Human Head And Neck Squamous Cell Carcinoma Cell Lines Research Articles

HOTTIP Functions as a Key Candidate Biomarker in Head and Neck Squamous Cell Carcinoma by Integrated Bioinformatic Analysis.

Background Accumulating evidence has demonstrated the pivotal role of long noncoding RNAs (lncRNAs) in competing endogenous RNA (ceRNA) networks for predicting survival and evaluating prognosis in cancer patients. However, the pathogenesis of head and neck squamous cell carcinoma (HNSCC) remains unclear, and prognostic biomarkers for HNSCC are still lacking. Methods A total of 546 RNA sequencing profiles of HNSCC patients with clinical outcome data were obtained from the Cancer Genome Atlas (TCGA) database, providing a large sample of RNA sequencing data. From these, 71 Long noncoding RNAs lncRNAs, 8 microRNAs (miRNAs), and 16 messenger RNAs (mRNAs) were identified to construct a HNSCC-specific ceRNA network (fold change >2, P < 0.05). Univariate and multivariate Cox proportional regression models were used to assess independent indicators of prognosis. Then the expression of lncRNAs harboring prognostic value was validated in human HNSCC cell lines and tumor samples from our cohort and another two datasets from GEO (Gene Expression Omnibus) databases. Results As a result, a 3-mRNA signature and 6-lncRNA signature were identified. The six-lncRNA signature exhibited the highest prognostic value. Notably, in the six lncRNAs, HOTTIP showed the greatest prognostic value and was significantly correlated with clinical stage and histological grade of HNSCC patients. Furthermore, it was proved that HOTTIP was upregulated in HNSCC cell lines and cancerous tissues compared with corresponding normal cell lines and normal tissues. Functional assessment analysis revealed that HOTTIP might play a key role in the oncogenesis and progression of HNSCC. Conclusion The present study deepened our understanding of the ceRNA-related regulatory mechanism in the pathogenesis of HNSCC and identified candidate prognostic biomarkers for clinical outcome prediction in HNSCC. HOTTIP may function as a key candidate biomarker in HNSCC and serve as a prognostic marker for HNSCC patients.

Inter- and intra-tumor heterogeneity of SMAD4 loss in head and neck squamous cell carcinomas.

Reports regarding the frequency of SMAD4 loss in human head and neck squamous cell carcinoma (HNSCC) vary significantly. We have shown that SMAD4 deletion contributes to HNSCC initiation and progression. Therefore, accurately detecting genetic SMAD4 loss is critical to determine prognosis and therapeutic interventions in personalized medicine. We developed a SMAD4 fluorescence in situ hybridization (FISH) assay to identify chromosomal SMAD4 loss at the single cell level of primary HNSCC specimens and patient derived xenograft (PDX) tumors derived from HNSCCs. SMAD4 heterozygous loss was detected in 35% of primary HNSCCs and 41.3% of PDX tumors. Additionally, 4.3% of PDX tumors had SMAD4 homozygous loss. These frequencies of SMAD4 loss were similar to those in The Cancer Genome Atlas (TCGA). However, we identified significant heterogeneities of SMAD4 loss (partial or complete) among cells within each tumor. We also found that aneuploidy (monosomy and polysomy) contributed greatly to how to define chromosomal SMAD4 deletion. Furthermore, in cultured PDX tumors, SMAD4 mutant cells outcompeted SMAD4 wildtype cells, resulting in establishing homogenous SMAD4 mutant HNSCC cell lines with partial or complete genomic SMAD4 loss, suggesting a survival advantage of SMAD4 mutant cells. Taken together, our study reveals inter- and intra-tumor heterogeneities of SMAD4 chromosomal loss in HNSCCs. Further, SMAD4 FISH assay provides a platform for future clinical diagnosis of SMAD4 chromosomal loss that potentially serves as a molecular marker for prognosis and therapeutic intervention in cancer patients.

Involvement of HPV Infection in the Release of Macrophage Migration Inhibitory Factor in Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV) infection has been well-established as a risk factor in head and neck squamous cell carcinoma (HNSCC). The carcinogenic effect of HPV is mainly due to the E6 and E7 oncoproteins, which inhibit the functions of p53 and pRB, respectively. These oncoproteins could also play a role in the Warburg effect, thus favoring tumor immune escape. Here, we demonstrated that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is expressed at higher levels in HPV-negative patients than in HPV-positive patients. However, the secretion of MIF is higher in HPV-positive human HNSCC cell lines, than in HPV-negative cell lines. In-HPV positive cells, the half inhibitory concentration (IC50) of MIF inhibitor (4-iodo-6-phenylpyrimidine (4-IPP)) is higher than that in HPV-negative cells. This result was confirmed in vitro and in vivo by the use of murine SCCVII cell lines expressing either E6 or E7, or both E6 and E7. Finally, to examine the mechanism of MIF secretion, we conducted proton nuclear magnetic resonance (1H-NMR) experiments, and observed that lactate production is increased in both the intracellular and conditioned media of HPV-positive cells. In conclusion, our data suggest that the stimulation of enzymes participating in the Warburg effect by E6 and E7 oncoproteins increases lactate production and hypoxia inducible factor 1α (HIF-1α) expression, and finally induces MIF secretion.

Niclosamide Induces Cell Cycle Arrest in G1 Phase in Head and Neck Squamous Cell Carcinoma Through Let-7d/CDC34 Axis.

Niclosamide is a traditional anti-tapeworm drug that exhibits potent anti-cancer activity. Our previous study showed that niclosamide induces cell cycle arrest in G1 phase. Nevertheless, the underlying mechanism remains unknown. The following study investigated the molecular mechanism through which niclosamide induced G1 arrest in head and neck squamous cell carcinoma (HNSCC) cell lines. The effect of niclosamide on human HNSCC cell line WSU-HN6 and CNE-2Z were analyzed using IncuCyte ZOOMTM assay, flow cytometry (FCM), real-time PCR and western blot. Luciferase assay was conducted to demonstrate the interaction between let-7d (a let-7 family member which functions as a tumor suppressor by regulating cell cycle) and 3′UTR of CDC34 mRNA. Xenografts tumor model was established to evaluate the niclosamide treatment efficacy in vivo. Briefly, an exposure to niclosamide treatment led to an increased let-7d expression and a decreased expression of cell cycle regulator CDC34, finally leading to G1 phase arrest. Moreover, an overexpression of let-7d induced G1 phase arrest and downregulated CDC34, while the knockdown of let-7d partially rescued the niclosamide-induced G1 phase arrest. Luciferase assay confirmed the direct inhibition of CDC34 through the targeting of let-7d. Furthermore, niclosamide markedly inhibited the xenografts growth through up-regulation of let-7d and down-regulation of CDC34. To sum up, our findings suggest that niclosamide induces cell cycle arrest in G1 phase in HNSCC through let-7d/CDC34 axis, which enriches the anti-cancer mechanism of niclosamide.

Downregulation of iASPP Expression Suppresses Proliferation, Invasion and Increases Chemosensitivity to Paclitaxel of Head and Neck Squamous Cell Carcinoma In Vitro.

Objective Our previous study has revealed that iASPP is elevated in human head and neck squamous cell carcinoma (HNSCC) and iASPP overexpression signifcantly correlates with tumor malignant progression and poor survival of HNSCC. This study investigated the function of iASPP playing in proliferation and invasion of HNSCC in vitro. Methods HNSCC cell line Tu686 transfected with Lentiviral vector-mediated iASPP-specific shRNA and control shRNA were named the shRNA-iASPP group and shRNA-NC group, respectively. The non-infected Tu686 cells were named the CON group. CCK-8 assay, flow cytometry, transwell invasion assay were performed to detect the effects of iASPP inhibition in vitro. Results Our results demonstrated that the proliferation of shRNA-iASPP cells at the time of 72 h (F=32.459, P=0.000), 96 h (F=51.407, P=0.000), 120 h (F=35.125, P=0.000) post-transfection, was significantly lower than that of shRNA-NC cells and CON cells. The apoptosis ratio of shRNA-iASPP cells was 9.42% ± 0.39% (F=299.490, P=0.000), which was significantly higher than that of CON cells (2.80% ± 0.42%) and shRNA-NC cells (3.18% ± 0.28%). The percentage of shRNA-iASPP cells in G0/G1 phase was 74.65% ± 1.09% (F=388.901, P=0.000), which was strikingly increased, compared with that of CON cells (55.19% ± 1.02%) and shRNA-NC cells (54.62% ± 0.88%). The number of invading cells was 56 ± 4 in the shRNA-iASPP group (F=84.965, P=0.000), which decreased significantly, compared with the CON group (111 ± 3) and the shRNA-NC group (105 ± 8). The survival rate of shRNA-iASPP cells administrated with paclitaxel was highly decreased, compared with CON cells and shRNA-NC cells (F=634.841, P=0.000). Conclusion These results suggest iASPP may play an important role in progression and aggressive behavior of HNSCC and may be an efficient chemotherapeutic target for the treatment of HNSCC.

Co-targeting EGFR and IKK\u03b2/NF-\u03baB signalling pathways in head and neck squamous cell carcinoma: a potential novel therapy for head and neck squamous cell cancer

BackgroundEpidermal growth factor receptor (EGFR) plays an important role in head and neck squamous cell carcinoma (HNSCC) proliferation and therapy resistance, but the efficacy of targeting of EGFR for therapy has been limited. Here, we explore the molecular link between EGFR and inhibitor of κB kinase β/nuclear factor-κB (IKKβ/NF-κB) signalling pathways in the regulation of HNSCC EGFR inhibitor resistance.MethodsWe performed in vitro experiments in eight human HNSCC cell lines and a patient-derived HNSCC cell line as well as in vivo xenografts in a human HNSCC cell line.ResultsWe found that treatment of all HNSCC cells with Gefitinib and Erlotinib, two Food Drug Administration-approved EGFR inhibitors, blocked the activity of Akt/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase, two crucial downstream effectors of EGFR, but up-regulated IKKβ/NF-κB signalling. In addition, induction of IKKβ/NF-κB by EGFR inhibitors required HER2 and HER3 expression. In keeping with these, IKKβ inhibitor CmpdA synergistically enhanced the efficacy of EGFR inhibitors to further inhibit in vitro HNSCC cell growth. Importantly, we demonstrated that the combination of Gefitinib with CmpdA inhibited xenograft tumour formation.ConclusionOur data demonstrated that co-targeting EGFR and IKKβ with Gefitinib and IKKβ inhibitors could provide a potential novel therapy for head and neck squamous cell cancer.

Subjugation of TGFβ Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End Joining.

Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFβ signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair. Human HNSCC cell lines (n = 9), patient-derived xenografts (n = 9), tissue microarray (n = 194), TCGA expression data (n = 279), and primary tumor specimens (n = 10) were used to define the relationship between TGFβ competency, response to DNA damage, and type of DNA repair. Analysis of HNSCC specimens in situ and in vitro showed that HPV associated with loss of TGFβ signaling that increased response to radiation or cisplatin. TGFβ suppressed miR-182, which inhibited both BRCA1, necessary for homologous recombination repair (HRR), and FOXO3, required for ATM kinase activity. TGFβ signaling blockade by either HPV or inhibitors released miR182 control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the HRR deficit in HPV-positive cells. Loss of TGFβ signaling unexpectedly increased repair by error prone, alternative end-joining (alt-EJ). HPV-positive HNSCC cells are unresponsive to TGFβ. Abrogated TGFβ signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFβ's role in DNA repair proficiency and further raises the translational potential of TGFβ inhibitors in cancer therapy.

Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) poses a significant challenge clinically, as it can invade facial bones and cause bone pain that is undertreated and poorly understood. Here we studied HNSCC bone pain (HNSCC-BP) in an intratibial mouse xenograft model that uses a human HNSCC cell line (SAS cells). These mice develop HNSCC-BP associated with an upregulation of phosphorylated ERK1/2 (pERK1/2), which is a molecular indicator of neuron excitation in the dorsal root ganglia (DRGs) of sensory nerve cell bodies. Our experiments demonstrated that the inhibition of monocarboxylate transporter 4 (MCT4) by short hairpin (shRNA) transduction suppressed the HNSCC-BP, the lactate level in bone marrow, and the pERK1/2 expression in DRG. The sensory nerves also expressed increased levels of the acid-sensing receptor TRPV1. DRG neurons co-cultured with SAS cells showed increased neurite outgrowth, and were inhibited by MCT4 silencing with shRNA. Collectively, our results show that HNSCC induced an acidic bone microenvironment that evokes HNSCC-BP via MCT4 expression.

Appropriate Sequence for Afatinib and Cisplatin Combination Improves Anticancer Activity in Head and Neck Squamous Cell Carcinoma.

Despite a better understanding in head and neck tumors pathogenesis as well as improvements in radiotherapy and surgery, locally advanced head and neck squamous cell carcinoma (HNSCC) remains of poor prognosis. One promising target is the epidermal growth factor receptor (EGFR), which is overexpressed in the majority of HNSCC and is associated to tumor progression and resistance to treatment. However, in several clinical trials, the combination of EGFR inhibitors with chemotherapy and/or radiotherapy generates moderate results. In this study, we investigated the anti-tumor activity of afatinib, an irreversible pan-EGFR inhibitor, combined to cisplatin in different schedules of exposure. For that, we used two human EGFR wild-type HNSCC cell lines and we evaluated the cytotoxicity of the two drugs combined in different sequences. The efficiency of each strategy was assessed by evaluating the effects on cell cycle distribution, DNA damage, cell death and downstream pathways of ErbB family receptors. We demonstrated that cisplatin treatment followed by afatinib exposure displayed more cytotoxic effects than the opposite timing or than simultaneous association. This higher anticancer activity is probably due to afatinib-induced cell cycle arrest, which prevents the repair of cisplatin-induced DNA damage and promotes cell death by various mechanisms including apoptosis. These data suggest the importance of an appropriate timing administration between an EGFR inhibitor and a conventional chemotherapy in order to obtain the best clinical benefit for patients with a head and neck cancer.

Inhibition of MEK with trametinib enhances the efficacy of anti-PD-L1 inhibitor by regulating anti-tumor immunity in head and neck squamous cell carcinoma

ABSTRACT Major histocompatibility complex (MHC) class I downregulation is the primary immune evasion mechanism associated with failure in anti-PD-1/PD-L1 blockade therapies for cancer. Here, we examined the role of MEK signaling pathway inhibition in head and neck squamous cell carcinoma (HNSCC) both in vitro and in vivo. We found that trametinib, a small molecule inhibitor of MEK, significantly enhanced MHC class I and PD-L1 expression in human HNSCC cell lines, and this occurred via STAT3 activation. Trametinib also further upregulated the increase in CXCL9 and CXCL10 expression caused by IFN-γ in HNSCC cells, which is associated with T cell infiltration in tumor tissues. Finally, we evaluated the therapeutic efficacy of trametinib combined with an anti-PD-L1 monoclonal antibody in vivo, using SCCVII mouse syngeneic tumor model for HNSCC. While neither PD-L1 blockade nor trametinib treatment alone affected tumor growth, the combined therapy significantly delayed tumor growth. Our results indicate that in the combined therapy trametinib increases CD8+ T cell infiltration in the tumor site and upregulates antigen presentation, and this may be associated with enhanced PD-L1 blockade efficacy. Furthermore, our results suggest that this combination would therapeutically benefit patients with HNSCC.

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

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

Abstract 1748: Head and neck cancer-derived exosomes facilitate carcinogenesis in the murine 4NQO chemically induced oropharyngeal cancer

Abstract Purpose: An increasing body of evidence suggests that tumor-derived exosomes (TEX) promote tumor formation and progression. This study aims to show that exosomes produced by the SCCVII murine tumor cell line promote progression of pre-malignant oropharyngeal lesions to malignant tumors. The 4-Nitroquinoline 1-oxide (4NQO) tumor model is chemically induced in immunocompetent mice. It recapitulates all the histological stages and molecular changes associated with of oral carcinogenesis described for human head and neck squamous cell carcinoma (HNSCC). Experimental design: Exosomes were isolated from supernatant of a murine as well as human HNSCC cell-lines by mini size-exclusion chromatography (miniSEC). Morphology, number, protein and the molecular cargo of the recovered exosomes were determined. Exosome-mediated in vitro inhibition of lymphocyte proliferation and induction of CD8+ T-cell apoptosis were demonstrated. C57BL/6 mice were treated with 4NQO supplied in drinking water (100ug/ml) for 16 weeks and all developed oral and esophageal pre-malignant lesions. Exosomes were administered via a single tail-vein or retro-orbital injection at week 18. Tumor sizes and overall tumor burden per mouse were measured with a caliper. Proliferation indexes of individual tumors were measured by immunohistochemistry. Splenocytes were harvested and were analyzed for the presence and frequency of myeloid derived suppressor cells (MDSC) and Tregs, and for in vitro responsiveness of T cells to exogenous activators. Results: SCCVII-derived exosomes were found to carry an immunosuppressive cargo, including CD39, CD73, PD-L1, FasL and COX-2. One-time 90ug injections (IV) of exosomes to groups of &amp;gt;8 mice induced a statistically significant increase in the number of developing tumors and in the overall tumor burden per mouse, relative to control mice injected with PBS. The average number of tumors per mouse was 6.3 versus 3.6, respectively (p &amp;lt; 0.005). TEX derived from human HNSCC cell lines also induced a greater though not statistically significant number of tumors in 4NQO-treated mice. The MDSC frequency was increased in the exosome-injected mice relative to controls. The Treg frequency was not altered. Conclusions: The data demonstrate that the delivery of Head and Neck Cancer-derived exosomes enhanced carcinogenesis of the 4NQO-induced tumors in mice. Augmented carcinogenesis was associated with systemic immunosuppression and increases in the frequency of MDSCs in spleens of mice treated with exosomes. Citation Format: Beatrice M. Razzo, Chang-Sook Hong, Kellsye P. Fabian, Nils Ludwig, Priyanka Sharma, Walter J. Storkus, Theresa L. Whiteside. Head and neck cancer-derived exosomes facilitate carcinogenesis in the murine 4NQO chemically induced oropharyngeal cancer [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 1748.

Abstract 55: Intrinsic molecular characteristics of bone marrow-disseminated tumor cells

Abstract Dormant or slow-cycling disseminated tumor cells (DTC) in bone marrow (BM) are known to persist in patients with various types of cancer including head and neck squamous cell carcinoma (HNSCC). BM-DTC has been thought to have possible metastatic stem cell traits although functional and clonal analysis of BM-DTC remain elusive. Recently, we reported that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis was crucial for drug resistance dependent on a slow-cycling state in BM-DTC but not in lung metastatic cells. This study aimed to elucidate intrinsic molecular characteristics in BM-DTC. We used the human HNSCC cell line HEp3-originated sublines [i.e. parental line (P-HEp3), BM-DTC-derived (BM-HEp3), and lung metastases-derived sublines (Lu-HEp3)]. Our transcriptome analyses for these sublines revealed that slow-cycling and drug-resistant BM-HEp3 cells had unique gene expression signatures. In addition, our investigation for phosphorylation of 39 kinases associated with tumor progression showed enhanced activation of only Src in BM-HEp3 compared with P- and Lu-HEp3. Importantly, Src inhibition by dasatinib in these sublines led to a remarkable reduction in cell viability only in BM-HEp3. These data suggest that intrinsic molecular characteristics of BM-DTC are distinct from primary and lung metastatic tumor cells. Src activation may be important for survival of dormant or slow-cycling BM-DTC. Further studies should contribute to better understanding of biology of minimal residual disease in BM and metastatic initiation. Citation Format: Manabu Maeshiro, Satoru Shinriki, Takuya Nakamura, Hirofumi Jono, Hideki Nakayama, Yukio Ando, Hirotaka Matsui. Intrinsic molecular characteristics of bone marrow-disseminated tumor 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 55.

Abstract 5481: Targeting the kynurenine pathway as a novel metabolic treatment for head and neck cancer

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) still claims 330,000 lives globally each year, indicating a critical need for novel therapies. Recently, we identified high levels of kynurenine (K) in HNSCC patients through global metabolic profiling of HNSCC tumor tissues via mass spectrometry (MS). Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3- dioxygenase (TDO) are enzymes that degrade tryptophan to K. Elevated K allows tumor cells to evade immune response more easily, promoting cancer progression, while increased IDO levels are associated with poorer prognosis. We hypothesize that restoring immune response to tumors by blocking IDO1 will be an effective therapeutic strategy for sensitizing HNSCC. Methods: Validated human HNSCC cell lines were grown in 2D culture. Kynurenine levels were measured by MS. IDO1, IDO2 and TDO levels in tissue samples were measured by Western blot (WB) analysis. siRNAs for IDO1, IDO2, and TDO were used to knock down tryptophan pathway enzymes (non-targeting siRNA controls). Knockdown efficiency was evaluated by immunocytochemistry. After knockdown of IDO1, RNA was isolated, reverse transcribed and cancer associated pathways were analyzed using RT2 cancer pathway profiler (data analyzed using GeneGlobe; Qiagen). Results: Evaluation of the expression levels of enzymes that play a critical role in kynurenine pathway by WB analysis indicated upregulation of IDO1, IDO2 and TOD in both primary and metastatic tumor samples. siRNA-mediated knockdown of these enzymes indicated that only IDO1 knockdown was able to significantly reduce the levels of kynurenine by more than 80% (p&amp;lt;0.001), whereas knockdown of IDO2 and TDO reduced the kynurenine levels only by 10-25% (p&amp;gt;0.05%). Regulation of IDO1 function evaluated by the RT2 cancer pathway finder indicated downregulation of several genes involved in angiogenesis (ANGPT1, MCP-1, FGF2, TEK, VEGF, SERPINF1), pro-survival (c-IAP2 and XIAP), EMT transition (CDH2, OCLN and SNAI1), and DNA repair (LIG4, POLB, ERCC3 and DDB2), whereas genes involved in glycolysis (ACLY, G6PD,COX5A,LPL and PFKL), DNA damage (GADD34, GADD45G etc) and apoptosis (CASP7, CASP9, BCL2L11) were upregulated. Conclusion: Kynurenine is highly upregulated in HNSCC with checkpoint inhibition of IDO1 leading to increased apoptosis in vitro as well as increases in several glycolytic enzymes. Further translational analysis of glycolytic influences on the tryptophan pathway and immune checkpoint inhibition of IDO1 will provide mechanistic support for development of novel metabolic therapies for HNSCC. Citation Format: Chitra Subramanian, Thekkelnaycke M. Rajendiran, Tanu Soni, Mark S. Cohen. Targeting the kynurenine pathway as a novel metabolic treatment for head and neck cancer [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 5481.

In vitro assessment of the role of DpC in the treatment of head and neck squamous cell carcinoma.

The present study aimed to investigate the antitumor efficacy of di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) on head and neck squamous cell carcinoma (HNSCC) cells. The proliferation and apoptosis of HNSCC cells treated with the iron chelators DpC and Dp44mT were detected. The mechanism of DpC-induced apoptosis on HNSCC cells was investigated. The human HNSCC cell lines FaDu, Cal-27 and SCC-9 were cultured in vitro and exposed to gradient concentrations of DpC and Dp44mT. A Cell Counting Kit-8 assay was used to detect the viability of FaDu, Cal-27, SCC-9 cells. Double staining with annexin V and propidium iodide was performed for the detection of the proportion of apoptotic FaDu, Cal-27 and SCC-9 cells following treatment. The nuclear damage to Cal-27 cells that were treated with DpC was detected by Hoechst staining. Finally, western blot analysis was used to detect the expression of proteins associated with the DNA damage pathway in Cal-27 cells that were treated with DpC. The CCK-8 assay showed that treatment with DpC and Dp44mT was able to markedly inhibit the viability of FaDu, Cal-27 and SCC-9 cells in a concentration-dependent manner. In comparison to Dp44mT, treatment with DpC exhibited a more effective inhibitory effect on the viability of HNSCC cells. The proportion of apoptotic cells detected by flow cytometry increased in a dose-dependent manner in all cell lines following DpC and Dp44mT treatment, with the proportion of apoptotic HNSCC cells induced by DpC treatment being significantly higher compared with Dp44mT (P<0.05). The results of Hoechst staining revealed that the nuclei of Cal-27 cells exhibited morphological changes in response to DpC treatment, including karyopyknosis and nuclear fragmentation. The expression of DNA damage-associated proteins, including phosphorylated (p)-serine-protein kinase ATM, p-serine/threonine-protein kinase Chk1 (p-Chk-1), p-serine/threonine-protein kinase ATR (p-ATR), p-Chk-2, poly (ADP-ribose) polymerase, p-histone H2AX, breast cancer type 1 susceptibility protein, p-tumor protein P53, increased with increasing concentration of DpC in Cal-27 cells. Treatment with DpC and Dp44mT markedly inhibited cell viability and increased the apoptotic rates in human HNSCC cells in a concentration-dependent manner. DpC exhibited a stronger antitumor effect compared with Dp44mT, potentially inducing the apoptosis of HNSCC cells via the upregulation of DNA damage repair-associated proteins.

Abstract 33: CEACAM1 blockade increases NK cell cytotoxicity in head and neck squamous cell carcinoma

Abstract In conjunction with advances made in cytotoxic chemotherapy, radiation, and surgery, immunotherapy has emerged in recent years as a promising fourth modality of treatment for head and neck squamous cell carcinoma (HNSCC). Cetuximab, an anti-EGFR antibody, is now routinely used as an adjunct to radiation therapy and in the recurrent/metastatic setting following treatment failure with concurrent cisplatin and radiation. Understanding the fundamental mechanisms by which HNSCC evades the immune system will aid the development of new therapies and strategies to augment an antitumor immune response. Carcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is a cell surface receptor that is expressed on HNSCC cells, as well as on lymphocytes, such as NK cells. Overexpression of CEACAM1 in HNSCC has been associated with a poor prognosis, higher clinical stage, recurrence, and lymph node metastasis. A phase I trial (NCT02346955) of an anti-CEACAM1 antibody is currently underway for a variety of non-HNSCC malignancies. We demonstrate that in mice inoculated with TUBO-EGFR, a murine cancer cell line, treatment with an anti-CEACAM1 antibody rescues NK cell effector function from exhaustion resulting in reduced tumor growth and enhanced antibody-dependent cell-mediated cytotoxicity. The addition of Cetuximab has an additive effect and results in additional tumor shrinkage. Thus, these data indicate that CEACAM1 is a novel checkpoint molecule on NK cells. CEACAM1 is also expressed on tumor cells themselves; its expression in HNSCC is associated with suppression of NK cell cytotoxicity. In vitro co-incubation of NK cells with PCI-13, a human HNSCC cell line, results in IFNγ-mediated upregulation of CEACAM1 on the tumor cells. IFNγ-exposed cells are more resistant to NK cytotoxicity compared to untreated cells, suggesting that immune pressure induces upregulation of NK inhibitors. Importantly, CEACAM1+ NK cells have decreased cytotoxicity against HNSCC tumor cells compared to CEACAM1- NK cells, but the addition of an anti-CEACAM1 antibody restores NK cell effector function. Together, these data indicate that CEACAM1 acts as a checkpoint molecule and that a blocking antibody against CEACAM1 releases its immunosuppressive effects and could serve as a novel immunotherapy for HNSCC. Citation Format: Kenric Tam, Yunqin Lee, David Schoppy, John B. Sunwoo. CEACAM1 blockade increases NK cell cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 33.

Abstract 52: Dynamic rewiring of apoptotic signaling networks by combined EGFR/FRGR inhibition enhances killing of head and neck squamous tumor by DNA damage

Abstract We have previously shown that a subset of triple-negative breast cancers can be dramatically sensitized to killing by DNA-damaging chemotherapy by time-staggered inhibition of EGFR. This sensitization effect results from dynamic re-wiring of apoptotic signaling pathways to engage a caspase-8-dependent cell death mechanism (Figure 1). Here we explored whether combined EGFR and/or FGFR inhibition could enhance the killing of head and neck squamous cell cancers by DNA damaging treatments through a similar re-wiring mechanism. The epidermal growth factor receptor (EGFR) and FGFR pathways are two of the most dysregulated molecular pathways in human head-and-neck squamous cell carcinoma (HNSCC). Despite overexpression in approximately 90% of HNSCC tumors, EGFR inhibitors alone have not exerted a major therapeutic impact on tumor response or patient survival. A recent study provides evidence that the insensitivity of the majority of HNSCC to EGFR inhibitors is mediated by dominant activity of alternative receptor tyrosine kinase (RTK) systems such as FGFR signaling (Clinical Cancer Research 2011). We have found that time-staggered administration of either EGFR inhibitors, or FGFR inhibitors, can dramatically enhance the apoptotic response of HNSCCs to DNA damage from doxorubicin. Importantly, combined inhibition of both the EGFR and FGFR pathways was more effective than inhibition of either pathway alone, at inducing sensitization to cytotoxic chemotherapy through dynamic network re-rewiring. Enhanced cell death by dual EGFR/FGFR inhibition and subsequent genotoxic injury results from greater activation of Caspase-8 than that seen by either EGFR or FGFR inhibitors alone. This enhanced Caspase-8 acitivty, together with Caspase-9 activation, accounts for the majority of apoptotic death. Our data further suggest that Src family kinases are a critical downstream node of EGFR/FGFR activity since treatment of HNSCC cell lines with Src kinase inhibitors resulted in similar sensitization to doxorubicin treatment. In summary, our data extend the utility of therapeutic dynamic rewiring for the targeting of tumors with redundant receptor signaling pathways as well as their critical downstream nodes. Citation Format: Yogesh Dayma, Michael B. Yaffe. Dynamic rewiring of apoptotic signaling networks by combined EGFR/FRGR inhibition enhances killing of head and neck squamous tumor by DNA damage [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 52.

MiR-206 inhibits Head and neck squamous cell carcinoma cell progression by targeting HDAC6 via PTEN/AKT/mTOR pathway

As a kind of endogenous noncoding small RNA, microRNA (miRNA) plays important roles of regulation to various physiological functions, while its affections on senescence of human Head and neck squamous cell carcinoma (HNSCC) are still unknown. The objective of this study was to investigate the effect of miR-206 in HNSCC tissues, adjacent normal tissues and cell lines, and explore its biological functions in HNSCC.In our study, the level of miR-206 in HNSCC tissues and adjacent normal tissues was detected via real-time qPCR. The effect of miR-206 on cell proliferation was tested by MTT assay, colony formation and cell cycle assays. In order to explore the effect of miR-206 on HNSCC cell migration and invasion, we performed wound healing assays and transwell assays. Luciferase reporter assays were designed to identify the interaction between 3′UTR of HDAC6 and miR-206. The level of signaling pathway-related proteins was determined by western blot. The expression of miR-206 was found to be observably decreased in HNSCC tissues and cell lines through real time-PCR. Restoration of miR-206 weaked cell proliferation, invasion and migration in HNSCC cells and the cell cycle was arrest in S phase. Further explores have shown that miR-206 could inhibit HNSCC cells proliferation by targeting the HDAC6 via PTEN/AKT/mTOR pathway. Taken together, our results demonstrated that miR-206 plays a critical role in HNSCC progression by targeting HDAC6 via PTEN/AKT/mTOR pathway, which might be a potential target for diagnostic and therapeutic in HNSCC.

Abstract 652: PD-L1 and MHC class 1 expression levels are modulated by MEK inhibition on head and neck squamous cell carcinoma

Abstract Background: To overcome immunosuppression, multiple pathways can be targeted by counter strategies, including elimination of suppressive signals (such as PD-L1 and CTLA-4) and promotion of antigen presentation from tumor cells, and of T cell infiltration into tumor sites. The purpose of this study is to investigate the change in expression of PD-L1 and MHC class I as well as of T cell chemoattractants, CXCL9 and CXCL10, in human head and neck squamous cell carcinoma (HNSCC) cell lines after treatment with MEK inhibitor. In addition, for evaluation of immunologic effects of MEK inhibitor in vivo, we employed SCC VII mouse squamous cell carcinoma (SCC) model. Methods: Six human HNSCC cell lines (SNU-1041, SNU-1066, SNU-1076, Detroit 562, FaDu, and HN31) and a mouse squamous cell carcinoma cell line (SCC VII) were used. Trametinib was purchased from Selleckchem. We conducted cell viability assay using these cell lines after 72 h incubation with MEK inhibitor trametinib. PD-L1 and MHC class I expression levels were analyzed by flow cytometry after treatment with trametinib and/or interferon-gamma (IFN-γ). Expression of PD-L1, p-Erk1/2, and p-STAT1/3 were analyzed by western blot. STAT3 was knocked down by siRNA transfection. To determine the levels of CXCL9 and CXCL10 transcripts after trametinib treatment, reverse-transcription PCR was carried out. Results: The growth inhibition by trametinib treatment was variable between cell lines with moderate sensitivity (10 nM &amp;lt; IC50 &amp;lt; 100 nM in five of seven cells). IC50 values were over 10 μM in Detroit 562 and FaDu. All the cell lines have no upstream RAS or RAF mutation. Although trametinib treatment was not shown to be enough for inhibiting growth of these cell lines, it up-regulated MHC class I expression in all cells either alone (&amp;gt; 2-fold) or when combined with IFN-γ (&amp;gt; 2-fold of IFN-γ alone). At the same time, we observed PD-L1 expression was elevated by trametinib treatment when combined with IFN-γ in all cells except SNU-1066, in which trametinib treatment down-regulate IFN-γ-induced increase of PD-L1 expression. Furthermore, we observed that trametinib alone and/or with IFN-γ enhanced mRNA expression of CXCL9 and CXCL10 in all human cells. Mechanistically, trametinib treatment enhanced STAT1 phosphorylation by IFN-γ in five of six human cells, and directly phosphorylated STAT3. When we silenced STAT3, enhanced expression of MHC class I by trametinib treatment was not observed in SNU-1041 cell. Conclusion: Our results suggest that MEK pathway is closely related with modulating the expression of PD-L1 and MHC class I as well as of CXCL9 and CXCL10 in HNSCC cell lines. Up-regulated levels of MHC class I and T cell-recruiting chemokines imply that MEK inhibitor may mediate potentiating T cell responses. Moreover, simultaneous increase of PD-L1 expression suggests the possible synergistic role of MEK inhibitor when combined with anti-PD-1/PD-L1 immunotherapy in HNSCC. Citation Format: Seong-Ho Kang, Bhumsuk Keam, Soyeon Kim, Miso Kim, Yong-Oon Ahn, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. PD-L1 and MHC class 1 expression levels are modulated by MEK inhibition on head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 652. doi:10.1158/1538-7445.AM2017-652

Abstract 843: CLDN1 mediates tumor invasion in HNSCC by regulation of EMT through AMPK-TGF-β signalling

Abstract Background: Claudin-1 (CLDN1), a major component of tight junction complexes in epithelium, maintains cellular polarity and plays critical roles in cell-to-cell communication and epithelial cell homeostasis. Although, the role of CLDN1 in cancer has been generally studied in cancers, the exact regulatory mechanisms and downstream pathways in cancer have been controversial and are not completely understood. Materials and Methods: After, analysed mutational profiling of tumor/non-tumor paired tissue in the patients with head and neck squamous cell carcinoma (HNSCC) by next-generation sequencing (NGS), CLDN1 expression levels were measured in human HNSCC cell lines and tissues from HNSCC patients using next-generation sequencing. For functional in vitro analysis, cell proliferation testing using a WST-1 assay, an invasion assay, and Western blotting was performed after CLDN1 expression regulation. In vivo validations were performed using nude mouse and patient-derived tumor xenograft (PDTX) model. Results: Mutational profiling of tumor/non-tumor paired HNSCC tissue by NGS revealed that HNSCC tumor tissues and cell lines had relatively high levels of CLDN1 expression. Although, CLDN1 regulation did not affect proliferation of HNSCC cell lines, knock down of CLDN1 inhibited invasive characteristics (migration/invasion) with down-regulation of EMT associated proteins (vimentin and slug). We also found that CLDN1 negatively correlated with AMPK phosphorylation and AMPK activator (AICAR) showed the same effect with CLDN1 suppression on HNSCC cells without change in CLDN1 expression which lead to inhibition of TGF-β-induced Smad phosphorylation and their transcriptional activity, suggesting that CLDN1/AMPK/TGF-β cascade. Then, we confirmed antitumorigenic effects of suppression of CLDN1 in HNSCC in a nude mouse and PDTX model. Conclusions: Our findings suggest that CLDN1 acts as oncogenic driver in HNSCC via regulating EMT through AMPK/TGF-β signalling and if validated in further studies, a CLDN1-targeting agent partnered with an existing anti-TGF-β modality could be a novel tactic to overcome HNSCC. Citation Format: Jae Won Chang, Seung-Nam Jung, Lihua Liua, Jin Man Kim, Myung Jin Ban, Yeon Soo Kim, Bok-Soon Lee, Yang Eun Kim, Ho Ryun Won, Chul-Ho Kim, Bon Seok Koo. CLDN1 mediates tumor invasion in HNSCC by regulation of EMT through AMPK-TGF-β signalling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2017-843