Head And Neck Squamous Cell Carcinoma Cell Research Articles

Abstract 3859: HSP90 blockade activates antitumor immunity by suppressing tumor glycolytic flux in head and neck squamous cell carcinoma

Abstract Accumulating evidence suggests that failure of antitumor immunity may be due to dysregulation of energy metabolism. Head and neck squamous cell carcinoma (HNSCC) exhibits increased activity of two key enzymes, pyruvate kinase M2 (PKM2) and platelet phosphofructokinase (PFKP), which are central to the glycolytic pathway responsible for the conversion of glucose to ATP energy. HSP90 is a molecular chaperone that plays a critical role in the folding, stabilization, and regulation of a wide range of client proteins involved in various cellular processes, including metabolism. However, whether HSP90 signaling contributes to tumor metabolism rewiring and antitumor immune response remains unknown. Using ganetespib as a drug model for HSP90 inhibition and combining results from clinical trials and animal treatment, our study demonstrated that HSP90 inhibition causes an obstruction of glycolytic flux in HNSCC cells by simultaneously suppressing both PKM2 and PFKP at the transcriptional and post-translational levels. In addition, ganetespib enhances T cell-driven antitumor immune responses by promoting CD8+ T cell infiltration into the tumor. Mechanistically, ganetespib downregulates IL8 signaling by inhibiting PKM2 and PFKP-mediated glycolysis in HNSCC cells, which in turn contributes to ganetespib-mediated T cell tumor infiltration. These findings provide a novel molecular basis for the use of effective HSP90 inhibitors to improve the outcome of HNSCC patients. Citation Format: Fanghui Chen, Chris Tang, Fan Yang, Asari Ekpenyong, Richard Qin, Nabil F. Saba, Yong Teng. HSP90 blockade activates antitumor immunity by suppressing tumor glycolytic flux in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3859.

Abstract 1219: In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables ATAD3A gene depletion to enhance RAS-targeted therapy

Abstract Targeting mitochondrial oncoproteins presents a new concept in developing effective cancer therapeutics. ATAD3A is a nuclear-encoded mitochondrial enzyme that contributes to mitochondrial dynamics, cholesterol metabolism, and signal transduction. Our previous study has demonstrated elevated expression of ATAD3A in head and neck squamous cell carcinoma (HNSCC) primary tissues and cultured cell lines. Loss of function suppresses HNSCC cell growth and elicits tumor regression in orthotopic tumor-bearing mice. Mechanistically, ATAD3A interacts with ERK1/2 in the mitochondria of HNSCC cells in the presence of VDAC1, and the ATAD3A-ERK1/2 signaling axis drives HNSCC development in a RAS-independent fashion. CRISPR-Cas9 has revolutionized genome engineering and has been intensively used to modify cancer-associated genes. However, challenges remain as CRISPR editing components must be transported into cells to exert their function. There is a need to explore effective in vivo delivery systems to accelerate the clinical use of CRISPR-Cas9. As no drug is available to target ATAD3A directly, we developed CRISPR-based ATAD3A-targeting nanodrugs by loading Cas9 mRNA and anti-ATAD3A sgRNAs into lipid nanoparticles (LNPs) that have become clinically acceptable delivery system due to low toxicity and high delivery efficacy and biocompatibility. As expected, treatment tongue tumors established in NSG mice with LNP-sgATAD3A remarkably repressed tumor growth. Most interestingly, the addition of LNP-sgATAD3A to the RAS inhibitor salirasib potentiated anticancer activity for HNSCC compared with the efficacy resulting from single-arm treatment. These novel and significant findings demonstrate a novel approach to inhibiting ATAD3A in tumor cells and provide a possible therapeutic strategy to enhance the success of multimodality therapy in cancer patients. Citation Format: Fanghui Chen, Liwei Lang, Yamin Li, Wei Zheng, Yong Teng. In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables ATAD3A gene depletion to enhance RAS-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1219.

Abstract 6646: Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models

Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is often marked by an immunosuppressive tumor microenvironment, which contributes to the dismal 10-20% response rate to immune checkpoint inhibitor (ICI) therapy in HNSCC patients. Radiotherapy (RT) is a currently standard of care and frontline therapy for HNSCC, yet resistance is common. We sought to examine the combination therapy of RT followed by ICI therapy to test if RT induces an in situ vaccination which enhances the efficacy of subsequent ICI. Methods: We utilized SCC cell lines derived from K15.KrasG12D/Smad4−/− SCCs, which mimic the progression of HPV-unrelated HNSCC, and transplanted SCC cells into immune-competent C57BL/6J mouse recipients. Mice were subjected to RT followed by anti-PD-L1/anti-TGFb therapy. CD8+ splenic T cells of responder or non-responder mice were examined by paired single-cell VDJ sequencing and single cell RNA sequencing (TCR/sc-RNAseq) to look for adaptations of the cytotoxic T cell compartment following combination therapy. Responder and non-responder tumor lines or tumors were treated with RT alone in vitro and in vivo, respectively, to examine major histocompatibility complex I (MHC-I) levels on tumor cells. Results: Responders to this combination therapy exhibited complete tumor eradication while non-responders displayed rapid tumor progression. When re-challenged with same HNSCCs cells, responders rejected implanted tumor cells while naïve recipients rapidly developed HNSCCs. Single-cell TCR sequencing of splenic CD8+ T cells revealed that responders, but not naïve recipients exhibited multiclonal CD8+ T cell expansion specifically of memory cell populations. Responder tumor cells exhibited greater levels of MHC-I protein level presentation than non-responders when stimulated with RT. Conclusions: Our data suggest that 1) elevated MHC-I by SCC tumor cells presentation driven by RT and ICI combination therapy facilitate a systemic expansion of memory CD8+ T cell clonotypes which drive anti-tumor immunity and 2) MHC-I expression at baseline or when induced by RT may serve as a predictive marker for therapeutic response to RT and ICI combination therapy in HNSCCs. Citation Format: Hanne Lind, Alexander Strait, Spencer Hall, Jack B. Goon, John D. Aleman, Samantha Chen, Philip Owens, Jing H. Wang, Christian Young, Xiao-Jing Wang. Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6646.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6956.

Abstract 3061: A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness

Abstract Both GLS1 and c-Myc are upregulated in head and neck squamous cell carcinoma (HNSCC) primary tumors and further increased in metastatic tumors. However, the regulation between these two molecules remains largely unknown. GLS1 is a critical enzyme that regulates glutamate, which plays an important role in cancer metabolism that supports cancer growth and survival. Our bioinformatic analysis of the TCGA HNSCC cohort revealed a strong correlation between c-Myc and GLS1 expression. We describe the importance of c-Myc in regulating GLS1 and vice versa. c-Myc protein directly binds to the promoter of the GLS1 gene and upregulates its expression at the transcriptional level. Interestingly, blocking GLS1 signaling in HNSCC cells by lentiviral shRNA knockdown or CB-839 treatment downregulates USP1, one of the best characterized human DUBs, which in turn reduces c-Myc protein stability via the ubiquitin-proteasome pathway. The GLS1-c-Myc axis thus represents a novel positive feedback loop that is critical for driving the aggressiveness of HNSCC. As the treatment of HNSCC remains a major challenge, these novel findings provide the molecular basis for combining GLS1-specific inhibitors with c-Myc-targeted therapy for the treatment of HNSCC patients. Citation Format: Jianqiang Yang, Fanghui Chen, Fan Yang, Yong Teng. A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3061.

Abstract 4517: Novel chemical degraders targeting NSD2 in head and neck squamous cell carcinoma (HNSCC)

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in treatments and significant efficacy of immunotherapy drugs in HNSCC, no effective targeted therapies have been approved in this disease yet. NSD2 is a histone lysine methyltransferase, which is overexpressed in HNSCC and is implicated in tumor growth, cell cycle regulation, epithelial-mesenchymal transition (EMT) and metastasis not only in HNSCC, but also in many other solid tumors. Development of inhibitors to the catalytic activity of NSD2 has been rather limited to date, which necessitates an alternative approach. To target NSD2, we have recently developed a series of novel proteolysis targeting chimeras (PROTACs), which chemically degrade NSD2 utilizing native cellular machinery - ubiquitin-proteasome system - with VHL as an E3 ligase. A series of >40 compounds were tested in HNSCC cell lines (JHU-011 and FaDu) and NSD2-dependent multiple myeloma cell line (KMS11) for degradation activity against NSD2 protein, and H3K36me2 chromatin mark reduction, using Western blot. We also tested their anti-proliferative activity in cell viability assay (CTB) and clonogenic assay. Compounds NUCC-0227381, NUCC-0227384, NUCC-0227414 and NUCC-0227383 show promising results by reducing NSD2 and H3K36me2 levels. NUCC-0227383 compound was highly promising, as it was the most potent in reducing NSD2/K36me2 levels without affecting NSD1/NSD3 levels and had profound effects on HNSCC cell growth. Lead compound NUCC-0227383 was chosen for further testing and in preliminary testing, showed a dose-dependent, VHL-mediated, and proteasome dependent function. Proteomics analysis, testing for specificity of NUCC-0227383 against a panel of histone methyltransferases, and in vivo testing of this promising compound against HNSCC is planned. Citation Format: Amr Ismail, Ekaterina Roshchina, Iuliia Topchu, Gary Schiltz, Yanis Boumber. Novel chemical degraders targeting NSD2 in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4517.

Abstract 5143: Molecular investigation of a polymorphic variant in JMJD1C that associates with chemoradiotherapy outcomes in locally advanced rectal and head and neck cancer

Abstract Background: Neoadjuvant chemoradiotherapy (nCRT) is standard therapy for locally advanced rectal cancer (LARC). While only a minority of patients are expected to have a complete response, all are at risk of toxicity. Currently no biomarkers reliably predict response to nCRT in LARC. Further, standard therapy in locally advanced head and neck squamous cell carcinoma (HNSCC) is adjuvant CRT. A major cause of poor outcomes in HNSCC is regional persistence or recurrence post-CRT. No biomarkers, apart from HPV, predict CRT response in HNSCC. CRT causes double-strand breaks (DSBs) and methylation of histones on lysine residues is known to affect chromatin, influencing DSB repair. We hypothesized that single nucleotide polymorphisms (SNP) in the histone 3 lysine 9 (H3K9) demethylase-encoding JMJD1C influenced CRT response. Methods: LARC patients (n=84) were divided into poor (PoR) and complete (CR) responders based on Neoadjuvant Rectal score. Lymphocyte DNA was sequenced, and LARC patient-derived cells were treated with DSB-inducing agents to assess viability. Sequencing was confirmed in an independent cohort of CRT-treated HNSCC patients (n=90) divided into: disease-free (n=30), recurrent disease (n=28), and never-disease-free (n=32). HNSCCs were in the larynx or oral cavity. Not all patients received HPV-testing; p16 expression was positive for 19/43 tumors. CRISPR/Cas9 editing of HPV-negative HNSCC lines (SCC9, Cal27) generated JMJD1C WT or SNP isogenic pairs. Bulk RNA-sequencing, colony survival, and immunofluorescence studies were performed. Results: In LARC patients, CRs were enriched for a coding, missense germline SNP in JMJD1C versus PoRs (discovery n=30, p<0.00001; validation n=54, p=0.001, Fisher’s Exact). When subjected to DSB, LARC patient-derived cells with the SNP displayed greater sensitivity than their WT counterparts (n=12, p<0.001, Mann-Whitney). In HNSCC, a correlation was identified between the same JMJD1C SNP and no recurrence (p=0.0003, Fisher’s Exact). HNSCC SNP cells showed changes in mRNA levels of DNA repair and immune response genes versus HNSCC WT cells (q-value<0.05). Upon irradiation, kinetics of foci resolution of the DNA damage response protein MDC1, specific to DSBs, were slower in SNP versus WT HNSCC cells (p<0.05, Mann Whitney). Further, there was reduced colocalization of BRCA1 with RAP80, a crucial factor for homology-directed DSB repair, in SNP versus WT HNSCC cells (p<0.01, Mann-Whitney).Targeted introduction of the SNP into HNSCC cell lines reduced viability and radiation response. Conclusion: Our study identifies a polymorphic coding variant in JMJD1C, as a predictive biomarker for CRT effectiveness in LARC and HNSCC, and defines a DNA repair mechanism by which JMJD1C influences response to irradiation. Citation Format: Adria Hasan, Elena V. Demidova, Philip Czyzewicz, Shreya M. Shah, Karthik Devarajan, Thomas J. Galloway, Margret B. Einarson, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Molecular investigation of a polymorphic variant in JMJD1C that associates with chemoradiotherapy outcomes in locally advanced rectal and head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5143.

Abstract 7585: Targeting of 7 or 24 dehydrocholesterol reductase decreases cancer progression in head and neck cancer

Abstract The elevated expression of cholesterol metabolism has been associated with many cancer including head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms between cholesterol biosynthesis and head and neck cancer have not yet been studied. In this study, we identified upregulation of dehydrocholesterol reductase such as DHCR7 or DHCR24 in HNSCC compared with adjacent normal tissues from the same patients using RNA sequencing data. We observed protein expression level of DHCR7 or DHCR24 in head and neck cancer cell lines through Western blotting. We also checked high expression of DHCR7 or DHCR24 in HNSCC tissues from patients using immunohistochemistry assays. Yet, when DHCR7 or DHCR24 was knocked down using siRNA, the viability of HNSCC cells was decreased in MTT assays. In parallel, we adapted small inhibitory drugs; AY9944 for DHCR7 inhibition and Triparanol or SH-42 for DHCR24 inhibition. Treatment of AY9944, Triparanol, or SH-42 also resulted in decreased viability of HNSCC cells. Moreover, when we used a cholesterol blocker, MCD, the cell viability was decreased, whereas the cells overcame cell death by adding the combination of cholesterol and LDL. Taken together, we concluded that head and neck cancer requires DHCR7 and DHCR24 activity involved in cholesterol metabolism for cancer growth. Our data suggest that targeting DHCR7 or DHCR24 using AY9944, Triparanol or SH-42 is one of the potential strategies that will definitely help with cancer treatment for head and neck cancer. Citation Format: Bok-Soon Lee, Ji-Hye Choi, Hyo Jeong Kim, Jin Roh, Joselyn Padilla, Kennedy Davis Brock, Jeon Yeob Jang, Hyun Goo Woo, Chul-Ho Kim, Jiyoung Lee. Targeting of 7 or 24 dehydrocholesterol reductase decreases cancer progression in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7585.

Abstract 5595: Multifaceted role Cornulin in head and neck squamous cell carcinoma: From tumor suppressor pathophysiology to prognosticating biomarker

Abstract Background: In our previous study, based on LC-MS/MS analysis, the salivary Cornulin, a relatively less explored protein, was found to be ~10-fold downregulated in Head and Neck Squamous Cell Carcinoma (HNSCC) patients. However, the exact role of Cornulin in the pathophysiology of HNSCC is not reported. Here, we investigated its role in the pathophysiology of HNSCC and elucidated its potential as a prognostic marker as well as its antitumour effect. Methods: Sandwich ELISA and immunohistochemistry were performed to estimate the salivary and tissue levels of Cornulin, respectively, in HNSCC patients, along with an analysis of the effects of Cornulin levels on disease outcome. To investigate the antitumor effects ,its levels were upregulated through lentiviral transduction in low expressing Cal27, FaDu cell lines. Subsequently, various assays were conducted to examine the antitumor effects of Cornulin upregulation. The molecular signalling pathway was elucidated by analysing transcriptome. Additionally, the effects of increased Cornulin expression were 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) and 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) compared to the tumor-free margin (n=72,H-score=139.6±10.34). The patients showing complete clinical response regained normal salivary levels 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), the cellular viability, migration, and invasion showed a significant decrease. These indicate its potential as a tumor suppressor. RNAseq data of Cornulin overexpressed cell lines and further Gene set enrichment analysis, the G2/M checkpoint pathway was found to be positively enriched. Cornulin overexpression also arrests the G1/S phase of the cell cycle. GSEA and in vitro experiments revealed Cornulin to be involved in the PI3K/Akt/mTOR signalling pathway. In the nude mice xenograft model, decreased tumor progression, and tumor volume, was documented after Cornulin overexpression. There was an increase in differentiation in the xenograft tumors formed by Cornulin overexpressed cells. Conclusion: Here, we report for the first time that intrinsic Cornulin has a role as a potent antitumor and has potential as a prognostic marker. However the exact molecular mechanism needs to be further evaluated. Citation Format: Rajandeep Kaur, Anshika Chauhan, Sushmita Ghoshal, Jaimanti Bakshi, Radhika Srinivasan, Arnab Pal. Multifaceted role Cornulin in head and neck squamous cell carcinoma: From tumor suppressor pathophysiology to prognosticating biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5595.

Abstract 5409: Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion

Abstract Although head and neck squamous cell carcinomas (HNSCCs), including tongue, vocal cord, and buccal cancers, are one of the most common cancers worldwide with a mortality rate of more than 40%, there have been no solid standard biomarkers for effectively diagnosing HNSCCs at early stages and no effective protocols available to support in vitro expansion of HNSCC cells. To establish a cell culture method to support in vitro self-renewal of HNSCC cells, patient-derived tumor was enzymatically digested, and the tongue cancer epithelial cells were seeded onto feeder cells with the Rho kinase inhibitor (Y-27632). Additionally, to test our hypothesis that free thiol groups in the cells, generated by a mild reduction of cell surface proteins using the reducing agent tris(2-carboxyethyl) phosphine hydrochloride (TCEP), would modulate cancer cell adhesion and migration, we assessed the expression levels of integrins, focal adhesion kinase (FAK), and phosphorylated FAK (pFAK) of tongue cancer epithelial cells cultured on both soft and rigid polyacrylamide (PAAm) hydrogel-based cell culture substrates. Patient-derived tongue cancer epithelial cells exhibited self-renewal ability and maintained cancerous phenotypes, with high expressions of P53, Ki67, E-cadherin, CD44, and AHDH1A1. Treatment with the reducing agent (TCEP) increased the degree of cell adhesion through the significantly upregulated integrin α5β1-mediated phosphorylation of FAK, resulting in inhibited single-cell and collective cell migration as well as invasion. Additionally, when these cells were cultured on stiff PAAm hydrogels mimicking the tumor microenvironment, the cells exhibited accelerated cell migration behaviors, compared to cells on soft hydrogels. These results highlight the synergetic contributions of feeder cells and Rho kinase inhibitor to support long-term self-renewal of tongue cancer patient-derived epithelial cells. Citation Format: Joo Hyun Kim, Laurensia D. Anggradita, Sung Sik Hur, Jae Hong Park, Sang-Heon Kim, Myung Jin Ban, Yongsung Hwang. Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5409.

Abstract 1729: SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) poses a significant clinical challenge with limited treatment options. SUV420H1, which encodes for a protein lysine methyltransferase (PMT) that methylates H4K20 (H4K20me3), is recurrently amplified in ~5% of HPV-negative HNSCC tumors and is the second most frequently amplified PMT in this disease, suggesting an oncogenic function in this subset of HPV-negative HNSCC patients. Gene set enrichment analysis using the TCGA database of HPV-negative HNSCC tumors revealed that SUV420H1 overexpressing tumors showed enrichment in the mitotic spindle related pathways, and repression of immune-related pathways. To assess whether SUV420H1 depletion affects the proliferation of HPV-negative HSNCC cells, CCK8 and colony formation assays were pursued after siRNA-mediated knockdown of SUV420H1 in five SUV420H1-overexpressing HPV-negative HNSCC cell lines and revealed a significant reduction in cell proliferation and colony forming capacity. SUV420H1 CRISPR knockout (KO) HPV-negative HNSCC human and mouse cell lines were generated. Proliferation and colony formation assays with the SUV420H1 KO cell lines are ongoing to validate the aforementioned phenotypes. To identify oncogenic mechanism of SUV420H1, genome-wide mapping of H4K20me3 after siRNA-mediated depletion or enzymatic inhibition of SUV420H1 (A-196) was pursued using CUT&RUN assays combined with RNA-seq in a SUV420H1 amplified cell line (SCC-151), and analysis is ongoing. Preliminary RNA-seq analysis revealed upregulation of a number of type I IFN response genes after siRNA-mediated knockdown of SUV420H1. In vivo mouse experiments using SUV420H1 KO mouse cell lines are planned in a syngeneic HPV-negative HNSCC flank mouse model (MOC1) with or without anti-PD-1 treatment with the goal to assess whether SUV420H1 depletion affects tumor growth and/or sensitizes cancer cells to anti-PD-1 therapy. These data provide preliminary support for the function of SUV420H1 as an oncogene in HPV-negative HNSCC. Citation Format: Arfa Moshiri, Sohyoung Kim, Marie Luff, Vassiliki Saloura. SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1729.

Abstract 2771: Increased mtDNA copy number via EPOR signaling: A potential mechanism for metastatic dissemination in head and neck squamous cell carcinoma

Abstract Introduction: Head and Neck Squamous cell carcinoma (HNSCC) is characterized by high degree of invasion, locoregional metastasis, relapse which leads to overall poor prognosis. The molecular mechanism responsible for aggressiveness is poorly understood. A better understanding of molecular pathways involved shall precede the identification of newer diagnostic tools and therapeutic targets. We explored EPOR signaling pathway in Circulating tumor cells (CTCs) and HNSCC cell lines. Methods and Results: By using a validated in-house CTC isolation protocol, which addresses various drawbacks of currently available techniques and isolates CTCs suitable for various downstream molecular analysis, we isolated CTCs from HNSCC patients. The whole transcriptome analysis was performed from CTCs using ultra-low cell number RNA sequencing and corresponding primary tumors in 24 paired samples. We found a significant transcriptomic heterogeneity in CTCs, which are associated with overall disease outcome in HNSCC patients. To explore the plausible molecular pathways, we also identified 521 differentially expressed genes in CTCs with respect to primary tumors and various canonical pathways including oxidative phosphorylation, TCA cycle, cytokine-cytokine receptor interaction signaling, and ECM receptor interaction were enriched in CTC samples. Overexpression of erythropoietin receptor (EPOR) along with various genes encoded by mitochondrial genome and genes involved in maintenance of mtDNA in CTCs as compared to primary tumors was an important finding from our data. When analyzed in depth, there was a significant positive correlation between various genes involved in mtDNA maintenance (ABAT, NT5M) and encoded by mitochondrial genome (MT-ND1, MT-ND2, MT-ND4, MT-ND5, MT-ND6, MT-CO1, MT-CO2, MT-ATP6, MT-CYTB) with EPOR expression in our RNA seq data which is also substantiated by analyzing TCGA data. We confirmed these findings experimentally in HNSCC cell lines Cal 27 and FaDu. It was also observed that metastatic cell line FaDu has significantly higher mtDNA content as compared to non-metastatic cell line Cal 27, which was correlated with EPOR expression. Treatment with low dose Erythropoietin (0.1IU/mL) which a natural ligand for EPOR, enhanced mtDNA content in both the cell lines at low doses and boosted the metastatic capability while minimal effect on cell proliferation was observed. Conclusion: EPOR signaling might promote the metastatic ability of cancer cells and may act as a potential target for cancer dissemination and metastasis. Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Increased mtDNA copy number via EPOR signaling: A potential mechanism for metastatic dissemination in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2771.

Abstract 2882: Investigating mechanisms of radioresistance in TYK2 knockout head and neck cancer cells

Abstract Radiation therapy is the primary form of treatment of head and neck squamous cell carcinoma (HNSCC). However, more than half of patients experience disease progression with standard of care treatment. To investigate novel therapeutic targets, a CRISPR screen was done to identify genes that are associated with radioresistance. The screening results indicated that JAK1 and TYK2 knockout (KO) resulted in radioresistance in HNSCC. Preliminary data suggests that loss of JAK1 causes an enhanced G2/M arrest phenotype and enhanced genome stability that contribute to radioresistance. To investigate whether TYK2 KO caused changes to the cell cycle and genome stability as observed in JAK1 KO, Western blots, cell cycle assays, and micronuclei staining experiments were performed. Cal27, a HNSCC cell line, was used. Cal27 cells were transduced with a non-targeting control (NT2) guide RNA (gRNA) or two different gRNAs targeting TYK2 to generate TYK2 KO cells. Western Blot analysis was performed to determine changes in signaling upon TYK2 KO. Additional conditions included radiation and treatments with Interferon gamma, Interferon alpha and Interleukin-6 to activate the canonical JAK-STAT pathways. Overall, there were minimal changes in downstream signaling between the treatments and the knockouts. Cell cycle analysis via flow cytometry revealed no changes between the NT2 and TYK2 KO cells, potentially corroborating signaling results. Forty percent of TYK2 KO cells were arrested at the G2/M checkpoint at 8 hrs and 24 hrs after radiation treatment, but twenty percent returned to the G1 phase at 24 hrs. This indicates that loss of TYK2 is not responsible for cell cycle arrest. Lastly, micronuclei formation was observed to measure genomic instability. Twenty thousand Cal27 NT2 and TYK2 KO cells were seeded. They were allowed to incubate for 48 hrs before treatment with 4 Gy. Micronuclei staining revealed minimal differences between NT2 and TYK2 KO cell lines, although there were significant differences between the control and the radiation groups. Overall, radioresistance by TYK2 KO is not caused by changes in cell cycle or genomic instability as demonstrated with JAK1 KO. To understand how TYK2 is causing radioresistance in HNSCC, next steps would be to see if there are changes in proliferation. If MTT assays demonstrate significant changes, downstream signaling in growth pathways will be measured. If there is no increased proliferation, we will examine changes in DNA damage repair. Citation Format: Nicole Mayerli Constante, Vanessa Kelley, Joseph Contessa. Investigating mechanisms of radioresistance in TYK2 knockout head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2882.

Abstract 1734: Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC

Abstract Background: Approximately 75% of Human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors carry genetic and expression alterations in the family of protein methyltransferases and demethylases, underscoring the importance of these enzymes in the pathogenesis of HNSCC. SMYD3 a lysine methyltransferase that is overexpressed in HPV-negative HNSCC. We recently reported that nuclear SMYD3 plays dual role in immune-related gene regulation through H3K4me3 or H4K20me3, leading to gene activation or silencing, respectively in HPV-negative HNSCC. While SMYD3 has been reported to write and bind to H3K4me3 activating target gene expression, its function in gene silencing is understudied. This study aims to determine the mechanism(s) through which SMYD3 functions both as a transcriptional activator and a repressor within the same cell context, specifically in HPV-negative HNSCC. Methods: immunoprecipitation (IP) of SMYD3 was performed from nuclear extracts isolated from HN-6 cells to identify candidate interacting partners of SMYD3. The nuclear immunoprecipitates were sent for MS analysis to identify candidate nuclear interacting partners of SMYD3. CUT&RUN assays for selected candidate interacting proteins and SMYD3 will be performed in control HN-6 cells versus SMYD3 KO cells to assess genomic co-occupancy with SMYD3. Further, CUT&RUN assays for repressive marks H4K20me3, H3K27me3 and H3K9me3 have been performed in HN-6 and SMYD3 KO cells to assess co-occupancy with SMYD3 as well as its effect of SMYD3 on the genome-wide mapping of these marks. Finally, CUT&RUN assays for SMYD3 followed by re-CUT&RUN of repressive histone marks are ongoing to identify co-occupied loci. Results: Preliminary analysis of our nuclear SMYD3 IP results has identified a chromatin modifier with known repressive function. Co-immunoprecipitation experiments to validate this interaction are planned. CUT&RUN assays for repressive histone marks in HN-6 versus SMYD3 KO cells have revealed impact on H4K20me3 and H3K27me3. Conclusions: Our preliminary results suggest that SMYD3 may exert its silencing function by regulating the genome-wide deposition of H4K20me3.Our nuclear SMYD3 IP analysis has identified candidate interacting partners that may assist SMYD3 in promoting the deposition of these repressive marks. CUT&RUN assays for SMYD3 followed by re-CUT&RUN for H4K20me3 and H3K27me3 bound chromatin may provide further insight about whether SMYD3 co-occupies specific genomic regions together with these repressive marks. This project is expected to provide mechanistic insights into the bifaceted functions of SMYD3 within the same cell context using HPV-negative HNSCC cell lines as an experimental model system.. Citation Format: Jawad Akhtar, Sohyoung Kim, Vassiliki Saloura. Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1734.

Involvement of TGFBI-TAGLN axis in cancer stem cell property of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a significant healthcare burden globally. Previous research using single-cell transcriptome analysis identified TGFBI as a crucial marker for the partial-epithelial-mesenchymal transition (partial-EMT) program. However, the precise role of TGFBI in HNSCC progression remains unclear. Therefore, our study aimed to clarify the impact of TGFBI on the malignant behavior of HNSCC cells. Through RNA-sequencing data from the TCGA database, we validated that increased TGFBI expression correlates with a higher occurrence of lymph node metastasis and unfavorable prognosis in HNSCC cases. Functional experiments demonstrated that TGFBI overexpression enhances the ability of sphere formation, indicating stem-cell-like properties. Conversely, TGFBI depletion reduces sphere formation and suppresses the expression of cancer stem cell (CSC) markers. RNA-sequencing analysis of TGFBI-overexpressing and control HNSCC cells revealed TAGLN as a downstream effector mediating TGFBI-induced sphere formation. Remarkably, TAGLN depletion abolished TGFBI-induced sphere formation, while its overexpression rescued the suppressed sphere formation caused by TGFBI depletion. Moreover, elevated TAGLN expression showed correlations with the expression of TGFBI and partial-EMT-related genes in HNSCC cases. In conclusion, our findings suggest that TGFBI may promote CSC properties through the upregulation of TAGLN. These novel insights shed light on the involvement of the TGFBI-TAGLN axis in HNSCC progression and hold implications for the development of targeted therapies.

CELSR3 is a prognostic marker in HNSCC and correlates with immune cell infiltration in the tumor microenvironment

PurposeTo look at the diagnostic value of the CELSR receptor 3 (CELSR3) gene in head and neck squamous cell carcinoma (HNSCC) and its effect on tumor immune invasion, which is important for enhancing HNSCC treatment.MethodsSeveral bioinformatics tools were employed to investigate CELSR3’s putative oncogenic pathway in HNSCC, and datasets from The Tumor Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER), Gene Expression Profile Interaction Analysis (GEPIA) and LinkedOmics were extracted and evaluated. CELSR3 has been linked to tumor immune cell infiltration, immunological checkpoints, and immune-related genes. CELSR3’s putative roles were investigated using Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and pathway enrichment analysis. The expression level of CELSR3 in HNSCC tissues and cells was detected by RT-qPCR. The effects of CELSR3 on proliferation of HNSCC cells were detected by CCK-8 assay.ResultsCELSR3 was shown to be expressed differently in different types of cancer and normal tissues. CELSR3 gene expression was linked to pN-stage and pM-stage. Patients with high CELSR3 expression also have a well prognosis. CELSR3 expression was found to be an independent predictive factor for HNSCC in both univariate and multivariate Cox regression analyses. We discovered the functional network of CELSR3 in HNSCC using GO and KEGG analysis. CELSR3 expression levels were found to be favorably associated with immune cell infiltration levels. Furthermore, CELSR3 expression levels were significantly correlated with the expression levels of many immune molecules, such as MHC genes, immune activation genes, chemokine receptors, and chemokines. CELSR3 is highly expressed in HNSCC tissues and cells. CELSR3 overexpression significantly inhibited the proliferation of HNSCC cells. CELSR3 expression may affect the immune microenvironment and, as a result, the prognosis of HNSCC.ConclusionCELSR3 expression is elevated in HNSCC tumor tissues, and high CELSR3 expression is associated with well prognosis, which inhibited the proliferation of NHSCC cells. CELSR3 has the potential to influence tumor formation by controlling tumor-infiltrating cells in the tumor microenvironment (TME). As a result, CELSR3 may have diagnostic significance in HNSCC.

Exercise Suppresses Head and Neck Squamous Cell Carcinoma Growth via Oncostatin M.

Major advances have been made in cancer treatment, but the prognosis for elderly cancer patients with sarcopenia and frailty remains poor. Myokines, which are thought to exert preventive effects against sarcopenia, have been reported to be associated with the prognosis of various cancers, but their effect on head and neck squamous cell carcinoma (HNSCC) is unknown. The aim of this study was to clarify the influence of exercise on the control of HNSCC and to examine the underlying mechanism involved. Mice were injected with HSC-3-M3 cells, a human cell line of highly metastatic and poorly differentiated tongue cancer, at the beginning of the study. Just prior to transplantation, blood was collected from the mice, and the levels of myokines were measured by ELISA. Oncostatin M (OSM), a selected myokine, was added to HSC-3-M3 cells, after which the cell proliferation ability, cell cycle, and protein expression were analyzed in vitro. Tumor cell viability was lower (control: 100%, exercise: 75%), tumors were smaller (control: 26.2 mm3, exercise: 6.4 mm3), and survival was longer in the exercise group than in the control group in vivo. OSM inhibited HSC-3-M3 cell proliferation in a concentration-dependent manner in vitro. The addition of OSM increased the proportion of cells in the G0/G1 phase, decreased the proportion of cells in the G2/M phase, and increased the expression of the CDK inhibitors p21 and p27. These results indicate that exercise may directly inhibit the proliferation of HNSCC cell lines via OSM.

PIP4K2B Protein Regulation by NSD1 in HPV-Negative Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) ranks among the most prevalent global cancers. Despite advancements in treatments, the five-year survival rate remains at approximately 66%. The histone methyltransferase NSD1, known for its role in catalyzing histone H3 lysine 36 di-methylation (H3K36me2), emerges as a potential oncogenic factor in HNSCC. Our study, employing Reverse Phase Protein Array (RPPA) analysis and subsequent validation, reveals that PIP4K2B is a key downstream target of NSD1. Notably, PIP4K2B depletion in HNSCC induces downregulation of the mTOR pathway, resulting in diminished cell growth in vitro. Our investigation highlights a direct, positive regulatory role of NSD1 on PIP4K2B gene transcription through an H3K36me2-dependent mechanism. Importantly, the impact of PIP4K2B appears to be context-dependent, with overexpression rescuing cell growth in laryngeal HNSCC cells but not in tongue/hypopharynx cells. In conclusion, our findings implicate PIP4K2B as a novel NSD1-dependent protein in HNSCC, suggesting its potential significance for laryngeal cancer cell survival. This insight contributes to our understanding of the molecular landscape in HNSCC and establishes PIP4KB as a promising target for drug development.

Nicotine downregulates miR-375–3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375–3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375–3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375–3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375–3p specifically binds to 3′ untranslated region (3’UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375–3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

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

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