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.

Hsp47 promotes cancer metastasis by enhancing collagen-dependent cancer cell-platelet interaction.

Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell-platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell-platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell-platelet interaction and cancer colonization in secondary organs.

ITCH nuclear translocation and H1.2 polyubiquitination negatively regulate the DNA damage response.

The downregulation of the DNA damage response (DDR) enables aggressive tumors to achieve uncontrolled proliferation against replication stress, but the mechanisms underlying this process in tumors are relatively complex. Here, we demonstrate a mechanism through which a distinct E3 ubiquitin ligase, ITCH, modulates DDR machinery in triple-negative breast cancer (TNBC). We found that expression of a nuclear form of ITCH was significantly increased in human TNBC cell lines and tumor specimens. Phosphorylation of ITCH at Ser257 by AKT led to the nuclear localization of ITCH and ubiquitination of H1.2. The ITCH-mediated polyubiquitination of H1.2 suppressed RNF8/RNF168-dependent formation of 53BP1 foci, which plays important roles in DDR. Consistent with these findings, impaired ITCH nuclear translocation and H1.2 polyubiquitination sensitized cells to replication stress and limited cell growth and migration. AKT activation of ITCH-H1.2 axis may confer TNBC cells with a DDR repression to counteract the replication stress and increase cancer cell survivorship and growth potential.

POMC maintains tumor-initiating properties of tumor tissue-derived long-term-cultured breast cancer stem cells.

The identification and understanding of the molecular network of cancer stem cells (CSCs) have had a profound impact on our view of carcinogenesis and treatment strategy. Unfortunately, a major problem is that serial passages of CSCs from clinical solid tumor specimens currently are not available in any lab, and thus, reported data are difficult to confirm and intensively interrogated. Here, we have generated two tumor tissue-derived breast CSC (BCSC) lines that showed prolonged maintenance over 20 serial passages in vitro, while retaining their tumor-initiating biological properties. We then deciphered the intrinsic mechanism using analyses of mRNA expression array profiles. It has been determined that pro-opiomelanocortin (POMC) is closely related with protein phosphorylation mediated by G-protein-coupled estrogen receptor (GPER) in BCSC. Following, knockdown of POMC inhibits properties of mammosphere formation, CD44+ CD24- population, CD44 expression, and clonogenicity ability in BCSC. We found that inhibition of POMC attenuates phosphorylation of AKT2 and GSK3ß in BCSC. Further in vivo investigations demonstrated that POMC interference regulates proliferation of BCSC-bearing tumors. Combination of the clinical results that POMC positive expression is frequently upregulated in human breast cancer and POMC positivity correlated with a poor prognosis, POMC is a potential therapeutic target for BCSC. In conclusion, we have successfully established two long-term-cultured BCSC from clinical specimens. We further indicated that POMC acts as a potential therapeutic target and prognostic marker for future treatment of BCSC.

Autophagy regulation in the development and treatment of breast cancer.

Autophagy is a major catabolic process in which intracellular membrane structures, protein complexes, and lysosomes are formed as lysoautophagosome to degrade and renew cytoplasmic components. Autophagy is physiologically a strategy and mechanism for cellular homeostasis as well as adaptation to stress, and thus alterations in the autophagy machinery may lead to diverse pathological conditions. The role of autophagy in cancer is complex, and the current literature reflects this as a 'double-edged sword'. Autophagy shows promise as a novel therapeutic target in various types of breast cancer, inhibiting or increasing treatment efficacy in a context- and cell-type-dependent manner. This review aims to summarize the recent advances in the understanding of the mechanisms by which key modulators of autophagy participate in cancer metastasis, highlight different autophagy-deficient murine models for breast cancer study, and provide further impetus for the modulation of autophagy in anticancer therapy.

The impact of low-dose carcinogens and environmental disruptors on tissue invasion and metastasis.

The purpose of this review is to stimulate new ideas regarding low-dose environmental mixtures and carcinogens and their potential to promote invasion and metastasis. Whereas a number of chapters in this review are devoted to the role of low-dose environmental mixtures and carcinogens in the promotion of invasion and metastasis in specific tumors such as breast and prostate, the overarching theme is the role of low-dose carcinogens in the progression of cancer stem cells. It is becoming clearer that cancer stem cells in a tumor are the ones that assume invasive properties and colonize distant organs. Therefore, low-dose contaminants that trigger epithelial-mesenchymal transition, for example, in these cells are of particular interest in this review. This we hope will lead to the collaboration between scientists who have dedicated their professional life to the study of carcinogens and those whose interests are exclusively in the arena of tissue invasion and metastasis.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer.

BACKGROUND: GPRC5A is a retinoic acid inducible gene that is preferentially expressed in lung tissue. Gprc5a- knockout mice develop spontaneous lung cancer, indicating Gprc5a is a lung tumor suppressor gene. GPRC5A expression is frequently suppressed in majority of non-small cell lung cancers (NSCLCs), however, elevated GPRC5A is still observed in a small portion of NSCLC cell lines and tumors, suggesting that the tumor suppressive function of GPRC5A is inhibited in these tumors by an unknown mechanism. METHODS: In this study, we examined EGF receptor (EGFR)-mediated interaction and tyrosine phosphorylation of GPRC5A by immunoprecipitation (IP)-Westernblot. Tyrosine phosphorylation of GPRC5A by EGFR was systematically identified by site-directed mutagenesis. Cell proliferation, migration, and anchorage-independent growth of NSCLC cell lines stably transfected with wild-type GPRC5A and mutants defective in tyrosine phosphorylation were assayed. Immunohistochemical (IHC) staining analysis with specific antibodies was performed to measure the total and phosphorylated GPRC5A in both normal lung and lung tumor tissues. RESULT: We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y350, at the C-terminal tail of GPRC5A. EGF induced phosphorylation of GPRC5A, which disrupted GPRC5A-mediated suppression on anchorage-independent growth of NSCLC cells. On contrary, GPRC5A-4 F, in which the four tyrosine residues have been replaced with phenylalanine, was resistant to EGF-induced phosphorylation and maintained tumor suppressive activities. Importantly, IHC analysis with anti-Y317/Y320-P sites showed that GPRC5A was non-phosphorylated in normal lung tissue whereas it was highly tyrosine-phosphorylated in NSCLC tissues. CONCLUSION: GPRC5A can be inactivated by receptor tyrosine kinase via tyrosine phosphorylation. Thus, targeting EGFR can restore the tumor suppressive functions of GPRC5A in lung cancer.

The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1.

Epithelial-mesenchymal transition (EMT) is a transdifferentiation programme. The mechanism underlying the epigenetic regulation of EMT remains unclear. In this study, we identified that Snail1 interacted with histone lysine-specific demethylase 1 (LSD1). We demonstrated that the SNAG domain of Snail1 and the amine oxidase domain of LSD1 were required for their mutual interaction. Interestingly, the sequence of the SNAG domain is similar to that of the histone H3 tail, and the interaction of Snail1 with LSD1 can be blocked by LSD1 enzymatic inhibitors and a histone H3 peptide. We found that the formation of a Snail1-LSD1-CoREST ternary complex was critical for the stability and function of these proteins. The co-expression of these molecules was found in cancer cell lines and breast tumour specimens. Furthermore, we showed that the SNAG domain of Snail1 was critical for recruiting LSD1 to its target gene promoters and resulted in suppression of cell migration and invasion. Our study suggests that the SNAG domain of Snail1 resembles a histone H3-like structure and functions as a molecular hook for recruiting LSD1 to repress gene expression in metastasis.

NF-κB represses retinoic acid receptor-mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia.

Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulation plays a critical role. Lung tumor suppressor G protein-coupled receptor, family C, member 5A (GPRC5A), is repressed in most non-small cell lung cancer (NSCLC); however, the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induced GPRC5A repression both in vitro and in vivo. Intriguingly, transactivation of NF-κB downstream targets was not required, but the transactivation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in the GPRC5A promoter. Instead, p65 was complexed with retinoic acid receptor α/ß (RARα/ß) and recruited to the RA response element site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complexing and suppressed transcription. Notably, phosphorylation on serine 276 of p65 was required for interaction with RARα/ß and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression was through suppression of acetylated histone H3K9 (H3K9ac), but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a histone deacetylase inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via a complex with RARα/ß and mediates epigenetic repression via suppression of H3K9ac.

beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer.

beta-catenin plays an important role in development and homeostasis. Deregulated beta-catenin is involved in oncogenesis. In this study, we found that beta-catenin can physically complex with NF-kappa B, resulting in a reduction of NF-kappa B DNA binding, transactivation activity, and target gene expression. Repressed NF-kappa B activity is found in human colon cancer cells in which beta-catenin is activated. Importantly, activated beta-catenin was found to inhibit the expression of NF-kappa B target genes, including Fas and TRAF1. Furthermore, a strong inverse correlation was identified between the expression levels of beta-catenin and Fas in colon and breast tumor tissues, suggesting that beta-catenin regulates NF-kappa B and its targets in vivo. Thus, beta-catenin may play an important role in oncogenesis through the crossregulation of NF-kappa B.

Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis.

The receptor tyrosine kinase HER2 enhances tumor metastasis; however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the movement of malignant cancer cells to specific organs. Here, we show that HER2 enhances the expression of CXCR4, which is required for HER2-mediated invasion in vitro and lung metastasis in vivo. HER2 also inhibits ligand-induced CXCR4 degradation. Finally, a significant correlation between HER2 and CXCR4 expression was observed in human breast tumor tissues, and CXCR4 expression correlated with a poor overall survival rate in patients with breast cancer. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and establish a functional link between HER2 and CXCR4 signaling pathways.

Identification of Active Bronchioalveolar Stem Cells as the Cell of Origin in Lung Adenocarcinoma.

While initiation is established as a critical step in tumorigenesis, the identity of the cell of origin for lung adenocarcinoma and the mechanism controlling susceptibility to initiation remain elusive. Here we show that lung tumor suppressor Gprc5a-knockout (KO) mice are susceptible to initiation of lung tumorigenesis. Bronchioalveolar stem cells (BASC) and alveolar type 2 (AT2) cells were aberrantly expanded in Gprc5a-KO mouse lungs compared with those in wild-type (WT) mice, suggesting that Gprc5a-KO might confer susceptibility to initiation by increasing the cell of origin in mouse lungs. BASCs from Gprc5a-KO mice (KO-BASC) exhibited significantly increased stemness and self-renewal potential and reduced differentiation capacity compared with BASCs from WT mice (WT-BASC). AT2 cells did not possess self-renewal potential regardless of Gprc5a status. KO-BASCs expressed a stem-like gene profile with upregulated Abcg2, EGFR, and NF-κB signaling compared with WT-BASCs. Blockade of EGFR and NF-κB signaling inhibited both expansion of BASC and AT2 cells and lung tumorigenesis. Abcg2 was expressed in active KO-BASCs as well as in lung tumor cells but not in quiescent WT-BASCs or AT2 cells, supporting that lung adenocarcinoma cells are derived from Abcg2-positive KO-BASCs (active). Taken together, Gprc5a deletion leads to expansion of active BASCs via dysregulated EGFR and NF-κB signaling that confers susceptibility to initiation of lung tumorigenesis, marking Abcg2-positive BASCs as candidate cell of origin for lung adenocarcinoma. SIGNIFICANCE: Identification of active bronchioalveolar stem cells as lung adenocarcinoma cells of origin provides insights into mechanisms of lung tumorigenesis and could facilitate development of effective strategies for cancer prevention and therapy. See related commentary by Osborne and Minna, p. 972.

Activation of ß-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters.

BACKGROUND: Epithelial-mesenchymal transition (EMT) not only confers tumor cells with a distinct advantage for metastatic dissemination, but also it provides those cells with cancer stem cell-like characters for proliferation and drug resistance. However, the molecular mechanism for maintenance of these stem cell-like traits remains unclear. METHODS: In this study, we induced EMT in breast cancer MCF7 and cervical cancer Hela cells with expression of Twist, a key transcriptional factor of EMT. The morphological changes associated with EMT were analyzed by immunofluorescent staining and Western blotting. The stem cell-like traits associated with EMT were determined by tumorsphere-formation and expression of ALDH1 and CD44 in these cells. The activation of ß-catenin and Akt pathways was examined by Western blotting and luciferase assays. RESULTS: We found that expression of Twist induced a morphological change associated with EMT. We also found that the cancer stem cell-like traits, such as tumorsphere formation, expression of ALDH1 and CD44, were significantly elevated in Twist-overexpressing cells. Interestingly, we showed that ß-catenin and Akt pathways were activated in these Twist-overexpressing cells. Activation of ß-catenin correlated with the expression of CD44. Knockdown of ß-catenin expression and inhibition of the Akt pathway greatly suppressed the expression of CD44. CONCLUSIONS: Our results indicate that activation of ß-catenin and Akt pathways are required for the sustention of EMT-associated stem cell-like traits.

Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition.

The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. Here we find that Snail is highly unstable, with a short half-life about 25 min. We show that GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT.

Epithelial-mesenchymal transition in breast cancer progression and metastasis.

Breast cancer is the most common cancer in women, and approximately 90% of breast cancer deaths are caused by local invasion and distant metastasis of tumor cells. Epithelial-mesenchymal transition (EMT) is a vital process for large-scale cell movement during morphogenesis at the time of embryonic development. Tumor cells usurp this developmental program to execute the multi-step process of tumorigenesis and metastasis. Several transcription factors and signals are involved in these events. In this review, we summarize recent advances in breast cancer researches that have provided new insights in the molecular mechanisms underlying EMT regulation during breast cancer progression and metastasis. We especially focus on the molecular pathways that control EMT.

The Context-Dependent Impact of Integrin-Associated CD151 and Other Tetraspanins on Cancer Development and Progression: A Class of Versatile Mediators of Cellular Function and Signaling, Tumorigenesis and Metastasis.

As a family of integral membrane proteins, tetraspanins have been functionally linked to a wide spectrum of human cancers, ranging from breast, colon, lung, ovarian, prostate, and skin carcinomas to glioblastoma. CD151 is one such prominent member of the tetraspanin family recently suggested to mediate tumor development, growth, and progression in oncogenic context- and cell lineage-dependent manners. In the current review, we summarize recent advances in mechanistic understanding of the function and signaling of integrin-associated CD151 and other tetraspanins in multiple cancer types. We also highlight emerging genetic and epigenetic evidence on the intrinsic links between tetraspanins, the epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), and the Wnt/ß-catenin pathway, as well as the dynamics of exosome and cellular metabolism. Finally, we discuss the implications of the highly plastic nature and epigenetic susceptibility of CD151 expression, function, and signaling for clinical diagnosis and therapeutic intervention for human cancer.

Erratum: Twist promotes tumor metastasis in basal-like breast cancer by transcriptionally upregulating ROR1: Erratum.

[This corrects the article DOI: 10.7150/thno.21477.].

Dysregulated Glutamate Transporter SLC1A1 Propels Cystine Uptake via Xc- for Glutathione Synthesis in Lung Cancer.

Cancer cells need to generate large amounts of glutathione (GSH) to buffer oxidative stress during tumor development. A rate-limiting step for GSH biosynthesis is cystine uptake via a cystine/glutamate antiporter Xc-. Xc- is a sodium-independent antiporter passively driven by concentration gradients from extracellular cystine and intracellular glutamate across the cell membrane. Increased uptake of cystine via Xc- in cancer cells increases the level of extracellular glutamate, which would subsequently restrain cystine uptake via Xc-. Cancer cells must therefore evolve a mechanism to overcome this negative feedback regulation. In this study, we report that glutamate transporters, in particular SLC1A1, are tightly intertwined with cystine uptake and GSH biosynthesis in lung cancer cells. Dysregulated SLC1A1, a sodium-dependent glutamate carrier, actively recycled extracellular glutamate into cells, which enhanced the efficiency of cystine uptake via Xc- and GSH biosynthesis as measured by stable isotope-assisted metabolomics. Conversely, depletion of glutamate transporter SLC1A1 increased extracellular glutamate, which inhibited cystine uptake, blocked GSH synthesis, and induced oxidative stress-mediated cell death or growth inhibition. Moreover, glutamate transporters were frequently upregulated in tissue samples of patients with non-small cell lung cancer. Taken together, active uptake of glutamate via SLC1A1 propels cystine uptake via Xc- for GSH biosynthesis in lung tumorigenesis. SIGNIFICANCE: Cellular GSH in cancer cells is not only determined by upregulated Xc- but also by dysregulated glutamate transporters, which provide additional targets for therapeutic intervention.