Targeting Serine Peptidase Inhibitor, Kazal Type 1 (SPINK1) as a Potential Treatment for Hepatocellular Carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) ranks fourth as the most common cancer worldwide with high prevalence in Southeast Asia and Africa and the number of new cases in America and Europe is on the rise. HCC patients usually have poor prognosis partly due to the limitations of current treatments. For instance, surgical resection as the mainstream treatment for HCC patients is not suitable for those with advanced disease and poor liver functions. In addition, a subgroup of HCC patients suffers from post-treatment recurrence. All these factors make HCC as the third leading cause of cancer-related mortality. To address this clinical dismal, it is important to research for new therapeutic targets for HCC. Our previous gene profiling analysis implicated a secretory trypsin inhibitor named serine peptidase inhibitor, Kazal type 1 (SPINK1) as a potential molecular target for HCC because of its high expression in liver tumors. Other studies also revealed high serum level of SPINK1 in patients with cancers of various types including HCC. In view of these observations, this study aims to study the role of SPINK1 in liver tumorigenesis. Objectives: To study the role of SPINK1 in liver tumorigenesis Methods: High expression of SPINK1 was validated using quantitative polymerase chain reaction (qPCR) in tumor (T) and adjacent non-tumor (NT) tissues of 60 HCC patients. In examining the involvement of SPINK1 in HCC, loss-of-function approach by short hairpin RNA (shRNA)-based RNA interference (RNAi) was performed to suppress SPINK1 expression in HCC cells. Tumorigenic phenotypes like proliferation, migration and colony formation of these SPINK1-suppressed cells were examined using various in vitro assays. The abilities of these cells to form subcutaneous tumors in nude mice were also assessed. Finally, the downstream signaling pathway associated with SPINK1 suppression was delineated. Results: qPCR results demonstrated the up-regulation of SPINK1 (T/NT ≥ 2) in 68% (41/60) of HCC cases. Such high expression was positively correlated with poor tumor differentiation (Edmondson grading, p = 0.034) and late tumor stage (pTNM staging, p = 0.046). This clinical analysis revealed the involvement of SPINK1 in severe HCC condition. When SPINK1 was suppressed in HCC cells using shRNA, a reduction in proliferation, migration and colony formation ability of these cells was demonstrated. When these cells were used to inoculate into nude mice to form subcutaneous tumors, tumor xenografts derived from SPINK1-suppressed cells did not associate with growth advantage. These reduced tumorigenic properties and tumorigenicity of HCC cells after SPINK1 suppression led to an inactivation of Raf/MEK/ERK pathway, such that a down-regulation of the phosphorylated forms of Raf, MEK, ERK and Akt was observed in SPINK1-suppressed cells. Conclusion: High expression of SPINK1 in human liver tumors indicates severe tumor progression. Suppressing SPINK1 expression in HCC cells alleviates tumor phenotypes and tumorigenicity in vitro and in vivo. Findings reported here support SPINK1 as a potential biotherapeutic for HCC and implicate knockdown of its expression as a treatment for this liver disease.