RNA interference mediated silencing of SPINK reduces invasion and proliferation of prostate cancer cells

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Serine protease inhibitors (SPI) regulate the activity of proteases that are known to promote tumor invasion and metastasis. The expression of protease inhibitors has been generally considered to be an anti-malignant phenotype. However, recent studies indicate that certain SPI are linked to a worse prognosis for cancer patients, thus adding a new dimension to their functions in Tumor Biology. Recently, a SPI belonging to the Kazal type family, known as Serine Protease Inhibitor Kazal type 1 (SPINK) has been shown to be overexpressed in various malignancies (1-4). In prostate cancer, SPINK is overexpressed by more than 100-fold compared to normal prostate according to Serial Analysis of Gene Expression.. We aimed to investigate the biological significance of the increase in SPINK and its mode of action in prostate cancers based on the hypothesis that downregulation of SPINK mRNA levels could affect cell proliferation and invasion. We transfected human prostate cancer cell lines (LNCaP, PC3 and DU145) with short interfering RNA (siRNA) against SPINK and verified the gene knockdown effect (68%) with real-time RT-PCR. Using Matrigel invasion and colorimetric cell viability (MTS) assays, we found that the knockdown of SPINK caused >34% decrease (*P<0.05) in cancer cell invasion as well as growth of DU145 (46%) and PC3 (39%) cells. A reduction of thymidine incorporation indicates that the decline in cell numbers observed by MTS assay could be due to decreased proliferation rates. Furthermore, this invasion and growth promoting effect of SPINK is independent of its protease-inhibitory activity as the commercial trypsin inhibitors Leupeptin and SBTI did not have the same effect on invasion and cell growth. While the mechanism underlying the invasion and growth-promoting effect of SPINK in prostate cancers is yet to be established, we can conclude that SPINK promotes cell invasion and cell growth in culture. These studies indicate that SPINK may be a possible target for treatment of cancer and the knowledge of its mode of action could provide important information for future investigations. 1. Tomita, N. et al (1987) FEBS letters, 225, 113-119. 2. Ogawa, M. et al (1987) Res Commun Chem Pathol Pharmacol, 55, 137-140. 3. Paju, A. et al (2001) Journal of Urology, 165, 959-962. 4. Diggle, C.et al (2003) American Journal of Pathology, 163, 493-504.