Oncogenic Epithelial-mesenchymal Transition Research Articles

The novel epithelial-mesenchymal transition-related proteins and their therapeutic targets in cholangiocarcinoma: a narrative review.

Cholangiocarcinoma (CCA), a liver cancer of bile duct epithelial cells, is a severe health issue in northeastern Thailand. The epithelial-mesenchymal transition (EMT) is a crucial process in the development of CCA. To comprehend oncogenic EMT in CCA, several newly found EMT factors are being explored in these underlying pathways. This narrative review explained the latest in vitro and in vivo findings on the molecular mechanisms of 21 new EMT-related proteins that affect CCA progression. We evaluated the PubMed database for relevant articles that fulfilled our criteria for investigating the molecular pathways of the novel EMT markers involved in oncogenic EMT and how they contribute to CCA development, including cell proliferation, apoptosis, invasion, migration, and chemoresistance. We discuss the potential of these new EMT markers as diagnostic, prognostic, and therapeutic indicators for CCA and describe their underlying mechanisms in the development of the disease. The discovery of several oncogenic EMT proteins and their key signaling pathways and downstream targets will also broaden novel paths of investigation into the diagnosis and targeted treatment of CCA. The EMT-related proteins that were found are good sources of knowledge and interesting information for future research. The possible ways to treat CCA that could be tested in clinical trials were also discussed.

Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib.

Cancer-associated fibroblasts (CAFs) play an important role in the induction of chemo-resistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma (HCC). We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively, to identify key molecules that might induce resistance to TKIs. We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms. The associations of plasma secreted phosphoprotein 1 (SPP1) expression levels before sorafenib/lenvatinib treatment with progression-free survival (PFS) and overall survival (OS) of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis. Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance. SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo. CAF-derived SPP1 activated rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) through the integrin-protein kinase C-alpha (PKCα) signaling pathway and promoted epithelial-to-mesenchymal transition (EMT). A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS (P = 0.026) and OS (P = 0.047) in patients with advanced HCC after TKI treatment. CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion. Its inhibition represents a promising therapeutic strategy against TKI resistance in HCC. Moreover, plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

Slow skeletal muscle troponin T acts as a potential prognostic biomarker and therapeutic target for hepatocellular carcinoma

Slow skeletal muscle troponin T (TNNT1) as a poor prognostic indicator is upregulated in colon and breast cancers. However, the role of TNNT1 in the disease prognosis and biological functions of hepatocellular carcinoma (HCC) is still unclear. The Cancer Genome Atlas (TCGA), real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to evaluate the TNNT1 expression of human HCC. The impact of TNNT1 levels on disease progression and survival outcome was studied using TCGA analysis. Moreover, the bioinformatics analysis and HCC cell culture were used to investigate the biological functions of TNNT1. Besides, the immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) were used to detect the extracellular TNNT1 of HCC cells and circulating TNNT1 of HCC patients, respectively. The effect of TNNT1 neutralization on oncogenic behaviors and signaling was further validated in the cultured hepatoma cells. In this study, tumoral and blood TNNT1 was upregulated in HCC patients based on the analyses using bioinformatics, fresh tissues, paraffin sections, and serum. From the multiple bioinformatics tools, the TNNT1 overexpression was associated with advanced stage, high grade, metastasis, vascular invasion, recurrence, and poor survival outcome in HCC patients. By the cell culture and TCGA analyses, TNNT1 expression and release were positively correlated with epithelial-mesenchymal transition (EMT) processes in HCC tissues and cells. Moreover, TNNT1 neutralization suppressed oncogenic behaviors and EMT in hepatoma cells. In conclusion, TNNT1 may serve as a non-invasive biomarker and drug target for HCC management. This research finding may provide a new insight for HCC diagnosis and treatment.

Non-genetic adaptive resistance to KRASG12C inhibition: EMT is not the only culprit.

Adaptions to therapeutic pressures exerted on cancer cells enable malignant progression of the tumor, culminating in escape from programmed cell death and development of resistant diseases. A common form of cancer adaptation is non-genetic alterations that exploit mechanisms already present in cancer cells and do not require genetic modifications that can also lead to resistance mechanisms. Epithelial-to-mesenchymal transition (EMT) is one of the most prevalent mechanisms of adaptive drug resistance and resulting cancer treatment failure, driven by epigenetic reprogramming and EMT-specific transcription factors. A recent breakthrough in cancer treatment is the development of KRASG12C inhibitors, which herald a new era of therapy by knocking out a unique substitution of an oncogenic driver. However, these highly selective agents targeting KRASG12C, such as FDA-approved sotorasib (AMG510) and adagrasib (MRTX849), inevitably encounter multiple mechanisms of drug resistance. In addition to EMT, cancer cells can hijack or rewire the sophisticated signaling networks that physiologically control cell proliferation, growth, and differentiation to promote malignant cancer cell phenotypes, suggesting that inhibition of multiple interconnected signaling pathways may be required to block tumor progression on KRASG12C inhibitor therapy. Furthermore, the tumor microenvironment (TME) of cancer cells, such as tumor-infiltrating lymphocytes (TILs), contribute significantly to immune escape and tumor progression, suggesting a therapeutic approach that targets not only cancer cells but also the TME. Deciphering and targeting cancer adaptions promises mechanistic insights into tumor pathobiology and improved clinical management of KRASG12C-mutant cancer. This review presents recent advances in non-genetic adaptations leading to resistance to KRASG12C inhibitors, with a focus on oncogenic pathway rewiring, TME, and EMT.

LACTB exerts tumor suppressor properties in epithelial ovarian cancer through regulation of Slug.

Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.

VEGF-C mediates tumor growth and metastasis through promoting EMT-epithelial breast cancer cell crosstalk.

It is well established that a subset of cells within primary breast cancers can undergo an epithelial to mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via non-canonical paracrine-mediated activation of GLI activity that is dependent on SIX1 expression in the EMT cancer cells. However, the mechanism by which these SIX1-expressing EMT cells activate GLI signaling remained unclear. In this study, we demonstrate a novel mechanism for activation of GLI-mediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C. We show that VEGF-C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine-mediated increases in proliferation, migration and invasion of epithelial breast cancer cells, via non-canonical activation of GLI-signaling. We further show that the aggressive phenotypes, including metastasis, imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2, a receptor which interacts with VEGF-C, in neighboring epithelial cancer cells. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, demonstrating that VEGF-C strongly correlates with activation of Hedgehog signaling and EMT in the human disease. Our study suggests that the VEGF-C/NRP2/GLI axis is a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease.

Butyrate-containing structured lipids inhibit RAC1 and epithelial-to-mesenchymal transition markers: a chemopreventive mechanism against hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers. The rising incidence of HCC worldwide and its resistance to pharmacotherapy indicate that the prevention of HCC development may be the most impactful strategy to improve HCC-related morbidity and mortality. Among the broad range of chemopreventive agents, the use of dietary and nutritional agents is an attractive and promising approach; however, a better understanding of the mechanisms of their potential cancer suppressive action is needed to justify their use. In the present study, we investigated the underlying molecular pathways associated with the previously observed suppressive effect of butyrate-containing structured lipids (STLs) against liver carcinogenesis using a rat “resistant hepatocyte” model of hepatocarcinogenesis that resembles the development of HCC in humans. Using whole transcriptome analysis, we demonstrate that the HCC suppressive effect of butyrate-containing STLs is associated with the inhibition of the cell migration, cytoskeleton organization, and epithelial-to-mesenchymal transition (EMT), mediated by the reduced levels of RACGAP1 and RAC1 proteins. Mechanistically, the inhibition of the Racgap1 and Rac1 oncogenes is associated with cytosine DNA and histone H3K27 promoter methylation. Inhibition of the RACGAP1/RAC1 oncogenic signaling pathways and EMT may be a valuable approach for liver cancer prevention.

Developing Novel Means to Inhibit Metastasis Through Targeting Tumor Heterogeneity and EMT

It is well established that a subset of cells within primary breast cancers can undergo an epithelial to mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated a novel mechanism by which EMT contributes to metastasis, via mediating tumor cell crosstalk. In this seminar, I will discuss how breast cancer cells that have undergone an oncogenic EMT can increase metastasis of neighboring epithelial cancer cells via non‐canonical paracrine‐mediated activation of GLI activity that is dependent on Six1 expression in the EMT cancer cells. I will discuss how VEGF‐C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine‐mediated increases in proliferation, migration, invasion, and metastasis of epithelial breast cancer cells, via non‐canonical activation of GLI‐signaling. Importantly, the aggressive phenotypes imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF‐C in EMT cells or by knocking down NRP2, which interacts with VEGF‐C, in neighboring epithelial cancer cells both in vitro and in vivo. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, as VEGF‐C and GLI pathway genes significantly positively correlate. Our study suggests that the VEGF‐C/NRP2/GLI axis may be a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease. In addition, because SIX1 activates VEGF‐C expression which leads to enhancement of metastasis via cell autonomous and non‐cell autonomous means, we have developed an inhibitor that targets SIX1 action and thereby inhibits metastatic disease. We describe results using this inhibitor in cell culture, where it inhibits SIX1 function and EMT, and in animals, where it inhibits metastasis without an effect on primary tumor growth.Support or Funding InformationColorado Cancer Translational Research Accelerator (CCTRA) PI Heide L. Ford, Co‐I Rui ZhaoNCI R01 CA224867 PIs Heide L. Ford and Michael T. Lewis

KRAS Mutation-Responsive miR-139-5p inhibits Colorectal Cancer Progression and is repressed by Wnt Signaling.

Introduction: Colorectal cancer (CRC) frequently harbors KRAS mutations that result in chemoresistance and metastasis. MicroRNAs (miRNAs) are usually dysregulated and play important regulatory roles in tumor progression. However, the KRAS mutation-responsive miRNA profile in CRC remains uninvestigated.Methods: miR-139-5p was identified and evaluated by small RNA sequencing, qRT-PCR and in situ hybridization. The roles of miR-139-5p in CRC cells with and without KRAS mutation were determined by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and transwell assays in vitro and by tumorigenesis and metastasis assays in vivo. Microarrays followed by bioinformatic analyses, luciferase reporter assays and Western blotting were applied for mechanistic studies.Results: miR-139-5p was significantly downregulated in KRAS-mutated CRC cells and tissues compared with their wild-type counterparts. Low miR-139-5p expression was associated with aggressive phenotypes and poor prognosis in CRC patients. miR-139-5p overexpression inhibited CRC cell proliferation, migration and invasion in vitro, sensitized tumors to chemotherapy, and impaired tumor growth and metastasis in vivo. Transcriptomic profiling identified multiple modulators in the Ras (JUN and FOS) and Wnt (CTNNB1 and DVL1) signaling pathways and the epithelial-to-mesenchymal transition (EMT) process (ZEB1) as direct targets of miR-139-5p, and inverse correlations were confirmed in CRC clinical tissues. Aberrantly activated Wnt signaling in KRAS-mutant cells was demonstrated to transcriptionally repress miR-139-5p through TCF4, forming a miR-139-5p/Wnt signaling double-negative feedback loop.Conclusions: We identified miR-139-5p as a KRAS-responsive miRNA and demonstrated its involvement in CRC progression. KRAS mutation disrupted the miR-139-5p/Wnt signaling reciprocal negative feedback mechanism, which might cause miR-139-5p downregulation and derepression of oncogenic signaling pathways and EMT. These results reveal a transcriptional regulatory mode of KRAS-driven malignant transformation and highlight miR-139-5p as a novel regulator of crosstalk between the Ras and Wnt signaling pathways in CRC.

Ormeloxifene Suppresses Prostate Tumor Growth and Metastatic Phenotypes via Inhibition of Oncogenic β-catenin Signaling and EMT Progression.

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0-G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267-80. ©2017 AACR.

The N-terminal polypeptide derived from viral macrophage inflammatory protein II reverses breast cancer epithelial-to-mesenchymal transition via a PDGFRα-dependent mechanism.

NT21MP, a 21-residue peptide derived from the viral macrophage inflammatory protein II, competed effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in breast cancer. Its role in tumor epithelial-to-mesenchymal transition (EMT) regulation remains unknown. In this study, we evaluated the reversal of EMT upon NT21MP treatment and examined its role in the inhibition of EMT in breast cancer. The parental cells of breast cancer (SKBR-3 and MCF-7) and paclitaxel-resistant (SKBR-3 PR and MCF-7 PR) cells were studied in vitro and in combined immunodeficient mice. The mice injected with SKBR-3 PR cells were treated with NT21MP through the tail vein or intraperitoneally with paclitaxel or saline. Sections from tumors were evaluated for tumor weight and EMT markers based on Western blot. In vitro, the effects of NT21MP, CXCR4 and PDGFRα on tumor EMT were assessed by relative quantitative real-time reverse transcription–polymerase chain reaction, western blot and biological activity in breast cancer cell lines expressing high or low levels of CXCR4. Our results illustrated that NT21MP could reverse the phenotype of EMT in paclitaxel-resistant cells. Furthermore, we found that NT21MP governed PR-mediated EMT partly due to controlling platelet-derived growth factors A and B (PDGFA and PDGFB) and their receptor (PDGFRα). More importantly, NT21MP down-regulated AKT and ERK1/2 activity, which were activated by PDGFRα, and eventually reversed the EMT. Together, these results indicated that CXCR4 overexpression drives acquired paclitaxel resistance, partly by activating the PDGFA and PDGFB/PDGFRα autocrine signaling loops that activate AKT and ERK1/2. Inhibition of the oncogenic EMT process by targeting CXCR4/PDGFRα-mediated pathways using NT21MP may provide a novel therapeutic approach towards breast cancer.

Grainyhead-like 2 Reverses the Metabolic Changes Induced by the Oncogenic Epithelial-Mesenchymal Transition: Effects on Anoikis.

Resistance to anoikis is a prerequisite for tumor metastasis. The epithelial-to-mesenchymal transition (EMT) allows tumor cells to evade anoikis. The wound-healing regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses/reverses EMT, accompanied by suppression of the cancer stem cell (CSC) phenotype and by resensitization to anoikis. Here, the effects of GRHL2 upon intracellular metabolism in the context of reversion of the EMT/CSC phenotype, with a view toward understanding how these effects promote anoikis sensitivity, were investigated. EMT enhanced mitochondrial oxidative metabolism. Although this was accompanied by higher accumulation of superoxide, the overall level of reactive oxygen species (ROS) declined, due to decreased hydrogen peroxide. Glutamate dehydrogenase 1 (GLUD1) expression increased in EMT, and this increase, via the product α-ketoglutarate (α-KG), was important for suppressing hydrogen peroxide and protecting against anoikis. GRHL2 suppressed GLUD1 gene expression, decreased α-KG, increased ROS, and sensitized cells to anoikis. These results demonstrate a mechanistic role for GRHL2 in promoting anoikis through metabolic alterations. Mol Cancer Res; 14(6); 528-38. ©2016 AACR.

Abstract POSTER-BIOL-1301: The receptor tyrosine kinase AXL modulates oncogenic signaling and epithelial mesenchymal transition in epithelial ovarian cancer

Abstract Metastasis in epithelial ovarian cancer (EOC) is lethal, and results into a 5-year-survival rate lower than 30%. In EOC, tumor cells undergo Epithelial-Mesenchymal Transition (EMT) to attain the invasive and migratory potential crucial for metastasis. The analysis of molecular heterogeneity in EOC also demonstrated the important role of EMT. Our group has previously classified EOC tumors and cell lines into 5 distinct molecular subtypes based on their global gene expression patterns. Among these subtypes, the Mesenchymal subtype is believed to be driven by EMT. Receptor Tyrosine Kinases (RTKs), which play a pivotal role in proliferation and survival pathways, have been strongly indicated to mediate signals in EMT. Upon interrogating the kinome expression patterns by determining the top 10 ranked kinases in each molecular subtype, the RTK AXL was revealed to have a significantly high ranking in the Mesenchymal subtype. The purpose of this study is to elucidate the role of AXL in oncogenic signaling and EMT in EOC. Western Blotting of total Axl protein in a panel of 40 EOC cell lines revealed a distinctive pattern among the 5 molecular subtypes. Upon ligand activation by Gas6 in Axl-expressing cell lines, pERK and pAkt, two signaling molecules downstream of Axl, displayed a unique temporal signaling pattern specific to the Mesenchymal subtype. This resulted into an implication of the preferential sensitivity to Axl-specific inhibitors in the Mesenchymal subtype. The IC-50 of R428, one small molecule inhibitor specific to Axl, was significantly lower in the Mesenchymal cell lines compared to the other subtypes (Average of 2.91 μM versus 9.38 μM; p-value=0.01) Stable clones were generated by using shRNAs to knockdown AXL in Mesenchymal EOC lines. shAXL SKOV3 cells demonstrated a reversal of EMT evident by restoration of Ecadherin. siRNA mediated transient silencing of AXL showed a reduction in cell motility with decreased total displacement and velocity measured over 12 hours (31.98 μm compared to 44.59 μm, p-value=0.01; and 17.35 μm/second compared to 23.15 μm/second, pvalue= 0.03) measured by using single cell tracking. Survival analysis showed that AXL over expression correlated positively with poor overall survival of EOC in both univariate (HR=2.2 p-value=6.46e−11), and multivariate analysis (HR=1.513 p-value=0.0012). In conclusion, AXL is significantly over-expressed and displays a unique signaling pattern in the Mesenchymal subtype of EOC. Knocking down AXL in the Mesenchymal cell lines affects their EMT phenotype and migratory properties. AXL is an independent prognostic factor for EOC and is a clinically relevant target that is potentially involved in cross talks between RTK oncogenic signaling and EMT. Citation Format: Jane Antony, Tuan Zea Tan, Andrew Paterson, Chiara Recchi, Hani Gabra, Jean Paul Thiery, Ruby Yun-Ju Huang. The receptor tyrosine kinase AXL modulates oncogenic signaling and epithelial mesenchymal transition in epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1301.

Interleukin-6 expression by interactions between gynecologic cancer cells and human mesenchymal stem cells promotes epithelial-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) facilitates the invasion and metastasis of cancer cells. EMT seems to be mediated by the interaction between cancer cells and human mesenchymal stem cells (hMSCs) in the tumor microenvironment. The present study is intended to identify specific cytokines as potent inducers of EMT associated hMSCs-tumor interactions. We used ovarian cancer cell lines (SKOV-3 and IGROV-1), endometrial cancer cell line (Ishikawa) and hMSCs (bone marrow MSC, amniotic membrane MSC and decidua MSC). The expressions of EMT markers (E-cadherin, Snail, Twist and N-cadherin) were analyzed using quantitative RT-PCR, immunofluorescence and western blot analysis. Matrix metalloproteinases (MMP-2 and MMP-9), Matrigel invasion assay, and wound healing assay were used to analyze cell migration and invasion. Gynecologic cancer cells directly co-cultured with hMSCs had contact-dependent altered morphology and growth patterns. IL-6 was elevated in all co-cultures using a human cytokine array. After IL-6 treatment of cancer cell lines, RT-PCR and western blot analysis indicated a decrease in an epithelial marker and an increase in mesenchymal markers. Also, cancer cells with IL-6 significantly increase in MMP-2 and MMP-9 and significantly enhance the migration ability compared to untreated cells (P<0.05), as shown by wound healing assay. On Matrigel invasion assay, treated cells displayed significantly increased invasiveness compared to untreated cancer cells. Gyneocologic cancer cells exposed to IL-6 acquired mesenchymal properties that facilitated metastasis and invasion by promoting EMT. The present study suggests that IL-6 of the tumor microenvironment has a critical role in oncogenic EMT.

Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis and epithelial‑to‑mesenchymal transition in colon and breast cancers.

The Nef-M1 peptide competes effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in colon cancer (CRC) and breast cancer (BC). Its role in tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) regulation, key steps involved in tumor growth and metastasis, are unknown. We evaluated the angioinhibitory effect of Nef-M1 peptide and examined its role in the inhibition of EMT in these cancers. Colon (HT29) and breast (MDA-MB231) cancer cells expressing CXCR4 were studied in vitro and in xenograft tumors propagated in severe combined immunodeficient mice. The mice were treated intraperitoneally with Nef-M1 or scrambled amino acid sequence of Nef-M1 (sNef-M1) peptide, a negative control, starting at the time of tumor implantation. Sections from tumors were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial markers. In vitro tumor angiogenesis was assessed by treating human umbilical vein endothelial cells with conditioned media from the tumor cell lines. A BC cell line (MDA-MB 468) which does not express CXCR4 was used to study the actions of Nef-M1 peptide. Western blot and immunofluorescence analyses assessed the effect of Nef-M1 on tumor angiogenesis and EMT in both tumors and cancer cells. Metastatic lesions of CRC and BC expressed more CXCR4 than primary lesions. It was also found that tumors from mice treated with sNef-M1 had well established vascularity, while Nef-M1 treated tumors had very poor vascularization. Indeed, the mean MVD was lower in tumors from Nef-M1 treated mice than in sNef-M1 treated tumors. Nef-M1 treated tumor has poor morphology and loss of endothelial integrity. Although conditioned medium from CRC or BC cells supported HUVEC tube formation, the conditioned medium from Nef-M1 treated CRC or BC cells did not support tube formation. Western blot analyses revealed that Nef-M1 effectively suppressed the expression of VEGF-A in CRC and BC cells and tumors. This suggests that Nef-M1 treated CRC and BC cells are more consistent with E-cadherin signature, and thus appears more epithelial in nature. Our data indicate that Nef-M1 peptide inhibits tumor angiogenesis and the oncogenic EMT process. Targeting the chemokine receptor, CXCR4, mediated pathways using Nef-M1 may prove to be a novel therapeutic approach for CRC and BC.

Epithelial Plasticity in Cancer: Unmasking a MicroRNA Network for TGF-β-, Notch-, and Wnt-Mediated EMT.

Epithelial-to-mesenchymal transition (EMT) is a reversible process by which cancer cells can switch from a sessile epithelial phenotype to an invasive mesenchymal state. EMT enables tumor cells to become invasive, intravasate, survive in the circulation, extravasate, and colonize distant sites. Paracrine heterotypic stroma-derived signals as well as paracrine homotypic or autocrine signals can mediate oncogenic EMT and contribute to the acquisition of stem/progenitor cell properties, expansion of cancer stem cells, development of therapy resistance, and often lethal metastatic disease. EMT is regulated by a variety of stimuli that trigger specific intracellular signalling pathways. Altered microRNA (miR) expression and perturbed signalling pathways have been associated with epithelial plasticity, including oncogenic EMT. In this review we analyse and describe the interaction between experimentally validated miRs and their target genes in TGF-β, Notch, and Wnt signalling pathways. Interestingly, in this process, we identified a “signature” of 30 experimentally validated miRs and a cluster of validated target genes that seem to mediate the cross talk between TGF-β, Notch, and Wnt signalling networks during EMT and reinforce their connection to the regulation of epithelial plasticity in health and disease.

Ras promotes the epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) has been implicated in cancer invasion and metastasis. Depending on the cellular context, oncogenic Ras can induce EMT in the presence of transforming growth factor-b (TGF-b). By synergistically promoting the EMT in cooperation with TGF-b, oncogenic Ras promotes cell migration in vitro and tumor invasion and metastasis in vivo . Despite these observations, the mechanism by which oncogenic Ras contributes to the EMT is not well understood. We recently demonstrated that BLT2, a G protein-coupled receptor for the inflammatory lipid mediator leukotriene B 4 (LTB 4 ), lies downstream of Ras and mediates oncogenic Ras-induced EMT in mammary epithelial cells. Thus, these inextricable networks involving the Ras oncogene, EMT and inflammatory lipid mediators play critical roles in the tumor microenvironment.

Mutant p53 promotes epithelial-mesenchymal plasticity and enhances metastasis in mammary carcinomas of WAP-T mice.

To study the postulated mutant p53 (mutp53) "gain of function" effects in mammary tumor development, progression and metastasis, we crossed SV40 transgenic WAP-T mice with mutant p53 transgenic WAP-mutp53 mice. Compared to tumors in monotransgenic WAP-T mice, tumors in bitransgenic WAP-T x WAP-mutp53 mice showed higher tumor grading, enhanced vascularization, and significantly increased metastasis. Bitransgenic tumors revealed a gene signature associated with the oncogenic epithelial-mesenchymal transition pathway (EMT gene signature). In cultures of WAP-T tumor-derived G-2 cancer cells, which are comprised of subpopulations displaying "mesenchymal" and "epithelial" phenotypes, this EMT gene signature was associated with the "mesenchymal" compartment. Furthermore, ectopic expression of mutp53 in G-2 cells sufficed to induce a strong EMT phenotype. In contrast to these in vitro effects, monotransgenic and bitransgenic tumors were phenotypically similar suggesting that in vivo the tumor cell phenotype might be under control of the tumor microenvironment. In support, orthotopic transplantation of G-2 cells as well as of G-2 cells expressing ectopic mutp53 into syngeneic mice resulted in tumors with a predominantly epithelial phenotype, closely similar to that of endogenous primary tumors. We conclude that induction of an EMT gene signature by mutp53 in bitransgenic tumors primarily promotes tumor cell plasticity, that is, the probability of tumor cells to undergo EMT processes under appropriate stimuli, thereby possibly increasing their potential to disseminate and metastasize.

Decoding the Knots of Initiation of Oncogenic Epithelial-Mesenchymal Transition in Tumor Progression

Oncogenic epithelial-mesenchymal transition (oncEMT) plays important roles in the genesis of cancer stem cells (CSCs), malignant tumor initiation and progression, cancer metastasis, and drug resistance. Although the role of oncEMT in tumorigenesis has recently been extensively studied, the initiation of oncEMT is not clearly understood, and its mechanisms of action are still unknown. Emerging evidence suggests that oncEMT is a complex process, which involves multiple endogenous and exogenous factors. Overexpression of several oncogenes and reprogramming factors in precancerous and cancerous cells, including Ras, Myc, Bmi-1, Oct4, Nanog, Slug, Twist, Zeb1, and Zeb2, may initiate oncEMT and tumorigenesis. Defects in key tumor suppressors, such as p53, PTEN, CCN6 protein, and p21 also are associated with oncEMT. MicroRNA (miRNA) may also play a role in the oncEMT. Furthermore, exogenous factors, including chemical carcinogens, viruses, radiation, hypoxia, and acidic microenvironment, can drive oncEMT. Moreover, various growth factors derived from either malignant tumor cells or tumor-associated non-tumor cells in the cancer microenvironment can promote oncEMT. Together, the endogenous and exogenous factors, as well as a hostile cancer microenvironment, initiate the oncEMT program through diverse signaling pathways and networks. However, the dynamic process of initiating oncEMT and the mechanisms are still incompletely understood. Further characterization of the dynamics and mechanisms of the oncEMT will provide new insights into oncogenesis, as well as identify specific oncEMT markers and targets for early diagnosis of cancer and novel anti-cancer drug discovery.

English

Abstract Introduction Invasion and metastasis are the main causes for the death of patients with breast cancers. Epithelial–mesenchy-mal transition is implicated as a vital process in the invasion and metasta -sis of breast cancers, with endowing migratory and invasive cancer cells associated with metastatic capabil-ity. Increasing evidences demon -strated that epithelial–mesenchymal tions. However, this process is retransition-initiating cells possessed mesenchymal features and stem-like traits that are resistant to chemother-apy. In this review, we summarise the physiological and pathological roles of epithelial–mesenchymal transi-tions, new insights in the molecular mechanisms of regulating epithe-lial–mesenchymal transition during breast cancer invasion and metasta -sis, and its implication in chemother-apy resistance. Conclusion Therefore, it is challenging to probe and uncover the mechanistic regu -lation of oncogenic epithelial–mes-enchymal transition, which will contribute to our understanding of metastatic dissemination and the role of targeting epithelial–mesenchymal transition with existing therapy as well as developing new drugs, in order to prevent metastasis and to diminish distant recurrence in pa -tients with breast cancers.