Human Head And Neck Squamous Cell Carcinoma Cell Lines Research Articles

Tobacco smoke exposure is a driver of altered oxidative stress response and immunity in head and neck cancer.

Exposomes are critical drivers of carcinogenesis. However, how they modulate tumor behavior remains unclear. Extensive clinical data link cigarette smoke as a key exposome that promotes aggressive tumors, higher rates of metastasis, reduced response to chemoradiotherapy, and suppressed anti-tumor immunity. We sought to determine whether smoke itself can modulate aggressive tumor behavior in head and neck squamous cell carcinoma (HNSCC) through reprogramming the cellular reductive state. Using established human and murine HNSCC cell lines and syngeneic mouse models, we utilized conventional western blotting, steady state and flux metabolomics, RNA sequencing, quantitative proteomics and flow cytometry to analyze the impact of smoke exposure on HNSCC tumor biology. Cigarette smoke persistently activated Nrf2 target genes essential for maintenance of the cellular reductive state and survival under conditions of increased oxidative stress in HNSCC regardless of HPV status. In contrast to e-cigarette vapor, conventional cigarette smoke mobilizes cellular metabolism toward oxidative stress adaptation, resulting in development of cross-resistance to cisplatin. In parallel, smoke exposure modulates both expression of PDL1 and the secretory phenotype of HNSCC cells through activation of NF-κB resulting in an altered tumor immune microenvironment (TIME) in syngeneic mouse models and altered PBMC differentiation that includes downregulated expression of antigen presentation and costimulatory genes in myeloid cells. Cigarette smoke exposome is a potent activator of the Nrf2 pathway and is a likely primary trigger for the tripartite phenotype of aggressive HNSCC consisting of: 1) reduced chemotherapy sensitivity, 2) enhanced metastatic potential and 3) suppressed anti-tumor immunity. The smoke exposome drives aggressive tumor behavior, treatment resistance and suppressed immunity through coordinated metabolic reprogramming. Successfully targeting this adaptation is critical to improving survival in smokers with head and neck cancer.

Cisplatin promotes TNF-α autocrine to trigger RIP1/RIP3/MLKL-dependent necroptosis of human head and neck squamous cell carcinoma cells

To investigate whether cisplatin induces tumor necrosis factor-α (TNF-α) secretion in human head and neck squamous cell carcinoma (HNSCC) cells to trigger RIP1/RIP3/MLKL-dependent necroptosis of the cells. HNSCC cell lines HN4 and SCC4 treated with cisplatin (CDDP) or the combined treatment with CDDP and z-VAD-fmk (a caspase inhibitor) or Nec-1 (a necroptosis inhibitor) for 24 h were examined for changes in cell viability using CCK8 assay and expressions of caspase-8 and necroptosis pathway proteins (RIP1/RIP3/MLKL) using Western blotting. The changes in migration of the cells were assessed with cell scratch assay, and the expressions of epithelial-mesenchymal transition (EMT) marker proteins N-cadherin, vimentin, and E-cadherin as well as the expressions of NF-κB (p65) and TNF-α were detected with Western blotting. The IC50 of cisplatin was 10 μg/mL in HN4 cells and 15 μg/mL in SCC4 cells. Cisplatin treatment significantly decreased the expressions of caspase-8, N-cadherin and vimentin and increased the expressions of Ecadherin, the necroptosis pathway proteins (RIP1/RIP3/MLKL), TNF-α, and NF-κB (p65), and these changes were obviously inhibited by treatment with Nec-1. Cisplatin stimulation also significantly lowered migration of the cells, and this inhibitory effect was strongly attenuated by Nec-1 treatment. Cisplatin activates nuclear factor-κB signaling in HNSCCs to promote TNF-α autocrine and induce RIP1/RIP3/MLKL-dependent necroptosis, thus leading to inhibition of cell proliferation.

Effects of α‑solanine on human head and neck squamous cell carcinoma cells and human umbilical vein endothelial cells in vitro.

α-solanine is a glycoalkaloid that is commonly found in nightshades (Solanum) and has a toxic effect on the human organism. Among other things, it is already known to inhibit tumor cell proliferation and induce apoptosis in tumor cell lines. Due to its potential as a tumor therapeutic, the current study investigated the effect of α-solanine on head and neck squamous cell carcinoma (HNSCC). In addition, genotoxic and antiangiogenic effects on human umbilical vein endothelial cells (HUVECs) were evaluated at subtoxic α-solanine concentrations. Cytotoxicity and apoptosis rates were measured in two human HNSCC cell lines (FaDu pharynx carcinoma cells and CAL-33 tongue carcinoma cells), as well as in HUVECs. MTT and Annexin V analyses were performed 24 h after α-solanine treatment at increasing doses up to 30 µM to determine cytotoxic concentrations. Furthermore, genotoxicity at subtoxic concentrations of 1, 2, 4 and 6 µM in HUVECs was analyzed using single-cell gel electrophoresis (comet assay). The antiangiogenic effect on HUVECs was evaluated in the capillary tube formation assay. The MTT assay indicated an induction of concentration-dependent viability loss in FaDu and CAL-33 cancer cell lines, whereas the Annexin V test revealed α-solanine-induced cell death predominantly independent from apoptosis. In HUVECs, the cytotoxic effect occurred at lower concentrations. No genotoxicity or inhibition of angiogenesis were detected at subtoxic doses in HUVECs. In summary, α-solanine had a cytotoxic effect on both malignant and non-malignant cells, but this was only observed at higher concentrations in malignant cells. In contrast to existing data in the literature, tumor cell apoptosis was less evident than necrosis. The lack of genotoxicity and antiangiogenic effects in the subtoxic range in benign cells are promising, as this is favorable for potential therapeutic applications. In conclusion, however, the cytotoxicity in non-malignant cells remains a severe hindrance for the application of α-solanine as a therapeutic tumor agent in humans.

P4HA2 contributes to head and neck squamous cell carcinoma progression and EMT through PI3K/AKT signaling pathway

Head and neck squamous cell carcinoma (HNSCC) can be defined as a deadly illness with a dismal prognosis in advanced stages. Therefore, we seek to examine P4HA2 expression and effect in HNSCC, along with the underlying mechanisms. This study utilized integrated bioinformatics analyses to evaluate the P4HA2 expression pattern, prognostic implication, and probable function in HNSCC. The study conducted various in vitro experiments, including colony formation, CCK-8, flow cytometry, wound healing, and transwell assays, on the human HNSCC cell line CAL-27 to examine the involvement of P4HA2 in HNSCC progression. Moreover, western blotting was used to investigate epithelial-mesenchymal transition (EMT) markers and PI3K/AKT pathway markers to elucidate the underlying mechanisms. P4HA2 expression was significantly enhanced in HNSCC, and its overexpression was correlated to tumor aggressiveness and a poor prognosis in patients. Based on in vitro experiments, the overexpressed P4HA2 enhanced cell proliferation, migration, invasion, as well as EMT while reducing apoptosis, whereas P4HA2 silencing exhibited the reverse effect. P4HA2 overexpression enhanced PI3K/AKT phosphorylation in HNSCC cells. Moreover, LY294002 was observed to counteract the effects of upregulated P4HA2 on proliferation, migration, invasion, and EMT in HNSCC. Collectively, we indicated that P4HA2 promoted HNSCC progression and EMT via PI3K/AKT signaling pathway.

ELOVL6 promotes the progression of head and neck squamous cell carcinoma via activating WNT/β-catenin pathway.

This study was to explore the role of ELOVL6 in the development of head and neck squamous cell carcinoma (HNSCC). Considering its previously identified oncogenic role in hepatocellular carcinoma. ELOVL6 gene expression, clinicopathological analysis, enrichment analysis, and immune infiltration analysis were based on the data from Gene Expression Omnibus and The Cancer Genome Atlas, with additional bioinformatics analyses performed. Human HNSCC tissue microarray and cell lines were used. The expression of ELOVL6 in HNSCC was detected by quantitative polymerase chain reaction, immunohistochemistry assay, and western blot analysis. The proliferation ability of HNSCC cells, invasion, and apoptosis were evaluated using cell counting kit-8 method, Transwell assay, and flow cytometry, respectively. Based on the data derived from the cancer databases and our HNSCC cell and tissue studies, we found that ELOVL6 was overexpressed in HNSCC. Moreover, ELOVL6 expression level had a positive correlation with clinicopathology of HNSCC. Gene set enrichment analysis showed that ELOVL6 affected the occurrence of HNSCC through WNT signaling pathway. Functional experiments demonstrated that ELOVL6 knockdown inhibited the proliferation and invasion of HNSCC cells while promoting apoptosis. Additionally, compound 3f, an agonist of WNT/β-catenin signaling pathway, enhances the effect of ELOVL6 on the progression of HNSCC cells. ELOVL6 is upregulated in HNSCC and promotes the development of HNSCC cells by inducing WNT/β-catenin signaling pathway. ELOVL6 stands a potential target for the treatment of HNSCC and a prognosis indicator of human HNSCC.

Efficacy of depatuxizumab mafodotin (ABT-414) in preclinical models of head and neck cancer.

Epidermal growth factor receptor (EGFR) is highly expressed in 80-90% of head and neck squamous cell carcinomas (HNSCCs), making it an ideal target for antibody-drug conjugates. Depatuxizumab mafodotin (ABT-414), is an EGFR-targeting ADC comprised of the monoclonal antibody ABT-806 conjugated to monomethyl auristatin F, a tubulin polymerization inhibitor. This study assessed the in vivo efficacy of ABT-414 in HNSCC. The effects of ABT-414 on HNSCCs were determined using in vitro cytotoxicity assays and in vivo flank xenograft mouse models. The distribution of ABT-414 was assessed ex vivo via optical imaging methods using a conjugate of ABT-414 to the near-infrared agent IRDye800. In vitro treatment of high EGFR-expressing human HNSCC cell lines (UMSCC47 and FaDu) with ABT-414 (0-3.38 nM) resulted in dose-dependent cell death (IC50 values of 0.213 nM and 0.167 nM, respectively). ABT-414 treatment of the FaDu mouse xenografts displayed antitumor activity (P = 0.023) without a change in body mass (P = 0.1335), whereas treatment of UMSCC47 did not generate a significant response (P = 0.1761). Fluorescence imaging revealed ABT-414-IRDye800 accumulation in the tumors of both FaDu and UMSCC47 cell lines, with a signal-to-background ratio of >10. ABT-414 treatment yielded antitumor activity in FaDu tumors, but not in UMSCC47, highlighting the potential for ABT-414 efficacy in high EGFR-expressing tumors. Although ABT-414-IRDye800 localized tumors in both cell lines, the differing antitumor responses highlight the need for further investigation into the role of the tumor microenvironment in drug delivery.

Gold Nanoparticles Enhance the Tumor Growth-Suppressing Effects of Cetuximab and Radiotherapy in Head and Neck Cancer In Vitro and In Vivo

Introduction: Head and neck squamous cell carcinoma (HNSCC) treatment includes surgery, radiotherapy, and immunotherapy with the aim of eradicating cancer cells without affecting normal tissues. HNSCC expresses epidermal growth factor receptor (EGFR) and cetuximab, an IgG1 monoclonal antibody targeting epidermal growth factor receptor, has been approved for the treatment of HNSCC. However, cetuximab has low reactivity and induces serious side effects. Gold nanoparticles (AuNPs) were reported to enhance the local antitumor effects of radiotherapy without damaging normal cells. Methods and Results: This study investigated the in vitro effects of single and combination therapy with AuNPs (1.0 nM), cetuximab (30 nM), and radiotherapy (4 Gy) on a human HNSCC cell line, HSC-3. Combination treatment of AuNPs + cetuximab + radiotherapy markedly reduced HSC-3 numbers and proliferation and enhanced apoptosis compared with single and double combination treatments. Furthermore, the in vivo combination treatment (AuNPs + cetuximab + radiotherapy) of a xenograft model of HSC-3 cells transplanted into nude mice (BALB/cAJcl-nu/nu) reduced the tumor volume compared with the controls. Scanning electron microscopy demonstrated the presence of AuNPs in tumor tissues and toxicity analysis indicated that AuNPs had no toxic effect on normal tissues. Conclusions: This study showed that AuNPs alone do not have a tumor-suppressing effect, but they sensitize tumors to radiotherapy and bind to cetuximab, leading to enhanced antitumor effects.

Pre-Clinical Validation of Targeting Cholesterol Metabolism for Radiotherapy Sensitization in Head and Neck Cancer

This study examines the potential benefits of targeting cholesterol metabolism in combination with radiation therapy (RT) for treating Head and Neck Squamous Cell Carcinoma (HNSCC). Despite advancements in RT delivery, 60% of patients still experience a relapse within 2 years, highlighting the need for improved treatment strategies. Our hypothesis is that a specific, biological response to RT by a subset of tumor cells drives radio-resistance and recurrence, and that targeting cholesterol metabolism may be an effective way to improve RT efficacy. To identify radiation-induced biological responses, whole-genome RNA sequencing analysis was carried out on Cal27 (HPV-) and UMSCC47 (HPV+) cell lines after X-Ray (6MeV Linac) or proton (pencil beam scanning, center part of SOBP) radiations. Potential radio-resistance targets were identified by comparing RT-induced transcriptomic changes with publicly available GEO and TCGA transcriptomic data from human HNSCC samples. Cholesterol metabolism as a driver of HNSCC radio-resistance was validated in vitro by pharmacological and genetic approaches. Transcriptomic analysis revealed a significant increase in 11 genes related to sterol and cholesterol in both the Cal27 and UMSCC47 cell lines after radiation exposure (pathway functional enrichment analysis, p = 2.05e-05). High expression of these RT-induced cholesterol gene signatures correlated with decreased overall survival among HNSCC patients based on the TCGA database (log rank p = 0.022, HR = 1.4). Moreover, a Gene Set Enrichment Analysis of the GSE23036 dataset (pre-treatment HNSCC tumors: locoregional recurrence vs. nonrecurrence) also linked cholesterol metabolism and local recurrence (p = 0.002, FDR = 0.07, NES = 1.72). Single-cell RNA sequencing data from the GSE164690 dataset revealed high expression of cholesterol metabolism genes in human HNSCC epithelial and mesenchymal fractions. In vitro studies demonstrated that RT increased the nuclear translocation of the master transcription factors controlling cholesterol gene expression SREBP2 and increased the expression of the SREBP2 target gene SQLE at the protein level. In accordance with this result, RT increased cellular cholesterol content and uptake in human (p = 0.027) and murine (p = 0.015) HNSCC cell lines. Drug or genetic targeting of SREBP2 reduced cellular cholesterol levels in HNSCC cells and increased toxicity when combined with RT in both 2D and 3D cell cultures compared to RT alone. Targeting cholesterol metabolism in combination with RT could be beneficial for treating HNSCC. In vivo validation in immunocompetent mouse models and orthotopic patient-derived xenograft models is ongoing.

Abstract PO-053: Chemical suppression of DHFR induces NRF2 inhibition and tumor regression

Abstract Background: Recurrence in head and neck squamous cell carcinoma (HNSCC) is common due to primary resistance and acquired resistance arising during therapy. Recent studies have highlighted a critical role of NRF2 in radiation (RT) resistance. Constitutive activation of the NRF2 oxidative stress response pathway occurs in 20-30% of primary HNSCC. As a ROS-activated transcription factor that is frequently mutated and active in cancer, NRF2 is central to both intrinsic and acquired determinants of RT resistance. We recently showed that high NRF2 activity is predictive for locoregional failure and poor outcomes following RT in HNSCC. The downstream effectors of NRF2 responsible for RT resistance are poorly defined. Therapeutic strategies to inhibit NRF2 may improve HNSCC patient outcomes, yet remain unproven. Methods: We sought to study NRF2 activation and biology in two model systems: MOC1 syngeneic HNSCC cells and mouse embryonic fibroblasts (MEFs) derived from a NRF2E79Q genetically engineered mouse model. We defined a transcriptional signature associated with an activating NRF2 mutation (NRF2E79Q) and characterized the effect of NRF2 on cell growth and RT response. We also investigated a novel NRF2 inhibitor in the syngeneic mouse MOC1 model. MEFs were isolated from a pregnant mouse, immortalized and selectively infected with 500-800 MOI of Adenovirus Cre-Recombinase for in vitro proliferation and RT assays. Drug studies were performed at 50mM in vitro or 20mg/kg PO daily starting day 7 in vivo. RT was delivered at 0, 4Gy, or 8Gy in vitro or 3Gy daily x 5 days starting day 17 in vivo. Results: RNA isolated from NRF2E79Q MEFs demonstrated activation of numerous oxidative stress pathway transcripts including canonical targets HMOX1, NQO1, BLVRB, and SLC71a11. NRF2E79Q MEFs demonstrated significantly enhanced baseline growth kinetics and RT resistance relative to the NRF2-wildtype line. We recently reported that pyrimethamine—an FDA-approved antiparasitic drug that targets one carbon metabolism— [1.2uM] induced NRF2 ubiquitylation and degradation in vitro and in vivo. Through structure-activity relationship optimization we have produced a novel analogue, [WCDD115; 50nM], with efficacious DHFR suppression and NRF2 inhibition in human and mouse HNSCC cell lines. In vitro, WCDD115 produced growth suppression in the MOC1 cell line which was enhanced in the setting of RT relative to either treatment alone. MOC1-bearing mice treated with WCDD115 or RT as monotherapy had significant reduction in growth kinetics relative to vehicle control. WCDD115 and RT combination therapy produced the greatest effect with significant growth suppression and 25% tumor rejection without weight loss or toxicity. Conclusions: Our study demonstrates that NRF2E79Q mutation is associated with enhanced growth kinetics and RT resistance along with upregulation of oxidative stress pathway targets. Drug targeting with the novel NRF2 inhibitor, WCDD115, has significant tumor suppressive activity in vitro and in vivo and warrants further investigation as a RT-sensitizing agent. Citation Format: Paul Zolkind, Harit Panda, Caroline Krall, Brittany Bowman, Nathan Wamsley, Bernard Weissman, Ben Major. Chemical suppression of DHFR induces NRF2 inhibition and tumor regression [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-053.

Downregulation of ribosomal RNA (rRNA) genes in human head and neck squamous cell carcinoma (HNSCC) cells correlates with rDNA promoter hypermethylation

Eukaryotes carry hundreds of ribosomal RNA (rRNA) genes as tandem arrays, which generate rRNA for protein synthesis. Humans carry ∼ 400 rRNA gene copies and their expression is epigenetically regulated. Dysregulation of rRNA synthesis and ribosome biogenesis are characteristic features of cancers. Targeting aberrant rRNA expression for cancer therapy is being explored. Head and neck squamous cell carcinoma (HNSCC) is among the most prevalent cancers globally. Using quantitative PCR and bisulfite sequencing, we show that rRNA genes are downregulated and their promoters are hypermethylated in HNSCC cell lines. These findings may have relevance for prognosis and diagnosis of HNSCC.

Abstract 377: RON receptor tyrosine kinase in head and neck cancer: a potential target for overcoming resistance to anti-EGFR therapy

Abstract Background: Although the epidermal growth factor receptor (EGFR) is overexpressed in 80-90% of Head and Neck Squamous Cell Carcinomas (HNSCCs), response to anti-EGFR targeted therapy remains low. Trans-activation of other receptor tyrosine kinases is a common mechanism of resistance to EGFR directed therapy. The RON receptor tyrosine kinase (Macrophage stimulating 1-receptor/MST1R) is a member of the c-MET oncogene family. While limited, prior studies by our group and others have reported elevated RON expression in over 60% of primary HNSCC samples and that receptor cross talk exists with RON and EGFR. Based on this information, we hypothesize that the pro-tumorigeneic role of RON and its crosstalk with EGFR contributes to HNSCC growth and to anti-EGFR therapeutic failure. To test this hypothesis, we examined the expression level of RON and EGFR in human and mouse HNSCC cell lines and in HNSCC patient samples from publicly available databases. Further, we investigated the role of RON in radiation resistance as radiation is a primary treatment modality for HNSCC. Results: Immunoblotting demonstrated strong RON expression in eight of nine human HNSCC cell lines tested and strong EGFR expression in six HNSCC cell lines. In exploring mRNA expression of RON using the Kaplan-Meier Plotter HNSCC RNA-seq dataset, high RON expression was associated with poorer overall survival (P = 0.054) in patients with Stage 3 HNSCC and low mutation burden. To assess the relationship between RON and EGFR, mRNA expression was mined in the GSE6631 dataset from the NCBI gene expression omnibus (GEO). Our analysis found RON to be significantly overexpressed in HNSCC samples compared to normal matched controls (P = 0.029). Interestingly, EGFR mRNA expression was negatively correlated (r = -0.2) with RON mRNA expression from the same patients. This inverse correlation between RON and EGFR expression was corroborated by immunoblotting data of HNSCC lines, UM-SCC-090 and 93-UV-147T which demonstrated low levels of EGFR and high levels of RON. As ionizing radiation is a pillar of HNSCC treatment and is often delivered concurrent with anti-EGFR therapy, we next examined the relative radiosensitivity in multiple murine and human cancer cell lines with and without RON expression using colony formation assays. Preliminary studies demonstrated significantly increased colony formation in high RON-expressing cells relative to low RON-expressing cells. Conclusion: RON is expressed at high levels in multiple HNSCC cell lines and in human samples. Protein expression data and in silica analyses support an inverse association between EGFR and RON. RON expression is associated with radiation resistance. Future studies will investigate interactions between RON and EGFR, and whether RON inhibition can increase anti-EGFR treatment and radiation sensitivity in HNSCC cells. Citation Format: Zhixin Lu, Vinita Takiar, Susan E. Waltz. RON receptor tyrosine kinase in head and neck cancer: a potential target for overcoming resistance to anti-EGFR therapy [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 377.

Abstract 2832: Plk1 signaling as a therapeutic target for HPV- head and neck cancer

Abstract Current frontline chemotherapy and radiotherapy for head and neck cancer (HNC) is insufficient as ~60% of patients relapse within two years, indicating a need to identify novel targeted therapies to improve survival outcomes for HNC patients. Human papilloma virus (HPV) incidence is a major cause of head and neck squamous cell carcinoma (HNSCC) in addition to tobacco use. HPV- HNSCC patients are less responsive to frontline treatment than HPV+ HNSCC patients. However, HPV- and HPV+ HNSCC patients are treated similarly. The cell cycle serine-threonine kinase polo-like kinase 1 (Plk1) is known to be overexpressed in HNC. The Cancer Genome Atlas (TCGA) revealed that high expression of Plk1 correlates with worse survival in HNSCC patients (p<0.05). Yet, Plk1 is expressed at similar levels when comparing HPV- and HPV+ HNSCC patients. Therefore, in this study we sought to establish if Plk1 inhibition with onvansertib (currently in Phase I/II clinical trials) alone and in combination with radiation would inhibit HPV- and HPV+ HNSCC growth. We used two HPV+ (UMSCC47, UDSCC2), and two HPV- (HN5, Cal27) human HNSCC cell lines. First, we confirmed that Plk1 inhibition with onvansertib stalls HNSCC cells at the G2/M phase of the cell cycle (n=3). Then, we determined that HNSCC cell viability is reduced in a dose-dependent manner after Plk1 inhibition with onvansertib. Surprisingly, HPV- HNSCC cells exhibited a 2-fold decrease in cell viability as compared to HPV+ HNSCC cells (n=3). These findings were confirmed when assessing 3D spheroid growth of HNSCC cells. Plk1 inhibition with onvansertib (25nM) reduced HPV- HNSCC spheroid growth more than HPV+ (n=2). Combining Plk1 inhibition with radiation also reduced colony formation (n=3, p<0.0001) and increased G2/M stalling (n=3) compared to either single agent in HPV- HNSCC cells. Functionally, onvansertib induces cleaved PARP and pPlk1 expression in HNSCC cells. However, pPlk1 is induced to a higher degree in HPV+ HNSCC cells potentially indicating a positive feedforward loop leading to decreased sensitivity to Plk1 inhibition. In HPV- but not HPV+ HNSCC cells, Plk1 expression is also increased after radiation emphasizing Plk1 as an attractive target to mitigate radioresistance in HPV- HNSCCs (n=3). Our findings that HPV+ HNSCC cells are more resistant to Plk1 inhibition than HPV- HNSCC cells provides novel mechanistic insight into these disease subtypes. As HPV- HNSCC is typically more resistant to standard therapies, these results support a novel combinatorial approach using an already approved Plk1 inhibitor with radiation to improve HPV- HNSCC patient outcomes. Citation Format: Julianna Korns, Samuel Thompson, Trisha Wise-Draper, Vinita Takiar. Plk1 signaling as a therapeutic target for HPV- head and neck cancer [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 2832.

Abstract 3601: Tumor MK2 signaling regulates cell migration and invasion in head and neck squamous cell carcinoma

Abstract Poor 5-year overall survival in head and neck cancer (HNC) can be attributed to high rates of locoregional and distant metastasis. We have previously demonstrated that high phosphorylation/activation of MAPK-activated protein kinase 2 (MK2), a stress-activated kinase directly downstream of p38 MAPK, in head and neck squamous cell carcinoma (HNSCC) is associated with worse overall survival and inhibition of the MK2 pathway enhances in vivo HNSCC radiosensitivity. Our prior work also demonstrates that radiotherapy-induced epithelial-to-mesenchymal transition (EMT) gene expression can be suppressed in cells when MK2 is inhibited. Several studies have shown that EMT plays a prominent role in HNSCC tumor invasion, treatment resistance, and locoregional metastases to lymph nodes. In this study, we investigate the impact of MK2 activity on HNSCC tumor progression by examining its contribution to cancer cell morphology. We used lentiviral-shRNA and the CRISPR-Cas9 system to knock down/out the MK2 gene in multiple human and murine HNSCC cell lines. We measured tumor cell migration and invasion in both classic 2D in vitro culture as well as with 3D spheroids. We performed unbiased analysis via RNAseq in human HNSCC cell lines comparing MK2 shRNA vs scramble cell lines. Substantial gene and pathway alterations were examined using a combination of immunoblot and RT-qPCR. Our data shows knocking down/out MK2 in HNSCC cells significantly diminishes cell migration and invasion in 2D and 3D in vitro culture assays. RNAseq analysis demonstrated loss of MK2 leads to a significant increase in many cell adhesion molecules, including E-cadherin, and a decrease in N-cadherin, suggesting a shift from mesenchymal to more epithelial phenotype. Furthermore, we note expression of multiple matrix metalloproteinases and EMT markers (i.e., Snail) are substantially reduced in MK2 shRNA and KO cell lines. These results indicate that the MK2 pathway is involved in HNSCC motility and invasion. We also observe the loss of MK2 contributes to a shift in the epithelial:mesenchymal ratio potentially by suppressing cancer EMT plasticity. All in all, this study provides evidence suggesting tumor MK2 activation mediates HNC progression. Citation Format: Dakota D. Okwuone, Deri Morgan, Hannah M. Smith, Grace Millington, Kiersten L. Berggren, Christopher E. Lominska, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 signaling regulates cell migration and invasion in head and neck squamous cell carcinoma. [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 3601.

Abstract 3628: RAC1 aberrations in head and neck cancer affect immune microenvironments

Abstract RAC1 is a Rho GTPase well-identified as an oncogene overexpressed in various cancer types. RAC1 amplification or gain account for ~40% of head and neck squamous cell carcinoma (HNSCC) while mutation of RAC1 occurs at ~3% according to TCGA-HNSCC cohort. HNSCC patients with RAC1 aberrations, including RAC1 mutations and amplification or gain, are associated with poor overall and disease-free survival in HNSCC. Significantly higher tumor mutational burden is observed in RAC1-mutated group (p=0.0336) or in RAC1-amplified/gain group (p=0.0109) vs. wildtype patients in TCGA-HNSCC cohort. As increases in TMB are often associated with immune cell infiltration in cancers, we hypothesized that RAC1 aberrations impact the tumor immune microenvironment (TIME) of HNSCC, potentially contributing to disease progression.PD-L1 (CD274) is an important immunosuppressive immune checkpoint molecule expressed in tumor cells. Here, we first reported that ectopic overexpression of RAC1 p.P29S and p.A159V mutations in a human HNSCC cell line (PECAPJ41 clone D2, ATCC, USA) could cause upregulation of PD-L1 at both mRNA (by RNA-seq) and protein levels (by Western blotting), first demonstrating a direct effect of RAC1 mutations on potential HNSCC TIME modulation via PD-L1. Furthermore, by tumor immune estimation resources (TIMER) analysis, we found that RAC1-mutated tumors have significantly higher level of neutrophils immune infiltration of 38.63% (p=0.027) compared to the wildtype tumors. From the CIBERSORTx analysis, M2 Macrophages was significantly increased by 40.42% in RAC1-mutated tumors vs wildtype in the TCGA-HNSCC cohort (p=0.031). We also validated this finding by immunofluorescent staining of PD-L1 and M2 Macrophages in immunocompetent mouse HNSCC xenografts models expressing RAC1 p.P29S and p.A159V mutations as compared to controls. Our in silico, in vitro and in vivo findings first uncover an important role of RAC1 aberrations in HNSCC TIME immunosuppression by regulating PD-L1 expression, neutrophil and M2 macrophages infiltrations. Clinical activities of PD-L1 inhibitors in RAC1-mutated HNSCC patients worth future investigations. Acknowledgement: This research is funded by the General Research Fund (Ref. No.:14168517) and the Research Impact Fund (Ref. No.: R4015-19) from the Research Grants Council in Hong Kong. VWYL is supported by Start-up Fund, Georgia Cancer Center, Medical College of Georgia at Augusta University. Citation Format: Helen Hoi Yin Chan, Yuchen Liu, Hoi Lam Ngan, Vivian Wai Yan Lui. RAC1 aberrations in head and neck cancer affect immune microenvironments. [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 3628.

DNA 6mA demethylase ALKBH1 regulates DDX18 expression to promote proliferation of human head and neck squamous cell carcinoma.

Human head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Currently, surgical resection plus a combination of chemotherapy and radiotherapy is the standard treatment for HNSCC, and the 5-year survival rate of patients with HNSCC remains very low because of the higher incidence of metastasis with consequent recurrence. Here, we aimed to investigate the potential role of DNA N6-methyladenine (6mA) demethylase ALKBH1 in tumor cell proliferation in HNSCC. The expression of ALKBH1 in 10 pairs of HNSCC/normal tissues and 3 HNSCC cell lines were measured by qRT‒PCR and western blotting. Colony formation, flow cytometry, patient-derived HNSCC organoid assays were used to assess the role of ALKBH1 in HNSCC cell proliferation in cell lines and human HNSCC patients. MeDIP-seq, RNA sequencing, Dot blotting and western blotting were used to evaluate the regulatory effect of ALKBH1 on the expression of DEAD-box RNA helicase DDX18. A dual-luciferase reporter assay was used to assess the putative effect of DNA 6mA levels on DDX18 transcription. ALKBH1 was highly expressed in HNSCC cells and patient tissues. Functional experiments revealed that ALKBH1 knockdown in SCC9, SCC25, and CAL27 cells inhibited their proliferation in vitro. Using patient-derived HNSCC organoid assay, we found that knockdown of ALKBH1 inhibited the proliferation and colony formation of HNSCC patients-derived organoids. Moreover, we found that ALKBH1 can enhance DDX18 expression by erasing DNA 6mA level and regulating its promoter activity. ALKBH1 deficiency blocked tumor cell proliferation by inhibiting DDX18 expression. Exogenous overexpression of DDX18 rescued the cell proliferation arrest caused by ALKBH1 knockdown. Our data reveal the important role of ALKBH1 in regulating proliferation of HNSCC.

Tumor treating fields as novel combination partner in the multimodal treatment of head and neck cancer.

We aimed to demonstrate the effects of tumor treating fields (TTFields) in head and neck squamous cell carcinoma (HNSCC) cells when combined with radiotherapy (RT) and chemotherapy. Two human HNSCC cell lines (Cal27, FaDu) received five different treatments: TTFields, RT +/- TTFields and RT + simultaneous cisplatin +/- TTFields. Effects were quantified using clonogenic assays and flow cytometric analyses of DAPI, caspase-3 activation and γH2AX foci. Treatment with RT + TTFields decreased the clonogenic survival as strong as treatment with RT + simultaneous cisplatin. The triple combination of RT + simultaneous cisplatin + TTFields even further decreased the clonogenic survival. Accordingly, combination of TTFields with RT or RT + simultaneous cisplatin increased cellular apoptosis and DNA double-strand breaks. TTFields therapy seems a promising combination partner in the multimodal treatment of locally advanced HNSCC. It could be used to intensify chemoradiotherapy or as alternative to chemotherapy.

Identification and verification of eight cancer-associated fibroblasts related genes as a prognostic signature for head and neck squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) can exert their immunosuppressive effects by secreting various effectors that are involved in the regulation of tumor-infiltrating immune cells as well as other immune components in the tumor immune microenvironment (TIME), thereby promoting tumorigenesis, progression, metastasis, and drug resistance. Although a large number of studies suggest that CAFs play a key regulatory role in the development of head and neck squamous cell carcinoma (HNSCC), there are limited studies on the relevance of CAFs to the prognosis of HNSCC. In this study, we identified a prognostic signature containing eight CAF-related genes for HNSCC by univariate Cox analysis, lasso regression, stepwise regression, and multivariate Cox analysis. Our validation in primary cultures of CAFs from human HNSCC and four human HNSCC cell lines confirmed that these eight genes are indeed characteristic markers of CAFs. Immune cell infiltration differences analysis between high-risk and low-risk groups according to the eight CAF-related genes signature hinted at CAFs regulatory roles in the TIME, further revealing its potential role on prognosis. The signature of the eight CAF-related genes was validated in different independent validation cohorts and all showed that it was a valid marker for prognosis. The significantly higher overall survival (OS) in the low-risk group compared to the high-risk group was confirmed by Kaplan-Meier (K-M) analysis, suggesting that the signature of CAF-related genes can be used as a non-invasive predictive tool for HNSCC prognosis. The low-risk group had significantly higher levels of tumor-killing immune cell infiltration, as confirmed by CIBERSORT analysis, such as CD8+ T cells, follicular helper T cells, and Dendritic cells (DCs) in the low-risk group. In contrast, the level of infiltration of pro-tumor cells such as M0 macrophages and activated Mast cells (MCs) was lower. It is crucial to delve into the complex mechanisms between CAFs and immune cells to find potential regulatory targets and may provide new evidence for subsequently targeted immunotherapy. These results suggest that the signature of the eight CAF-related genes is a powerful indicator for the assessment of the TIME of HNSCC. It may provide a new and reliable potential indicator for clinicians to predict the prognosis of HNSCC, which may be used to guide treatment and clinical decision-making in HNSCC patients. Meanwhile, CAF-related genes are expected to become tumor biomarkers and effective targets for HNSCC.

Dysregulation and Epigenetic Reprogramming of NRF2 Signaling Axis Promote Acquisition of Cisplatin Resistance and Metastasis in Head and Neck Squamous Cell Carcinoma.

Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1-NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Differential Immunomodulatory Effects of Head and Neck Cancer-Derived Exosomes on B Cells in the Presence of ATP.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy. Tumor-derived exosomes (TEX) have immunoregulatory properties. Adenosine triphosphate (ATP) and its immunosuppressive precursor adenosine (ADO) have been found in cancerous tissue. We investigated the effect of TEX on B cells in the presence of ATP. TEX were isolated from human HNSCC cell line (PCI-13) cultures and co-cultured with peripheral blood B cells of healthy donors, with or without TEX in different concentrations and with or without a low (20 µM) or high (2000 µM) ATP dose. We were able to demonstrate that TEX inhibit B-cell proliferation. The addition of TEX to either ATP concentration showed a decreasing trend in CD39 expression on B cells in a dose-dependent manner. High ATP levels (2000 µM) increased apoptosis and necrosis, and analysis of apoptosis-associated proteins revealed dose-dependent effects of ATP, which were modified by TEX. Altogether, TEX exhibited dual immunomodulatory effects on B cells. TEX were immunosuppressive by inhibiting B-cell proliferation; they were immunostimulatory by downregulating CD39 expression. Furthermore, TEX were able to modulate the expression of pro- and anti-apoptotic proteins. In conclusion, our data indicate that TEX play an important, but complex, role in the tumor microenvironment.

The EXTREME Regimen Associating Cetuximab and Cisplatin Favors Head and Neck Cancer Cell Death and Immunogenicity with the Induction of an Anti-Cancer Immune Response.

(1) Background: The first line of treatment for recurrent/metastatic Head and Neck Squamous Cell Carcinoma (HNSCC) has recently evolved with the approval of immunotherapies that target the anti-PD-1 immune checkpoint. However, only about 20% of the patients display a long-lasting objective tumor response. The modulation of cancer cell immunogenicity via a treatment-induced immunogenic cell death is proposed to potentially be able to improve the rate of patients who respond to immune checkpoint blocking immunotherapies. (2) Methods: Using human HNSCC cell line models and a mouse oral cancer syngeneic model, we have analyzed the ability of the EXTREME regimen (combination therapy using the anti-EGFR cetuximab antibody and platinum-based chemotherapy) to modify the immunogenicity of HNSCC cells. (3) Results: We showed that the combination of cetuximab and cisplatin reduces cell growth through both cell cycle inhibition and the induction of apoptotic cell death independently of p53. In addition, different components of the EXTREME regimen were found to induce, to a variable extent, and in a cell-dependent manner, the emission of mediators of immunogenic cell death, including calreticulin, HMGB1, and type I Interferon-responsive chemokines. Interestingly, cetuximab alone or combined with the IC50 dose of cisplatin can induce an antitumor immune response in vivo, but not when combined with a high dose of cisplatin. (4) Conclusions: Our observations suggest that the EXTREME protocol or cetuximab alone are capable, under conditions of moderate apoptosis induction, of eliciting the mobilization of the immune system and an anti-tumor immune response in HNSCC.