TβRII Expression Research Articles

Kindlin-2 regulates the oncogenic activities of integrins and TGF-β in triple-negative breast cancer progression and metastasis

Kindlin-2, an adapter protein, is dysregulated in various human cancers, including triple-negative breast cancer (TNBC), where it drives tumor progression and metastasis by influencing several cancer hallmarks. One well-established role of Kindlin-2 involves the regulation of integrin signaling, achieved by directly binding to the cytoplasmic tail of the integrin β subunit. In this study, we present novel insights into Kindlin-2’s involvement in stabilizing the β1-Integrin:TGF-β type 1 receptor (TβRI) complexes, acting as a physical bridge that links β1-Integrin to TβRI. Loss of Kindlin-2 results in the degradation of this protein complex, leading to the inhibition of downstream oncogenic pathways. We used a diverse range of in vitro assays, including CRISPR/Cas9 gene editing, cell migration, 3D-tumorsphere formation and invasion, solid binding, co-immunoprecipitation, cell adhesion and spreading assays, as well as western blot and flow cytometry analyses, utilizing MDA-MB-231 and 4T1 TNBC cell lines. Additionally, preclinical in vivo mouse models of TNBC tumor progression and metastasis were employed to substantiate our findings. Our studies established the direct interaction between Kindlin-2 and β1-Integrin and between Kindlin-2 and TβRI. Disruption of these interactions, via CRISPR/Cas9-mediated knockout of Kindlin-2, led to the degradation of β1-Integrin and TβRI, resulting in the inhibition of oncogenic pathways downstream of both proteins, subsequently hindering tumor growth and metastasis. Treatment of Kindlin-2-deficient cells with the proteasome inhibitor MG-132 restored the expression of both β1-Integrin and TβRI. Furthermore, the rescue of Kindlin-2 expression reinstated their oncogenic activities in vitro and in vivo, while Kindlin-2 lacking domains involved in the interaction of Kindlin-2 with β1-Integrin or TβRI did not. This study identifies a novel function of Kindlin-2 in stabilizing the β1-Integrin:TβRI complexes and regulating their downstream oncogenic signaling. The translational implications of these findings are substantial, potentially unveiling new therapeutically targeted pathways crucial for the treatment of TNBC tumors.

Abstract 4381: Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC

Abstract Background: Kindlins are a small gene family of FERM domain-containing adaptor proteins that function as essential drivers of integrin activation. Kindlin-2 (K2) is the most widely expressed member of the Kindlin family; its homozygous deletion in mice is embryonically lethal, K2 expression is also dysregulated in several human cancers, including the breast. We established K2 as a major driver of TNBC tumors progression and metastasis through the regulation of several hallmarks of cancer. A well-established pathway whereby K2 regulates TNBC oncogenic behavior is through the regulation of integrin inside-out signaling by directly binding to the cytoplasmic tail of integrin β subunit. Interestingly, our recent studies found K2 to play a major role in activating the TGF-β-mediated regulation of the CSF1/EGF signaling through macrophage polarization to the M2 tumorigenic state and their increased tumor infiltration, as well as inhibiting tumor infiltration of cytotoxic lymphocytes, therefore, driving tumor immune evasion. Here we present novel findings involving K2 in the stabilization of the β1-Integrin:TβRI complexes by establishing a physical bridge that links β1-Integrin to TβRI. Loss of K2 results in the degradation of this protein complex and therefore, the inhibition of the oncogenic pathways downstream of these two major regulators of several hallmarks of cancer. Methods: We applied a combination of in vitro assays; CRISPR/Cas9 gene editing, cell migration, 3D tumor-sphere, solid binding, co-immunoprecipitation, cell adhesion and spreading, as well as western blot and flow cytometry analyses with MDA-MB-231 and 4T1 TNBC cell lines. We also used preclinical in vivo mouse models of TNBC tumor progression and metastasis to support or investigations. Results: We found that the K2/β1-Integrin direct interaction is mediated through the C-terminal F3 domain of K2, while the K2/TβRI direct interaction is mediated through the F2 domain of K2. Disruption of this bridge, via CRISPR/Cas9-mediated knockout of K2, leads to β1-Integrin and TβRI degradation, and inhibition of the oncogenic pathways downstream of both β1-Integrin and TβRI, and inhibited tumor growth and metastasis in levels comparable to loss of expression of either β1-Integrin and TβRI. Treatment of the K2-deficent cells with the proteasome inhibitor MG-132 restored expression of both β1-Integrin and TβRI, confirming that K2 is required for the stabilization of the β1-integrin/TβR1 complexes at the protein level. Rescue of K2 expression in the K2-KO cells restored their oncogenic activities both in vitro and in vivo. Conclusion: We identified K2 to play a novel and major in the stabilization of the β1-integrin/TβR1 complexes and the regulation of their downstream oncogenic signaling. These studies have a significant translational impact as they may identify new therapeutically targeted pathways that are desperately needed for the treatment of TNBC tumors. Citation Format: Neelum A. Yousaf Zai, Lamyae El Khalki, Wei Wang, Justin Szpendyk, Khalid Sossey-Alaoui. Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4381.

Androgen receptor inhibition suppresses anti-tumor neutrophil response against bone metastatic prostate cancer via regulation of TβRI expression

Bone metastatic disease of prostate cancer (PCa) is incurable and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone neutrophils initially inhibit bone metastatic PCa growth yet metastatic PCa becomes resistant to neutrophil response. Further, neutrophils isolated from tumor-bone lost their ability to suppress tumor growth through unknown mechanisms. With this study, our goal was to define the impact of metastatic PCa on neutrophil function throughout tumor progression and to determine the potential of neutrophils as predictive biomarkers of metastatic disease. Using patient peripheral blood polymorphonuclear neutrophils (PMNs), we identified that PCa progression dictates PMN cell surface markers and gene expression, but not cytotoxicity against PCa. Importantly, we also identified a novel phenomenon in which second generation androgen deprivation therapy (ADT) suppresses PMN cytotoxicity via increased transforming growth factor beta receptor I (TβRI). High dose testosterone and genetic or pharmacologic TβRI inhibition rescued androgen receptor-mediated neutrophil suppression and restored neutrophil anti-tumor immune response. These studies highlight the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa.

Ubiquitin-specific peptidase 11 promotes development of keloid derived fibroblasts by de-ubiquitinating TGF-β receptorII

BackgroundKeloid scars occur as a result of abnormal wound healing caused by trauma or inflammation of the skin. The progression of keloids is dependent on genetic and environmental influences. The incidence is more prevalent in people with darker skin tones (African, Asian and Hispanic origin). Studies have demonstrated that transforming growth factor (TGF) β/Smad signalling has an essential function in keloid as well as that USP11 could modulate the activation of TGFβ/Smad signalling and impact the progression of the fibrotic disease. Nonetheless, the potential mechanisms of USP11 in keloid were still unclear. The authors postulated that USP11 up-regulates and augments the ability of proliferation, invasion, migration and collagen deposition of keloid-derived fibroblasts (KFBs) through deubiquitinating TGF-β receptor II (TβRII). MethodsFibroblast cells were isolated from keloid scars in vitro. Lentivirus infection was utilized to knockdown and over-express the USP11 in KFBs. Influence of USP11 on proliferation, invasion and migration of KFBs, and expression level of TβRII, Smad2, Smad3, α-SMA, collagen1 and collagen3 were assayed by CCK8, scratching, transwell, Western blot and real-time quantitative polymerase chain reaction. The interactions between USP11 and TβRII were examined using ubiquitination assays and co-immunoprecipitation. To further confirm the role of USP11 in keloid growth, we performed animal experiments. ResultsResults show that down-regulated USP11 markedly suppressed the ability of proliferation, invasion and migration of keloid derived-fibroblasts in vitro and reduce the expression of TβRII, Smad2, Smad3, αSMA, collagen1 and collagen3. In addition, over-expression of USP11 demonstrated the contrary tendency. Ubiquitination experiments and co-immunoprecipitation demonstrated that USP11 was interacting with TβRII and deubiquitinated TβRII. Interferences with USP11 inhibited growth of keloid in vivo. Additionally, we have verified that knockdown of USP11 has no significant effect on normal skin fibroblasts. ConclusionUSP11 elevates the ability of proliferation, collagen deposition, invasion and migration of keloid–derived fibroblasts by deubiquitinating TβRII.

Sildenafil Inhibits the Growth and Epithelial-to-mesenchymal Transition of Cervical Cancer via the TGF-β1/Smad2/3 Pathway.

The study aims to explore new potential treatments for cervical cancer. Cervical cancer is the second most common cancer in women, causing >250,000 deaths worldwide. Patients with cervical cancer are mainly treated with platinum compounds, which often cause severe toxic reactions. Furthermore, the long-term use of platinum compounds can reduce the sensitivity of cancer cells to chemotherapy and increase the drug resistance of cervical cancer. Therefore, exploring new treatment options is meaningful for cervical cancer. The present study was to investigate the effect of sildenafil on the growth and epithelial-tomesenchymal transition (EMT) of cervical cancer. HeLa and SiHa cells were treated with sildenafil for different durations. Cell viability, clonogenicity, wound healing, and Transwell assays were performed. The levels of transforming growth factor-β1 (TGF-β1), transforming growth factor-β type I receptor (TβRI), phosphorylated (p-) Smad2 and p-Smad3 in cervical cancer samples were measured. TGF-β1, Smad2 or Smad3 were overexpressed in HeLa cells, and we measured the expression of EMT marker proteins and the changes in cell viability, colony formation, etc. Finally, HeLa cells were used to establish a nude mouse xenograft model with sildenafil treatment. The survival rate of mice and the tumor size were recorded. High concentrations of sildenafil (1.0-2.0 μM) reduced cell viability, the number of HeLa and SiHa colonies, and the invasion/migration ability of HeLa and SiHa cells in a dose- and time-dependent manner. The expression of TGF-β1, TβRI, p-Smad2 and p-Smad3 was significantly enhanced in cervical cancer samples and cervical cancer cell lines. Sildenafil inhibited the expression of TGF-β1-induced EMT marker proteins (Snail, vimentin, Twist, E-cadherin and N-cadherin) and p-Smad2/3 in HeLa cells. Overexpression of TGF-β1, Smad2, and Smad3 reversed the effect of sildenafil on EMT, viability, colony formation, migration, and invasion ability of HeLa cells. In the in vivo study, sildenafil significantly increased mouse survival rates and suppressed xenograft growth. Sildenafil inhibits the proliferation, invasion ability, and EMT of human cervical cancer cells by regulating the TGF-β1/Smad2/3 pathway.

Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism.

Expression of the nerve growth factor NGF is increased in pulmonary hypertension (PH). We have here studied whether oxidative stress and inflammation, two pathological conditions associated with transforming growth factor-β1 (TGF-β1) in PH, may trigger NGF secretion by pulmonary arterial (PA) cells. Effects of hydrogen peroxide (H2O2) and interleukin-1β (IL-1β) were investigated ex vivo on rat pulmonary arteries, as well as in vitro on human PA smooth muscle (hPASMC) or endothelial cells (hPAEC). TβRI expression was assessed by Western blotting. NGF PA secretion was assessed by ELISA after TGF-β1 blockade (anti-TGF-β1 siRNA, TGF-β1 blocking antibodies, TβRI kinase, p38 or Smad3 inhibitors). TβRI PA expression was evidenced by Western blotting both ex vivo and in vitro. H2O2 or IL-1β significantly increased NGF secretion by hPASMC and hPAEC, and this effect was significantly reduced when blocking TGF-β1 expression, binding to TβRI, TβRI activity, or signaling pathways. In conclusion, oxidative stress and inflammation may trigger TGF-β1 secretion by hPASMC and hPAEC. TGF-β1 may then act as an autocrine factor on these cells, increasing NGF secretion via TβRI activation. Since NGF and TGF-β1 are relevant growth factors involved in PA remodeling, such mechanisms may therefore be relevant to PH pathophysiology.

Novel Soy Peptide CBP: Stimulation of Osteoblast Differentiation via TβRI-p38-MAPK-Depending RUNX2 Activation.

DEDEQIPSHPPR, the calcium-binding peptide (CBP) identified in soy yogurt, was proven to be a potential cofactor in osteoporosis prevention in our previous study, but the mechanism was unknown. In this study, the activity of alkaline phosphatase (ALP) and osteocalcin (OCN), the regulation of RUNX2, and the expression of TβRI were investigated to elucidate the underlying mechanism. The results show that CBP upregulated ALP activity and OCN concentration and increased the expression of RUNX2 and the activation of the MAPK signaling pathway. Similarly, the expression of osteogenesis-related genes in osteoblasts also increased upon CBP treatment. Moreover, the CBP-induced enhancement of ALP activity and phosphorylation levels in the p38 pathway was inhibited by treatment with a p38 inhibitor (SB203538) and TβRI inhibitor (SB431542), respectively, suggesting that p38 and TβRI were involved in the osteogenic action. Based on the signaling pathways, the intracellular calcium concentration was significantly elevated by CBP, which was correlated with the increased behavioral functions and the relative fluorescence intensity of the bone mass. These findings suggest that CBP stimulates osteoblast differentiation and bone mineralization through the activation of RUNX2 via mechanisms related to the TβRI-p38-MAPK signaling pathways, further highlighting CBP’s important potential for treating osteoporosis.

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation.

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.

USP15 Enhances the Proliferation, Migration, and Collagen Deposition of Hypertrophic Scar-Derived Fibroblasts by Deubiquitinating TGF-βR1 In Vitro.

Background:Hypertrophic scar is a fibroproliferative disorder caused by skin injury. The incidence of hypertrophic scar following trauma or burns is 40 to 70 percent or 70 percent, respectively. It has been shown that transforming growth factor (TGF) β1/Smad signaling plays a crucial role in hypertrophic scar, and that USP15 can regulate the activity of TGFβ1/Smad signaling to affect the progression of the disease. However, the underlying mechanism of USP15 in hypertrophic scar remains unclear. The authors hypothesized that USP15 was up-regulated and enhanced the proliferation, migration, invasion, and collagen deposition of hypertrophic scar–derived fibroblasts by deubiquitinating TGF-β receptor I (TβRI) in vitro.Methods:Fibroblasts were isolated from human hypertrophic scars in vitro. The knockdown and overexpression of USP15 in hypertrophic scar–derived fibroblasts were performed using lentivirus infection. The effect of USP15 on hypertrophic scar–derived fibroblast proliferation, migration, and invasion, and the expression of TβRI, Smad2, Smad3, α-SMA, COL1, and COL3, were detected by Cell Counting Kit-8, scratch, invasion, quantitative real-time polymerase chain reaction, and Western blot assays. The interaction between USP15 and TβRI was detected by co-immunoprecipitation and ubiquitination assays.Results:The authors demonstrated that USP15 knockdown significantly inhibited the proliferation, migration, and invasion of hypertrophic scar–derived fibroblasts in vitro and down-regulated the expression of TβRI, Smad2, Smad3, α-SMA, COL1, and COL3; in addition, USP15 overexpression showed the opposite trends (p < 0.05). Co-immunoprecipitation and ubiquitination assays revealed that USP15 interacted with TβRI and deubiquitinated TβRI.Conclusion:USP15 enhances the proliferation, migration, invasion, and collagen deposition of hypertrophic scar–derived fibroblasts by deubiquitinating TβRI in vitro.

SHOX2 promotes migration, invasion and stemness of bladder cancer cells in vitro

To explore the role of human short stature homeobox 2 (SHOX2) in regulating the migration, invasion and stemness of human bladder cancer cells. We analyzed SHOX2 gene expression in bladder cancer and adjacent tissues based on TCGA database. Univariate survival analysis of SHOX2 gene expression in TCGA-BLCA data was performed using GEPIA. The probable function of SHOX2 was predicted using GSEA. Human bladder cancer T24 cell models of SHOX2 knockdown or overexpression were assessed for changes in migration and invasion abilities using wound healing assay and Transwell assay, and their cancer stem cell-like characteristics were evaluated using tumorsphere formation assay and colony formation assay. Western blotting was used to detect the expressions of epithelial mesenchymal transition (EMT) markers Ecadherin and vimentin and the TGF-β signaling network component TβR-I in the cells. SHOX2 expression was significantly higher in bladder cancer tissues than in the adjacent tissues (P < 0.05), especially in paired tissue specimens (P < 0.01), and was negatively correlated with the overall survival of the patients (P < 0.05). SHOX2 gene expression was correlated positively with EMT-related (P < 0.05) and stemness-related gene signatures (P < 0.01). In T24 cells, SHOX2 knockdown significantly suppressed cell migration and invasion, which was significantly enhanced by SHOX2 overexpression (P < 0.01). The cancer stem cell-like characteristics of T24 cells was repressed by SHOX2 knockdown but significantly enhanced by SHOX2 overexpression (P < 0.01). SHOX2 knockdown induced morphological changes of the cells into epithelioid cells, whereas SHOX2 overexpression induced a mesenchymal morphology of the cells. SHOX2 knockdown increased E-cadherin expression and decreased vimentin and TβR-I expression, while SHOX2 overexpression increased the expressions of vimentin and TβR-I in the cells. SHOX2 promotes the migration, invasion and stemness of human bladder cancer cells possibly by regulating EMT via the TGF-β signaling pathway.

Overexpression of TGF-β1 and SDF-1 in cervical cancer-associated fibroblasts promotes cell growth, invasion and migration

The purpose of this study was to investigatethe effect of overexpression of transforming growth factor β1 (TGF-β1) and stromal cell-derived factor 1 (SDF-1) in cervical cancer-associated fibroblasts (CAFs) on regulating cell growth, invasion and migration. CAF cells and normal fibroblast cells (NFs) were obtained from patients with cervical squamous cell carcinoma andmultiple uterine leiomyomas, respectively. Immunofluorescence assay and western blot were used to determine the expression of Vimentin andα-smooth muscle actin (α-SMA). CCK-8 assay was used to detect cell viability.Giemsa dyer was used to detect the colony formation. Flow cytometry was used to detect the growth state of cells. Transwell assayswereused to detect the migration and invasion. Vimentin and α-SMAexpressionin CAFs were significantly increased than those in NFs.In addition, TGF-β1 and SDF-1 expression were notably increased,and transforming growth factor beta receptor 2 (TβRII) expression wasmarkedly decreased in CAF cellsthan those in NFs. Similarly, TGF-β1 and SDF-1expressionin the co-culture of CAFs and Hela cells were significantly increased,and cell proliferation, migration, invasion, colony formation and cell cycle progression were also promoted, while cell apoptosis was decreased.Those phenomena were reversed in the co-culture system with neutralizing antibodies to TGF-β1 and SDF-1.Furthermore, exogenous TGF-β1and SDF-1enhanced proliferation, colony formation, cell cycle progression, migration and invasion while decreased apoptosis of cells. These phenomena were also reversed by the addition of neutralizing antibodiesto TGF-β1 and SDF-1. Overexpression ofTGF-β1and SDF-1in CAFscan promote the growth, invasion and migration of cervical cancer cells.

Garcimultiflorone K from Garcinia multiflora attenuates hepatocellular carcinoma metastasis by suppressing transforming growth factor-β signaling

Transforming growth factor‑β (TGF-β) signaling is a crucial inducer of tissue fibrosis and extracellular matrix accumulation and a vital suppressor of epithelial cell proliferation and cancer metastasis. The nature of this multifunctional cytokine has prompted the development of TGF-β signaling inhibitors as therapeutic agents. Our research group has recently isolated the polyprenylated polycyclic acylphloroglucinol garcimultiflorone K (GMK) from the stems of Garcinia multiflora; GMK exhibits antiangiogenic activity in endothelial cells. In the current study, we aimed to explore the antitumor effect and detailed mechanisms of Garcimultiflorone K in hepatocellular carcinoma cells. Cell proliferation and viability were evaluated using the MTT assay. The migratory ability of HepG2 cells was measured using wound healing assays. The inhibitory effect of GMK against the nuclear translocation of Smad by TGF-β was assessed through immunofluorescence staining and Western blotting. To investigate TGF-β-dependent gene expression profiles upon GMK stimulation, RNA transcript levels were determined using reverse transcription polymerase chain reaction. The effects of GMK in Smad2-driven transcriptomic activities were studied using a reporter gene assay. Protein levels were detected using Western blotting. Our data revealed that GMK inhibited TGF-β-induced cellular responses, including Smad protein phosphorylation, cell migration, and extracellular matrix production, during epithelial-mesenchymal transition (EMT). Mechanistic studies further demonstrated that GMK suppressed TGF-β signaling by downregulating TGF-β receptor II (TβRII). These findings elucidate that TβRII expression in hepatic cells can be specifically suppressed by GMK to attenuate metastasis and the disease-promoting effects of EMT, representing a therapeutic approach.

EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-\u03b2/SMAD signalling

BackgroundEukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD).MethodsImmunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP).ResultsIn this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD.ConclusionsThese findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

Up-regulation of TβRIII facilitates the osteogenesis of supraspinous ligament-derived fibroblasts from patients with ankylosing spondylitis.

Spinal supraspinous ligament (SL) osteogenesis is the key risk of ankylosing spondylitis (AS), with an unclear pathogenesis. We previously found that transforming growth factor β1 (TGF‐β1), bone morphogenetic proteins (eg BMP2) and type III TGF‐β1 receptor (TβRIII) expression were markedly up‐regulated in AS‐SLs. However, the roles of these closely related molecules in AS are unknown. Here, we showed that BMP2, TGF‐β1, TβRIII and S100A4 (a fibroblast marker) were abundant in active osteogenic AS‐SL tissues. In vitro, AS‐SL fibroblasts (AS‐SLFs) showed high BMP2, TGF‐β1 and TβRIII expression and auto‐osteogenic capacity. We further evaluated the role of TβRIII in the osteogenesis of normal SLFs. BMP2 combined with TGF‐β1 induced the osteogenesis of TβRIII‐overexpressing SLFs, but the activity was lost in SLFs upon TβRIII knockdown. Moreover, our data suggested that BMP2 combined with TGF‐β1 significantly activated both TGF‐β1/Smad signalling and BMP2/Smad/RUNX2 signalling to induce osteogenesis of SLFs with TβRIII up‐regulation. Furthermore, our multi‐strategy molecular interaction analysis approach indicated that TGF‐β1 presented BMP2 to TβRIII, sequentially facilitating BMP2 recognition by BMPR1A and promoting the osteogenesis of TβRIII‐overexpressing SLFs. Collectively, our results indicate that TGF‐β1 combined with BMP2 may participate in the osteogenic differentiation of AS‐SLF by acting on up‐regulated TβRIII, resulting in excessive activation of both TGF‐β1/Smad and BMP2/BMPR1A/Smad/RUNX2 signalling.

Prognostic significance of transforming growth factor β receptor II in clinical stage III breast cancer patients - a pilot study.

Transforming growth factor-β (TGFβ) has a dual function in breast cancer, having a tumor suppressor activity in early carcinomas while enhancing tumor metastasis in advanced breast carcinoma. Consequently, the prognostic role of TGFβ and its signaling cascade in breast cancer remain unclear. To investigate the relationship between TβRII expression, clinic-pathological characteristics, and prognostic significance of TβRII expression in clinical stage III breast cancer. Biopsy from the primary tumor was obtained from 30 newly diagnosed clinical stage III breast cancer patients before receiving any therapy. Expression of TβRII, ER, PR, Her2 and Ki-67 was assessed by immunohistochemistry. TβRII expression was positive in 66.7% of cases and was significantly associated with advanced nodal stage and distant metastases. After a median follow up of 42.3 months, TβRII was associated with poor disease-free survival and it was an independent factor for predicting the poor outcome for breast cancer patients, especially in node positive tumors, ER/PR positive and Her2-negative tumors. These findings suggest the usage of therapeutic drugs that target TGFβ in advanced breast cancer patients may be effective. Nevertheless, blockage of the tumor promoting and sparing of the tumor suppressor effect of TGFβ pathway should be taken into consideration. We suggest that these therapies might have more benefit in ER and PR positive tumors.

Nogo-B receptor is required for stabilizing TGF-β type I receptor and promotes the TGF-β1-induced epithelial-to-mesenchymal transition of non-small cell lung cancer.

Background and Objective: Metastasis is the leading cause of death in patients with advanced non-small cell lung cancer (NSCLC), and epithelial-mesenchymal transition (EMT) is a crucial event in the metastasis of NSCLC. Our previous works demonstrated that NgBR promoted EMT in NSCLC. However, the molecular mechanism was unclear.Methods: TGF-β1 was used to induce EMT process of NSCLC cells. The biological functions of NgBR in promoting TGF-β1-induced NSCLC metastasis were studied by gain- and loss-of-function assays both in vitro and in vivo. The underlying mechanisms were studied using molecular biology assays.Results: We found that knockdown of NgBR inhibited TGF-β1-induced cell migration and invasion in NSCLC cells. In contrast, NgBR overexpression promoted TGF-β1-induced EMT of A549 cells. Mechanically, we found that knockdown of NgBR facilitated ubiquitination and degradation of TβRI, leading to downregulation of TβRI expression in NSCLC cells. Moreover, we confirmed a positive correlation between NgBR and TβRI in NSCLC tissues.Conclusions: Our findings provide a novel role of NgBR in modulating TGF-β1-induced EMT and propose NgBR as a new therapeutic target for treating NSCLC patients.

Clinicopathological and prognostic significance of ubiquitin‐specific peptidase 15 and its relationship with transforming growth factor‐β receptors in patients with pancreatic ductal adenocarcinoma

Ubiquitin-specific peptidase 15 (USP15) has been correlated to aggressive oncogenic behavior in several types of carcinomas, but its function in pancreatic ductal adenocarcinoma (PDAC) has not been clarified. This study aimed to evaluate the clinicopathological and prognostic value of USP15 and its relationship with transforming growth factor-β (TGF-β) receptors (TβRs) in PDAC. By immunohistochemical staining of tissue microarrays, the expression patterns of USP15 and TβRs were retrospectively analyzed in 287 PDAC patients who underwent radical surgical resection without neoadjuvant therapy. Cancer-specific survival was compared based on USP15 expression, and the correlations between USP15 and TβRs were analyzed. Ubiquitin-specific peptidase 15 expression in tumor tissues was significantly higher than that in para-tumor tissues (P<0.0001), and high USP15 expression was associated with the pathological N (pN) stage (P=0.033). In addition, high USP15 expression was significantly associated with shorter cancer-specific survival (P=0.019). Univariate analyses showed that high USP15 expression (P=0.024), a poor histopathological grade (P=0.003), and the pN1 stage (P=0.009) were significantly correlated with shorter survival. Although the independent prognostic value of USP15 alone was not established, the combination of USP15 and the histological grade was identified as an independent prognostic factor in multivariate analyses (P=0.015). USP15 expression was correlated with TβR-I, TβR-II, or TβR-III expression in PDAC. High USP15 expression is a potential prognostic indicator in patients with PDAC, and it might affect the TGF-β signaling pathway in PDAC.

Hydroxypropyl‑β‑cyclodextrin attenuates the epithelial‑to‑mesenchymal transition via endoplasmic reticulum stress in MDA‑MB‑231 breast cancer cells.

The epithelial-to-mesenchymal transition (EMT) has been reported to serve vital roles in regulating the progress of cancer metastasis. In addition, lipid rafts enriched in sphingolipids and cholesterol serve important roles in physiological and biochemical processes as a signaling platform. The present study explored the effects of hydroxypropyl-β-cyclodextrin (HP-β-CD), a cholesterol-depleting agent of lipid rafts, on the transforming growth factor (TGF)-β/Smad signaling pathway and endoplasmic reticulum (ER) stress in mediating EMT in MDA-MB-231 breast cancer cells. HP-β-CD treatment inhibited TGF-β1-induced EMT, based on increased expression of E-cadherin and decreased expression of vimentin. HP-β-CD reduced the expression of the TGF receptor TβRI and blocked the phosphorylation of Smad2. In addition, HP-β-CD increased the expression of ER stress-related proteins (binding immunoglobulin protein and activating transcription factor 6), but TGF-β1 could reverse these changes. Sodium 4-phenylbutyrate, an inhibitor of ER stress, suppressed these effects of HP-β-CD on EMT and TGF-β/Smad signaling pathway inhibition in breast cancer cells. Thus, HP-β-CD can block the TGF-β/Smad signaling pathway via diminishing the expression of TβRI which helps to activate ER stress and attenuate EMT in MDA-MB-231 cells, highlighting a potential target of lipid rafts for breast cancer treatment.

Increasing the effectiveness of tyrosine kinase inhibitor (TKI) in combination with a statin in reducing liver fibrosis.

It has been shown that both nilotinib as a tyrosine kinase inhibitor, and atorvastatin as a rho-kinase inhibitor, have antifibrotic effects. Therefore, considering the relationship between these two pathways, this study aimed to investigate the effects of their co-treatment against hepatic stellate cells (HSCs) activation and liver fibrosis. For this purpose, the activation of HSCs coincided with these therapies. Also, liver fibrosis by carbon tetrachloride (CCl4 ) was induced in male Wistar rats and treated simultaneously with these compounds. The expression of alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), Ras homolog gene family, and member A (RhoA)/Rho-associated protein kinase (ROCK) in HSCs were measured. The expression of transforming growth factor beta-1 (TGF-β1), its receptor (TβRII), CTGF, and platelets derived growth factor (PDGF), in the livers, were also investigated, all by real-time PCR and western blot analysis. Also, histopathologic and immunohistochemical evaluations were performed to evaluate changes in liver fibrosis during treatment. The results indicated the down-regulation of RhoA/ROCK, CTGF, and α-SMA, and inhibition of the HSCs activation toward myofibroblasts. The results also showed that the combined use of atorvastatin and nilotinib has significantly higher inhibitory effects. The antifibrotic effects of atorvastatin and nilotinib co-administration were also observed by histopathologic and immunohistochemical observations, and inhibiting the expression of TGF-β1, TβRII, CTGF, and PDGF. Taken together, this study revealed that co-administration of nilotinib-atorvastatin has novel antifibrotic effects, by inhibiting RhoA/ROCK, and CTGF pathway. Therefore, the importance of the common pathway of RhoA/ROCK and CTGF, in reducing fibrosis may almost be concluded.

Berberine inhibits epithelial-mesenchymal transition and promotes apoptosis of tumour-associated fibroblast-induced colonic epithelial cells through regulation of TGF-β signalling

Tumour-associated fibroblasts (TAFs) mediate the differentiation of adjacent stromal cells. Berberine (BBR), a monomer of traditional Chinese herbs, exhibits a potent therapeutic effect against cancer. However, the effects of BBR on the differentiation of normal colonic epithelial cells induced by TAFs have not been determined. In the present study, we selected the TAF-like myofibroblast cell line CCD-18Co. CCD-18Co-derived conditioned medium (CM) and co-culture induced epithelial-mesenchymal transition (EMT) changes in colonic epithelial HCoEpiC cells with decreased E-cadherin and increased vimentin and α-SMA expression. In addition, CCD-18Co stimulated the expression of ZEB1 and Snail and promoted motility. We used LY364947, a TGF-β receptor kinase type I (TβRI) inhibitor, and BBR. Our results showed that LY364947 and BBR inhibited these phenomena. BBR decreased the expression of ZEB1 and Snail, and this effect was concentration dependent. BBR also downregulated the expression of TβRI, TβRII, Smad2/p-Smad2 and Smad3/p-Smad3. In addition, BBR induced apoptosis in EMT-like HCoEpiC cells in a concentration-dependent manner with upregulation of Bax and downregulation of Bcl-2. However, VX-702, an inhibitor of p38 MAPK, significantly suppressed the apoptosis rate. BBR promoted the expression of p38 MAPK and phosphorylated p38 MAPK. In conclusion, berberine inhibits EMT and promotes apoptosis in TAF-induced colonic epithelial cells through mediation of the Smad-dependent and SMAD-independent TGF-β signalling pathways.