Development of 14-gene signature prognostic model based on metastasis for colorectal cancer.

BACKGROUND: Metastasis is the main cause of death of colorectal tumors, in our study a prognosis model was built by analyzing the differentially expressed genes between metastatic and non-metastatic colorectal cancer (CRC). We used this feature to predict CRC patient prognosis and explore the causes of colorectal tumor metastasis by characterizing the immune status alteration. METHODS: CRC patient data were obtained from TCGA and GEO databases. We constructed a risk prognostic model by using Cox regression and the least absolute shrinkage and selection operator (LASSO) based on CRC metastasis-related genes. We also obtained a nomogram to predict the prognosis of CRC patients. Finally, we explored the underlying mechanism of these metastasis-related genes and CRC prognosis using immune infiltration analysis and experimental verification. RESULTS: According to our prognostic model, in TCGA, the area under the curve (AUC) values of the training and test sets were 0.72 and 0.76, respectively, and 0.68 for the GEO external data set. This suggested that the treatment and prognosis of patients could be effectively determined. At the same time, we found that the B and T cells in both tissues and peripheral blood of high MR-risk score patients were mostly in immune static or inactivated states compared with those of low MR-risk score patients. CONCLUSIONS: MR-risk score has a direct correlation with CRC patient prognosis. It is useful for predicting the prognosis and patient immune status for these patients.

Exosomal microRNAs in the DLK1-DIO3 imprinted region derived from cancer-associated fibroblasts promote progression of hepatocellular carcinoma by targeting hedgehog interacting protein.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and third leading cause of cancer-related death worldwide in 2020. Exosomes derived from cancer-associated fibroblasts (CAFs-exo) can promote tumor progression in various human cancers. However, the underlying regulatory mechanism controlling how CAFs-exo can promote HCC progression remains poorly understood. METHODS: CAFs and para-cancer fibroblasts (PAFs) were isolated from HCC tissues and corresponding para-cancer tissues, then were cultured in vitro. CAFs and PAFs were characterized by immunofluorescence and western blot (WB) assays. Exosomes were isolated by ultracentrifugation, and characterized by transmission electron microscopy, nanoflow cytometry, and WB assay. The internalization of exosomes by HCC cells was observed under a fluorescence microscope. Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Wound healing and transwell assays were used for migration and invasion experiments. RT-PCR assay was used to examine differentially expressed microRNAs (miRNAs) in exosomes and HCC cells. The TargetScan database was used to predict miRNA target genes. Hedgehog interacting protein (HHIP) expression analysis, prognostic analysis, and enrichment analysis of HHIP-related co-expressed genes were performed using the TIMER, UALCAN, Kaplan-Meier plotter, and LinkedOmics databases. RESULTS: CAFs-exo were internalized by HCC cells. CAFs-exo contributed to the aggressive phenotype of HCC cells, while inhibiting exosome secretion reversed these effects. Mechanistically, miRNAs in the DLK1-DIO3 imprinted region (miR-329-3p, miR-380-3p, miR-410-5p, miR-431-5p) were increased in HCC cells co-cultured with CAFs-exo compared with PAFs-exo. Expression of HHIP, a possible miR-431-5p target gene, was significantly downregulated in HCC cells. Low HHIP expression level in tumor tissues could predict poor prognosis in HCC patients. HHIP-related co-expressed genes were mainly associated with cell adhesion molecules. CONCLUSIONS: CAFs-exo can promote HCC progression by delivering miRNAs in the DLK1-DIO3 imprinted region to HCC cells, subsequently inhibiting HHIP expression. HHIP is a potential prognostic biomarker in HCC.

Modulation of the p38 MAPK Pathway by Anisomycin Promotes Ferroptosis of Hepatocellular Carcinoma through Phosphorylation of H3S10.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide. Ferroptosis is emerging as an effective target for tumor treatment as it has been shown to potentiate cell death in some malignancies. However, it remains unclear whether histone phosphorylation events, an epigenetic mechanism that regulates transcriptional expression, are involved in ferroptosis. Our study found that supplementation with anisomycin, an agonist of p38 mitogen-activated protein kinase (MAPK), induced ferroptosis in HCC cells, and the phosphorylation of histone H3 on serine 10 (p-H3S10) was participated in anisomycin-induced ferroptosis. To investigate the anticancer effects of anisomycin-activated p38 MAPK in HCC, we analyzed cell viability, colony formation, cell death, and cell migration in Hep3B and HCCLM3 cells. The results showed that anisomycin could significantly suppress HCC cell colony formation and migration and induce HCC cell death. The hallmarks of ferroptosis, such as abnormal accumulation of iron and elevated levels of lipid peroxidation and malondialdehyde, were detected to confirm the ability of anisomycin to promote ferroptosis. Furthermore, coincubation with SB203580, an inhibitor of activated p38 MAPK, partially rescued anisomycin-induced ferroptosis. And the levels of p-p38 MAPK and p-H3S10 were successively increased by anisomycin treatment. The relationship between p-H3S10 and ferroptosis was revealed by ChIP sequencing. The reverse transcription PCR and immunofluorescence results showed that NCOA4 was upregulated both in mRNA and protein levels after anisomycin treatment. And by C11-BODIPY staining, we found that anisomycin-induced lipid reactive oxygen species was reduced after NCOA4 knockdown. In conclusion, the anisomycin-activated p38 MAPK promoted ferroptosis of HCC cells through H3S10 phosphorylation.

ARHGAP24 represses ß-catenin transactivation-induced invasiveness in hepatocellular carcinoma mainly by acting as a GTPase-independent scaffold.

Rationale: Accumulating evidence shows that Rho-GTPase-activating proteins (RhoGAPs) exert suppressive roles in cancer cell proliferation and metastasis. However, no study has systematically investigated the clinical significance of RhoGAPs and analyzed the functions of ARHGAP24 in hepatocellular carcinoma (HCC). Methods: The relationship between RhoGAP expression and HCC prognosis was investigated via using The Cancer Genome Atlas and Gene Expression Omnibus databases. ARHGAP24 expression was detected by reverse transcription-polymerase chain reaction, western blot and immunohistochemistry staining assays. Moreover, in vitro assays including cell counting kit-8, colony formation, wound healing and Transwell assays, and in vivo tumor growth and pulmonary metastases evaluations were conducted to evaluate the biological function of ARHGAP24 in HCC. Liquid chromatography-tandem mass spectrometry, co-immunoprecipitation, GTPase activation, ubiquitination, and luciferase reporter assays and bioinformatics analysis were carried out to gain insights into the mechanisms underlying the tumor-suppressive function of ARHGAP24. Results: ARHGAP24 expression was dramatically decreased in HCC tissues, and low ARHGAP24 expression was an independent poor prognostic indicator for progression-free survival in HCC patients. ARHGAP24 overexpression significantly inhibited cell proliferation, migration and invasion, while knockdown of ARHGAP24 exerted the opposite effects. Through Gene Set Enrichment Analysis (GSEA), we found ARHGAP24 mainly suppressed HCC cell proliferation and invasion by attenuating ß-catenin transactivation and blocking ß-catenin signaling could effectively abolish the promotional effects of ARHGAP24 knockdown in HCC cells. Notably, GAP-deficient mutant of ARHGAP24 exerted similar inhibitory effects as the wild-type did, indicating suppressive function of ARHGAP24 was independent of its RhoGAP activity. Moreover, we identified pyruvate kinase M2 (PKM2) as a new binding partner of ARHGAP24, which recruited a novel E3 ligase (WWP1) and subsequently promoted PKM2 degradation. WWP1 knockdown significantly reduced the inhibitory function of ARHGAP24, and the C-terminal fragments of ARHGAP24 (amino acids 329 - 430 and 631 - 748) bound directly to WWP1 and PKM2 (amino acids 388 - 531), respectively. Conclusions: Our data indicate that ARHGAP24 may be an independent prognostic indicator for HCC. It is a critical suppressor of HCC that recruits WWP1 for PKM2 degradation. Targeting the ARHGAP24/WWP1/PKM2/ß-catenin axis may provide new insights into HCC prevention and treatment.

CD155/SRC complex promotes hepatocellular carcinoma progression via inhibiting the p38 MAPK signalling pathway and correlates with poor prognosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a prevalent malignancy with poor prognosis. As a cell adhesion molecule, poliovirus receptor (PVR/CD155) is abnormally overexpressed in tumour cells, and related to tumour proliferation and invasion. However, the potential role and mechanism of CD155 have not yet been elucidated in HCC. METHODS: Immunohistochemistry, RT-PCR and Western blot assays were used to determine CD155 expression in HCC cell lines and tissues. Cell Counting Kit-8 and colony formation assays were used to examine cell proliferation. Transwell and wound healing assays were used to evaluate cell migration and invasion. Cell apoptosis and cycle distribution were assessed by flow cytometry. Cox regression and Kaplan-Meier analyses were performed to explore the clinical significance of CD155. The role of CD155 in vivo was evaluated by establishing liver orthotropic xenograft mice model. RNA sequencing, bioinformatics analysis and co-immunoprecipitation assay were used to explore the downstream signalling pathway of CD155. RESULTS: CD155 was upregulated in HCC tissues and represented a promising prognostic indicator for HCC patients (n = 189) undergoing curative resection. High CD155 expression enhanced cell proliferation, migration and invasion, and contributed to cell survival in HCC. CD155 overexpression also induced epithelial-mesenchymal transition in HCC cells. CD155 function in HCC involved SRC/p38 MAPK signalling pathway. CD155 interacted with SRC homology-2 domain of SRC and promoted SRC activation, further inhibiting the downstream p38 MAPK signalling pathway in HCC. CONCLUSIONS: CD155 promotes HCC progression via the SRC/p38 MAPK signalling pathway. CD155 may represent a predictor for poor postsurgery prognosis in HCC patients.

A novel prognostic model for hepatocellular carcinoma based on 5 microRNAs related to vascular invasion.

BACKGROUND: Hepatocellular carcinoma (HCC) is prevalent worldwide with a high mortality rate. Prognosis prediction is crucial for improving HCC patient outcomes, but effective tools are still lacking. Characteristics related to vascular invasion (VI), an important process involved in HCC recurrence and metastasis, may provide ideas on prognosis prediction. METHODS: Tools, including R 4.0.3, Funrich version 3, Cytoscape 3.8.2, STRING 11.5, Venny 2.1.0, and GEPIA 2, were used to perform bioinformatic analyses. The VI-related microRNAs (miRNAs) were identified using Gene Expression Omnibus HCC miRNA dataset GSE67140, containing 81 samples of HCC with VI and 91 samples of HCC without VI. After further evaluated the identified miRNAs based on The Cancer Genome Atlas database, a prognostic model was constructed via Cox regression analysis. The miRNAs in this model were also verified in HCC patients. Moreover, a nomogram was developed by integrating risk score from the prognostic model with clinicopathological parameters. Finally, a potential miRNA-mRNA network related to VI was established through weighted gene co-expression network analysis of HCC mRNA dataset GSE20017, containing 40 samples of HCC with VI and 95 samples of HCC without VI. RESULTS: A prognostic model of 5 VI-related miRNAs (hsa-miR-126-3p, hsa-miR-148a-3p, hsa-miR-15a-5p, hsa-miR-30a-5p, hsa-miR-199a-5p) was constructed. The area under receiver operating characteristic curve was 0.709 in predicting 5-year survival rate, with a sensitivity of 0.74 and a specificity of 0.63. The nomogram containing risk score could also predict prognosis. Moreover, a VI-related miRNA-mRNA network covering 4 miRNAs and 15 mRNAs was established. CONCLUSION: The prognostic model and nomogram might be potential tools in HCC management, and the VI-related miRNA-mRNA network gave insights into how VI was developed.

Hsa_circ_0003945 promotes progression of hepatocellular carcinoma by mediating miR-34c-5p/LGR4/ß-catenin axis activity.

Accumulating evidence suggests that circular RNAs (circRNAs) play essential roles in regulating cancer progression, but many circRNAs in hepatocellular carcinoma (HCC) remain unknown. Dysregulated circRNAs in HCC were identified through bioinformatics analysis of Gene Expression Omnibus data sets. Quantitative real-time PCR (qRT-PCR), Sanger sequencing, RNase R digestion and actinomycin D treatment were conducted to confirm the characterization of circRNAs. CCK-8, wound-healing and Transwell assays were performed to assess the functional roles of Hsa_circ_0003945 (Circ_0003945) in HCC cell lines. Subcellular fractionation and fluorescence in situ hybridization (FISH) were performed to locate Circ_0003945 in HCC cells. Dual-luciferase reporter assay was executed to verify the binding of Circ_0003945 to microRNAs (miRNAs) or the miRNAs to their target genes. In this study, we found that Circ_0003945 was upregulated in HCC tissue, and higher Circ_0003945 expression was positively correlated with tumour size and tumour stage. Furthermore, high plasma levels of circulating Circ_0003945 were confirmed in HCC patients compared with those in non-HCC groups. The functional experiments revealed that overexpression or knockdown of Circ_0003945 promoted or attenuated tumour growth and migration, respectively. Mechanistically, Circ_0003945 might exert as a miR-34c-5p sponge to upregulate the expression of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4), activating the ß-catenin pathway, and finally facilitating HCC progression. Additionally, a ß-catenin activator could reverse the effect of Circ_0003945 knockdown. In conclusion, Circ_0003945 exerts a tumour-promoting role in HCC cells by regulating the miR-34c-5p/LGR4/ß-catenin axis, which may be a potential target for HCC therapy.

High serum soluble CD155 level predicts poor prognosis and correlates with an immunosuppressive tumor microenvironment in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies with poor prognosis. There is no research about the clinical significance of serum soluble CD155 (sCD155) level for HCC. We aim to explore the prognostic and diagnostic value of sCD155 in HCC patients undergoing curative resection. METHODS: Serum sCD155 level in HCC patients was determined by enzyme-linked immunosorbent assay. The prognostic significance of sCD155 was evaluated by Cox regression and Kaplan-Meier analyses. CD155 expression and biomarkers of immune cells in HCC tissues were detected by immunohistochemistry staining. The diagnostic significance of sCD155 was evaluated using receiver operating characteristic curve. RESULTS: Serum sCD155 level was significantly increased in HCC patients and predicted poor prognosis. The prognostic value of sCD155 remained in low recurrent risk subgroups of HCC. Serum sCD155 level was positively related to CD155 expression in HCC tissues. High serum sCD155 level was associated with decreased numbers of CD8+ T cells and CD56+ NK cells and increased number of CD163+ M2 macrophages. Serum sCD155 level had better performance in distinguishing HCC patients from healthy donors and patients with chronic liver conditions than α-fetoprotein. Among patients with α-fetoprotein ≤ 20 ng/ml, serum sCD155 level could differentiate HCC patients from non-HCC patients. CONCLUSION: Serum sCD155 level represents a promising biomarker for diagnosis and prognosis of HCC. High serum sCD155 level may reflect an immunosuppressive tumor microenvironment in HCC.

The diagnostic value of plasma exosomal hsa_circ_0070396 for hepatocellular carcinoma.

Aim: We aimed to identify novel exosomal circular RNAs for hepatocellular carcinoma (HCC) diagnosis. Materials & methods: Exosomes were extracted and characterized. The expression level of exosomal circRNAs were verified via quantitative real-time PCR. The diagnostic value of candidate circRNAs was evaluated according to the receiver operating characteristic curve analysis. Results: The exosomal circ_0070396 significantly elevated in HCC patients than other control groups and it performed better in distinguishing HCC patients from healthy donors than that of α-fetoprotein. Combination of two above markers exerted greater diagnostic performance. Exosomal circ_0070396 could discriminate HCC individuals from patients with chronic hepatitis B and liver cirrhosis. Intriguingly, exosomal circ_0070396 was positively correlated with HCC progression. Conclusion: Exosomal circ_0070396 may be a potential biomarker for HCC detection and management.

BCL11B suppresses tumor progression and stem cell traits in hepatocellular carcinoma by restoring p53 signaling activity.

Accumulating evidence indicates that hepatocellular carcinoma (HCC) tumorigenesis, recurrence, metastasis, and therapeutic resistance are strongly associated with liver cancer stem cells (CSCs), a rare subpopulation of highly tumorigenic cells with self-renewal capacity and differentiation potential. Previous studies identified B cell leukemia/lymphoma-11b (BCL11B) as a novel tumor suppressor with impressive capacity to restrain CSC traits. However, the implications of BCL11B in HCC remain unclear. In this study, we found that low BCL11B expression was an independent indicator for shorter overall survival (OS) and time to recurrence (TTR) for HCC patients with surgical resection. In vitro and in vivo experiments confirmed BCL11B as a tumor suppressor in HCC with inhibitory effects on proliferation, cell cycle progression, apoptosis, and mobility. Furthermore, BCL11B could suppress CSC traits, as evidenced by dramatically decreased tumor spheroid formation, self-renewal potential and drug resistance. A Cignal Finder Array and dual-luciferase activity reporter assays revealed that BCL11B could activate the transcription of P73 via an E2F1-dependent manner. Thus, we concluded that BCL11B is a strong suppressor of retaining CSC traits in HCC. Ectopic expression of BCL11B might be a promising strategy for anti-HCC treatment with the potential to cure HBV-related HCC regardless of P53 mutation status.

Phosphorylase Kinase ß Represents a Novel Prognostic Biomarker and Inhibits Malignant Phenotypes of Liver Cancer Cell.

Glycogen phosphorylase kinase ß-subunit (PHKB) is a regulatory subunit of phosphorylase kinase (PHK), involving in the activation of glycogen phosphorylase (GP) and the regulation of glycogen breakdown. Emerging evidence suggests that PHKB plays a role in tumor progression. However, the function of PHKB in HCC progression remains elusive. Here, our study revealed that the expression of PHKB significantly decreased in HCC tissues, and the low expression of PHKB could serve as an independent indicator for predicting poor prognosis in HCC. Functional experiments showed that PHKB knockdown significantly promoted cell proliferation both in vitro and in vivo, whereas PHKB overexpression resulted in opposing effects. Additionally, in vitro assays revealed that the over (or high) expression of PHKB greatly hindered HCC cell invasion and increased apoptosis rates. Also, we found that the over (or high) expression of PHKB effectively suppressed the epithelial-mesenchymal transition, which was further confirmed by our clinical data. Intriguingly, the biological function of PHKB in HCC was independent of glycogen metabolism. Mechanically, PHKB could inhibit AKT and STAT3 signaling pathway activation in HCC. Collectively, our data demonstrate that PHKB acts as a novel prognostic indicator for HCC, which exerts its suppression function via inactivating AKT and STAT3. Our data might provide novel insights into progression and facilitate the development of a new therapeutic strategy for HCC.

CD73 promotes hepatocellular carcinoma progression and metastasis via activating PI3K/AKT signaling by inducing Rap1-mediated membrane localization of P110ß and predicts poor prognosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. METHODS: CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110ß and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. RESULTS: In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110ß to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CONCLUSIONS: CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.