Liver is the most common site of metastasis from colorectal cancers, and liver of patients with liver colorectal metastasis have abnormal levels of the proprotein convertases (PCs). These proteases are involved in the activation and/or expression of various colon cancer-related mediators, making them promising targets in colorectal liver metastasis therapy. Here, we revealed that the serpin Spn4 from Drosophila melanogaster inhibits the activity of all the PCs found in the constitutive secretory pathway and represses the metastatic potential of the colon cancer cells HT-29 and CT-26. In these cells, Spn4A inhibited the processing of the PCs substrates IGF-1R and PDGF-A that associated their reduced anchorage-independent growth, invasiveness and survival in response to apoptotic agents. In vivo, Spn4A-expressing tumor cells showed repressed subcutaneous tumor development and liver metastases formation in response to their intrasplenic inoculation. In these cells Spn4A induced the expression of molecules with anti-metastatic functions and inhibited expression of pro-tumorigenic molecules. Taken together, our findings identify Spn4A as the only endogenous inhibitor of all the constitutive secretory pathway PCs, which is able to repress the metastatic potential of colon cancer cells. These results suggest the potential use of Spn4A and/or derivates as a useful adduct colorectal liver metastasis prevention.

Metformin inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which is frequently upregulated in hepatocellular carcinoma (HCC). Metformin has also been shown to induce apoptosis in this cancer. Here, we investigate whether metformin-induced apoptosis in HCC is mediated by the downstream mTORC1 effectors eukaryotic initiation factor 4E and (eIF4E)-binding proteins (4E-BPs). Further, we ask whether changes in 4E-BPs activity during metformin treatment negatively regulate translation of the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) mRNA. A genetic HCC mouse model was employed to assess the ability of metformin to reduce tumor formation, induce apoptosis, and control 4E-BP1 activation and Mcl-1 protein expression. In parallel, the HCC cell line Huh7 was transduced with scrambled shRNA (control) or shRNAs targeting 4E-BP1 and 4E-BP2 (4E-BP knock-down (KD)) to measure differences in mRNA translation, apoptosis, and Mcl-1 protein expression after metformin treatment. In addition, immunohistochemical staining of eIF4E and 4E-BP1 protein levels was addressed in a HCC patient tissue microarray. We found that metformin decreased HCC tumor burden, and tumor tissues showed elevated apoptosis with reduced Mcl-1 and phosphorylated 4E-BP1 protein levels. In control but not 4E-BP KD Huh7 cells, metformin induced apoptosis and repressed Mcl-1 mRNA translation and protein levels. Immunostaining of HCC patient tumor tissues revealed a varying ratio of eIF4E/4E-BP1 expression. Our results propose that metformin induces apoptosis in mouse and cellular models of HCC through activation of 4E-BPs, thus tumors with elevated expression of 4E-BPs may display improved clinical chemopreventive benefit of metformin.