Head And Neck Squamous Cell Carcinoma Cancer Cells Research Articles

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.

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.

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.

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 & 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.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC(HNSCC) and anal SCC(ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virusnegative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone)binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Pentagalloyl Glucose and Cisplatin Combination Treatment Exhibits a Synergistic Anticancer Effect in 2D and 3D Models of Head and Neck Carcinoma.

Although cisplatin is a first-line chemotherapy drug for head and neck squamous cell carcinoma (HNSCC), its therapeutic efficacy is limited owing to serious side effects and acquired drug resistance. This study determined whether combining pentagalloyl glucose (PGG) and cisplatin enhanced their anti-tumor activities on HNSCC cell lines. We investigated the anticancer effect of PGG combined with cisplatin in 2D and 3D multicellular spheroid cell culture. The results revealed that PGG combined with cisplatin inhibited cell viability and produced synergistic effects. PGG potentiates the anticancer effect of cisplatin by promoting apoptosis and inhibiting cell migration. The western blot and molecular docking analysis revealed that the synergistic effect of the combination treatment may be related to the PGG-mediated reduced expression of phosphorylated STAT3 and phosphorylated Akt. Furthermore, we found that the combined treatment of PGG and cisplatin’s effect on 3D multicellular spheroid size was more potent than the monotherapies. Our findings indicated that the combination therapy of PGG and cisplatin synergistically inhibited HNSCC cancer cell viability and induced apoptosis in 2D and 3D models. The present results suggested that PGG may be a promising adjunct drug used with cisplatin for a practical therapeutic approach to head and neck cancer.

CD73 in small extracellular vesicles derived from HNSCC defines tumour-associated immunosuppression mediated by macrophages in the microenvironment.

Research on tumour cell‐derived small extracellular vesicles (sEVs) that regulate tumour microenvironment (TME) has provided strategies for targeted therapy of head and neck squamous cell carcinoma (HNSCC). Herein, we demonstrated that sEVs derived from HNSCC cancer cells carried CD73 (sEVsCD73), which promoted malignant progression and mediated immune evasion. The sEVsCD73 phagocytosed by tumour‐associated macrophages (TAMs) in the TME induced immunosuppression. Higher CD73high TAMs infiltration levels in the HNSCC microenvironment were correlated with poorer prognosis, while sEVsCD73 activated the NF‐κB pathway in TAMs, thereby inhibiting immune function by increasing cytokines secretion such as IL‐6, IL‐10, TNF‐α, and TGF‐β1. The absence of sEVsCD73 enhanced the sensitivity of anti‐PD‐1 therapy through reversed immunosuppression. Moreover, circulating sEVsCD73 increased the risk of lymph node metastasis and worse prognosis. Taken together, our study suggests that sEVsCD73 derived from tumour cells contributes to immunosuppression and is a potential predictor of anti‐PD‐1 responses for immune checkpoint therapy in HNSCC.

EGFR Regulates the Hippo pathway by promoting the tyrosine phosphorylation of MOB1

The Hippo pathway is frequently dysregulated in cancer, leading to the unrestrained activity of its downstream targets, YAP/TAZ, and aberrant tumor growth. However, the precise mechanisms leading to YAP/TAZ activation in most cancers is still poorly understood. Analysis of large tissue collections revealed YAP activation in most head and neck squamous cell carcinoma (HNSCC), but only 29.8% of HNSCC cases present genetic alterations in the FAT1 tumor suppressor gene that may underlie persistent YAP signaling. EGFR is overexpressed in HNSCC and many other cancers, but whether EGFR controls YAP activation is still poorly understood. Here, we discover that EGFR activates YAP/TAZ in HNSCC cells, but independently of its typical signaling targets, including PI3K. Mechanistically, we find that EGFR promotes the phosphorylation of MOB1, a core Hippo pathway component, and the inactivation of LATS1/2 independently of MST1/2. Transcriptomic analysis reveals that erlotinib, a clinical EGFR inhibitor, inactivates YAP/TAZ. Remarkably, loss of LATS1/2, resulting in aberrant YAP/TAZ activity, confers erlotinib resistance on HNSCC and lung cancer cells. Our findings suggest that EGFR-YAP/TAZ signaling plays a growth-promoting role in cancers harboring EGFR alterations, and that inhibition of YAP/TAZ in combination with EGFR might be beneficial to prevent treatment resistance and cancer recurrence.

Tipifarnib enhances anti-EGFR activity of cetuximab in non-HRas mutated head and neck squamous cell carcinoma cancer (HNSCC)

ObjectiveTo test the potential ability of tipifarnib to impair proliferation and to enhance the activity of the EGFR inhibitor cetuximab in wild-type H-Ras HNSCC, which accounts for the majority of HNSCC. Materials and MethodsCell growth, apoptosis and signaling changes in HNSCC cells following tipifarnib exposure in vitro were assessed by SRB, colony formation assay, annexin V staining and Western blot, respectively. A patient-derived xenograft (PDX) animal model was adopted to evaluate the efficacy of tipifarnib in vivo with and without cetuximab. ResultsTreatment of wild-type H-Ras HNSCC cell lines in vitro with tipifarnib reduced cell growth and increased levels of defarnesylated H-Ras in a dose-dependent manner. In a PDX mouse model, treatment with single-agent tipifarnib led to only near-significant growth inhibition. The addition of cetuximab resulted in increased anti-proliferative effect both in culture and in PDX models, which was also mirrored by Western blot and apoptosis assay results. ConclusionTipifarnib has only a moderate ability to slow tumor growth as a single agent in HNSCC with wild type H-Ras, despite specifically inhibiting the farnesyltransferase upon which the function of H-Ras depends. The combination of cetuximab and tipifarnib appears to enhance the anti-proliferative effect of single-agent tipifarnib and marginally enhance that of single agent cetuximab. These findings deserve further evaluation.

Abstract 1552: Single cell expression analysis of PIK3 genes to direct solid tumor treatment with the dual PI3K-δ,γ inhibitor duvelisib

Abstract Duvelisib is a dual PI3K-δ,γ inhibitor that targets malignant B and T cells in hematologic malignancies and modulates key non-malignant immune cells in the tumor microenvironment. PI3K-δ and PI3K-γ are thought to govern tumor immune modulation through reduction of immunosuppressive Tregs and myeloid cells in solid tumors (Ali, 2014, Kaneda, 2016; De Henau, 2016). Yet, a potential role of these isoforms in directly targeting cancer cells of solid tumors is not understood. Expression levels of PI3K-δ (PIK3CD) and PI3K-γ (PIK3CG) were analyzed in 9 major tumor types (BRCA, HNSCC, LUSC, PAAD, STAD, COAD, LUAD, SKCM and THCA), and it was found that PIK3CD reproducibly had higher expression levels than PIK3CG. For example, in head and neck squamous cell carcinoma (HNSCC), PIK3CD had a median expression of 9.51 RNA-Seq Expectation-Maximization (RSEM) log2 and PIK3CG had a median of 5.56 RSEM log2; each of which are higher than the levels in normal cells (8.40 RSEM log2 & 5.38 RSEM log2, respectively) (FireBrowse). By clustering solid tumors into ′Hot′ (inflamed) and ′Cold′ subgroups with an inflammatory/immunosuppressive gene signature (Givechian, 2018; TCGA, n=1687); a correlation was observed between higher PIK3CD and PIK3CG expression in ′Hot′ (median ≈11 log2 Transcripts Per Million (TPM) & ≈9.2 log2 TPM) compared to ′Cold′ tumors (median ≈9.4 log2 TPM & ≈6.6 log 2 TPM). Using CIBERSORT deconvolution, most ′Hot′ cluster tumors contained higher fractions of CD8+ T cells, M1-type macrophages, and lower M2-type macrophages; with Tregs at similar abundances in both ′Hot′ and ′Cold′ clusters. To evaluate cellular distribution, single-cell RNA expression of PIK3C genes in a HNSCC dataset (Puram, 2017) was examined at >0.3 log2 TPM. PIK3CD and PIK3CG expression was observed in T cells (32% & 11%, respectively), B cells (13% & 56%), dendritic cells (15% & 40%) and macrophages (24% & 29%) in HNSCC tumors. PIK3A and PIK3B were less frequently expressed in macrophages (10% & 17%) and T cells (3% & 4%) than PIK3D. A significant proportion of the HNSCC cancer cells expressed PIK3CD (20%) and PIK3CB (27%), whereas PIK3CA (14%) and PIK3CG (0%) were less frequently expressed. Most individual tumors had cancer cells expressing PIK3CD (6%-63%; >50 cells/tumor). Importantly, PIK3CD expression was mostly mutually exclusive with PIK3CG, PIK3CA, and PIK3CB in the cancer cells. Cancer cells expressing PIK3CD showed striking co-associated differentially expressed genes (p<0.009). These results indicate that PI3Kδ expression is observed in both the immune cells which are fashioning the suppressive microenvironment and in cancer cells. These findings suggest an approach to identify patients for treatment guided by cell type expression of PI3K isoforms, and provide strategies for duvelisib use as monotherapy and in combinations with PD-1 checkpoint inhibitors and other agents. Citation Format: Samantha Hidy, Jonathan A. Pachter, David T. Weaver. Single cell expression analysis of PIK3 genes to direct solid tumor treatment with the dual PI3K-δ,γ inhibitor duvelisib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1552.

CHK1/2 Inhibitor Prexasertib Suppresses NOTCH Signaling and Enhances Cytotoxicity of Cisplatin and Radiation in Head and Neck Squamous Cell Carcinoma.

Platinum-based chemoradiotherapy is a mainstay of organ-preserving therapy for patients with head and neck squamous cell carcinoma cancer (HNSCC). However, the disease eventually becomes resistant to treatment necessitating new therapies. Checkpoint kinase 1 and 2 (CHK1/2) are serine/threonine kinases that activate cell-cycle checkpoints and serve a critical role in the DNA-damage response (DDR). As resistance to cisplatin and radiation may involve a heightened DDR, we hypothesized that prexasertib, an inhibitor of CHK1/2, may enhance the cytotoxicity induced by cisplatin and irradiation in HNSCC. In this study, we found that combining prexasertib with cisplatin and radiation significantly decreased the in vitro survival fraction in HNSCC cell lines both with and without radiotherapy. Reduced survival was accompanied by inhibition of DNA repair checkpoint activation, which resulted in persistent DNA damage and increased apoptosis. In addition, NanoString analysis with the PanCancer Pathways Panel revealed that prexasertib downregulated NOTCH signaling target genes (NOTCH1, NOTCH2, and NOTCH3) and their associated ligands (JAG1, JAG2, SKP2, MAML2, and DLL1). Prexasertib also reduced NOTCH1, NOTCH3 and HES1 protein expression. Importantly, a significant tumor growth delay was observed in vivo in both human papillomavirus (HPV)-positive UM-SCC47 and HPV-negative UM-SCC1 cell line xenografts treated with prexasertib, cisplatin, and radiotherapy without increased toxicity as measured by mouse body weight. Taken together, prexasertib reduced NOTCH signaling and enhanced the in vitro and in vivo response of HNSCCs to cisplatin and radiation, suggesting combination therapy may increase clinical benefit. A clinical trial has recently completed accrual (NCT02555644).

Abstract A10: Fusobacteria upregulates PD-L1 in head and neck cancer cell lines: Potential mechanism for immune evasion

Abstract Objectives: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide with a poor prognosis of ~40% at 5 years for HPV-negative disease. Recently, immune checkpoint inhibitors (specifically, anti-PD-1 checkpoint inhibitors that block the PD-L1/PD-1 immunosuppressive axis) have been approved as a treatment for patients with recurrent/metastatic HNSCC. However, most patients (>80%) fail to respond to checkpoint inhibitors. PD-L1 expression on tumors plays an important role in immune evasion and can also influence response to checkpoint inhibitors. Tumor PD-L1 expression has been shown to correlate with checkpoint response. Factors that might affect PDL1 expression in HNSCC remain unclear. In this study, we explored the effect of periodontal bacteria, which has been associated with oral cancer, on PD-L1 expression in oral cancer cells. Materials and Methods: 303 HNSCC were compared to 37 adjacent control tissues in the TCGA mRNA expression database (RNASeq V2); the analysis was done based on tumor types. RT-PCR and flow cytometry were used to measure the base line expression of PD-L1 in seven head and neck cancer cell lines and human oral tissues. OQ01, an oral cancer cell line, was infected with four periodontal bacteria (Tanneralla forsynthia, Treponema denticola, Porphyromonas gingivalis, and Fusobacterium nucleatum). OQ01 and RPMI2650 cells were infected with two different strains of Fusobacteria (F. periodonticum, F. vincentii). The relative changes in expression of PLD1 was measured using RT-PCR in cell lysate after RNA isolation. PD-L1 protein level on the cell surface was measured using flow cytometry. Results: TCGA data analysis revealed that PDL1 was significantly (P< 0.0005) elevated in total cancer tissues compared to total controls. In the same data analysis, PDL1 expression was significantly (P< 0.05) elevated in oral cavity tumor compared to normal control. Expression of PD-L1 was variable among head and neck cancer patients and cell lines. Both F. nucleatum and P. gingivalis were able to enhance PD-L1 expression in OQ01 cells (10 and 15 fold, respectively). Two strains of Fusobacteria tested (F. periodonticum, F. vincentii) were also able to induce PD-L1 both at the mRNA (P< 0.005 and P< 0.05 respectively) and protein level in OQ01 and RPMI 2650 cells. Interestingly, the same strains of Fusobacteria enhanced MYC expression in OQ01 and RPMI 2650 cells. As a transcription factor MYC has been reported to regulate PDL1 transcription. These data along with our results suggest that Fusobacterial upregulation of PD-L1 expression might be through MYC enhanced expression. Conclusions: This study revealed that Fusobacteria enhance PD-L1 expression on HNSCC cancer cell lines, possibly through upregulation of MYC. This may have implications on cancer cell immune evasion and checkpoint inhibitor treatment response. Note: This abstract was not presented at the conference. Citation Format: Amani Harrandah, Rekha Rani Grag, Yuk Pheel Park, Sasanka S. Chukkapalli, Nikhita Yadlapalli, Edward K. L. Chan, Natalie Silver. Fusobacteria upregulates PD-L1 in head and neck cancer cell lines: Potential mechanism for immune evasion [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A10.

MiR-488-3p sponged by circ-0000495 and mediated upregulation of TROP2 in head and neck squamous cell carcinoma development.

TROP2 (trophoblast cell surface antigen 2) overexpression has been reported in many human cancers. The correlation between TROP2 and tumor aggressiveness has implied it could be a prognostic indicator. However, the roles of TROP2 and their underlying mechanisms remain of great interest in head and neck squamous cell carcinoma (HNSCC) biology. In the current study, the prognostic significance of TROP2 in HNSCC archival samples was determined using immunohistochemistry. Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the phenotypic effects of TROP2 knockdown, miR-488-3p re-expression, and circRNAs expression. Cell viability, migration/invasion as well as in vivo tumor formation assays were accessed. The interactions of miRNAs-TROP2 or circRNAs-miRNAs were determined by qRT-PCR, western blot analysis and luciferase assays. TROP2 was demonstrated overexpression in HNSCC patients and cancer cell lines. High expression of TROP2 was significantly associated with patient relapse. TROP2 promoted tumor cell proliferation, migration, invasion, and tumor growth, through AKT and MAPK pathways. Further investigation revealed that TROP2 is a direct target of miR-488-3p, while circ-0000495 bounds to miR-488-3p. Our study unraveled a novel mechanism by which down-regulation of miR-488-3p sponged by circ-0000495 releases its epigenetic silencing to TROP2. The increased TROP2 promotes tumor proliferation, therefore, providing evidence in support of targeting the circ-0000495/miR-488-3p/TROP2 axis in contributing to HNSCC therapy and preventing tumor metastasis.

Abstract 4507: Study of CCCTC-binding factor (CTCF) genetic aberrations and dysregulation in head and neck squamous cell carcinoma (HNSCC)

Abstract CCCTC-binding Factor (CTCF) is a DNA-binding protein that acts as a global genome organizer in higher eukaryotes. CTCF folds the genome into spatial domains that is crucial for regulation of genes, including oncogenes and tumor suppressor genes. Recently, Bornstein et al. has reported that CTCF truncating mutations are associated with progression of Head and Neck Squamous Cell Carcinoma (HNSCC), suggesting a potential role of CTCF and its aberrations in HNSCC progression. Survival analysis of the US-HNSCC The Cancer Genome Atlas (TCGA) Provisional cohort (N=502) revealed that CTCF mutations are associated with poor overall survival (P = 0.0206, Median survival of15 months vs 56.9 months) and disease-free survival (DFS; P = 0.0078, median DFS of 12.975 months vs 71.22 months). Furthermore, CTCF truncating mutations are associated with poor overall survival (P = 0.0153, Median survival of 14.98 months vs 56.9 months) and disease-free survival (DFS; P = 0.0010, median DFS of 8.97 months vs 71.22 months). Intriguingly, RNA-seq analysis of 43 pairs of normal-tumor tissues from the TCGA reveal a tumor-specific upregulation of CTCF mRNA (P < 0.0001). Subsequent Western blot analyses demonstrated at least 2.5-fold and 1.2-fold CTCF protein upregulation in HNSCC patient primary cultures and HNSCC cancer cell lines relative to normal head and neck squamous cell and normal immortalized head and neck squamous cell line respectively. Based on these preliminary genomic findings, we hypothesized that CTCF upregulation may have pro-tumorigenic effects in HNSCC. Therefore, cancer cell proliferation was monitored following exogenous overexpression by retroviral introduction of the human CTCF gene or specific knockdown (by shRNA) of CTCF in HNSCC cell lines. We found that exogenous overexpression of CTCF promoted HNSCC cell proliferation by about 40% (N=6). However, with multiple trials, specific knockdown of CTCF only resulted in about 10% growth inhibition of HNSCC cells (N=6) with minimal apparent knockdown of CTCF protein. This unexpected challenge of CTCF protein knockdown could likely be due to potentially long CTCF protein half-life in HNSCC. In fact, we found that CTCF protein is highly stable in HNSCC cell line and HNSCC normal immortalized cell line, with protein level reduction by only 20 to 40% after two to three days of cycloheximide inhibition. Our results demonstrated a growth-promoting role of CTCF in HNSCC, and this CTCF protein appears to be highly stable with overexpression in HNSCC, potentially serving as a stable push for tumorigenesis. Citation Format: Sze Man Chan, Hoi-Lam Ngan, Yuchen Liu, Wenying Piao, Chin Wang Lau, Jason Ying Kuen Chan, Yu Xiong Su, Vivian Wai Yan Lui. Study of CCCTC-binding factor (CTCF) genetic aberrations and dysregulation in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4507.

Combined expression and prognostic significance of PPFIA1 and ALG3 in head and neck squamous cell carcinoma.

PPFIA family members and ALG3 play important roles in tumorigenesis and tumor progression. However, the exact roles of distinct PPFIA family members and ALG3 in head and neck squamous cell carcinoma (HNSCC) remain unclear. We studied the mRNA expressions of PPFIA family members and ALG3 in a variety of tumor types compared with the normal controls using the Oncomine database along with meta-analyses of their expressions in HNSCC cancer cell line. The mRNA expressions of PPFIA family members and ALG3 in laryngeal squamous cell carcinoma cell line and normal laryngeal cell line were detected by quantitative real-time polymerase chain reaction. Based on the cBioportal database, we further studied mRNA expression alterations and co-occurrence relationships of the PPFIA family members and ALG3 in HNSCC. The relationship between PPFIA1 and ALG3 mRNA expression alterations and prognoses in patients with HNSCC was explored. We found that PPFIA1 and ALG3 were distinctively overexpressed at the mRNA level in HNSCC tissues compared with normal tissues, they had a significant co-occurrence relationship, their mRNA expressions were significantly higher than other PPFIA family members in laryngeal squamous cell carcinoma cell line, and their mRNA expressions were also significantly higher in laryngeal carcinoma cell line than in normal laryngeal cell line. Patients without both PPFIA1 and ALG3 mRNA expression alterations had better overall survival and disease/progression-free survival compared with patients with both PPFIA1 and ALG3 alterations. Based on these findings, PPFIA1 and ALG3 may play roles in oncogene expression in HNSCC. Their combined overexpression is significantly associated with poor survival outcomes. The relationship between them and the mechanism of action in head and neck cancers deserve further investigation.

Optimal targeting of BCL-family proteins in head and neck squamous cell carcinoma requires inhibition of both BCL-xL and MCL-1.

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares – Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9μM (range 6.6μM – 13.9μM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7μM (range, 8.8μM to 12.7μM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.

Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769-82. ©2018 AACR.

CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma

ObjectivesIn accordance with the Precision Medicine Initiative, new treatment strategies for head and neck squamous cell carcinoma (HNSCC) are needed to yield better therapeutic outcomes. The purpose of this study was to establish and validate chimeric antigen receptor (CAR)-T cells targets in HNSCC. MethodsPutative CAR-T antigens were identified in The Cancer Genome Atlas database. To validate antigen suitability, quantitative RT-PCR, flow cytometry, and immunofluorescent staining were performed. A retroviral human CD70 CAR construct, using truncated CD27 conjugated with 4-1BB and CD3-zeta costimulatory molecules, was used to transduce activated human T cells to generate CD70 CAR-T cells. Cell-based cytotoxicity and cytokine ELISAs were used to measure efficacy of killing. ResultsNine potential CAR-T targets (CD276, EGFR, MICA, MICB, MAGE-A4, FAP, EPCAM, CD70, B4GALNT1) were identified based on their high expression in tumors compared to flanking control tissues. CD70 was selected for further proof-of-principle analysis based on its differential expression in several tumor subtypes, and showed substantial heterogeneity in individual tumors analyzed. Cell surface CD70 protein and CD70 mRNA were detected from low to high levels in established HNSCC cancer cell lines. CD70 was highly expressed in 4 of 21 tumor biopsies (19%), and 3 of 4 specimens showed strong CD70 expression on the tumor cell surface. CD70-specific CAR-T cells were generated and further demonstrated to recognize and kill CD70-positive HNSCC cells efficiently, but not CD70-negative cancer cells. ConclusionCD70-specific CAR-T cells specifically recognized and efficiently eliminated CD70-positive HNSCC cells. This study provides the basis for further investigation into CD70 and other CAR-T targets.

Anthelmintic mebendazole enhances cisplatin's effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC)

Head and neck squamous cell carcinoma (HNSCC) is one of the most common and aggressive types of human cancers worldwide. Nearly a half of HNSCC patients experience recurrence within five years of treatment and develop resistance to chemotherapy. Thus, there is an urgent clinical need to develop safe and novel anticancer therapies for HNSCC. Here, we investigate the possibility of repurposing the anthelmintic drug mebendazole (MBZ) as an anti-HNSCC agent. Using the two commonly-used human HNSCC lines CAL27 and SCC15, we demonstrate MBZ exerts more potent anti-proliferation activity than cisplatin in human HNSCC cells. MBZ effectively inhibits cell proliferation, cell cycle progression and cell migration, and induces apoptosis of HNSCC cells. Mechanistically, MBZ can modulate the cancer-associated pathways including ELK1/SRF, AP1, STAT1/2, MYC/MAX, although the regulatory outcomes are context-dependent. MBZ also synergizes with cisplatin in suppressing cell proliferation and inducing apoptosis of human HNSCC cells. Furthermore, MBZ is shown to promote the terminal differentiation of CAL27 cells and keratinization of CAL27-derived xenograft tumors. Our results are the first to demonstrate that MBZ may exert its anticancer activity by inhibiting proliferation while promoting differentiation of certain HNSCC cancer cells. It's conceivable the anthelmintic drug MBZ can be repurposed as a safe and effective agent used in combination with other frontline chemotherapy drugs such as cisplatin in HNSCC treatment.

Notch Signaling Activation Is Associated with Patient Mortality and Increased FGF1-Mediated Invasion in Squamous Cell Carcinoma of the Oral Cavity.

Oral squamous cell carcinoma (OSCC) is a cancer subtype that lacks validated prognostic and therapeutic biomarkers, and human papillomavirus status has not proven beneficial in predicting patient outcomes. A gene expression pathway analysis was conducted using OSCC patient specimens to identify molecular targets that may improve management of this disease. RNA was isolated from 19 OSCCs treated surgically at the University of Alabama at Birmingham (UAB; Birmingham, AL) and evaluated using the NanoString nCounter system. Results were confirmed using the oral cavity subdivision of the Head and Neck Squamous Cell Carcinoma Cancer (HNSCC) study generated by The Cancer Genome Atlas (TCGA) Research Network. Further characterization of the in vitro phenotype produced by Notch pathway activation in HNSCC cell lines included gene expression, proliferation, cell cycle, migration, invasion, and radiosensitivity. In both UAB and TCGA samples, Notch pathway upregulation was significantly correlated with patient mortality status and with expression of the proinvasive gene FGF1 In vitro Notch activation in HNSCC cells increased transcription of FGF1 and induced a marked increase in cell migration and invasion, which was fully abrogated by FGF1 knockdown. These results reveal that increased Notch pathway signaling plays a role in cancer progression and patient outcomes in OSCC. Accordingly, the Notch-FGF interaction should be further studied as a prognostic biomarker and potential therapeutic target for OSCC. Patients with squamous cell carcinoma of the oral cavity who succumb to their disease are more likely to have upregulated Notch signaling, which may mediate a more invasive phenotype through increased FGF1 transcription. Mol Cancer Res; 14(9); 883-91. ©2016 AACR.