Targeted inactivation of ?1 integrin induces ?3 integrin switching, which drives breast cancer metastasis by TGF-?.
ABSTRACT: Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-? (TGF-?) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated ?1 and ?3 integrins with TGF-? stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-? remain unknown. We demonstrate that inactivation of ?1 integrin impairs TGF-? from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of ?3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory ?3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-?. Of importance, compensatory expression of ?3 integrin rescues the growth and pulmonary metastasis of ?1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of ?3 integrin. Collectively our findings demonstrate that inactivation of ?1 integrin elicits metastatic progression via a ?3 integrin-specific mechanism, indicating that dual ?1 and ?3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.
Project description:Transforming growth factor (TGF)-beta suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells (MECs). During the course of mammary tumorigenesis, genetic and epigenetic changes negate the cytostatic actions of TGF-beta, thus enabling TGF-beta to promote the acquisition and development of metastatic phenotypes. The molecular mechanisms underlying this conversion of TGF-beta function remain poorly understood but may involve signaling inputs from integrins.beta3 Integrin expression or function in MECs was manipulated by retroviral transduction of active or inactive beta3 integrins, or by transient transfection of small interfering RNA (siRNA) against beta3 integrin. Altered proliferation, invasion, and epithelial-mesenchymal transition (EMT) stimulated by TGF-beta in control and beta3 integrin manipulated MECs was determined. Src involvement in beta3 integrin mediated alterations in TGF-beta signaling was assessed by performing Src protein kinase assays, and by interdicting Src function pharmacologically and genetically.TGF-beta stimulation induced alphavbeta3 integrin expression in a manner that coincided with EMT in MECs. Introduction of siRNA against beta3 integrin blocked its induction by TGF-beta and prevented TGF-beta stimulation of EMT in MECs. beta3 integrin interacted physically with the TGF-beta receptor (TbetaR) type II, thereby enhancing TGF-beta stimulation of mitogen-activated protein kinases (MAPKs), and of Smad2/3-mediated gene transcription in MECs. Formation of beta3 integrin:TbetaR-II complexes blocked TGF-beta mediated growth arrest and increased TGF-beta mediated invasion and EMT. Dual beta3 integrin:TbetaR-II activation induced tyrosine phosphorylation of TbetaR-II, a phosphotransferase reaction mediated by Src in vitro. Inhibiting Src activity in MECs prevented the ability of beta3 integrin to induce TbetaR-II tyrosine phosphorylation, MAPK activation, and EMT stimulated by TGF-beta. Lastly, wild-type and D119A beta3 integrin expression enhanced and abolished, respectively, TGF-beta stimulation of invasion in human breast cancer cells.We show that beta3 integrin alters TGF-beta signaling in MECs via Src-mediated TbetaR-II tyrosine phosphorylation, which significantly enhanced the ability of TGF-beta to induce EMT and invasion. Our findings suggest that beta3 integrin interdiction strategies may represent an innovative approach to re-establishing TGF-beta mediated tumor suppression in progressing human breast cancers.
Project description:Transforming growth factor-? (TGF-?) regulates all stages of mammary gland development, including the maintenance of tissue homeostasis and the suppression of tumorigenesis in mammary epithelial cells (MECs). Interestingly, mammary tumorigenesis converts TGF-? from a tumor suppressor to a tumor promoter through molecular mechanisms that remain incompletely understood. Changes in integrin signaling and tissue compliance promote the acquisition of malignant phenotypes in MECs in part through the activity of lysyl oxidase (LOX), which regulates desmoplastic reactions and metastasis. TGF-? also regulates the activities of tumor reactive stroma and MEC metastasis. We show here that TGF-?1 stimulated the synthesis and secretion of LOX from normal and malignant MECs in vitro and in mammary tumors produced in mice. The ability of TGF-?1 to activate Smad2/3 was unaffected by LOX inactivation in normal MECs, whereas the stimulation of p38 MAPK by TGF-?1 was blunted by inhibiting LOX activity in malignant MECs or by inducing the degradation of hydrogen peroxide in both cell types. Inactivating LOX activity impaired TGF-?1-mediated epithelial-mesenchymal transition and invasion in breast cancer cells. We further show that increasing extracellular matrix rigidity by the addition of type I collagen to three-dimensional organotypic cultures promoted the proliferation of malignant MECs, a cellular reaction that was abrogated by inhibiting the activities of TGF-?1 or LOX, and by degrading hydrogen peroxide. Our findings identify LOX as a potential mediator that couples mechanotransduction to oncogenic signaling by TGF-?1 and suggest that measures capable of inactivating LOX function may prove effective in diminishing breast cancer progression stimulated by TGF-?1.
Project description:Mammary tumorigenesis is associated with the increased expression of several proteins in the focal adhesion complex, including focal adhesion kinase (FAK) and various integrins. Aberrant expression of these molecules occurs concomitant with the conversion of TGF-beta function from a tumor suppressor to a tumor promoter. We previously showed that interaction between beta3 integrin and TbetaR-II facilitates TGF-beta-mediated oncogenic signaling, epithelial-mesenchymal transition (EMT), and metastasis. However, the molecular mechanisms by which the focal adhesion complex contributes to beta3 integrin:TbetaR-II signaling and the oncogenic conversion of TGF-beta remain poorly understood.FAK expression and activity were inhibited in normal and malignant mammary epithelial cells (MECs) either genetically by using lentiviral-mediated delivery of shRNAs against FAK, or pharmacologically through in vitro and in vivo use of the FAK inhibitors, PF-562271 and PF-573228. Altered Smad2/3 and p38 MAPK activation, migration, EMT, and invasion in response to TGF-beta1 were monitored in FAK-manipulated cells. TbetaR-II expression was increased in metastatic breast cancer cells by retroviral transduction, and the metastasis of FAK- and TbetaR-II-manipulated tumors was monitored by using bioluminescent imaging.TGF-beta stimulation of MECs stabilized and activated FAK in a beta3 integrin- and Src-dependent manner. Furthermore, by using the human MCF10A breast cancer progression model, we showed that increased FAK expression in metastatic breast cancer cells mirrored the acquisition of enhanced activation of p38 MAPK by TGF-beta. Administering FAK inhibitors or rendering metastatic breast cancer cells FAK deficient abrogated the interaction between beta3 integrin and TbetaR-II, thereby preventing TGF-beta from (a) activating p38 MAPK; (b) stimulating MEC invasion, migration, and EMT; and (c) inducing early primary tumor dissemination to the lungs. Finally, in contrast to FAK depletion, adjuvant FAK chemotherapy of mammary tumors decreased their growth in part by diminished macrophage tumor infiltration.Our studies identify an essential function for FAK in mediating the interaction between beta3 integrin and TbetaR-II, and thus in facilitating the oncogenic conversion of TGF-beta required for mammary tumor metastasis. Furthermore, this study establishes chemotherapeutic targeting of FAK as an effective, two-pronged approach in preventing tumor progression both by decreasing innate immune cell infiltration, and by inhibiting early TGF-beta-dependent metastasis.
Project description:Epithelial-mesenchymal transition (EMT) plays a pivotal role for tumor progression. Recent studies have revealed the existence of distinct intermediate states in EMT (partial EMT); however, the mechanisms underlying partial EMT are not fully understood. Here, we demonstrate that αvβ3 integrin induces partial EMT, which is characterized by acquiring mesenchymal phenotypes while retaining epithelial markers. We found αvβ3 integrin to be associated with poor survival in patients with lung adenocarcinoma. Moreover, αvβ3 integrin-induced partial EMT promoted migration, invasion, tumorigenesis, stemness, and metastasis of lung cancer cells in a TGF-β-independent fashion. Additionally, TGF-β1 promoted EMT progression synergistically with αvβ3 integrin, while a TGF-β signaling inhibitor showed no effect on αvβ3 integrin-induced partial EMT. Meanwhile, the microRNA-200 family abolished the αvβ3 integrin-induced partial EMT by suppressing αvβ3 integrin cell surface expression. These findings indicate that αvβ3 integrin is a key inducer of partial EMT, and highlight a new mechanism for cancer progression.
Project description:Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing ?5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBP? expression to directly induce ?5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBP?-dependent ?5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal-epithelial interactions.
Project description:BACKGROUND:Integrin-mediated platelet-tumor cell contacting plays an important role in promoting epithelial-mesenchymal transition (EMT) transformation of tumor cells and cancer metastasis, but whether it occurs in breast cancer cells is not completely clear. OBJECTIVE:The purpose of this study was to investigate the role of integrin ?2?1 in platelet contacting to human breast cancer cell line MCF-7 and its effect on the EMT and the invasion of MCF-7 cells. METHODS:Human platelets were activated by thrombin, and separated into pellets and releasates before the co-incubation with MCF-7 cells. Cell invasion was evaluated by transwell assay. The surface integrins on pellets and MCF-7 cells were inhibited by antibodies. The effect of integrin ?2?1 on Wnt-?-catenin pathway was assessed by integrin ?2?1-silencing and Wnt-?-catenin inhibitor XAV. The therapeutic effect of integrin ?2?1-silencing was confirmed in the xenograft mouse model. RESULTS:Pellets promote the invasion and EMT of MCF-7 cells via direct contacting of surface integrin ?2?1. The integrin ?2?1 contacting activates Wnt-?-catenin pathway and promotes the expression of EMT proteins in MCF-7 cells. The activated Wnt-?-catenin pathway also promotes the autocrine of TGF-?1 in MCF-7 cells. Both Wnt-?-catenin and TGF-?1/pSmad3 pathways promote the expression of EMT proteins. Integrin ?2?1-silencing inhibits breast cancer metastasis in vivo. CONCLUSIONS:The direct interaction between platelets and tumor cells exerts its pro-metastatic function via surface integrin ?2?1 contacting and Wnt-?-catenin activation. Integrin ?2?1-silencing has the potential effect of inhibiting breast cancer metastasis.
Project description:Transforming growth factor-beta (TGF-beta) is a ubiquitous cytokine with dual roles in tumor suppression and promotion, and these dichotomous functions have frustrated the development of therapies targeting oncogenic signaling by TGF-beta. In comparison, Abl is well established as an initiator of hematopoietic cancers; however, a clear role for Abl in regulating solid tumor development remains elusive. Here, we investigated the role of Abl in TGF-beta-mediated epithelial-mesenchymal transition (EMT) in normal and metastatic mammary epithelial cells (MECs). In doing so, we identified Abl as an essential regulator of MEC morphology and showed that Abl inactivation was sufficient to induce phenotypic and transcriptional EMT in normal MECs. Increasing Abl activity in metastatic MECs resulted in their complete morphological reversion, restored their cytostatic response to TGF-beta, and blocked their secretion of matrix metalloproteinases induced by TGF-beta. Constitutively active Abl expression blocked TGF-beta-responsive mammary tumor growth in mice, while Imatinib therapy afforded no clinical benefit in mice bearing mammary tumors. Collectively, this investigation establishes Abl as a potent mediator of MEC identity, and as a suppressor of oncogenic TGF-beta signaling during mammary tumorigenesis. Notably, our findings strongly caution against the use of pharmacological Abl antagonists in the treatment of developing and progressing mammary tumors.
Project description:Transforming growth factor-?1 (TGF-?1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-?1 enhances ?(1) integrin levels in NMuMG cells, the detailed molecular mechanisms underlying TGF-?1-induced ?(1) integrin gene expression and the role of ?(1) integrin during EMT in the renal system are still unclear. In this study, we examined the role of ?(1) integrin in TGF-?1-induced EMT both in vitro and in vivo. TGF-?1-induced augmentation of ?(1) integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-?1-induced augmentation of ?(1) integrin. TGF-?1 triggered ?(1) integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the ?(1) integrin promoter markedly reduced TGF-?1-induced ?(1) integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-?1 enhanced Smad3 binding to the ?(1) integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of ?(1) integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of ?(1) integrin and ?-smooth muscle actin in tubule epithelia. Blockade of ?(1) integrin signaling dampened the progression of fibrosis. Taken together, ?(1) integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of ?(1) integrin is a possible therapeutic target for prevention of renal fibrosis.
Project description:Alterations in the composition and architecture of the extracellular matrix (ECM) can influence cancer growth and dissemination. During epithelial-mesenchymal transition (EMT), epithelial cells assume a mesenchymal cell phenotype, changing their adhesion profiles from cell-cell contacts to cell-matrix interactions, contributing to metastasis. Breast cancer cells present at different stages of differentiation, producing distinct ECMs in the same tumor mass. However, the contribution of ECM derived from metastatic tumor cells to EMT is unclear. Here, we showed the mechanisms involved in the interaction of MCF-7, a low-metastatic, epithelial breast cancer cell line, with the ECM produced by a high metastatic breast tumor cell, MDA-MB-231 (MDA-ECM). MDA-ECM induced morphological changes in MCF-7 cells, decreased the levels of E-cadherin, up-regulated mesenchymal markers, and augmented cell migration. These changes were accompanied by the activation of integrin-associated signaling, with increased phosphorylation of FAK, ERK, and AKT and activation canonical TGF-? receptor signaling, enhancing phosphorylation of SMAD2 and SMAD4 nuclear translocation in MCF-7 cells. Treatment with Kistrin (Kr), a specific ligand of integrin ?v?3 EMT induced by MDA-ECM, inhibited TGF-? receptor signaling in treated MCF-7 cells. Our results revealed that after interaction with the ECM produced by a high metastatic breast cancer cell, MCF-7 cells lost their characteristic epithelial phenotype undergoing EMT, an effect modulated by integrin signaling in crosstalk with TGF-? receptor signaling pathway. The data evidenced novel potential targets for antimetastatic breast cancer therapies.
Project description:Overexpression of MYC transforms cells in culture, elicits malignant tumours in experimental animals and is found in many human tumours. We now report the paradoxical finding that this powerful oncogene can also act as a suppressor of cell motility, invasiveness and metastasis. Overexpression of MYC stimulated proliferation of breast cancer cells both in culture and in vivo as expected, but inhibited motility and invasiveness in culture, and lung and liver metastases in xenografted tumours. We show further that MYC represses transcription of both subunits of ?v?3 integrin, and that exogenous expression of ?3 integrin in human breast cancer cells that do not express this integrin rescues invasiveness and migration when MYC is downregulated. These data uncover an unexpected function of MYC, provide an explanation for the hitherto puzzling literature on the relationship between MYC and metastasis, and reveal a variable that could influence the development of therapies that target MYC.