Tumoral Skin Invasion Is an Independent Predictor of Rapid Recurrence in Head and Neck Cancer.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. To identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant intercell line differences for 42 kinases (p < 0.05), and loss of function experiments using siRNA in high and low expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1, and EPHA2 involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib, and erlotinib as well as siRNA led to strong reduction of viability in high but not low expressing lines, confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.

Head and Neck Cancer

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&amp;lt;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&amp;lt;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.

Understanding of p53 family protein (p53, p63 and p73) networks in head and neck squamous cell carcinoma (HNSCC), can positively influence in cancer screening, diagnosis, treatment and prevention. P53 family proteins are regulating diverse cell signalling pathways at diverse conditions to determine cell fate. At each condition of cells state, how these proteins are controlling cell fate is more complexed due to the presense of several isoforms for each p53 family proteins and their multifaceted interactions. Like other solid tumours, the p53 pathway is disabled by several mechanisms in HNSCC. Despite of the convincing evidence of a high frequency of p53 mutations in HNSCC, a subset of cancers arise in the absence of mutations. The molecular mechaninsms through which p53 lacking mutations subvert its tumor supressor functions in HNSCC still remain uncertain. In fact, some cancers and established cancer cell lines are over-expressing wild type p53 protein makes questionable of p53 role as only a tumor suppressor or it has some other additional functions, even in cancer cells. For an answer of this hypothesis, we have performed detailed molecular characterization, in an invitro model of head and neck cancer, in a broad panel of 12 newly established HNSCC cell lines. In our studies, we found that some head and neck cancer cell lines are accumulating wild p53 protein hyperphosphorylated at serine15 and 392 and it leads to the over expression of DNp73, the mechanism already reported from our laboratory in HPV38 immortalized keratinocytes. To better understand the functions of accumulated wild p53 role in HPV positive and negative cancer cell lines, we have performed p53 knock down by siRNA and the results have showed that p53 knock down inhibited cell proliferation in both HPV positive and negative cancer cells. Moreover, p53 knock down induced significant morphological changes and senescence associated beta galactosidase activity in these cells. Our results pinpoint, wild-type p53 protein accumulated in transformed cell lines has an additional role in cell proliferation other than its well known tumor supressor activity. Moreover, the key role of p53 family network in modulating epidermal growth factor receptor (EGFR) expression and in controlling cell proliferation and apoptosis arises the question of whether p53 family proteins status influences the efficacy of EGFR inhibitors, investigating its ability to reduce cell growth, to induce apoptosis and to modulate cell cycle and various EGFR pathwayrelated targets in head and neck cancer. Because, recently improved understanding of the pathogenesis of human head and neck squamous cell carcinoma has led to the development of new, molecular based therapeutic strategies, one of the most promising is the utilization of tyrosine kinase (TK) inhibitors, targeting EGFR. The comparison between the targets analysed and gefitinib effectiveness evidenced the absence of a clear relationship, exluding them as predictive factors for gefitinib efficacy. Our results confirmed the in vitro efficacy of an anti-EGFR approach, but other targets than those analysed here should be characterized in order to identify valid predictive factors for gefitinib utilization in head and neck cancer. We have also performed chemoresistance analysis in our invitro model of HNSCC cell lilnes and found that p53 family network has less predictive role in HNSCC chemoresistance but ABCG2, a multidrug resistance protein, has over-expressed in HNSCC.

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

Head and neck cancer is the sixth most common cancer worldwide and associated with a poor clinical prognosis, due to development of recurrent tumors and metastasis. Tumor recurrence and low patient survival are strongly linked with the ability of tumor cells to invade and infiltrate the surrounding tissue. Stress-activated protein kinases (SAPK), particularly p38, are known to regulate a wide range of cellular phenotypes, including cell invasion via the activity of secreted proteases. The proliferation-associated Forkhead box protein M1 (FOXM1) transcription factor, a p38 downstream target, plays a role in the development and growth of many cancer types. However, only very little is known about the role of p38 and FOXM1 in invasive processes of head and neck cancer and the exact mechanism underlying this process. In this work we examined the downstream events of p38 signaling primarily focusing on the role of FOXM1 transcription factor in regulation of the urokinase-type plasminogen activator (uPA) gene and invasion of head and neck squamous cell carcinoma (HNSCC) cells. Using different HNSCC cell lines, we confirm that p38 regulates FOXM1 expression and provide evidence that p38 signaling driven in vitro invasion of HNSCC cells requires FOXM1 expression. Furthermore, siRNA-mediated FOXM1 knockdown is sufficient to inhibit the invasive behavior of HNSCC cells in vitro. By using reporter gene assays, bioinformatical analysis of the publically available ChIP-Seq data, chromatin immunoprecipitation assays, and transplantation-based mouse model of oral cancer, we identified the molecular mechanism of FOXM1-mediated invasion of HNSCC cells. FOXM1 controls the uPA-dependent invasion via activation of c-Fos and thus drives AP-1 activity on the uPA promoter, which enhances its expression and proteolytic activity. Further, an activated Ras signaling is necessary for a potent FOXM1-mediated uPA activity and tumor formation. The data are supported by a bioinformatical study, demonstrating concomitant up-regulation of FOXM1 and uPA in oral dysplasia and SCCs of head and neck, oesophagus, lung and cervix. In the mouse model of oral cancer we show that uPA expression is upregulated in recurrent tumors compared to primary tumors, giving further evidence for a crucial role of the p38-FOXM1-uPA axis in the development of recurrent tumors. Taken together, we conclude that the stress signalling cascade requires a FOXM1-dependent intermediate step preceding the activation of AP-1 transcription factor to enhance invasive behaviour of tumor cells. This novel mechanism promotes invasion of HNSCC and may provide a potential target for the adjuvant therapy of these highly invasive cancers.

The American Joint Committee on Cancer staging system (Cancer Staging Manual, 8th Edition) for head and neck squamous cell carcinoma (HNSCC) now categorizes human papillomavirus (HPV)-negative HNSCC in a single positive lymph node smaller than 3 cm with pathologic extranodal extension (ENE) as N2a. The standard of care for pathologic ENE is adjuvant chemoradiation therapy (CRT). Whether adding chemotherapy concurrent with adjuvant radiation therapy improves survival in this clinical scenario is unknown. To assess whether adjuvant CRT relative to radiation therapy alone is associated with improved survival among patients with pN2a HPV-negative HNSCC with ENE. This retrospective cohort study included 504 patients with pN2a HPV-negative HNSCC with ENE who had undergone margin-negative surgery and adjuvant therapy. The patients were identified from the National Cancer Database from January 1, 2004, to December 31, 2015. Statistical analyses were conducted from September 1, 2019, to April 16, 2020. The primary end point was overall survival. The association of adjuvant CRT with overall survival was analyzed using univariate and multivariate Cox proportional hazards regression analyses. Planned subset analyses were conducted in patients younger than 70 years with no comorbidities (the subset most likely to be eligible for a clinical trial of cisplatin-based chemoradiation) and in patients with pT3/T4 disease classification. Of 504 patients (mean [SD] age, 60.5 [12.7] years; 319 [63.3%] men; 434 [86.1%] White) with pN2a HPV-negative HNSCC with ENE who had undergone margin-negative surgery and adjuvant therapy, 298 patients (59.1%) received adjuvant CRT. For the overall cohort of patients with pN2a ENE, adjuvant CRT was not associated with improved overall survival relative to adjuvant radiation therapy alone in a multivariate analysis (adjusted hazard ratio, 0.98; 95% CI, 0.74-1.30). Adjuvant CRT was still not associated with improved overall survival in a subset analysis of 304 patients younger than 70 years with no comorbidities (adjusted hazard ratio, 0.98; 95% CI, 0.66-1.45) nor in a subset of 220 patients with pT3/T4 disease classification (adjusted hazard ratio, 1.03; 95% CI, 0.70-1.54). This study found that for patients with pN2a HPV-negative HNSCC with ENE who underwent margin-negative surgery and adjuvant therapy, adding chemotherapy concurrent with adjuvant radiation therapy was not associated with improved overall survival. Additional research is necessary to identify the optimal treatment paradigm for this clinical scenario.

HNSCC is a common, deadly, and disfiguring disease. While combinations of radiotherapy, surgery, and chemotherapy are highly effective in HNSCC, there is significant morbidity associated with the disease and recurrence is common. There is great interest in identifying molecular events and pathways which drive HNSCC progression in order that targeted therapies may be developed. The loss of the tumor suppressor function such as p53 and NOTCH1, as well as activation of the STAT3 and STAT5 pathways, has been implicated in HNSCC progression. We have identified interactions between STAT3, STAT5, and the novel tumor suppressor SOCS2 to be important in HNSCC. To extend these findings to humans, we performed experiments to identify prognostic markers in HNSCC and to investigate the expression of STAT3, STAT5, and SOCS2 in HNSCC tissue. Immunohistochemistry was performed on tissue microarrays from resected tumor specimens of 123 stage I-IVB oral cavity SCC patients (treated with surgery +/- adjuvant radiation therapy) with annotated clinical outcome information from a median follow up of 76 months collected at the UT/MD Anderson Cancer Center. The array was screened with antibodies against STAT5, STAT3, and SOCS2, and scored in a blinded fashion by a pathologist. SOCS2 expression (present vs. absent) correlated significantly with recurrence-free survival in both the univariate (hazard ratio 0.24, p=0.0003; Cox proportional hazards model) and multivariate (hazard ratio 0.24, p=0.0004) analyses. Notably all patients who lacked SOCS2 tumor expression recurred within 45 months. There was a trend towards a correlation of SOCS2 expression with overall survival (HR 0.50, p=0.11) and disease-specific survival (HR 0.52, p=0.28). SOCS2 expression did not significantly correlate with tumor stage and extracapsular extension emphasizing that its absence is an independent marker of poor prognosis. Our prior published work demonstrates that STAT5 drives SOCS2 expression. The expression of SOCS2 in these patient samples positively correlated with total and phosphoSTAT5 (r=0.29 and 0.21 respectively; p&amp;lt;0.05) as well as total and phospho-STAT3 (r=0.27 and 0.26, respectively; p&amp;lt;0.05). Likewise, we measured total and phosphoSTAT3, total and phosphoSTAT5, and SOCS2 in a panel of HNSCC cell lines. SOCS2 protein levels and phosphorylation of STAT3 and STAT5 varied among cell lines, however cells with elevated phosphoSTAT3 and STAT5 also showed elevated SOCS2 protein. This is the first study which identifies SOCS2 as an independent marker of prognosis in HNSCC. In support of its role as a tumor suppressor, the lack of SOSC2 expression was associated with universal recurrence. This work was supported The University of Texas SPORE in Head and Neck Cancer (P50 CA097007), The Cancer Center Support Grant, and the ASCO Cancer Foundation Young Investigator Award (WW). Citation Format: Courtney Nicholas, Maria I. Nunez, Nusrat Harun, J. Jack Lee, Jeffrey Myers, Ignacio I. Wistuba, Banibrata Sen, Adel K. El-Naggar, Stephen Y. Lai, Faye M. Johnson, William N. William. SOCS2 (suppressor of cytokine signaling protein 2) is a prognostic indicator of progression-free survival in head and neck squamous cell carcinoma (HNSCC) patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2013-5578

Introduction: Head and neck squamous cell carcinomas (HNSCCs) afflict over half a million patients annually worldwide. In the absence of disease at distant sites, salvage treatment may provide durable disease control in only ∼15% of such patients. Our research goal is to improve radiotherapy for aggressive HNSCCs by identifying novel targets for radiosensitization. The EphB4 receptor is ubiquitously expressed in HNSCCs and has been shown to promote tumorigenic and invasive properties of HNSCCs but the effect of EphB4 on cellular radiosensitization has not been investigated. We hypothesize that knockdown of EphB4 receptor will enhance radiosensitization of HNSCCs by inhibiting EphB4 targets involved in radioresistance. Materials/Methods: To determine the in vitro radiosensitization effect following EphB4 knockdown, we used EphB4-targeting siRNA in clonogenic assays in HNSCC cell lines. Effects of EphB4-siRNA on cell cycle progression, DNA damage response, and cell death pathways were also investigated. For in-vivo testing, an EphB4 blocking protein was used to investigate the radiosensitization in a patient derived xenograft (PDX) model of HNSCC. Results: We observed a decrease in the survival fractions in HNSCC cell lines following knockdown of EphB4 at increasing doses of radiation. Cell cycle analysis showed an enhanced G2 arrest of HNSCC cells following EphB4 knockdown and radiation exposure. We also observed an increase in the expression of p-H2AX, a DNA damage marker protein, in HNSCC cells suggesting activation of DNA damage response pathway following EphB4 knockdown and radiation exposure. This was further accompanied by enhanced percentage of apoptotic cells as evident in TUNEL assay and modulation of key apoptotic markers. Several pro-survival markers, including, p-AKT, p-EGFR, EGFR, and Bcl-XL were markedly decreased in the EphB4-knockdown and radiation combination group. Data using an in-vivo PDX animal model of HNSCC showed reduction in tumor growth following administration with an optimal dose of EphB4 blocking protein or XRT alone. This reduction in tumor growth was significantly augmented when EphB4 blocking protein was administered in combination with radiation demonstrating radiosensitization effect in an in vivo PDX model of HNSCC. Conclusions: Our findings support the hypothesis that EphB4 promotes resistance of HNSCCs to ionizing radiation and its targeted inhibition will therefore result in enhanced radiosensitization both in vitro and in vivo. This data should serve as the basis for a clinical trial design for combination therapy of anti-EphB4 with radiation in patients with locally advanced head and neck cancer. Citation Format: Shilpa Bhatia, Kellen Hirsch, Jaspreet Sharma, Stephen Keysar, Antonio Jimeno, Parkash Gill, Xiao-Jing Wang, Sana D. Karam. Role of EphB4 in radiosensitization of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-127.

6053 Background: Several studies have demonstrated a negative prognostic impact of tumor cell budding (TCB) in HPV- head and neck squamous cell carcinoma (HNSCC), but analyses of its prognostic impact in HPV+ HNSCC and of the underlying molecular alterations are lacking. Methods: The study cohort included 331 HPV- and HPV+ HNSCC from TCGA with digitalized H&amp;E-stained slides available. Corresponding mutation, methylation, and gene expression data were obtained from the pan-cancer atlas web page. Tumor buds were defined as clusters of up to four tumor cells separated from the tumor mass. The numbers of tumor buds were evaluated in ten digital high-power fields by a senior pathologist. A two-tier cellular dissociation grading system was introduced by separating tumors with six or more tumor buds (TCB-high) from tumors with fewer tumor buds (TCB-low). The impact of TCB on overall survival (OS) was analyzed using Cox proportional hazard models. Results with p &lt; 0.05 were considered significant. Association of TCB with mutations was analyzed using the Wilcoxon test. Association of TCB with gene expression and methylation was analyzed using Spearman correlations. Lists of altered genes were compiled correcting the p-values with the Benjamin-Hochberg method and controlling the FDR at 5%. Results: In a univariate analysis, OS was significantly shorter in TCB-high tumors compared to TCB-low tumors in HPV- HNSCC (HR = 1.4, 95% CI 1.0-2.3) and HPV+ HNSCC (HR = 5.0, 95% CI 1.9-13.7). Shorter OS in TCB-high HNSCC was confirmed in a multivariate analysis including age, sex, HPV status, tumor site, tumors stage, and tumor margin status (HR = 1.7, 95% CI 1.2-2.4). Significant association of TCB with mutations was detected for two genes: NSD1 mutations correlated negatively with TCB in HPV- HNSCC, while TP53 mutations correlated positively with TCB in HPV+ HNSCC. Methylation of 126 (1%) genes was associated with TCB in HPV- HNSCC, while methylation of 511 (3%) genes was associated with TCB in HPV+ HNSCC. Expression of 422 (2%) genes was associated with TCB in HPV- HNSCC, while expression of 786 (4%) genes was associated with TCB in HPV+ HNSCC. Among these genes, those annotated to the epithelial mesenchymal transition were highly significantly enriched in both HPV- HNSCC (5.5-fold enrichment) and HPV+ HNSCC (2.7-fold enrichment). Conclusions: Evaluation of TCB based on digital HE slides was conducted in a large clinically and molecularly characterized HNSCC cohort. The prognostic impact of TCB could be validated in HPV- HNSCC, while a prognostic impact of TCB could be demonstrated for the first time in HPV+ HNSCC. TCB correlated with mutations of only two genes and theses correlations were imperfect. A plethora of genes correlated with TCB on the level of methylation and gene expression level. These genes should be further analyzed and prioritized for the evaluation of therapeutic targeting.

6047 Background: Increasing use of transoral robotic surgery (TORS) for human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) is likely to impact recurrence patterns and outcomes. Profiling HPV+ HNSCC recurrences after TORS and identifying features predictive of lethal outcome would facilitate tailoring adjuvant therapy and guide surveillance post-therapy. This study uses long term follow-up of patients at the first institution to bring TORS into clinical use to describe the recurrence patterns, distinguish outcomes associated with distinct patterns, and create a risk model for lethal recurrence. Methods: This retrospective cohort study at a single academic tertiary center analyzed 634 consecutive, treatment-naïve HPV+ HNSCC patients receiving TORS and neck dissection for clinical features at presentation and pathologic traits identified by surgical resection. The main outcomes were distant metastatic recurrence (DMR) and locoregional recurrence (LRR). Multivariate logistic regression with backward stepwise elimination was used to identify features associated with recurrence. Results: 6.5% of patients developed DMR at a median of 12.4 months after surgery and had a 5-year overall survival (OS) of 52.5% (95% CI, 33.9%-68.2%), whereas the 6.2% patients developing LRR alone had 5-year OS of 83.3% (95% CI, 66.2%-92.2%; P =.01). After recurrence, 5-year progression-free survival was 24.7% (95% CI, 11.4%-40.7%) for DMR cases and 85.7% (95% CI, 65.1-94.6%) for cases with LRR alone (P &lt;.001). Comparing recurrent cases to recurrence-free controls showed DMR to be independently associated with positive surgical margins (AOR 5.7; 95% CI, 2.1-15.7) and advanced clinical stage at presentation (AOR 6.5; 95% CI, 1.9-23.0). Positive margins increased DMR risk by 4.2-fold and reduced 5-year disease-free survival (P &lt;.001) in early-stage cases (Table), which comprised 95% of the cohort. By contrast, isolated LRR was associated with failure to receive indicated adjuvant therapy and was usually controllable by salvage therapy. Conclusions: Based on the largest single institution cohort reported to date, long term oncologic outcomes for HPV+ HNSCCs after TORS are excellent overall. While DMR is often fatal, LRR is salvageable with durable disease control. In addition to standard staging criteria, positive margins indicate substantially higher risk of DMR but not LRR. A risk model for DMR that incorporates margin status after TORS is relevant for guiding clinical trial design and whole-body surveillance.[Table: see text]