Induction Of Snail Research Articles

Collective cell migration and metastases induced by an epithelial-to-mesenchymal transition in Drosophila intestinal tumors

Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to the lack of models in vivo. Here we show that expression of the EMT master inducer Snail in primary adult Drosophila intestinal tumors leads to the dissemination of tumor cells and formation of macrometastases. Snail drives an EMT in tumor cells, which, although retaining some epithelial markers, subsequently break through the basal lamina of the midgut, undergo a collective migration and seed polyclonal metastases. While metastases re-epithelialize over time, we found that early metastases are remarkably mesenchymal, discarding the requirement for a mesenchymal-to-epithelial transition for early stages of metastatic growth. Our results demonstrate the formation of metastases in adult flies, and identify a key role for partial-EMTs in driving it. This model opens the door to investigate the basic mechanisms underlying metastasis, in a powerful in vivo system suited for rapid genetic and drug screens.

Resistin induces breast cancer cells epithelial to mesenchymal transition (EMT) and stemness through both adenylyl cyclase-associated protein 1 (CAP1)-dependent and CAP1-independent mechanisms

Breast cancer incidence and metastasis in postmenopausal women are known to associate with obesity, but the molecular mechanisms behind this association are largely unknown. We investigated the effect of adipokine resistin on epithelial to mesenchymal transition (EMT) and stemness in breast cancer cells in vitro. Previous reports demonstrated that the inflammatory actions of resistin are mediated by the adenylyl cyclase-associated protein 1 (CAP1), which serves as its receptor. As a model for our study, we used MCF-7 and MDA-MB-231 breast cancer and MCF-10A breast epithelial cells. We showed that in MCF-7 cells resistin increases the migration of MCF-7 and MDA-MB-231 cells and induces the formation of cellular protrusions through reorganization of F-actin filaments. Resistin upregulated the expression of mesenchymal markers involved in EMT (SNAIL, SLUG, ZEB1, TWIST1, fibronectin, and vimentin), and downregulated those of epithelial markers (E-cadherin and claudin-1). Resistin also potentiated the nuclear translocation of SNAIL protein, indicating initiation of EMT reprogramming. We further induced EMT in non-carcinogenic breast epithelial MCF-10A cells demonstrating that the effects of resistin on EMT were not breast cancer cell specific. In order to assess whether resistin-induced EMT depends on CAP1, we used siRNA approach to silence CAP1 gene in MCF-7 cells. Results demonstrated that when CAP1 was silenced, the induction of SNAIL, ZEB1 and vimentin expression by resistin as well as SNAIL and ZEB1 nuclear translocation, were abolished. Additionally, CAP1 silencing resulted in a suppression of MCF-7 cells migration. We performed quantitative PCR array profiling the expression of 84 genes related to cancer stem cells (CSC), pluripotency and metastasis and selected a set of genes (ALDH1A1, ITGA4, LIN28B, SMO, KLF17, PTPRC, PROM1, SIRT1, and PECAM1) that were modulated by resistin. Further experiments demonstrated that the effect of resistin on the expression of some of these genes (PROM1, PTPRC, KLF17, SIRT1, and PECAM1) was also dependent on CAP1. Our results demonstrate that resistin promotes the metastatic potential of breast cancer cells by inducing EMT and stemness and some of these effects are mediated by CAP1.

Abstract P5-08-06: Differential effects of eribulin on key transcription factors snail and slug

Abstract Microtubule targeting agents (MTAs) are a mainstay in the treatment of breast cancer and a growing body of evidence demonstrates that they have non-mitotic effects that contribute to their anticancer actions. Even after decades of clinical use, there is much to still be learned about the mechanisms of action these drugs, and differences among them, for optimal utility. MTAs rapidly alter microtubule dynamics, often within minutes, leading to significant changes in oncogenic cellular signaling. We evaluated the early effects of eribulin on key oncogenic signaling pathways following a 2 h incubation using clinically relevant concentrations and compared the effects to those initiated by other MTAs. These studies led to the identification of novel mechanisms by which MTAs disrupt oncogenic signaling and contribute to the reversal of epithelial to mesenchymal transition (EMT), including unanticipated differences among drugs. The TGF-β-mediated expression of the Snail transcription factor is a key driver pathway of EMT in breast cancer. The effects of eribulin and other MTAs on TGF-β-induced, Smad-dependent expression of Snail were evaluated. Our results show that eribulin and vinorelbine, but not paclitaxel or ixabepilone, inhibit the ability of TGF-β to promote the transcriptional induction of Snail by impeding the nuclear transport of Smad2/3 proteins in 4 triple-negative breast cancer cell lines. This study begins to explain how microtubule disruption might contribute to the eribulin-mediated EMT reversal observed in vitro, in vivo, and in patients. Slug is another member of the Snail family of transcription factors that plays a central role in breast cancer EMT. Although Snail and Slug are often grouped together due to functional similarities, it is becoming increasingly clear that Slug has an independent role in regulating stemness and cancer cell survival during partial EMT. In contrast to our findings with Snail, eribulin and vinorelbine, but not paclitaxel or ixabepilone, induced Slug expression independent of TGF-β stimulation in a subset of triple-negative breast cancer cell lines. Studies are ongoing to identify the molecular pathways and consequences of eribulin and vinorelbine-induced Slug induction, which might begin to identify biomarkers of patient response to different MTAs. This work highlights the multifaceted nature of MTA-mediated effects on EMT-associated signaling pathways in breast cancer cells and prompts a reevaluation of their differential efficacy in tumors with distinct molecular profiles. These studies are supported by Eisai Inc. Citation Format: Kaul R, Risinger AL, Mooberry SL. Differential effects of eribulin on key transcription factors snail and slug [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-06.

MiR-363 inhibits cisplatin chemoresistance of epithelial ovarian cancer by regulating snail-induced epithelial-mesenchymal transition

Chemoresistance is a major barrier to successful cisplatin-based chemotherapy for epithelial ovarian cancer (EOC), and emerging evidences suggest that microRNAs (miRNAs) are involved in the resistance. In this study, it was indicated that miR-363 downregulation was significantly correlated with EOC carcinogenesis and cisplatin resistance. Moreover, miR-363 overexpression could resensitise cisplatin-resistant EOC cells to cisplatin treatment both in vitro and in vivo. In addition, data revealed that EMT inducer Snail was significantly upregulated in cisplatin-resistant EOC cell lines and EOC patients and was a functional target of miR-363 in EOC cells. Furthermore, snail overexpression could significantly attenuate miR-363-suppressed cisplatin resistance of EOC cells, suggesting that miR-363-regulated cisplatin resistance is mediated by snail-induced EMT in EOC cells. Taken together, findings suggest that miR-363 may be a biomarker for predicting responsiveness to cisplatin-based chemotherapy and a potential therapeutic target in EOC.

Acidic bile salts induces mucosal barrier dysfunction through let-7a reduction during gastric carcinogenesis after Helicobacter pylori eradication.

Gastric cancer (GC) after eradication for Helicobacter pylori (H.pylori) increases, but its carcinogenesis is not elucidated. It is mainly found in acid non-secretion areas (ANA), as mucosal regeneration in acid secretory areas (AA) after eradication changes the acidity and bile toxicity of gastric juice. We aimed to clarify the role of barrier dysfunction of ANA by the stimulation of pH3 bile acid cocktail (ABC) during carcinogenesis. We collected 18 patients after curative endoscopic resection for GC, identified later than 24 months after eradication, and took biopsies by Congo-red chromoendoscopy to distinguish AA and ANA (UMIN00018967). The mucosal barrier function was investigated using a mini-Ussing chamber system and molecular biological methods. The reduction in mucosal impedance in ANA after stimulation was significantly larger than that in AA, 79.6% vs. 87.9%, respectively. The decrease of zonula occludens-1 (ZO-1) and let-7a and the increase of snail in ANA were significant compared to those in AA. In an in vitro study, the restoration of ZO-1 and let-7a as well as the induction of snail were observed after stimulation. High mobility group A2 (HMGA2)-snail activation, MTT proliferation, and cellular infiltration capacity were significantly increased in AGS transfected with let-7a inhibitor, and vice versa. Accordingly, using a mini-Ussing chamber system for human biopsy specimens followed by an in vitro study, we demonstrated for the first time that the exposure of acidic bile salts to ANA might cause serious barrier dysfunction through the let-7a reduction, promoting epithelial-mesenchymal transition during inflammation-associated carcinogenesis even after eradication.

Upregulation of lactate-inducible snail protein suppresses oncogene-mediated senescence through p16INK4a inactivation

BackgroundThe preferential use of aerobic glycolysis by tumor cells lead to high accumulation of lactate in tumor microenvironment. Clinical evidence has linked elevated lactate concentration with cancer outcomes. However, the role and molecular mechanisms of lactate in cellular senescence and tumor progression remain elusive.MethodsThe function of Snail in lactate-induced EMT in lung cancer cells was explored by wound healing assay and cell invasion assay. The qRT-PCR and dual luciferase reporter assay were performed to investigate how lactate regulates Snail expression. The level of TGF-β1 in culture supernatant of cells was measured by ELISA for its correlation with extracellular levels of lactate. Ras activity assay and SA-β-gal activity assay were established to determine the effect of lactate on oncogene-induced senescence in human lung epithelial cells. ChIP assays were conducted to determine the binding of snail to p16INK4a promoter. Two TCGA data sets (TCGA-LUAD and TCGA-LUSC) were used to explore the correlations between SNAI1 and CDKN2A expression.ResultsIn this study, we showed the invasive and migratory potential of lung cancer cells was significantly enhanced by lactate and was directly linked to snail activity. We also demonstrated that extracellular acidification itself is a direct cause of the increased snail expression and physiologically coupled to LDHA-dependent conversion of pyruvate to lactate. Mechanistically, lactate exerts its central function in induction of snail and EMT by directly remodeling ECM and releasing activated TGF-β1. We also demonstrated that Snail help premalignant cells to escape the oncogene-induced senescence by directly targeting and inhibiting p16INK4a expression.ConclusionsOur study extends the understanding of EMT in tumorigenesis by uncovering the role of snail in cellular senescence. This study also reveals lactate may be a potent tumor-promoting factor and provides the basis for the development of lactate-targeted therapy.

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a ‘multi hit’ effect may be achieved through the aberrant activation of just a single gene.

Hypoxia-mediated translational activation of ITGB3 in breast cancer cells enhances TGF-β signaling and malignant features in vitro and in vivo.

Breast cancer is the most prevalent malignancy in women and there is an urgent need for new therapeutic drugs targeting aggressive and metastatic subtypes, such as hormone-refractory triple-negative breast cancer (TNBC). Control of protein synthesis is vital to cell growth and tumour progression and permits increased resistance to therapy and cellular stress. Hypoxic cancer cells attain invasive and metastatic properties and chemotherapy resistance, but the regulation and role of protein synthesis in this setting is poorly understood. We performed a polysomal RNA-Seq screen in non-malignant breast epithelial (MCF10A) and TNBC (MDA-MB-231) cells exposed to normoxic or hypoxic conditions and/or treated with an mTOR pathway inhibitor. Analysis of both the transcriptome and the translatome identified mRNA transcripts translationally activated or repressed by hypoxia in an mTOR-dependent or -independent manner. Integrin beta 3 (ITGB3) was translationally activated in hypoxia and its knockdown increased apoptosis and reduced survival and migration, particularly under hypoxic conditions. Moreover, ITGB3 was required for sustained TGF-β pathway activation and for the induction of Snail and associated epithelial-mesenchymal transition markers. ITGB3 downregulation significantly reduced lung metastasis and improved overall survival in mice. Collectively, these data suggest that ITGB3 is translationally activated in hypoxia and regulates malignant features, including epithelial-mesenchymal transition and cell migration, through the TGF-β pathway, revealing a novel angle for the treatment of therapy-resistant hypoxic tumours.

Abstract 5458: Oncostatin M induces epithelial-mesenchymal transition and stem cell properties in senescent human mammary epithelial cells

Abstract The Interleukin-6 (IL-6) cytokine family member Oncostatin M (OSM) induces epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties in transformed cancer cells. Paradoxically, OSM induces a growth-inhibiting senescence response in normal human mammary epithelial cells (HMEC). Senescence is induced in normal cells by various stresses, such as aberrant oncogene signaling, and serves as a major tumor-suppressive barrier that must be bypassed for cellular transformation in vitro and tumorigenesis in vivo. Previous reports show that aberrant reactivation of EMT-inducing transcription factors (EMT-TFs) promotes senescence bypass. Thus, aberrant EMT may drive tumor initiation in addition to providing cancer cells with mesenchymal cell and stem cell properties that drive tumor progression, metastasis, and tumor recurrence. Conversely, our studies here indicate that much like aberrant oncogene signaling, aberrant EMT and CSC-properties induced by OSM may actually engage senescence in normal cells. Infecting Primary HMEC isolated from reduction mammoplasty tissue with lentivirus encoding shRNA to p16 created indefinite lifespan “Normal” HMEC. Subsequent infections with viruses encoding shRNA to p53, then constitutive expression of MYC and RAS created “Transformed” HMEC. After treating HMEC with recombinant OSM, proliferation and senescence were assessed by growth assays and qRT-PCR analysis of senescence-associated β-galactosidase (GLB1) expression. EMT and CSC-properties were assessed by western and qRT-PCR analysis of an epithelial marker E-cadherin (CDH1), a mesenchymal marker Vimentin (VIM), the EMT-TF Snail (SNAI1), and the CSC markers CD44 and HAS2. Flow cytometry examining BrdU-incorporation and CD24/CD44 surface expression also assessed proliferation and a CSC phenotype. OSM reduced growth by 80% and induced GLB1 expression in Normal HMEC, but not in Transformed HMEC. OSM-induced senescence was accompanied by Snail induction, changes in EMT-associated markers (reduced E-cadherin/increased Vimentin), and CSC marker (CD44 and HAS2) induction. Notably, OSM induced immediate SNAI1 and HAS2 expression; and a majority of the OSM-induced CD44+ population consisted of non-proliferating (BrdU-) cells. Unexpectedly, Snail and CD44 were also induced during oncogenic RAS-induced senescence; and Snail expression alone reduced growth and induced CD44 in normal HMEC. These results suggest that much like aberrant oncogene signaling, aberrantly induced EMT and CSC-properties may engage senescence in normal cells rather than promoting senescence bypass. Defining the mechanism linking senescence, EMT, and CSC-properties will identify targets for novel “pro-senescence” therapies that may inhibit metastasis and tumor recurrence, and expand our repertoire of therapies that enhance cancer patient outcomes. Citation Format: Benjamin L. Bryson, Damian J. Junk, Mark W. Jackson. Oncostatin M induces epithelial-mesenchymal transition and stem cell properties in senescent human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5458. doi:10.1158/1538-7445.AM2017-5458

Transforming growth factor-β1 suppresses bone morphogenetic protein-2-induced mesenchymal-epithelial transition in HSC-4 human oral squamous cell carcinoma cells via Smad1/5/9 pathway suppression

Squamous cell carcinoma is the most common cancer in the oral cavity. We previously demonstrated that transforming growth factor-β1 (TGF-β1) promotes the epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (hOSCC) cells; however, it remains to be clarified whether the TGF-β superfamily member bone morphogenetic protein (BMP) affects this process in hOSCC cells. Here, we examined the independent and collective effects of TGF-β1 and BMP-2 on EMT and mesenchymal-epithelial transition (MET) in a panel of four hOSCC cell lines. Notably, we found that HSC-4 cells were the most responsive to BMP-2 stimulation, which resulted in the upregulation of Smad1/5/9 target genes such as the MET inducers ID1 and cytokeratin 9 (CK9). Furthermore, BMP-2 downregulated the mesenchymal marker N-cadherin and the EMT inducer Snail, but upregulated epithelial CK9 expression, indicating that BMP-2 prefers to induce MET rather than EMT. Moreover, TGF-β1 dampened BMP-2-induced epithelial gene expression by inhibiting Smad1/5/9 expression and phosphorylation. Functional analysis revealed that TGF-β1 and BMP-2 significantly enhanced HSC-4 cell migration and proliferation, respectively. Collectively, these data suggest that TGF-β positively regulates hOSCC invasion in the primary tumor, whereas BMP-2 facilitates cancer cell colonization at secondary metastatic sites. Thus, the invasive and metastatic characteristics of hOSCC appear to be reciprocally regulated by BMP and TGF-β.

Serum response factor induces endothelial-mesenchymal transition in glomerular endothelial cells to aggravate proteinuria in diabetic nephropathy.

We investigated the expression and function of serum response factor (SRF) in endothelial-mesenchymal transition (EndMT) in glomerular endothelial cells (GEnCs) of diabetic nephropathy (DN). The expression of SRF, endothelial markers (VE-cadherin, CD31), and mesenchymal markers (α-SMA, FSP-1, fibronectin) was examined in GEnCs following high glucose or in renal cortex tissues of DN rats. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 (a small molecule inhibitor of SRF) to investigate how SRF influenced EndMT in GEnCs of DN. Streptozocin (STZ) was used to generate diabetes mellitus DM in rats. In GEnCs after high glucose treatment and in renal cortex tissues of diabetic rats, SRF, α-SMA, FSP-1, and fibronectin increased, while VE-cadherin and CD31 declined. SRF overexpression in GEnCs induced expression of Snail, an important transcription factor mediating EndMT. Blockade of SRF reduced Snail induction, protected GEnCs from EndMT, and ameliorated proteinuria. Together, increased SRF activity provokes EndMT and barrier dysfunction of GEnCs in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

Abstract 3024: Targeting TMEPAI/PMEPA1 inhibits triple negative breast cancer cell growth and metastasis by enhancing growth suppressive TGF-β signaling

Abstract Triple negative breast cancers (TNBC) that lack estrogen receptor (ER), progesterone receptor (PR) and hormone epidermal growth factor receptor 2 (HER-2/neu) are aggressive and cause high mortality among breast cancer patients. Transforming growth factor beta (TGF-β) dependency for their aggressive behavior (growth and metastasis) has been well established in many of these tumors. Although targeting TGF-β signaling has major potential to treat TNBCs, however it carries the risk of disturbing the tumor suppressive effects of TGF-β in early tumors and its homeostatic control of normal tissues. Hence there is a need to identify novel targets to impede tumor progression without compromising the beneficial effects of TGF-β signaling. To satisfy this unmet need, earlier we identified that transmembrane prostate androgen induced (TMEPAI/PMEPA1), a direct target gene of TGF-β could act as a “molecular switch” that converts TGF-β from a tumor suppressor to promoter in TNBC. Thus, we undertook the present study that identified a novel compound that blocked TMEPAI expression. We identified terpenoid derivatives (TD) which inhibited the expression of TMEPAI, enhanced TGF-β signaling and blocked proliferation, migration and invasion of several triple negative breast cancer cells in vitro. Our results showed that TD increased phosphorylation of Smad2/3 and increased PTEN, p21 and p27 proteins that cause growth suppression. Concomitantly, TD decreased Akt phosphorylation and reduced Snail and Slug that are required for cell growth and metastasis. Interestingly, TD did not affect the growth of normal human mammary epithelial cells and failed to cause associated molecular changes that can result in growth suppression. Notably, using mice models (nude mice and syngeneic BALB/c mice), we showed that TD reduced tumor burden significantly with little or no toxicity. Consistent with its ability to inhibit Akt phosphorylation and induction of Snail and Slug proteins, TD suppressed lung metastasis of TNBC in mice model. Importantly, reduced tumors derived from TD treated mice exhibited increased expression of pSmad2/3, PTEN, p21, p27 and reduced expression of pAkt compared to tumors obtained from vehicle treated mice. These results suggest that drugs that target TMEPAI will restore homeostatic functions of TGF-β to inhibit growth and metastasis of TNBC and thus prevent new tumor development. Citation Format: Prajjal Kanti Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam, Pothana Saikumar. Targeting TMEPAI/PMEPA1 inhibits triple negative breast cancer cell growth and metastasis by enhancing growth suppressive TGF-β signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3024.

Abstract 1593: Stat3 signaling in erbB3-mediated epithelial-mesenchymal transition in erbB2-positive breast cancer cells

Abstract Introduction: Understanding the mechanism of metastatic program is essential to reducing the mortality of cancer patients. It is well known that epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Recently, numerous studies have documented the role of erbB3-mediated oncogenic signaling in breast cancer progression. In this study, we have focused on investigating the effect of Stat3 activation on EMT-induced by erbB3 receptor in erbB2-positive (erbB2+) breast cancer cells. Methods: Immunofluorescence analyses were performed to study the localization and expression of EMT markers in erbB2+ breast cancer cells. Lentiviral vector containing shRNA was used to specifically knockdown erbB3. Western blot analyses were performed to assess the expression and activation of proteins. Cell growth (MTS) assays were used to determine cell viability. Wound healing and transwell assays were used to examine the motility and migration of cells. Results: We discovered that ectopic expression of erbB3 facilitated EMT, as evidenced by change of cell morphology, induction of mesenchymal markers Vimentin, Snail, Slug, ZEB1 and repression of the epithelial marker E-cadherin in erbB2+ breast cancer cells. Wound healing and transwell assays indicated that the capability of cell motility and migration was enhanced. In contrast, specific knockdown of erbB3 abrogated the observations described above. Moreover, elevated expression of erbB3 potently stimulated activation of Stat3, which has been validated to act as a promoter of tumor invasion and metastasis in many type cancers. Suppression of Stat3 signaling via its inhibitor, S3I-201, significantly alleviated EMT and cell motility/migration that were triggered by activation of erbB3. To further assess the clinical relevance of erbB3 signaling in cancer metastasis, we took advantage of a publicly accessible portal that allows analysis of the effect of genes on survival using 4,142 breast cancer samples (http://kmplot.com/analysis/). Kaplan-Meier survival curves showed that the erbB2+ breast cancer patients with erbB3-high expression tended to have a shorter Distance Metastasis-free Survival (DMFS). Conclusion: Activation of Stat3 signaling by erbB3 promotes EMT and shifts the cellular phenotype toward a mesenchymal state that is advantageous for cancer metastasis. Our data shed a new sight on erbB3's role in erbB2+ breast cancer metastasis and may facilitate the development of novel strategy for treatment of tumor progression. Keywords: ErbB3, Stat3, EMT, Metastasis, Breast Cancer Citation Format: Hui Lyu, Ying Wu, Yan Zhou, Bolin Liu. Stat3 signaling in erbB3-mediated epithelial-mesenchymal transition in erbB2-positive breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1593.

Abstract 2889: Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail

Abstract The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular mechanisms underlying the translational activation for cancer metastatic progression remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer snail followed by repression of E-cadherin expression and promotion of cell migration and invasion as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates snail expression and inhibits cell migratory/invasive capacities and metastasis. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the eIF4F translation initiation also profoundly attenuates snail translation/expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings reveal a novel role of 4E-BP1 in the regulation of EMT and metastasis through translational control of snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking snail-mediated metastatic potential of cancer. Citation Format: Qing Ye, Weijia Cai, Qing-Bai She. Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2889.

Serum response factor provokes epithelial-mesenchymal transition in renal tubular epithelial cells of diabetic nephropathy.

We investigated the role of serum response factor (SRF) in epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs) in diabetic nephropathy (DN). The expression of SRF, epithelial markers (E-cadherin and ZO-1), and mesenchymal markers (fibronectin, collagen-1, α-SMA, FSP-1) was examined in human proximal renal tubular epithelial cells (HK-2 cells) or renal medulla tissues following high glucose. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 (a small molecule inhibitor of SRF) to investigate how SRF influenced EMT in TECs of DN. Streptozotocin was used to generate DM in rats. In HK-2 cells after high-glucose treatment and renal medulla tissues of diabetic rats, SRF, fibronectin, collagen-1, α-SMA, and FSP-1 increased, while E-cadherin and ZO-1 declined. SRF overexpression in HK-2 cells induced expression of Snail, an important transcription factor mediating EMT. Blockade of SRF by CCG-1423 reduced Snail induction and protected TECs from EMT both in vitro and in vivo. Together, increased SRF activity promotes EMT in TECs and dysfunction in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension.

Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH.

Suppression of Spry1 inhibits triple-negative breast cancer malignancy by decreasing EGF/EGFR mediated mesenchymal phenotype.

Sprouty (Spry) proteins have been implicated in cancer progression, but their role in triple-negative breast cancer (TNBC), a subtype of lethal and aggressive breast cancer, is unknown. Here, we reported that Spry1 is significantly expressed in TNBC specimen and MDA-MB-231 cells. To understand Spry1 regulation of signaling events controlling breast cancer phenotype, we used lentiviral delivery of human Spry1 shRNAs to suppress Spry1 expression in MDA-MB-231, an established TNBC cell line. Spry1 knockdown MDA-MB-231 cells displayed an epithelial phenotype with increased membrane E-cadherin expression. Knockdown of Spry1 impaired MDA-MB-231 cell migration, Matrigel invasion, and anchorage-dependent and -independent growth. Tumor xenografts originating from Spry1 knockdown MDA-MB-231 cells grew slower, had increased E-cadherin expression, and yielded fewer lung metastases compared to control. Furthermore, suppressing Spry1 in MDA-MB-231 cells impaired the induction of Snail and Slug expression by EGF, and this effect was associated with increased EGFR degradation and decreased EGFR/Grb2/Shp2/Gab1 signaling complex formation. The same phenotype was also observed in the TNBC cell line MDA-MB-157. Together, our results show that unlike in some tumors, where Spry may mediate tumor suppression, Spry1 plays a selective role in at least a subset of TNBC to promote the malignant phenotype via enhancing EGF-mediated mesenchymal phenotype.

Abstract P3-14-09: Targeting TMEPAI/ PMEPA1 inhibited triple negative breast cancer cell growth and metastasis through growth suppressive TGF-β signaling

Abstract Background: Triple negative breast cancers (TNBC) that lack estrogen receptor (ER), progesterone receptor (PR) and hormone epidermal growth factor receptor 2 (HER-2/neu) are aggressive and cause high mortality among breast cancer patients. Transforming growth factor beta (TGF-β) dependency for their aggressive behavior (growth and metastasis) has been well established in many of these tumors. Although targeting TGF-β signaling has major potential to treat TNBCs, however it carries the risk of disturbing the tumor suppressive effects of TGF-β in early tumors and its homeostatic control of normal tissues. Hence there is a need to identify novel targets to impede tumor progression without compromising the beneficial effects of TGF-β signaling. To satisfy this unmet need, earlier we identified that transmembrane prostate androgen induced (TMEPAI/PMEPA1), a direct target gene of TGF-β could act as a "molecular switch" that converts TGF-β from a tumor suppressor to promoter in TNBC. Thus, we undertook the present study that identified a novel compound that blocked TMEPAI expression. Materials and Methods: All cell lines were cultured according to the ATCC recommendations. Cell proliferation was measured by quantitation of total cellular DNA. Immunoblotting, migration, invasion, tumor xenografts and lung metastasis were performed using standard methods. Results: We identified a terpenoid derivative (TD) which inhibited the expression of TMEPAI, enhanced TGF-β signaling and blocked proliferation, migration and invasion of several triple negative breast cancer cells in vitro. Our results showed that TD increased phosphorylation of Smad2/3 and increased PTEN, p21 and p27 proteins that cause growth suppression. Concomitantly, TD decreased Akt phosphorylation and reduced Snail and Slug that are required for cell growth and metastasis. Interestingly, TD did not affect the growth of normal human mammary epithelial cells and failed to cause associated molecular changes that can result in growth suppression. Notably, using mice models (nude mice and syngeneic BALB/c mice), we showed that TD reduced tumor burden significantly with little or no toxicity. Consistent with its ability to inhibit Akt phosphorylation and induction of Snail and Slug proteins, TD suppressed lung metastasis of TNBC in mice model. Importantly, reduced tumors derived from TD treated mice exhibited increased expression of pSmad2/3, PTEN, p21, p27 and reduced expression of pAkt compared to tumors obtained from vehicle treated mice. Conclusions: Our results have two important dimensions: On one hand, TD inhibited TMEPAI induction to promote growth suppressive TGF-β dependent Smad signaling in TNBC but not in normal cells. On the other hand, TD also inhibited non-Smad signaling that promotes growth and metastasis of TNBC both in vitro and in vivo. Hence our findings suggest that drugs that target TMEPAI will not only inhibit growth and metastasis of TNBC but will also restore homeostatic functions of TGF-β to prevent new tumor development. Citation Format: Singha PK, Pandeswara S, Venkatachalam MA, Saikumar P. Targeting TMEPAI/ PMEPA1 inhibited triple negative breast cancer cell growth and metastasis through growth suppressive TGF-β signaling. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-09.

Role of the Slug Transcription Factor in Chemically-Induced Skin Cancer

The Slug transcription factor plays an important role in ultraviolet radiation (UVR)-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT) occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2) pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results.

Helicobacter pylori induces Snail expression through ROS-mediated activation of Erk and inactivation of GSK-3β in human gastric cancer cells.

Helicobacter pylori (H. pylori) infection has been known to be implicated in human gastric carcinogenesis. Snail, the zinc-finger transcription factor known as a key inducer of changes in the cell shape and morphogenetic movement, is aberrantly overexpressed and correlates with lymph node metastasis in gastric cancer. In the present study, we investigated whether H. pylori could induce Snail activation to provoke these changes. Using a cell scatter assay, we noticed that human gastric cancer AGS cells infected with H. pylori underwent morphological changes as well as disruption of cell-cell interaction, which was then reversed by silencing of Snail by use of small interfering RNA (siRNA). In addition, infection with H. pylori resulted in an increased intracellular level of Snail in gastric cancer cells, which was abrogated in the presence of U0126 and LY294002, inhibitors of MEK/Erk and PI3K/Akt pathways, respectively. Cycloheximide pulse-chase experiments coupled with immunocytochemical analysis revealed that the induction of Snail by H. pylori was regulated at multiple levels, including increased transcription of Snail mRNA, inhibition of protein degradation, and enhancement of nuclear translocation of Snail. Pre-treatment of AGS cells with N-acetylcysteine, a well-known reactive oxygen species (ROS) scavenger, attenuated the H. pylori-induced activation of Erk, its binding to Snail promoter, inactivation of GSK-3β, and accumulation of Snail. Collectively, these findings suggest that the upregulation of Snail expression induced by H. pylori and transformation to a spindle-like shape as a consequence in gastric cancer cells are attributable to ROS-mediated activation of Erk and the inhibition of GSK-3β signaling. © 2016 Wiley Periodicals, Inc.