Bad Tumors Made Worse: SPINK1

Abstract

Serine protease inhibitor Kazal type 1 (SPINK1) is a small secreted protein with dual roles—in the pancreas, it is a protective trypsin inhibitor, while in the context of the tumor microenvironment, it is a cell growth and survival factor that promotes tumor progression. While the mechanism by which SPINK1 protects the pancreas is long established and well-understood, the mechanisms that underlie its tumor promoting properties are complex and multifaceted, with major questions remaining to be answered. In this Opinion article, we briefly overview the known functions and mechanisms of SPINK1 both in health and in disease, and then seek to highlight several of the mechanistic “missing links,” with the aim of identifying research opportunities and stimulating new lines of investigation. SPINK1, also known as pancreatic secretory trypsin inhibitor (PSTI), is a 6.2 kDa secreted serine protease inhibitor that is produced by pancreatic acinar cells. In the pancreas, SPINK1 plays a physiological role as an inhibitor of digestive trypsins (Figure 1A) (Rinderknecht, 1986; Paju and Stenman, 2006). It is co-secreted in zymogen granules with trypsinogen, the trypsin precursor protein, allowing inhibitory intervention in case of early activation of trypsinogen to trypsin, and preventing organ damage of the pancreas or duct system due to autodigestion. The importance of SPINK1 for pancreatic health is demonstrated by the association of SPINK1 gene mutations (N34S, P55S, IVS3 + 2TC, and others) with increased risk for several forms of chronic pancreatitis (Pfützer et al., 2000; Witt et al., 2000; Raphael and Willingham, 2016). Most pathogenic SPINK1 mutations reduce function of the protein by interfering with folding and/or secretion (Kiraly et al., 2007a,b; Kereszturi et al., 2009), while the N34S mutation does not appear intrinsically deleterious, but is associated with another mutation in the 5′ regulatory region of the gene that can diminish mRNA expression (Kereszturi and Sahin-Toth, 2017). On the other hand, homozygous mutations causing complete loss of SPINK1 function were found to be responsible for several cases of severe early-onset exocrine pancreatic insufficiency (Venet et al., 2017). Figure 1. Roles of SPINK1. (A) In the pancreas, SPINK1 acts as an important regulator of protease activity. SPINK1 is co-expressed with trypsinogen by the pancreatic acinar cells and secreted from zymogen granules into the pancreatic duct. Within the acinar cells or the duct, SPINK1 quenches prematurely activated trypsin to prevent further protease activation and organ damage. (B) Tumor cell secreted SPINK1 inhibits unknown serine protease(s) to induce anoikis resistance, tumor cell survival and metastatic disease. (C) Tumor cell secreted SPINK1 activates EGFR kinase pathways and leads to tumor cell proliferation; the direct receptor of SPINK1 in this context requires further definition. (D) Sequence alignment using Clustal Omega comparing human, mouse, and rat EGF with human, mouse, and rat SPINK1 (ISK1) homologs. Identified are sequence identity between hEGF and hSPINK1, sequence identities across all three species, and disulfide bond pattern. Outside of the normal pancreas, aberrant expression of SPINK1 plays a role in cancer. SPINK1 was originally named tumor associated tissue inhibitor (TATI) when it was first isolated from the urine of ovarian cancer patients (Huhtala et al., 1982). Since then SPINK1 has been found to be overexpressed by multiple types of tumor cells, including breast, ovarian, prostate, pancreas, liver, and colon (reviewed Itkonen and Stenman, 2014; Rasanen et al., 2016). More recently, SPINK1 has also been found to be expressed by the tumor stroma after chemotherapy, where it may contribute to chemoresistance and increased risk of recurrence (Chen et al., 2018). SPINK1 tumor cell expression and possible prognostic value have been most studied in prostate cancer, where SPINK1 positive tumors form a subgroup of about 10–15% of prostate cancers (Tomlins et al., 2008; Ateeq et al., 2011, 2015). Prostate tumors that express SPINK1 have been reported to show a significantly more aggressive phenotype and poorer progression-free survival (Tomlins et al., 2008; Leinonen et al., 2010). In other tumor types, multiple studies have explored the potential utility of SPINK1 expression as a biomarker through analysis of tumor tissues, urine, and serum (Halila et al., 1988; Inaudi et al., 1991; de Bruijn et al., 1993; Paju et al., 2007). Tumor tissue staining for SPINK1 has been associated with poorer survival in non-serous ovarian cancers (Mehner et al., 2015) and in estrogen receptor- positive breast cancer (Soon et al., 2011), and there is potential for SPINK1 to serve as a diagnostic marker for hepatocellular carcinoma (Marshall et al., 2013). Studies in experimental model systems have demonstrated significant effects of SPINK1 in promoting tumor cell growth and survival (Rasanen et al., 2016), the mechanisms of which remain to be fully elucidated. Unlike in the normal pancreas, in tumors SPINK1 appears to be expressed independently of trypsin, and little is known about the direct target(s) of SPINK1 in the context of cancer. Normal epithelial cells require contact to other cells or the extracellular matrix to ensure their function and survival; if they detach, intracellular mechanisms drive the apoptosis protocol called anoikis resulting in cell death. Tumor cell metastasis often involves circulation as isolated cells, and thus anoikis resistance is believed to be a common feature of metastatic dissemination (Frisch and Francis, 1994; Simpson et al., 2008; Kim et al., 2012). We have shown that SPINK1 plays an essential role in ovarian cancer cell survival under attachment free...