T?RIII/?-arrestin2 regulates integrin ?5?1 trafficking, function, and localization in epithelial cells.
ABSTRACT: The type III TGF-? receptor (T?RIII) is a ubiquitous co-receptor for TGF-? superfamily ligands with roles in suppressing cancer progression, in part through suppressing cell motility. Here we demonstrate that T?RIII promotes epithelial cell adhesion to fibronectin in a ?-arrestin2 dependent and TGF-?/BMP independent manner by complexing with active integrin ?5?1, and mediating ?-arrestin2-dependent ?5?1 internalization and trafficking to nascent focal adhesions. T?RIII-mediated integrin ?5?1 trafficking regulates cell adhesion and fibronectin fibrillogenesis in epithelial cells, as well as ?5 localization in breast cancer patients. We further demonstrate that increased T?RIII expression correlates with increased ?5 localization at sites of cell-cell adhesion in breast cancer patients, while higher T?RIII expression is a strong predictor of overall survival in breast cancer patients. These data support a novel, clinically relevant role for T?RIII in regulating integrin ?5 localization, reveal a novel crosstalk mechanism between the integrin and TGF-? superfamily signaling pathways and identify ?-arrestin2 as a regulator of ?5?1 trafficking.
Project description:Cell adhesion and migration are tightly controlled by regulated changes in the actin cytoskeleton. Previously we reported that the TGF? (transforming growth factor ?) superfamily co-receptor, T?RIII (type III TGF? receptor; also known as ?glycan), regulates cell adhesion, migration and invasion, and suppresses cancer progression, in part, through activation of the small GTPase Cdc42 (cell division cycle 42), and Cdc42-dependent alterations to the actin cytoskeleton. In the present study we demonstrate that T?RIII specifically promotes filopodial formation and extension in MCF10A and HMEC (human mammary epithelial cell) mammary epithelial cells. Mechanistically, cell-surface T?RIII and Cdc42 co-localize to filopodial structures and co-complex in a ?-arrestin2-dependent, and a T?RI/T?RII-independent manner. The ?-arrestin2-mediated interaction between T?RIII and Cdc42 increases complex formation between the Cdc42 effectors IRSp53 with N-WASP (neuronal Wiskott-Aldrich syndrome protein) to increase filopodial formation. We demonstrate a function link between filopodial structures and epithelial cell adhesion as regulated by the T?RIII-Cdc42 interaction. The present studies identify T?RIII as a novel regulator of IRSp53/N-WASP via Cdc42 to regulate filopodial formation and cell adhesion.
Project description:The type III transforming growth factor ? (TGF-?) receptor (T?RIII), also known as betaglycan, is the most abundantly expressed TGF-? receptor. T?RIII suppresses breast cancer progression by inhibiting migration, invasion, metastasis, and angiogenesis. T?RIII binds TGF-? ligands, with membrane-bound T?RIII presenting ligand to enhance TGF-? signaling. However, T?RIII can also undergo ectodomain shedding, releasing soluble T?RIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound T?RIII on TGF-? signaling and breast cancer biology, we defined T?RIII mutants with impaired (?Shed-T?RIII) or enhanced ectodomain shedding (SS-T?RIII). Inhibiting ectodomain shedding of T?RIII increased TGF-? responsiveness and abrogated T?RIII's ability to inhibit breast cancer cell migration and invasion. Conversely, expressing SS-T?RIII, which increased soluble T?RIII production, decreased TGF-? signaling and increased T?RIII-mediated inhibition of breast cancer cell migration and invasion. Of importance, SS-T?RIII-mediated increases in soluble T?RIII production also reduced breast cancer metastasis in vivo. Taken together, these studies suggest that the ratio of soluble T?RIII to membrane-bound T?RIII is an important determinant for regulation of T?RIII- and TGF-?-mediated signaling and biology.
Project description:There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-?) pathway-associated genes relative to other subtypes, including the TGF-? receptor type III (T?RIII). We hypothesize that T?RIII is tumor promoter in mesenchymal-stem like TNBC cells.Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of T?RIII in the MSL subtype. We stably expressed short hairpin RNAs specific to T?RIII (T?RIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine T?RIII expression patterns across all TNBC subtypes.T?RIII was the most differentially expressed TGF-? signaling gene in the MSL subtype. Silencing T?RIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when T?RIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed T?RIII-dependent phenotypes, we discovered that integrin-?2 was expressed at higher level in MSL cells after T?RIII-KD. Stable knockdown of integrin-?2 in T?RIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells.We have found that T?RIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that T?RIII-KD elevates expression of integrin-?2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both T?RIII and integrin-?2. Overall, our results indicate a potential mechanism in which T?RIII modulates integrin-?2 expression to effect MSL cell migration, invasion, and tumorigenicity.
Project description:The ?5?1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for ?5?1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and ?5?1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of ?1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated ?5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of ?5?1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.
Project description:Bone morphogenetic proteins (BMPs) are members of the TGF-? superfamily that are over-expressed in breast cancer, with context dependent effects on breast cancer pathogenesis. The type III TGF-? receptor (T?RIII) mediates BMP signaling. While T?RIII expression is lost during breast cancer progression, the role of T?RIII in regulating BMP signaling in normal mammary epithelium and breast cancer cells has not been examined. Restoring T?RIII expression in a 4T1 murine syngeneic model of breast cancer suppressed Smad1/5/8 phosphorylation and inhibited the expression of the BMP transcriptional targets, Id1 and Smad6, in vivo. Similarly, restoring T?RIII expression in human breast cancer cell lines or treatment with sT?RIII inhibited BMP-induced Smad1/5/8 phosphorylation and BMP-stimulated migration and invasion. In normal mammary epithelial cells, shRNA-mediated silencing of T?RIII, T?RIII over-expression, or treatment with sT?RIII inhibited BMP-mediated phosphorylation of Smad1/5/8 and BMP induced migration. Inhibition of T?RIII shedding through treatment with TAPI-2 or expression of a non-shedding T?RIII mutant rescued T?RIII mediated inhibition of BMP induced Smad1/5/8 phosphorylation and BMP induced migration and/or invasion in both in normal mammary epithelial cells and breast cancer cells. Conversely, expression of a T?RIII mutant, which exhibited increased shedding, significantly reduced BMP-mediated Smad1/5/8 phosphorylation, migration, and invasion. These data demonstrate that T?RIII regulates BMP-mediated signaling and biological effects, primarily through the ligand sequestration effects of sT?RIII in normal and cancerous mammary epithelial cells and suggest that the ratio of membrane bound versus sT?RIII plays an important role in mediating these effects.
Project description:Increasing evidence is emerging highlighting the role of parathyroid hormone-related protein (PTHrP) during metastasis by regulating cell adhesion. The current study demonstrated that modulation of PTHrP expression by PTHrP overexpression and small interfering RNA-induced silencing resulted in changes in cell adhesion and integrin expression. RNA interference of endogenous PTHrP caused a significant reduction in cell adhesion of a breast cancer cell line to collagen type I, fibronectin and laminin (P<0.05) and of a colon cancer cell to collagen type I and fibronectin (P<0.05). Overexpression of PTHrP induced a significant increase in cell adhesion of colon (P<0.0001) and breast (P<0.05) cancer cells to the same extracellular matrix proteins. These PTHrP-mediated effects were attributed to changes in integrin expression as the differences in adhesion profile correlated with the integrin expression profile. In an attempt to elucidate the mechanism whereby PTHrP regulates integrin expression, promoter activity of the integrin alpha5 subunit was analysed and significant increases in transcriptional activity were observed in PTHrP overexpressing cells (P<0.0001), which was dependent on nuclear localisation. These results indicate that modulation of cell adhesion is a normal physiological action of PTHrP, mediated by increasing integrin gene transcription.
Project description:CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of ?1-integrin receptors, and increased ?5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an ?5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the ?1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with ?1-integrin and repressed HA-induced, CD44-mediated activation of ?1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and ?1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.
Project description:Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin ?5?1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin ?5?1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin ?5?1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin ?5?1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin ?5?1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.
Project description:Transforming growth factor-? (TGF-?) plays an important role in regulating hematopoiesis, inhibiting proliferation while stimulating differentiation when appropriate. We previously demonstrated that the type III TGF-? receptor (T?RIII, or betaglycan) serves as a novel suppressor of cancer progression in epithelial tumors; however, its role in hematologic malignancies is unknown. Here we demonstrate that T?RIII protein expression is decreased or lost in the majority of human multiple myeloma specimens. Functionally, restoring T?RIII expression in myeloma cells significantly inhibited cell growth, proliferation, and motility, largely independent of its ligand presentation role. In a reciprocal fashion, shRNA-mediated silencing of endogenous T?RIII expression enhanced cell growth, proliferation, and motility. Although apoptosis was not affected, T?RIII inhibited proliferation through induction of the cyclin-dependent kinase inhibitors p21 and p27. T?RIII further regulated myeloma cell adhesion, increasing homotypic myeloma cell adhesion while decreasing myeloma heterotropic adhesion to bone marrow stromal cells. Mechanistically, live cell imaging of myeloma and stroma cell cocultures revealed that T?RIII-mediated inhibition of heterotropic adhesion was associated with decreased duration of myeloma/bone marrow stromal cell interaction. These results suggest that loss of T?RIII expression during multiple myeloma progression contributes to disease progression through its functional effects on increased cell growth, proliferation, motility, and adhesion.