Therapeutic Targeting of Fibrotic Epithelial-Mesenchymal Transition–An Outstanding Challenge

Back in 1995, a landmark paper was published, which shaped the fibrosis literature for many years to come. During the characterization of a fibroblast-specific marker (FSP1) in the kidneys, an observation was made, which gave rise to the hypothesis that “fibroblasts in some cases arise from the local conversion of epithelium.” In the following years, epithelial-mesenchymal transition was in the spotlight of fibrosis research, especially in the kidney. However, the hypothesis came under scrutiny following some discouraging findings from lineage tracing experiments and clinical observations. In this review, we provide a timely overview of the current position of the epithelial-mesenchymal transition hypothesis in the context of fibrosis (with a certain focus on renal fibrosis) and highlight some of the potential hurdles and pitfalls preventing therapeutic breakthroughs targeting fibrotic epithelial-mesenchymal transition

Trends in Mortality and Morbidity in Infants Under 500 Grams Birthweight: Observations from Our Neonatal Intensive Care Unit (NICU)

BACKGROUND In our previous study, some changes were presented in obstetric care and we studied the morbidity and mortality trends of infants with <500 grams birth weight. Several neonatal protocol changes occurred during the study period. The aim of this study was to analyze the changes in mortality and morbidity of premature infants in light of changing neonatal protocols. MATERIAL AND METHODS We performed a retrospective study of premature infants with <500 grams birth weight, born at our department between 2006 and 2015. We divided the study period into two 5-year epochs and compared mortality and morbidity rates. We calculated the duration of mechanical ventilation and non-invasive respiratory support, and also investigated the potential impact of the differences in clinical practice. RESULTS The survival rate was 30.8% during first epoch, which was significantly lower than the 70.4% survival rate during second epoch. There was no difference in the rate of complications between the 2 epochs. The total number of ventilator and non-invasive ventilation days was significantly lower in the second epoch. CONCLUSIONS We found significant differences in survival rates but no change in the incidence of morbidities between the 2 epochs. Therefore, although the number of neonates surviving with morbidities has increased, so did the number of those with intact survival. The increased survival of infants born with <500 grams birth weight is not associated with increased rate of morbidities. Protocol changes may have contributed to these findings; however, in a retrospective study it is not possible to separate the impact of individual changes

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-beta, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-beta 1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, beta 1-integrin, calponin and a-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-beta signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in trans-endothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-beta-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-beta-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation

Forward Genetic Screens as Tools to Investigate Role and Mechanisms of EMT in Cancer

Epithelial–mesenchymal transition (EMT) is a process of cellular plasticity regulated by complex signaling networks. Under physiological conditions, it plays an important role in wound healing and organ repair. Its importance for human disease is given by its central role in chronic fibroproliferative diseases and cancer, which represent leading causes of death worldwide. In tumors, EMT is involved in primary tumor growth, metastasis and therapy resistance. It is therefore a major requisite to investigate and understand the role of EMT and the mechanisms leading to EMT in order to tackle these diseases therapeutically. Forward genetic screens link genome modifications to phenotypes, and have been successfully employed to identify oncogenes, tumor suppressor genes and genes involved in metastasis or therapy resistance. In particular, transposon-based insertional mutagenesis screens and CRISPR-based screens are versatile and easy-to-use tools applied in recent years to discover and identify novel cancer-related mechanisms. Here, we review the contribution of forward genetic screens to our understanding of how EMT is regulated and how it is involved in various aspects of cancer. Based on the current literature, we propose these methods as additional tools to investigate EMT

Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation

Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation

TGF-β1 induces EndMT in primary rat BECs.

<p><b>(a)</b> BECs were subjected to 48 hrs TGF-β1 treatment, then fixed and stained for claudin-5 and VE-cadherin. Control cells showed claudin-5 and VE-cadherin at their intercellular borders, which was depleted in TGF-β1 treated cells (400x magnification). BECs were treated with TGF-β1 for 48 hrs, and were analyzed for endothelial, mesenchymal and myogenic marker expression by Western blot. TGF-β1 treatment led to the <b>(b)</b> down-regulation of tight and adherens junction protein expression (claudin-5, occludin, VE-cadherin), it induced β1-integrin expression and <b>(c)</b> and led to a marked increase in N-cadherin expression (2.18±0.48 fold based on three independent experiments, where N-cadherin expression increased 2.11, 1.52 and 2.89 fold respectively, as compared to controls). TGF-β1 treated cells showed a robust expression of fibronectin <b>(d)</b>, SMA and calponin <b>(e)</b>. SMA expression was observed by immunofluorescence microscopy (400x magnification) under similar conditions as well <b>(f)</b>.</p

Activated breast cancer cell conditioned medium induces SMA expression through TGF-β signaling in HUVECs.

<p>FBS-free M199 medium was conditioned with MDA-MB231 <b>(a)</b> or SK-BR3 <b>(b)</b> human breast cancer cells for 24 hrs. A half of the conditioned media were subjected heat activation, and then both conditioned (CM) and activated conditioned media (ACM) were supplemented with FBS. Cells were pre-treated with 10 μM SB-431542 for 60 min and then treated with CM or ACM for 72 hrs, in the presence of the inhibitor. Samples were analyzed by Western blot. SB-431542 inhibited MDA-MB231 or SK-BR3 ACM induced SMA expression. <b>(c) Co-culture with melanoma cells leads to expressional changes characteristic to EndMT in HUVECs</b>. Gene expression profiles of HUVECs and HUVECs co-cultured with 1205Lu human metastatic melanoma cells were analyzed and compared. In HUVECs co-cultured with melanoma cells there was a marked decrease in expression levels of several endothelial markers (KRT7, KRT18, TJP2), as well as a severe decrease in FST expression. In parallel, co-cultured HUVECs exhibited elevated expression levels of EndMT markers (FN1, COL3A1, S100A4, MMP2, COL1A2) and transcriptional regulators (ZEB1, Wnt5a, TWIST1, Snai2).</p