Quercetin as a Bitter Taste Receptor Agonist with Anticancer Effects in Head and Neck Cancer Cells.

Background/Objectives: Quercetin is a bitter compound with demonstrated anticancer effects in preclinical models of head and neck squamous cell carcinoma (HNSCC). In taste transduction, bitter compounds activate bitter taste receptors (T2Rs), a group of G protein-coupled receptors with downstream signaling that includes cytosolic calcium (Ca2+) release. T2Rs are expressed in HNSCC cells, where their activation induces apoptosis in vitro. Increased T2R expression in HNSCC also correlates with improved patient survival. The objective of this study was to investigate the role of quercetin as an anticancer T2R agonist in HNSCC cells in vitro and ex vivo. Methods: Quercetin-mediated Ca2+ responses were assessed using live cell Ca2+ imaging in the presence of the T2R14 antagonist LF1 and G-protein inhibitor YM-254980 (YM) in UM-SCC-47 and FaDu HNSCC cell lines. Cell viability was evaluated using crystal violet assays in cell lines and MTS assays in patient-derived tumor slices. Mitochondrial depolarization was measured with TMRE in the presence and absence of T2R pathway inhibitors. Results: Quercetin induced a Ca2+ response in HNSCC cells, which was significantly reduced by LF1 and YM. Quercetin also decreased cell viability in vitro. Ex vivo experiments showed a decrease in viability that was not statistically significant. Finally, quercetin caused mitochondrial depolarization, which was reduced in the presence of LF1 but not by YM. Conclusions: In HNSCC cells, quercetin causes a Ca2+ response that is likely mediated by T2R14, although genetic knockdown or knockout models are needed to more definitively support this hypothesis. Additionally, quercetin decreases viability in vitro and causes mitochondrial depolarization.

PurposeLong non-coding RNAs (lncRNAs) play critical roles in cancer onset and development, including head and neck squamous cell carcinoma (HNSCC). This study aimed to investigate the biological role of LINC00460 and the mechanisms underlying epithelial-mesenchymal transition (EMT) in HNSCC.MethodsAberrantly LINC00460 expression in HNSCC and overall survival outcomes were constructed using the TCGA database. Quantitative real-time polymerase chain reaction (RT-qPCR) was applied to examine the LINC00460 expression level in HNSCC cell lines. The role of LINC00460 knockdown on HNSCC cell growth, migration, invasion, and EMT was investigated in vitro using cell counting kit-8 (CCK-8), colony formation, transwell assay, and Western blot assay. Besides, bioinformatics prediction, dual-luciferase reporter assay, and RNA immunoprecipitation (RIP) were performed to reveal the interaction among LINC00460 and its target genes. The function of the LINC00460/miR-320a/BGN axis in HNSCC cells was clarified by rescue assays. Furthermore, the in vivo effects of LINC00460 on tumor growth were investigated using mice xenograft models.ResultsIn this study, LINC00460 was upregulated in HNSCC tissues and cells and was associated with poor clinical prognosis. Further functional analysis showed that LINC00460 knockdown decreased HNSCC cell proliferation, migration, invasion, as well as EMT in vitro. Mechanistic investigation indicated that LINC00460 sponged miR-320a to upregulate Biglycan (BGN) expression, thereby facilitating HNSCC progression and induced EMT. Moreover, knockdown of LINC00460 significantly suppressed the progression of HNSCC cells in vivo.ConclusionTaken together, LINC00460 mediates miR-320a/BGN signaling axis to promote cell proliferation, migration, invasion, and induce the EMT process in HNSCC cells. Our findings elucidated a novel mechanism underlying the progression of HNSCC. LINC00460 could serve as a potential therapeutic target for the treatment of HNSCC.

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.

The presence of regional metastases in head and neck squamous cell carcinoma (HNSCC) patients is a common and adverse event associated with poor prognosis. Understanding the molecular mechanisms that mediate HNSCC metastasis may enable identification of novel therapeutic targets. E-cadherin plays a key role in epithelial intercellular adhesion; its downregulation is a hallmark of the epithelial-to-mesenchymal transition (EMT) (an essential process during tumor progression); and it is associated with invasion, metastasis, and decreased survival. Inflammatory cytokines have been implicated in the progression of HNSCC. Herein, the mechanisms by which the inflammatory mediator, Interleukin-1β (IL-1β), might contribute to EMT in HNSCC is investigated. The pathways involved in E-cadherin regulation in HNSCC had not previously been defined. It is hypothesized that 1) inflammatory mediators upregulate cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2), which then in turn regulate E-cadherin expression in HNSCC; and 2) PGE2 downregulates E-cadherin via transcriptional repressors of E-cadherin (such as Snail) in HNSCC. The outcome of the proposed research will allow us to define how resistance to epidermal growth factor receptor (EGFR)-selective tyrosine kinase inhibitors is mediated and whether the benefits of combination therapy are due to the capacity of COX-2 inhibitors to increase E-cadherin expression and thus create a more sensitive target for EGFR TK inhibition. Basic science, molecular biology, animal model, immunohistochemistry. We evaluated the effect of IL-1β on the molecular events of EMT in surgical specimens and HNSCC cell lines. We examined the correlation with tumor histologic features, and a severely compromised immunodeficient (SCID) xenograft model was used to assess the effects in vivo. COX-2-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-2 and E-cadherin was demonstrated in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1β caused the downregulation of E-cadherin expression and upregulation of COX-2 expression. This effect was blocked in the presence of COX-2 small hairpin RNA (shRNA). IL-1β -treated HNSCC cell lines demonstrated a significant decrease in E-cadherin messenger RNA (mRNA) and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1β exposure led to enhanced Snail binding at the chromatin level. ShRNA-mediated knockdown of Snail interrupted the capacity of IL-1β to downregulate E-cadherin. Snail overexpression in normal oral keratinocytes and HNSCC cells is sufficient to drive EMT and confers resistance to erlotinib. In a SCID xenograft model, HNSCC Snail overexpressing cells demonstrated significantly increased primary and metastatic tumor burdens. The inflammatory mediator IL-1β modulates Snail and thereby regulates COX-2-dependent E-cadherin expression in HNSCC. This is the first report indicating the role of Snail in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy. N/A.

BackgroundThere is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC).MethodsWe conducted whole‐transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression.ResultsIn this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27‐JNK/P38 mitogen‐activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27‐JNK/P38 MAPK signaling and promoted tumor progression.ConclusionsOur results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27‐JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

Head and neck squamous cell carcinoma (HNSCC) ranks among the top most common cancers with a poor prognosis. The mechanism of chemoresistance is still not well known. This study is to investigate the programmed death‐ligand 1 (PD‐L1) expression in HNSCC, and test the effect of lactoferricin B (LfcinB) on chemoresistance and its mechanism. We analyzed 510 HNSCC patients in TCGA database and investigated how CD274 expression was related to patient prognosis. PD‐L1 was verified from HNSCC samples at local hospital with immunohistochemistry. PD‐L1 expression in the acquired cisplatin‐resistant HNSCC cells was examined by PCR and WB in order to test PD‐L1‐induced chemoresistance. LfcinB inoculation in cisplatin‐resistant HNSCC cells and in the nude mice was introduced to test the effect of LfcinB on targeting cisplatin resistance and its mechanism. High CD274 mRNA (>125 FPKM) from TCGA database had a significantly reduced 5‐year survival rate, and a lower 5‐year survival rate in the chemotherapy and radiotherapy‐treated patients (P < .05). PD‐L1 overexpression was further supported from analysis of 40 HNSCC specimens. PD‐L1 and IL‐6 in the established cisplatin‐resistant HNSCC cells were shown significantly higher (P < .05). IL‐6 and PD‐L1 expression were partially inhibited by the anti‐IL‐6/STAT3 antibody. LfcinB displayed a direct cytotoxic effect on cisplatin‐resistant HNSCC cells and HNSCC xenografts of cisplatin‐resistant cells in the nude mice displayed significant reduction in tumor volume after LfcinB injection (P < .05). Besides, the increase of IL‐6 and PD‐L1 in cisplatin‐resistant HNSCC cells was abolished in vitro by LfcinB (P < .05). PD‐L1 expression in HNSCC cells correlates with poor prognosis and chemoresistance, and LfcinB might provide therapeutic potential in HNSCC patients through modulating IL‐6 and PD‐L1.

Introduction: HNSCC is an immunosuppressive disease, with multiple functional and quantitative defects contributing to immune evasion and tumor escape. One of the identified areas for therapy development is the Programmed Death-1 (PD-1)/Programmed Death Ligand 1 (PD-L1) pathway, as this pathway has been hypothesized to allow cancer cells to evade the immune system by promoting T cell anergy and apoptosis. Pembrolizumab, a PD-L1 inhibitor, has recently been approved for the treatment of recurrent/metastatic HNSCC. As regulation of PD-L1 expression could play an important role in the effectiveness of therapy, we further explored the regulation of PD-L1 expression in HNSCC cells. Specifically, we targeted our investigation by evaluating the effects on expression of one of the most frequently mutated genes in HNSCC, PIK3CA. We evaluated this with and without interferon-γ, which has previously been shown to affect PD-L1 expression, possibly through activation of the STAT1 pathway. Methods: HPV+ and HPV- HNSCC cell lines were grown in cell culture and treated with selective and non-selective PI3K inhibitors, in combination with interferon-γ. After 72 hours, cells were harvested and flow cytometry was used to measure the expression of PD-L1. Protein expression of various pathway intermediaries was evaluated via western blot to better delineate the mechanism of PD-L1 upregulation. Results: Treatment with selective PI3K inhibitors in combination with interferon-γ in several cell lines significantly increased expression of PD-L1, beyond the increase noted after treatment with interferon-γ alone. Maintenance of STAT1 phosphorylation correlated with upregulation of PD-L1 expression, while total STAT1 expression remained stable. The majority of the cell lines maintaining STAT1 phosphorylation were HPV+, but a few HPV- cell lines also maintained this phosphorylation with a correlating upregulation in PD-L1 expression. Additionally, mutation in PIK3Ca despite HPV- status was noted to maintain phosphorylation of STAT1 with upregulation of PD-L1 expression. Conclusions: Treatment with PI3K inhibitors in combination with interferon-γ significantly upregulated PD-L1 expression in several cell lines, suggesting a possible synergistic effect. Since PD-L1 expression correlated with maintenance of phosphorylation of STAT1, these results suggest a pivotal link between PIK3CA signaling, STAT1 activity and PD-L1 expression in PIK3CA aberrant HNSCC. Citation Format: Rebecca C. Hoesli, Nicole L. Michmerhuizen, Vivek Nair, Chloe Matovina, Elizabeth Leonard, Matthew E. Spector, Carol R. Bradford, Mark E. Prince, Andrew C. Birkeland, J Chad Brenner. Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC) [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 3712. doi:10.1158/1538-7445.AM2017-3712

Objective GIT1 is identified as a novel tumor oncogene in breast cancer. In this article, we aimed to explore the role of GIT1 in the progression of head and neck squamous cell carcinoma (HNSCC). Methods GIT1 expression in HNSCC was detected by RT-qPCR, immunohistochemistry assay, and Western blot. HNSCC cell proliferation, migration, and invasion were examined by CCK-8 assay, Wound healing assay, and Transwell assay, respectively. Cell apoptosis was detected by flow cytometric analysis. Results In our study, GIT1 was notably upregulated in HNSCC tissues and cells. Moreover, GIT1 expression level had positive corelation with pathological grade and nodal status of HNSCC. Functional experiments showed that knockdown of GIT1 restrained HNSCC proliferation, invasion, migration, and EMT and facilitated cell apoptosis. Furthermore, GIT1 knockdown was found to restrain HNSCC tumor growth and lung metastasis. Additionally, PI3K/AKT/mTOR signal pathway inhibitors suppressed the effect of GIT1 on HNSCC cell progression. Conclusion GIT1 was upregulated in HNSCC and facilitated HNSCC cell progression by inducing PI3K/AKT/mTOR signal pathway. Therefore, we suggested that GIT1 might be a potential target for HNSCC treatment.

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.

Head and neck squamous cell carcinoma (HNSCC) refers to the malignancy of squamous cells in the head and neck region. Ranked as the seventh most common cancer worldwide, HNSCC has a very low survival rate, highlighting the importance of finding therapeutic targets for the disease. Integrins are cell surface receptors that play a crucial role in mediating cellular interactions with the extracellular matrix (ECM). Within this protein family, Integrin αV (ITGAV) has received attention for its important functional role in cancer progression. In this study, we first demonstrated the upregulation of ITGAV expression in HNSCC, with higher ITGAV expression levels correlating with significantly lower overall survival, based on TCGA (the Cancer Genome Atlas) and GEO datasets. Subsequent in vitro analyses revealed an overexpression of ITGAV in highly invasive HNSCC cell lines UM1 and UMSCC-5 in comparison to low invasive HNSCC cell lines UM2 and UMSCC-6. In addition, knockdown of ITGAV significantly inhibited the migration, invasion, viability, and colony formation of HNSCC cells. In addition, chromatin immunoprecipitation (ChIP) assays indicated that SOX11 bound to the promoter of ITGAV gene, and SOX11 knockdown resulted in decreased ITGAV expression in HNSCC cells. In conclusion, our studies suggest that ITGAV promotes the progression of HNSCC cells and may be regulated by SOX11 in HNSCC cells.

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

Simple SummaryHead and neck squamous cell carcinoma (HNSCC) arises in the mucosal membranes in the head and neck and constitutes 3.9% of new cancer cases annually. Its pathogenesis is stepwise; the formation of invasive and metastatic cancer is preceded by premalignant mucosal changes. With little improvement in overall survival over the last decade, HNSCC remains an important cause of patient morbidity. Therapy options include surgery, radiochemotherapy, and immunotherapy, but resistance is common. A way to overcome drug resistance is to target cellular processes present in all cells, such as protein biosynthesis, i.e., translation. This study found that eukaryotic translation initiation factor 2α (EIF2α) is highly expressed in HNSCC in comparison to normal tissue, and its expression mirrors the progression to malignancy. Pharmacological targeting of EIF2α by inhibition of its dephosphorylation with salubrinal decreases cell viability, the ability to form colonies, and disrupts the cell cycle. Salubrinal also works synergistically with standard chemotherapeutics.Drug resistance is a common cause of therapy failure in head and neck squamous cell carcinoma (HNSCC). One approach to tackling it is by targeting fundamental cellular processes, such as translation. The eukaryotic translation initiation factor 2α (EIF2α) is a key player in canonical translation initiation and integrates diverse stress signals; when phosphorylated, it curbs global protein synthesis. This study evaluates EIF2α expression and phosphorylation in HNSCC. A small-molecule inhibitor of EIF2α dephosphorylation, salubrinal, was tested in vitro, followed by viability assays, flow cytometry, and immunoblot analyses. Patient-derived 3D tumor spheres (PD3DS) were cultured with salubrinal and their viability assessed. Lastly, salubrinal was evaluated with standard-of-care chemotherapeutics. Our analysis of RNA and proteomics data shows elevated EIF2α expression in HNSCC. Immunohistochemical staining reveals increasing EIF2α abundance from premalignant lesions to invasive and metastatic carcinoma. In immunoblots from intraoperative samples, EIF2α expression and steady-state phosphorylation are higher in HNSCC than in neighboring normal tissue. Inhibition of EIF2α dephosphorylation decreases HNSCC cell viability and clonogenic survival and impairs the G1/S transition. Salubrinal also decreases the viability of PD3DS and acts synergistically with cisplatin, 5-fluorouracil, bleomycin, and proteasome inhibitors. Our results indicate that pharmacological inhibition of EIF2α dephosphorylation is a potential therapeutic strategy for HNSCC.

To characterize the HER-2/neu oncogene in head and neck squamous cell carcinoma (HNSCC) cell lines and tumor tissue specimens. Molecular analysis of HER-2/neu oncogene amplification and expression in HNSCC cell lines by Southern, Northern, and Western blot techniques, and HER-2/neu oncoprotein expression in HNSCC tumor tissue sections by immunohistochemical analysis. Eleven HNSCC cell lines, eight paired samples of frozen HNSCC tumor tissue specimens and adjacent nonmalignant mucosa, and 38 paraffin-embedded slides derived from HNSCC tumor specimens (including those from which the cell lines were derived) were analyzed. Southern blot analysis showed twofold HER-2/neu gene amplification in two (18%) of the 11 HNSCC cell lines, MDA-1386 and Tu-167. Northern blot analysis showed messenger RNA overexpression in the same two cell lines, and to a lesser degree in MDA-1483. Western blot analysis showed high levels of HER-2/neu oncoprotein expression in two (18%) of the 11 cell lines (MDA-1386 and Tu-167), a moderate level of protein expression in one cell line (9%) (MDA-1483), and low levels of protein expression in eight cell lines (73%). Some HER-2/neu protein expression was seen in all of the HNSCC cell lines. Immunohistochemical analysis of the tumor tissue sections from which the cell lines were derived corroborated the Western blot results. Western blot analysis of frozen primary tumor specimens showed HER-2/neu oncoprotein overexpression in two (25%) of eight specimens. Immunohistochemical analysis showed high levels of protein expression in six (16%) of the 38 tumor tissue slides, moderate levels in 12 (31%), and low levels in 20 (53%). The HER-2/neu oncogene is overexpressed in a subset of HNSCC tumors and cell lines. The results from Western blot and immunohistochemical analyses underscore a variable HER2/neu oncoprotein expression in HNSCC. Gene amplification was observed in a few of the cell lines, suggesting a potential mechanism of oncoprotein overexpression. Messenger RNA overexpression, however, can be seen in the absence of gene amplification, indicating that transcriptional or posttranscriptional control mechanisms must be involved. Further studies are indicated to determine the biologic role of HER-2/neu expression in the clinical progression of these lesions and to further define the molecular basis regulating its expression.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent disease worldwide, and the survival of HNSCC has not improved significantly over the last few decades. MicroRNAs (miRNAs) have an important regulatory role during carcinogenesis. Our study investigated the pathogenic implications of miR-134 in head and neck carcinogenesis. The clinicopathologic implications of miR-134 in HNSCC were investigated using expression assays and the functional role of miR-134 in HNSCC pathogenesis was determined using ectopic expression, knockdown and reporter assay experiments. Xenographic tumorigenesis and orthotopic nodal metastasis were assayed in mouse models. In situ hybridization and immunohistochemistry were used to detect the expression of miR-134 and the WWOX gene in human HNSCC. The results indicated that miR-134 was upregulated in HNSCC tissues relative to control mucosa. High expression of miR-134 was associated with nodal metastasis and mortality of patients. Decreased plasma miR-134 levels after tumor ablation indicated a better prognosis for patients. Multivariate analysis showed that high miR-134 expression in HNSCC was an independent predictor of poor survival. Ectopic miR-134 expression significantly enhanced in vitro oncogenic phenotypes and the orthotopic growth and metastasis of HNSCC cells. miR-134 targeted WW domain-containing oxidoreductase (WWOX) gene and cell invasion enhanced by miR-134 expression was abrogated by ectopic WWOX expression in HNSCC cells. miR-134 expression was reversely associated with the WWOX expression in HNSCC tissues. Evidences from our study substantiated that miR-134 expression contributes to head and neck carcinogenesis by targeting the WWOX.

UBE2C promotes the progression of head and neck squamous cell carcinoma