SPOCK2 is downregulated due to hypermethylation in PDAC and might inhibit cell proliferation and migration of pancreatic cancer cells

Background and aims SPOCK2, Sparc/Osteonection, cwcv, and kazal-like domains proteoglycan2, is a member of the SPOCK protein family and has been recently reported in several malignancies, such as prostate, breast, and colon cancer. In the present study, we aimed to investigate the potential role of SPOCK2 in pancreatic ductal adenocarcinoma (PDAC).

Methods Data for the mRNA expression levels of SPOCK2 in the patients with PDAC were obtained from Oncomine database using three different patient cohorts. Expression level of SPOCK2 in PDAC and normal pancreas was evaluated by quantitative real-time PCR (RT-PCR) in 7 PDAC cell lines and 1 normal pancreatic epithelial cell line (HPDE). Moreover, 4 PDAC cell lines were treated with 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and expression of SPOCK2 was analyzed using RT-PCR. The results were validated in 2 PDAC cell lines with western blot analysis. In order to further study the biological role of SPOCK2 in PDAC, SPOCK2 was knocked down by using siRNA transfection in one PDAC cell line, Capan2 and the knockdown efficiency was evaluated through qRT-PCR and Western Blot. Subsequently, we invastigated the effect of the inhibition of SPOCK2 on cell proliferation rate and migration ability using MTT and transwell assays, respectively. Additionally, th correlation between SPOCK2 mRNA expression and overall survival (OS) in PDAC patients was studied using KM-Plotter bioinformatics tool.

Results According to the Oncomine data, SPOCK2 was significantly downregulated in PDAC in contrast to pancreatic cancer precursors in Bucholz cohort. However, there was no significant difference between mRNA expression of SPOCK2 in PDAC and normal pancreas in both Badeas and Pei Pancreas cohorts. Interestingly, in contrast to normal pancreatic cell line, SPOCK2 mRNA level was significantly downregulated in all seven investigated PDAC cell lines. Treatment with DAC, led to increase of SPOCK2 expression in all four PDAC cell lines tested. Moreover, we could validate these results in two PDAC cell lines at protein level using western blot analysis. Importantly, compared with control cells, si-SPOCK2 cells exhibited increased growth rates and more migration abilty. Finally, using KM-Plotter, we found that a high SPOCK2 expression level correlated with longer OS in PDAC.

Conclusion These data imply that, SPOCK2 is downregulated in PDAC due to hypermethylation and might act as a tumor supressor and prognostic marker in PDAC.

Publication History

Article published online:
19 August 2022