The role of metformin in dysplastic mucosa of the larynx: A follow up study.

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

Background: Metformin, an oral anti-hyperglycemic drug which inhibits mitochondrial complex I and oxidative phosphorylation has been reported to correlate with improved outcomes in head and neck squamous cell carcinoma (HNSCC) and other cancers. This effect is postulated to occur through disruption of tumor-driven metabolic and immune dysregulation in the tumor microenvironment (TME). We report new findings on the impact of metformin on the tumor and immune elements of the TME from a clinical trial of metformin in HNSCC.Methods: Human papilloma virus—(HPV–) tobacco+ mucosal HNSCC samples (n = 12) were compared to HPV+ oropharyngeal squamous cell carcinoma (OPSCC) samples (n = 17) from patients enrolled in a clinical trial. Apoptosis in tumor samples pre- and post-treatment with metformin was compared by deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Metastatic lymph nodes with extra-capsular extension (ECE) in metformin-treated patients (n = 7) were compared to archival lymph node samples with ECE (n = 11) for differences in immune markers quantified by digital image analysis using co-localization and nuclear algorithms (PD-L1, FoxP3, CD163, CD8).Results: HPV–, tobacco + HNSCC (mean Δ 13.7/high power field) specimens had a significantly higher increase in apoptosis compared to HPV+ OPSCC specimens (mean Δ 5.7/high power field) (p < 0.001). Analysis of the stroma at the invasive front in ECE nodal specimens from both HPV—HNSCC and HPV+ OPSCC metformin treated specimens showed increased CD8+ effector T cell infiltrate (mean 22.8%) compared to archival specimens (mean 10.7%) (p = 0.006). Similarly, metformin treated specimens showed an increased FoxP3+ regulatory T cell infiltrate (mean 9%) compared to non-treated archival specimens (mean 5%) (p = 0.019).Conclusions: This study presents novel data demonstrating that metformin differentially impacts HNSCC subtypes with greater apoptosis in HPV—HNSCC compared to HPV+ OPSCC. Moreover, we present the first in vivo human evidence that metformin may also trigger increased CD8+ Teff and FoxP3+ Tregs in the TME, suggesting an immunomodulatory effect in HNSCC. Further research is necessary to assess the effect of metformin on the TME of HNSCC.

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

CHK1 plays a critical role in DNA damage repair (DDR) pathways as well as in coordinating DNA replication. Selective CHK1/CHK2 compounds are being tested in clinical trials but predictive biomarkers of patient response are lacking. A phase 1b expansion cohort study (I4D-MC-JTJA, NCT01115790) with the CHK 1 inhibitor, prexasertib, included patients with advanced, metastatic head and neck squamous cell carcinoma (HNSCC) or squamous cell carcinoma of the anus (SCCA). To identify genomic biomarkers associated with single-agent drug response, pre-treatment tissues (archived or biopsy) from 71 consented patients (HNSCC=47, SCCA=24) were subjected to next-generation sequencing (NGS) using the FoundationOne gene panel. In this subset of patients, the disease control rate (DCR) (Complete Response (CR) + Partial Response (PR) + Stable Disease (SD) based on RECIST Criteria (v 1.1)) was 60% (28/47) and 75% (18/24), respectively. We present here the observed genetic alterations corresponding to three pathways, Cell Cycle, DNA Damage Repair (DDR) and PI3K. In addition, patients’ human papillomavirus (HPV) carrier status was inferred from DNA sequencing using HPV-specific capture probes. HPV+ was 47% for HNSCC and 87% for SCCA. HPV+ and TP53 mutations were mutually exclusive across the two patient cohorts. In HNSCC patients with evaluable progression-free survival (PFS) data, greater clinical efficacy was observed in the HPV+ cohort (median PFS: 4.5 vs 1.4 months, log-rank p = 0.0008). Known or likely loss-of-function (LOF) mutations in FBXW7 and PARK2, two genes implicated in Cyclin E1 proteolysis, were noted in patients with favorable response in both tumor types. Across both HNSCC and SCCA cohorts, mutations and/or germline variants in the DDR genes BRCA1, BRCA2, MRE11A and ATR but not in Fanconi (FANC) pathway genes, were found in patients with treatment benefit. Whereas PIK3CA mutations were infrequent in the HNSCC cohort, in SCCA, mutations occurred in 5/8 (63%) patients with disease control vs 1/6 (17%) with PD. All 7 PI3KCA mutations observed in HPV+ HNSCC and SCCA patients mapped to the helical domain suggestive of Apobec-induced mutagenesis as their source of origin. The enhanced clinical benefit to prexasertib associated with HPV+ in HNSCC may reflect a prognostic effect. Alternatively, the clinical biomarker findings may support the hypothesis that oncogene-induced replication stress (RS) (i.e. arising from HPV E6/E7 and/or FBXW7 loss-dependent Cyclin E1 dysregulation) in the context of attenuated DDR (i.e. BRCA1/2, MRE11A mutations) may sensitize patients to prexasertib monotherapy. Citation Format: Ricardo Martinez, Sameera R. Wijayawardana, David Hong, Johanna Bendell, Anna Maria Russell, Richard P. Beckmann, Aimee Bence Lin. A clinical genomic biomarker study of the CHK1 inhibitor prexasertib in advanced head and neck squamous cancer and squamous cell carcinoma of the anus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2017-1778

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

Heat Shock Protein 27 as a New Therapeutic Target for Radiation Sensitization of Head and Neck Squamous Cell Carcinoma

Introduction: Head and neck squamous cell carcinoma (HNSCC) incidence and survivorship has been thoroughly described in the literature. Most of the literature has mainly focused on older adults. However, there are documented cases of early-onset HNSCC, typically in adolescents and young adults (AYA) aged 15-39 years. In fact, 12% of all pediatric cancers are in the head and neck region. As individuals live longer in the general population, it will become more important to track AYA cancer cases due to the potential for treatment effects, late toxicities, and comorbidities after cancer survivorship. Additionally, the emergence of the human papillomavirus (HPV)-associated oropharyngeal cancer as a dominant head and neck cancer means that more HNSCC patients are diagnosed at a younger age than previously known. These patients with HPV-associated oropharyngeal cancer are likely to live longer since HPV-associated HNSCC typically has better prognosis than other HNSCC. While numerous studies have investigated HNSCC survivorship in older adults and the elderly, limited research exists describing the incidence and survivorship in adolescents and young adults. The aim of this study is to characterize survivorship of HNSCC in the AYA population. Methods: In this retrospective study, we utilized a patient cohort of 3,366 first primary HNSCC cases from the Surveillance, Epidemiology, and End Results (SEER) 18 database diagnosed between ages 15-39 and the years 2000-2014. Actuarial survival curves stratified by age group (15-29, 30-34, 35-39) indicated differences in HNSCC survival among groups with a log-rank test. Patient characteristics including age, sex, race/ethnicity, HNSCC site, stage, and treatment modality were utilized in a Fine and Gray competing risk proportional hazard model to examine their impact on HNSCC death in this cohort. Results: The cohort was mostly male (65.1%) with an average age of 33.6. There was no significant HNSCC survival difference between the age-stratified survival curves (log-rank p=0.83). The Fine and Gray model also did not find a significant effect for age. All race/ethnicity groups had a significantly increased hazard of HNSCC death compared with non-Hispanic whites, with non-Hispanic American Indians/Alaska Natives having the highest increased hazard (aHR=4.01, 95% CI: 2.18, 7.38). Each increasing year of diagnosis was associated with a 5% decrease in hazard of death from HNSCC (aHR=0.95, 95% CI: 0.93, 0.97). Regional (aHR=3.90, 95% CI: 3.13, 4.86) and distant (aHR=6.77, 95% CI: 5.27, 8.70) stage had a higher HNSCC death hazard compared with localized stage. Patients who did not receive surgery had a 93% (aHR=1.93, 95% CI: 1.60, 2.33) increased hazard of HNSCC death compared with those who received surgery. Compared with oropharyngeal cancer, hypopharyngeal, oral cavity, and sinonasal cancers were associated with significantly increased hazard of HNSCC death, while nasopharynx was associated with a decreased hazard (aHR=0.68, 95% CI 0.52, 0.90). Conclusions: We found no survival differences between adolescents and young adults based on age at diagnosis; however, minorities, especially American Indians/Alaskan Natives, had the worst survival outcomes. Also, patients who did not have the definite treatment of surgery and had regional or distant stage faced increased death from HNSCC. More research needs to be done to understand the mechanisms underlying the survival disparities noted among minorities, especially American Indians/Alaskan Natives. Efforts should also focus on establishing risk factor awareness and educational interventions, earlier screening measures, and patient navigator programs to improve prognosis of HNSCC in the American Indian/Alaskan Natives and other high-risk populations. Citation Format: Sai D. Challapalli, Eric Adjei Boakye, Matthew C. Simpson, Nosayaba Osazuwa-Peters. Head and neck squamous cell carcinoma in adolescents and young adults: Survivorship patterns and disparities [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A33.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies with a low survival rate for its ability of metastasis. Therefore, finding the potential biomarkers for the diagnostic, as well as therapy and prognostic of HNSCC metastasis is extremely urgent. Previous studies have demonstrated that microRNAs, a 21-23 nucleotide strands of endogenous single-stranded RNAs, which could regulate the expression of numerous genes post-transcriptionally, played a key role in the invasion and metastasis of human cancer. In this study, we attempted to explore the function and mechanism of microRNAs in invasion and migration of HNSCC, and hoped to provide a novel diagnostic and therapeutic strategy for HNSCC patient with metastasis. Materials and Methods: In order to identify a new microRNA/gene pathway that regulates the invasion and metastasis in HNSCC, we performed a microarray assay to explore the microRNAs expression profiling in HNSCC cells with varying invasiveness. Quantitative RT (qRT)-PCR and functional analysis were used to confirm the role of microRNA, which we focused on, in HNSCC invasion. Then we predicted the targeted gene of microRNA through a bioinformatics approach and defined it by luciferase assay and western blot. At last, we utilized the buccal mucosa-lymph node metastasis model to detect the effect of microRNA in HNSCC metastasis in vivo. Results: Here, we established the role and mechanisms of miR-223 in metastasis of HNSCC. First, we profiled microRNA expression in four HNSCC cells with varying invasive capacity. MiR-223 was highly down regulated in cells with highly invasive capacity. The negative correlation between miR-223 expression and invasiveness was confirmed in another validation cohort of seven HNSCC cells. Functional analysis showed that ectopic expression of miR-223 repressed HNSCC cell invasiveness; on the other hand, miR-223 inhibition resulted in increased HNSCC cell invasiveness. Furthermore, the expression level of TCF7L2, an important transcription factor in Wnt pathway, was repressed when overexpressing the miR-223 expression in HNSCC cells, whereas miR-223 silencing increased its expression. Next, we showed that miR-223 could bind to the 3′-untranslated region of TCF7L2 by luciferase assay. Moreover, we demonstrated that TCF7L2 enhanced the invasion of HNSCC cells, and the silencing of TCF7L2 inhibited invasion. Most importantly, increased miR-223 expression level in HNSCC cell repressed the metastasis of lymph node in vivo, and decreased the miR-223 expression enhanced the metastasis. Conclusions: Taken together, these data indicated that miR-223 played a key role in HNSCC invasion and metastasis by targeting to the 3′UTR of TCF7L2. And our results presented evidences for a potential anti-metastasis therapeutic value of miR-223 in HNSCC patients. Citation Format: Yun Wang, Zhi Wang, Bin Cheng. Role and mechanisms of miR-223 in the invasion and metastasis of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 177. doi:10.1158/1538-7445.AM2015-177

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.

INTRODUCTION: FAT1 alterations with somatic mutations or deletions are observed in head and neck squamous cell carcinomas (HNSCC). These alterations lead to loss of function of FAT1. FAT1 loss of function was known to be associated with aberrant Wnt activation, cell migration and invasion. In this study, we present analysis and outcomes of co-mutations seen in FAT1-mutation positive HNSCC. METHODS: TCGA data were analyzed for mutation, gene expression data and overall survival. We examined mutation data in 415 HPV negative samples and 72 HPV positive samples. The Fisher's Exact test was used to determine if differences in mutation frequency existed between the different cohorts. Mantel-Cox method was used for overall survival (OS) comparison between the cohorts. RESULTS: FAT1 somatic mutation was the third most commonly mutated gene in HPV-negative HNSCC while it was not common in HPV-positive HNSCC. FAT1 mutation was seen in 25.8% of HPV negative HNSCC (107/415), while 4.2% (3/72) HPV positive HNSCC had FAT1 mutation. We then analyzed the frequency of co-mutations in FAT1-mutated HPV-negative HNSCC. CDKN2A, NOTCH1, CASP8, FBXW7, ZFHX4, HRAS, DCC, ATR mutations were more common in FAT1-mutated cancers. CASP8 mutation was seen in 24.30% (26 out of 107 samples) of FAT1-mutations positive HPV negative HNSCC, while it was less common in FAT1-mutation negative HNSCC (9.08% 28/308) (P=0.002). The patients with FAT1 and CASP8 co-mutation were associated with shorter overall survival when compared with patients with FAT1-mutation (19.8 months vs 52.3 months P=0.02). FAT1 only or CASP8 only mutation without co-mutation did not affect survival. To determine the mutation impact of the FAT1 and CASP8 mutations in HNSCC, differentially expressed genes between mutated and non-mutated genes were analyzed in HNSCC. CASP8 mutation was the top gene among the genes with somatic mutation in HNSCC and associated with gene expression changes in more than 6000 genes. FAT1 was ranked 11th resulting in differential gene expression in more than 1000 genes. DISCUSSION: FAT1 somatic mutations are commonly seen in HPV negative HNSCC. We have demonstrated for the first time that CASP8 and FAT1 co-mutations are associated with poor prognosis with decreased overall survival in HPV negative HNSCC. Both CASP8 and FAT1 mutations are among the top genes with mutation impact on differential gene expression in HNSCC suggesting significance of these mutations in HNSCC. Citation Format: Emrullah Yilmaz, David Lee, Andrew Cowan, Gregory Gan, Michelle Ozbun, Yan Guo. FAT1 and CASP8 co-mutations are associated with poor prognosis in HPV negative head and neck squamous cell carcinoma [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 6474.

Head and neck squamous cell carcinoma (HNSCC) has a high mortality rate, and with rising incidences in Asia. Previously, we demonstrated that the PI3K pathway is the most frequently altered pathway in HNSCC (Lui et al, Cancer Discovery, 2013). However, cumulative PI3K pathway aberrations, nor PIK3CA aberrations, are not associated with clinical outcomes in HNSCC patients (P=n.s.). In this study, we investigate the prognostic significance of RAPTOR (Regulatory associated protein of mTOR), a key component of the PI3K signaling pathway, in HNSCC. To examine the clinical relevance of RAPTOR levels in human cancers, including HNSCC, we performed pan-cancer analyses of RAPTOR protein levels in the Cancer Proteome Atlas datasets (TCPA, USA). Strikingly, across 28 tumor types examined, we found that RAPTOR protein overexpression is most significantly associated with poor patient survival in HNSCC (P=0.00162***), followed by renal clear cell carcinoma (P=0.00198), and stomach cancer (P=0.0443; Log-Rank test; Table 1). We then extracted the original quantification data of RAPTOR protein array of HNC (TCPA, USA), and performed RAPTOR expression-survival correlation analyses on quantitative basis by Kaplan-Meier analysis with Log-Rank test. We found that HNSCC patients with RAPTOR protein overexpression in their primary tumors (mean+5SEM) have significantly poorer overall survival (median OS=27.56 months) than patients with RAPTOR protein underexpression (mean-5SEM; median OS=68.43 months). Moreover, this group of RAPTOR-high patients are found to have a higher risk of recurrences, with an Odd Ratio of 1.75 (P=0.05; Fisher’s Exact test). Notably, this poor OS of RAPTOR-high HNSCC is much shorter than that of TP53-mutated HNSCC in the same cohort (45.8 months; TCGA Provisional,N=510). Further, patients with extreme-high levels of RAPTOR protein expression (top 5% cases) have an even worst OS of only 11.56 months vs 48.16 months (the remaining 95% cases). Using our small cohort of Asian HNSCC tumors as a validation cohort, we show that RAPTOR protein overexpression is very common in Asian HNSCC (37.5%; 6/16 cases), suggestive of its likely contribution to HNSCC in Asia as well. In conclusion, RAPTOR-high status is likely to be a useful predictive of very poor HNSCC patient survival, possibly superior than the previously known survival-related biomarker,TP53 mutation. RAPTOR is one of the first components of the PI3K pathway shown to be associated with patient outcome in HNSCC. Acknowledgements:VWYL receives fundings from Health and Medical Research Fund(#15160691), Research Grant Council, Hong Kong(GRF#1711484,#17121616,# 14168571;T12-401/13-R), UICP(UIM/329;Innovation and Technology Fund); Hong Kong Cancer Fund. YCL is supported by Postdoctoral Hub(UIM/329) from Innovation and Technology Fund, Hong Kong government.J YKC supported by Dr Stanley Ho Medical Foundation. Citation Format: Hui Yan Poon, Yuchen Liu, Yu Xiong SU, Jason Y.k. Chan, Chin Wang Lau, Vivian W y Lui. RAPTOR protein overexpression is predictive of poor clinical outcomes in head and neck squamous cell carcinoma (HNSCC) patients [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 4933.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with approximately 650,000 new cases diagnosed annually and 400,000 HNSCC-related deaths worldwide each year. HNSCC is typically diagnosed when already at an advanced stage. Despite advancements in surgery, radiation therapy, and chemotherapy, patients with advanced HNSCC have a poor prognosis. The median overall survival time for patients with recurrence and metastasis is 10-13 months in the setting of first-line chemotherapy and 6 months in the second-line setting. Many studies have shown that noncoding RNAs encoded by the human genome are functional and play critical roles in various cellular processes, e.g., cell growth, migration, invasion, and apoptosis. Among noncoding RNAs, microRNAs (miRNAs) are endogenous single-stranded RNA molecules comprising 19-22 nucleotides that function as fine-tuners of RNA expression. A single miRNA regulates many RNA transcripts, and bioinformatics studies have shown that more than half of protein-coding genes are controlled by miRNAs. Aberrantly expressed miRNAs are closely associated with cancer pathogenesis via the disruption of RNA networks within cancer cells. To identify novel oncogenic targets in head and neck squamous cell carcinoma (HNSCC), we have analyzed antitumor microRNAs (miRNAs) and their controlled molecular networks in HNSCC cells. Based on our miRNA signature in HNSCC, both strands of the miR-99a-duplex (miR-99a-5p: the guide strand, and miR-99a-3p: the passenger strand) are downregulated in cancer tissues. Moreover, low expression of miR-99a-5p and miR-99a-3p significantly predicts poor prognosis in HNSCC, and these miRNAs regulate cancer cell migration and invasion. We previously showed that passenger strands of miRNAs have antitumor functions. Here, we screened miR-99a-3p-controlled oncogenes involved in HNSCC pathogenesis. Thirty-two genes were identified as miR-99a-3p-regulated genes, and 10 genes (STAMBP, TIMP4, TMEM14C, CANX, SUV420H1, HSP90B1, PDIA3, MTHFD2, BCAT1, and SLC22A15) significantly predicted 5-year overall survival. Notably, among these genes, STAMBP, TIMP4, TMEM14C, CANX, and SUV420H1 were independent prognostic markers of HNSCC by multivariate analyses. We further investigated the oncogenic function of STAMBP in HNSCC cells using knockdown assays. Our data demonstrated that the aggressiveness of phenotypes in HNSCC cells was attenuated by siSTAMBP transfection. Moreover, aberrant STAMBP expression was detected in HNSCC clinical specimens by immunochemistry. Our strategy, i.e., identification of antitumor miRNAs and their targets, maybe an attractive tool to reveal novel prognostic and therapeutic targets in HNSCC. Citation Format: Reona Okada, Keiichi Koshizuka, Yasutaka Yamada, Shogo Moriya, Naoko Kikkawa, Takashi Kinoshita, Toyoyuki Hanazawa, Naohiko Seki. Regulation of oncogenic targets by miR-99a-3p (the passenger strand of the miR-99a-duplex) in head and neck squamous cell carcinoma [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 3711.

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis to ensure delivery of oxygen, nutrients, and growth factors to tumor cells and provide access to the systemic circulation. Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes that mediate many of the adaptive responses to decreased oxygen concentration, such as enhanced glucose uptake and formation of new blood vessels. Acting through Plexin-B1 on endothelial cells, Semaphorin 4D (Sema4D) has been shown to promote angiogenesis and enhance invasive growth and proliferation in some tumors. Here we show that the gene for Sema4D, the product of which is elevated in head and neck squamous cell carcinoma (HNSCC) cells, contains upstream hypoxia response elements (HRE) and is strongly induced in hypoxia in a HIF-1-dependent manner. Knocking down Sema4D expression with short hairpin (sh) RNA reduces in vitro endothelial cell migration and growth and vascularity of HNSCC xenografts expressing a degradation resistant HIF-1alpha subunit. We also demonstrate a correlation between HIF-1 activity and Sema4D expression in HNSCC specimens. These findings indicate that Sema4D is induced by hypoxia in a HIF-1-dependent manner and influences endothelial cell migration and tumor vascularity. Expression of Sema4D may be a strategy by which carcinomas promote angiogenesis and therefore could represent a therapeutic target for these malignancies.