Epithelial to Mesenchymal Transition: A Challenging Playground for Translational Research. Current Models and Focus on TWIST1 Relevance and Gastrointestinal Cancers

Resembling the development of cancer by multistep carcinogenesis, the evolution towards metastasis involves several passages, from local invasion and intravasation, encompassing surviving anoikis into the circulation, landing at distant sites and therein establishing colonization, possibly followed by the outgrowth of macroscopic lesions. Within this cascade, epithelial to mesenchymal transition (EMT) works as a pleiotropic program enabling cancer cells to overcome local, systemic, and distant barriers against diffusion by replacing traits and functions of the epithelial signature with mesenchymal-like ones. Along the transition, a full-blown mesenchymal phenotype may not be accomplished. Rather, the plasticity of the program and its dependency on heterotopic signals implies a pendulum with oscillations towards its reversal, that is mesenchymal to epithelial transition. Cells in intermixed EÛM states can also display stemness, enabling their replication together with the epithelial reversion next to successful distant colonization. If we aim to include the EMT among the hallmarks of cancer that could modify clinical practice, the gap between the results pursued in basic research by animal models and those achieved in translational research by surrogate biomarkers needs to be filled. We review the knowledge on EMT, derived from models and mechanistic studies as well as from translational studies, with an emphasis on gastrointestinal cancers (GI).

Epithelial-to-mesenchymal transition lowers the cholesterol pathway, wich influences colon tumors differentiation.

Colorectal cancer (CRC) is the second cause of death worldwide. Up to 70% of CRC patients will metastasize at one point. Understanding the chain of events that lead to metastasis occurrence is urgent to identify new biomarkers of progression or new targets to prevent and delay disease evolution. Epithelial to mesenchymal transition (EMT) is a major program engaged during metastasis. EMT is extremely complex to analyze in situ due to the broad involvement of its transcription factors. We hypothesized that a relevant and dynamic in vitro model of pure cancer cells will reveal a combination of new genes that might further identify signs of EMT in cancer tissues. We treated HT-29 cells grown in 3D with an EMT-inducing factor, but also looked at reverse changes after EMT-inducing factor removal. For each condition, pan-transcriptomic analyses were done. Genes that were both induced upon EMT induction and inhibited upon EMT release (mesenchymal to epithelial transition or MET) were selected. Consistent with our hypothesis, we identified new genes for the EMT-MET programs. These genes were used to build a metagene that, when applied to a large database of transcriptomic data from primary colorectal tumors (n= 2,239), had an independent prognosis value. Finally, we submitted this metagene to CMap and identified drugs that might affect EMT-MET programs. Statins, well-known inhibitors of cholesterol synthesis, were among them and effectively delayed MET in vitro. These data show that cholesterol and EMT pathways are opposite regulators and impact differently tumors differentiation and outcome.

The Impact of SOD3 on Prostatic Diseases: Elevated SOD3 Serves as a Novel Biomarker for the Diagnosis of Chronic Nonbacterial Prostatitis

BACKGROUND: Prostate is the most common gland for the three major diseases in male, such as chronic nonbacterial prostatitis (CNP), benign prostatic hyperplasia (BPH) and prostate cancer (PCa). However, there is lack of ideal biomarker for diagnosis with prostatic diseases, especially CNP. METHODS AND RESULTS: Extracellular superoxide dismutase (SOD3) levels in serum or expressed prostatic secretion (EPS) with CNP, and in the prostate tissues of rat CNP modules, BPH, and PCa was quantified, which showed that SOD3 was significantly increased in CNP and BPH, but decreased in PCa compared to controls. Receiver operating characteristic curve (ROC) analysis suggested that SOD3 was an efficient diagnosis biomarker discriminating CNP versus normal controls (accuracy= 0.831, 95%CI: 0.726-0.937, P＜0.001), CNP III versus CNP IV (accuracy=0.868, 95%CI: 0.716-0.940, P＜0.001). SOD3 were associated with the clinical characteristics of patients with CNP including pelvic pain, blood pressure, and lecithin/leukocyte in EPS. Multiple bioinformatic analysis showed that SOD3 mainly participated in superoxide radicals degradation, apoptotic execution phase, and mesenchymal-to-epithelial transition, etc. Furthermore, the structural features of SOD3 and the interacting proteins were evaluated by molecular docking, and hotspot analysis indicated that better affinities between SOD3 and its interacting molecules were associated with the presence of Arginine (Arg) in the binding site. CONCLUSIONS: According to the results, it can be concluded that SOD3 plays an important role in prostatic diseases, and it may potentially serve as an ideal diagnostic biomarker for CNP.

Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts

Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.

Forkhead box family transcription factors as versatile regulators for cellular reprogramming to pluripotency

Forkhead box (Fox) transcription factors play important roles in mammalian development and disease. However, their function in mouse somatic cell reprogramming remains unclear. Here, we report that FoxD subfamily and FoxG1 accelerate induced pluripotent stem cells (iPSCs) generation from mouse fibroblasts as early as day4 while FoxA and FoxO subfamily impede this process obviously. More importantly, FoxD3, FoxD4 and FoxG1 can replace Oct4 respectively and generate iPSCs with germline transmission together with Sox2 and Klf4. On the contrary, FoxO6 almost totally blocks reprogramming through inhibiting cell proliferation, suppressing the expression of pluripotent genes and hindering the process of mesenchymal to epithelial transition (MET). Thus, our study uncovers unexpected roles of Fox transcription factors in reprogramming and offers new insights into cell fate transition.

A reappraisal of mesenchymal-to-epithelial transition within the endometrium

A controversial topic, mesenchymal-epithelial transition (MET) is thought to be a mechanism involved in regeneration of the uterine epithelial layer following pregnancy and menstruation. Little is known about this process though, requiring further exploration. Previously, MET was thought to only occur as a damage-repair mechanism following parturition and menses-like events in mouse models. However, in the current study we hypothesized that MET would also occur in other endometrial epithelialization events as a mechanism of homeostatic epithelial turnover. To identify mesenchymal-derived cells within the adult uterine epithelium, an Amhr2-Cre; Rosa26-EYFP reporter mouse line was used. Mice were staged by vaginal cytology prior to the isolation of uterine epithelial and stromal cells, which were then stained with EpCAM to identify epithelial cells and analyzed by flow cytometry. EpCAM+YFP+ cells were identified in all stages of the estrous cycle except diestrus, indicating a proportion of epithelial cells were derived from the stroma (i.e., mesenchyme). Up to 80 percent of the uterine epithelia was EpCAM+YFP+ during estrogen-dominant stages (proestrus and estrus) of the cycle, with negligible amounts found during progesterone-dominant stages (metestrus and diestrus), suggesting this population may be responsive to estrogen. Uteri were also evaluated direct fluorescence and immunofluorescence in tissue sections. Immunofluorescence for EpCAM, forkhead box protein A2 (FOXA2), Ki67, estrogen receptor alpha (ESR1), and progesterone receptor (PGR) was performed to assess epithelial characteristics and potential functionality. To further investigate the role of MET in epithelialization and assess the temporal origin of mesenchymal-derived epithelial cells, we evaluated key postnatal (P) developmental time points using Amhr2-Cre; Rosa26-tTA; H2B-GFP reporter mice. Flow cytometry data indicated that MET may initially occur immediately after birth at P 0.5, with results varying from negligible amounts (0.21 percent) to approximately 82 percent. Similar results were found at P 3, but with decreasing variation; the highest EpCAM+GFP+ population representing approximately 50 percent of the epithelium. Between P 3 and early adenogenesis at P 8, this population decreased to average less than 2 percent. By the completion of adenogenesis initiation at P 14, approximately 10 percent of epithelial cells were EpCAM+GFP+, suggesting MET may occur during adenogenesis initiation and is maintained through P 21. Together, these results suggest that MET may be a more ubiquitous mechanism of epithelialization than originally thought and is likely hormone regulated. This research will help elucidate the role of MET in uterine epithelialization with potential for insights into dysregulation of MET in diseases such as endometrial adenocarcinoma and endometriosis.

The mutual repression between Pax2 and Snail factors regulates the epithelial/mesenchymal state during intermediate mesoderm differentiation

The pronephros is the first renal structure in the embryo, arising after mesenchymal to epithelial transition (MET) of the intermediate mesoderm, where Pax2 induces epithelialization of the mesenchyme. Here we show that, in the early embryo, Snail1 directly represses Pax2 transcription maintaining the intermediate mesoderm in an undifferentiated state. Reciprocally, Pax2 directly represses Snail1 expression to induce MET upon receiving differentiation signals. We also show that BMP7 acts as one such signal by downregulating Snail1 and upregulating Pax2 expression. This, together with the Snail1/Pax2 reciprocal repression, establish a regulatory loop in a defined region along the anteroposterior axis, the bi-stability domain within the transition zone, where differentiation of the neural tube and the somites is known to occur. Thus, we show that the antagonism between Snail1 and Pax2 determines the epithelial/mesenchymal state during the differentiation of the intermediate mesoderm and propose that the bi-stability zone extends to the intermediate mesoderm, synchronizing the differentiation of tissues aligned along the mediolateral embryonic axis.