The protease inhibitor gabexate mesylate targets Raf kinase inhibitory protein (RKIP) and reverses epithelial-mesenchymal transition in triple negative breast cancer cells

Marzia Deserti¹Simone Sabbioni¹Valeria Relli¹Francesca Poli²Francesco Vasuri³Giovanni Brandi¹Simona Tavolari^4*

• ¹Universita degli Studi di Bologna Dipartimento di Scienze Mediche e Chirurgiche, Bologna, Italy
• ²Azienda Unita Sanitaria Locale di Imola, Imola, Italy
• ³Ospedale Santa Maria delle Croci, Ravenna, Italy
• ⁴IRCCS University Hospital of Bologna Sant Orsola Polyclinic, Bologna, Italy

The prognosis of triple negative breast cancer (TNBC) still remains poor, mainly due to the occurrence of early metastases and the lack of effective treatments. The RAF-kinase inhibitor protein (RKIP) is a tumor metastasis suppressor frequently downregulated in human cancers. Here we report that RKIP expression is lost in the tumor tissue of TNBC patients. Treatment with the protease inhibitor Gabexate mesilate (GM) increased RKIP expression and reverted epithelial-mesenchymal transition (EMT) phenotype in MDA-MB-231 cells, a well-established in vitro TNBC model. This phenotypic change was concomitant to upregulation of the epithelial markers E-Cadherin and Claudin-1, and to downregulation of the mesenchymal ones N-Cadherin, Vimentin and nuclear β-catenin. Furthermore, GM significantly decreased TNBC cell motility and invasiveness, along with MMP-2 and MMP-9 protein expression and activity. At mechanistic level, RKIP upregulation inhibited p42/44 MAPK and NF-kB signalling that, in turn, down-regulated the expression of the two EMT transcription factors Snail and Slug. Despite these preliminary findings need to be confirmed in more representative ex-vivo (such as patient-derived primary cells or patient-derived organoids) and in vivo TNBC models, they provide evidence that EMT process could be pharmacologically reverted by the protease inhibitor GM in a highly metastatic TNBC in vitro model, deserving further investigation in future studies.