HOXA10 controls proliferation, migration and invasion in oral squamous cell carcinoma.

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.

Anti-heparanase aptamers as potential diagnostic and therapeutic agents for oral cancer.

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.

Cathepsin K is present in invasive oral tongue squamous cell carcinoma in vivo and in vitro.

OBJECTIVES: Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens. MATERIALS AND METHODS: OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated. RESULTS: Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively). CONCLUSIONS: Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.

Heterogeneous presence of myofibroblasts in hereditary gingival fibromatosis.

BACKGROUND/AIM: Hereditary gingival fibromatosis (HGF) fibroblasts are characterized by an increased production of collagen and transforming growth factor-beta1 (TGF-beta1), resulting in a fibrotic enlargement of the gingiva of affected patients. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. The goal of this article is to describe the presence of myofibroblasts in HGF in order to elucidate the mechanisms underlying HGF gingival overgrowth. MATERIALS AND METHODS: Fibroblast cell lines and gingival samples from patients of two distinct families affected by HGF and from normal gingiva (NG) were included in this study. To characterize the presence of myofibroblasts, the expression of specific myofibroblast marker smooth muscle isoform of alpha-actin (alpha-SMA) was examined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and flow cytometric analysis. Immunohistochemistry against the alpha-SMA antigen was performed in the gingival tissue samples. RESULTS: Our results demonstrated a significant increase in the expression of the myofibroblast marker alpha-SMA in cells from one HGF family (designed as HGF Family 2), which are also characterized by an elevated expression of type I collagen, TGF-beta1 and connective tissue growth factor (CTGF). Additionally, alpha-SMA-positive cells were broadly detected in the gingival tissue samples from HGF Family 2 patients. In contrast, alpha-SMA expression by HGF Family 1 cells was quite similar to NG cells and no myofibroblasts were detected immunohistochemically, despite the higher levels of TGF-beta1 and type I collagen in HGF Family 1 fibroblasts than in NG cells. The expression of CTGF, which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation, by HGF Family 1 cultures was significantly lower compared with HGF Family 2 and similar to NG control cells. CONCLUSIONS: Our results suggest that the presence of myofibroblasts in HGF could be dependent on CTFG expression levels, and different biological mechanisms may account for the gingival overgrowth observed in HGF patients. This could be an underlying reason for the high variable clinical expressivity of disease.