Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Interleukin-3 production by basal-like breast cancer cells is associated with poor prognosis.

Breast cancer represents a collection of pathologies with different molecular subtypes, histopathology, risk factors, clinical behavior, and responses to treatment. "Basal-like" breast cancers predominantly lack the receptors for estrogen and progesterone (ER/PR), lack amplification of human epidermal growth factor receptor 2 (HER2) but account for 10-15% of all breast cancers, are largely insensitive to targeted treatment and represent a disproportionate number of metastatic cases and deaths. Analysis of interleukin (IL)-3 and the IL-3 receptor subunits (IL-3RA + CSF2RB) reveals elevated expression in predominantly the basal-like group. Further analysis suggests that IL-3 itself, but not the IL-3 receptor subunits, associates with poor patient outcome. Histology on patient-derived xenografts supports the notion that breast cancer cells are a significant source of IL-3 that may promote disease progression. Taken together, these observations suggest that IL-3 may be a useful marker in solid tumors, particularly triple negative breast cancer, and warrants further investigation into its contribution to disease pathogenesis.

Aberrant post-translational modifications in endosomal trafficking are potential therapeutic targets to avert therapy resistance in solid cancers: Dysregulation of PTM-regulated endosomal interactions presents an opportunity to block oncogenic signalling from multiple receptors by targeting common trafficking pathways: Dysregulation of PTM-regulated endosomal interactions presents an opportunity to block oncogenic signalling from multiple receptors by targeting common trafficking pathways.

Drugs targeting a single TK/RTK in the treatment of solid cancers has not had the same success seen in blood cancers. This is, in part, due to acquired resistance in solid cancers arising from a range of mechanisms including the upregulation of compensatory RTK signalling. Rather than attempting to inhibit individual compensatory RTK-requiring knowledge of which RTKs are upregulated in any given tumour-strategies to universally inhibit signalling from multiple RTKs may represent an effective alternative. Endosomal trafficking of RTKs is a common conduit that can regulate signalling from multiple RTKs simultaneously. As such, we posit that targeting endosomal trafficking-in particular, aberrant post-translational modifications in cancers that contribute to dysregulated endosomal trafficking-could inhibit oncogenic signalling driven by multiple RTKs and pave the way for the development of a novel class of inhibitors that shift the trafficking of RTKs to inhibit tumour growth.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Stathmin levels alter PTPN14 expression and impact neuroblastoma cell migration.

BACKGROUND: Stathmin mediates cell migration and invasion in vitro, and metastasis in vivo. To investigate stathmin's role on the metastatic process, we performed integrated mRNA-miRNA expression analysis to identify pathways regulated by stathmin. METHODS: MiRNA and gene arrays followed by miRNA-target-gene integration were performed on stathmin-depleted neuroblastoma cells (CtrlshRNA vs. Stmn Seq2shRNA). The expression of the predicted target PTPN14 was evaluated by RT-qPCR, western blot and immunohistochemistry. Gene-silencing technology was used to assess the role of PTPN14 on proliferation, migration, invasion and signalling pathway. RESULTS: Stathmin levels modulated the expression of genes and miRNA in neuroblastoma cells, leading to a deregulation of migration and invasion pathways. Consistent with gene array data, PTPN14 mRNA and protein expression were downregulated in stathmin- depleted neuroblastoma cells and xenografts. In two independent neuroblastoma cells, suppression of PTPN14 expression led to an increase in cell migration and invasion. PTPN14 and stathmin expression did not act in a feedback regulatory loop in PTPN14- depleted cells, suggesting a complex interplay of signalling pathways. The effect of PTPN14 on YAP pathway activation was cell-type dependent. CONCLUSIONS: Our findings demonstrate that stathmin levels can regulate PTPN14 expression, which can modulate neuroblastoma cell migration and invasion.

PTPN14 phosphatase and YAP promote TGFß signalling in rheumatoid synoviocytes.

OBJECTIVE: We aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFß-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity. CONCLUSION: In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.

Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell-like state.

The miR-200 family is a key regulator of the epithelial-mesenchymal transition, however, its role in controlling the transition between cancer stem-cell-like and non-stem-cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial (HMLE) cells to investigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable of spontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression. Stem-like cell fractions isolated from metastatic breast cancers also displayed loss of miR-200 indicating similar molecular changes may occur during breast cancer progression. The phenotypic change observed in HMLE cells was directly controlled by miR-200 because restoration of its expression decreased stem-like properties while promoting a transition to an epithelial phenotype. Investigation of the mechanisms controlling miR-200 expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In particular, in the stem-like phenotype, the miR-200b-200a-429 cluster was silenced primarily through polycomb group-mediated histone modifications whereas the miR-200c-141 cluster was repressed by DNA methylation. These results indicate that the miR-200 family plays a crucial role in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therapy targeted against miR-200 family members and epigenetic modifications might therefore be applicable to breast cancer.

The secretome in cancer progression.

The secretome is the collection of all macromolecules secreted by a cell, and is a vital aspect of cell-cell communication in eukaryotes. In cancer, tumour cells often display secretomes with altered composition compared to the normal tissue from which they are derived. These changes can contribute to the acquisition and maintenance of the recognised hallmarks of cancer. In addition, evidence is emerging for a more sophisticated role for the tumour secretome in cancer, with significant implications for malignant disease progression. In this review, we highlight recent advances in our understanding of factors contributing to secretome alterations in cancer, including genetic mutations, microRNA-based regulation and the influence of the tumour microenvironment. The contribution of secreted factors in maintenance and function of cancer stem cells, and of tumour-derived factors in specification of a pre-metastatic niche are also discussed. Collectively, evidence from the current literature suggests that the tumour secretome, consisting of factors derived from cancer stem cells, non-stem cells and the surrounding stroma, plays a deterministic role in cancer progression, and may constitute a key therapeutic target in many cancers. This article is part of a Special Issue entitled: An Updated Secretome.

Basonuclin-2 regulates extracellular matrix production and degradation.

Epithelial-mesenchymal transition is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the ECM-a complex environment of proteoglycans and fibrous proteins essential for tissue homeostasis, signaling in response to chemical and biomechanical stimuli, and is often dysregulated under conditions such as cancer, fibrosis, and chronic wounds. Here, we demonstrate that basonuclin-2 (BNC2), a mesenchymal-expressed gene, that is, strongly associated with cancer and developmental defects across genome-wide association studies, is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases, and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the production and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers, which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.

The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development.

Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.

A proinflammatory role for proteolytically cleaved annexin A1 in neutrophil transendothelial migration.

Neutrophil extravasation, a critical component of innate immunity must be tightly regulated to prevent inadvertent or prolonged inflammation and subsequent tissue damage. We have shown previously that endothelial ERK1/2 signaling essential for neutrophil transendothelial migration is induced by a soluble factor produced by activated neutrophils. In this study, we demonstrate that the soluble neutrophil factor is a truncated form of annexin A1 (AnxA1) that can be generated by calpain 1 cleavage of the N terminus, thus identifying a novel proinflammatory function to AnxA1. In contrast, neither the full-length protein nor the N-terminal 26 aa peptide, previously shown to be antiinflammatory, were able to activate Erk. Our data suggest that two different fragments of AnxA1 have opposing functions in inflammation. We also provide evidence that C-terminal AnxA1 functions by increasing ICAM1 clustering around adherent neutrophils to anchor them to the endothelium and promote transmigration through the transcellular route.

Quantifying EGFR endosomal recycling via immunofluorescence in breast cancer cells.

Previously published protocols for quantification of endosomal recycling are limited by the use of radioactive reagents, washing of cells in reducing buffers, or the requirement for large numbers of cells. Here, we describe a protocol for quantification of endosomal recycling using immunofluorescence that is optimized for EGFR in BT-549 breast cancer cells but could be applied to other RTKs and cell lines. Our protocol enables quick assessment of recycling and uses a relatively small number of cells. For complete details on the use and execution of this protocol, please refer to Lonic et al. (2021).

The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease.

Kidney disease progression can be affected by Na+ abundance. A key regulator of Na+ homeostasis is the ubiquitin ligase NEDD4-2 and its deficiency leads to increased Na+ transport activity and salt-sensitive progressive kidney damage. However, the mechanisms responsible for high Na+ induced damage remain poorly understood. Here we show that a high Na+ diet compromised kidney function in Nedd4-2-deficient mice, indicative of progression toward end-stage renal disease. Injury was characterized by enhanced tubule dilation and extracellular matrix accumulation, together with sustained activation of both Wnt/ß-catenin and TGF-ß signaling. Nedd4-2 knockout in cortical collecting duct cells also activated these pathways and led to epithelial-mesenchymal transition. Furthermore, low dietary Na+ rescued kidney disease in Nedd4-2-deficient mice and silenced Wnt/ß-catenin and TGF-ß signaling. Our study reveals the important role of NEDD4-2-dependent ubiquitination in Na+ homeostasis and protecting against aberrant Wnt/ß-catenin/TGF-ß signaling in progressive kidney disease.

Phosphorylation of PKCδ by FER tips the balance from EGFR degradation to recycling.

Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.

The microRNA-200 family regulates epithelial to mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is implicated in metastasis initiation and has recently been shown to be regulated by the miRNA-200 family and miR-205. Expression of these miRNAs was lost in invasive breast cancer cell lines displaying mesenchymal-like morphology suggesting these microRNAs may play a role in cancer metastasis.

The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin.

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.

Network-Based Approaches to Understand the Roles of miR-200 and Other microRNAs in Cancer.

microRNAs (miRNA) are well suited to the task of regulating gene expression networks, because any given miRNA has the capacity to target dozens, if not hundreds, of genes. The simultaneous targeting of multiple genes within a pathway may enable miRNAs to more strongly regulate the pathway, or to achieve more subtle control through the targeting of distinct subnetworks of genes. Therefore, as our capacity to discover miRNA targets en masse increases, so must our consideration of the complex networks in which these genes participate. We highlight recent studies in which the comprehensive identification of targets has been used to elucidate miRNA-regulated gene networks in cancer, focusing especially upon miRNAs such as members of the miR-200 family that regulate epithelial-mesenchymal transition (EMT), a reversible phenotypic switch whereby epithelial cells take on the more invasive properties of their mesenchymal counterparts. These studies have expanded our understanding of the roles of miRNAs in EMT, which were already known to form important regulatory loops with key transcription factors to regulate the epithelial or mesenchymal properties of cells.