Purpose Recurrence in early-stage laryngeal carcinoma after radiation therapy often necessitates a total salvage laryngectomy (TSL). Understanding the genetic landscape that influences cancer progression after radiation therapy may help future treatments. We investigated early-stage laryngeal carcinoma patients treated with radiotherapy to identify mutation patterns that predispose to disease recurrence. Methods The patient cohort had 35 early-stage laryngeal head and neck squamous cell carcinoma (HNSCC) patients (T1=15, T2=20,) treated with radiotherapy (RT). We stratified patients: Responders (no recurrence, N=14), non-responders (recurrence within 12 months, N=21), and non-responders undergoing TSL (N=18). We employed whole exome sequencing to characterize gene mutations using the Genome Analysis Toolkit (GATK) for variant detection and impact on critical biological pathways and post-radiotherapy patient outcomes. Pathway analysis Ingenuity and Reactome Pathway Analysis tools were used to explore the mutated gene pathways. Results – Differential mutation analysis was performed in the respective groups to find driver genes. In the pre-treatment samples, KCNT2 and AGAP6 mutations were exclusively found in non-responders (OR=0, P=0.005 and OR=0, P=0.027, respectively), while ADAMTS7 mutations were solely present in responders (OR=inf, P=0.019). PLEC mutations were more prevalent in responders (OR=11.6, P=0.006). Pathway analysis revealed that significant genes were involved in the RND2 GTPase cycle, protein O-glycosylation-related diseases, and apoptotic pathways. Post-treatment analysis in patients undergoing TSL had enrichment of mutations in apoptosis regulation pathways. Conclusions – The study reveals that mutations in apoptosis controlling genes were predominantly represented in the larynx non-responder patients both in the pre-radiotherapy and post-TSL populations.

Higher abundance of oral human papilloma virus (HPV), Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) correlate with worse survival of head and neck squamous cell carcinoma (HNSCC). One-third of males globally are HPV-positive, one-fifth have high-risk HPV-16 and is transmitted through oral sex. In our ex vivo clinical study of HNSCC patients, HPV ELISA detected viruses in 100% of saliva and 75% of oral-rinse samples (n = 44). Cell culture studies showed 1000-fold higher Pg/Fn in their saliva and 100-fold higher in oral-rinse samples than non-cancer control participants. Bean-lectin (FRIL) gum (trapped) aggregated 93% of HPV in saliva and 80% in oral-rinse samples in ELISA. Single dose of bean gum + protegrin-1 (antimicrobial peptide) reduced Pg/Fn > 99% in saliva and oral-rinse samples (n = 42). Although protegrin was effective against anaerobic Pg/Fn, it did not kill capsule forming bacteria including Streptococci, providing selectivity and protection of oral commensal bacteria. These findings from ex vivo clinical studies support potential value of advancing these biologics to clinical trials as adjuncts to current therapeutic strategies or as prophylaxis to control pathogenic microbes associated with HNSCC.

HNSCC presents a significant health challenge due to its high mortality resulting from treatment resistance and locoregional invasion into critical structures in the head and neck region. Understanding the invasion mechanisms of HNSCC has the potential to guide targeted therapies, improving patient survival. Previously, we demonstrated the involvement of doublecortin like kinase 1 (DCLK1) in regulating HNSCC cell invasion. Here, we investigated the hypothesis that DCLK1 modulates proteins within invadopodia, specialized subcellular protrusions that secrete matrix metalloproteinases to degrade the ECM. We employed tandem mass tag (TMT)-based proteomics to identify the role of DCLK1 in regulating proteins involved in HNSCC invasion and validated the findings using immunoblotting. The Cancer Genome Atlas (TCGA) database was interrogated to correlate DCLK1 expression with tumor stage, grade, and invasion-associated proteins. In vitro invasion was assessed using a Boyden chamber assay, and immunohistochemistry on patient samples determined DCLK1's distribution within tumors. Gelatin invadopodia assay was used to establish DCLK1 localization to invadopodia related gelatin degradation. Super-resolution confocal microscopy demonstrated colocalization of DCLK1 with invadopodia markers and MMP trafficking proteins. ECM degradation by MMPs in HNSCC cells with wild-type and knockdown DCLK1 was evaluated using a dye-quenched tracer, while gel zymography and MMP array identified secreted proteases. Proximity ligation assay (PLA) and co-immunoprecipitation assays were used to confirm interactions between DCLK1, MMP9, KIF16B, and RAB40B. Proteomic analysis demonstrate DCLK1's role in regulating proteins involved in cytoskeletal and ECM remodeling. Clinically, rising DCLK1 levels correlate with higher histological grade and lymph node metastasis, with heightened expression observed at the leading edge of HNSCC patient tissue. DCLK1 is localized with markers of mature invadopodia including TKS4, TKS5, cortactin, and MT1-MMP. Knockdown of DCLK1 led to reductions in invadopodia numbers and decreased in vitro invasion and ECM degradation. MMP9 colocalizes with DCLK1 within invadopodia structures and its secretion is disrupted by DCLK1 knockdown. Further, PLA and co-immunoprecipitations studies demonstrate DLCK1 complexes with KIF16B and RAB40B enabling trafficking of degradative MMP9 cargo along the invadopodia to degrade local ECM. This work unveils a novel function of DCLK1 in regulating KIF16B and RAB40B to traffic matrix degrading MMP9 cargo to the distal end of the invadopodia facilitating HNSCC invasion.

For patients with locally advanced, p16-negative head and neck squamous cell carcinoma (HNSCC), overall survival remains poor due to primary locoregional failure and distant metastasis following curative therapy. We aimed to understand how MAPKAPK2 (MK2) regulates HNSCC tumor cell migration and invasion, important first steps in cancer metastases. The TCGA database and HNSCC tissue microarrays were used to show that MK2 expression was associated with more advanced cancers and faster cancer recurrence rates. We observed that silencing of tumor MK2 in human cell lines (shRNA) caused a significant reduction in tumor cell migration-invasion in a complex HNSCC microphysiologic system used to recapitulate the tumor microenvironment. Murine cells (Ly2) with MK2 silenced (CRISPR-Cas9) also demonstrated reduced migration and invasion using 2D and 3D monoculture cell migration-invasions assays. Ly2 cells are orthotopic p16-negative murine metastatic cells that spontaneously metastasize, and we observed that MK2 inhibition via genetic (Cas9/CRISPR) or pharmacologic (PF-3644022) methods led to a significant reduction in the number of circulating tumor cells, fewer lymph node and lung metastases, and MK2 inhibited mice showed improved overall survival. Our findings suggest that HNSCC MK2 regulates tumor cell migration-invasion and may be a promising therapeutic target to reduce metastases.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Abstract Background: Head and neck squamous carcinoma (HNSCC) results in poor patient outcomes driven primarily by locoregional and distant metastatic tumor spread. We have demonstrated that highly phosphorylated/active MAPKAPK-2 (MK2) is a poor prognostic factor in HNSCC patients. MK2 is associated with the expression of pro-inflammatory cytokines that are linked with creating a pro-tumorigenic microenvironment. Nevertheless, how MK2 contributes to HNSCC development has not been established. In this study, we aimed to identify the effect of MK2 on cancer progression, immune infiltration, and cytokine secretion. Method: To assess the effect of tumor MK2 in vivo, we used CRISPR-Cas9 to knock out (KO) MK2 in a metastatic murine HNSCC cell line, Ly2, and orthotopically implanted wildtype (WT) or MK2 KO cells into the floor-of-mouth of Balb/c mice. At the endpoint of the experiment, the tumors were excised, and myeloid and lymphocyte immune profiling was done via flow cytometry. Mouse lungs and cervical lymph nodes were dissected, paraffin-fixed, sectioned, and H&E stained, and the presence/quantity of metastatic foci was evaluated by a blinded board-certified pathologist. We broadly evaluated the tumor secretome via a 105-cytokine dot blot array using 8 mg of the same resected tumor sample and performed densitometric analysis comparing WT and KO tumors. Results: Loss of MK2 significantly reduced primary tumor growth, as well as lymph node and lung metastasis, compared to WT tumors. Tumor immunophenotyping by flow cytometry revealed that MK2 KO tumors had increased overall immune cell (CD45+) infiltration, but decreased quantities of immunosuppressive neutrophils (CD11b+Ly6G+Arg1+Ly6C-) and a shift in macrophage polarity favoring M1 (anti-tumor) macrophages (CD11b+Ly6G-F4-80+CD80+). In addition, there were increased quantities of anti-tumor immune cells including natural killer (NK)-T, CD4+, and CD8+ T cells in the MK2-KO tumors, while the proportion of CD4+CD25+ Tregs and exhausted CD8 T cells (CD8+PD1+) was decreased compared to WT tumors. Furthermore, cytokine array analysis revealed that secreted levels of several cytokines, including CXCL1, CXCL2, GM-CSF, G-CSF, IL-1alpha, and IL-6, were significantly lower in MK2 KO tumors compared to WT tumors. Conclusion: These results show that tumoral MK2 signaling promotes tumor growth, metastasis, immunosuppressive inflammation, and pro-tumorigenic cytokine release which substantiates future therapeutic targeting of this pathway. Citation Format: Dakota D.D. Okwuone, Deri Morgan, Alyssa Schmidt, Devin Shrock, Yuting Lin, Hao Gao, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C010.

Abstract Despite current therapy, head and neck squamous cell carcinoma (HNSCC), the 7th most common cancer worldwide, is associated with high morbidity and mortality. Response to current therapies could be improved with a better understanding of the tumor microenvironment of HNSCC. We previously reported that cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the HNSCC microenvironment, have elevated levels of basal autophagy than oral fibroblasts from cancer-free subjects (NF). Disrupting autophagy in CAFs with Beclin-1 siRNA reduced secretion of IL-6, IL-8, and other factors known to promote HNSCC progression. The recent emerging role of autophagy in secretion of tumor promoting factors has high impact because (i) the mechanism can provide a new target to block the secretion of protumor factors from CAFs that contribute to HNSCC progression, and (ii) these cytokines can serve as potential indicators for the efficacy of autophagy-targeted therapies. To this end, we carried out an unbiased assessment of the proteins associated with autophagosomes in primary CAFs from HNSCC samples using mass spectrometry. We performed immunoprecipitation of LC3B from the isolated small vesicles of the CAFs, identified and validated the association of several trafficking proteins in secretory autophagy including VAMP3 and SNAP23. Further, the tripartite motif (TRIM) proteins have been shown to mediate the fate of autophagosomes either for degradation or secretion. We hypothesized that TRIM proteins are involved in transport of secretory autophagosomes. Our data demonstrate CAFs express higher levels of TRIM16 mRNA than NF. We demonstrate immunogold labeling of TRIM16 or IL-6 localized to the autophagosomes using transmission electron microscopy. Further, using immunofluorescence, we demonstrated the colocalization of TRIM16 and IL-6, as well as IL-6 and LC3B in cytoplasmic puncta. In addition, we used proximity ligation assays to confirm the interaction of TRIM16 and IL-6, as well as IL-6 and LC3B. TRIM16 knockdown using siRNA in CAFs reduced the secretion of IL-6 as measured by ELISA. In conclusion, we demonstrate an important role of TRIM16 in secretory autophagy making it a potential therapeutic target to mitigate CAF-mediated HNSCC growth. Citation Format: Thuc Ly, Bailey Pickard, Avisha Pandey, Noraida Martinez-Rivera, Eduardo Rosa-Molinar, Michael Washburn, Wen-Xing Ding, Sufi Mary Thomas. TRIM16 regulates IL-6 secretion in head and neck cancer-associated fibroblasts [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 6803.

PDF file, 289KB, Phosphorylated MAPK and AKT abrogated in C225-treated PDKM xenografts.

PDF file, 201KB, Biochemical effect of PDK1 targeting strategies on Akt and p70S6K.

PDF file - 122K, STAT3 decoy treatment does not inhibit cell viability or STAT3 target gene expression in A4 STAT3 null cells

CAF autophagy inhibition significantly decreases HNSCC in vitro progression.

<p>HNSCC Cells have Higher Glycolytic Capacity than CAFs and CAFs have Higher Maximal Respiration than HNSCC.</p>

Aim: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with a survival rate below fifty percent. Addressing meager therapeutic options, a series of small molecule inhibitors were screened for antitumor efficacy. The most potent analog, acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD; A-DiFiD), demonstrated strong cellular JUN proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (JUN, c-Jun) antagonism. c-Jun, an oncogenic transcription factor, promotes cancer progression, invasion, and adhesion; high (JUN) mRNA expression correlates with poorer HNSCC survival. Methods: Four new small molecules were generated for cytotoxicity screening in HNSCC cell lines. A-DiFiD-treated HNSCC cells were assessed for cytotoxicity, colony formation, invasion, migration, and adhesion. Dot blot array was used to identify targets. Phospho-c-Jun (p-c-Jun) expression was analyzed using immunoblotting. The Cancer Genome Atlas (TCGA) head and neck cancer datasets were utilized to determine overall patient survival. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets interfaced with University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) were analyzed to determine protein levels of c-Jun in HNSCC patients and correlate levels with patient. Results: Of the small molecules tested, A-DiFiD was the most potent in HNSCC lines, while demonstrating low half-maximal drug inhibitory concentration (IC50) in non-malignant Het-1A cells. Additionally, A-DiFiD abrogated cell invasion, migration, and colony formation. Phospho-kinase in vitro array demonstrated A-DiFiD reduced p-c-Jun. Likewise, a time dependent reduction in p-c-Jun was observed starting at 3 min post A-DiFiD treatment. TCGA Firehose Legacy vs. recurrent and metastatic head and neck cancer reveal a nearly 3% DNA amplification in recurrent/metastatic tumor compared to below 1% in primary tumors that had no lymph node metastasis. CPTAC analysis show higher tumor c-Jun levels compared to normal. Patients with high JUN expression had significantly reduced 3-year survival. Conclusions: A-DiFiD targets c-Jun, a clinical HNSCC driver, with potent anti-tumor effects.

Ovarian cancer (OvCa) is a deadly gynecologic malignancy that presents many clinical challenges due to late-stage diagnoses and the development of acquired resistance to standard-of-care treatment protocols. There is an increasing body of evidence suggesting that STATs may play a critical role in OvCa progression, resistance, and disease recurrence, and thus we sought to compile a comprehensive review to summarize the current state of knowledge on the topic. We have examined peer reviewed literature to delineate the role of STATs in both cancer cells and cells within the tumor microenvironment. In addition to summarizing the current knowledge of STAT biology in OvCa, we have also examined the capacity of small molecule inhibitor development to target specific STATs and progress toward clinical applications. From our research, the best studied and targeted factors are STAT3 and STAT5, which has resulted in the development of several inhibitors that are under current evaluation in clinical trials. There remain gaps in understanding the role of STAT1, STAT2, STAT4, and STAT6, due to limited reports in the current literature; as such, further studies to establish their implications in OvCa are necessitated. Moreover, due to the deficiency in our understanding of these STATs, selective inhibitors also remain elusive, and therefore present opportunities for discovery.