Cancer-associated fibroblast-derived Gremlin 1 promotes breast cancer progression

Background: Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. Methods: In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. Results: Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer ce

Ras enhances TGF-β signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1

Abstract Background Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial. Methods Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion. Results Ras interacts with the SPSB1’s SPRY domain to enhance TGF-β signaling. Ras interacts and colocalizes with the TGF-β type II receptor’s (TβRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion. Conclusion Ras positively cooperates with TGF-β signaling by reducing the cellular protein levels of TβRII negative regualtor SPSB1

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Abstract Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-β signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-β biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-β signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV’s unique role, TGF-β signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-β signaling to nearly-normal levels. Otherwise, TGF-β signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-β signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-β signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-β signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-β biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-β signaling levels in breast cancer cells

Additional file 2: of Ras enhances TGF-β signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1

Figure S7. v-Ha-Ras N85A and v-Ha-Ras N86A increase the degradation rate of SPSB1. 293 T cells were co-transfected with indicated FLAG-SPSB1 and v-Ha-Ras/v-Ha-Ras mutant/pcDNA3 control vector for 24 h, then cells were treated with TGF-β (2 ng/ml). 36 h post-transfection, cells were exposed to cycloheximide (20 μg/ml) for indicated periods and lysed. Whole cell lysates were then examined for indicated proteins by immunoblotting (IB). Results are representative of experiments repeated at least once. Figure S8. TGF-β receptors levels regulate TGF-β signaling sensitivity and duration. MDCK cells were co-transfected with pCAGA-luc and indicated TβRII and/or TβRI and/or pcDNA3 control vector. 24 h later, cells were treated with ± TGF-β at indicated concentration for a further 24 h and lysed. Luciferase activity was determined as desribed in Fig. 6. Data are expressed as mean relative Smad3 luciferase activity (fold-induction) and error bars represent S.D. from representative experiments performed 3 times. * P < 0.05. Figure S9 & 10. Induced expression of SPSB1 suppresses TGF-β signaling in Ras transformed 21D1 cells through destabilizing TβRII. Doxycycline inducible FLAG-SPSB1 21D1 cells were cultured in ± doxycycline (2 μg/ml) for 2 (S.10) or 7 days (S.9). Whole cell lysates (S.9) were then examined for indicated proteins by immunoblotting (IB). Cells (S.10) were then transfected with pCAGA-luc. 24 h post-transfection, cells were treated with ± TGF-β (0.2 ng/ml) for a further 24 h and lysed. Luciferase activity was determined as desribed in Fig. 6. Data are expressed as mean relative Smad3 luciferase activity (fold-induction) and error bars represent S.D. from representative experiments performed 3 times. * P < 0.05. In all case, each experiment was repeated at least once, one representing result is showing. Figure S11 & 12. FLAG-SPSB1 and eGFP co-expression in the cells. 293 T cells (S.11) and 21D1 cells (S.12) were co-transfected with eGFP construct and FLAG-SPSB1/MYC-SPSB1 Y129A as indicated for 48 h. Fixed cells were stained with Hoechst dye. FLAG-SPSB1 was immunostained with mouse anti-FLAG antibody followed by Alexa546-conjugated secondary anti-mouse IgG. MYC-SPSB1 Y129A was immunostained with mouse anti-MYC antibody followed by Alexa546-conjugated secondary anti-mouse IgG. The expression of SPSB1/SPSB1 mutant (red) and eGFP was analyzed by fluorescent microscope (magnification = 20×) in 4 random fields. (PPT 3720 kb

Additional file 1: of Ras enhances TGF-β signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1

Figure S1. EGF stimulation has no effect on SPSB1 protein degradation. 293 T cells were transfected with FLAG-SPSB1. 40 h post-transfection, cells were exposed to cycloheximide (20 μg/ml) for indicated periods with or without EGF (50 μg/ml, 5 mins pretreated) and lysed. Cell lysates were examined for indicated proteins by immunoblotting (IB). Results are representative of experiments repeated at least once. Figure S2. EGF stimulation does not alter the interaction between endogenouse Ras and SPSB1. 293 T cells were transfected with FLAG-SPSB1. 48 h post-transfection, indicated cells were stimulated with EGF (50 μg/ml) for 10 min and lysed. Thereafter, cell lysates were immunoprecipitated (IP) with anti-Ras antibody conjugated with protein G beads. Both whole cell lysates and immunoprecipatates were examined for indicated proteins by immunoblotting (IB). Results are representative of experiments repeated at least once. Figure S3–6. v-Ha-Ras N85A, v-Ha-Ras N86A and v-Ha-Ras D120A, R124A mutants do not disrupt their ability to interact with SPSB1. 293 T cells (S.3, 4, 5, 6) were transfected with indicated DNA constructs for 48 h. Thereafter, cell lysates were immunoprecipitated (IP) with anti-SPSB1 anibody (S.4) or anti-MYC antibody (S.5) or anti-Ras antibody (S.6) conjugated with protein G beads. Both whole cell lysates and immunoprecipatates were examined for indicated proteins by immunoblotting (IB). In all case, each experiment was repeated at least once, one representing result is shown. (PPT 3970 kb

Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin

Abstract Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists