Hoxa10 controls proliferation, migration and invasion in oral squamous cell carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previous studies we analyzed the expression profile of the members of the HOX family of homeobox genes in oral samples of normal mucosa and squamous cell carcinoma (OSCC) and identified differently expressed genes such as HOXA10. The present study aimed to validate the increased expression of HOXA10 in OSCCs, and to investigate the effects arising from its knockdown in OSCC cells. The levels of HOXA10 mRNA were determined in human OSCC samples and cell lines by quantitative PCR, and HOXA10- mediated effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), migration and invasion were studied in HSC-3 tongue carcinoma cells by using retrovirus-mediated RNA interference. Higher expression of HOXA10 mRNA was observed in OSCC cell lines and in tumor tissues compared to normal controls. HOXA10 knockdown significantly reduced the proliferation of the tumor cells which was accompanied by increased levels of p21. HOXA10 silencing also significantly induced the expression of EMT markers and enhanced the adhesion, migration and invasion of HSC-3 cells. No effects on cell death were observed after HOXA10 knockdown. The results of the current study confirm the overexpression of HOXA10 in OSCCs, and further demonstrate that its expression is functionally associated with several important biological processes related to oral tumorigenesis, such as proliferation, migration and invasion.Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previou8436133623FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)sem informaçãosem informaçãoAlthough HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previo

Cathepsin K Is Present in Invasive Oral Tongue Squamous Cell Carcinoma In Vivo and In Vitro

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HOXA1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis

<p>Abstract</p> <p>Background</p> <p>HOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. However, members of this family demonstrated oncogenic properties in some malignancies. The present study investigated whether genes of the HOXA cluster play a role in oral cancer.</p> <p>Methods</p> <p>In order to identify differentially expressed HOXA genes, duplex RT-PCR in oral samples from healthy mucosa and squamous cell carcinoma was used. The effects of HOXA1 on proliferation, apoptosis, adhesion, invasion, epithelial-mesenchymal transition (EMT) and anchorage-independent growth were assessed in cells with up- and down-regulation of HOXA1. Immunohistochemical analysis using a tissue microarray (TMA) containing 127 oral squamous cell carcinomas (OSCC) was performed to determine the prognostic role of HOXA1 expression.</p> <p>Results</p> <p>We showed that transcripts of HOXA genes are more abundant in OSCC than in healthy oral mucosa. In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas. Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it. Enforced HOXA1 expression in HaCAT cells was not capable of modulating other events related to tumorigenesis, including apoptosis, adhesion, invasion, EMT and anchorage-independent growth. A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival. In multivariate analysis, HOXA1 was an independent prognostic factor for OSCC patients (HR: 2.68; 95% CI: 1.59-2.97; p = 0.026).</p> <p>Conclusion</p> <p>Our findings indicate that HOXA1 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and suggest that HOXA1 expression might be helpful as a prognostic marker for patients with OSCC.</p

Anti-Heparanase Aptamers as Potential Diagnostic and Therapeutic Agents for Oral Cancer

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Cathepsin inhibitor E64 reduces HSC-3 tongue carcinoma cell invasion in the myoma organotypic invasion model.

<p>The effect of cathepsin inhibitor on the invasion of HSC-3 cells in the myoma organotypic assay was studied. E64 at 10 µM could prevent HSC-3 invasion as compared with the controls (A–B). Overall HSC-3 cell invasion depth (C) as well as invasion index (the ratio between the invaded area and the total area of AE1/AE3-stained HSC-3 cells) (D) were quantified. The invasion depth was significantly reduced (***p<0.0001), but the invasion index shows no significant reduction (p = 0.114). Scale bars 200 µm.</p

Cathepsin K expression in the myoma organotypic model.

<p>Invasive HSC-3 cells grown on myoma show intensive cathepsin K immunohistological staining (A). HSC-3 cells grown in type I collagen organotypic culture discs with embedded fibroblasts show cathepsin K staining in all cells present (B). Myoma tissue (without HSC-3 cells) as well as fibroblasts embedded in the collagen gel also stained with cathepsin K antibody (D–E). A negative control for immunostaining is shown (C). A Western blot confirmed that the monolayer cultures of HSC-3 cells (G, lane 2) and two distinct myoma tissue samples (without added carcinoma cells) contained cathepsin K (G, lanes 3 and 4). HSC-3 cells microdissected from the organotypic myoma model (F) of both formalin-fixed paraffin-embedded blocks (FFPE) and OCT-embedded frozen blocks (fresh frozen), as well as HSC-3 cells cultured <i>in vitro</i> in monolayers, express cathepsin K mRNA, as detected by RT-PCR (H). A differentiated human osteoclast progenitor cell line (Osteo) was used as a positive control for cathepsin K mRNA expression, represented by (+). Negative controls, where no sample was used, are demonstrated by (−) Scale bars 200 µm.</p

Weaker staining of cathepsin K in the invasive front correlates with shorter recurrence-free survival and increased mortality.

<p>Survival analyses show a shorter overall recurrence-free interval in patients with a lower cathepsin K staining gradient (cat-K gradient −) compared with those with either higher (cat-K gradient +) or no gradient (gradient 0) of cathepsin K staining intensities in the invasive front compared with the upper part of the tumor (A), especially seen in patients with stage 1 or 2 OTSCC (B). Disease-specific survival of patients with stages 1 and 2 OTSCC tumors was better in patients with a positive or no gradient of cathepsin K staining compared with those with a negative gradient (C).</p

Cathepsin K immunostaining pattern in lymph node metastases of OTSCC is similar to the primary tumor.

<p>Metastatic carcinoma cells were identified with pancytokeratin staining (A–B), followed by cathepsin K staining of the paired primary OTSCC (C) and lymph node metastases (D) (n = 10, the paired sections were taken from the same patient). The arrows indicate similar cathepsin K staining patterns in both the primary tumor and in the lymph node metastases. Lymph nodes without metastases contain some cathepsin K positive cells (E), but the stromal reaction drastically changes in the presence of cathepsin K positive OTSCC cells (F). Scale bars 200 µm.</p

Data retrieved from the files of patients whose samples were used in this study.

<p>Data retrieved from the files of patients whose samples were used in this study.</p

Cathepsin K immunostaining in invasive tongue cancer tissues and dysplastic oral epithelium.

<p>Cathepsin K in OTSCC tumors is localized in a few areas of dysplastic epithelium (dp) surrounding the cancer tissue (SCC) (A–B). A no staining area within a tumor slide with a score of (0) is shown by the first arrow (B). Other arrows, from left to right, show weak epithelial staining (+) and moderate stromal staining (++) (B). Cathepsin K shows a membranous, defined staining pattern in invading OTSCC carcinoma cells (C). The top arrow indicates an area with strong (+++) epithelial diffuse staining and moderate (++) stromal staining and the arrow below indicates stromal staining at the invasive front (D). At the invasive front, carcinoma cells show a cytoplasmic diffuse staining pattern. Big arrows indicate strong (+++) epithelial membranous staining. Empty arrows highlight individual cells in the stroma with moderate staining (++) (E). Arrows indicate different intensities of cathepsin K staining in superficial and invasive areas of tumor from weak (+) to moderate (++), illustrating the expression gradient observed in the samples (F). Example of strong stromal staining (full arrows) (+++) when epithelial staining is only moderate or weak (empty arrows) (G). Scale bars 200 µm.</p