Background: Hepsin (HPN) is an extracellular serine-protease which dysregulation leads to tumor invasion and malignancy. Our group previously described HPN increases protumor and prothrombotic phenotypes in colorectal cancer (CRC) cells, and promotes invasion in zebrafish models. We also identified higher HPN plasma levels in metastatic vs. localized CRC patients, and in patients with thrombosis vs. no thrombosis.

Aims: To identify new HPN inhibitors that reduced its protumor and prothrombotic effects in CRC cells.

Methods: We performed a virtual screening to identify potential compounds that blocked HPN active site. The most outstanding compounds were tested in a fluorogenic assay that measured HPN proteolytic activity. Then, we measured HPN inhibitors effect on CRC cell line Caco-2 transfected with HPN-expression vector (Caco-2-HPN; HPN-overexpression) and negative-control vector (Caco-2; HPN-endogenous expression). We conducted wound confluence, gelatin matrix, cell cytometry and thrombin generation assays (TGA) for measuring migration, invasion, proliferation and procoagulant cell phenotypes, respectively. Finally, zebrafish models were used to test whether HPN inhibitors interfered with distant tumor dissemination.

Results: Virtual screening highlighted suramin as the most effective HPN inhibitor, with a docking score= -12 Kcal/mol. Fluorogenic assay confirmed suramin inhibited HPN in an irreversible and dose-dependent way (IC50=0.66 µM; 95%IC=0.42-1.32). In Caco-2 and Caco-2-HPN cells, suramin reduced migration and, significantly, invasion phenotypes (Figure 1A,B). These effects were more pronounced in Caco-2-HPN cells. Also in both cell types, suramin increased lag time and reduced endogenous thrombin potential and thrombin peak in TGA. Moreover, suramin increased time to peak and reduced velocity to peak in Caco-2-HPN cells (Figure 2A). Finally, suramin reduced Caco-2-HPN invasion to Caco-2 levels in zebrafish (Figure 2B).

Conclusion(s): Suramin is a HPN inhibitor that reduces invasive, migratory and procoagulant characteristics of CRC cells in vitro. In addition, suramin reduces the invasion phenotype in a zebrafish model. These results point suramin as a potential therapeutic agent in CRC.

Figure 1

Suramin reduces both migration and invasion phenotypes of Caco-2 cells. A- %wound confluence. B- %degradation area of gelatin matrix. Asterisks indicated significant differences. -: no incubation with suramin; +: preincubation with suramin.

Figure 2

Suramin suppresses in vitro HPN procoagulant effect and in vivo HPN proinvasive effect in CRC cells. A- Endogenous thrombin potential, thrombin generation peak, thrombin generation lag time, time to thrombin generation peak and velocity to reach thrombin generation peak for Caco-2 and Caco-2-HPN cells. Asterisks indicated significant differences. B- Percentage of larvae with invasive tumor cells in distant organs from injection site; comparison among Caco-2, Caco-2-HPN and Caco-2-HPN cells preincubated with 0.66 µM suramin. Asterisks indicated significant differences. ETP: Endogenous thrombin potential; -: no incubation with suramin; +: preincubation with suramin; S: Suramin; n: number of total larvae.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/suramin-is-a-hepsin-inhibitor-which-reduces-its-protumor-and-prothrombotic-effects-in-colorectal-cancer-cells/