Secreted chimeric proteins as a strategy to correct SOX17 deficiency and attenuate the malignant phenotype of cholangiocarcinoma cells: a proof-of-concept study.

Cholangiocarcinoma (CCA) is a biliary cancer with a poor prognosis and marked chemoresistance. The transcription factor SOX17, which is essential for cholangiocyte differentiation, is frequently downregulated in CCA, as is the case with several tumor suppressor genes. This study provides a proof-of-concept for further development of cellular therapeutic strategies to restore SOX17 expression in CCA cells. For this aim, we used EGI-1 cells as the target cell model derived from extrahepatic CCA. Recombinant Tat-SOX17 protein was first produced in bacteria and purified using Ni-NTA affinity columns and asymmetric flow-field-flow fractionation. Tat-SOX17 entered EGI-1 cells and reached their nucleus. The addition of the α-fetoprotein signal peptide (AFPsp) to the chimeric protein enabled the efficient secretion of the fusion protein formed by AFPsp, SOX17, and reverse Tat (TatR) by donor cells. For the production of secretome enriched in AFPsp-SOX17-TatR protein, HEK293T cells, derived from embryonic kidney, were used. In EGI-1 cells cultured in the presence of this secretome, target gene expression, cell cycle progression, apoptosis, proliferation, colony formation, and cell migration were assessed. Protein expression and localization were analyzed by Western blotting and immunofluorescence. AFPsp-SOX17-TatR enters EGI-1 cells, reaches their nucleus, and modulates the expression of SOX17-dependent genes, such as ABCB1 and ABCG2. Moreover, a marked reduction in proliferation and colony-forming ability was found. In contrast, no significant effect on cell cycle progression, apoptosis, or cell migration was observed. Similar treatment of immortalized human cholangiocytes also increased their SOX17 content, resulting in upregulation of the cholangiolar marker cytokeratin 7 (CK7), but did not affect their proliferation rate. In conclusion, using chimeric proteins such as AFPsp-SOX17-TatR, which contain components for secretion from donor cells and entry into target cancer cells, can provide a promising approach for treating tumors such as CCA, which are characterized by reduced expression of tumor suppressor genes, including SOX17.