TGFβ1 and RGD Cooperatively Regulate SMAD 2/3 Mediated Oncogenic Effects in Prostate Cancer Cells in Bioorthogonally Constructed Hydrogels

To recapitulate prostate cancer metastasis, DU145 cells were cultured in a hyaluronic acid-based, bioorthogonally constructed, protease-degradable hydrogel. In the presence of covalently conjugated integrin-binding peptide (GRGDSP), DU145 cells formed tumoroids and exhibited small protrusions. Upon addition of soluble transforming growth factor beta 1 (TGF{beta}1), cells underwent morphological changes to form extended interconnected cellular networks. Contrarily, in RGD-free hydrogels, cells maintained spherical structures even in the presence of TGF{beta}1. In RGD-conjugated hydrogels, TGF{beta}1 induced nuclear localization of SMAD2/3, upregulating a wide range of TGF{beta}1 target genes and proteins. Prolonged exposure to TGF{beta}1 led to matrix remodeling and induced epithelial-to-mesenchymal transition in DU145 cells, with loss of epithelial markers and gain of mesenchymal markers. TGF{beta}RI/ALK5 inhibitor SB-431542 attenuated TGF{beta}1-induced morphological changes, abrogated nuclear localization of SMAD2/3, and restored the expression of key epithelial markers. Our findings highlight the cooperative role of TGF{beta}1 signaling and integrin binding peptide in the acquisition of aggressive phenotype and promoting tumor progression. TEASERPhysiologically relevant 3D cell culture platforms enabled mechanistic investigation of growth factor signaling related to prostate cancer metastasis.