Fibroblast Growth Factor Receptor Splice Variants are Stable Markers of Oncogenic Transforming Growth Factor β1 Signaling in Metastatic Breast Cancers.

Introduction Epithelial–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. Methods An 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 Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-β1, thus completing one full EMT:MET cycle.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. Results Metastatic 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 BC cells treated and withdrawn from TGF-β stably upregulate a truncated FGFR1-β splice variant that lacks the outermost extracellular immunoglobulin domain.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. Conclusion The 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 inhibitor

The Antitumorigenic Function of EGFR in Metastatic Breast Cancer is Regulated by Expression of Mig6

Numerous studies by our lab and others demonstrate that epidermal growth factor receptor (EGFR) plays critical roles in primary breast cancer (BC) initiation, growth and dissemination. However, clinical trials targeting EGFR function in BC have lead to disappointing results. In the current study we sought to identify the mechanisms responsible for this disparity by investigating the function of EGFR across the continuum of the metastatic cascade. We previously established that overexpression of EGFR is sufficient for formation of in situ primary tumors by otherwise nontransformed murine mammary gland cells. Induction of epithelial-mesenchymal transition (EMT) is sufficient to drive the metastasis of these EGFR-transformed tumors. Examining growth factor receptor expression across this and other models revealed a potent downregulation of EGFR through metastatic progression. Consistent with diminution of EGFR following EMT and metastasis EGF stimulation changes from a proliferative to an apoptotic response in in situ versus metastatic tumor cells, respectively. Furthermore, overexpression of EGFR in metastatic MDA-MB-231 BC cells promoted their antitumorigenic response to EGF in three dimensional (3D) metastatic outgrowth assays. In line with the paradoxical function of EGFR through EMT and metastasis we demonstrate that the EGFR inhibitory molecule, Mitogen Induced Gene-6 (Mig6), is tumor suppressive in in situ tumor cells. However, Mig6 expression is absolutely required for prevention of apoptosis and ultimate metastasis of MDA-MB-231 cells. Further understanding of the paradoxical function of EGFR between primary and metastatic tumors will be essential for application of its targeted molecular therapies in BC

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

INTRODUCTION: Transforming 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. METHOD: Retroviral 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. RESULTS: CystC 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. CONCLUSION: Our 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

SPARC Inhibits Epithelial Cell Proliferation in Part through Stimulation of the Transforming Growth Factor-β–Signaling System

Secreted protein, acidic and rich in cysteine (SPARC) is a multifunctional secreted protein that regulates cell–cell and cell–matrix interactions, leading to alterations in cell adhesion, motility, and proliferation. Although SPARC is expressed in epithelial cells, its ability to regulate epithelial cell growth remains largely unknown. We show herein that SPARC strongly inhibited DNA synthesis in transforming growth factor (TGF)-β–sensitive Mv1Lu cells, whereas moderately inhibiting that in TGF-β–insensitive Mv1Lu cells (i.e., R1B cells). Overexpression of dominant-negative Smad3 in Mv1Lu cells, which abrogated growth arrest by TGF-β, also attenuated growth arrest stimulated by SPARC. Moreover, the extracellular calcium-binding domain of SPARC (i.e., SPARC-EC) was sufficient to inhibit Mv1Lu cell proliferation but not that of R1B cells. Similar to TGF-β and thrombospondin-1, treatment of Mv1Lu cells with SPARC or SPARC-EC stimulated Smad2 phosphorylation and Smad2/3 nuclear translocation: the latter response to all agonists was abrogated in R1B cells or by pretreatment of Mv1Lu cells with neutralizing TGF-β antibodies. SPARC also stimulated Smad2 phosphorylation in MB114 endothelial cells but had no effect on bone morphogenetic protein-regulated Smad1 phosphorylation in either Mv1Lu or MB114 cells. Finally, SPARC and SPARC-EC stimulated TGF-β–responsive reporter gene expression through a TGF-β receptor- and Smad2/3-dependent pathway in Mv1Lu cells. Collectively, our findings identify a novel mechanism whereby SPARC inhibits epithelial cell proliferation by selectively commandeering the TGF-β signaling system, doing so through coupling of SPARC-EC to a TGF-β receptor- and Smad2/3-dependent pathway

CystC and Δ14CystC inhibit actin cytoskeletal rearrangements stimulated by TGF-β in NMuMG cells

<p><b>Copyright information:</b></p><p>Taken from "The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β"</p><p>Breast Cancer Research 2005;7(5):R844-R853.</p><p>Published online 23 Aug 2005</p><p>PMCID:PMC1242164.</p><p>Copyright © 2005 Sokol et al.; licensee BioMed Central Ltd.</p> NMuMG cells were infected with ecotropic retrovirus encoding either green fluorescent protein (GFP; control), CystC, or Δ14CystC, and subsequently were isolated by fluorescence-activated cell sorting for GFP expression to yield stable polyclonal populations of control, CystC-expressing, and Δ14CystC-expressing cells as indicated (upper panel). The expression and secretion of recombinant CystC proteins by infected NMuMG cells were monitored by immunoblotting conditioned medium with anti-CystC antibodies (lower panel). Control, CystC-expressing, and Δ14CystC-expressing NMuMG cells were incubated in the absence or presence of transforming growth factor-β1 (TGF-β1; 5 ng/ml) for 8 hours, whereupon alterations in actin cytoskeletal architecture were revealed by direct rhodamine–phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. NMuMG cells were stimulated with TGF-β1 (5 ng/ml) for 0 to 24 hours in the presence of either glutathione S-transferase (GST), GST–CystC, or GST–Δ14CystC (each at 10 μg/ml) as indicated. Afterward, altered actin cytoskeletal architecture was revealed by direct rhodamine–phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated once with identical results

CystC and Δ14CystC inhibit TGF-β signaling in human MDA-MB-231 breast cancer cells

<p><b>Copyright information:</b></p><p>Taken from "The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β"</p><p>Breast Cancer Research 2005;7(5):R844-R853.</p><p>Published online 23 Aug 2005</p><p>PMCID:PMC1242164.</p><p>Copyright © 2005 Sokol et al.; licensee BioMed Central Ltd.</p> Control, CystC-expressing, or Δ14CystC-expressing MDA-MB-231 cells were transiently transfected with p3TP-luciferase and pCMV-β-Gal cDNAs, and were subsequently stimulated with increasing concentrations of transforming growth factor-β1 (TGF-β1; from 0 to 5 ng/ml) for 24 hours. Afterward, luciferase and β-Gal activities contained in detergent-solubilized cell extracts were measured. Values are luciferase activities (means ± SEM) observed in two independent experiments normalized to maximal reporter gene expression in TGF-β-stimulated cells expressing green fluorescent protein. CystC and Δ14CystC both significantly inhibit reporter gene expression stimulated by TGF-β (*< 0.05; Student's -test). MDA-MB-231 cells were transiently transfected with p3TP-luciferase and pCMV-β-Gal cDNAs. Afterward, the transfectants were treated with 25 μg/ml recombinant glutathione S-transferase (GST; G), CystC (C), or Δ14CystC (D) and immediately stimulated with TGF-β1 (1 ng/ml) for 24 hours before determination of the luciferase and β-Gal activities contained in detergent-solubilized cell extracts. Values are luciferase activities (means ± SEM) observed in four independent experiments normalized to maximal reporter gene expression stimulated by TGF-β in GST-treated cells. MDA-MB-231 cells were treated with 25 μg/ml recombinant GST (G), CystC (C), or Δ14CystC (D) for 2 hours before their stimulation with TGF-β (1 ng/ml) for 30 min. Afterward, the activation status of Smad2 was determined by immunoblot analysis with phospho-specific Smad2 antibodies. Differences in protein loading were monitored by reprobing stripped membranes with antibodies against extracellular signal-related kinase 1. Data are from a representative experiment that was repeated once with similar results

CystC and Δ14CystC antagonize E-cadherin downregulation stimulated by TGF-β in NMuMG cells

<p><b>Copyright information:</b></p><p>Taken from "The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β"</p><p>Breast Cancer Research 2005;7(5):R844-R853.</p><p>Published online 23 Aug 2005</p><p>PMCID:PMC1242164.</p><p>Copyright © 2005 Sokol et al.; licensee BioMed Central Ltd.</p> Control, CystC-expressing, and Δ14CystC-expressing NMuMG cells were incubated in the absence or presence of transforming growth factor-β1 (TGF-β1; 5 ng/ml) for 36 hours, whereupon alterations in E-cadherin expression was monitored by immunoprecipitation and subsequent immunoblotting with anti-E-cadherin antibodies. The representative immunoblot depicts the downregulation of E-cadherin expression induced by TGF-β in NMuMG cells. The lower panel shows the alterations in E-cadherin expression (means ± SEM) induced by TGF-β relative to their untreated counterparts observed in three independent experiments. TGF-β significantly downregulated E-cadherin expression in NMuMG cells (*< 0.05; Student's -test). NMuMG cells were stimulated with TGF-β1 (5 ng/ml) for 36 hours in the presence of glutathione S-transferase (GST) fusion proteins (10 μg/ml) as indicated. E-cadherin expression was monitored by indirect immunofluorescence with anti-E-cadherin antibodies. Shown are representative images from a single experiment that was repeated once with identical results