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

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

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

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