Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition

The TGF-beta 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-beta 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-beta effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-beta 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-13 treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-beta 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-beta 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-beta have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.

Automated summary

The TGF-beta signaling pathway and AGR2 downregulation play important roles in focal adhesion formation and cancer cell migration and invasion. In addition, AGR2 is identified as a crucial component of the arachidonic acid metabolic pathway.