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BACKGROUND Tumor-associated trypsin inhibitor (TATI) was originally isolated from the urine of a patient with ovarian cancer. It was later shown to be produced by many other tumors and several normal tissues. It had earlier been isolated from the pancreas and was hence called pancreatic secretory trypsin inhibitor (PSTI). It belongs to a family of protease inhibitors presently called serine peptidase inhibitor Kazal type (SPINK). In the SPINK family TATI/PSTI is SPINK1, which is the name used in this review. CONTENT In addition to being a protease inhibitor, SPINK1 also acts as an acute-phase reactant and a growth factor. Furthermore, it has been shown to modulate apoptosis. Overexpression of SPINK1 predicts an unfavorable outcome in several cancers and determination of SPINK1 in serum can be used to identify patients at increased risk of aggressive disease. Thus serum SPINK1 can be used as a prognostic tumor marker. Because SPINK1 acts as a growth factor and an inhibitor of apoptosis in some cancers, it has also been suggested that it can be a therapeutic target in cancer. However, because SPINK1 is the major physiological inhibitor of trypsin, inhibition of SPINK1 may increase the risk of pancreatitis. SUMMARY Taking into account the many functions of SPINK1, assessing the role of SPINK1 in cancer has several potentially important clinical applications ranging from a biomarker to a potential new target for cancer therapy.

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Squamous cell carcinomas (SCCs) with an infiltrative invasion pattern carry a higher risk of treatment failure. Such infiltrative invasion may be mediated by a mesenchymal-like subpopulation of malignant cells that we have previously shown to arise from epithelial to mesenchymal transition (EMT) and resist epidermal growth factor receptor (EGFR) targeting. Here we demonstrate that SCCs with infiltrative, high risk invasion patterns contain abundant mesenchymal-like cells, which are rare in tumors with low risk patterns. This cellular heterogeneity was modeled accurately in three dimensional culture using collagen-embedded SCC spheroids, which revealed distinct invasive fronts created by collective migration of E-cadherin-positive cells versus infiltrative migration of individual mesenchymal-like cells. Because EGFR expression by mesenchymal-like cells was diminished in the spheroid model and in human SCCs, we hypothesized that SCCs shift toward infiltrative invasion mediated by this subpopulation during anti-EGFR therapy. Anti-EGFR treatment of spheroids using erlotinib or cetuximab enhanced infiltrative invasion by targeting collective migration by E-cadherin-positive cells while sparing mesenchymal-like cells; by contrast, spheroid invasion in absence of mesenchymal-like cells was abrogated by erlotinib. Similarly, cetuximab treatment of xenografts containing mesenchymal-like cells created an infiltrative invasive front comprised of this subpopulation, whereas no such shift was observed upon treating xenografts lacking these cells. These results implicate mesenchymal-like SCC cells as key mediators of the infiltrative invasion seen in tumors with locally aggressive behavior. They further demonstrate that EGFR inhibition can promote an infiltrative invasion front comprised of mesenchymal-like cells preferentially in tumors where they are abundant prior to therapy.

Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over-expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL-6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL-6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT-29 cells express and secrete SPINK1, and both fibroblast-derived and recombinant IL-6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL-6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL-6 was found in stromal cells. In Colo205 cells IL-6 also stimulated the secretion of trypsin-1 and -2, the key targets of SPINK1 protease inhibition, whereas in HT-29 cells trypsin-1 and -2 levels remained constantly low. Functionally, both IL-6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL-6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin-1 and -2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer. © 2015 Wiley Periodicals, Inc.

Epithelial-mesenchymal transition (EMT) is a key contributor in tumor progression and metastasis. EMT produces cellular heterogeneity within head and neck squamous cell carcinomas (HNSCC) by creating a phenotypically distinct mesenchymal subpopulation that is resistant to conventional therapies. In this study, we systematically characterized differences in the secretomes of E-cadherin high epithelial-like and E-cadherin low mesenchymal-like subpopulations using unbiased and targeted proteomics. A total 1765 proteins showed significant changes with 177 elevated in the epithelial subpopulation and 173 elevated in the mesenchymal cells. Key nodes in affected networks included NF B, Akt, and ERK, and most implicated cellular components involved various aspects of the extracellular matrix. In particular, large changes were observed in multiple collagens with most affected collagens at much higher abundance levels in the mesenchymal subpopulation. These cells also exhibited a secretome profile resembling that of cancer-associated fibroblastic cells (CAF). S100A4, a commonly used marker for cancer-associated fibroblastic cells, was elevated more than 20-fold in the mesenchymal cells and this increase was further verified at the transcriptome level. S100A4 is a known mediator of EMT, leading to metastasis and EMT has been proposed as a potential source of cancer-associated fibroblastic cells in solid tumors. S100A4 knockdown by small interfering RNA led to decreased expression, secretion and activity of matrix metalloproteinase 2, as verified by quantitative PCR, multiple reaction monitoring and zymography analyses, and reduced invasion in collagen-embedded spheroids. Further confirmation in three-dimensional organotypic reconstructs showed less invasion and advanced differentiation in the S100A4 RNA interference samples. Head and neck squamous cell carcinoma (HNSCC) 1 is the sixth most common cancer worldwide with high morbidity and mortality. Treatment options are limited; surgery, radiation and conventional chemotherapy are generally associated with considerable impairment to quality of life (1). Currently cetuximab, the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is the only FDA-approved molecularly targeted drug for HNSCC. Several clinical trials are underway that target EGFR or other tyrosine kinases, but many have failed to progress beyond Phase II (2). One reason behind the failure of new treatment options for HNSCC is resistance to therapy, which tumor heterogeneity is thought to provide.