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The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1 , COL4A2 , FLNA , VAV3 , VEGFA , and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2 , PTGS2 , and PLA2G4A . Since PGE 2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2 -knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway., Competing Interests: The authors declare no conflict of interest.

Background: During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance., Methods: The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay., Results: Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2., Conclusion: Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.