Molly A. Taylor

The Pathophysiology of Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor-β in Normal and Malignant Mammary Epithelial Cells

Epithelial-mesenchymal transition (EMT) is an essential process that drives polarized, immotile mammary epithelial cells (MECs) to acquire apolar, highly migratory fibroblastoid-like features. EMT is an indispensable process that is associated with normal tissue development and organogenesis, as well as with tissue remodeling and wound healing. In stark contrast, inappropriate reactivation of EMT readily contributes to the development of a variety of human pathologies, particularly those associated with tissue fibrosis and cancer cell invasion and metastasis, including that by breast cancer cells. Although metastasis is unequivocally the most lethal aspect of breast cancer and the most prominent feature associated with disease recurrence, the molecular mechanisms whereby EMT mediates the initiation and resolution of breast cancer metastasis remains poorly understood. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is intimately involved in regulating numerous physiological processes, including cellular differentiation, homeostasis, and EMT. In addition, TGF-β also functions as a powerful tumor suppressor in MECs, whose neoplastic development ultimately converts TGF-β into an oncogenic cytokine in aggressive late-stage mammary tumors. Recent findings have implicated the process of EMT in mediating the functional conversion of TGF-β during breast cancer progression, suggesting that the chemotherapeutic targeting of EMT induced by TGF-β may offer new inroads in ameliorating metastatic disease in breast cancer patients. Here we review the molecular, cellular, and microenvironmental factors that contribute to the pathophysiological activities of TGF-β during its regulation of EMT in normal and malignant MECs.

Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

Transforming growth factor β and its regulation of epithelial mesenchymal transition contribute to the initiation of pulmonary metastatic outgrowth specifically through the down-regulation of epithelial cadherin.

Noncanonical TGF-β Signaling During Mammary Tumorigenesis

Breast cancer is a heterogeneous disease comprised of at least five major tumor subtypes that coalesce as the second leading cause of cancer death in women in the United States. Although metastasis clearly represents the most lethal characteristic of breast cancer, our understanding of the molecular mechanisms that govern this event remains inadequate. Clinically, ~30% of breast cancer patients diagnosed with early-stage disease undergo metastatic progression, an event that (a) severely limits treatment options, (b) typically results in chemoresistance and low response rates, and (c) greatly contributes to aggressive relapses and dismal survival rates. Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that regulates all phases of postnatal mammary gland development, including branching morphogenesis, lactation, and involution. TGF-β also plays a prominent role in suppressing mammary tumorigenesis by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Genetic and epigenetic events that transpire during mammary tumorigenesis conspire to circumvent the tumor suppressing activities of TGF-β, thereby permitting late-stage breast cancer cells to acquire invasive and metastatic phenotypes in response to TGF-β. Metastatic progression stimulated by TGF-β also relies on its ability to induce epithelial-mesenchymal transition (EMT) and the expansion of chemoresistant breast cancer stem cells. Precisely how this metamorphosis in TGF-β function comes about remains incompletely understood; however, recent findings indicate that the initiation of oncogenic TGF-β activity is contingent upon imbalances between its canonical and noncanonical signaling systems. Here we review the molecular and cellular contributions of noncanonical TGF-β effectors to mammary tumorigenesis and metastatic progression.

Fibroblast growth factor receptor splice variants are stable markers of oncogenic transforming growth factor β1 signaling in metastatic breast cancers

IntroductionEpithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle.MethodsAn in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor β1 (TGF-β1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-β1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398.ResultsMetastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-β1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-β stably upregulate a truncated FGFR1-β splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-β was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth.ConclusionThe results of our study strongly suggest that FGFR1-β is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.

Abstract B46: The role of integrin switching and Deptor in metastatic breast cancer

Mammary tumorigenesis converts TGF-β; from a tumor suppressor in normal mammary epithelial cells (MECs) to a tumor promoter in cancerous MECs, likely via Epithelial-Mesenchymal Transiton (EMT). Integrin signaling plays a critical role in this conversion, although the underlying molecular mechanisms remain elusive. Previously, both β1 and β3 integrins have been associated with TGF-β; mediated EMT and metastasis in breast cancer, making both integrins attractive therapeutic targets. Importantly, the interplay and requirement for β1 and β3 integrins in mediating metastatic disease and the downstream effectors that effectuate the malignancy associated with these intergrins have not been fully evaluated. Here, we describe an integrin switching mechanism where functional disruption of β1 integrin mediates compensatory upregulation of β3 integrin expression, which augments TGF-β; signaling and mediates 3D outgrowth of metastatic breast cancer cells. We demonstrate that sole targeting of β1 integrin is insufficient in reducing tumor burden, while sole targeting of β3 integrin significantly alleviates the disease. Furthermore, we identify Deptor, the endogenous inhibitor of mTOR, to be downregulated in β3 integrin expressing cells, suggesting that attenuated Deptor expression may engender the malignancy associated with β3 integrin. Importantly, functional disruption of Deptor in systemically invasive 4T07 cells induces their acquisition of mesenchymal characteristics, while attenuating their ability to proliferate. Collectively, we demonstrate that targeting β1 integrin alone is insufficient in alleviating metastatic breast cancer, while sole targeting of β3 integrin significantly attenuates the disease. Furthermore, our data identifies Deptor as a potential downstream effector in β3 integrin-dependent malignancies. Citation Format: Jenny G. Parvani, Molly A. Taylor, William P. Schiemann. The role of integrin switching and Deptor in metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B46.

Abstract C34: WAVE3 is required for TGF-β-mediated EMT

Breast cancer (BC) is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90% of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that mediate essential roles in regulating cell shape/morphology, actin polymerization and cytoskeleton remodeling, and cell motility and invasion. Accordingly, we have shown that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human BCs. Along these lines, we also established that members of the miR-200 family, together with miR-31 suppress WAVE3 expression, thereby limiting its prometastatic behavior. We show here that Transforming Growth Factor-beta (TGFβ;) robustly decreases miR-31 expression and induces WAVE3 expression through a Smad2 and β3-integrin dependent mechanism. Moreover, WAVE3 expression is required for BC cells to undergo epithelial-mesenchymal transition (EMT) in response to TGF-β;. Accordingly, stable depletion of WAVE3 expression in metastatic MDA-MB-231 or 4T1 BC cells prevented TGF-β; from inducing the EMT as determined by monitoring changes in the actin cytoskeleton and expression of EMT markers by real-time PCR. Along these lines, WAVE3 depletion significantly inhibited the ability of 4T1 cells to migrate and proliferate in response to TGF-β;. Moreover, WAVE3 deficiency abrogated the outgrowth of MDA-MB-231 and 4T1 cell organoids in 3D organotypic cultures. In vivo bioluminescent imaging indicated that WAVE3 knockdown also decreased tumor growth and metastasis in 4T1 tumors that possess enhanced autocrine TGF-β; signaling. Collectively, these findings indicate that WAVE3 activity is required for TGF-β;-mediated EMT and suggest that measures capable of inactivating WAVE3 may alleviate BC metastasis stimulated by TGF-β;. Citation Format: Molly A. Taylor, Gangarao Davuluri, Brian McCue, Edward F. Plow, William P. Schiemann, Khalid Sossey-Alaoui. WAVE3 is required for TGF-β-mediated EMT. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C34.

Abstract A33: The role of microRNA-181a in TGF-β-mediated breast cancer progression

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that functions to inhibit breast cancer development in the normal mammary epithelium by inducing cell cycle arrest, and maintaining microenvironmental homeostasis through extracellular matrix (ECM) deposition and remodeling. Mammary tumorigenesis causes a loss in TGF-β-mediated cytostasis and leads to a switch in TGF-β function, whereby TGF-β promotes epithelial-mesenchymal transition (EMT), proliferation, invasion and metastasis. This switch in TGF-β function, from tumor suppressor to tumor promoter, is referred to as the “TGF-β paradox” and occurs through molecular mechanisms that remain incompletely understood. Moreover, this switch in TGF-β function is often accompanied by desmoplastic and fibrotic reactions, leading to the formation of a more rigid microenvironment, which in and of itself has been associated with tumor progression. We have shown that tissue compliance and extracellular matrix rigidity mediate how cells sense and respond to TGF-β. Indeed, we can recapitulate the “TGF-β paradox” by exposing metastatic breast cancer cells to compliant ECM signals, which reinstates the cytostatic activities of TGF-β. As a possible mechanism for the influence of matrix rigidity on TGF-β signaling, we sought to define the miRNA expression profiles induced by TGF-β in metastatic cells as compared to their non-metastatic counterparts when grown in either compliant or rigid conditions. We show that ECM rigidity regulates the miRNA expression signature induced by TGF-β in breast cancer cells. From this miRNA signature, we have identified the miR-181 family as a miR family that is highly upregulated in metastatic cells in response to TGF-β. Additionally, over-expression of a miR-181a mimic enhances TGF-β-mediated invasion. Conversely, treating cells with a hairpin inhibitor directed against miR-181a inhibits TGF-β-mediated invasion, EMT, and survival signaling. Furthermore, inhibition of miR-181a abrogates pulmonary outgrowth and enhances overall survival in vivo. Collectively, our findings identify the miR-181a family as a novel effector of TGF-β-driven tumor progression and suggest that measures capable of diminishing miR-181a levels may alleviate breast cancer progression. [FY09-DOD-BCRP-Predoctoral Traineeship Award]. Citation Format: Molly A. Taylor, William P. Schiemann. The role of microRNA-181a in TGF-β-mediated breast cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A33.