Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

<div><p>Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.</p></div

The role of agrin and perlecan in cell viability.

<p>The viability of SCC-9 (n = 2, A) and SCC-9 LN-1 (n = 3, B) was significantly reduced after siRNA-knockdown of agrin, but no difference in viability was observed in perlecan knockdown. The viability of A431 was not altered neither by agrin knockdown nor by perlecan knockdown (n = 3, C) (One-way ANOVA followed by Tukey’s test, different letters indicate statistically difference at p<0.05).</p

Treatment with chondroitinase ABC decreased SCC-9 LN-1 cell adhesion to extracellular matrix, but not SCC-9 LN-1 cell migration and increased sensibility of SCC-9 LN-1, SCC-9 and A431 cells to cisplatin.

<p>(A) SCC-9 LN-1 had a lower ability to adhere to extracellular matrix proteins (Matrigel) after treatment with 0.1 U/ml of chondroitinase for 4 h/37°C in serum free media (n = 3, triplicate, Student’s <i>t</i>-test, * indicates p<0.05). (B) SCC-9 LN-1 was treated with 0.1 U/ml for 4 h/37°C in serum free media were seeded in the upper chamber of 96-well transwell plates (n = 3, triplicate). RPMI media, which was supplemented with 1% FBS, was added in the lower chamber. (C) SCC-9 LN-1, SCC-9 and A431 cells treated with increasing concentrations of cisplatin (0–100 µM) for 48 h in the presence of 0.1 U/ml of chondroitinase showed increased sensibility to cisplatin, calculated by a non-linear regression of a dose-response curves (log[µM cisplatin] vs normalized response). Data are expressed as means ± SD from one independent experiment.</p

mRNA expression levels of agrin and perlecan.

<p>(A) Validation of higher expression of agrin and perlecan by qRT-PCR in OSCC tumor tissues<b>.</b> Agrin and Perlecan showed higher mRNA expression levels in human OSCC tumor tissues compared to control tissues by qRT-PCR (Kruskal-Wallis followed by Dunn’s test, n = 16, *p<0.05). (B) Agrin showed higher mRNA expression levels in SCC-9 compared to A431 and SCC-9 LN-1 cell lines. (C) Perlecan showed higher mRNA expression levels in SCC-9 LN-1 compared with A431 and SCC-9 cell lines. The data were normalized with glyceraldehyde-3-phosphate dehydrogenase gene, used as internal reference). Each bar represents means ± SD of at least two independent experiments in triplicates (one-way ANOVA followed by Tukey’s test. Different letters indicate statistically difference at p<0.05).</p

The role of agrin and perlecan in cell adhesion.

<p>Knockdown of agrin decreased adhesion to Matrigel in SCC-9 (n = 2, A) and SCC-9 LN-1 (n = 3, B), while no difference was observed in A431 (n = 3, C). When perlecan was silenced by siRNA no difference was observed in SCC-9 adhesion to Matrigel (n = 2, D) but a significant reduction was observed in SCC-9 LN-1 (n = 3, E) and A431 (n = 3, F) adhesion to Matrigel (Student’s <i>t</i>-test, * indicates p<0.05).</p

Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

<div><p>Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.</p></div

Knockdown of activin A increases cyclin-dependent kinase inhibitors p16, p21 and p27.

<p>Cells were harvested, lysed and proteins were subjected to western blot analysis using specific antibodies against p16, p21, p27, CDK2, CDK4, CDK6, cyclin D1, cyclin E and phospho-RB. β-actin was used as loading control. A strong increase in expression of p16, p21 and p27, concomitant with decrease in phosphorylation of RB, was observed in shINHBA cells in comparison with shControl cells. Beta-actin was used as loading control.</p

Expression of INHBA is inversely correlated with miR-143 and miR-145 expression in OSCC cells lines and tumor samples.

<p>(A) Spearman correlation analysis between the expressions of INHBA and miR-143 and miR-145 in 7 OSCC cell lines (A) and in 11 OSCC fresh samples (B). A significant inverse correlation was observed between both miR-143 and miR-145 and INHBA expression levels.</p

Downregulation of activin A leads to a decrease in proliferation.

<p>Treatment with recombinant activin A was not able to promote proliferation of HaCat cells, as revealed by bromodeoxyuridine (BrdU) incorporation index (A) and cell cycle analysis (B). (C) Follistatin at 100 ng/ml significantly blocked BrdU incorporation in SCC-9 ZsGreen LN-1 cells, but no effects on cell cycle distribution were observed (D). Knockdown of activin A significantly decreased proliferation (E), enhancing the number of cells at G0/G1 and reducing the number in the S phase of cell cycle (F). Bars represent the means ± SD of three independent experiments. *p<0.01, **p<0.001, ***p<0.0001.</p

INHBA mRNA is target by miR-143 and miR-145.

<p>(A) SCC-9 and SCC-9 ZsGreen LN-1 cells were exposed to Scramble control or miR-143 and miR-145 mimics. qPCR analysis demonstrated that levels of miR-143 and miR-145 were dramatically increased after introduction of the mimics. (B) Levels of activin A mRNA and protein were clearly decreased in both OSCC cell lines, demonstrating that miR-143/miR-145 cluster regulates INHBA mRNA levels.</p