Tgm-2 Mediated Downregulation Of Fibronectin During Tgfb-Induced Epithelial-Mesenchymal Transition In Lung Cancer Nci-H358 Cells

The epithelial to mesenchymal transition (EMT), which converts epithelial cells into an elongated, motile, and invasive phenotype, is considered to be a critical step in the dissemination of tumor cells. During EMT, the loss of E-cadherin (ECad) and upregulation of N-cadherin (NCad) or fibronectin (FN) have been most frequently described. FN binds integrin Alpha5 and is known for its upregulation in aggressive tumor cells. In addition, FN binds tissue transglutaminase (TGM-2) with high affinity (∼in nM) and plays a role in TGM-2 secretion. TGM-2, a multifunctional enzyme with transamidation or crosslinking, and GTP/ATP binding activities, is up-regulated in various malignant diseases, including lung cancer. In breast, cervical and ovarian cancers, TGM-2 has been shown to play a role in chemotherapy resistance and tumor metastasis during induction of EMT. TGM-2 overexpression results in the loss of epithelial features, up-regulation of mesenchymal markers, increased migration and invasion, and up-regulation of transcriptional repressors (e.g., ZEB1/2, Snail) known to mediate the EMT process. However, most of these studies were performed in aggressive tumor cells. In this study, we investigated the role of TGM-2 in TGFbeta-induced EMT process using NSCLC H358 cells. H358 cells express high levels of epithelial genes and are relatively resistant to TGFbeta induced EMT when compared to A549 cells. Particularly, H358 expresses barely detectable TGM-2 and FN. To investigate the role of TGM-2 during TGFbeta induced EMT, we constructed three isogenic cell lines with doxycycline (Dox)-inducible TGM-2 or C277A (an inactive mutant of TGM-2 with no transamidating activity) using InVitrogen9s Flp-in TRex system. We found continuing treatment of 10 ng/ml TGFbeta for 6 days inducing H358 flp-in (vector control) cells to mescenchymal phenotype as demonstrated by the disappearance of Ecad, and the increased expression of NCad and FN. Under the same condition, Dox inducible TGM-2 and C277A facilitate the disappearance of Ecad, and Occludin and the appearance of Ncad. However, the expression of FN was undetectable in H358/TGM-2 and H358/C277A cells. As a result, H358/TGM-2 and H358/C277A remained localized and failed to migrate as demonstrated by a scratch wound healing assay. Seahorse mitochondria stress assay also demonstrated H358/TGM-2 and H358/C277A utilize more oxidative phosphorylation pathway than glycolytic pathway when compared with H358/flp-in, an indication of shifting cells to more normal phenotype. In summary, overexpression of TGM-2 (or inactive C277A mutant) inhibited the expression of FN and resulted in a less migratory H358 cells. Our results demonstrated that the role of TGM-2 during EMT in less aggressive epithelial cells such as H358 is different from its role in promoting EMT in other aggressive tumor cell; the suppression of FN might be a critical factor. Note: This abstract was not presented at the meeting. Citation Format: Thung S. Lai, Pin-Tsen Lin, Charles S. Greenberg, Harry A. Drabkin, Robert Gemmill. TGM-2 mediated downregulation of fibronectin during TGFb-induced epithelial-mesenchymal transition in lung cancer NCI-H358 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2015-5192