The Complex Interplay of TGF-ß and Notch Signaling in the Pathogenesis of Fibrosis.

Fibrosis is a major medical challenge, as it leads to irreversible tissue remodeling and organ dysfunction. Its progression contributes significantly to morbidity and mortality worldwide, with limited therapeutic options available. Extensive research on the molecular mechanisms of fibrosis has revealed numerous factors and signaling pathways involved. However, the interactions between these pathways remain unclear. A comprehensive understanding of the entire signaling network that drives fibrosis is still missing. The TGF-ß and Notch signaling pathways play a key role in fibrogenesis, and this review focuses on their functional interplay and molecular mechanisms. Studies have shown synergy between TGF-ß and Notch cascades in fibrosis, but antagonistic interactions can also occur, especially in cardiac fibrosis. The molecular mechanisms of these interactions vary depending on the cell context. Understanding these complex and context-dependent interactions is crucial for developing effective strategies for treating fibrosis.

Transient downregulation of NR4A1 leads to impaired osteoblast differentiation through the TGF-ß pathway, and Elesclomol (STA-4783) rescues this phenotype.

Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway.

miR-335-5p inhibits endochondral ossification by directly targeting SP1 in TMJ OA.

OBJECTIVE: During the development of temporomandibular joint osteoarthritis, endochondral ossification is compromised which leads to condylar degeneration; miR-335-5p in endochondral ossification in osteoarthritic condylar cartilage tissue remains unclear. METHODS: Up-regulated microRNA and its target gene were searched for endochondral ossification in osteoarthritis articular cartilage. The effect of increased or decreased miR-335-5p on endochondral ossification was evaluated by transfecting miR-335-5p mimics or miR-335-5p inhibitor in vitro in chondrocytes C28/I2. Finally, we injected the temporomandibular joint of rats intra-articularly with agomiR-335 in a unilateral anterior crossbite rat model to determine the in vivo regulation of miR-335. RESULTS: After the onset of temporomandibular joint osteoarthritis, miR-335-5p levels were gradually up-regulated, whereas endochondral ossification-related genes were down-regulated in condylar cartilage specimens. Our results showed that miR-335 inhibited endochondral ossification after administration of a miR-335 antagonist into the temporomandibular joint articular cavity of a unilateral anterior crossbite rat model. AgomiR-335, a miR-335 agonist, inhibited matrix mineralization in fibrocartilage stem cells in vitro and then miR-335-5p negatively regulated chondrocyte activity by directly targeting SP1 via promoting transforming growth factor-ß/Smad signalling. CONCLUSION: miR-335-5p can significantly inhibit endochondral ossification; therefore, its inhibition may be beneficial for the treatment of temporomandibular joint osteoarthritis.

Changes in the Expression of TGF-Beta Regulatory Pathway Genes Induced by Vitamin D in Patients with Relapsing-Remitting Multiple Sclerosis.

Vitamin D is an environmental factor related to multiple sclerosis that plays a significant role in immune regulation. TGF-ß is a superfamily of cytokines with an important dual effect on the immune system. TGF-ß inhibits the Th1 response while facilitating the preservation of regulatory T cells (FOXP3+) in an immunoregulatory capacity. However, when IL-6 is present, it stimulates the Th17 response. Our aim was to analyze the regulatory effect of vitamin D on the in vivo TGF-ß signaling pathway in patients with relapsing-remitting multiple sclerosis (RRMS). A total of 21 patients with vitamin D levels < 30 ng/mL were recruited and supplemented with oral vitamin D. All patients were receiving disease-modifying therapy, with the majority being on natalizumab. Expression of SMAD7, ERK1, ZMIZ1, BMP2, BMPRII, BMP4, and BMP5 was measured in CD4+ lymphocytes isolated from peripheral blood at baseline and one and six months after supplementation. SMAD7 was overexpressed at six months with respect to baseline and month one. ERK1 was overexpressed at six months with respect to month one of treatment. No significant differences in expression were observed for the remaining genes. No direct correlation was found with serum vitamin D levels. BMPRII expression changed differentially in non-natalizumab- versus natalizumab-treated patients. Changes were observed in the expression of ERK1, BMP2, and BMP5 based on disease activity measured using the Rio-Score, BMP2 in patients who had relapses, and BMP5 in those whose EDSS worsened. Our results suggest indirect regulation of vitamin D in TGF-ß pathway genes in patients with RRMS.

The geometry-dependent regulation of hepatic stellate cells by graphene oxide nanomaterials.

Nanomaterials are widely used in biomedical applications such as drug delivery, bioimaging, and photothermal therapy. For example, graphene oxide (GO) nanomaterials are among the most popular drug delivery vehicles in treating liver diseases due to their tunable chemical/physical properties, and biocompatibility. However, it has been reported that nanomaterials tend to accumulate in livers. The biophysical impact of the accumulation in liver cells remains unclear, and it may cause the liver fibrosis in the long run. The activation of hepatic stellate cells (HSCs) is one of the key initial steps of liver fibrosis. In this paper, we explored the geometric effect (nanosheets vs. quantum dots) of GO nanomaterials on human HSCs, in terms of cell viability, fibrotic degree, mobility and regulation pathways. Our study showed that GO nanosheets could significantly reduce HSCs cell viability and mobility. The protein expression levels of TGFßRⅡ/Smad2/Smad3 decreased, corresponding to a trend of attenuating fibrotic degree. However, the expression level of α-SMA, a maker protein of fibrosis, increased and contradicted with the projection. Further investigation on mitochondria showed that GO nanosheets disrupted mitochondria membrane and membrane potentials. We found that while modulating fibrotic effect through the TGF-ß pathway, GO nanosheets induced oxidative stress and activated HSCs through reactive oxygen species(ROS)pathway. This was confirmed by the decreased expression level of α-SMA after co-incubation of GO nanosheets and n-acetyl cysteine (NAC) with HSCs. GO quantum dots decreased α-SMA expression level at 100 mg/l, along with decrease in GAPDH expression level and constant expression level of ß-actin. The correlation between GAPDH and α-SMA remains to be explored. Our study suggested that the biophysical impacts of GO nanomaterials on HSCs are geometry-dependent. Both GO nanosheets and quantum dots can be adapted for attenuating liver fibrosis with further investigation on mechanisms.

Lactobacillus plantarum-derived extracellular vesicles protect against ischemic brain injury via the microRNA-101a-3p/c-Fos/TGF-ß axis.

Currently, the reported source of extracellular vesicles (EVs) for the treatment of ischemic stroke（IS）is limited to mammals. Moreover, these EVs are restricted to clinical translation by the high cost of cell culture. In this respect, Lactobacillus plantarum culture is advantaged by low cost and high yield. However, it is poorly understood whether Lactobacillus plantarum-derived EVs (LEVs) are applicable for the treatment of IS. Here, our results demonstrated that LEVs reduced apoptosis in ischemic neuron both in vivo and in vitro. As revealed by high-throughput sequencing, miR-101a-3p expression was significantly elevated by LEV treatment in OGD/R-induced neurons, as confirmed in the tMCAO mice treated with LEVs. Mechanistically, c-Fos was directly targeted by miR-101a-3p. In addition, c-Fos determined ischemia-induced neuron apoptosis in vivo and in vitro through the TGF-ß1 pathway, miR-101a-3p inhibition aggravated ischemia-induced neuron apoptosis in vitro and in vivo, and miR-101a-3p overexpression produced the opposite results. Hsa-miR-101-3p was downregulated in the plasma of patients with IS but upregulated in the patients with neurological recovery after rt-PA intravenous thrombolysis. In conclusion, Our results demonstrated for the first time that LEVs might inhibit neuron apoptosis via the miR-101a-3p/c-Fos/TGF-ß axis, and has-miR-101-3p is a potential marker of neurological recovery in IS patients.

circEHBP1 promotes lymphangiogenesis and lymphatic metastasis of bladder cancer via miR-130a-3p/TGFßR1/VEGF-D signaling.

Lymphatic metastasis constitutes a leading cause of recurrence and mortality in bladder cancer. Accumulating evidence indicates that lymphangiogenesis is indispensable to trigger lymphatic metastasis. However, the specific mechanism is poorly understood. In the present study, we revealed a pathway involved in lymphatic metastasis of bladder cancer, in which a circular RNA (circRNA) facilitated lymphangiogenesis in a vascular endothelial growth factor C (VEGF-C)-independent manner. Novel circRNA circEHBP1 was markedly upregulated in bladder cancer and correlated positively with lymphatic metastasis and poor prognosis of patients with bladder cancer. circEHBP1 upregulated transforming growth factor beta receptor 1 (TGFBR1) expression through physically binding to miR-130a-3p and antagonizing the suppression effect of miR-130a-3p on the 3' UTR region of TGFBR1. Subsequently, circEHBP1-mediated TGFßR1 overexpression activated the TGF-ß/SMAD3 signaling pathway, thereby promoting the secretion of VEGF-D and driving lymphangiogenesis and lymphatic metastasis in bladder cancer. Importantly, administration of VEGF-D neutralizing antibodies remarkably blocked circEHBP1-induced lymphangiogenesis and lymphatic metastasis in vivo. Our findings highlighted that the circEHBP1/miR-130a-3p/TGFßR1/VEGF-D axis contributes to lymphangiogenesis and lymphatic metastasis of bladder cancer independent of VEGF-C, which might lead to the development of circEHBP1 as a potential biomarker and promising therapeutic target for lymphatic metastasis in bladder cancer.

Inhibin, beta A regulates the transforming growth factor-beta pathway to promote malignant biological behaviour in colorectal cancer.

Inhibin, beta A (INHBA) is a member of the transforming growth factor (TGF-ß) family. The carcinogenic mechanisms of INHBA during the development of colorectal cancer (CRC) remain unclear. In the present study, we further elucidated the role of INHBA in CRC. We analysed the expression of INHBA in CRC and its relationship with patient prognosis using data from public databases. INHBA expression was evaluated in CRC tissues and cell lines using immunohistochemistry and western blotting. After inhibiting the expression of INHBA, the effect of INHBA on the function of CRC cells was evaluated in vitro. We found that INHBA was upregulated in CRC. High INHBA expression is closely related to poor prognosis in patients with CRC. Knockdown of INHBA in vitro can inhibit the proliferation, migration, and invasion of CRC cells. In terms of mechanism, we found that high INHBA expression activates the TGF-ß pathway. SIGNIFICANCE OF THE STUDY: INHBA acts as an oncogene in the progression of CRC and may, therefore, be a potential therapeutic target for CRC.

Arecoline enhances miR-21 to promote buccal mucosal fibroblasts activation.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is an irreversible fibrosis disease and a potentially malignant disorder in the oral cavity. Various studies have shown that miR-21 was implicated in the fibrogenesis and carcinogenesis, but its functional role in the development of OSF has not been investigated. METHODS: The expression levels of miR-21 in arecoline-stimulated normal buccal mucosal fibroblasts (BMFs) and OSF specimens were determined by qRT-PCR. Exogenous administration of TGF-ß and its inhibitor (SB431542) were utilized to examine the involvement of TGF-ß signaling in miR-21 alteration. Collagen gel contraction, transwell migration, and invasion assays were used to assess the myofibroblast activities. The relationship between α-SMA and miR-21 was calculated using the Pearson correlation coefficient. RESULTS: MiR-21 expression was induced in BMFs by arecoline treatment in a dose-dependent manner. Our results showed that this upregulation was mediated by TGF-ß signaling. Subsequently, we demonstrated that the administration of the miR-21 inhibitor suppressed the arecoline-induced myofibroblast characteristics, including a higher collagen gel contractility and cell motility, in normal BMFs. Furthermore, inhibition of miR-21 was sufficient to attenuate the myofibroblast features in fibrotic BMFs. Besides, we showed that the expression of miR-21 was aberrantly upregulated in the OSF tissues and there was a positive correlation between miR-21 and myofibroblast marker, α-SMA. CONCLUSION: MiR-21 overexpression in OSF may be due to the stimulation of areca nut, which was mediated by the TGF-ß pathway. Our data suggested that the repression of miR-21 was a promising direction to palliate the development and progression of OSF.

Silencing ZEB2 Induces Apoptosis and Reduces Viability in Glioblastoma Cell Lines.

BACKGROUND: Glioma is an aggressive type of brain tumor that originated from neuroglia cells, accounts for about 80% of all malignant brain tumors. Glioma aggressiveness has been associated with extreme cell proliferation, invasion of malignant cells, and resistance to chemotherapies. Due to resistance to common therapies, glioma affected patients' survival has not been remarkably improved. ZEB2 (SIP1) is a critical transcriptional regulator with various functions during embryonic development and wound healing that has abnormal expression in different malignancies, including brain tumors. ZEB2 overexpression in brain tumors is attributed to an unfavorable state of the malignancy. Therefore, we aimed to investigate some functions of ZEB2 in two different glioblastoma U87 and U373 cell lines. METHODS: In this study, we investigated the effect of ZEB2 knocking down on the apoptosis, cell cycle, cytotoxicity, scratch test of the two malignant brain tumor cell lines U87 and U373. Besides, we investigated possible proteins and microRNA, SMAD2, SMAD5, and miR-214, which interact with ZEB2 via in situ analysis. Then we evaluated candidate gene expression after ZEB2-specific knocking down. RESULTS: We found that ZEB2 suppression induced apoptosis in U87 and U373 cell lines. Besides, it had cytotoxic effects on both cell lines and reduced cell migration. Cell cycle analysis showed cell cycle arrest in G0/G1 and apoptosis induction in U87 and U373 cell lines receptively. Also, we have found that SAMAD2/5 expression was reduced after ZEB2-siRNA transfection and miR-214 upregulated after transfection. CONCLUSIONS: In line with previous investigations, our results indicated a critical oncogenic role for ZEB2 overexpression in brain glioma tumors. These properties make ZEB2 an essential molecule for further studies in the treatment of glioma cancer.

A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity.

A key feature in the pathogenesis of heart failure is cardiac fibrosis, but effective treatments that specifically target cardiac fibrosis are currently not available. A major impediment to progress has been the lack of reliable in vitro models with sufficient throughput to screen for activity against cardiac fibrosis. Here, we established cell culture conditions in micro-well format that support extracellular deposition of mature collagen from primary human cardiac fibroblasts - a hallmark of cardiac fibrosis. Based on robust biochemical characterization we developed a high-content phenotypic screening platform, that allows for high-throughput identification of compounds with activity against cardiac fibrosis. Our platform correctly identifies compounds acting on known cardiac fibrosis pathways. Moreover, it can detect anti-fibrotic activity for compounds acting on targets that have not previously been reported in in vitro cardiac fibrosis assays. Taken together, our experimental approach provides a powerful platform for high-throughput screening of anti-fibrotic compounds as well as discovery of novel targets to develop new therapeutic strategies for heart failure.

LncRNA SNHG3 is activated by E2F1 and promotes proliferation and migration of non-small-cell lung cancer cells through activating TGF-ß pathway and IL-6/JAK2/STAT3 pathway.

Recently, long noncoding RNAs (lncRNAs) have been widely reported to play pivotal roles in the regulation of human cancers. Although the oncogenic property of lncRNA small nucleolar RNA host gene 3 (SNHG3) has been revealed in a variety of cancers, functions and regulatory mechanism of SNHG3 in non-small-cell lung cancer (NSCLC) remain to be investigated. In this study, we detected the upregulated expression of SNHG3 in NSCLC tissues as well as cells through quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Using Kaplan-Meier analysis, we determined that a high-level of SNHG3 was associated with a low overall survival rate of patients with NSCLC. Through gain and loss of function experiments, we demonstrated that SNHG3 had a significantly positive effect on NSCLC cell proliferation and migration. Mechanistic investigations revealed that SNHG3 was a predicted direct transcriptional target of E2F1. We observed that the transcriptional activation of SNHG3 could be induced by E2F1. To explore the mechanism, rescue experiments were carried out, which revealed that the cotreatment with SB-431542, JSI-124, or JSI-124 + SB-431542 rescued the effects brought by the overexpression of SNHG3 on NSCLC cell proliferation, migration, and epithelial-mesenchymal transition process. Our results suggested that E2F1 activated SNHG3 and promoted cell proliferation and migration in NSCLC via transforming growth factor-ß pathway and interleukin-6/janus-activated kinase 2/signal transducer and activator of transcription 3 pathway, which implied that SNHG3 may be a biomarker for the treatment of patients with NSCLC.

Glycoprotein M6B Interacts with TßRI to Activate TGF-ß-Smad2/3 Signaling and Promote Smooth Muscle Cell Differentiation.

Smooth muscle cells (SMCs), which form the walls of blood vessels, play an important role in vascular development and the pathogenic process of vascular remodeling. However, the molecular mechanisms governing SMC differentiation remain poorly understood. Glycoprotein M6B (GPM6B) is a four-transmembrane protein that belongs to the proteolipid protein family and is widely expressed in neurons, oligodendrocytes, and astrocytes. Previous studies have revealed that GPM6B plays a role in neuronal differentiation, myelination, and osteoblast differentiation. In the present study, we found that the GPM6B gene and protein expression levels were significantly upregulated during transforming growth factor-ß1 (TGF-ß1)-induced SMC differentiation. The knockdown of GPM6B resulted in the downregulation of SMC-specific marker expression and repressed the activation of Smad2/3 signaling. Moreover, GPM6B regulates SMC Differentiation by Controlling TGF-ß-Smad2/3 Signaling. Furthermore, we demonstrated that similar to p-Smad2/3, GPM6B was profoundly expressed and coexpressed with SMC differentiation markers in embryonic SMCs. Moreover, GPM6B can regulate the tightness between TßRI, TßRII, or Smad2/3 by directly binding to TßRI to activate Smad2/3 signaling during SMC differentiation, and activation of TGF-ß-Smad2/3 signaling also facilitate the expression of GPM6B. Taken together, these findings demonstrate that GPM6B plays a crucial role in SMC differentiation and regulates SMC differentiation through the activation of TGF-ß-Smad2/3 signaling via direct interactions with TßRI. This finding indicates that GPM6B is a potential target for deriving SMCs from stem cells in cardiovascular regenerative medicine. Stem Cells 2018 Stem Cells 2019;37:190-201.

Targeting TGF-ß-Mediated SMAD Signaling Pathway via Novel Recombinant Cytotoxin II: A Potent Protein from Naja naja oxiana Venom in Melanoma.

Since the current treatments have not resulted in the desired outcomes for melanoma patients, there is a need to identify more effective medications. Together with other snake venom proteins, cytotoxin-II has shown promising results in tumoral cells. In this study, recombinant cytotoxin-II (rCTII) was expressed in SHuffle® T7 Express cells, while the epitope mapping of rCTII was performed to reveal the antibody-binding regions of rCTII. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess the viability of SK-MEL-3 and HFF-2 cells after treating these cells with rCTII. The qRT-PCR was performed to evaluate the expression levels of matrix metallopeptidase 3 (MMP-3), SMAD2, SMAD3, caspase-8, caspase-9, and miR-214 in order to reveal the rCTII-induced signaling pathways in melanoma. Our results have shown that two regions of amino acids, 6-16 and 19-44, as predicted epitopes of this toxin, are essential for understanding the toxicity of rCTII. Treating the melanoma cells with rCTII substantially inhibited the transforming growth factor-beta (TGF-ß)-SMAD signaling pathway and down-regulated the expression of MMP-3 and miR-214 as well. This cytotoxin also restored apoptosis mainly via the intrinsic pathway. The down-regulation of MMP-3 and miR-214 might be associated with the anti-metastatic property of rCTII in melanoma. The inhibitory effect of rCTII on the TGF-ß signaling pathway might be associated with increased apoptosis and decreased cancer cell proliferation. It is interesting to see that the IC50 value of rCTII has been lower in the melanoma cells than non-tumoral cells, which may indicate its potential effects as a drug. In conclusion, rCTII, as a novel medication, might serve as a potent and efficient anticancer drug in melanoma.

miR-335-5p induces insulin resistance and pancreatic islet ß-cell secretion in gestational diabetes mellitus mice through VASH1-mediated TGF-ß signaling pathway.

Multiple studies have reported different methods in treating gestational diabetes mellitus (GDM); however, the relationship between miR-335-5p and GDM still remains unclear. Here, this study explores the effect of miR-335-5p on insulin resistance and pancreatic islet ß-cell secretion via activation of the TGFß signaling pathway by downregulating VASH1 expression in GDM mice. The GDM mouse model was established and mainly treated with miR-335-5p mimic, miR-335-5p inhibitor, si-VASH1, and miR-335-5p inhibitor + si-VASH1. Oral glucose tolerance test (OGTT) was conducted to detect fasting blood glucose (FBG) fasting insulin (FINS). The OGTT was also used to calculate a homeostasis model assessment of insulin resistance (HOMA-IR). A hyperglycemic clamp was performed to measure the glucose infusion rate (GIR), which estimated ß-cell function. Expressions of miR-335-5p, VASH1, TGF-ß1, and c-Myc in pancreatic islet ß-cells were determined by RT-qPCR, western blot analysis, and insulin release by ELISA. The miR-335-5p mimic and si-VASH1 groups showed elevated blood glucose levels, glucose area under the curve (GAUC), and HOMA-IR, but a reduced GIR and positive expression of VASH1. Overexpression of miR-335-5p and inhibition of VASH1 contributed to activated TGFß1 pathway, higher c-Myc, and lower VASH1 expressions, in addition to downregulated insulin and insulin release levels. These findings provided evidence that miR-335-5p enhanced insulin resistance and suppressed pancreatic islet ß-cell secretion by inhibiting VASH1, eventually activating the TGF-ß pathway in GDM mice, which provides more clinical insight on the GDM treatment.

Polycomb complex mediated epigenetic reprogramming alters TGF-ß signaling via a novel EZH2/miR-490/TGIF2 axis thereby inducing migration and EMT potential in glioblastomas.

Recent advancement in understanding cancer etiology has highlighted epigenetic deregulation as an important phenomenon leading to poor prognosis in glioblastoma (GBM). Polycomb repressive complex 2 (PRC2) is one such important epigenetic modifier reportedly altered in GBM. However, its defined mechanism in tumorigenesis still remains elusive. In present study, we analyzed our in-house ChIPseq data for H3k27me3 modified miRNAs and identified miR-490-3p to be the most common target in GBM with significantly downregulated expression in glioma patients in both TCGA and GBM patient cohort. Our functional analysis delineates for the first time, a central role of PRC2 catalytic unit EZH2 in directly regulating expression of this miRNA and its host gene CHRM2 in GBM. In accordance, cell line treatment with EZH2 siRNA and 5-azacytidine also confirmed its coregulation by CpG and histone methylation based epigenetic mechanisms. Furthermore, induced overexpression of miR-490-3p in GBM cell lines significantly inhibited key hallmarks including cellular proliferation, colony formation and spheroid formation, as well as epithelial-to-mesenchymal transition (EMT), with downregulation of multiple EMT transcription factors and promigratory genes (MMP9, CCL5, PIK3R1, ICAM1, ADAM17 and NOTCH1). We also for the first time report TGFBR1 and TGIF2 as two direct downstream effector targets of miR-490-3p that are also deregulated in GBM. TGIF2, a novel target, was shown to promote migration and EMT that could partially be rescued by miR-490-3p overexpression. Overall, this stands as a first study that provides a direct link between epigenetic modulator EZH2 and oncogenic TGF-ß signaling involving novel miR-490-3p/TGIF2/TGFBR1 axis, that being targetable might be promising in developing new therapeutic intervention strategies for GBM.

Association of pathway mutation with survival after recurrence in colorectal cancer patients treated with adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

BACKGROUND: Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR. METHODS: Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-ß, and PI3K). RESULTS: Mutation of genes within the WNT, P53, RTK-RAS, TGF-ß, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-ß pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-ß pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p <  0.001). Mutations in genes within other critical pathways were not associated with SAR. When MAC was excluded as a covariate, multivariate analysis revealed TGF-ß pathway mutation and curative resection after distant recurrence as an independent prognostic factor for SAR. The impact of TGF-ß pathway mutations were predicted using the PROVEAN, SIFT, and PolyPhen-2. Among 25 mutations, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation. CONCLUSIONS: Mutation in genes within TGF-ß pathway may have negative prognostic role for SAR in CRC. Other pathway mutations were not associated with SAR.

Asiatic Acid Attenuates Bone Loss by Regulating Osteoclastic Differentiation.

Anti-resorptive agents like bisphosphonates have been widely used for the treatment of postmenopausal osteoporosis. However, their long-term safety and efficacy are still controversial. This study is to examine the effect of Asiatic acid (AA) in osteoclastic differentiation, and further to investigate its effect on bone quality in animals. Effect of AA on osteoclastic differentiation was measured by Tartrate-resistant acid phosphatase stain, bone resorption pit assays, and quantitative real-time polymerase chain reaction. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor-ß (TGF-ß) signaling were measured by western blot before and after AA treatment. Ovariectomized (OVX) wild-type or Smad7 partially knock out mice were used to evaluate the effects of AA on bone quality by micro-computed tomography, mechanical test, and histomorphometry. Results revealed a dose-dependent inhibitory effect of AA on osteoclastic differentiation. After AA treatment, Smad7 was upregulated, while NF-κB and TGF-ß signaling were inhibited during osteoclastic differentiation. Results from animal study revealed that AA prevented bone from further loss caused by OVX and increased the mechanical properties of femur in wild-type animals. AA also prevented bone loss in the Smad7-deficient animals. When combining with OVX in the Smad7-deficient mice, AA could only partially preserve their bone mass. Taken together, we found that AA effectively inhibited osteoclastic differentiation and attenuated osteoporosis, which effects may be through TGF-ß and NF-κB pathways. This study reveals that AA may be a potential anti-resorptive agent for postmenopausal osteoporosis.

Galunisertib modifies the liver fibrotic composition in the Abcb4Ko mouse model.

Transforming growth factor (TGF)-ß stimulates extracellular matrix (ECM) deposition during development of liver fibrosis and cirrhosis, the most important risk factor for the onset of hepatocellular carcinoma. In liver cancer, TGF-ß is responsible for a more aggressive and invasive phenotype, orchestrating remodeling of the tumor microenvironment and triggering epithelial-mesenchymal transition of cancer cells. This is the scientific rationale for targeting the TGF-ß pathway via a small molecule, galunisertib (intracellular inhibitor of ALK5) in clinical trials to treat liver cancer patients at an advanced disease stage. In this study, the hypothesis that galunisertib modifies the tissue microenvironment via inhibition of the TGF-ß pathway is tested in an experimental preclinical model. At the age of 6 months, Abcb4ko mice-a well-established model for chronic liver disease development and progression-are treated twice daily with galunisertib (150 mg/kg) via oral gavage for 14 consecutive days. Two days after the last treatment, blood plasma and livers are harvested for further assessment, including fibrosis scoring and ECM components. The reduction of Smad2 phosphorylation in both parenchymal and non-parenchymal liver cells following galunisertib administration confirms the treatment effectiveness. Damage-related galunisertib does not change cell proliferation, macrophage numbers and leucocyte recruitment. Furthermore, no clear impact on the amount of fibrosis is evident, as documented by PicroSirius red and Gomori-trichome scoring. On the other hand, several fibrogenic genes, e.g., collagens (Col1α1 and Col1α2), Tgf-ß1 and Timp1, mRNA levels are significantly downregulated by galunisertib administration when compared to controls. Most interestingly, ECM/stromal components, fibronectin and laminin-332, as well as the carcinogenic ß-catenin pathway, are remarkably reduced by galunisertib-treated Abcb5ko mice. In conclusion, TGF-ß inhibition by galunisertib interferes, to some extent, with chronic liver progression, not by reducing the stage of liver fibrosis as measured by different scoring systems, but rather by modulating the biochemical composition of the deposited ECM, likely affecting the fate of non-parenchymal cells.

Heteronemin Induces Anti-Proliferation in Cholangiocarcinoma Cells via Inhibiting TGF-ß Pathway.

A marine sesterterpenoid-type natural product, heteronemin, retains anticancer effects. In the current study, we investigate the antitumor mechanism of heteronemin in cholangiocarcinoma cells and further explore its molecular targets. Initially, heteronemin exhibited potent cytotoxic effects against cholangiocarcinoma HuccT1 and SSP-25 cells. In vitro, heteronemin altered the abilities of cell adhesion and cell migration in HuccT1 and SSP-25 cell lines. It repressed messenger ribonucleic acid (mRNA) expression levels of transforming growth factor (TGF)-ß, mothers against decapentaplegic homolog (SMAD) and Myc, whose protein products play important roles in regulating cell growth, angiogenesis, and metastasis. In addition, heteronemin altered several signaling pathways. The results indicate that heteronemin was able to modulate cell adhesion, the expression of extracellular matrix (ECM) receptors, the TGF-ß pathway, cell motility, the membrane integration, metastasis response, matrix metalloproteinase (MMP) remodeling, the regulation of metabolism, sprouting angiogenesis, transcription factors, and vasculogenesis in cholangiocarcinoma cell lines. The results also suggest that it activated multiple signal transduction pathways to induce an anti-proliferation effect and anti-metastasis in cholangiocarcinoma. In conclusion, heteronemin may be used as a potential medicine for anticancer therapy.