Epithelial To Mesenchymal Transition In Breast Cancer Cells Research Articles

P66ShcA promotes breast cancer plasticity by inducing an epithelial-to-mesenchymal transition.

Breast cancers are stratified into distinct subtypes, which influence therapeutic responsiveness and patient outcome. Patients with luminal breast cancers are often associated with a better prognosis relative to that with other subtypes. However, subsets of patients with luminal disease remain at increased risk of cancer-related death. A critical process that increases the malignant potential of breast cancers is the epithelial-to-mesenchymal transition (EMT). The p66ShcA adaptor protein stimulates the formation of reactive oxygen species in response to stress stimuli. In this paper, we report a novel role for p66ShcA in inducing an EMT in HER2(+) luminal breast cancers. p66ShcA increases the migratory properties of breast cancer cells and enhances signaling downstream of the Met receptor tyrosine kinase in these tumors. Moreover, Met activation is required for a p66ShcA-induced EMT in luminal breast cancer cells. Finally, elevated p66ShcA levels are associated with the acquisition of an EMT in primary breast cancers spanning all molecular subtypes, including luminal tumors. This is of high clinical relevance, as the luminal and HER2 subtypes together comprise 80% of all newly diagnosed breast cancers. This study identifies p66ShcA as one of the first prognostic biomarkers for the identification of more aggressive tumors with mesenchymal properties, regardless of molecular subtype.

Abstract 1046: Molecular link between HER2 and epithelial-to-mesenchymal transition in breast cancer

Abstract Breast cancer is a second most lethal form of cancer. Metastasis is responsible for about 90% of cancer- related deaths. Metastasis is a multi-step process and epithelial to mesenchymal transition (EMT) is the initiating step. HER2 is overexpressed in about 25-30% of breast cancer patients and responsible for poor prognosis. Our results show a novel role of HER2 in inducing EMT in breast cancer cells. To delineate the mechanism of EMT and role of HER2, we used MDA-MB-231, MCF-7 breast cancer cells with their syngeneic variants having stable HER2 overexpression (referred to as high HER2 or HH cells) and 4T-1 cells. The Boyden's chamber assay showed that HH cells were more invasive than the parent cells. The higher invasive potential of cells was accompanied with increased expression of mesenchymal markers like N-cadherin, along with suppression of epithelial markers like Cytokeratin-18. HER2 overexpression was also associated with enhanced expression of transcription factors like SNAIL and ZEB-1 that orchestrate the process of EMT. It is known that TGFβ is a master regulator of EMT process in cancer cells. Interestingly, HH cells exhibited higher levels of TGFβ protein and mRNA levels along with increased expression of SMADs relative to the parent cell lines. SMADs are the classical downstream effector molecules for TGFβ. Hence these results indicate that HER2 overexpression modulates TGFβ signaling to promote the process of EMT. In addition, induction of SNAIL, TGFβ and SMAD4 by heregulin treatment further established the role of HER2 in EMT process. To confirm the requirement of HER2 in the maintenance of mesenchymal traits, we silenced HER2 in MDA-MB-231 and MDA-MB-231 (HH) cells. HER2 silencing suppressed the invasion and mesenchymal markers indicating a direct connection of EMT with HER2 in breast cancer cells. Taken together our results for the first time provides a molecular evidence for the regulation of EMT by HER2 through TGFβ signaling. Further mechanistic and pre-clinical in vivo studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Parul Gupta, Sanjay K. Srivastava. Molecular link between HER2 and epithelial-to-mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2014-1046

Abstract 1032: Ormeloxifene attenuates metastatic and glycolytic pathways in breast cancer cells

Abstract Background: Breast cancer is the second most deadly cancer in female, resulting in over 39,000 deaths in year 2012. Triple negative breast cancer is a subtype of breast cancer characterized by its poor outcome and a lack of targeted therapies. New targeted therapies are therefore an urgent unmet medical need for this patient population. Recent investigations into anti-cancer drug are targeted on repurposing pre-approved and safe drugs for cancer treatment. We report for the first time that a non-steroidal, selective estrogen receptor inhibitor, ormeloxifene (ORM) inhibits Metastasis-associated protein 1 (MTA1) and glycolytic pathway through AMPKα 1/2 inhibition in triple negative breast cancer cells. They are both involved in promoting cancer cell survival, proliferation and metastasis. MTA1 is an initiator of epithelial to mesenchymal transition (EMT) which is responsible for metastasis and poor prognosis. AMPKα 2 is important for glycolysis and promotes tumor survival. Methods: Herein, we investigated the use of ORM for the treatment of breast cancer. We used MB-231 and MCF-7 breast cell lines as prototypical model and investigated the effect of ORM treatment on MTA1 and AMPKα 2 expression pattern. We investigated the effect of ORM on cell proliferation (MTS), clonogenic potential (anchorage dependent growth) of the cells and analyzed its effect on markers of EMT through Western blotting. Further, we performed glycolytic function analysis using Seahorse analyzer and mitochondrial membrane potential analysis by Tetramethylrhodamine, ethyl ester (TMRE). We performed Western blotting to investigate the role of ORM on MTA1 and AMPKα inhibition. Results: Our results suggest that ORM inhibits proliferation of both MB-231 and MCF-7 breast cancer cells at low concentrations. Investigation of the EMT marker profile revealed a dose dependent decrease in the expression of MTA1 in both the cells on ORM treatment. This leads to inhibition of motility and invasion of cells in a concentration dependent manner. Additionally, treatment of MB-231 cells with ORM suppresses vimentin expression, a marker for EMT that aids in rapid metastasis. ORM inhibits transcription factors, Snail and Slug, known to promote epithelial-mesenchymal transitions during tumor development. Importantly, it was observed that ORM also inhibits glycolytic pathway in breast cancer cells independent of mitochondrial membrane depolarization. ORM inhibits total AMPKα and dephosphorylates AMPKα 2 in breast cancer cells. Conclusions: Our findings suggest that ORM effectively targets MTA1 and glycolytic pathway and inhibits breast cancer cell growth and EMT. Overall, this study suggests that ORM can be a potential therapeutic modality for triple negative breast cancer treatment. Citation Format: Aditya Ganju, Vasudha Sundram, Keith Miskimins, Rishi Gara, Sheema Khan, Man Mohan Singh, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene attenuates metastatic and glycolytic pathways in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2014-1032

Abstract 3591: 15-Deoxy-Δ12,14-prostaglandin J2-induced epithelial-to-mesenchymal transition in human breast cancer cells promotes fibroblast activation

Abstract In inflammation-associated carcinogenesis, cyclooxygenase-2 (COX-2) is markedly overexpressed, resulting in accumulation of various prostaglandins. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and prostahlandin E2 (PGE2) are the predominant COX-2-derived prostaglandins (PGs) with oncogenic potential. In the present study, we examined the effects of these to PGs on epithelial-to-mesenchymal transition (EMT), a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties. 15d-PGJ2 induced EMT to a greater than PGE2 in human breast cancer (MCF-7) cells. Snail and ZEB1 are known to bind to the E-boxes present in the E-cadherin promoter, thereby repressing E-cadherin transcription. Treatment of MCF-7 cells with 30 μM of 15d-PGJ2 increased the expression of Snail and ZEB1, followed by the down-regulation of E-cadherin. Nuclear extract from 15d-PGJ2-treated MCF-7 cells showed the binding of Snail and ZEB1 to E-box sequences in the E-cadherin promoter. In addition, among various cytokines/chemokines examined, the mRNA level of interleukin-8 (CXCL8) was highly elevated in 15d-PGJ2-stimulated MCF-7 cells. 15d-PGJ2-induced up-regulation of CXCL8 expression was abrogated by the introduction of Snail short interfering RNA. These findings suggest that CXCL8 may function as a positive regulator of EMT in 15d-PGJ2-treated MCF-7 cells. Moreover, the addition of conditioned medium of MCF-7 cells undergoing EMT activates normal fibroblast cells to cancer-associated fibroblasts (CAFs). Co-culture of normal fibroblasts with 15d-PGJ2-treaed-MCF-7 cells also induced the expression of α-SMA and FAPα, representative markers of CAFs. As a corresponding receptor of CXCL8, CXCR2 was overexpressed in activated fibroblasts co-cultured with 15d-PGJ2-treated MCF-7 cells. Unlike 15d-PGJ2, its non-electrophilic analogue 9,10-dihydro-15d-PGJ2 failed to induce EMT, suggesting that the α,β-unsaturated carbonyl group located in the cyclopentenone ring of 15d-PGJ2 is essential for EMT induction. Notably, activation of Rho kinase through direct binding of ArhGEF5, a guanine nucleotide exchange factor, with 15d-PGJ2 seems to contribute to the EMT induction. Taken together, above findings suggest that 15d-PGJ2 induces EMT through up-regulation of Snail expression and subsequent production of CXCL8 which may contribute to increased EMT of human breast cancer cells. Citation Format: Do-Hee Kim, Jeehye Choi, Jin-Young Suh, Hye-Kyung Na, Young-Joon Surh. 15-Deoxy-Δ12,14-prostaglandin J2-induced epithelial-to-mesenchymal transition in human breast cancer cells promotes fibroblast activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3591. doi:10.1158/1538-7445.AM2014-3591

VDR Status Arbitrates the Prometastatic Effects of Tumor-Associated Macrophages

The relationship between tumor-associated macrophages (TAM) and epithelial-to-mesenchymal transition (EMT) during the initiation and progression of metastasis is still unclear. Here, a role for the vitamin D receptor (VDR) in metastasis was identified, as well as a role in the relationship between TAMs and EMT. First, the expression level of VDR was examined in clinical tissue from human patients with breast cancer or a mouse model of breast cancer with differential metastasis. These results revealed that VDR expression negatively correlates with metastasis in breast cancer. Second, coculture of VDR-overexpressing breast cancer cells with a macrophage cell line demonstrated that overexpression of VDR alleviated the prometastatic effect of cocultured macrophages on breast cancer cells. Furthermore, VDR overexpression abrogated the induction of EMT in breast cancer cells by cocultured macrophage cells, as measured by a loss of E-cadherin (CDH1) and induction of α-smooth muscle actin (α-SMA). TNFα in macrophage conditioned media inhibited VDR expression, whereas downregulation of VDR further mediated the promotion of TGFβ-induced EMT by TNFα. In addition, β-catenin expression was inhibited in VDR-overexpressing breast cancer cells and tumor xenografts. Finally, administration of calcitriol [1,25-(OH)2D3], an active vitamin D metabolite, exerted similar antimetastatic effects in breast cancer cells in vitro and a mouse model of breast cancer in vivo with preservation of VDR and suppression of β-catenin. VDR suppression by TNFα mediates the prometastatic effect of TAMs through enhancement of the β-catenin pathway.

The tetraindole SK228 reverses the epithelial-to-mesenchymal transition of breast cancer cells by up-regulating members of the miR-200 family.

The results of recent studies have shown that metastasis, the most common malignancy and primary cause of mortality promoted by breast cancer in women, is associated with the epithelial-to-mesenchymal transition (EMT). The results of the current study show that SK228, a novel indole containing substance, exhibits anti-cancer activity. In addition, the effects of SK228 on the regulation of EMT in breast cancer cells as well as the underlying mechanism have been explored. SK228 was observed to induce a fibroblastoid to epithelial-like change in the appearance of various breast cancer cell lines and to suppress the migration and invasion of these cancer cells in vitro. Moreover, expression of E-cadherin was found to increase following SK228 treatment whereas ZEB1 expression was repressed. Expression of other major EMT inducers, including ZEB2, Slug and Twist1, is also repressed by SK228 as a consequence of up-regulation of members of the miR-200 family, especially miR-200c. The results of animal studies demonstrate that SK228 treatment leads to effective suppression of breast cancer growth and metastasis in vivo. The observations made in this investigation show that SK228 reverses the EMT process in breast cancer cells via an effect on the miR-200c/ZEB1/E-cadherin signalling pathway. In addition, the results of a detailed analysis of the in vivo anti-cancer activities of SK228, carried out using a breast cancer xenograft animal model, show that this substance is a potential chemotherapeutic agent for the treatment of breast cancer.

SHOX2 Is a Direct miR-375 Target and a Novel Epithelial-to-Mesenchymal Transition Inducer in Breast Cancer Cells

MicroRNAs have added a new dimension to our understanding of tumorigenesis and associated processes like epithelial-to-mesenchymal transition (EMT). Here, we show that miR-375 is elevated in epithelial-like breast cancer cells, and ectopic miR-375 expression suppresses EMT in mesenchymal-like breast cancer cells. We identified short stature homeobox 2 (SHOX2) as a miR-375 target, and miR-375–mediated suppression in EMT was reversed by forced SHOX2 expression. Ectopic SHOX2 expression can induce EMT in epithelial-like breast cancer cells, whereas SHOX2 knockdown diminishes EMT traits in mesenchymal-like breast cancer cells, demonstrating SHOX2 as an EMT inducer. We show that SHOX2 acts as a transcription factor to upregulate transforming growth factor β receptor I (TβR-I) expression, and TβR-I inhibitor LY364947 abolishes EMT elicited by ectopic SHOX2 expression, suggesting that transforming growth factor β signaling is essential for SHOX2-induced EMT. Manipulating SHOX2 abundance in breast cancer cells impact in vitro invasion and in vivo dissemination. Analysis of breast tumor microarray database revealed that high SHOX2 expression significantly correlates with poor patient survival. Our study supports a critical role of SHOX2 in breast tumorigenicity.

Lin28 Induces Epithelial-to-Mesenchymal Transition and Stemness via Downregulation of Let-7a in Breast Cancer Cells

The RNA-binding protein Lin28 is known to promote malignancy by inhibiting the biogenesis of let-7, which functions as a tumor suppressor. However, the role of the Lin28/let-7 axis in the epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer has not been clearly expatiated. In our previous study, we demonstrated that let-7 regulates self-renewal and tumorigenicity of breast cancer stem cells. In the present study, we demonstrated that Lin28 was highly expressed in mesenchymal (M) type cells (MDA-MB-231 and SK-3rd), but it was barely detectable in epithelial (E) type cells (MCF-7 and BT-474). Lin28 remarkably induced the EMT, increased a higher mammosphere formation rate and ALDH activity and subsequently promoted colony formation, as well as adhesion and migration in breast cancer cells. Furthermore, we demonstrated that Lin28 induced EMT in breast cancer cells via downregulation of let-7a. Strikingly, Lin28 overexpression was found in breast cancers that had undergone metastasis and was strongly predictive of poor prognoses in breast cancers. Given that Lin28 induced the EMT via let-7a and promoted breast cancer metastasis, Lin28 may be a therapeutic target for the eradication of breast cancer metastasis.

Expression of Cox-2 in human breast cancer cells as a critical determinant of epithelial-to-mesenchymal transition and invasiveness

Introduction: Cyclooxygenase-2 (COX-2) is overexpressed in several malignancies and is implicated in breast cancer progression.Objectives: We investigated whether changes in COX-2 expression may affect epithelial-to-mesenchymal transition (EMT) and then invasive potential of human breast cancer cells, in relationship with hypoxia. COX-2-null MCF-7 human breast cancer cells, MCF-7 cells transiently expressing COX-2 and COX-2-expressing MDA-MB-231 cells were employed.Results: COX-2 overexpression resulted in downregulation of E-cadherin and β-catenin, upregulation of vimentin, N-cadherin and SNAI1, suggesting EMT occurrence. COX-2-overexpressing MCF-7 cells were also characterized by increased invasiveness and release of matrix-metalloproteinase-9. The above-mentioned characteristics, homologous to those detected in highly invasive MDA-MB-231 cells, were reverted by treatment of COX-2-overexpressing MCF-7 cells with celecoxib, a COX-2-specific inhibitor, partly through the inhibition of COX-2-related intracellular generation of reactive oxygen species. Hypoxia further exacerbated COX-2 expression, EMT changes and invasive ability in both COX-2-overexpressing MCF-7 cells and MDA-MB-231 cells. Finally, immunohistochemistry performed on samples from normal and neoplastic human breast tissues revealed that COX-2-positive malignant cells were also positive for EMT-related antigens, hypoxia-inducible factor (HIF)-2α and the oxidative stress marker heme oxygenase.Conclusions: These findings support the existence of a direct link between COX-2 overexpression, EMT and invasiveness in human breast cancer cells, emphasizing the role of hypoxic microenvironment.

MCF-7 Cells Expressing Nuclear Associated Lysyl Oxidase-like 2 (LOXL2) Exhibit an Epithelial-to-Mesenchymal Transition (EMT) Phenotype and Are Highly Invasive in Vitro

LOXL2 is a copper- and lysine tyrosylquinone-dependent amine oxidase that has been proposed to function both extracellularly and intracellularly to activate oncogenic signaling pathways leading to EMT and invasion of breast cancer cells. In this study, we selected MCF-7 cells that stably express forms of recombinant LOXL2 differing in their subcellular localizations and catalytic competencies. This enabled us to dissect the molecular functions of intracellular and extracellular LOXL2s and examine their contributions to breast cancer metastasis/invasion. We discovered that secreted LOXL2 (~100-kDa) is N-glycosylated at Asn-455 and Asn-644, whereas intracellular LOXL2 (~75-kDa) is nonglycosylated and N-terminally processed, and is primarily associated with the nucleus. Both forms of LOXL2 can oxidize lysine in solution. However, we found that expression of intracellular LOXL2 is more strongly associated with EMT and invasiveness than secreted LOXL2 in vitro. The results indicate that nuclear associated LOXL2 contributes to the stabilization of Snail1 transcription factor at the protein level to induce EMT and promote invasion in vitro, through repression of E-cadherin, occludin, and estrogen receptor-α, and up-regulation of vimentin, fibronectin, and MT1-MMP.

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial. MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms. ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin. ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.

FOXM1 promotes the epithelial to mesenchymal transition by stimulating the transcription of Slug in human breast cancer

The Forkhead Box M1 (FOXM1) transcription factor is involved in tumorigenesis and tumor progression in multiple human carcinomas. In this study, we found that FOXM1 promoted the epithelial to mesenchymal transition (EMT) in human breast cancer. We observed a strong correlation between the expression levels of FOXM1 and the mesenchymal phenotype. Knockdown of FOXM1 inhibited the mesenchymal phenotype, whereas stable overexpression of FOXM1 induced EMT in breast cancer cells. FOXM1 was found to endogenously bind to and stimulate the promoter of Slug that is crucial for EMT progression. The knockdown of Slug abolished the EMT-inducing function of FOXM1. The stable overexpression of FOXM1 promoted metastasis of breast cancer cells in vivo. This study confirmed that FOXM1 promoted EMT in breast cancer cells by stimulating the transcription of EMT-related genes such as Slug.

Cooperative involvement of NFAT and SnoN mediates transforming growth factor-β (TGF-β) induced EMT in metastatic breast cancer (MDA-MB 231) cells

Epithelial to mesenchymal transition (EMT) is a secondary phenomenon concomitantly associated with the tumor progression. The regulatory signals and mechanistic details of EMT are not fully elucidated. Here, we shared a TGF-β mediated mechanism of EMT in breast cancer (MDA-MB 231) cells. Initial exposure of TGF-β for 48 h, enhanced the rate of cell proliferation and associated with EMT of MDA-MB 231 cells. The EMT was characterized by observing the increased N-cadherin, fibronectin, Snail expression and associated with the morphological change with a reduced E-cadherin expression. NFAT, a transcription factor, alters tumor suppressive function of TGF-β towards tumor progression. Up regulation of NFAT, coupled with a foremost translocation of one oncogenic protein SnoN from cytoplasm to nucleus was noticed during this TGF-β mediated EMT. Silencing of NFAT also showed the inhibition of TGF-β mediated EMT characterized by down regulation of N-cadherin and associated with reduced expression of SnoN. In addition, it was also observed that NFAT sequestering the Smad3 prevents the proteasome mediated degradation of SnoN and this SnoN has a role on the regulation of MMP-2, MMP-9 activity. Increased Smad3-SnoN interaction and proteasome mediated degradation of SnoN were detected after silencing of NFAT with a reduced MMP-2, MMP-9 activity. All of these observations provide a fresh mechanism in which by a twofold involvement of NFAT and SnoN plays a crucial role in TGF-β mediated EMT by recruiting the effector molecules N-cadherin and MMP-2, MMP-9.

Abstract 3671: The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells.

Abstract We have shown previously that breast cancer chemoprevention by benzyl isothiocyanate (BITC) in mouse mammary tumor virus-neu transgenic mice is associated with induction of E-cadherin protein expression. Loss of E-cadherin expression concomitant with induction of mesenchymal markers (eg, vimentin and fibronectin) is a biochemical hallmark of epithelial-to-mesenchymal transition (EMT). EMT is a developmental process but implicated in progression of cancer to aggressive state. We have also shown previously that BITC treatment inhibits EMT in cultured MDA-MB-231 human breast cancer cells. The present study extends these observations and investigates the mechanism by which BITC inhibits EMT. Initially, we designed experiments using another triple-negative human breast cancer cell line (SUM159) to determine generality of the BITC-mediated inhibition of EMT. Exposure of SUM159 cells to pharmacologically-relevant concentrations of BITC (2.5 and 5 μM) resulted in induction of E-cadherin protein expression, which was accompanied by suppression of vimentin protein level especially at the 5 μM dose. Protein levels of slug and snail, which are transcriptional repressors of E-cadherin, were also decreased markedly after treatment of SUM159 cells with BITC, but this effect was relatively more pronounced on slug protein level compared with snail. Similar to MDA-MB-231 cells, the BITC-mediated induction of E-cadherin protein expression in SUM159 cells was accompanied by its transcriptional upregulation as revealed by E-cadherin luciferase reporter assay. Immunofluorescence microscopy confirmed induction of E-cadherin protein and suppression of vimentin protein expression in BITC-treated SUM159 cells. BITC treatment resulted in downregulation of Forkhead Box Q1 transcription factor (FOXQ1) protein and FOXQ1 mRNA in MDA-MB-231, SUM159, and MDA-MB-468 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with suppression of FOXQ1 protein level. Ectopic expression of FOXQ1 in a mammary epithelial cell line conferred marked protection against BITC-mediated inhibition of EMT as well as cell migration. In conclusion, the present study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells. This study was supported by the grant CA129347-06 awarded by the National Cancer Institute. Citation Format: Anuradha Sehrawat, Su-Hyeong Kim, Andreas Vogt, Shivendra V. Singh. The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3671. doi:10.1158/1538-7445.AM2013-3671

Abstract 2687: Krüppel-like factor 8 targets matrix metalloproteinase-14 to promote breast cancer invasion and metastasis .

Abstract Krüppel-like factor 8 (KLF8) regulates critical gene transcription and invasion events associated with cancer, including epithelial to mesenchymal transition (EMT) and extracellular matrix degradation. Here, we report a novel KLF8-to- matrix metalloproteinase-14 (MMP14) signaling that promotes human breast cancer cell EMT, invasion and metastasis. Cell-surface protein biotinylation and streptavidin pull-down showed that overexpression of KLF8 induced cell-surface accumulation of MMP14 in MCF-10A cells in a FAK-dependent manner, which was correlated with increased MMP14 activity and the ectodomain shedding of E-cadherin. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced expression and activity of MMP14 on the cell surface. Promoter reporter assays and chromatin and oligonucleotide precipitations determined that KLF8 activated the human MMP14 gene promoter both directly and indirectly via beta-catenin. Importantly, Matrigel invasion assays showed that MMP14 was required for the KLF8-induced MCF-10A cell invasion in vitro. Experiments using nude mice demonstrated that MMP-14 overexpression downstream of KLF8 in the MDA-MB-231 cells makes a significant contribution to the invasion and angiogenesis in the local primary tumors and lung metastasis. Taken together, this work identified a novel role and mechanisms for MMP14 downstream of KLF8 in the progression of breast cancer metastasis. Citation Format: Heng Lu, Liu Hu, Lin Yu, Xianhui Wang, Chao Shen, Tianshu Li, Debarati Mukherjee, Satadru Lahiri, Melissa Wason, Jihe Zhao. Krüppel-like factor 8 targets matrix metalloproteinase-14 to promote breast cancer invasion and metastasis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2013-2687

Correction: Grhl2 Determines the Epithelial Phenotype of Breast Cancers and Promotes Tumor Progression

[This corrects the article on p. e50781 in vol. 7.].

Brk/PTK6 cooperates with HER2 and Src in regulating breast cancer cell survival and epithelial-to-mesenchymal transition

Breast tumor kinase (Brk)/protein tyrosine kinase-6 (PTK-6) is a nonreceptor PTK commonly expressed at high levels in breast cancer. Brk interacts closely with members of the human epidermal growth factor receptor (HER) family in breast cancer but the functional role of this interaction remains to be determined. Here, we provide novel mechanistic insights into the role of Brk in regulating cell survival and epithelial-to-mesenchymal transition (EMT) in the context of HER2-positive breast cancer cells. Overexpression of HER2 in MCF7 breast cancer cells (MCF7HER2) led to a higher level of Brk protein and concomitantly reduced Src Y416-phosphorylation, and the cells became mesenchymal in morphology. An in vivo selection of MCF7HER2 cells in nude mice resulted in a subline, termed EMT1, that exhibited not only mesenchymal morphology but also enhanced migration potential. Compared with MCF7HER2 cells, EMT1 cells maintained a similar level of HER2 protein but had much higher level of activated HER2, and the increase in Brk protein and the decrease in Src Y416-phosphorylation were less in EMT1 cells. EMT1 cells exhibited increased sensitivity to both pharmacological inhibition of HER2 and knockdown of Brk than did MCF7HER2 cells. Knockdown of Brk induced apoptosis and partially reversed the EMT phenotype in EMT1 cells. Overexpression of a constitutively active STAT3, a known substrate of Brk, overcame Brk knockdown-induced effects in EMT1 cells. Together, our findings support a new paradigm wherein Brk plays both a complementary and a counterbalancing role in cooperating with HER2 and Src to regulate breast cancer cell survival and EMT.

ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR

IntroductionEpithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.MethodsBasal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.ResultsHere we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.ConclusionsTaken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.

Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into multiple mesoderm lineages in the course of normal tissue homeostasis or during injury. We have previously shown that MSCs migrate to sites of tumorigenesis, where they become activated by cancer cells to promote metastasis. However, the molecular and phenotypic attributes of the MSC-induced metastatic state of the cancer cells remained undetermined. Here, we show that bone marrow-derived human MSCs promote de novo production of lysyl oxidase (LOX) from human breast carcinoma cells, which is sufficient to enhance the metastasis of otherwise weakly metastatic cancer cells to the lungs and bones. We also show that LOX is an essential component of the CD44-Twist signaling axis, in which extracellular hyaluronan causes nuclear translocation of CD44 in the cancer cells, thus triggering LOX transcription by associating with its promoter. Processed and enzymatically active LOX, in turn, stimulates Twist transcription, which mediates the MSC-triggered epithelial-to-mesenchymal transition (EMT) of carcinoma cells. Surprisingly, although induction of EMT in breast cancer cells has been tightly associated with the generation of cancer stem cells, we find that LOX, despite being critical for EMT, does not contribute to the ability of MSCs to promote the formation of cancer stem cells in the carcinoma cell populations. Collectively, our studies highlight a critical role for LOX in cancer metastasis and indicate that the signaling pathways controlling stroma-induced EMT are distinct from pathways regulating the development of cancer stem cells.

Abstract 959: ERα1 represses breast cancer EMT by inhibiting EGFR-mediated downregulation of E-cadherin

Abstract Although epithelial to mesenchymal transition (EMT) is associated with specific breast cancer phenotypes, such as the basal-like and claudin-low tumors, there is a lack of validated markers, which can distinguish tumors with poor clinical outcome and high metastatic potential. Estrogen receptor beta (ERα) has been shown to regulate invasion in prostate cancer. However, it is still unclear if ERα by influencing invasion can predict metastasis in breast cancer. Here we show that wild-type ERα (ERα1) inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. This correlates with an ERα1-mediated upregulation of miR-200a, miR-200b and miR-429 and the subsequent repression ZEB1 and SIP1, which results in increased expression of E-cadherin. Downregulation of the basal marker EGFR was found to be involved in the ERα1-mediated mesenchymal to epithelial transition since induction of EGFR expression or treatment with EGF abolished the ability of ERα1 to sustain the epithelial phenotype. This inverse correlation of ERα1 with EGFR and its positive association with epithelial markers was additionally observed in breast cancer specimens. These results strengthen the association of ERα1 with the regulation of EMT and propose the receptor as potential crucial marker in predicting survival and metastasis in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 959. doi:1538-7445.AM2012-959