Head And Neck Squamous Cell Carcinoma Cancer Research Articles

Oncogenic H-RAS induces metformin resistance in head and neck cancer by promoting glycolytic metabolism.

Metformin administration has recently emerged as a candidate strategy for the prevention of head and neck squamous cell carcinoma (HNSCC). However, the intricate relationship between genetic alterations in HNSCC and metformin sensitivity is still poorly understood, which prevents the stratifications of patients harboring oral premalignant lesions that may benefit from the chemopreventive activity of metformin. In this study, we investigate the impact of prevalent mutations in HNSCC in response to metformin. Notably, we found that the expression of oncogenic HRAS mutants confers resistance to metformin in isogenic HNSCC cell systems and that HNSCC cells harboring endogenous HRAS mutations display limited sensitivity to metformin. Remarkably, we found that metformin fails to reduce activation of the mTOR pathway in HRAS oncogene expressing HNSCC cells in vitro and in vivo, correlating with reduced tumor suppressive activity. Mechanistically, we found that this process depends on the ability of HRAS to enhance glycolytic metabolism, thereby suppressing the requirement of oxidative phosphorylation to maintain the cellular energetic balance. Overall, our study revealed that HNSCC cells with oncogenic HRAS mutations exhibit diminished metformin sensitivity, thus shedding light on a potential mechanism of treatment resistance. This finding may also help explain the limited clinical responses to metformin in cancers with RAS mutations. Ultimately, our study underscores the importance of understanding the impact of the genetic landscape in tailoring precision cancer preventive approaches in the context of HNSCC and other cancers that are characterized by the presence of a defined premalignant state and, therefore, amenable for cancer interception strategies.

Type I Interferon Drives a Cellular State Inert to TCR-Stimulation and Could Impede Effective T-Cell Differentiation in Cancer.

Head and neck squamous cell carcinoma (HNSCC) is linked to human papillomavirus (HPV) infection. HPV-positive and HPV-negative HNSCC exhibit distinct molecular and clinical characteristics. Although checkpoint inhibitors have shown efficiency in recurrent/metastatic HNSCC, response variability persists regardless of HPV status. This study aimed to explore the CD8+ T-cell landscape in HPV-negative HNSCC. We performed simultaneous single-cell RNA and TCR sequencing of CD8+ tumor-infiltrating lymphocytes (TILs) from treatment-naïve HPV-negative HNSCC patients. Additionally, cells were stimulated ex vivo, which allowed for the tracking of clonal transcriptomic responses. Our analysis identified a subset of CD8+ TILs highly enriched for interferon-stimulated genes (ISG). TCR analysis revealed ISG cells are clonally related to a population of granzyme K (GZMK)-expressing cells. However, unlike GZMK cells, which exhibited rapid effector-like phenotypes following stimulation, ISG cells were transcriptionally inert. Additionally, ISG cells showed specific enrichment within tumor and were found across multiple tumor entities. ISG-enriched CD8+ TILs are a consistent feature of various tumor entities. These cells are poorly understood but possess characteristics that may impact antitumor immunity. Understanding the unique properties and functionality of ISG cells could offer innovative treatment approaches to improve patient outcomes in HPV-negative HNSCC and other cancer types.

Erlotinib and curcumin-loaded nanoparticles embedded in thermosensitive chitosan hydrogels for enhanced treatment of head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) remain a major oncological challenge with significant morbidity and mortality rates. Erlotinib (Er) and Curcumin (Cm) are potential therapeutic agents for HNSCC, yet they are hindered by poor solubility and bioavailability. This study explored the optimization of poly(lactic-co-glycolic acid) nanoparticles co-loaded with Er and Cm (Er/Cm-NP), prepared via a D-optimal response surface design-guided nanoprecipitation process. The optimized formulation, optEr/Cm-NP, was then incorporated into chitosan/β-glycerophosphate hydrogels (optEr/Cm-NP-HG) to create an injectable intratumoral (IT) nanocomposite hydrogel (HG) delivery system. Physicochemical properties of the formulations, including gelation time, injectability, mechanical strength and drug release profiles were assessed alongside hemolytic activity. Compared to optEr/Cm-NP alone, the NP-loaded HG formulation exhibited a more pronounced modulation effect, enabling sustained and controlled drug release. The cytotoxicity of the developed formulations was evaluated using the FaDu HNSCC cancer cell line. Both optEr/Cm-NP and optEr/Cm-NP-HG21 displayed enhanced cytotoxicity compared to free drugs. Confocal laser microscopy and flow cytometry confirmed superior cellular uptake of Er and Cm when delivered via NPs or NP-loaded HG. Furthermore, a significant increase in apoptotic cell death upon treatment with optEr/Cm-NP was observed, highlighting its potential for HNSCC therapy. In vivo studies conducted on a xenograft HNSCC mouse model revealed the significant capacity of the intratumorally-injected optEr/Cm-NP-HG21 formulation to retard the tumor growth. Conclusively, the results presented herein report the successful development of a nanocomposite HG system incorporating NPs co-loaded with Er and Cm that could be efficiently utilized in the treatment of HNSCC.

Cancer Cell Small Molecule Secretome Induces the Immune Checkpoint NKG2A and Dysfunction of Human CD8+ T Cells.

PD-1 blockade has been approved for head and neck squamous cell carcinoma (HNSCC) patients. However, many HNSCC patients do not respond to this treatment, and other tumor microenvironmental factors may promote resistance to PD-1 blockade. We previously identified increased expression of the inhibitory receptor NKG2A on CD8+ T cells in HNSCC tumors compared with T cells in matching PBMC samples. Mechanisms that promote NKG2A expression and the role of NKG2A on human T cells in the tumor microenvironment, however, are uncertain. In this study, we show that tumor-conditioned media (TCM) of HNSCC cancer cell lines or ascites fluid from colorectal carcinoma patients is sufficient to induce the expression of NKG2A and other inhibitory receptors on activated CD8+ T cells isolated from PBMCs of healthy donors. Boiling or small molecular mass cutoff filtering did not eliminate the effect of TCM, suggesting that a small molecule promotes NKG2A. T cell activation in TCM decreased the basal and maximal mitochondrial respiration to metabolically restrain CD8+ T cells. Functionally, T cell activation in TCM reduced CD8+ T cell cytotoxicity as shown by lower production of cytokines, granzyme B, and perforin. Furthermore, TCM prevented CD8+ T cells from killing cancer cells in response to an anti-CD19/anti-CD3 bispecific T cell engager. Thus, a small secreted molecule from HNSCC cells can induce NKG2A expression and promote T cell dysfunction. Our findings may lead to targets for novel cancer therapies or biomarkers for NKG2A blockade response and provide a model to study T cell dysfunction and impaired metabolism.

Abstract 6882: Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types

Abstract Background: The aryl hydrocarbon receptor (AhR) is a transcription factor that performs various biological functions upon ligand activation. While there have been extensive studies and development of AhR-targeting anticancer therapies, including active clinical trials, research on the expression of AhR, a key biomarker, in the TME remain limited. Herein, we evaluated the nuclear and cytosolic AhR expression across five solid cancer types: head and neck squamous cell carcinoma (HNSCC), bladder cancer, colorectal cancer, esophageal cancer, and non-small cell lung cancer (NSCLC). Methods: Multiplexed immunohistochemistry (mIHC) and image cytometry were employed to investigate the AhR expression and distribution in 513 patient samples, of which 292 were from patients with one of five solid cancer types. For quantification of AhR expression levels, FCS files were generated, and the resulting data was analyzed using the R program. Results: AhR expression was predominantly high in cancer cells, followed by T cells and macrophages across all cancer types. All 513 patient samples were categorized into three clusters based on distinct expression and localization patterns of AhR. Cluster 1 exhibited high AhR expression in the cancer cell nucleus, whereas cluster 3 showed the lowest. AhR expression in the stromal macrophage nucleus was the most pronounced in cluster 2. In cluster 1, NSCLC was entirely absent, with esophageal cancer representing the largest proportion. In contrast, cluster 2 was notably different from cluster 1, with a 5% prevalence of NSCLC and a relatively higher proportion of HNSCC and colorectal cancer. The AhR expression in macrophages within cluster 2 was noticeably higher than in cluster 1. Cluster 3, which had the highest NSCLC proportion, generally displayed low AhR expression compared to other clusters, but also exhibited AhR expression in regulatory T cells (Tregs), a pattern which was not observed in other cancer types. Notably, a positive correlation was observed between the nuclear and cytosolic expressions of AhR, suggesting that AhR expression as a biomarker is independent of its subcellular localization. Furthermore, there was no clear correlation between AhR expression levels and cancer stage, grade, and metastatic status. Conclusion: We elucidated the expression profile of AhR within the TME across five cancer types, then classified the patient samples into three clusters based on their distinct AhR expression patterns. By demonstrating different expression patterns of AhR in cancer cells and immune cells, our findings are anticipated to provide a fundamental basis for clinical and immunological research on AhR-targeting therapies. Citation Format: Dong Kwon Kim, Chai Young Lee, Yu Jin Han, So Young Park, Heekyung Han, Kwangmin Na, Mi Hyun Kim, Seung Min Yang, Sujeong Baek, Youngtaek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Min Hee Hong, Sun Min Lim, Jii Bum Lee, Jae Hwan Kim, Kyoung-Ho Pyo, Byoung Chul Cho. Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types [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 6882.

Cytotoxic response of tumor-infiltrating lymphocytes of head and neck cancer slice cultures under mitochondrial dysfunction.

Head and neck squamous cell carcinomas (HNSCC) are highly heterogeneous tumors. In the harsh tumor microenvironment (TME), metabolic reprogramming and mitochondrial dysfunction may lead to immunosuppressive phenotypes. Aerobic glycolysis is needed for the activation of cytotoxic T-cells and the absence of glucose may hamper the full effector functions of cytotoxic T-cells. To test the effect of mitochondrial dysfunction on cytotoxic T cell function, slice cultures (SC) of HNSCC cancer were cultivated under different metabolic conditions. Tumor samples from 21 patients with HNSCC were collected, from which, SC were established and cultivated under six different conditions. These conditions included high glucose, T cell stimulation, and temporarily induced mitochondrial dysfunction (MitoDys) using FCCP and oligomycin A with or without additional T cell stimulation, high glucose and finally, a control medium. Over three days of cultivation, sequential T cell stimulation and MitoDys treatments were performed. Supernatant was collected, and SC were fixed and embedded. Granzyme B was measured in the supernatant and in the SC via immunohistochemistry (IHC). Staining of PD1, CD8/Ki67, and cleaved-caspase-3 (CC3) were performed in SC. Hematoxylin eosin stains showed that overall SC quality remained stable over 3 days of cultivation. T cell stimulation, both alone and combined with MitoDys, led to significantly increased granzyme levels in SC and in supernatant. Apoptosis following T cell stimulation was observed in tumor and stroma. Mitochondrial dysfunction alone increased apoptosis in tumor cell aggregates. High glucose concentration alone had no impact on T cell activity and apoptosis. Apoptosis rates were significantly lower under conditions with high glucose and MitoDys (p=0.03). Stimulation of tumor-infiltrating lymphocytes in SC was feasible, which led to increased apoptosis in tumor cells. Induced mitochondrial dysfunction did not play a significant role in the activation and function of TILs in SC of HNSCC. Moreover, high glucose concentration did not promote cytotoxic T cell activity in HNSCC SC.

Prognostic significances of systemic inflammatory response markers in patients with synchronous esophageal and head and neck cancers.

Patients with head and neck squamous cell carcinoma (HNSCC) frequently develop synchronous esophageal cancer (ESCC), but there is a lack of clinical predictors. The neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR), and lymphocyte to monocyte ratios (LMRs), reflect the balance between pro-cancer inflammation and anti-cancer immune responses, but their role in HNSCC and synchronous cancer remain uncertain. The study consecutively enrolled a total of 717 patients with newly diagnosed HNSCC who received pre-treatment esophageal endoscopic screening. The pretreatment NLR, LMR and PLRs were calculated and analyzed in comparison with the clinical factors. A total of 103 patients (14.4%) were found to have synchronous ESCCs, and were associated with a significantly lower absolute lymphocyte count (p < 0.001), higher NLRs (p = 0.044) and lower LMRs (p = 0.001), but not PLRs (p = 0.49). The ROC curve for the presence of synchronous ESCC verified the optimal cutoff value as 2.5 for NLRs and 4.0 for LMRs. Multivariable logistic regression revealed that a LMR <4 (OR 2.22; 95% CI 1.27-3.88, p = 0.005), alcohol consumption (OR 4.19; 95% CI 1.47-11.91, p = 0.007), tumor location over the pharynx (OR 1.68; 95% CI 1.07-2.64, p = 0.025), and low body mass index (OR 0.94; 95% CI 0.88-0.99, p = 0.039) were risk factors for developing synchronous ESCC. A low-LMR was significantly associated with decreases in overall survival (p < 0.0001), in both synchronous and non-synchronous groups. Multivariate analysis demonstrated that LMR <4 (HR 1.97; 95% CI 1.38-2.81, p < 0.001), a low-BMI (HR 0.96; 95% CI 0.93-0.99, p = 0.044) and presence of synchronous ESCC (HR 1.56; 95% CI 1.10-2.22, p = 0.013) were independent prognostic factors for HNSCC patients. Incorporation of LMR into other identified risk factors, such as alcohol consumption, tumor location over pharynx, and low-BMI, may establish a more efficient screening program for esophageal exploration in HNSCC patients. The significances of LMR also suggest that anti-cancer immunity may play a role in the filed cancerization to initiate multiple cancers, and the immunotherapy may have potentials for prevention or as an adjuvant treatment for synchronous SCC in the future.

Diallyl Trisulfide Induces ROS-Mediated Mitotic Arrest and Apoptosis and Inhibits HNSCC Tumor Growth and Cancer Stemness.

Despite advances in therapeutic approaches, the five-year survival rate for head and neck squamous cell carcinoma (HNSCC) patients is still less than fifty percent. Research has indicated that the consumption of Allium vegetables or processed garlic containing diallyl trisulfide (DATS) can lower the risk of multiple types of cancer. Nevertheless, the effectiveness and underlying mechanisms of DATS against HNSCC have not been thoroughly explored until the current study. In this research, it was found that DATS notably curtailed the growth and viability of HNSCC cells. Additionally, DATS triggered a significant G2/M cell cycle arrest in these cells, accumulating cyclin B1, Cip1/p21, and Ser-10 phospho-histone H3-this was indicative of mitotic arrest attenuated by NAC pretreatment, suggesting the role of reactive oxygen species (ROS) induction. The production of ROS induced by DATS led to DNA damage and apoptosis, a process associated with elevated levels of cleaved caspase-3 and cleaved PARP, along with reduced XIAP. When HNSCC cells were exposed to pharmacological concentrations of DATS, it resulted in the suppression of cancer stem cell (CSC) populations, as indicated by a decrease in the CD133high/CD44high cell fraction, reduced aldehyde dehydrogenase 1 (ALDH1) activity, inhibited spheroid formation and downregulated SOX2 and Oct4 expression. Furthermore, the administration of DATS to tumor xenografts demonstrated its in vivo capacity to hinder CSCs. Further, DATS treatment inhibited the growth of UMSCC-22B head and neck cancer tumor xenograft in immunocompromised mice. Overall, DATS inhibited cell proliferation; induced cell cycle mitotic arrest and apoptosis involving DNA damage through ROS generation; reduced the CSC fraction and spheroid formation; and downregulated SOX2 and Oct4 expression. More importantly, DATS inhibited HNSCC tumor growth and CSC fraction in vivo. Thus, DATS could be a potential anticancer agent that can be used against head and neck cancer.

Pharmacological inhibition of MYC to mitigate chemoresistance in preclinical models of squamous cell carcinoma.

Rationale: Cisplatin-based chemotherapy is the first-line treatment for late-stage head and neck squamous cell carcinoma (HNSCC). However, resistance to cisplatin has become a major obstacle for effective therapy. Cancer stem cells (CSCs) are critical for tumor initiation, growth, metastasis, and chemoresistance. How to effectively eliminate CSCs and overcome chemoresistance remains a key challenge. Herein, we confirmed that MYC plays critical roles in chemoresistance, and explored targeting MYC to overcome cisplatin resistance in preclinical models. Methods: The roles of MYC in HNSCC cisplatin resistance and cancer stemness were tested in vitro and in vivo. The combined therapeutic efficiency of MYC targeting using the small molecule MYC inhibitor MYCi975 and cisplatin was assessed in a 4‑nitroquinoline 1-oxide-induced model and in a patient-derived xenograft model. Results: MYC was highly-expressed in cisplatin-resistant HNSCC. Targeting MYC using MYCi975 eliminated CSCs, prevented metastasis, and overcame cisplatin resistance. MYCi975 also induced tumor cell-intrinsic immune responses, and promoted CD8+ T cell infiltration. Mechanistically, MYCi975 induced the DNA damage response and activated the cGAS-STING-IRF3 signaling pathway to increase CD8+ T cell-recruiting chemokines. Conclusions: Our findings suggested that targeting MYC might eliminate CSCs, prevent metastasis, and activate antitumor immunity to overcome cisplatin resistance in HNSCC.

Efficacy of natural killer cell therapy combined with chemoradiotherapy in murine models of head and neck squamous cell carcinoma

Background aimsNatural killer (NK) cell-based cancer immunotherapy is effective when combined with other treatment modalities such as irradiation and chemotherapy. NK cell's antitumor function to treat solid tumor, including head and neck squamous cell carcinoma (HNSCC), has been targeted recently. This study assessed NK cell recruitment in response to chemoradiation therapy (CRT) in HNSCC. MethodsEx vivo expansion of NK cell, flow cytometry, cell viability assay, cytotoxicity assay, immunohistochemistry, and animal model were performed. ResultsMouse NK cells were recruited to the tumor site by CRT in a nude mouse model. Furthermore, expanded and activated human NK cells (eNKs) were recruited to the tumor site in response to CRT, and CRT enhanced the anti-tumor activity of eNK in an NOD/SCID IL-2Rγnull mouse model. Various HNSCC cancer cell lines exhibited different NK cell ligand activation patterns in response to CRT that correlated with NK cell-mediated cytotoxicity. ConclusionsIdentifying the activation patterns of NK cell ligands during CRT might improve patient selection for adjuvant NK cell immunotherapy combined with CRT. This is the first study to investigate the NK cell's antitumor function and recruitment with CRT in HNSCC mouse model.

SOX2-OT Binds with ILF3 to Promote Head and Neck Cancer Progression by Modulating Crosstalk between STAT3 and TGF-β Signaling.

Long non-coding RNA (lncRNA) is involved in the progression of head and neck squamous cell carcinoma (HNSCC). The molecular mechanism of lncRNA SOX2-OT in HNSCC remains unclear. Therefore, we aimed to elucidate the oncogenic role of SOX2-OT in HNSCC. QRT-PCR analysis was performed in 61 pairs of HNSCC cancer tissues, adjacent normal tissues, and 68 plasma samples confirmed that lncRNA SOX2-OT was overexpressed in cancer tissues and plasma samples, which served as a poor prognostic factor for HNSCC. The FISH assay demonstrated that SOX2-OT was localized in the nucleus and cytoplasm of HNSCC cell lines. Further, the cell function assay confirmed that SOX2-OT promoted cell proliferation and metastasis in vitro and in vivo. RNA pulldown and RIP assay results revealed that SOX2-OT bonds with ILF3 in HNSCC, and the rescue assay confirmed that SOX2-OT played an oncogenic role depending on ILF3 protein expression. Ingenuity pathway analysis and Western blotting indicated that SOX2-OT regulated HNSCC progression by promoting STAT3 phosphorylation and modulating the crosstalk between STAT3 and TGF-β signaling. These results reveal evidence for the role of SOX2-OT in HNSCC progression and metastasis by binding to ILF3, which may serve as a therapeutic target and prognostic biomarker in HNSCC.

Abstract PR011: Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers

Abstract Somatic gain of function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor or loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1) E3 ligase, which regulates NRF2 protein levels, are frequently identified in many solid cancers such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), and head and neck squamous cell carcinoma (HNSCC). These genomic alterations drive the activation of cytoprotective NRF2 transcriptional programs. In addition to mutational activation, NRF2 pathway activation has been documented with high frequency in patients without mutations in the pathway. Since tumor cells with hyperactivated NRF2 have been demonstrated to be dependent on this pathway for survival, targeting NRF2 represents an attractive therapeutic approach in tumors with aberrant NRF2 activation. Here, we report the identification of VVD-065, a first-in-class NRF2 inhibitor that acts via an unprecedented mechanism of action. Covalent modification of sensor cysteines on KEAP1 during electrophilic or oxidative stress is known to inhibit KEAP1 mediated NRF2 degradation. In complete contrast, covalent modification of a KEAP1 sensor cysteine by VVD-065 dramatically enhances NRF2 degradation. At low nM concentrations, VVD-065 covalently targets KEAP1 E3 ligase, and allosterically increases the affinity between KEAP1 and CUL3, thereby promoting the formation of active KEAP1-CUL3 E3 ligase complexes and increasing the rate of NRF2 degradation. VVD-065 profoundly reduces NRF2 protein levels in WT KEAP1/NRF2 settings, as well as various KEAP1 and NRF2 mutant settings. Consequently, VVD-065 substantially inhibits NRF2-dependent gene expression and proliferation of NRF2-dependent cell lines. In vivo, oral administration of VVD-065 results in robust degradation of NRF2 and decreased expression of NRF2 target genes. Tumor growth inhibition studies with cell-line derived, and patient derived NSCLC/ESCC xenograft (CDX/PDX) models revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of any overt toxicity. Since constitutive NRF2 activation often contributes to chemotherapeutic resistance, we also conducted TGI studies with 100+ PDX models to assess combination opportunities with chemotherapeutic agents. A marked combination response was achieved with cisplatin and nab-paclitaxel in various solid tumor types such as NSCLC, ESCC, HNSCC, and uterine cancers. Combination with chemotherapy was safe and well-tolerated in these mouse studies. In summary, we have identified VVD-065, a potent and selective KEAP1 activator that promotes the degradation of NRF2. Degradation of NRF2 led to robust monotherapy response in NRF2-activated cancers and also chemo-sensitized chemo-refractory tumors. A related molecule, acting through the same mechanism of action has now entered into Phase I clinical trials. Citation Format: Nil Roy, Tine Wyseure, I-Chung Lo, Jordon Inloes, Aaron Snead, Steffen Bernard, Justine Metzger, Jonathan Pollock, Stephanie Grabow, Christie Eissler, Melaminah Williams, Sarah Jacinto, Gabe Simon, Todd Kinsella, David Weinstein, Matt Patricelli. Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR011.

Emerging Prognostic and Predictive Significance of Stress Keratin 17 in HPV-Associated and Non HPV-Associated Human Cancers: A Scoping Review.

A growing body of literature suggests that the expression of cytokeratin 17 (K17) correlates with inferior clinical outcomes across various cancer types. In this scoping review, we aimed to review and map the available clinical evidence of the prognostic and predictive value of K17 in human cancers. PubMed, Web of Science, Embase (via Scopus), Cochrane Central Register of Controlled Trials, and Google Scholar were searched for studies of K17 expression in human cancers. Eligible studies were peer-reviewed, published in English, presented original data, and directly evaluated the association between K17 and clinical outcomes in human cancers. Of the 1705 studies identified in our search, 58 studies met criteria for inclusion. Studies assessed the prognostic significance (n = 54), predictive significance (n = 2), or both the prognostic and predictive significance (n = 2). Altogether, 11 studies (19.0%) investigated the clinical relevance of K17 in cancers with a known etiologic association to HPV; of those, 8 (13.8%) were focused on head and neck squamous cell carcinoma (HNSCC), and 3 (5.1%) were focused on cervical squamous cell carcinoma (SCC). To date, HNSCC, as well as triple-negative breast cancer (TNBC) and pancreatic cancer, were the most frequently studied cancer types. K17 had prognostic significance in 16/17 investigated cancer types and 43/56 studies. Our analysis suggests that K17 is a negative prognostic factor in the majority of studied cancer types, including HPV-associated types such as HNSCC and cervical cancer (13/17), and a positive prognostic factor in 2/17 studied cancer types (urothelial carcinoma of the upper urinary tract and breast cancer). In three out of four predictive studies, K17 was a negative predictive factor for chemotherapy and immune checkpoint blockade therapy response.

Heparan-6-O-Endosulfatase 2 Promotes Invasiveness of Head and Neck Squamous Carcinoma Cell Lines in Co-Cultures with Cancer-Associated Fibroblasts.

Local invasiveness of head and neck squamous cell carcinoma (HNSCC) is a complex phenomenon supported by interaction of the cancer cells with the tumor microenvironment (TME). We and others have shown that cancer-associated fibroblasts (CAFs) are a component of the TME that can promote local invasion in HNSCC and other cancers. Here we report that the secretory enzyme heparan-6-O-endosulfatase 2 (Sulf-2) directly affects the CAF-supported invasion of the HNSCC cell lines SCC35 and Cal33 into Matrigel. The Sulf-2 knockout (KO) cells differ from their wild type counterparts in their spheroid growth and formation, and the Sulf-2-KO leads to decreased invasion in a spheroid co-culture model with the CAF. Next, we investigated whether a fucosylated chondroitin sulfate isolated from the sea cucumber Holothuria floridana (HfFucCS) affects the activity of the Sulf-2 enzyme. Our results show that HfFucCS not only efficiently inhibits the Sulf-2 enzymatic activity but, like the Sulf-2 knockout, inhibits Matrigel invasion of SCC35 and Cal33 cells co-cultured with primary HNSCC CAF. These findings suggest that the heparan-6-O-endosulfatases regulate local invasion and could be therapeutically targeted with the inhibitory activity of a marine glycosaminoglycan.

Abstract PO-077: High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC

Abstract Cancer metastasis is a complex cascade that involves activation of cancer cell invasion and migration. This activation is greatly attributed by multiple factors, including the tumor-microenvironment (TME). Cancer-associated fibroblasts (CAF), a prominent cell type within the TME, have been shown to promote cancer cell invasion and migration, causing metastasis in various cancers including head and neck squamous cell carcinoma (HNSCC). The molecular mechanisms of how CAFs promote HNSCC invasion remain elusive. Our research goal is to understand how CAFs influence HNSCC cells to become invasive and potentially metastatic. Using a top-down research approach, our aim is to identify novel therapeutic chemical probe/drug regimens that potentially target CAF-dependent HNSCC cancer cell-invasion. In-order to perform high-throughput small molecule screens, we have established a 384-well format, three-dimensional (3D) spheroid invasion assay, as a powerful tool to study CAF-dependent HNSCC cancer cell invasion. This platform is currently being used to screen small molecule libraries and identify putative molecular targets, providing insights into underlying mechanisms of CAF-induced cancer cell invasion and candidate therapeutic strategies. Citation Format: Kunal Karve, Stephanie Poon, Panagiotis Prinos, Laurie Ailles. High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-077.

Multi-Omics Identification of Genetic Alterations in Head and Neck Squamous Cell Carcinoma and Therapeutic Efficacy of HNC018 as a Novel Multi-Target Agent for c-MET/STAT3/AKT Signaling Axis.

Amongst the most prevalent malignancies worldwide, head and neck squamous cell carcinoma (HNSCC) is characterized by high morbidity and mortality. The failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy, demands the need for in-depth understanding of the complex signaling networks involved in the development of treatment resistance. A tumor's invasive growth and high levels of intrinsic or acquired treatment resistance are the primary causes of treatment failure. This may be a result of the presence of HNSCC's cancer stem cells, which are known to have self-renewing capabilities that result in therapeutic resistance. Using bioinformatics methods, we discovered that elevated expressions of MET, STAT3, and AKT were associated with poor overall survival in HNSCC patients. We then evaluated the therapeutic potential of our newly synthesized small molecule HNC018 towards its potential as a novel anticancer drug. Our computer-aided structure characterization and target identification study predicted that HNC018 could target these oncogenic markers implicated in HNSCC. Subsequently, the HNC018 has demonstrated its anti-proliferative and anticancer activities towards the head and neck squamous cell carcinoma cell lines, along with displaying the stronger binding affinities towards the MET, STAT3, and AKT than the standard drug cisplatin. Reduction in the clonogenic and tumor-sphere-forming ability displays HNC018's role in decreasing the tumorigenicity. Importantly, an vivo study has shown a significant delay in tumor growth in HNC018 alone or in combination with cisplatin-treated xenograft mice model. Collectively with our findings, HNC018 highlights the desirable properties of a drug-like candidate and could be considered as a novel small molecule for treating head and neck squamous cell carcinoma.

A comprehensive pan-cancer analysis of PD-L1 expression using clone E1L3N in Chinese patients with cancer.

2656 Background: Immune checkpoint inhibitors (ICIs) benefit patients with multiple cancer types, and the expression of programmed death ligand 1 (PD-L1) protein assessed by immunohistochemistry (IHC) has been correlated with response and survival benefit from anti-PD-1/PD-L1 ICI therapies in several cancer types. IHC assay with E1L3N clone is currently approved by the NMPA for PD-L1 detection in non-small cell lung cancer (NSCLC), but its performance in other cancer types needs to be further validated. Thus, we performed this study to examine the prevalence of PD-L1 expression in a wide variety of tumor types in Chinese pan-cancer patients using the E1L3N antibody clone. Methods: From 2019 to 2022, a total of 13,647 patient across multiple tumor types were analyzed. Tumor tissue samples were subjected to IHC. PD-L1 expression was tested using the E1L3N PD-L1 IHC assay (Amoy Diagnostics, Xiamen, China), and scored with the tumor proportion score (TPS) and combined positive score (CPS). PD-L1 expression status was identified as positive and negative according to TPS or CPS. For NSCLC: negative (TPS &lt; 1%), low expression (TPS 1-49%), and high expression (TPS ≥ 50%). For gastric/gastroesophageal junction carcinoma (GC/GJC): negative (CPS &lt; 1), low expression (CPS 1-5), and high expression (CPS ≥ 5). For other cancer: negative (CPS &lt; 1), low expression (CPS 1-10), and high expression (CPS ≥ 10). Results: In total, 14 cancer types were enrolled in the present study, including 68.2% NSCLC, 6.9% colorectal carcinoma (CRC), and 3.8% GC/GJC, and the remaining 21.1% include esophageal cancer (EC), breast cancer, cervical cancer (CC), biliary tract cancer (BTC), pancreatic cancer (PC), ovarian cancer, renal cancer, small cell lung cancer, urothelial carcinoma (UC), bladder cancer, head and neck squamous cell carcinoma (HNSCC), etc. PD-L1 positivity by TPS was observed in 52.5% NSCLC patients, similar to the frequency of PD-L1 expression in Chinese NSCLC patients detected using 22C3 clone. 76.0% and 36.7% of GC/GJC patients with PD-L1-positive tumors at a CPS cutoff of ≥ 1 and ≥ 5, respectively. PD-L1 positive expression frequency was higher than that in western GC/GJC patients detected using 22C3 and 28-8 clone (as reported from previous research: PD-L1 positivity with CPS ≥ 1 was 45.5% using the 22C3 assay and 49.1% using the 28-8 assay). 73.6% and 17.5% of CRC patients with PD-L1-positive tumors at a CPS cutoff of ≥ 1 and ≥ 10, respectively. In addition, 8 tumors (EC, CC, breast cancer, PC, BTC, UC, HNSCC, and bladder cancer) had the prevalence of PD-L1 CPS ≥ 1 greater than 50%. Conclusions: E1L3N can be used as a reliable alternative antibody clone to evaluate PD-L1 expression status not only in NSCLC patients but also in other cancer types.

Abstract 1563: Concomitant inhibition of Aurora kinase A and WEE1 kinases results in synergistic tumor control and heightens DNA replication stress in head and neck and lung carcinomas

Abstract Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) and lung cancer mainly harbor disruptive mutations in TP53 and/or CDKN2A tumor suppressor genes, which are associated with refractoriness to treatment as well as poor prognosis and survival. Mutational loss of TP53 function fosters elevated expression of Aurora kinase A (AURKA) that plays a crucial role in mitotic progression at G2/M and stabilizes DNA replication forks at S phase. We have previously demonstrated that combined inhibition of AURKA and WEE1, a G2/M checkpoint kinase induced by replication stress, results in synergistic anti-tumor effects in HNSCC in vitro and in vivo. We have now further confirmed synergy using the highly selective AURKA inhibitor VIC1911 in HNSCC and lung cancers harboring TP53 mutations and explored mechanisms of cell death. VIC1911 and adavosertib combination synergistically suppressed cell growth and survival in both 2D- and 3D-culture systems relative to vehicle or single-agent treatment in TP53-mutated HNSCC FaDu and CAL27, and lung cancer A549 and NCI-H358 cells, with no observable toxicity in normal cells, predicting a favorable therapeutic index. Furthermore, combination treatment accelerated mitotic entry and resulted in accumulation of mitotic catastrophe, as demonstrated by time-lapse imaging, and apoptotic cell death as evidenced by cleaved PARP assays. Notably, we found that combination VIC1911 and adavosertib in HNSCC cells drastically slowed and stalled progression of replication forks in DNA fiber analysis (Mean: DMSO=3.117; adavosertib=1.495; VIC1911=0.9757; combination=0.5548 kb/min; P &amp;lt;0.0001) and amplified DNA damage as measured by γH2AX, indicating induction of severe replication stress. In vivo, this combination resulted in significant tumor regression in both HNSCC and lung adenocarcinoma patient-derived and cancer cell-derived xenografted mice compared with either vehicle or single-agent treatment. Taken together, these results suggest that AURKA inhibition increases dependency on WEE1 by enhancing replication stress and mitotic catastrophe, and support clinical evaluation of combined AURKA and WEE1 inhibition as a novel and effective treatment for HNSCC and lung cancer patients with elevated AURKA expression. Citation Format: Jong Woo Lee, Sundong Kim, Sebastian Cruz-Gomez, Jackie Shi, Cindy Yang, Barbara Burtness. Concomitant inhibition of Aurora kinase A and WEE1 kinases results in synergistic tumor control and heightens DNA replication stress in head and neck and lung carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1563.

Abstract 384: Secondhand smoke exposure alters cisplatin-induced cell death and alters cell death and survival pathway in HNSCC

Abstract BACKGROUND: Secondhand smoke (SHS) contains more than 7000 chemicals, 70of which are carcinogens and it is estimated that every one in four Americans areinvoluntarily exposed to SHS. Carcinogenesis induced by SHS exposure isunclear, though SHS exposure is associated with various cancers including headand neck squamous cell carcinoma (HNSCC). Recently, it has been shown thatSHS exposure is a significant independent predictor of HNSCC recurrence. Wehave shown that in vitro exposure to SHS increases HNSCC cisplatin resistance. AIMS: Our study examines the effect of SHS smoke exposure on cisplatin-induced HNSCC cancer cell death and the possible mechanisms behind thisobservation. METHODS: Sidestream smoke (SS), the main component of SHS,was extracted as previously described. Authenticated HNSCC cell lines (UM-SCC1 &amp; WSU-HN6) were exposed to SS extract for 48 hours at doses mimickingthe nicotine levels observed in the saliva of passive smokers. Then, cancer cellswere treated with cisplatin in the presence or absence of SS extract. Cisplatin-induced cancer cell death was measured using Incucyte® S3 Live-Cell imaginganalysis. Transcriptomic analysis following RNA-sequencing was performedusing Ingenuity Pathway Analysis. Data were subjected to ANOVA. RESULTS: Our live-cell imaging system revealed that HNSCC cells exposed to SS extractsignificantly decreased cisplatin-induced cancer cell death at various time points compared with cells treated with cisplatin alone in both cell lines. The HNSCCcells treated only with SS extract did not show any change in cell death at 0, 12,24, 48, 72, and 96 hours. RNA-sequencing data revealed that cell death andsurvival pathways are the most changed pathways in the SS extract-treatedgroup compared with unexposed HNSCC cells. CONCLUSIONS: Our studydocuments for the first time that even short-term exposure to SHS allows cells toevade cisplatin-induced cell death and can lead to head and neck cancercisplatin resistance. At the molecular level, SHS exposure alters genes that areassociated with cell death and survival. Further, HNSCC patients-basedobservations and mechanistic studies are warranted. FUNDING: This work was partially supported by the National Cancer Institute of the NationalInstitutes of Health (R33CA202898, R01CA242168, and P30CA225520), theTSET Health Promotion Cancer Center, and the Oklahoma Center forAdvancement of Science and Technology (HR16-007). Dr. Queimado holds aPresbyterian Health Foundation Endowed Chair in Otorhinolaryngology Position. Citation Format: Balaji Sadhasivam, Jimmy Manyanga, Vengatesh Ganapathy, Célia Bouharati, David Rubenstein, Jonathan Wren, Pawan Acharya, Daniel Zhao, Lurdes Queimado. Secondhand smoke exposure alters cisplatin-induced cell death and alters cell death and survival pathway in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 384.

Amphipathic barbiturates as marine product mimics with cytolytic and immunogenic effects on head and neck squamous cell carcinoma cell lines.

The incidence of head and neck squamous cell carcinoma (HNSCC) is increasing and the conventional treatments for this form of cancer can be tough. Despite the success of existing immunotherapies in some HNSCC patients, many do not respond to this type of treatment. Thus, the development of novel anti-cancer therapies should be prioritized. In the current study, the anticancer activity of a panel of novel compounds, herein termed marine product mimics (MPMs), against HNSCC cell lines is explored. The previously reported compound MPM-1, which is structurally related to the novel MPMs, was shown to have promising effects on the HNSCC cell line HSC-3. The results from the current study indicate that the novel MPMs are more potent than MPM-1 but cause a similar type of cell death. The results indicated that the MPMs must cross through the cell membrane to exert their action and that they are lysosomotropic. Further experiments showed that some of the MPMs could induce phosphorylation of eukaryotic initiation factor 2α (eIF2α) in HSC-3 and UT-SCC-24A cells, which indicates that they can activate the integrated stress response that is strongly associated with immunogenic cell death. Cell surface expression of calreticulin and release of HMGB1 and ATP, which are all hallmarks of immunogenic cell death, was also demonstrated in HSC-3 and UT-SCC-24A cells treated with MPMs. This suggests that the MPMs are interesting candidates for future HNSCC cancer therapies.