The epithelial mesenchymal transition (EMT) plays a central role in both normal physiological events (e.g., embryonic development) and abnormal pathological events (e.g., tumor formation and metastasis). The processes that occur in embryonic development are often reactivated under pathological conditions such as oncogenesis. Therefore, defining the regulatory networks (both gene and protein levels) involved in the EMT during embryonic development will be fundamental in understanding the regulatory networks involved in tumor development, as well as metastasis. There are many molecules, factors, mediators and signaling pathways that are involved in the EMT process. Although the EMT is a very old topic with numerous publications, recent new technologies and discoveries give this research area some new perspective and direction. It is now clear that these important processes are controlled by a network of transcriptional and translational regulators in addition to post-transcriptional and post-translational modifications that amplify the initial signals. In this review article, we will discuss some key concepts, historical findings, as well as some recent progresses in the EMT research field.

Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells. Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β1. Tumor growth in vivo were analyzed using athymic nude mice. Claudin-7 co-localizes and forms a stable complex with integrin β1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β1 expression and increases cell-matrix adhesion. Integrin β1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells. In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β1.

About a third of tumors have activating mutations in HRAS, NRAS, or KRAS, genes encoding guanosine triphosphatases (GTPases) of the RAS family. In these tumors, wild-type RAS cooperates with mutant RAS to promote downstream effector activation and cell proliferation and transformation, suggesting that upstream activators of wild-type RAS are important modulators of mutant RAS-driven oncogenesis. The guanine nucleotide exchange factor (GEF) SOS1 mediates KRAS-driven proliferation, but little is understood about the role of SOS2. We found that RAS family members have a hierarchical requirement for the expression and activity of SOS2 to drive cellular transformation. In mouse embryonic fibroblasts (MEFs), SOS2 critically mediated mutant KRAS-driven, but not HRAS-driven, transformation. Sos2 deletion reduced epidermal growth factor (EGF)–dependent activation of wild-type HRAS and phosphorylation of the kinase AKT in cells expressing mutant RAS isoforms. Assays using pharmacological inhibitors revealed a hierarchical requirement for signaling by phosphoinositide 3-kinase (PI3K) in promoting RAS-driven cellular transformation that mirrored the requirement for SOS2. KRAS-driven transformation required the GEF activity of SOS2 and was restored in Sos2^−/− MEFs by expression of constitutively activated PI3K. Finally, CRISPR/Cas9-mediated deletion of SOS2 reduced EGF-stimulated AKT phosphorylation and synergized with MEK inhibition to revert the transformed phenotype of human KRAS mutant pancreatic and lung tumor cells. These results indicate that SOS2-dependent PI3K signaling mediates mutant KRAS-driven transformation, revealing therapeutic targets in KRAS-driven cancers. Our data also reveal the importance of three-dimensional culture systems in investigating the mediators of mutant KRAS.

Methyl tertiary-butyl ether (MTBE) is a solvent and fuel additive included in reformulated gasoline to increase combustion efficiency. While widespread use in motor fuels in the U.S. was discontinued after MTBE was detected in surface and ground waters due to concerns about environmental persistence and water quality, it is still manufactured in the U.S. for export. Questions concerning the etiology of rat Leydig cell and mouse liver tumors identified in extremely high dose cancer studies have led to an interest in evaluating potential hormonal imbalances and endocrine system involvement. To address the possibility that MTBE or its metabolite, tert-butanol (TBA), are interacting with components of the endocrine system that are involved in steroidogenesis a number of targeted experiments were performed focusing mostly on the primary gonadal steroids, estradiol and testosterone. The goal of the experiments was to gain a better understanding of potential interactions with the steroidogenic pathway, including effects specifically on aromatase, the P450 enzyme that converts testosterone to estradiol. In three GLP-compliant in vitro guideline studies, MTBE and TBA were classified as non-binders to the androgen receptor, were classified negative for effects on testosterone and estradiol in the steroidogenesis assay, and were classified as non-inhibitors of aromatase activity. In three 14-day in vivo experiments involving gavaging of male Sprague-Dawley rats with doses of MTBE ranging from 400 to 1,500 mg/kg bw/day, the lack of definitive and consistent supporting statistically significant findings in steroid hormone measurements and aromatase activity and mRNA measured in liver and testis microsomes further suggested that it is unlikely that MTBE is interacting with the endocrine system directly. Evidence of other underlying systemic effects were also seen, including reduced body weight gain, increased adrenal weights, and elevated corticosterone suggestive of a more general stress response. Taken together, the results from these studies suggest that MTBE and TBA do not directly impact the steroidogenic pathways involved in estrogen and androgen production.