Barbara J. Schiemann

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.

Epithelial to Mesenchymal Transition Promotes Breast Cancer Progression via a Fibronectin-dependent STAT3 Signaling Pathway

Background: Cells perceive their environment through soluble growth factors and in response to extracellular matrix. Results: STAT3 signaling can be activated by multiple pathways during breast cancer progression. Conclusion: Fibronectin:STAT3 signaling promotes three-dimensional outgrowth of breast cancer cells. Significance: This study demonstrates a novel mechanism by which STAT3 becomes activated by the extracellular matrix independent of the canonical EGF receptor signaling network. We previously established that overexpression of the EGF receptor (EGFR) is sufficient to induce tumor formation by otherwise nontransformed mammary epithelial cells, and that the initiation of epithelial-mesenchymal transition (EMT) is capable of increasing the invasion and metastasis of these cells. Using this breast cancer (BC) model, we find that in addition to EGF, adhesion to fibronectin (FN) activates signal transducer and activator of transcription 3 (STAT3) through EGFR-dependent and -independent mechanisms. Importantly, EMT facilitated a signaling switch from SRC-dependent EGFR:STAT3 signaling in pre-EMT cells to EGFR-independent FN:JAK2:STAT3 signaling in their post-EMT counterparts, thereby sensitizing these cells to JAK2 inhibition. Accordingly, human metastatic BC cells that failed to activate STAT3 downstream of EGFR did display robust STAT3 activity upon adhesion to FN. Furthermore, FN enhanced outgrowth in three-dimensional organotypic cultures via a mechanism that is dependent upon β1 integrin, Janus kinase 2 (JAK2), and STAT3 but not EGFR. Collectively, our data demonstrate that matrix-initiated signaling is sufficient to drive STAT3 activation, a reaction that is facilitated by EMT during BC metastatic progression.

The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β

IntroductionTransforming growth factor-β (TGF-β) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-β type II receptor antagonist that inhibits TGF-β binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-β, particularly its stimulation of mammary epithelial–mesenchymal transition (EMT), can be prevented by CystC.MethodRetroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Δ14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-β stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-β stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-β.ResultsCystC and Δ14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-β by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-β-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Δ14CystC also inhibited TGF-β signaling in two tumorigenic human breast cancer cell lines.ConclusionOur findings show that TGF-β stimulation of initiating metastatic events, including decreased cell polarization, reduced cell–cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-β.

Targeted inactivation of β1 integrin induces β3 integrin switching, which drives breast cancer metastasis by TGF-β

Chemotherapeutic targeting of β1 integrin has been proposed as a way to alleviate breast cancer metastasis. It is shown here that inactivation of β1 integrin elicits compensatory expression of β3 integrin, which rescues mammary tumor growth and metastasis, as well as promoting oncogenic TGF-β signaling in late-stage breast cancer.

TGF-β stimulates Pyk2 expression as part of an epithelial-mesenchymal transition program required for metastatic outgrowth of breast cancer

Epithelial-mesenchymal transition (EMT) programs are essential in promoting breast cancer invasion, systemic dissemination and in arousing proliferative programs in breast cancer micrometastases, a reaction that is partially dependent on focal adhesion kinase (FAK). Many functions of FAK are shared by its homolog, protein tyrosine kinase 2 (Pyk2), raising the question as to whether Pyk2 also participates in driving the metastatic outgrowth of disseminated breast cancer cells. In addressing this question, we observed Pyk2 expression to be (i) significantly upregulated in recurrent human breast cancers; (ii) differentially expressed across clonal isolates of human MDA-MB-231 breast cancer cells in a manner predictive for metastatic outgrowth, but not for invasiveness; and (iii) dramatically elevated in ex vivo cultures of breast cancer cells isolated from metastatic lesions as compared with cells that produced the primary tumor. We further show that metastatic human and murine breast cancer cells robustly upregulate their expression of Pyk2 during EMT programs stimulated by transforming growth factor-β (TGF-β). Genetic and pharmacological inhibition of Pyk2 demonstrated that the activity of this protein tyrosine kinase was dispensable for the ability of breast cancer cells to undergo invasion in response to TGF-β, and to form orthotopic mammary tumors in mice. In stark contrast, Pyk2-deficiency prevented TGF-β from stimulating the growth of breast cancer cells in 3D-organotypic cultures that recapitulated pulmonary microenvironments, as well as inhibited the metastatic outgrowth of disseminated breast cancer cells in the lungs of mice. Mechanistically, Pyk2 expression was inversely related to that of E-cadherin, such that elevated Pyk2 levels stabilized β1 integrin expression necessary to initiate the metastatic outgrowth of breast cancer cells. Thus, we have delineated novel functions for Pyk2 in mediating distinct elements of the EMT program and metastatic cascade regulated by TGF-β, particularly the initiation of secondary tumor outgrowth by disseminated cells.

Biochemical and Functional Characterization of Six SIX1 Branchio-oto-renal Syndrome Mutations

Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1.

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.

The WAVE3-YB1 interaction regulates cancer stem cells activity in breast cancer

Resistance to therapy is the main cause of tumor recurrence and metastasis and cancer stem cells (CSCs) play a crucial role in this process, especially in triple-negative breast cancers (TNBCs). Unfortunately, no FDA-approved treatment is currently available for this subtype of BC, which explains the high rate of mortality in patients with TNBC tumors. WAVE3, a member of the WASP/WAVE actin-cytoskeleton remodeling family of protein, has been established as a major driver of tumor progression and metastasis of several solid tumors, including those originating in the breast. Our recently published studies found WAVE3 to mediate the process of chemoresistance in TNBCs. The molecular mechanisms whereby WAVE3 regulates chemoresistance in TNBC tumors remains largely unknown, as does the role of WAVE3 in CSC maintenance. Here we show that WAVE3 promotes CSC self-renewal and regulates transcription of CSC-specific genes, which, in part, provides a mechanistic explanation for the function of WAVE3 in chemoresistance in TNBCs. Our data show that WAVE3 is enriched in the CSC-subpopulation of TNBC cell lines. Knockout of WAVE3 via CRISPR/Cas9 significantly attenuates the CSC-subpopulation and inhibits transcription of CSC transcription factors. Mechanistically, we established a link between WAVE3 and the Y-box-binding protein-1 (YB1), a transcription factor and CSC-maintenance gene. Indeed, the interaction of WAVE3 with YB1 is required for YB1 translocation to the nucleus of cancer cells, and activation of transcription of CSC-specific genes. Our findings identify a new WAVE3/YB1 signaling axis that regulates the CSC-mediated resistance to therapy and opens a new therapeutic window for TNBCs treatment.

Abstract A159: The inherent resistant of breast cancer to EGFR-targeted therapies is driven by EMT-induced expression of particular FGFR splice variants.

Epithelial-mesenchymal transition (EMT) and the reverse process of mesenchymal-epithelial transition (MET) are critical processes in cancer metastasis and resistance to molecular targeted therapies. However, the molecular events that facilitate these processes remain to be fully elucidated. Using an in vivo reporter system for the promoter activity of Epithelial-Cadherin (E-cad), we quantify a robust EMT that takes place upon orthotopic engraftment of highly metastatic breast cancer cells. Moreover, using this reporter system together with immunohistochemistry we demonstrate that E-cad expression does not return until after initiation of pulmonary metastatic outgrowth. Using an epidermal growth factor receptor (EGFR)-driven model of in situ breast cancer that is highly sensitive to Erlotinib, we show that induction of EMT is sufficient to drive metastasis and that following the MET process these cells become inherently resistant to EGFR-inhibition. Microarray analyses of these cells demonstrate that following EMT:MET there is critical pathway switch from EGFR to aberrant growth factor signaling that is mediated by a truncated splice variant of fibroblast growth factor receptor 1 (FGFR1). In silico and RT-PCR analyses of patient tumor samples revealed a potent upregulation of the truncated FGFR1 isoform. Indeed, while EGFR expression is initially required for aberrant maintenance of EMT-induced FGFR expression, EGFR and several EGFR ligands are potently down regulated during the MET process. These findings are consistent with previous studies from our group and others demonstrating loss of EGFR expression in late-stage breast cancers and the clinical failure of EGFR-targeted therapies in the treatment of breast cancer. shRNA-mediated depletion of FGFR1 in an EGFR-null model of pulmonary metastatic outgrowth dramatically inhibited pulmonary tumor formation. Moreover, in vivo treatment of mice with the FGFR kinase inhibitor BGJ-398 similarly decreased pulmonary tumor outgrowth. Finally, ectopic expression of the full-length form of FGFR1 also prevented tumor outgrowth following tail vein inoculation. Overall, our data demonstrate the dynamic flux between EGFR and FGFR during particular stages of breast cancer progression. Moreover, our findings demonstrate the plausible utility of identifying truncated FGFR1 splice variants as a predictive biomarker for FGFR targeted molecular therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A159. Citation Format: Michael K. Wendt, Jenny Parvani, Barbara Schiemann, Khalid Sossey-Aiaoui, William Schiemann. The inherent resistant of breast cancer to EGFR-targeted therapies is driven by EMT-induced expression of particular FGFR splice variants. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A159.

Abstract A17: Regulation of EGFR: Stat3 signaling by Mig6 is required for metastatic outgrowth in triple-negative breast cancer

Stat3 is an oncogene that enriches for tumor initiating breast cancer cells. Stat3 activity is driven by numerous signaling inputs, including those initiated by EGFR. Herein we shown that Stat3 is critically involved in EGFR-driven tumorigenesis as EGFR molecules harboring a mutation in the juxtamembrane domain that specifically prevents activation of Stat3 fail to transform mammary epithelial cells. Using this model of EGFR transformation, we previously established that the induction of epithelial-mesenchymal transition (EMT) enhances pulmonary tumor formation. Surprisingly, EMT drastically diminishes the capacity of EGFR to signal to Stat3. Indeed, using the MCF-10A progression series, we show that constitutive activation of Stat3 is consistent with the acquisition of a mesenchymal phenotype but independent of EGFR activity. As a compensatory mechanism, we find that EMT increases the expression and ability of fibronectin to directly activate Stat3 in an EGFR-independent, Jak2-dependent manor. Indeed, human triple-negative breast cancer (TNBC) cells that fail to activate Stat3 in response to EGF robustly activate Stat3 upon fibronectin adhesion. Consistent with these results, TNBC outgrowth in pulmonary 3D-organotypic cultures is enhanced by the addition of fibronectin, a process that is absolutely dependent on β1-integrin, Jak2 and Stat3, but independent of EGFR. Underscoring the importance of this switch from EGFR-mediated to fibronectin-mediated Stat3 activation in metastatic TNBC cells, we demonstrate that shRNA-mediated diminution of the EGFR inhibitory molecule MIG6 results in increased apoptosis and consequently, decreased pulmonary tumor formation by TNBC cells. Our data clearly demonstrate that matrix-initiated signaling is sufficient to drive Stat3 activation, a reaction that likely contributes to the resistance of metastatic TNBCs to molecular therapies, particularly those that target EGFR. Citation Format: Mike K. Wendt, Nikolas Balanis, Barbara Schiemann, Cathleen Carlin, William Schiemann. Regulation of EGFR: Stat3 signaling by Mig6 is required for metastatic outgrowth in triple-negative 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 A17.