unfavorable prognosis in HNSCC, highlighting that WWTR1-AS1 might be a potential oncogenic lncRNA during HNSCC progression.

Overexpression of ZEB2-AS1 promotes epithelial-to-mesenchymal transition and metastasis by stabilizing ZEB2 mRNA in head neck squamous cell carcinoma.

The long noncoding RNAs (lncRNAs) have been increasingly appreciated as key players underlying tumourigenesis and hold great potentials as prognostic biomarkers and therapeutic targets. However, their roles in head neck squamous cell carcinoma (HNSCC) have remained incompletely known. Here, we sought to reveal the oncogenic roles and clinical significance of a tumour‐associated lncRNA, zinc finger E‐box binding homeobox 2 antisense RNA 1 (ZEB2‐AS1), in HNSCC. ZEB2‐AS1 was aberrantly overexpressed in a fraction of HNSCC samples. Its overexpression significantly associated with large tumour size, cervical node metastasis and reduced overall and disease‐free survival. Antisense oligonucleotides (ASO)‐mediated ZEB2‐AS1 depletion markedly inhibited cell proliferation, migration and invasion while triggered apoptosis in HNSCC cells in part via modulating ZEB2 mRNA stability. Enforced overexpression of ZEB2 largely attenuated the phenotypic changes resulted from ZEB2‐AS1 inhibition except the impaired cell proliferation. In addition, ZEB2‐AS1 was required for TGF‐β1‐induced epithelial‐mesenchymal transition (EMT) in vitro. Significantly reduced tumour growth and lung metastasis were observed in ZEB2‐AS1‐depleted cells in HNSCC xenograft animal models. Taken together, our findings reveal that overexpression of ZEB2‐AS1 associates with tumour aggressiveness and unfavourable prognosis by serving as a putative oncogenic lncRNA and a novel prognostic biomarker in HNSCC.

Abstract 5047: Exosomes derived from HIV-1-infected T cells activate ERK 1/2 through EGFR and TLR3 in cancer cells

Abstract Exosomes secreted from HIV-1-infected T cells play important roles in coordinating the immune responses and disease processes in HIV-infected people who are treated with antiretroviral therapy. We hypothesize that exosomes from HIV-infected T cells can promote growth and progression of non-AIDS-defining cancer cells. Activation of the MAPK ERK 1/2 is important for cancer cell survival, proliferation and invasion. We investigated the ability of exosomes from HIV-1-infected T cells to activate ERK 1/2 in head neck squamous cell carcinoma (HNSCC) and lung cancer cells for cancer cell proliferation and progression. Exosomes were isolated from Jurkat and J1.1, a latently HIV-1-infected Jurkat cells, using differential ultracentrifugation and quantified by acetylcholinesterase (AchE) activity. HNSCC cell line HSC3 and lung cancer cell lines NCI-H1299 and NCI-H1437 cells were treated with exosomes. To test the involvement of EGFR and TLR3 in ERK 1/2 activation by exosomes, EGFR neutralizing antibody Cetuximab or the TLR3/dsRNA inhibitor complex was added to cancer cells 30 min prior to adding exosomes. To test the entry of exosomes into cells, HEK293 cells were transiently transfected with EGFR-GFP, and seeded into glass bottom 35 mm dishes, time lapse Z-stacking imaging was performed and deconvolution was used to remove out of focus signals. By using Western blotting, we found that exosomes from J1.1 cells (HIV-1-infected), but not those from HIV-negative Jurkat cells, constantly induced ERK 1/2 phosphorylation in HNSCC and lung cancer cells. Cetuximab dramatically decreased ERK activation by exosomes from J1.1 cells, suggesting an EGFR-dependent ERK activation. While EGF induced a whole series of EGFR phosphorylation, exosomes from J1.1 cells failed to do so, suggesting that exosomes from J1.1 cells trigger ERK activation through a non-canonical EGFR pathway. Furthermore, ERK activation was significantly reduced in TLR3/dsRNA inhibitor pretreated cancer cells in response to exosomes from J1.1 cells, indicating exosomes triggered ERK activation is also TLR3 dependent. Time lapse imaging showed that exosomes quickly entered into cells within two minutes; however, Cetuximab greatly delayed exosome entry. To conclude, we found that exosomes from HIV-infected T cells can induce ERK activation in HNSCC and lung cancer cells through interaction with EGFR and TLR3. Citation Format: Zhimin Feng, Lechuang Chen, Hong Yue, Bingcheng Wang, Ge Jin. Exosomes derived from HIV-1-infected T cells activate ERK 1/2 through EGFR and TLR3 in cancer cells [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 5047.

Overexpression of WD repeat domain 5 associates with aggressive clinicopathological features and unfavorable prognosis in head neck squamous cell carcinoma.

WD repeat domain 5 (WDR5), a core member of Mixed lineage leukemia (MLL) and SET1 histone H3 lysine 4 (H3K4) methyltransferase complexes, is involved in multiple biological and pathological processes. Its deregulation in cancer and pro-tumorigenic roles has been increasingly appreciated. However, the expression pattern of WDR5 and its biological functions in head neck squamous cell carcinoma (HNSCC) have not been well established. The expression of WDR5 mRNA in HNSCC was determined by data mining and interrogation using publicly available databases. Its protein expression was measured by immunohistochemistry in a retrospective cohort of primary HNSCC samples. Moreover, the associations between WDR5 expression and various clinicopathological parameters and patient survival were assessed. The pro-tumorigenic roles of WDR5 in HNSCC were further delineated invitro by loss-of-function assay. Our bioinformatics analyses revealed that WDR5 mRNA was significantly overexpressed in 3 HNSCC cohorts. WDR5 protein was markedly upregulated in HNSCC samples as compared to normal counterparts and its overexpression significantly associated with large tumor size, advanced clinical stage (chi-square test, P=.048, .006) and reduced overall and disease-free survival (Kaplan-Mier analyses, Log-rank test, P=.0137, .0154). Univariate and multivariate survival analyses further revealed WDR5 protein abundance as an independent prognostic factor for patients' overall survival. Moreover, WDR5 knockdown significantly inhibited cell proliferation, migration and invasion, and induced cell apoptosis in HNSCC cells. Our findings reveal that WDR5 is aberrantly overexpressed in HNSCC and associates with aggressiveness and unfavorable prognosis, thus representing a novel diagnostic and prognostic biomarker for HNSCC.

Long Non-coding MIR205HG Depletes Hsa-miR-590-3p Leading to Unrestrained Proliferation in Head and Neck Squamous Cell Carcinoma.

Over 70% of head & neck squamous cell carcinoma (HNSCC) patients carry TP53 oncogenic mutations. Here we studied the role of specific tumor-derived mutant p53 proteins in the aberrant transcription of long non-coding (lnc) MIR205HG gene in head and neck cancer cells.Methods: To understand the role of lncMIR205HG, that we showed to be transcriptionally regulated by mutant p53 in HNSCC, we have employed siRNA and shRNA in CAL27 and FaDu HNSCC cell lines to suppress p53 gene expression in ChIP assays and RT-qPCR. We validated our findings in a cohort of 522 HNSCC patients from The Cancer Genome Atlas Data Portal (TCGA). We further evaluated our results in 63 HNSCC tumor samples collected at our institute, 32 of which were characterized by mutated TP53 (missense mutations) while 31 were characterized by wild-type TP53.Results: Maturation of pre-MIR205HG transcript produces two non-coding RNAs, lncMIR205HG and hsa-miR-205-5p. Down-regulation of lncMIR205HG expression significantly reduced cell proliferation, cell migration and clonogenic activity of head and neck cancer cells. Expression of MIR205HG was significantly increased in HNSCC with mutated TP53 when compared with matched non-tumoral tissues. Furthermore, MIR205HG expression levels were significantly higher in tumoral samples with mutant p53 than in tumoral tissues expressing wild-type p53. Mechanistically, MIR205HG depletes endogenous miR-590-3p leading to increased cyclin B, cdk1, and YAP protein expression.Conclusions: Taken together, these findings identify a transcriptional and post-transcriptional molecular network that includes mutant p53 protein, lncMIR205HG, YAP, and other proliferation-related genes, which are enriched in HNSCC patients with poor prognosis.

Pharmacological inhibition of Bmi1 by PTC-209 impaired tumor growth in head neck squamous cell carcinoma

BackgroundBmi1 (B lymphoma Mo-MLV insertion region 1 homolog) contributes to human tumorigenesis via epigenetic transcriptional silencing and represents a novel therapeutic target with great potentials. Here we sought to determine the therapeutic efficiency of PTC-209, a potent and selective Bmi1 inhibitor, in head neck squamous cell carcinoma (HNSCC) cells and a HNSCC xenograft model.MethodsThe mutation pattern, mRNA level of Bmi1 in HNSCC and its associations with clinicopathological parameters were determined through comprehensive data mining and interrogation using publicly available databases GENT, cBioPortal, Oncomine and TCGA. The PTC-209, a selective and potent Bmi1 inhibitor, was exploited and its effect on Bmi1 expression was measured in two HNSCC cell lines Cal27 and FaDu. The phenotypical changes of HNSCC cells were observed upon PTC-209 treatment in vitro. Moreover, the therapeutic effects of PTC-209 for HNSCC were determined in a xenograft animal model.ResultsThrough comprehensive data mining and interrogation, we found that Bmi1 mRNA was frequently overexpressed in a subset of HNSCC samples. Our data revealed that PTC-209 robustly reduced the expression of Bmi1 in Cal27 and FaDu cells presumably by post-transcriptional repression and ubiquitin-proteasomal degradation. PTC-209 treatment resulted in impaired cell proliferation, G1-phase cell cycle arrest, compromised migration and invasiveness, and increased cell apoptosis and chemosensitivity to 5-FU and cisplatin in vitro. Moreover, PTC-209 exposure reduced colony formation, tumorsphere formation and the percentage of ALDH1+ subpopulation in both Cal27 and FaDu cells. Importantly, in vivo PTC-209 administration significantly reduced tumor growth in a HNSCC xenograft model probably by Bmi1 inhibition and impaired cell proliferation.ConclusionsOur findings indicate that pharmacological inhibition of Bmi1 is a novel therapeutic strategy for HNSCC patients, especially with those with aberrant Bmi1 overexpression.

Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma.

Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways.

ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer.

The epidermal growth factor receptor (EGFR) contributes to the pathogenesis of head&neck squamous cell carcinoma (HNSCC). However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy. By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC. Using structural mutants of EGFR and ANO1 we identified the trans/juxtamembrane domain of EGFR to be critical for the interaction with ANO1. Our results show that ANO1 and EGFR form a functional complex that jointly regulates HNSCC cell proliferation. Expression of ANO1 affected EGFR stability, while EGFR-signaling elevated ANO1 protein levels, establishing a functional and regulatory link between ANO1 and EGFR. Co-inhibition of EGFR and ANO1 had an additive effect on HNSCC cell proliferation, suggesting that co-targeting of ANO1 and EGFR could enhance the clinical potential of EGFR-targeted therapy in HNSCC and might circumvent the development of resistance to single agent therapy. HNSCC cell lines with amplification and high expression of ANO1 showed enhanced sensitivity to Gefitinib, suggesting ANO1 overexpression as a predictive marker for the response to EGFR-targeting agents in HNSCC therapy. Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

Induction of BCL2-Interacting Killer, BIK, is Mediated for Anti-Cancer Activity of Curcumin in Human Head and Neck Squamous Cell Carcinoma Cells.

Naturally occurring diarylheptanoid curcumin (CUR), a principal component of the Asian spice turmeric, has been shown to have anti-cancer effects in many tumor types. However, a detailed mechanism regarding CUR induced tumor cell killing remain to be comprehensively explored. Using two head neck squamous cell carcinoma (HNSCC) cell lines FaDu (hypopharyngeal) and Cal27 (tongue), we demonstrated a novel mechanism by which CUR levies the cytotoxic effect. We found that CUR induced upregulation of pro-apoptotic Bik, down-regulation of survival signaling by AKT and NF-κB prior to the induction of the caspase-cascade reduction of cell proliferation, are primary mechanisms of CUR-induced cell death, thus providing insights into the anti-tumor activity of CUR in HNSCC cells.

1017O - Preclinical Rational, Safety, and Preliminary Efficacy Results of Weekly Everolimus, Carboplatin and Paclitaxel as An Induction Therapy for Patients with Unresectable Locally Advanced Head & Neck Squamous Cell Carcinoma (CAPRA): A GERCOR-IRC Phase I/II Study

ABSTRACT Background PI3K/mTOR is a survival pathway that was shown activated in 57-81% of patients (pts) with head & neck squamous cell carcinoma (HNSCC) and might drive resistance to cytotoxics. Methods We have previously shown synergistic cell-cycle dependent effects between rapamycin/everolimus and carboplatin, or paclitaxel in HNSCC cells (Aissat 2008). Based on this preclinical translational rationale, a two-step phase I/II trial was designed using 9 consecutive weekly (w) cycles of oral everolimus combined with carboplatin (AUC2) and paclitaxel (60mg/m2) followed by chemoradiotherapy in pts with untreated locally advanced HNSCC. Results A total of 27 pts with stage IV HNSCC (median age 58, range 46-84; ECOG 0-2) have been enrolled in this phase I/II study (42 pts scheduled for the phase II). Among the 7 pts enrolled in the everolimus dose escalation phase I step, 6 pts were evaluable for safety (3pts at 30 mg/w and 3pts at 50 mg/w). No dose-limiting toxicity (defined during the first 4 weeks of treatment) was reported. Transient asymptomatic grade 3 neutropenia (3 pts), anemia (3 pts), and thrombocytopenia (1 pt) were observed. The most frequently reported adverse events were mild to moderate asthenia, skin toxicity, and alopecia. The recommended dose of everolimus for the phase II step was 50 mg/w. So far, among pts treated in the phase I/II, 11/13 objective responses (1CR, 10 PR, 2SD) were observed in evaluable pts. Interestingly, several major responses (ranging -60 to -80% by RECIST) were observed in large necrotic primary tumors and lymph nodes of >6cm diameters. Preliminary genetic analysis of tumors showed neither KRAS, BRAF, PI3KCA, nor EGFR mutations in tumors responding to this combination. Conclusions Weekly 50 mg everolimus combined with carboplatin and paclitaxel was well tolerated with no DLT, allowing repeated cycles. Major clinical responses in pts with bulky, locally advanced and necrotic diseases deserve further evaluation of this combination in pts with HNSCC. Updated results will be presented at meeting. Disclosure S. Faivre: Novartis, honorarium, compensated E. Raymond: Novartis, honorarium, compensated K. Slimane: Novartis, employment, compensated All other authors have declared no conflicts of interest.

Abstract 841: Inhibition of head and neck squamous cell carcinoma growth by combination treatment of erlotinib and celecoxib: Potential chemopreventive agents

Abstract The interaction of epithelial growth factor receptor (EGFR) and cyclooxygenase 2 (COX-2) pathways plays an important role in tumorigenesis. Our recent Phase I chemoprevention trial of premalignant lesions including pathologically moderate or severe dysplasia and carcinoma in situ in oral cavity and larynx showed a 71% (5/7) estimated overall response rate after a 6-month-long combination treatment of Erlotinib and Celecoxib without significant toxicities (ASCO 2010 Abstract # 5535). The current preclinical study was designed to investigate the underlying mechanisms that contribute to the inhibitory effect on the disease progression by the combined treatment of Erlotinib and Celecoxib. Chemopreventive efficacy was determined using a head neck squamous cell carcinoma (HNSCC) xenograft model with Tu212 cell line. Animals received vehicle control (0.1% Tween 80 and 0.5% methylcellulose, n=6), Celecoxib at 50mg/kg body weight (n=6), Erlotinib at 75mg/kg body weight (n=7), or combination of Erlotinib and Celecoxib (n=8) with the same dose as the single agent by oral gavaging daily for 6 days before subcutaneous implantation of cancer cells. Animals were further treated daily for 5 days per week with a total of 4 weeks. The combination treatment significantly suppressed tumor growth compared with Erlotinib alone (p=0.01), Celecoxib alone (p<0.0001) or the vehicle control (p<0.0001). In vitro study was performed with Tu212 and Tu686 HNSCC cell lines. Cell cycle analysis revealed that G1 arrest was induced by either Erlotinib alone (59% and 67% for Tu212 and Tu686, respectively), or Celecoxib alone (50% and 58% for Tu212 and Tu686 cells, respectively). The combination treatment resulted in greater G1 arrest than either single agent alone (72% in Tu212 and 65% in Tu686 cells). Molecular analysis showed that the combined treatment augmented suppression of pERK, pAKT and pEGFR. In addition, Erlotinib suppressed mTOR pathway by diminishing pS6. These results support the findings in the phase I study. The study has progressed to Phase II clinical trial. In conclusion, augmented inhibition of pEGFR/pERK/pS6 pathways could contribute to the synergism by Erlotinib and Celecoxib in suppressing HNSCC tumor growth. (This study is supported by NIH grant U01 CA151802 and P50CA128613 to Dr. Shin and Georgia Cancer Coalition Distinguished Cancer Scholar award to Drs Shin and Chen. *: These authors had equal contribution.) 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 841. doi:10.1158/1538-7445.AM2011-841

Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.

Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of dominant negative constructs of the nbs1 protein

Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome show radiation sensitivity we hypothesized that tumor−specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. In order to test this hypothesis we have devised recombinant adenoviruses expressing various portions of the Nbs1 protein and assessed the ability of these viruses to increase the radiation sensitivity of HNSCC cells. Materials and Methods: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1(Nbs1-300, aa453 to aa754) into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line 011. These cells were evaluated for expression of the viral based constructs and assayed for growth rate and clonogenic survival following radiation exposure. Results: A constitutively expressed GFP gene in the viral backbone confirmed efficient uptake of the virus into the 011 cell line and Western blot confirmed the presence of the virally expressed Nbs1 and Nbs1-300. Following exposure to ionizing radiation cells infected with the Nbs1-300 virus showed a significant reduction in growth rate relative to cells infected with control virus. Surprisingly, this effect was even stronger with the full-length wild-type Nbs1 protein. Examination of clonogenic survival also demonstrated statistically significant sensitization, however the effects of the two constructs were distinct as Nbs1-300 expression resulted in reduction of the shoulder while expression of the full-length Nbs1 showed a change in the slope of the survival curve. Conclusion: Dominant negative constructs of the Nbs1 protein are able to sensitize cells to ionizing radiation exposure. Surprisingly expression of the full-length Nbs1 protein results in enhanced sensitivity as well. These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors.