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 PGE2 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.

• Keywords
• AGR2, EMT, RNAseq, TGF-β, arachidonic acid, focal adhesion,
• MeSH
• Cyclooxygenase 2 genetics   MeSH
• Epithelial-Mesenchymal Transition * genetics   MeSH
• Arachidonic Acid MeSH
• Cell Line, Tumor MeSH
• Cell Movement genetics   MeSH
• Prostaglandins E MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Transforming Growth Factor beta genetics   MeSH
• Vinculin genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cyclooxygenase 2 MeSH
• Arachidonic Acid MeSH
• Prostaglandins E MeSH
• Transforming Growth Factor beta MeSH
• Vinculin MeSH

There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2-knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP-scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2-knockout cells.

• Keywords
• AGR2, AMPK, ROS, autophagy, colorectal cancer, diabetes mellitus,
• Publication type
• Journal Article MeSH

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.

• Keywords
• AGR2, E-cadherin, EMT, Metastasis, TGF-β, Vimentin,
• MeSH
• Cell Adhesion genetics   MeSH
• Epithelial-Mesenchymal Transition drug effects   genetics   MeSH
• Gene Knockdown Techniques MeSH
• Cadherins metabolism   MeSH
• Humans MeSH
• Mucoproteins MeSH
• Cell Line, Tumor MeSH
• Oncogene Proteins MeSH
• Cell Movement genetics   MeSH
• Smad Proteins metabolism   MeSH
• Proteins genetics   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Signal Transduction drug effects   MeSH
• Transforming Growth Factor beta pharmacology   MeSH
• Vimentin metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AGR2 protein, human MeSH Browser
• Cadherins MeSH
• Mucoproteins MeSH
• Oncogene Proteins MeSH
• Smad Proteins MeSH
• Proteins MeSH
• Transforming Growth Factor beta MeSH
• Vimentin MeSH

The tumor suppressor p53 plays a key role in malignant transformation and tumor development. However, the frequency of p53 mutations within individual types of cancer is different, suggesting the existence of other mechanisms attenuating p53 tumor suppressor activity. Changes in upstream regulators of p53 such as MDM2 amplification and overexpression, expression of viral oncoproteins, estrogen receptor signaling, or changes in p53 transcriptional target genes were previously described in wild-type p53 tumors. We identified a novel pathway responsible for attenuation of p53 activity in human cancers. We demonstrate that AGR2, which is overexpressed in a variety of human cancers and provides a poor prognosis, up-regulates DUSP10 which subsequently inhibits p38 MAPK and prevents p53 activation by phosphorylation. Analysis of human breast cancers reveals that AGR2 specifically provides a poor prognosis in ER+ breast cancers with wild-type p53 but not ER- or mutant p53 breast cancers, and analysis of independent data sets show that DUSP10 levels also have prognostic significance in this specific sub-group of patients. These data not only reveal a novel pro-oncogenic signaling pathway mediating resistance to DNA damaging agents in human tumors, but also has implications for designing alternative strategies for modulation of wild-type p53 activity in cancer therapy.

• Keywords
• AGR2, Breast cancer, DUSP10, Drug resistance, p38 MAPK, p53,
• MeSH
• Enzyme Activation drug effects   MeSH
• Drug Resistance, Neoplasm MeSH
• Adult MeSH
• Mitogen-Activated Protein Kinase Phosphatases metabolism   MeSH
• Dual-Specificity Phosphatases metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• p38 Mitogen-Activated Protein Kinases metabolism   MeSH
• Mucoproteins MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Breast Neoplasms drug therapy   metabolism   pathology   MeSH
• Neoplasms drug therapy   metabolism   pathology   MeSH
• Oncogene Proteins MeSH
• Proteins metabolism   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Breast drug effects   metabolism   pathology   MeSH
• Aged MeSH
• Signal Transduction * drug effects   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AGR2 protein, human MeSH Browser
• DUSP10 protein, human MeSH Browser
• Mitogen-Activated Protein Kinase Phosphatases MeSH
• Dual-Specificity Phosphatases MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• Mucoproteins MeSH
• Tumor Suppressor Protein p53 MeSH
• Oncogene Proteins MeSH
• Proteins MeSH
• Antineoplastic Agents MeSH

Anterior gradient protein (AGR) 3 is a highly related homologue of pro-oncogenic AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumorigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly correlated with estrogen receptor, progesterone receptor (both P<0.0001) as well as low histological grade (P=0.003), and inversely correlated with the level of Ki-67 expression (P<0.0001). In the whole cohort, AGR3 expression was associated with longer progression-free survival (PFS), whereas AGR3-positive subgroup of low-histological grade tumors showed both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients.

• Keywords
• AGR3, ER-positive breast cancer, immuno histochemistry, patient survival, protein disulfide isomerase,
• Publication type
• Journal Article MeSH