Hepatocyte Growth Factor Activator Inhibitor Type 1 Regulates Epithelial to Mesenchymal Transition through Membrane-Bound Serine Proteinases

Abstract

Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-associated proteinase inhibitor that potently inhibits a variety of serine proteinases, including those that are membrane bound. Although HAI-1/SPINT1 is widely expressed by epithelial cells and cancer cells, its functional role is still unclear, particularly in cancer. Here, we show that stable knockdown of HAI-1/SPINT1 in the human pancreatic cancer cell line SUIT-2 induces an elongated spindle-like morphology associated with accelerated invasion, thereby mimicking an epithelial to mesenchymal transition (EMT). We found that HAI-1/SPINT1 knockdown significantly reduced the expression of E-cadherin and was accompanied by up-regulation of Smad-interacting protein 1 (SIP1), an E-cadherin transcriptional repressor. In addition, matrix metalloproteinase-9 (MMP-9) was up-regulated. Similar results were obtained in the HLC-1 lung carcinoma cell line. Moreover, a metastatic variant of SUIT-2 (S2-CP8) that showed loss of E-cadherin expression also showed a significantly reduced level of HAI-1/SPINT1. Engineered overexpression of HAI-1/SPINT1 in S2-CP8 resulted in reversion of E-cadherin expression and SIP1 down-regulation, which accompanied reestablishment of epithelial morphology in culture. The EMT caused by HAI-1/SPINT1 knockdown seemed to be mediated, at least partly, by membrane-bound serine proteinases, matriptase/ST14 and TMPRSS4, as knockdown of matriptase/ST14 or TMPRSS4 in HAI-1/SPINT1 knockdown SUIT-2 cells and HLC-1 cells resulted in reversion of SIP1 and/or MMP-9 expression levels. We suggest that interactions between HAI-1/SPINT1 and membrane-bound serine proteinases contribute to transcriptional and functional changes involved in EMT in certain carcinoma cells.

Hepatocyte growth factor activator inhibitor type-1 in cancer: Advances and perspectives (Review)

“…Thus, the net balance between HGFA/matriptase and HAI-1 was shifted in favor of HGFA/matriptase in various types of carcinoma, including breast, ovarian, renal, prostate and colorectal carcinoma (18)(19)(20)38,40,43,49,50). In addition, in vitro studies have validated the finding that HAI-1 knockdown-induced enhanced migration is partially reversed by silencing of matriptase or other serine protease expression (33,34,36).…”

“…Vimentin, SIP1 and matrix metalloproteinase (MMP)-9 expression was upregulated in these cells but E-cadherin expression was downregulated. The subsequent silencing of matriptase in these HAI-1 knockdown cells resulted in reversal in the expression levels of MMP-9 accompanied by a recovery of E-cadherin expression levels (34). In another study, HAI-1 overexpression resulted in a significant increase in E-cadherin expression levels but a reduction in Vimentin, SIP1, Snail and Twist expression levels in human endometrial cancer cell lines (30).…”

“…Such findings are important for the analysis of the pathogenic role of HAI-1 in cancer cells. However, whether HAI-1 suppresses or promotes proliferation of cancer cells remains elusive (28)(29)(30)(31)(32)(33)(34)37).…”

Membrane-Bound Serine Protease Inhibitor HAI-1 Is Required for Maintenance of Intestinal Epithelial Integrity

“…For quantitative real-time RT-PCR using cyber green, PCR was performed in a LightCycler (Roche Diagnostics Japan, Tokyo, Japan) as previously described. 14 For internal control, ␤-actin mRNA was also measured.…”

“…13 Even in neoplastic cells, short hairpin RNA knockdown of HAI-1/ SPINT1 induced epithelial to mesenchymal transition in certain human epithelial cancer cell lines with enhanced metastatic colonization capability. 14,15 These lines of evidence strongly suggest that HAI-1/SPINT1 has a significant functional role in epithelial biology.…”