BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

Abstract Background Autophagy is a conserved catabolic process with complicated roles in tumor development. Bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor (TGF-β) family of regulatory proteins, plays a crucial role in human malignancies. However, whether BMP4 contributes to the regulation of autophagy in hepatocellular carcinoma (HCC) progression remains elusive. Methods Functional analysis of BMP4 on HCC proliferation and autophagy was performed both in vitro and in vivo in HepG2 and HCCLM3 cells. Autophagic activity was estimated by Western blot for autophagic marker proteins and by transmission electron microscopy (TEM). Transfection of mRFP-GFP-LC3 adenovirus was applied to observe autophagic flux and high content screening was used for quantification. The signaling pathway of BMP4-regulated HCC proliferation and autophagy was investigated by Western blot. Results BMP4 treatment promoted HCC cells proliferation and induced autophagy. The in vivo xenograft model supported that BMP4 overexpression promoted the growth of HCC cells and autophagy induction while BMP4 knockdown exerted the opposite effect. 3-MA pre-treatment or knockdown of Beclin-1 (BECN1) blocked HCC autophagy by decreasing the expression of LC3-II and subsequently attenuated BMP4-induced autophagy and cells proliferation enhanced by BMP4 in vitro and in vivo. Mechanistic study revealed that the induction of autophagy by BMP4 was mediated through activating the JNK1/Bcl2 pathway. Furthermore, the JNK1 inhibitor and knockdown of JNK1 could attenuate autophagy induced by BMP4 and eliminated BMP4-promoted HCC cells growth. Conclusions BMP4 promoted HCC proliferation by autophagy activation through JNK1/Bcl-2 signaling

Additional file 5: Figure S2. of Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance

Nestin upregulation in the Bel-7402-Nestin cell line resulted in increased mRNA levels of survivin, c-myc, and Bcl-2 (proteins encoded by β-catenin target genes). (TIF 128 kb

Additional file 4: Figure S1. of Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance

The mRNA expression levels of EMT markers in different groups of Bel-7402 cells measured by quantitative real-time RT-PCR. (DOCX 13 kb

Additional file 3: Table S3. of Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance

The influence of Nestin on IC50s (ΟM/L) of anticancer drugs for HCC cells. (TIF 304 kb

Additional file 1: Table S1. of Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance

The primers sequences of qRT-PCR. (DOCX 14 kb

Additional file 2: Table S2. of Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance

IC50s (ΟM/L) of anticancer drugs for HCC cells. (DOCX 14 kb

A cancer-associated fibroblast subtypes-based signature enables the evaluation of immunotherapy response and prognosis in bladder cancer

Summary: Bladder cancer (BLCA) is one of the most prevalent and heterogeneous urinary malignant tumors. Previous researches have reported a significant association between cancer-associated fibroblasts (CAFs) and poor prognosis of tumor patients. However, uncertainty surrounds the role of CAFs in the BLCA tumor microenvironment, necessitating further investigation into the CAFs-related gene signatures in BLCA. In this study, we identified three CAF subtypes in BLCA according to single-cell RNA-seq data and constructed CAFs-related risk score (CRRS) by screening 102,714 signatures. The survival analysis, ROC curves, and nomogram suggested that CRRS was a valuable predictor in 2,042 patients from 9 available public datasets and Xiangya real-world cohort. We further revealed the significant correlation between CRRS and clinicopathological characteristics, genome alterations, and epithelial-mesenchymal transition (EMT). A high CRRS indicated a non-inflamed phenotype and a lower remission rate of immunotherapy in BLCA. In conclusion, the CRRS had the potential to predict the prognosis and immunotherapy response of BLCA patients

Additional file 1: of BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

Figure S1. Original blots of Western blot. Original blots of Fig. 1. (2) Original blots of Fig. 2. (3) Orinigal blots of Fig. 3. (4) Orinigal blots of Fig. 4. (5) Orinigal blots of Fig. 5. (6) Orinigal blots of Fig. 6. Figure S2. The effeciency of BMP4 expression manipulated by lentivirus. The effeciency of BMP4 overexpression in HepG2 cells was confirmed by qRT-PCR and Western blot. (b)The effeciency of BMP4 knockdown in HCCLM3 cells was confirmed by qRT-PCR and Western blot. Figure S3. BMP4 promoted HepG2 cells growth. (a) HepG2 cells were treated with various concentrations of human recombinant BMP4 (0, 25, 50, 100 and 150 ng/mL) for different lengths of time to assay the effects on HCC cells proliferation. Cell viability was determined by CCK-8 assays. (b) & (c) Effects of BMP4 or Noggin on long term colony formation in HepG2 cells. The numbers of colonies in the BMP4-treated (100 ng/mL) groups were significantly more than that in the blank groups in HepG2 cells (p < 0.001), while Noggin-treated (200 ng/mL) groups displayed significantly less colony numbers than the blank control groups (p < 0.05). n = 3, one-way ANOVA with post-hoc Tukey’s test. Table S1. Information of antibodies for western blot. Table S2. The sequences of siRNA targeting BMP4. (PDF 783 kb