Anti-SPINT2 monoclonal antibody, clone 280029

A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of proteins that are upregulated in the plasma of OC patients. Using binders targeting 368 different plasma proteins, we compared 20 plasma samples from HGSC patients (OC-plasma) with 20 plasma samples from individuals with non-malignant gynecologic disorders (N-plasma). We identified 176 proteins with significantly higher levels in OC-plasma compared to N-plasma by PEA (p < 0.05 by U-test; Benjamini-Hochberg corrected), which are mainly implicated in immune regulation and metastasis-associated processes, such as matrix remodeling, adhesion, migration and proliferation. A number of these proteins have not been reported in previous studies, such as BCAM, CDH6, DDR1, N2DL-2 (ULBP2), SPINT2, and WISP-1 (CCN4). Of these SPINT2, a protease inhibitor mainly derived from tumor cells within the HGSC microenvironment, showed the highest significance (p < 2 x 10(-7)) similar to the previously described IL-6 and PVRL4 (NECTIN4) proteins. Results were validated by means of the aptamer-based 1.3 k SOMAscan proteomic platform, which revealed a high inter-platform correlation with a median Spearman rho of 0.62. Likewise, ELISA confirmed the PEA data for 10 out of 12 proteins analyzed, including SPINT2. These findings suggest that in contrast to other entities SPINT2 does not act as a tumor suppressor in HGSC. This is supported by data from the PRECOG and KM-Plotter meta-analysis databases, which point to a tumor-type-specific inverse association of SPINT2 gene expression with survival. Our data also demonstrate that both the PEA and SOMAscan affinity proteomics platforms bear considerable potential for the unbiased discovery of novel disease-associated biomarkers.

Aberrant HGF-MET (hepatocyte growth factor-met proto-oncogene) signaling activation via interactions with surrounding stromal cells in tumor microenvironment has significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation, which is influenced by the tumor microenvironment, and its consequential effects on melanoma malignancy remain uncharacterized. In this study, we identified SPINT2 (serine peptidase inhibitor Kunitz type 2), a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which has a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared with those in early-stage primary melanomas, which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA-hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits the HGF-induced MET-AKT (v-Akt murine thymoma viral oncogene) signaling pathway and decreases malignant phenotype potential such as cell motility and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor-suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF, which is typically activated by tumor-stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression.