Triggering Epithelial-mesenchymal Transition Research Articles

A Novel Splice Variant of the Inhibitor of Growth 3 Lacks the Plant Homeodomain and Regulates Epithelial-Mesenchymal Transition in Prostate Cancer Cells.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial–mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

TFAP2A potentiates lung adenocarcinoma metastasis by a novel miR-16 family/TFAP2A/PSG9/TGF-\u03b2 signaling pathway

Transcription factor AP-2α (TFAP2A) was previously regarded as a critical regulator during embryonic development, and its mediation in carcinogenesis has received intensive attention recently. However, its role in lung adenocarcinoma (LUAD) has not been fully elucidated. Here, we tried to investigate TFAP2A expression profiling, clinical significance, biological function and molecular underpinnings in LUAD. We proved LUAD possessed universal TFAP2A high expression, indicating a pervasively poorer prognosis in multiple independent datasets. Then we found TFAP2A was not indispensable for LUAD proliferation, and exogenous overexpression even caused repression. However, we found TFAP2A could potently promote LUAD metastasis possibly by triggering epithelial–mesenchymal transition (EMT) in vitro and in vivo. Furthermore, we demonstrated TFAP2A could transactivate Pregnancy-specific glycoprotein 9 (PSG9) to enhance transforming growth factor β (TGF-β)-triggering EMT in LUAD. Meanwhile, we discovered suppressed post-transcriptional silencing of miR-16 family upon TFAP2A partly contributed to TFAP2A upregulation in LUAD. In clinical specimens, we also validated cancer-regulating effect of miR-16 family/TFAP2A/PSG9 axis, especially for lymph node metastasis of LUAD. In conclusion, we demonstrated that TFAP2A could pivotally facilitate LUAD progression, possibly through a novel pro-metastasis signaling pathway (miR-16 family/TFAP2A/PSG9/ TGF-β).

Intracellular and extracellular S100A9 trigger epithelial-mesenchymal transition and promote the invasive phenotype of pituitary adenoma through activation of AKT1.

Pituitary adenoma (PA) is mostly benign intracranial tumor, but it also displays invasive growth characteristics and provokes challenging clinical conditions. S100A9 protein enhances tumor progression. In this study, we firstly demonstrated that both intracellular and extracellular S100A9 promoted the expression of Vimentin and Intercellular cell adhesion molecule-1 (ICAM-1), coupled with reduced E-cadherin in PA. As a result, PA acquired the phenotype of Epithelial-Mesenchymal Transition (EMT), leading to proliferation, cell cycle progression, migration and invasion. In addition, we indicated S100A9-induced EMT was mediated by activation of AKT1. Furthermore, immunohistochemistry showed that S100A9 expression was higher in invasive PA than that in non-invasive PA. These data extended our understanding for the effects of S100A9 on PA invasion and contributed to further development of a promising therapeutic target for invasive PA.

Nerve growth factor (NGF)-TrkA axis in head and neck squamous cell carcinoma triggers EMT and confers resistance to the EGFR inhibitor erlotinib.

Understanding the molecular mechanisms regulating tumor dissemination and therapeutic resistance is of central importance for effective cancer therapies. Here, we report that nerve growth factor (NGF) and its receptor TrkA facilitate epithelial-mesenchymal transition (EMT) and EGFR inhibitor resistance via STAT3 activation in head and neck squamous cell carcinoma (HNSCC). Both NGF and TrkA expression were elevated in HNSCC, indicating poor clinical outcomes. NGF was highly expressed in cancer cells and nerves in perineural niche, whereas TrkA expression was higher in cancer cells with perineural invasion. The NGF/TrkA axis could promote HNSCC cell dissemination and trigger EMT via STAT3 activation. Moreover, we discovered that the NGF/TrkA axis conferred resistance to the EGFR inhibitor erlotinib via EMT processes in HNSCC cells. Blocking TrkA signaling markedly reversed EMT and sensitized HNSCC cells to erlotinib in both in vitro and in vivo models. Overall, our results demonstrate novel evidence that the paracrine NGF/TrkA axis favors EMT and confers EGFR-targeted therapeutic resistance in HNSCC.

WSZG inhibits BMSC-induced EMT and bone metastasis in breast cancer by regulating TGF-β1/Smads signaling

Bone metastasis of breast cancer causes severe skeletal-related events and poor prognosis. Wensheng Zhuanggu Formula (WSZG), a traditional Chinese prescription, is used to adjunctively treat breast cancer bone metastases in clinical practice. This study was undertaken to investigate the antibone-metastatic activities and mechanisms of WSZG extract by evaluating the effect of this formula on the cross-talk between bone marrow-derived mesenchymal stem cells (BMSCs) and breast cancer cells in triggering epithelial-mesenchymal transition (EMT) in vivo and in vitro. The results demonstrated that BMSCs might enhance the invasive and metastatic potentials of breast cancer cells as a consequence of EMT induction through direct cell-to-cell contact. WSZG treatment remarkably suppressed motility, invasion, EMT-related gene, and protein markers in BMSC-conditioned breast cancer cells and ameliorated bone metastases and damages in nude mice following co-injection of BMSCs and MDA-MB-231BO breast cancer cells. Further investigation showed that the transforming growth factor-β1 (TGF-β1)/Smads pathway was an important mechanism enabling BMSCs to induce EMT occurrence of breast cancer cells. WSZG treatment reversed BMSC-induced EMT by downregulating TGF-β1/Smads signaling. Thus, WSZG extracts may be regarded as a potential antibone-metastatic agent for breast cancer therapy.

Higher matrix stiffness as an independent initiator triggers epithelial-mesenchymal transition and facilitates HCC metastasis

BackgroundIncreased liver stiffness exerts a detrimental role in driving hepatocellular carcinoma (HCC) malignancy and progression, and indicates a high risk of unfavorable outcomes. However, it remains largely unknown how liver matrix stiffness as an independent cue triggers epithelial-mesenchymal transition (EMT) and facilitates HCC metastasis.MethodsBuffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness were used in the study to explore the effects of matrix stiffness on EMT occurrence and its underlying molecular mechanism. Clinical significance of liver stiffness and key molecules required for stiffness-induced EMT were validated in HCC cohorts with different liver stiffness.ResultsHCC xenografts grown in higher stiffness liver exhibited worse malignant phenotypes and higher lung metastasis rate, suggesting that higher liver stiffness promotes HCC invasion and metastasis. Cell tests in vitro showed that higher matrix stiffness was able to strikingly strengthen malignant phenotypes and independently induce EMT occurrence in HCC cells, and three signaling pathways converging on Snail expression participated in stiffness-mediated effect on EMT including integrin-mediated S100A11 membrane translocation, eIF4E phosphorylation, and TGF β1 autocrine. Additionally, the key molecules required for stiffness-induced EMT were highly expressed in tumor tissues of HCC patients with higher liver stiffness and correlated with poor tumor differentiation and higher recurrence.ConclusionsHigher matrix stiffness as an initiator triggers epithelial-mesenchymal transition (EMT) in HCC cells independently, and three signaling pathways converging on Snail expression contribute to this pathological process. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis.

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.

Abstract 1110: Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program

Abstract Purpose: Recent report from our laboratory showed a novel role of tumor derived ganglioside, GM2 in mediating tumor cell migration as well invasion. In the present study we report a novel role of GM2 in mediating anchorage independent growth (AIG) and epithelial-mesenchymal transition (EMT) leading to tumor growth, progression and metastasis, through a HIPPO-YAP dependent transcriptional program. Experimental procedures : Permanent GM2-synthase knockout (KO) cells were generated using targeted genome editing tool, TALEN. TALEN was used to generate Renca-vGM2-syn KO cells (from a GM2-over-expressing variant of a mouse kidney cancer cell line, Renca-v). Renca-vWT as well as Renca-vGM2-syn KO cells were used to define the role of GM2 in AIG, EMT and tumorigenesis both in vitro and in vivo. DNA microarray analysis was used to elucidate the mechanism behind GM2-mediated AIG and EMT. To define involvement of YAP in GM2-mediated EMT, real time PCR was used to validate YAP-target gene expression in GM2-synthase KO or silenced (siRNA) cells as well as in cells treated with exogenous GM2, in the presence or absence of specific inhibitors of the Hippo-YAP signaling pathway. CRISPR-Cas9 mediated YAP/TAZ-double KO (HelaYAP/TAZ KO) cells were used to conclusively prove the involvement of YAP/TAZ in GM2-mediated EMT. Results : Renca-vGM2-syn KO cells show reduced AIG, higher cell adhesion possibly through increased anoikis sensitivity, indicating a critical role of GM2-synthase and complex gangliosides in GM2-mediated EMT. Furthermore, Renca-vGM2-syn KO cell line shows reduced tumor growth potential, significant reduction in experimental lung metastasis with increased median survival in a syngeneic mice tumor model. Gene expression profiling using DNA microarray with GM2-synthase silenced cells reveal significant modulation of YAP target genes, suggesting a critical role of the Hippo-YAP signaling axis. Molecular insight reveals that GM2-synthase knockout or siRNA mediated knockdown results in significant downregulation of YAP activated genes namely Ctgf, Cyr 61, Pdgf-c, Lox, etc. and upregulation of YAP repressed gene Ddit4, while exogenous addition of GM2 in GM2 deficient cells (Renca, MCF-7 and Hela) show opposite results. Verteporfin mediated disruption of YAP transcriptional program abrogated GM2 mediated modulation of YAP target genes. Finally, significant decrease in YAP-target gene expression in YAP/TAZ-double KO cells, but not in either YAP or TAZ (paralogue of YAP) single KOs confirm the definitive involvement of YAP/TAZ in GM2-mediated EMT. Conclusion : Our findings confirm a novel role of GM2 in triggering EMT by targeting YAP, ultimately leading to increased tumorigenic potential and metastatic activity in tumor cells. Citation Format: Barun Mahata, Abhisek Sarkar, Elora Khamrui, Sohini Chakraborty, Zhumur Ghosh, Kaushik Biswas. Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1110.

Long term exposure to environmental concentrations of diesel exhaust particles does not impact the phenotype of human bronchial epithelial cells

Chronic exposure to diesel engine exhausts is associated with an increased risk of pulmonary diseases including lung cancer. Diesel engine exhausts contain large amounts of diesel exhaust particles (DEP) on which are adsorbed several carcinogenic compounds such as polycyclic aromatic hydrocarbons. Acute toxicity of high concentrations of DEP has been largely demonstrated in various in vitro cellular models. In contrast, the cellular and molecular impacts of low environmental concentrations of DEP on the phenotype of chronically exposed lung epithelial cells remain to be investigated. In the present study, we show that long term exposure (6 months) to 2 μg/ml (0.4 μg/cm2) DEP (standard reference material 1650b) increased cytochrome P4501A mRNA levels in the human bronchial epithelial BEAS-2B cell line. However, chronic exposure to DEP did not change cell morphology, trigger epithelial-mesenchymal transition or increase anchorage-independent cell growth. Moreover, DEP increase neither the levels of reactive oxygen species or those of γ-histone H2AX, nor the expression of interleukin-6 and interleukin-8. Our results thus demonstrate that the chronic exposure to low DEP concentrations could increase cytochrome P501A gene expression in BEAS-2B cells but did not induce molecular effects related to genotoxicity, oxidative stress or inflammation.

Autophagy contributes to hypoxia-induced epithelial to mesenchymal transition of endometrial epithelial cells in endometriosis.

Endometriosis is a benign gynecologic disorder, and presents with malignant characteristics, such as migration and invasion. Hypoxia has been implicated in triggering epithelial-mesenchymal transition (EMT). Hypoxia is also known to induce autophagy. However, the relationship between autophagy and EMT under hypoxia conditions in endometriosis remains unknown. In the present study, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), microtubule associated protein light chain 3 (LC3), and mesenchymal cell marker vimentin was significantly higher in ectopic endometrium from patients with endometriosis, along with decreased expression of epithelial cell marker E-cadherin. After hypoxia treatment, endometrial epithelial cells exhibited enhanced migration and invasion abilities, as well as promoted autophagy and the EMT phenotype. Our analyses also show that HIF-1α was responsible for induction of autophagy. Moreover, inhibition of autophagy by chemical or genetic approaches suppressed hypoxia triggered EMT and reduced cell migration and invasion. Collectively, our findings identify that autophagy is critical for the migration and invasion of endometrial cells through the induction of EMT and indicate that inhibition of autophagy may be a novel useful strategy in the treatment of endometriosis.

Galunisertib modifies the liver fibrotic composition in the Abcb4Ko mouse model.

Transforming growth factor (TGF)-β stimulates extracellular matrix (ECM) deposition during development of liver fibrosis and cirrhosis, the most important risk factor for the onset of hepatocellular carcinoma. In liver cancer, TGF-β is responsible for a more aggressive and invasive phenotype, orchestrating remodeling of the tumor microenvironment and triggering epithelial-mesenchymal transition of cancer cells. This is the scientific rationale for targeting the TGF-β pathway via a small molecule, galunisertib (intracellular inhibitor of ALK5) in clinical trials to treat liver cancer patients at an advanced disease stage. In this study, the hypothesis that galunisertib modifies the tissue microenvironment via inhibition of the TGF-β pathway is tested in an experimental preclinical model. At the age of 6months, Abcb4ko mice-a well-established model for chronic liver disease development and progression-are treated twice daily with galunisertib (150mg/kg) via oral gavage for 14 consecutive days. Two days after the last treatment, blood plasma and livers are harvested for further assessment, including fibrosis scoring and ECM components. The reduction of Smad2 phosphorylation in both parenchymal and non-parenchymal liver cells following galunisertib administration confirms the treatment effectiveness. Damage-related galunisertib does not change cell proliferation, macrophage numbers and leucocyte recruitment. Furthermore, no clear impact on the amount of fibrosis is evident, as documented by PicroSirius red and Gomori-trichome scoring. On the other hand, several fibrogenic genes, e.g., collagens (Col1α1 and Col1α2), Tgf-β1 and Timp1, mRNA levels are significantly downregulated by galunisertib administration when compared to controls. Most interestingly, ECM/stromal components, fibronectin and laminin-332, as well as the carcinogenic β-catenin pathway, are remarkably reduced by galunisertib-treated Abcb5ko mice. In conclusion, TGF-β inhibition by galunisertib interferes, to some extent, with chronic liver progression, not by reducing the stage of liver fibrosis as measured by different scoring systems, but rather by modulating the biochemical composition of the deposited ECM, likely affecting the fate of non-parenchymal cells.

OVOL2 antagonizes TGF-β signaling to regulate epithelial to mesenchymal transition during mammary tumor metastasis.

Great progress has been achieved in the study of the role of TGF-β signaling in triggering epithelial-mesenchymal transition (EMT) in a variety of cancers; however, the regulation of TGF-β signaling during EMT in mammary tumor metastasis has not been completely defined. In the present study, we demonstrated that OVOL2, a zinc finger transcription factor, inhibits TGF-β signaling-induced EMT in mouse and human mammary tumor cells, as well as in mouse tumor models. Data from the Oncomine databases indicated a strong negative relationship between OVOL2 expression and breast cancer progression. Moreover, our experiments revealed that OVOL2 inhibits TGF-β signaling at multiple levels, including inhibiting Smad4 mRNA expression and inducing Smad7 mRNA expression, blocking the binding between Smad4 and target DNA, and interfering with complex formation between Smad4 and Smad2/3. These findings reveal a novel mechanism that controls the TGF-β signaling output level in vitro and in vivo. The modulation of these molecular processes may represent a strategy for inhibiting breast cancer invasion by restoring OVOL2 expression.

N-Myc-interacting protein (NMI) negatively regulates epithelial-mesenchymal transition by inhibiting the acetylation of NF-κB/p65

The epithelial-mesenchymal transition (EMT) plays an essential role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and tumor progression. However, the mechanisms underlying this process are poorly understood. Many signaling pathways, including the NF-κB signaling pathway, trigger EMT during development and differentiation. In the present study, we report that N-Myc interactor (NMI) inhibits EMT progression by suppressing transcriptional activities of NF-κB in human gastric cancer cells. We show that the expression of NMI is significantly reduced in invasive gastric cancer cells and gastric cancer tissues. Overexpression of NMI inhibited cell migration and invasion, and this inhibition was enhanced after TNF-α stimulation. Tumorigenicity assay in nude mice support the notion that NMI inhibits EMT in cancer cells. Mechanistically, NMI promotes the interaction between NF-κB/p65 and histone deacetylases (HDACs) and inhibits the acetylation and transcriptional activity of p65. The expression of p65 rescues NMI-mediated inhibition of EMT and the inhibition of the acetylation of p65 mediated by NMI is HDACs-dependent. Taken together, these findings suggest that NMI can suppress tumor invasion and metastasis by inhibiting NF-κB pathways, providing an alternative mechanism for EMT inhibition in stomach neoplasm.

Signaling mechanisms of the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is an essential mechanism in embryonic development and tissue repair. EMT also contributes to the progression of disease, including organ fibrosis and cancer. EMT, as well as a similar transition occurring in vascular endothelial cells called endothelial-mesenchymal transition (EndMT), results from the induction of transcription factors that alter gene expression to promote loss of cell-cell adhesion, leading to a shift in cytoskeletal dynamics and a change from epithelial morphology and physiology to the mesenchymal phenotype. Transcription program switching in EMT is induced by signaling pathways mediated by transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP), Wnt-β-catenin, Notch, Hedgehog, and receptor tyrosine kinases. These pathways are activated by various dynamic stimuli from the local microenvironment, including growth factors and cytokines, hypoxia, and contact with the surrounding extracellular matrix (ECM). We discuss how these pathways crosstalk and respond to signals from the microenvironment to regulate the expression and function of EMT-inducing transcription factors in development, physiology, and disease. Understanding these mechanisms will enable the therapeutic control of EMT to promote tissue regeneration, treat fibrosis, and prevent cancer metastasis.

Overexpression of Snail in retinal pigment epithelial triggered epithelial–mesenchymal transition

Snail transcription factor has been implicated as an important regulator in epithelial–mesenchymal transition (EMT) during tumourigenesis and fibrogenesis. Our previous work showed that Snail transcription factor was activated in transforming growth factor β1 (TGF-β1) induced EMT in retinal pigment epithelial (RPE) cells and may contribute to the development of retinal fibrotic disease such as proliferative vitreoretinopathy (PVR). However, whether Snail alone has a direct role on retinal pigment epithelial–mesenchymal transition has not been investigated. Here, we analyzed the capacity of Snail to drive EMT in human RPE cells. A vector encoding Snail gene or an empty vector were transfected into human RPE cell lines ARPE-19 respectively. Snail overexpression in ARPE-19 cells resulted in EMT, which was characterized by the expected phenotypic transition from a typical epithelial morphology to mesenchymal spindle-shaped. The expression of epithelial markers E-cadherin and Zona occludin-1 (ZO-1) were down-regulated, whereas mesenchymal markers a-smooth muscle actin (a-SMA) and fibronectin were up-regulated in Snail expression vector transfected cells. In addition, ectopic expression of Snail significantly enhanced ARPE-19 cell motility and migration. The present data suggest that overexpression of Snail in ARPE-19 cells could directly trigger EMT. These results may provide novel insight into understanding the regulator role of Snail in the development of retinal pigment epithelial–mesenchymal transition.

Notch activation stimulates migration of breast cancer cells and promotes tumor growth

IntroductionDysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells.MethodsWe have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors.ResultsWe found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining.ConclusionsThese results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia.

Tubular cell dedifferentiation and peritubular inflammation are coupled by the transcription regulator Id1 in renal fibrogenesis

During renal fibrogenesis, tubular epithelial-mesenchymal transition is closely associated with peritubular inflammation; however, it is not clear whether these two processes are connected. We previously identified the inhibitor of differentiation-1 (Id1), a dominant negative antagonist of basic helix-loop-helix transcription factors, as a major trigger of tubular cell dedifferentiation after injury. Id1 was induced selectively in degenerated proximal tubule and collecting duct epithelia after injury and was present in both the cytoplasm and nucleus, suggesting shuttling between these two compartments. Interestingly, the upregulation of Id1 was associated with peritubular inflammation in mouse and human nephropathies. In vitro, Id1 potentiated NF-κB signaling and augmented RANTES expression in kidney epithelial cells, which led to an enhanced recruitment of inflammatory cells. Id1 also induced Snail1 expression and triggered tubular epithelial dedifferentiation. In vivo, genetic ablation of Id1 in mice reduced peritubular inflammation and decreased tubular expression of RANTES following ureteral obstruction. Mice lacking Id1 were also protected against myofibroblast activation and matrix expression, leading to a reduced total collagen deposition in obstructive nephropathy. Thus, these results indicate that Id1 shuttles between nucleus and cytoplasm and promotes peritubular inflammation and tubular epithelial dedifferentiation, suggesting that these two events are intrinsically coupled during renal fibrogenesis.

Abstract 3369: Macrophages and their induced cytokines drive the epithelial to mesenchymal transition in prostate cancer cells

Abstract Macrophages derived from circulating monocytic myeloid precursors are a major component of leukocyte infiltrate found in tumors and their role in prostate cancer progression is now emerging. Here we investigate the potential of macrophages to induce the epithelial-mesenchymal transition (EMT) in prostate cancer cells. EMT is a critical process for metastasis, and the elucidation of factors that initiate EMT would be beneficial in the development of treatments to halt the dissemination of cancer cells throughout the body. CD14+-peripheral blood monocytes were isolated from healthy donors and stimulated with either interferon gamma (INFγ) or interleukin (IL)-4, factors known to promote the differentiation of monocytes into M1 or M2-type macrophages, respectively. After 48 hours, cells from prostate cancer cell lines PC3 Luc, DU145 Luc, and ARCAP Luc were co-cultured with the macrophages for four days. The cells were passaged three times by trypsination and protein lysates were analyzed by Western Blot for the expression of EMT markers: E-cadherin and vimentin. Our results revealed that cells co-cultured with IL-4-stimulated macrophages expressed lower levels of E-cadherin and higher levels of vimentin compared to control epithelial cells. In addition, three mesenchymal-type subpopulations that were isolated from PC3 cells interacting with IL-4-treated CD14+-cells exhibited a complete loss of E-cadherin. After more than 20 passages, these cells maintained the mesenchymal characteristics in culture and showed a striking up-regulation of the transcription factor ZEB 1, whose expression has been previously correlated with Gleason grade in human prostate tumors. Concurrently, we set out to study the secreted factors by which macrophages may trigger EMT. We treated DU145 Luc and PC3 Luc cells with several cytokines found to be differentially expressed in media containing cancer cells co-cultured with IL-4-stimulated macrophages and evaluated whether any of these factors induced EMT. It was discovered that cells grown in media containing the cytokine, IL-1B, in combination with IL-4 showed a greater display of mesenchymal markers than control cells. In conclusion, these results establish that macrophages can act as potent EMT-inducers in prostate cancer cells, although the exact mediators of this function remain unknown. The data suggests that CD14+ cells stimulated with IL-4 are more effective at triggering EMT than non-stimulated or INFγ-stimulated macrophages, which may pinpoint M2-type macrophages as a mediator of EMT in vivo. Furthermore, the cytokine study revealed that macrophage-induced IL-1B in combination with IL-4 may be an important factor in the mechanism of macrophage-induced EMT. As this transition may represent a major mechanism of prostate cancer metastasis, future research will be focused on elucidating the molecules involved in order to develop novel therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2011-3369

Correlation of Snail expression and survival in patients with early-stage triple-negative breast cancer (TNBC).

10525 Background: In breast and other cancers, tumors reactivate a developmental program, the epithelial-mesenchymal transition (EMT), to increase cell motility and facilitate invasion and metastasis. Snail is a transcription factor that has been implicated in triggering EMT. We hypothesized that Snail expression is an important mediator of invasion, metastasis, and decreased survival in breast cancer. Methods: We analyzed protein levels of Snail and EMT markers using reverse-phase protein arrays (RPPA) of 334 primary breast cancers from the frozen tumor bank at M. D. Anderson, obtained from patients with early-stage disease. Recurrence-free survival (RFS) and overall survival (OS) were estimated by the Kaplan-Meier product and compared among groups via the log-rank test. Multivariate analysis was conducted via Cox proportional hazards models. Results: 267 patients had complete clinical data. Median age was 61 (range 27-88). 84% had stage I-II disease, 16% stage III. 66% of tumors were hormone- receptor (HR) positive, 3% HER2-positive, 31% TN. Prognostic factors were similar in the low- and high-Snail groups. At a median follow-up of 30.3 months, there were 65 recurrences and 65 deaths. Among TNBC patients, in a univariate model, estimated RFS was marginally significantly worse in the high-Snail vs. the low-Snail group (hazard ratio [hr], 2.11; 95% CI = 0.91, 4.88; p = 0.082). This became significant after adjustment for age and stage in a multivariate model (hr, 2.44; 1.01,5.88; p = 0.047). Strikingly, Snail was a marker of early relapse in TNBC patients, with a 2-year relapse rate of 41% in high-Snail vs. 19% in low-Snail patients. RFS was not different between high- and low-Snail groups in the HR-positive subset (p = 0.737). For all patients, high Snail expression correlated with an increase in the mesenchymal marker fibronectin (p < 0.001, Fisher's exact), consistent with the occurrence of EMT in high- Snail tumors. Conclusions: Snail expression is correlated with decreased RFS, in particular early relapse at < 2 years, in early-stage TNBC patients. TNBC patients with high Snail expression may be candidates for therapy to prevent metastases, in the form of inhibitors of the Snail pathway or EMT. No significant financial relationships to disclose.

The signaling mechanism of ROS in tumor progression

Reactive oxygen species (ROS) are recently proposed to be involved in tumor metastasis which is a complicated processes including epithelial-mesenchymal transition (EMT), migration, invasion of the tumor cells and angiogenesis around the tumor lesion. ROS generation may be induced intracellularly, in either NADPH oxidase- or mitochondria-dependent manner, by growth factors and cytokines (such as TGFbeta and HGF) and tumor promoters (such as TPA) capable of triggering cell adhesion, EMT and migration. As a signaling messenger, ROS are able to oxidize the critical target molecules such as PKC and protein tyrosine phosphates (PTPs), which are relevant to tumor cell invasion. PKC contain multiple cysteine residues that can be oxidized and activated by ROS. Inactivation of multiple PTPs by ROS may relieve the tyrosine phosphorylation-dependent signaling. Two of the down-stream molecules regulated by ROS are MAPK and PAK. MAPKs cascades were established to be a major signal pathway for driving tumor cell metastasis, which are mediated by PKC, TGF-beta/Smad and integrin-mediated signaling. PAK is an effector of Rac-mediated cytoskeletal remodeling that is responsible for cell migration and angiogenesis. There are several transcriptional factors such as AP1, Ets, Smad and Snail regulating a lot of genes relevant to metastasis. AP-1 and Smad can be activated by PKC activator and TGF-beta1, respectively, in a ROS dependent manner. On the other hand, Est-1 can be upregulated by H2O2 via an antioxidant response element in the promoter. The ROS-regulated genes relevant to EMT and metastasis include E-cahedrin, integrin and MMP. Comprehensive understanding of the ROS-triggered signaling transduction, transcriptional activation and regulation of gene expressions will help strengthen the critical role of ROS in tumor progression and devising strategy for chemo-therapeutic interventions.