Oral Squamous Cell Carcinoma Tumor Research Articles

Abstract 3847: Oral cavity squamous cell carcinoma xenografts display conservation of primary tumor genomic heterogeneity

Abstract Purpose: Patient derived xenograft (PDX) models represent a platform for defining therapeutic opportunities based on their ability to maintain the genomic and molecular features of their respective tumors. We used a comprehensive genomics approach to analyze PDXs for oral squamous cell carcinoma (OSCC), which had not been previously described. This involved confirming that the PDXs conserve the heterogeneous somatic landscape of the primary tumors from which they were derived. Methods: We established a large panel of OSCC PDX models by engrafting fresh, primary tumor samples into NOD scid gamma (NSG) mice. We performed whole genome sequencing (WGS; n=10), whole exome sequencing (WES; n=15), and transcriptome sequencing (RNAseq; n=15) on matched primary and first passage xenografts. Somatic events, including mutations, copy number alterations (CNAs), loss-of-heterozygosity (LOH) regions, and structural variation (SV), were detected independently in tumors and PDXs. Conservation was defined by the correlation of variant allele frequency (VAF) distributions and gene expression patterns, presence of larger somatic events, and predictions made about the subclonal architecture of the tumor and xenograft. Results: PDXs were established with an overall success rate of 47% (56/118). The somatic landscape of primary tumors was representative of the published genomic data on OSCC, including amplifications of CCND1 and EGFR family members, inactivating mutations and loss of TP53 and CDKN2A, and chromosome 3q amplification. These somatic events were also strongly conserved in matched xenograft samples. Mutations that were detected in either the xenograft or primary tumor, but not its matched sample, consisted primarily of low-frequency mutations. The predicted clonal architecture, however, revealed correlative subclonal heterogeneity between matched xenograft and primary tumor. This includes maintenance of founding clone mutations and consistent VAF distributions, indicating that a heterogeneous tumor population engrafted. Conclusions: This comprehensive approach establishes early-passage PDXs as high fidelity models for OSCC. Their ability to maintain the genomic heterogeneity detected in primary OSCC tumors presents a clinically relevant platform for understanding tumor biology and identifying novel therapeutic strategies. Citation Format: Katie M. Campbell, Tianxiang Lin, Ashley E. Winkler, Paul Zolkind, Zachary L. Skidmore, Erica K. Barnell, Ian Hagemann, Elaine R. Mardis, Malachi Griffith, Rebecca D. Chernock, Obi L. Griffith, Ravindra Uppaluri. Oral cavity squamous cell carcinoma xenografts display conservation of primary tumor genomic heterogeneity [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 3847. doi:10.1158/1538-7445.AM2017-3847

Establishing Fascin over-expression as a strategic regulator of neoplastic aggression and lymph node metastasis in oral squamous cell carcinoma tumor microenvironment.

Oral squamous cell carcinoma (OSCC) has an aggressive behaviour with high incidence of nodal metastasis, even in the early stages, leading to poor prognosis. For progression and metastasis of cancers, the dominant element considered is cell motility. Fascin, an actin-binding protein has emerged as a protein of general importance for a diverse set of cell protrusions with functions in cell adhesion, cell interactions, and cell migration. The role of Fascin in various carcinomas, including aggressive behaviour in OSCC has been documented, but its role as a key regulator in lymph nodes metastasis is yet to be validated. This study was piloted to evaluate and correlate Fascin expression in OSCC lymph nodes and understand the role of Fascin in contemptuous Lesional tissue, as a predictor of survival. A retrospective study designed with 40 archival OSCC cases was included as sample, 20 each of both lymph node metastasis +ve (Group 1) and -ve (Group 2) groups. All the participants were smokeless tobacco user and had tumor located at gingivo-buccal complex. We established that Fascin over-expression in lymph nodes were significantly associated with clinico-histopathological parameters like staging (p=0.01), tumor size (cT) (p=0.03) and differentiation; and furthermore it was highly significant in correlation to nodal status (cN) (*p≤0.001). Fascin over-expression in lymph node metastasis positive cases correlated with that of Fascin expression in contemptuous Lesional tissue signifying its role in promoting aggressive progression and metastasis. This association was found to be statistically significant (p value=0.05). Overall Survival Analysis of both lymph node metastasis +ve and -ve groups assessed by Kaplan-Meier analysis (taking death and recurrence into consideration) showed patients with high Fascin expression (in lymph node and Lesional tissue) had shorter overall survival than patients who had no to weak Fascin expression. Our findings thereby establish Fascin expression as a regulator of metastasis in OSCC tumor microenvironment and predictor of survival.

Identification and validation long non-coding RNAs of oral squamous cell carcinoma by bioinformatics method.

Gene markers of oral squamous cell carcinoma (OSCC) have great significance on early diagnosis and treatment of clinical oral cancer. In this study, we used RNA-Seq data from OSCC patients and filtered differentially-expressed long non-coding RNA (lncRNA) to further clarify the molecular mechanism. Firstly, we downloaded datasets of OSCC from National Center for Biotechnology Information(NCBI), which were predicted and analyzed by cufflinks and tophat. Then, differentially expressed lncRNA enrichment was performed with The Database for Annotation, Visualization and Integrated Discovery (DAVID). Finally, we verified the gene expression via in vitro assays. Results showed that 52 lncRNAs were significantly differentially expressed compared to those in normal oral tissues, three highly expressed genes (XLOC_002599, XLOC_002634 and XLOC_132858) were verified by RT-PCR, which was consistent with the prediction. XLOC_002634 (GAS5) transcript levels were reduced both in vivo and in vitro assays, which confirmed that the expression of GAS5 was comparatively low in OSCC. Over-expression of GAS5 in cancer cells inhibited cell proliferation. Moreover, the migration and invasion potential of cancer cells were inhibited compared to control groups. All in all, the study indicated that the decrease in GAS5 expression may contribute to OSCC tumor pathogenesis and serve as a potential target for cancer therapy.

Loss of NDRG2 Expression Confers Oral Squamous Cell Carcinoma with Enhanced Metastatic Potential.

Loss of the tumor suppressor NDRG2 has been implicated in the development of oral squamous cell carcinoma (OSCC), acting by modulating PI3K/AKT-mediated dephosphorylation of PTEN at S380/S382/T383 (STT). Here, we show that the majority of OSCC tumors with lymph node metastasis, a major prognostic factor, exhibit high levels of phosphorylated AKT-S473 and PTEN-STT and low levels of NDRG2 expression. In Ndrg2-deficient mice, which develop a wide range of tumors, we developed a model of OSCC by treatment with the tobacco surrogate 4-nitroquinoline-1-oxide (4-NQO). In this model, both the number and size of OSCC tumors were increased significantly by Ndrg2 deficiency, which also increased invasion of cervical lymph nodes. 4-NQO treatment of human OSCC cell lines exhibiting low NDRG2 expression induced epithelial-mesenchymal transition via activation of NF-κB signaling. Conversely, ectopic expression of NDRG2 reversed the EMT phenotype and inhibited NF-κB signaling via suppression of PTEN-STT and AKT-S473 phosphorylation. Our results show how NDRG2 expression serves as a critical determinant of the invasive and metastatic capacity of OSCC. Cancer Res; 77(9); 2363-74. ©2017 AACR.

Exhaled breath and oral cavity VOCs as potential biomarkers in oral cancer patients

Corporal mechanisms attributed to cancer, such as oxidative stress or the action of cytochrome P450 enzymes, seem to be responsible for the generation of a variety of volatile organic compounds (VOCs) that could be used as non-invasive diagnosis biomarkers. The present work presents an attempt to use VOCs from exhaled breath and oral cavity air as biomarkers for oral squamous cell carcinoma (OSCC) patients. A total of 52 breath samples were collected (in 3 L Tedlar bags) from 26 OSCC patients and 26 cancer-free controls. The samples were analyzed using solid-phase microextraction followed by gas chromatography—mass spectrometry detection. Different statistical strategies (e.g., Icoshift, SIMCA, LDA, etc) were used to classify the analytical data. Results revealed that compounds such as undecane, dodecane, decanal, benzaldehyde, 3,7-dimethyl undecane, 4,5-dimethyl nonane, 1-octene, and hexadecane had relevance as possible biomarkers for OSCC. LDA classification with these compounds showed well-defined clusters for patients and controls (non-smokers and smokers). In addition to breath analysis, preliminary studies were carried out to evaluate the possibility of lesion-surrounded air (analyzed OSCC tumors are in the oral cavity) as a source of biomarkers. The oral cavity location of the squamous cell carcinoma tumors constitutes an opportunity to non-invasively collect the air surrounding the lesion. Small quantities (20 ml) of air collected in the oral cavity were analyzed using the above methodology. Results showed that aldehydes present in the oral cavity might constitute potential OSCC biomarkers.

FKBP51 Immunohistochemical Expression: A New Prognostic Biomarker for OSCC?

Up-to-date, several molecular markers of prognosis have been studied in Oral Squamous Cell Carcinoma (OSCC), but none entered in the clinical setting. Therapy of OSCC tumors mainly relies on surgery, radiotherapy and partially on chemotherapy; there is an urgent need for biomarkers able to better stratify OSCC patients’ risk to address targeted therapeutic strategies. The role of immune response in the pathogenesis and biological behavior of OSCC has been investigated by several authors, and promising results have been obtained with immune checkpoint inhibitors. We already investigated the role of the immune modulator FK506-binding protein 51 (FKBP51), a FK506-binding immunophilin, in cutaneous melanoma biology, and its expression in several human solid tumors. In the present study, we aimed to assess the value of FKBP51 expression in OSCC tumor cells as a marker of outcome. We collected clinical data from 72 patients who underwent surgery for Squamous Cell Carcinoma (SCC) of the tongue, floor, lips and palate. FKBP51 expression was assessed by immunohistochemistry on paraffin-embedded tumor tissues. In addition, we evaluated the human papillomavirus (HPV) status of primary tumors by immunohistochemistry, viral subtyping and In Situ Hybridization (ISH) assay. We found that high FKBP51-expressing tumors characterized the OSCCs with the worst prognosis: the high immunohistochemical expression of FKBP51 associated with death occurring within five years from the diagnosis with a sensitivity of 88.46% and a specificity of 91.67%. The estimated positive predictive value of the test was 88.45% and negative predictive value 91.67%. We tested FKBP51 mRNA presence, by RT-PCR assay, in a selected series of OSCC tumors, and we found that mRNA correlated well to the protein expression and to the clinical outcome. Applying the Bayes formula, we estimated an 88% probability of dying within five years from the diagnosis of OSCC patients with a high FKBP51 immunohistochemical (IHC) test result (>51% of FKBP51 positive tumor cells). On the basis of our analysis, we propose tumor tissue expression of FKBP51 protein as a reliable prognostic marker for OSCC tumors.

Altered Expression of Sphingosine-1-Phosphate Metabolizing Enzymes in Oral Cancer Correlate With Clinicopathological Attributes

ABSTRACTWe have determined the gene expression of sphingosine-1-phosphate (S1P) metabolizing enzymes (SphK1, SphK2, SGPL1, SGPP1, SGPP2, PPAP2A, PPAP2B, and PPAP2C) by quantitative real-time polymerase chain reaction in tumor tissues and adjacent normal tissues of 50 oral squamous cell carcinoma (OSCC) patients. Expression of SphK1 and SGPP1 genes was up-regulated significantly in 70% and 75% OSCC tumors respectively. Importantly, expression of SphK2 and PPAP2B was down-regulated in the tumor tissues of 70% OSCC patients. Expression of SphK2 and PPAP2B negatively correlated with tumor-node-metastasis (TNM) staging and tumor volume respectively. Furthermore, LPP1 is an independent predictor of TNM staging and lymph node ratio.

Protein S drives oral squamous cell carcinoma tumorigenicity through regulation of AXL.

The TAM family of proto-oncogenic receptor protein tyrosine kinases, comprising of TYRO3, AXL, and MERTK, is implicated in many human cancers. Their activation leads to cancer cell proliferation, enhanced migration, invasion, and drug resistance; however how TAMs are activated in cancers is less understood. We previously showed that Protein S (PROS1) is a ligand of the TAM receptors. Here we identify PROS1 as a mediator of Oral Squamous Cell Carcinoma (OSCC) in proliferation, cell survival and migration. We demonstrate that excess PROS1 induces OSCC proliferation and migration. Conversely, blocking endogenous PROS1 expression using shRNA significantly inhibits cell proliferation and migration in culture. This inhibition was rescued by the addition of purified PROS1. Moreover, PROS1 knockdown reduced anchorage-independent growth in-vitro, reduced tumor xenograft growth in nude mice and altered their differentiation profile. Mechanistically, we identify the downregulation of AXL transcripts and protein following PROS1 knockdown. Re-introducing PROS1 rescues AXL expression both at the protein and transcriptional levels. The anti-proliferative effect of the AXL inhibitor R428 was significantly reduced following PROS1 inhibition, indicating the functional significance of PROS1-mediated regulation of AXL in OSCC. Taken together, we identify PROS1 as a driver of OSCC tumor growth and a modulator of AXL expression. Our results point to PROS1 as a potential novel anti-cancer therapeutic target.

Downregulation of miR-1 enhances tumorigenicity and invasiveness in oral squamous cell carcinomas.

Cumulative evidence suggest that microRNAs (miRNAs) function as biosignatures of oral squamous cell carcinomas (OSCC). However, the functional roles of miR-1 as well as its downstream targets in the regulation of tumorigenicity in OSCC remain unclear. miRNAs RT-PCR analysis was performed to identify miR-1 as a putative candidate on mediating invasiveness of OSCC cells. Consequently, we elucidated the tumorigenicity of OSCC cells with miR-1 downregulation or overexpression, respectively. Finally, miR-1 on OSCC tumor tissues was examined. miR-1 levels were significantly downregulated in the malignant OSCC cells. Overexpression of miR-1 significantly reduced migration/invasiveness of OSCC cells. In addition, overexpression of miR-1 decreased cancer stem cells properties. Conversely, downregulation of miR-1 promotes migration and invasiveness in OSCC cells. We have shown that miR-1 is able to target Slug, suppressing their expression. Clinically, lower miR-1 expression was found in patients with advanced nodal metastasis OSCC. miR-1 as novel biosignatures in OSCC lymph node metastatic patients, supporting the development of novel strategies for OSCC treatment.

Patients with advanced oral squamous cell carcinoma have high levels of soluble E-cadherin in the saliva

BackgroundThe objective of this study was to assess the potential clinical value of the concentration of soluble salivary E-cadherin (sE-cadherin) compared with the clinical value of the presence of membranous E-cadherin (mE-cadherin) in oral squamous cell carcinoma tumor tissues.Material and MethodsData regarding patient demographics, clinical stage, saliva and tumor tissue samples were collected. The saliva was analyzed for sE-cadherin protein levels and was compared to the mE-cadherin immunohistochemical expression levels in tumor tissues, which were assessed via the HercepTest® method. Patients without cancer were included in the study as a control group for comparisons of the sE-cadherin levels.ResultssE-cadherin levels in the saliva of patients without cancer were lower than those in patients with cancer, and the difference was statistically significant (p=0.031). Low mE-cadherin expression was statistically significantly associated with lymph node positivity (p=0.015) and advanced clinical stage (p=0.001). The inverse relationship between mE-cadherin and sE-cadherin was significant in terms of lymph node positivity (p=0.014) and advanced clinical stage (p=0.037).ConclusionsThe results suggest that sE-cadherin levels are significantly increased in patients with oral cancer and that its low expression within the membrane as well as the progression of the disease appear to be inversely associated with levels of sE-cadherin in the saliva. Key words:E-cadher.in, saliva, oral cancer.

Benefits of Multifaceted Chemopreventives in the Suppression of the Oral Squamous Cell Carcinoma (OSCC) Tumorigenic Phenotype.

Over one third of patients who have undergone oral squamous cell carcinoma (OSCC) surgical resections develop life-threatening and often untreatable recurrences. A variety of drugs, intended for management of recurrent or disseminated cancers, were designed to exploit cancer cells' reliance upon overexpressed receptors and gratuitous signaling. Despite their conceptual promise, clinical trials showed these agents lacked efficacy and were often toxic. These findings are consistent with evasion of pathway-targeted treatments via extensive signaling redundancies and compensatory mechanisms common to cancers. Optimal secondary OSCC chemoprevention requires long-term efficacy with multifaceted, nontoxic agents. Accordingly, this study evaluated the abilities of three complementary chemopreventives, that is, the vitamin A derivative fenretinide (4-HPR, induces apoptosis and differentiation, inhibits signaling proteins, and invasion), the estrogen metabolite 2-methoxyestradiol (2-ME, apoptosis-inducing, antiangiogenic), and the humanized mAb to the IL6R receptor tocilizumab (TOC, reduces IL6 signaling) to suppress OSCC gratuitous signaling and tumorigenesis. Modeling studies demonstrated 4-HPR's high-affinity binding at STAT3's dimerization site and c-Abl and c-Src ATP-binding kinase sites. Although individual agents suppressed cancer-promoting pathways including STAT3 phosphorylation, STAT3-DNA binding, and production of the trans-signaling enabling sIL6R, maximal chemopreventive effects were observed with agent combinations. OSCC tumor xenograft studies showed that locally delivered TOC, TOC+4-HPR, and TOC+4-HPR+2-ME treatments all prevented significant tumor growth. Notably, the TOC+4-HPR+2-ME treatment resulted in the smallest overall increase in tumor volume. The selected agents use diverse mechanisms to disrupt tumorigenesis at multiple venues, that is, intracellular, tumor cell-ECM, and tumor microenvironment; beneficial qualities for secondary chemopreventives. Cancer Prev Res; 10(1); 76-88. ©2016 AACR.

Cancer stem cell, cytokeratins and epithelial to mesenchymal transition markers expression in oral squamous cell carcinoma derived from ortothopic xenoimplantation of CD44high cells

Oral squamous cell carcinoma (OSCC) is the most prevalent neoplasia of oral cavity worldwide and prognosis remains unchanged in decades. Recently, different authors reported that head and neck squamous cell carcinomas have a subpopulation of tumor initiating cells that apparently correspond to cancer stem cells (CSC) and are also responsible for tumor growth and metastasis. The purpose of the present study was to investigate the microscopic and phenotypic characteristics of OSCC tumors induced after orthotopic xenoimplantation of SCC9WT cell line and CSC-enriched subpopulation isolated from SCC9 cell line based on high expression of the putative CSC marker CD44. Different numbers of FACS-sorted SCC9 CD44high and CD44low cells as well as SCC9WT (wild type) were transplanted into the tongue of BALB/C nude (NOD/SCID) mice to evaluate their tumorigenic potential. Sixty days post-induction, tumors were morphologically characterized and immunostained for CSC markers (CD44, Nanog and Bmi-1), epithelial-mesenchymal transition (Snail, Slug) and epithelial differentiating cell markers (cytokeratins 4, 13, 15, 17 and 19), as well as E-cadherin and β-catenin. The data presented here shows that SCC9 CD44high cells have higher ability to form tumors than SCC9 CD44low cells, even when significantly lower numbers of SCC9 CD44high cells were transplanted. Immunoassessment of tumors derived from SCC9 CD44high cells revealed high expression of cytokeratin CK19, β-catenin, E-cadherin and CD44, and negative or low expression of CK17, CK4, CK15, CK13, Nanog, Bmi-1, Snail and Slug. While tumors derived from SCC9WT showed high expression of CK17, CK19, CD44, Nanog, Bmi-1, Snail and Slug, and negative or low expression of CK4, CK15, CK13, β-catenin and E-cadherin. Thus, SCC9 CD44high cells were highly tumorigenic, capable of originating heterogeneous tumors and these tumors have a immunohistochemical profile different from those formed by the wild type cell line.

PTENp1, a natural sponge of miR-21, mediates PTEN expression to inhibit the proliferation of oral squamous cell carcinoma.

PTENp1, non-coding RNA (ncRNA) pseudogene, is involved in oral squamous cell carcinoma (OSCC). The precise effects mediated by PTENp1 transcripts within intricate regulatory networks involving molecular interactions with ancestral gene PTEN and tumorigenicity in OSCC remain unclear. Here, we found that PTENp1 was aberrantly expressed in OSCC. There was a positive correlation between the expression levels of PTENp1 and PTEN. Further, we showed that PTENp1 acted as a competing endogenous RNA that protects PTEN transcripts from being inhibited by miR-21, and consequently inhibited proliferation and colony formation and triggered S-G2/M cell cycle arrest through the AKT pathway. Also, the homogeneous relationship between expression of PTENp1 and PTEN was confirmed in OSCC tumor xenografts. Finally, low expression of PTENp1 and PTEN was negatively associated with histological differentiation and OSCC prognosis. The present work provided the first evidence for the extraordinary crosstalk among PTENp1, PTEN, and miR-21, and rendered a new light on the treatment of OSCC. © 2016 Wiley Periodicals, Inc.

1011P - High expression of FOXM1 is a potential prognostic marker for oral squamous cell carcinoma patients treated with docetaxel-containing regimens

Forkhead box protein M1 (FOXM1) is an oncoprotein that regulates cell growth and differentiation, angiogenesis, apoptosis and aging; and it is reported to play an important role in progression and drug sensitivity of various cancers. The purpose of this study was to determine whether FOXM1 expression could be a useful prognostic factor for oral squamous cell carcinoma (OSCC). FOXM1 expression was investigated by immunohistochemistry in tissue samples of 56 OSCC patients treated with docetaxel (DOC)-containing regimens. In this study, we investigated the relationship between FOXM1 expression and clinicopathological features of OSCC, as well as the prognosis of above patients. Moreover, we examined the expression of FOXM1 in DOC-resistant human tongue carcinoma cell lines (HSC2/DOC, HSC3/DOC and HSC4/DOC) in vitro. We established these DOC-resistant cell lines by exposing HSC2, HSC3 and HSC4 parental cells to increasing concentrations of DOC over approximately two years. FOXM1 was detected both in nucleus and cytoplasm of OSCC tumor cells. FOXM1 expression in tumor tissues was significantly correlated with N classification (P = 0.0395), stage (P = 0.004), therapeutic efficacy (P = 0.0113) and outcome of patient (P = 0.0134); although there was no correlation between FOXM1 expression and patient's gender, age or T classification. Moreover, high expression of FOXM1 in tumor cells was associated with shorter overall survival (OS, P = 0.0257). Multivariate analysis also revealed that high expression of FOXM1 was a predictor of reduced survival (P = 0.0327). Additionally, DOC-resistant OSCC cell lines showed significantly higher expression of FOXM1 compared to the parental cell lines in vitro. These findings suggest that high expression of FOXM1 in OSCC tumors is correlated with DOC resistance, as well as poor therapeutic effects and worse clinical outcomes in OSCC patients treated with DOC -containing regimen. Therefore, FOXM1 might have prognostic significance in oral squamous cell carcinoma patients.

MicroRNA-375 as a biomarker for malignant transformation in oral lesions

Malignant transformation of oral premalignant lesions is the key process in the progression to oral squamous cell carcinoma (OSCC). Previously, we identified miR-7 and miR-21 as candidate oncogenes and miR-375 and miR-494 as candidate tumor suppressors in OSCC. We aim to evaluate these microRNAs as biomarkers of malignant transformation in oral premalignant lesions. Formalin-fixed, paraffin-embedded samples from progressive premalignant lesions and paired sequential OSCC tumors at the same site were obtained from same patients (n=31). Total RNA was extracted and analyzed for microRNA levels using real-time polymerase chain reaction. MiR-375 expression in progressive lesions was clearly lower than in nonprogressive control lesions (average eightfold difference, P=.0004). Furthermore, the expression of miR-375 decreased significantly after the progression from premalignant lesion to OSCC (P<.0001). Receiver operating characteristic curve analysis revealed that miR-375 was able to differentiate between progressive and nonprogressive premalignant lesions (P<.0001). MiR-375 downregulation in oral premalignant lesions is associated with a higher risk of malignant transformation.

Abstract 953: miR-31 targets ARID1A to enhance the progression of oral carcinoma

Abstract miR-31 is an oncogenic microRNA in several malignancies including oral squamous cell carcinoma (OSCC). However, the function of miR-31 in modulating the stem cell properties in OSCC has not yet been explored. AT-rich interacting domain (ARID) containing protein members play pluripotent roles in modulating chromatin accessibility of the transcription machineries for gene expression. ARID1A has been shown suppressive to several malignancies and the driver mutations in ARID1A gene are noted in tumors. In this study, we identified that miR-31 targets ARID1A in OSCC, and a reverse expression between miR-31 and ARID1A was noted in OSCC tumors. Furthermore, the miR-31 induced migration, invasion, CSC cell population and stemness gene expression were rescued by ARID1A expression in OSCC cells. ARID1A suppressed the transcription activity of a panel of stemness markers through its affinity to A-T rich region in the promoters. A reverse correlation between ARID1A and these stem cell markers was also seen in OSCC tumors. miR-31+/ARID1A− OSCC cell subclone exhibited higher potency in sphere formation, as well as tumorigenesis and neck metastasis in nude mice relative to other cell subclones. In addition, tumors carrying high miR-31 expression and low ARID1A expression had the worst survival. In summary, this study provides novel mechanistic clues demonstrating that miR-31 targets ARID1A to enrich the stemness properties for OSCC progression. Citation Format: Wen-Cheng Lu, Shu-Chun Lin, Kuo-Wei Chang. miR-31 targets ARID1A to enhance the progression of oral 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 953.

Abstract 1108: MicroRNA-211 enhances the oncogenicity of oral carcinoma through targeting TCF12 and up-regulating FAM213A expression

Abstract To address the oncogenic roles of miR-211, we generated K14-EGFP-miR-211 transgenic mouse lines. Upon the 4-nitroquinoline 1-oxide (4NQO) induction, the transgenic mice exhibited more extensive and severe tumorigenesis in tongue compared to the controls. 4NQO up-regulated miR-211 expression in oral squamous cell carcinoma (OSCC) cells. miR-211 directly targeted transcription factor 12 (TCF12), which mediated suppressor activities and was drastically down-regulated in OSCC tumors. GeneChip analysis, bioinformatic algorithm, reporter assay and ChIP assay pinpointed that Family with Sequence Similarity 213, Member A (FAM213A), an anti-oxidative molecule, was transcriptionally repressed by TCF12. Knockdown of FAM213A decreased oncogenic activity and ALDH1-positive cell population, and increased oxygen stress in OSCC cells. The expression of TCF12 and FAM213A was inversely correlated in head and neck carcinoma samples according to The Cancer Genome Atlas. OSCC patients carrying tumors with high FAM213A expression tended to have worse survival. Furthermore, 4NQO treatment down-regulated TCF12 and up-regulated FAM213A through modulating miR-211. The transgenic mouse model recaptures the molecular and histopathological changes as seen in human OSCC pathogenesis. This study highlights that miR-211 responds to oncogenic stimuli to promote the neoplastic process of OSCC by targeting TCF12 and up-regulating FAM213A. Citation Format: Yi-Fen Chen, Kuo-Wei Chang, Shu-Chun Lin. MicroRNA-211 enhances the oncogenicity of oral carcinoma through targeting TCF12 and up-regulating FAM213A expression. [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 1108.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Intraoperative Sentinel Lymph Node Evaluation: Implications of Cytokeratin 19 Expression for the Adoption of OSNA in Oral Squamous Cell Carcinoma.

BackgroundIntraoperative analysis of sentinel lymph nodes would enhance the care of early-stage oral squamous cell carcinoma (OSCC). We determined the frequency and extent of cytokeratin 19 (CK19) expression in OSCC primary tumours and surrounding tissues to explore the feasibility of a “clinic-ready” intraoperative diagnostic test (one step nucleic acid amplification—OSNA, sysmex).MethodsTwo cohorts were assembled: cohort 1, OSCC with stage and site that closely match cases suitable for sentinel lymph node biopsy (SLNB); cohort 2, HNSCC with sufficient fresh tumour tissue available for the OSNA assay (>50 mg). CK19 assays included qRT-PCR, RNA in situ hybridisation (ISH), and immunohistochemistry (IHC), as well as OSNA.ResultsCK19 mRNA expression was detected with variable sensitivity, depending on method, in 60–80% of primary OSCC tumours, while protein expression was observed in only 50% of tumours. Discordance between different techniques indicated that OSNA was more sensitive than qRT-PCR or RNA-ISH, which in turn were more sensitive than IHC. OSNA results showed CK19 expression in 80% of primary cases, so if used for diagnosis of lymph node metastasis would lead to a false-negative result in 20% of patients with cervical lymph node metastases.ConclusionsOSNA in its current form is not suitable for use in OSCC SLNB due to inadequate expression of the CK19 target in all case. However, the same assay technology would likely be very promising if applied using a more ubiquitous squamous epithelial target.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-016-5337-6) contains supplementary material, which is available to authorized users.

Cortactin is a prognostic marker for oral squamous cell carcinoma and its overexpression is involved in oral carcinogenesis.

EMS1 (chromosome eleven, band q13, mammary tumor and squamous cell carcinoma-associated gene 1) gene amplification and the concomitant cortactin overexpression have been reported to associate with poor prognosis and tumor metastasis. In this study, we examined cortactin expression by immunohistochemistry in human oral tumors and murine tongue tumors which were induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). The immunostaining results show over- to moderate expression of cortactin in 85% (104/122) of oral squamous cell carcinoma (OSCC) tissues and in all 15 leukoplakia tissues examined. Further, statistical analysis indicates that cortactin overexpression appears to be a predictor for shorter survival and poorer prognosis in OSCC patients. In an animal model, cortactin is shown to upregulate in infiltrating squamous cell carcinoma, papilloma, and epithelia with squamous hyperplasia, indicating that cortactin induction is an early event during oral carcinogenesis. It is suggested that cortactin expression is mediated in the progression of pre-malignancy to papilloma, based on earlier cortactin induction in pre-malignancy preceding cyclin D1 in papilloma. In conclusion, cortactin overexpression is frequently observed in human OSCC and mouse tongue tumors. Thus, cortactin may have an important role in the development of oral tumors in human and mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 799-812, 2017.