TGF-βRII Expression Research Articles

Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human cancers. Transforming Growth Factor Beta (TGF-β) is a cytokine that switches from a tumor-suppressor at early stages to a tumor promoter in the late stages of tumor development, by yet unknown mechanisms. Tumor associated MUC1 is aberrantly glycosylated and overexpressed in >80% of PDAs and is associated with poor prognosis. MUC1 expression is found in the early stages of PDA development with subsequent increase in later stages. Analysis of human PDA samples from TCGA database showed significant differences in gene expression and survival profiles between low and high MUC1 samples. Further, high MUC1 expression was found to positively correlate to TGF-βRII expression and negatively correlate to TGF-βRI expression in PDA cell lines. We hypothesized that MUC1 overexpression induces TGF-β mediated non-canonical signaling pathways which is known to be associated with poor prognosis. In this study, we report that MUC1 overexpression in PDA cells directly activates the JNK pathway in response to TGF-β, and leads to increased cell viability via up-regulation and stabilization of c-Myc. Conversely, in low MUC1 expressing PDA cells, TGF-β preserves its tumor-suppressive function and inhibits phosphorylation of JNK and stabilization of c-Myc. Knockdown of MUC1 in PDA cells also results in decreased phosphorylation of JNK and c-Myc in response to TGF-β treatment. Taken together, the results indicate that overexpression of MUC1 plays a significant role in switching the TGF-β function from a tumor-suppressor to a tumor promoter by directly activating JNK. Lastly, we report that high-MUC1 PDA tumors respond to TGF-β neutralizing antibody in vivo showing significantly reduced tumor growth while low-MUC1 tumors do not respond to TGF-β neutralizing antibody further confirming our hypothesis.

Endoplasmic reticulum stress as a novel target to inhibit transdifferentiation of human retinal pigment epithelial cells.

The epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical event in the pathogenesis of proliferative vitreoretinopathy and neovascular age-related macular degeneration, which are the leading causes of severe vision loss. Endoplasmic reticulum (ER) stress has been implicated in the EMT of many cell types and various ocular diseases. However, the relationship between ER stress and EMT in RPE cells remains unknown. Therefore, in the study, we explored the impact of ER stress on EMT in RPE cells. Different concentrations of tunicamycin (TM) and thapsigargin (TG) were used to induce ER stress in human RPE cells. The expression of epithelial marker, mesenchymal markers and some of genes/proteins involved in TGF-β/Smad signaling were analized by qPCR, western blot or immunostaining at the condition with or without stimulation of TGF-β2 (10 ng/mL). Boyden chamber and scratch assay were used to evaluate the migration of RPE cells, while cell viability and apoptosis of RPE cells were measured by MTT and TUNEL assay, respectively. Treatment of RPE cells with TM and TG (24 h) reduced the expression of α -SMA and FN, and increased the expression of Occludin in a dose dependent manner at protein level, which was highly associated with the expression of GRP78. Treatment with TGF-β2 significantly increased the expression of α-SMA and FN, and decreased the expression of Occludin both in protein and mRNA levels, which was significantly inhibited by a 4h pre-treatment with TM. In addition, the expression of TGF-βRII and Smad2/3, and mRNAs of TGF-βRII and Smad3 were also decreased by the TM treatment. TM-induced ER stress inhibited RPE cell migration, and high concentrations of TM and TG reduced cell viability and induced apoptosis of RPE cells. Chemical induction of ER stress inhibited EMT and migration in RPE cells, possibly by inactivation of TGF-β signaling, suggesting that regulation of ER stress in RPE cells may be a new approach to prevent the development of intraocular fibrosis.

Systematic Pharmacology-Based Strategy to Explore the Molecular Network Mechanism of Modified Taohong Siwu Decoction in the Treatment of Premature Ovarian Failure.

Objective To explore the molecular network mechanism of modified Taohong Siwu Decoction (MTHSWD) to interfere with premature ovarian failure based on systematic pharmacological strategy. Methods The network pharmacology strategy was used to explore the potential mechanism of MTHSWD intervention in POF, and then it was verified through animal experiments. Mouse zona pellucida 3 was used as an antigen to subcutaneously immunize BALB/c female mice to establish an immune POF model. Mice were divided into MTHSWD low-, medium-, and high-dose groups, positive control group, model group, and normal group. After 30 days of drug intervention, ovarian tissue was taken for pathological hematoxylin-eosin (HE) staining, and immunohistochemical methods were used to detect the expression of TGF-β1 and TGF-βRII and Smad2/3 protein expression in follicular wall granular cells and ovarian tissue, respectively. Results Network pharmacology studies have shown that MTHSWD may interfere with the TGF-β signaling pathway. Animal experimental research shows that, compared with the model group, the number of ovarian mature follicles in the MTHSWD groups and the positive group was significantly increased, and the number of atresia follicles decreased. Immunohistochemistry showed that, compared with the control group, the expression of TGF-β1, TGF-βRII, and Smad2/3 in the follicular wall granulosa cells and ovarian tissues of MTHSWD groups was significantly higher than that of the model group (P < 0.05). Conclusion MTHSWD may improve the ovarian function of POF mice by upregulating the protein expression of granulosa cells TGF-β1, TGF-βRII, and Smad2/3.

Transforming growth factor-β receptors mediates matrix degradation and abnormal hypertrophy in T-2 toxin-induced hypertrophic chondrocytes

This study investigated whether transforming growth factor-β receptor I (TGF-βRI) and TGF-βRII mediate matrix degradation and abnormal hypertrophy in T-2 toxin-induced hypertrophic chondrocytes. Hypertrophic chondrocytes were exposed to TGF-βRI and TGF-βRII binding inhibitor (GW788388) for 24 h prior to exposure to different concentrations of T-2 toxin (0, 10, 25, and 50 ng/mL for 48 h). Hypertrophic chondrocytes were assessed based on the expression of matrix-degrading and terminal differentiation-related genes and cell viability. Matrix metalloproteinases (MMPs, MMP-13, MMP-1, and MMP-9) were reduced in the GW788388+T-2 toxin group compared to the T-2 toxin group. The expression of terminal differentiation-related genes (MMP-2, MMP-10, and collagen X) was increased in hypertrophic chondrocytes in the inhibited groups compared to that in the T-2 toxin group. The survival rate of chondrocytes decreased significantly in a dose-dependent manner. GW788388 did not significantly block the reduced cell viability in hypertrophic chondrocytes exposed to T-2 toxin. The upregulated expression of TGF-βRI and TGF-βRII mediates the abnormal chondrocyte hypertrophy and extracellular matrix degeneration observed in T-2 toxin-induced hypertrophic chondrocytes.

T-2 toxin induces articular cartilage damage by increasing the expression of MMP-13 via the TGF-β receptor pathway.

T-2 toxin pre-disposes individuals to osteoarthritis, Kashin-Beck disease (KBD). The major pathological change associated with KBD is the degradation of the articular cartilage matrix. Herein, we investigated the key molecules that regulate T-2 toxin-mediated cartilage degradation. Potential KBD treatments were also investigated. Sprague Dawley rats were divided into the T-2 toxin group and the control group. The T-2 toxin group received 100ng/g BW/day, whereas the control group received a similar dose of PBS. The expression of matrix metalloproteinase-13 (MMP-13) and TGF-β receptor I/II (TGF-βRI/II) was analyzed using immunohistochemical staining. C28/I2 chondrocytes were exposed to TGF-βRI/II binding inhibitor (GW788388) for 24h before incubation in different T-2 toxin concentrations (0, 6, 12, and 24ng/mL for 72h). The expression of mRNA for TGF-βRI/II, MMP-13 and proteins for MMP-13, and Smad-2 in chondrocytes were analyzed using RT-PCR and western blot, respectively. Safranin O staining revealed that T-2 toxin treatment modulated the expression of articular cartilage matrix. On the other hand, T-2 toxin treatment sharply increased the expression of MMP-13, TGF-βRI, and TGF-βRII in the rat cartilages. Interestingly, blocking the TGF-βRs-smad 2 signaling pathway using GW788388 abrogated the effect of T-2 toxin on upregulating MMP-13 expression. The expression of MMP-13 in chondrocytes induced with T-2 toxin is regulated via the TGF-βRs signaling pathway. As such, inhibiting the expression of TGF-βRs is a potential KBD treatment.

BMP15 regulates FSHR through TGF-β receptor II and SMAD4 signaling in prepubertal ovary of Rongchang pigs

Bone morphogenetic protein 15 (BMP15) and follicle-stimulating hormone (FSH) both play important roles in mammalian ovary and follicular development. The aim of the present study is to investigate the effects of BMP15 and FSH in the prepubertal ovary of Rongchang pigs considering a possible signaling mechanism involving TβRII/ SMAD4 and FSHR in granulosa cells. For this purpose, we quantified expression levels of BMP15, SMAD2, SMAD3, SMAD4, SMAD7, TGF-β1, TGF-β2, TGF-β3, TGFβRI, TGFβRII, and FSHR via qRT-PCR at different ages in prepubertal ovaries and cultured biopsy of 90-day-old ovary in Rongchang pig. Additionally, the protein levels of BMP15, FSHR, SMAD2, SMAD4, TGFβRI, TGFβRII, TGF-β1, TGF-β2 were quantified via Western blot and the localizations of BMP15, FSHR and TGFβRII were observed via immunofluorescence confocal microscope. The results showed that expression levels of BMP15, TGF-β1, TGFβRII and FSHR increased significantly at day 60 as compared to day 30 and reached peak value at day 90 in prepubertal ovary of Rongchang pigs. We observed that BMP15, TGFβRII and FSHR was highly presented, which TGFβRII and FSHR displayed co-localization in the follicles of the prepubertal ovaries of 90-day-old Rongchang gilts. Treatment with TGFβRI/II inhibitor LY2109761 significantly decreased the expression of TGFβRI, TGFβRII and SMAD4 and TGFβRI inhibitor LY2157299 decreased TGFβRI, but increased the TGFβRII, SMAD4 and FSHR expression levels. Furthermore, the addition of rBMP15 and rFSH group significantly increased the expression of TGFβRII and FSHR proteins (P < 0.01), but no significant change in the expression of TGFβRI (P > 0.05) was observed by Western blot. In conclusion, BMP15, TGFβRII and FSHR were increased significantly in the prepubertal ovarian follicles of Rongchang pigs and FSHR expression in GCs was regulated by BMP15 and FSH through the TGFβRII.

Dihydroartemisinin Promoted Bone Marrow Mesenchymal Stem Cell Homing and Suppressed Inflammation and Oxidative Stress against Prostate Injury in Chronic Bacterial Prostatitis Mice Model.

Although bone marrow mesenchymal stem cells (BMMSCs) are effective in treating chronic bacterial prostatitis (CBP), the homing of BMMSCs seems to require ultrasound induction. Dihydroartemisinin (DHA) is an important derivative of artemisinin (ART) and has been previously reported to alleviate inflammation and autoimmune diseases. But the effect of DHA on chronic prostatitis (CP) is still unclear. This study aims to clarify the efficacy and mechanism of DHA in the treatment of CBP and its effect on the accumulation of BMMSCs. The experimental CBP was produced in C57BL/6 male mice via intraurethrally administered E. coli solution. Results showed that DHA treatment concentration-dependently promoted the accumulation of BMMSCs in prostate tissue of CBP mice. In addition, DHA and BMMSCs cotreatment significantly alleviated inflammation and improved prostate damage by decreasing the expression of proinflammatory factors such as TNF-α, IL-1β, and chemokines CXCL2, CXCL9, CXCL10, and CXCL11 in prostate tissue of CBP mice. Moreover, DHA and BMMSCs cotreatment displayed antioxidation property by increasing the production of glutathione peroxidase (GSH-Px), SOD, and decreasing malondialdehyde (MDA) expression. Mechanically, DHA and BMMSCs cotreatment significantly inhibited the expression of TGFβ-RI, TGFβ-RII, phosphor (p)-Smad2/3, and Smad4 in a dose-dependent manner while stimulated Smad7 expression in the same manner. In conclusion, our findings provided evidence that DHA effectively eliminated inflammatory and oxidative stress against prostate injury, and this effect involved the TGF-β/Smad signaling pathway in CBP.

Dihydrotestosterone through blockade of TGF-β/Smad signaling mediates the anti-fibrosis effect under hypoxia in canine Sertoli cells

The hypoxic microenvironment of cryptorchidism is an important factor to induce the impairment of the structure and function of Sertoli cells and thus lead to spermatogenesis loss or tumorigenesis. Dihydrotestosterone (DHT), as a potent nonaromatizable 5α-reduced androgen, has both positive and negative effect on pathological fibrosis process. However, it is still unknown whether DHT can regulate hypoxia-induced fibrosis of Sertoli cells. Herein, in this study, we evaluate the DHT level, two 5α-reductase isoforms, 5α-red1 and 5α-red2, as well as HIF-1α expression pattern in canine cryptorchidism and contralateral normal testis. Results showed that the abdominal testes presented low DHT levels and 5α-red1 and 5α-red2 expression, while significantly higher HIF-1α expression and ECM production compared with the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl2), and found that DHT inhibited the fibrosis of Sertoli cells in a dose-dependent manner. Meanwhile, DHT interfered with the TGF-β signaling by reducing the expression of TGF-βRI and TGF-βRII and inhibiting the expression and phosphorylation of Smad2 and Smad3, while flutamide (androgen receptor inhibitor) inhibited these effects of DHT. Furthermore, use of LY2109761 (TGF-β receptor type I/II inhibitor) to interfere with the TGF-β/Smad pathway showed a similar effect with DHT suppression of the fibrosis in Sertoli cells. Our research data demonstrated that cryptorchidism is located in a hypoxic and DHT deficiency microenvironment. Moreover, supplementing DHT can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with AR regulating the inhibition of TGF-β/Smad signaling.

Phylogenetic and functional analysis of TGF-β/Smad2 pathway genes in cotton bollworm, Helicoverpa armigera

• Phylogenetic analysis of TGF-β/Smad2 pathway genes in H. armigera . • Smad2 is activated by TGFβRI, and binds to TGFβRI or Smad4. • TGF-β/Smad2 pathway is involved in pupal development or diapause. Transforming growth factor-beta (TGF-β) signaling pathway plays important roles in embryonic development, cell proliferation and tissue differentiation in vertebrates. Our previous studies demonstrated that TGF-β signal activates Smad1-POU-TFAM and PP2A-Akt pathways to regulate pupal diapause in Helicoverpa armigera . In this study, we investigated the function of TGF-β activates Smad2 pathway in H. armigera . Phylogenetic analysis of H. armigera TGF-β receptor I ( TGFβRI ), Smad2 , Smad4 genes showed high conservation across species. In vitro experiments showed that TGFβRI was localized in the cell membrane where it binds Smad2 leading to the phosphorylation of Smad2. Smad4 was mainly localized in the cytoplasm, and bind to Smad2. Protein expression analysis showed that expression of TGFβRI, Smad4, Smad2, p-Smad2 were lower in diapause-destined pupae compared with nondiapause-destined pupae. Notably, treatment with 20-hydroxyecdysone (20E) increased expression of the above proteins. Inhibition of TGF-β/Smad2 signaling pathway delayed pupal development. These findings indicate that TGF-β/Smad2 pathway is involved in pupal development or diapause in H. armigera .

Circulating Tumor Cells in Desmoid Tumors: New Perspectives.

IntroductionDesmoid tumor (DT) is a rare neoplasm with high local recurrence rates, composed of fibroblastic cells that are characterized by the expression of key molecules, including the intermediate filament vimentin, cyclooxygenase-2 (COX-2), and nuclear β-catenin, and lack of epithelial markers. Circulating tumor cells (CTCs) isolated from the peripheral blood of patients with sarcomas and other neoplasms can be used as early biomarkers of tumor invasion and dissemination. Moreover, CTCs can also re-colonize their tumors of origin through a process of “tumor self-seeding.”ObjectivesWe aimed to identify CTCs in the peripheral blood of patients with DT and evaluate their expression of β-catenin, transforming growth factor receptor I (TGF-βRI), COX-2, and vimentin proteins.Material and MethodsWe conducted a prospective study of patients with initial diagnosis or relapsed DT with measurable disease. Blood samples from each patient were processed and filtered by ISET® (Rarecells, France) for CTC isolation and quantification. The CTC expression of β-catenin, COX-2, TGF-βRI, and vimentin was analyzed by immunocytochemistry (ICC).ResultsA total of 18 patients were included, and all had detectable CTCs. We found a concordance of β-catenin expression in both CTCs and primary tumors in 42.8% (6/14) of cases by using ICC and immunohistochemistry, respectively.ConclusionsOur study identified a high prevalence of CTCs in DT patients. Concordance of β-catenin expression between primary tumor and CTCs brings new perspectives to assess the dynamics of CTCs in the blood compartment, opening new avenues for studying the biology and behavior of DT. In addition, these results open the possibility of using CTCs to predict DT dynamics at the time of disease progression and treatment. Further studies with larger sample sizes are needed to validate our findings.

Alterations of non-coding RNA expression and mitochondrial biogenesis in colorectal cancer tissue: Possible crosstalk with macrophage polarization

Colorectal cancer (CRC) is a common worldwide cancer associated with different genetic changes. The present study aimed to elucidate the possible crosstalk between colorectal cancer-derived non-coding RNAs (ncRNAs), tumor mitochondrial biogenesis, and macrophage polarization in colorectal cancer patients. Fresh tumor tissue samples with adjacent normal tissues were obtained from 62 CRC patients, quantitative real-time PCR (qRT-PCR) was used to detect the tissue expression of long non-coding RNAs (lncRNAs) such as PTENP1, H19, XIST, MALAT1, and HOTAIR as well as microRNA ((miR-133a)), mitochondrial markers as mtDNA copy number and Uncoupling protein 2 (UCP2), macrophage markers (INOS, CD80 and CD163) and other genes as TGFβRII and NRF-2. The expression levels of the 5 studied lncRNAs were upregulated in CRC tissues compared to the adjacent non-cancer tissues. HOTAIR expression was negatively correlated with CD80 (M1-polarization marker) and positively correlated with CD163 (M2-polarization marker) while miR-133a showed opposite association with these macrophage markers. Non-significant increase was observed in the tumor mtDNA copy number which showed a positive correlation with XIST. Moreover, a significant positive correlation was shown between CD163 and UCP2 expressions. Furthermore, TGFβRII expression was directly correlated with some lncRNAs (PTENP1, MALAT1, and HOTAIR) and tumor mtDNA copy number. The study highlighted significant crosstalk between ncRNA (HOTAIR and miR-133a) and M2-polarization. The obtained data suggest the potential roles of HOTAIR, miR-133a, UCP2 and TGFβRII in the development of CRC, suggesting their possible diagnostic and therapeutic potentials in CRC.

Teucrium polium Extract Enhances the Anti-Angiogenic Effect of Tranilast in a Three-Dimensional Fibrin Matrix Model.

Objective:Angiogenesis plays a dominant role in many pathophysiologic disorders, including cancer. Tranilast, which is an anti-fibrotic drug, is also suggested as an anti-angiogenesis agent. As Teucrium polium (TP) is known as an herbal medicine with antitumor properties, this study aimed to investigate the effects of TP and Tranilast on human umbilical vein endothelial cells (HUVECs), in vitro model of angiogenesis, as well as rat’s aortic ring ex vivo model. Methods:In this study, The HUVECs were treated with various doses of TP and Tranilast each one alone or in combination together. Cell survival test, aortic ring ex-vivo assay, and evaluating mRNA expressions of VEGFA and TGF-β ligands and receptors were performed. Results:The survival rate of HUVECs has significantly (p<0.05) reduced by TP and Tranilast. The combination of both TP and Tranilast significantly reduced cell viability as compared to the administration of TP or Tranilast alone. As well, the treatment of HUVECs with TP and/or Tranilast significantly (p<0.05) decreased TGF-β1, TGF-β 2, TGF-βRI, and TGF-βRII mRNA expression levels, but not the expression of TGF-β3 and TGF-βRIII in the TP-treated cells. Image analysis showed that TP and/or Tranilast inhibited vascular growth in the aortic ring assay. Conclusion:Our results strongly support the anti-angiogenic effects of the TP and Tranilast combination on both in vitro and ex vivo models of angiogenesis. However, further investigations in in vivo models and human studies are needed before human use.

Tofacitinib Ameliorates Lupus Through Suppression of T Cell Activation Mediated by TGF-Beta Type I Receptor.

Autoreactive T cells play a crucial role in the pathogenesis of systemic lupus erythematosus (SLE). TGF-β type I receptor (TGFβRI) is pivotal in determining T cell activation. Here, we showed that TGFβRI expression in naïve CD4+ T cells was decreased in SLE patients, especially in those with high disease activity. Moreover, IL-6 was found to downregulate TGFβRI expression through JAK/STAT3 pathway in SLE patients. In vitro, the JAK inhibitor tofacitinib inhibited SLE T cell activating by upregulating TGFβRI expression in a dose-dependent manner. In MRL/lpr mice, tofacitinib treatment ameliorated the clinical indicators and lupus nephritis, as evidenced by reduced plasma anti-dsDNA antibody levels, decreased proteinuria, and lower renal histopathological score. Consistently, tofacitinib enhanced TGFβRI expression and inhibited T cell activation in vivo. TGFβRI inhibitor SB431542 reversed the effects of tofacitinib on T cell activation. Thus, our results have indicated that tofacitinib can suppress T cell activation by upregulating TGFβRI expression, which provides a possible molecular mechanism underlying clinical efficacy of tofacitinib in treating SLE patients.

Inhibiting TGFβ Signaling in CAR T-Cells May Significantly Enhance Efficacy of Tumor Immunotherapy

The use of genetically engineered T cells expressing chimeric antigen receptors (CAR) against CD19 or BCMA to treat hematologic malignancies has demonstrated promising results in clinical trials. However, it is possible that the efficacy of CAR T cells could be limited in some clinical indications due to suppressive factors in the tumor microenvironment. Immunosuppressive cytokines (e.g., IL-6, IL-10 and TGFβ) provide a favorable environment for tumor growth and inhibit the tumoricidal activity of the endogenous T cells. In particular, transforming growth factor β (TGFβ) has been shown to suppress T-cell proliferation, inhibit the maturation of T helper cells and reduce their effector functions. Elevated levels of TGFβ have been detected in many human cancers, including multiple myeloma (MM). Here, we sought to modulate the activity of CAR T cell-based immunotherapy for MM by inhibiting TGFβ signaling in BCMA-specific CAR T cells (Budha et al., Cancer Cell, 2012).We evaluated two strategies to modulate the TGFβ pathway: (1) knocking out TGFβ Receptor II (TGFβRII) using CRISPR gene editing and (2) overexpressing a dominant negative TGFβRII in BCMA targeted CAR T cells. TGFβRII modulation inhibited TGFβ-dependent Smad2/3 phosphorylation. TGFβRII-modulated BCMA CAR T cells were insensitive to TGFβ-mediated inhibition of proliferation, cell cytotoxicity and cytokine production, both IFNg and granzyme B, in the context of primary and/or repeat antigen stimulation with BCMA+ve MM cell lines in vitro . We further evaluated the gene expression signatures of BCMA CAR T cells isolated from tumor and spleen in a NSG mouse xenograft model subcutaneously inoculated with myeloma cell line (RPMI8226). TGFβ pathway gene expression changes indicating TGFβ exposure were observed in BCMA CAR T cells isolated from tumor, but not spleen, suggesting that BCMA CAR T cells are encountering a TGFβ-enriched TME. However, both TGFβ pathway modulating strategies prevented the development of the TGFβ-induced gene expression phenotype.Together, these data suggest that inhibition of TGFβRII signaling may potentiate CAR T activity by directly inhibiting immunosuppressive effects of TGFβ in the tumor microenvironment. By specifically targeting TGFβ signaling in CAR T cells, it may be possible to avoid side effects associated with systemic administration of TGFβ antagonists. Gene editing to remove TGFβRII expression or overexpressing dominant negative TGFβ RII could modulate the activity of BCMA CAR T therapy in MM. DisclosuresVong:Juno Therapeutics: Employment, Equity Ownership. Nye:Juno Therapeutics: Employment, Equity Ownership. Hause:Juno Therapeutics: Employment, Equity Ownership. Clouser:Juno Therapeutics: Employment, Equity Ownership. Jones:Juno Therapeutics: Employment, Equity Ownership. Burleigh:Juno Therapeutics: Employment, Equity Ownership. Borges:Editas Medicine: Employment, Equity Ownership. Chin:Editas Medicine: Employment, Equity Ownership. Marco:Editas Medicine: Employment, Equity Ownership. Barrera:Editas Medicine: Employment, Equity Ownership. Da Silva:Editas Medicine: Employment, Equity Ownership. Harbinski:Editas Medicine: Employment, Equity Ownership. Giannoukos:Editas Medicine: Employment, Equity Ownership. Dhanapal:Editas Medicine: Employment, Equity Ownership. Jiang:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment, Equity Ownership. Wilson:Editas Medicine: Employment, Equity Ownership. Myer:Editas Medicine: Employment, Equity Ownership. Welstead:Editas Medicine: Employment, Equity Ownership. Bond:Juno Therapeutics: Employment, Equity Ownership. Sather:Juno Therapeutics: Employment, Equity Ownership.

Molecular and Dynamic Evaluation of Proteins Related to Resistance to Neoadjuvant Treatment with Chemoradiotherapy in Circulating Tumor Cells of Patients with Locally Advanced Rectal Cancer.

The heterogeneity of response to neoadjuvant chemoradiotherapy (NCRT) is still a challenge in locally advanced rectal cancer (LARC). The evaluation of thymidylate synthase (TYMS) and RAD23 homolog B (RAD23B) expression in circulating tumor cells (CTCs) provides complementary clinical information. CTCs were prospectively evaluated in 166 blood samples (63 patients) with LARC undergoing NCRT. The primary objective was to verify if the absence of RAD23B/TYMS in CTCs would correlate with pathological complete response (pCR). Secondary objectives were to correlate CTC kinetics before (C1)/after NCRT (C2), in addition to the expression of transforming growth factor-β receptor I (TGF-βRI) with survival rates. CTCs were isolated by ISET and evaluated by immunocytochemistry (protein expression). At C1, RAD23B was detected in 54.1% of patients with no pCR and its absence in 91.7% of patients with pCR (p = 0.014); TYMS− was observed in 90% of patients with pCR and TYMS+ in 51.7% without pCR (p = 0.057). Patients with CTC2 > CTC1 had worse disease-free survival (DFS) (p = 0.00025) and overall survival (OS) (p = 0.0036) compared with those with CTC2 ≤ CTC1. TGF-βRI expression in any time correlated with worse DFS (p = 0.059). To conclude, RAD23B/TYMS and CTC kinetics may facilitate the personalized treatment of LARC.

Osteoking Decelerates Cartilage Degeneration in DMM-Induced Osteoarthritic Mice Model Through TGF-β/smad-dependent Manner.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration. In recent years much attention has been paid to Traditional Chinese Medicine (TCM) since its treatments have shown efficacy for ameliorating cartilage degradation with mild side effects. Osteoking is a TCM prescription that has long been used in OA treatment. However, the exact mechanism of Osteoking are not fully elucidated. In the current study, destabilization of the medial meniscus (DMM)-induced OA mice was introduced as a wild type animal model. After 8 weeks of administration of Osteoking, histomorphometry, OARSI scoring, gait analysis, micro-CT, and immunohistochemical staining for Col2, MMP-13, TGFβRII and pSmad-2 were conducted to evaluate the chondroprotective effects of Osteoking in vivo. Further in vitro experiments were then performed to detect the effect of Osteoking on chondrocytes. TGFβRIICol2ER transgenic mice were constructed and introduced in the current study to validate whether Osteoking exerts its anti-OA effects via the TGF-β signaling pathway. Results demonstrated that in wild type DMM mice, Osteoking ameliorated OA-phenotype including cartilage degradation, subchondral bone sclerosis, and gait abnormality. Col2, TGFβRII, and pSmad-2 expressions were also found to be up-regulated after Osteoking treatment, while MMP-13 was down-regulated. In vitro, the mRNA expression of MMP-13 and ADAMTS5 decreased and the mRNA expression of Aggrecan, COL2, and TGFβRII were up-regulated after the treatment of Osteoking in IL-1β treated chondrocytes. The additional treatment of SB505124 counteracted the positive impact of Osteoking on primary chondrocytes. In TGFβRIICol2ER mice, spontaneous OA-liked phenotype was observed and treatment of Osteoking failed to reverse the OA spontaneous progression. In conclusion, Osteoking ameliorates OA progression by decelerating cartilage degradation and alleviating subchondral bone sclerosis partly via the TGF-β signaling pathway.

Inhibition of TGF-β1 on Gli2 expression was promoted by TNF-α in primary leukemia cells

Purpose Hedgehog (Hh) signaling pathway regulates a variety of tumors-related diseases including leukemia. Whether inhibition of TGF-β1 on Gli2 expression is promoted by TNF-α in primary leukemia cells remains to be determined. Methods Primary leukemia cells were treated with TGF-β1, TNF-α or SIS3 at different concentrations. Gli2 expression was detected by quantitative real-time PCR and western blot analyses. Results We found that TGF-β significantly decreased Gli2 expression, and co-treatment with TNF-αfurther decreased Gli2 expression in primary leukemia cells. TNF-α can increased TGF-βRI and TGF-βRII protein expression in primary leukemia cells, while SIS3 inhibited the effect of TGF-β. Conclusion Our results suggest that Gli2 expression in primary leukemia cells is induced by TGF-β in a Smad3-dependent manner, and independent of Hh receptor signaling.

Acer tataricum subsp. ginnala Inhibits Skin Photoaging via Regulating MAPK/AP-1, NF-κB, and TGFβ/Smad Signaling in UVB-Irradiated Human Dermal Fibroblasts.

Skin, the organ protecting the human body from external factors, maintains structural and tensile strength by containing many collagen fibrils, particularly type I procollagen. However, oxidative stress by ultraviolet (UV) exposure causes skin photoaging by activating collagen degradation and inhibiting collagen synthesis. Acer tataricum subsp. ginnala extract (AGE) is a herbal medicine with anti-inflammatory and anti-oxidative effects, but there is no report on the protective effect against skin photoaging. Therefore, we conducted research concentrating on the anti-photoaging effect of Acer tataricum subsp. ginnala (AG) in UVB (20 mJ/cm2)-irradiated human dermal fibroblasts (HDF). Then, various concentrations (7.5, 15, 30 µg/mL) of AGE were treated in HDF for 24 h following UVB irradiation. After we performed AGE treatment, the matrix metalloproteinase1 (MMP1) expression was downregulated, and the type I procollagen level was recovered. Then, we investigated the mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1) and nuclear factor-κB (NF-κB) pathway, which induce collagen breakdown by promoting the MMP1 level and pro-inflammatory cytokines. The results indicated that AGE downregulates the expression of the MAPK/AP-1 pathway, leading to MMP1 reduction. AGE inhibits nuclear translocation of NF-κB and inhibitor of nuclear factor-κB (IκB) degradation. Therefore, it downregulates the expression of MMP1 and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 increased by UVB. Besides, the TGFβ/Smad pathway, which is mainly responsible for the collagen synthesis in the skin, was also analyzed. AGE decreases the expression of Smad7 and increases TGFβRII expression and Smad3 phosphorylation. This means that AGE stimulates the TGFβ/Smad pathway that plays a critical role in promoting collagen synthesis. Thus, this study suggests that AGE can be a functional material with anti-photoaging properties.

The TGF-β1 pathway is early involved in neurogenic bladder fibrosis of juvenile rats.

This study investigated whole neurogenic bladder's progression changes, as well as the expression of TGF-β1 fibrosis pathway-related proteins in bilateral spinal nerve-amputated juvenile rats. Sixty-four 8-week-old rats (32 bilateral L6 + S1 spinal nerve amputated and 32 sham operated) were selected. Cystometry was performed. General assessments, Masson, Sirius red, immunohistochemical staining, and western blotting of fibrosis and TGF-β1 pathway-related proteins were conducted using bladder tissues. Cystometry results showed that the basal intravesical pressures and bladder capacities in nerve-amputated rats were significantly higher than those in sham-operated ones. Compared to the sham-operated groups, the bladder size and wall thickness in the nerve-amputated groups increased initially but then decreased over time. However, bladder weight continuously increased over time. Disintegration, thickening, and hypertrophy of the bladder wall were found over time in the amputated rats. Moreover, there was a significant increase in collagen III, and the ratio of collagen III/I was higher in amputated rats (P < 0.01). Finally, the expression of TGF-β1, TGF-βRI, Smad2, and collagen III and I increased in amputated bladder tissues, while Smad6 decreased over time. The main clinical features of pediatric neurogenic bladder (PNB) were detrusor paralysis and continuous intravesical pressure. Biological molecular findings are earlier than the pathophysiological findings. Therefore, early preventing bladder fibrosis by targeting TGF-β1/Smad pathway-related proteins once knowing the PNB diagnosis might be an alternative treatment for PNB. The study found that the main clinical features of PNB were detrusor paralysis, continuous intravesical pressure, and increased TGF-beta/Smad signal proteins over time. The study makes contributions to the literature because it suggests biological molecular findings are earlier than the pathophysiological findings by various staining in PNB. The study investigated whole neurogenic bladder's progression changes, as well as the expression of TGF-β1 fibrosis pathway-related proteins in the spinal nerve-injured PNB juvenile rat models, which suggests that early prevention of bladder fibrosis by targeting TGF-β1/Smad pathway-related proteins once knowing the PNB diagnosis might be an alternative treatment for pediatric neurogenic bladder.

Comparison of hormonal receptor expression and HER2 status between circulating tumor cells and breast cancer metastases

OBJECTIVES:Breast cancer (BC) is the most common neoplasm in women. Biopsy of metastatic lesions is recommended to confirm estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status as there are discrepancies in these patterns between primary tumors and metastases in up to 40% of the cases. Circulating tumor cells (CTCs) are related to BC outcomes and could potentially be an alternative to the invasive procedures of metastasis rebiopsy. ISET® technology is not currently employed to detect CTCs in patients with BC. Emerging data support that the characterization of CTC protein expression can refine its prognostic value. Transforming growth factor (TGF)-β plays a role in BC progression and invasiveness. Thus, in this study, we aimed to compare ER, PR, and HER2 expression in primary tumors, CTCs, and metastases and evaluate TGF-β type 1 receptor (TGF-β RI) expression in CTCs as prognostic factor for progression free survival (PFS) and overall survival (OS).METHODS:This prospective study was conducted at the A.C. Camargo Cancer Center, Brazil. Blood samples were processed in ISET® (Isolation by SizE of Tumors, Rarecells, France) before computed tomography-guided biopsy of suspected metastatic lesions. Protein expression levels in CTCs were compared to those in primary tumors/metastases (medical records).RESULTS:Of the 39 patients initially included, 27 underwent both biopsies of metastases and blood collection and were considered for analysis. The concordance rates for ER, PR, and HER2 expression between primary tumors and metastases were high. No loss of HER2 expression at any metastasis site and retention of the same pattern of protein expression in all triple-negative (TN) tumors (92.5%, 81.5% and 96.2% respectively) (p<0.0001) was observed. When metastases/CTCs were classified as TN/non-TN, CTCs showed high specificity (93%), accuracy (84.2%), and negative predictive value (88%). The median OS of patients without TGF-β RI expression in CTCs was 42.6 versus 20.8 months for TGF-β RI expression-positive ones (p>0.05).CONCLUSION:The role of CTCs detected by ISET has not yet been established in BC. Here, we suggest that this methodology may be useful to evaluate metastasis in non-TN cases as well as TGF-β RI expression in CTCs, which may impact patient survival. Due to sample limitations, future studies must focus on specific BC subtypes and an expansion of the cohort.