Protein profiles reveal MSH6/MSH2 as a potential biomarker for hepatocellular carcinoma with microvascular invasion.

AIM: Microvascular invasion (MVI) is an independent risk factor for postoperative recurrence and metastasis in hepatocellular carcinoma (HCC). However, the specific protein expression profiles that differentiate HCC with MVI from those without MVI remain unclear. METHODS: The profiles of proteins in early-stage HCC tissues and normal liver tissues were characterized by quantitative proteomics techniques. Immunohistochemical (IHC) staining was undertaken on tissue microarrays from 80 HCC patients to assess the expression of MSH2 and MSH6. Cell counting, colony formation, migration, and invasion assays were carried out in vitro. RESULTS: We identified 5164 proteins in both HCC tissues and adjacent normal liver tissues. Compared to HCC without MVI, 148 upregulated proteins and 97 downregulated proteins were found in HCC with MVI. Particularly noteworthy was the remarkable upregulation of MSH6/MSH2 among these dysregulated proteins in HCC with MVI. Further validation through bioinformatics prediction and IHC confirmed the elevated expression of MSH6/MSH2, which correlated with aggressive disease characteristics and poor prognosis. Receiver operating characteristic curve analyses revealed a substantial area under the curve of 0.761 (specificity 71.79%, sensitivity 73.17%) for the combined use of MSH6/MSH2. Knockdown of MSH6/MSH2 significantly inhibited HCC cell proliferation and invasion in vitro. CONCLUSIONS: Our study establishes MSH6 or MSH2 as an oncogene that is prominently overexpressed during HCC progression, which provides new targets for HCC with MVI.

Exosomes derived from exhausted CD8+ T cells impaired the anticancer function of normal CD8+ T cells.

BACKGROUND: Previous studies suggested that diverse cells in cancer microenvironment can interact with CD8+ T cells via exosomes. We designed this study to explore the potential interaction between exhausted CD8+ T cells and normal CD8+ T cells via exosome. METHODS: Fluorescence activated cell sorting was used to get PD1+TIM3+/PD1-TIM3-CD8+ T cells. Exosomes from the cell culture medium were collected by ultracentrifugation. Microarrays were performed to analyse the lncRNA expression profile in exosomes. RESULTS: Functional exhausted CD8+ T cells could secrete vast exosomes, which can be uptake by normal CD8+ T cells, and impaired their proliferation (Ki67), cell activity (CD69) and the production of cytokines such as interferon-γ and interleukin-2. Microarray detection identified 257 candidate lncRNAs differently expressed in exosomes derived from exhausted CD8+ T cells and non-exhausted CD8+ T cells. Functional enrichment analysis indicated that these lncRNAs actively participated in the regulation of diverse process of CD8+ T cell activity, like metabolism, gene expression, biosynthetic process and so forth. CONCLUSIONS: The exosomes derived from exhausted CD8+ T cells could be uptake by non-exhausted CD8+ T cells and subsequently impaired the function of receipt cells. Exosomes secreted from exhausted CD8+ T cells have distinct lncRNA expression profiles which are significantly different from those in exosomes secreted by non-exhausted CD8+ T cells.

Applications and advances of CRISPR-Cas9 in cancer immunotherapy.

Immunotherapy has emerged as one of the most promising therapeutic strategies in cancer. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system, as an RNA-guided genome editing technology, is triggering a revolutionary change in cancer immunotherapy. With its versatility and ease of use, CRISPR-Cas9 can be implemented to fuel the production of therapeutic immune cells, such as construction of chimeric antigen receptor T (CAR-T) cells and programmed cell death protein 1 knockout. Therefore, CRISPR-Cas9 technology holds great promise in cancer immunotherapy. In this review, we will introduce the origin, development and mechanism of CRISPR-Cas9. Also, we will focus on its various applications in cancer immunotherapy, especially CAR-T cell-based immunotherapy, and discuss the potential challenges it faces.

Comprehensive treatments for hepatocellular carcinoma with portal vein tumor thrombosis.

Portal vein tumor thrombosis (PVTT) is one of the most common complications in hepatocellular carcinoma (HCC). HCC with PVTT usually indicates poor prognosis, which has a number of characteristics including a rapidly progressive disease course, worse liver function, complications connected with portal hypertension, and poorer tolerance to treatment. The exact mechanisms of PVTT remain unknown, even though some concerned signal transduction or molecular pathways have been identified. In western countries, sorafenib is the only recommended therapeutic strategy regardless of PVTT types. However, multiple treatment options including transhepatic arterial chemoembolization, hepatectomy, radiotherapy, and sorafenib available in the clinic. In this review, we enumerate and discuss therapeutics against patients with HCC having PVTT available in the clinic and put forward directions for future research.

TOX promotes the exhaustion of antitumor CD8+ T cells by preventing PD1 degradation in hepatocellular carcinoma.

BACKGROUND & AIMS: The thymocyte selection-associated high mobility group box protein (TOX) plays a vital role in T cell development and differentiation, however, its role in T cell exhaustion was unexplored. Here, we aim to investigate the role of TOX in regulating the antitumor effect of CD8+ T cells in hepatocellular carcinoma. METHODS: Fully functional, partially and severely exhausted tumor-infiltrating CD8+ T cells were sorted by flow cytometry and subjected to transcriptome sequencing analysis. Upregulated TOX expression was validated by flow cytometry. The antitumor function of CD8+ T cells with TOX downregulation or overexpression was studied in a mouse HCC model and HCC patient-derived xenograft mouse model. Transcriptome sequencing analysis was performed in TOX-overexpressing and control CD8+ T cells. The mechanism underlying the TOX-mediated regulation of PD1 expression was studied by laser confocal detection, immune co-precipitation and flow cytometer. RESULTS: TOX was upregulated in exhausted CD8+ T cells in hepatocellular carcinoma. TOX downregulation in CD8+ T cells inhibited tumor growth, increased CD8+ T cell infiltration, alleviated CD8+ T cell exhaustion and improved the anti-PD1 response of CD8+ T cells. The mechanism behind this involved the binding of TOX to PD1 in the cytoplasm, which facilitated the endocytic recycling of PD1, thus maintaining abundant PD1 expression at the cell surface. High expression of TOX in peripheral CD8+ T cells correlated with poorer anti-PD1 responses and prognosis. CONCLUSIONS: TOX promotes CD8+ T cell exhaustion in hepatocellular carcinoma by regulating endocytic recycling of PD1. Downregulating TOX expression in CD8+ T cells exerts synergistic effects with anti-PD1 therapy, highlighting a promising strategy for cancer immunotherapy. LAY SUMMARY: Abundant TOX expression in CD8+ T cells impairs their antitumor function in hepatocellular carcinoma. Mechanically, TOX reduces PD1 degradation and promotes PD1 translocation to the cell surface in CD8+ T cells, thus maintaining high PD1 expression at the cell surface. Downregulating TOX expression improves the antitumor function of CD8+ T cells, which shows the synergetic role of anti-PD1 therapy, highlighting a promising strategy for enhancement of cancer immunotherapy.

MicroRNA-3178 ameliorates inflammation and gastric carcinogenesis promoted by Helicobacter pylori new toxin, Tip-α, by targeting TRAF3.

BACKGROUND: Helicobacter pylori infection is the main cause of chronic gastritis, peptic ulcer, and gastric cancer. Tip-α is a newly identified carcinogenic factor present in H. pylori. TRAF3 can activate NF-κB by both canonical and noncanonical signaling pathways. In this study, we found that the expression of TRAF3 and NF-κB was upregulated, while microRNA-3178 (miR-3178) was decreased in H. pylori-positive gastric tissues but not in H. pylori-negative tissues. MATERIALS AND METHODS: GES-1 cells were incubated with 12.5 µg/mL recombinant Tip-α (rTip-α) in RPMI1640 for 2 hours. After another 24 hours, the supernatant medium was designed as inflammatory-conditioned medium (ICM) and that from the untreated control cells was designed as untreated control medium. The release of proinflammatory cytokines from GES-1 cells and proliferation of gastric cancer cells was determined by ELISA and CCK-8 kits. Cells were transfected with the mimic, inhibitor, negative control of miR-3178, or TRAF3 siRNA control siRNA. The medium was then replaced with RPMI1640, 12.5 µg/mL rTip-α, and collected, and the total cellular RNA and protein were extracted for the following detection. RESULTS: MiR-3178 mimic prevented the increasement of TRAF3 and hence decreased activation of NF-κB signals, whereas miR-3178 inhibitor could not, in GES-1 cells with Tip-α treatment. The condition medium from miR-3178 mimic transfected GES-1 cells could inhibit proliferation and induce apoptosis of inflammation-related gastric cancer cells SGC7901 and MGC803 by decreasing the production of inflammatory cytokines TNF-α and IL-6, which were secreted by GES-1 cells. CONCLUSIONS: Taken all together, Tip-α might activate NF-κB to promote inflammation and carcinogenesis by inhibiting miR-3178 expression, which directly targeting TRAF3, during H. pylori infection in gastric mucosal epithelial cells.

Retraction Notice to: circRASGRF2 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1224 sponge.

[This retracts the article DOI: 10.1016/j.omtn.2020.10.035.].

TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) stands as the fifth most prevalent malignant tumor on a global scale and presents as the second leading cause of cancer-related mortality. DNA damage-based radiotherapy (RT) plays a pivotal role in the treatment of HCC. Nevertheless, radioresistance remains a primary factor contributing to the failure of radiation therapy in HCC patients. In this study, we investigated the functional role of transketolase (TKT) in the repair of DNA double-strand breaks (DSBs) in HCC. Our research unveiled that TKT is involved in DSB repair, and its depletion significantly reduces both non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair. Mechanistically, TKT interacts with PARP1 in a DNA damage-dependent manner. Furthermore, TKT undergoes PARylation by PARP1, resulting in the inhibition of its enzymatic activity, and TKT can enhance the auto-PARylation of PARP1 in response to DSBs in HCC. The depletion of TKT effectively mitigates the radioresistance of HCC, both in vitro and in mouse xenograft models. Moreover, high TKT expression confers resistance of RT in clinical HCC patients, establishing TKT as a marker for assessing the response of HCC patients who received cancer RT. In summary, our findings reveal a novel mechanism by which TKT contributes to the radioresistance of HCC. Overall, we identify the TKT-PARP1 axis as a promising potential therapeutic target for improving RT outcomes in HCC.

Non-coding RNAs in radiotherapy resistance: Roles and therapeutic implications in gastrointestinal cancer.

Radiotherapy has become an indispensable and conventional means for patients with advanced solid tumors including gastrointestinal cancer. However, innate or acquired radiotherapy resistance remains a significant challenge and greatly limits the therapeutic effect, which results in cancer relapse and poor prognosis. Therefore, it is an urgent need to identify novel biomarkers and therapeutic targets for clarify the biological characteristics and mechanism of radiotherapy resistance. Recently, lots of studies have revealed that non-coding RNAs (ncRNAs) are the potential indicators and regulators of radiotherapy resistance via the mediation of various targets/pathways in different cancers. These findings may serve as a potential therapeutic strategy to overcome radiotherapy resistance. In this review, we will shed light on the recent findings regarding the functions and regulatory mechanisms of ncRNAs following radiotherapy, and comprehensively discuss their potential as biomarkers and therapeutic targets in radiotherapy resistance of gastrointestinal cancer.

The cancer-testis lncRNA LINC01977 promotes HCC progression by interacting with RBM39 to prevent Notch2 ubiquitination.

Cancer-testis genes are involved in the occurrence and development of cancer, but the role of cancer-testis-associated lncRNAs (CT-lncRNAs) in hepatocellular carcinoma (HCC) remains to be explored. Here, we discovered a novel CT-lncRNA, LINC01977, based on the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases. LINC01977 was exclusively expressed in testes and highly expressed in HCC. High LINC01977 levels correlated with poorer overall survival (OS) in individuals with HCC. Functional assays showed that LINC01977 promoted HCC growth and metastasis in vitro and in vivo. Mechanistically, LINC01977 directly bound to RBM39 to promote the further entry of Notch2 into the nucleus, thereby preventing the ubiquitination and degradation of Notch2. Furthermore, the RNA binding protein IGF2BP2, one of the m6A modification readers, enhanced the stability of LINC01977, resulting in its high level in HCC. Therefore, the data suggest that LINC01977 interacts with RBM39 and promotes the progression of HCC by inhibiting Notch2 ubiquitination and degradation, indicating that LINC01977 may be a potential biomarker and therapeutic target for HCC patients.

A pan-cancer analysis of the prognostic and immunological roles of matrix metalloprotease-1 (MMP1) in human tumors.

Objective: Cancer remains the leading killer of human health worldwide. It has been shown that matrix metalloproteinase-1(MMP1) is related to poor prognosis in cancers such as BRCA, CESC and COAD. However, systematic pan-cancer analysis about the prognostic and immunological roles of MMP1 has not been explored. Here, the purpose of this study was to investigate the prognostic and immunological roles of MMP1 in pan-cancer and confirm cancer-promoting effect in pancreatic cancer. Methods: In our study, bioinformatics were first used to analyze data from multiple databases. Then, several bioinformatics tools were utilized to investigate the role of MMP1 in 33 tumor types. Finally, molecular biology experiments were carried out to prove the cancer-promoting effect of MMP1 in pancreatic cancer. Results: MMP1 expression was higher in tumor tissues than in control tissues in most tumor types. High expression of MMP1 was associated with poor overall survival (OS) and disease-free survival (DFS) in some tumor types. Further analysis of MMP1 gene mutation data showed that MMP1 mutations significantly influenced the prognosis of STAD. In addition, MMP1 expression was closely related to cancer-associated fibroblast (CAFs) infiltration in a variety of cancers and played an important role on immune infiltration score, tumor mutational burden (TMB) and microsatellite instability (MSI). Gene Ontology enrichment analysis indicated that these 20 genes were mainly related to extracellular structure organization/extracellular matrix organization/extracellular matrix disassembly/collagen metabolic process in the enriched biological processes. Finally, molecular biology experiments confirmed the cancer-promoting effect of MMP1 in pancreatic cancer. Conclusions: Our pan-cancer analysis comprehensively proved that MMP1 expression is related with clinical prognosis and tumor immune infiltration, and MMP1 can become a prognostic and immunological biomarker.

The cancer-testis lncRNA lnc-CTHCC promotes hepatocellular carcinogenesis by binding hnRNP K and activating YAP1 transcription.

Cancer-testis (CT) genes participate in the initiation and progression of cancer, but the role of CT-associated long non-coding RNAs (CT-lncRNAs) in hepatocellular carcinoma (HCC) is still elusive. Here, we discovered a conserved CT-lncRNA, named lnc-CTHCC, which was highly expressed in the testes and HCC. A lnc-CTHCC-knockout (KO) mouse model further confirmed that the global loss of lnc-CTHCC inhibited the occurrence and development of HCC. In vitro and in vivo assays also showed that lnc-CTHCC promoted HCC growth and metastasis. Mechanistically, lnc-CTHCC bound to heterogeneous nuclear ribonucleoprotein K (hnRNP K), which was recruited to the YAP1 promoter for its activation. Additionally, the N6-methyladenosine (m6A) modification was mediated by N6-adenosine-methyltransferase 70-kDa subunit (METTL3) and recognized by insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)/IGF2BP3, which maintained lnc-CTHCC stability and increased its expression in HCC. Together, our results show that lnc-CTHCC directly binds to hnRNP K and promotes hepatocellular carcinogenesis and progression by activating YAP1 transcription, suggesting that lnc-CTHCC is a potential biomarker and therapeutic target of HCC.

APAF1-Binding Long Noncoding RNA Promotes Tumor Growth and Multidrug Resistance in Gastric Cancer by Blocking Apoptosome Assembly.

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease-activating factor 1 (APAF1)-binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA-binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3-mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase-9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

circRASGRF2 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1224 sponge.

Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the contributions and molecular mechanisms of circRNAs to hepatocellular carcinoma (HCC) remain largely unknown. In the present study, we compared the expression of circRNAs between five paired HCC and adjacent noncancerous liver (ANL) tissues by using RNA-sequencing (RNA-seq). circRASGRF2 (a circRNA located on chromosome 5 and derived from RASGRF2, hsa_circ_0073181) was identified and validated by quantitative reverse transcriptase PCR. The role of circRASGRF2 in HCC progression was assessed both in vitro and in vivo. Mechanistically, RNA immunoprecipitation and luciferase reporter assays were performed to confirm the interaction between circRASGRF2 and miR-1224 in HCC. circRASGRF2 was found to be significantly upregulated in HCC tissues and HCC cell lines compared with paired ANL tissues and normal cells. Our in vivo and in vitro data indicated that knockdown of circRASGRF2 inhibits the proliferation and migration of HCC cells. Mechanistically, we found that circRASGRF2 could promote the expression of focal adhesion kinase (FAK) by sponging miR-1224. Our data showed that circRASGRF2 is a central component linking circRNAs to progression of HCC, making it a potential therapeutic target.

Prognostic and predictive role of a metabolic rate-limiting enzyme signature in hepatocellular carcinoma.

OBJECTIVES: Abnormal expression of metabolic rate-limiting enzymes drives the occurrence and progression of hepatocellular carcinoma (HCC). This study aimed to elucidate the comprehensive model of metabolic rate-limiting enzymes associated with the prognosis of HCC. MATERIALS AND METHODS: HCC animal model and TCGA project were used to screen out differentially expressed metabolic rate-limiting enzyme. Cox regression, least absolute shrinkage and selection operation (LASSO) and experimentally verification were performed to identify metabolic rate-limiting enzyme signature. The area under the receiver operating characteristic curve (AUC) and prognostic nomogram were used to assess the efficacy of the signature in the three HCC cohorts (TCGA training cohort, internal cohort and an independent validation cohort). RESULTS: A classifier based on three rate-limiting enzymes (RRM1, UCK2 and G6PD) was conducted and serves as independent prognostic factor. This effect was further confirmed in an independent cohort, which indicated that the AUC at year 5 was 0.715 (95% CI: 0.653-0.777) for clinical risk score, whereas it was significantly increased to 0.852 (95% CI: 0.798-0.906) when combination of the clinical with signature risk score. Moreover, a comprehensive nomogram including the signature and clinicopathological aspects resulted in significantly predict the individual outcomes. CONCLUSIONS: Our results highlighted the prognostic value of rate-limiting enzymes in HCC, which may be useful for accurate risk assessment in guiding clinical management and treatment decisions.

Long noncoding RNA GMAN promotes hepatocellular carcinoma progression by interacting with eIF4B.

Gastric cancer metastasis associated long noncoding RNA (GMAN), a long noncoding RNA, is associated with metastasis in gastric cancer. However, its underlying mechanisms in hepatocellular carcinoma (HCC) are unclear. We found that lncRNA-GMAN was significantly overexpressed in HCC tissues. GMAN expression is associated with vascular invasion, histological grade, tumor, node, metastasis (TNM) stage, short overall survival, and disease-free survival. Knockdown of GMAN induced apoptosis and suppressed invasive and migration potential in vitro and vivo, whereas ectopic GMAN expression produced the opposite effect. We also found that the inhibition of apoptosis, rather than promotion of proliferation, was responsible for GMAN-enhanced cellular viability. Mechanistic analyses indicated that GMAN directly combined with eukaryotic translation initiation factor 4B (eIF4B) and promoted its phosphorylation at serine-422 by preventing eIF4B binding and dephosphorization of the protein phosphatase 2A subunit B. The results demonstrated the stability of p-eIF4B and the elevation of mRNA translation and anti-apoptosis-related protein expression, which further induced proliferation and metastasis of HCC. The current study demonstrates that GMAN regulates the progression of HCC by inhibiting apoptosis and promoting the survival of cancer cells.

T Cell Dysfunction in Cancer Immunity and Immunotherapy.

In cancer, T cells become dysfunctional owing to persistent antigen exposure. Dysfunctional T cells are characterized by reduced proliferative capacity, decreased effector function, and overexpression of multiple inhibitory receptors. Due to the presence of various inhibitory signals in the complex tumor microenvironment, tumor-specific T cells have distinct dysfunction states. Therapeutic reactivation of tumor-specific T cells has yielded good results in cancer patients. Here, we review the hallmarks of T cell dysfunction in cancer. Also, we discuss the relationship between T cell dysfunction and cancer immunotherapy.

Genomic and epigenomic perspectives of T-cell exhaustion in cancer.

In chronic infection and cancer, T cells gradually become exhausted because of the persistent stimulation by antigens. In this process, the overexpression of multiple inhibitory receptors is induced, the production of effective cytokines decreases and the cytotoxicity and proliferation of T cells impairs, all contributing to the failure of T cells in fighting against cancer. Reversing T-cell exhaustion is a promising immunotherapy for cancer that has yielded encouraging results. In this review, we discuss the genomic and epigenomic landscape of T-cell exhaustion in cancer. Also, we introduce the relevant therapeutic interventions for T-cell exhaustion in clinical trials.

Chimeric-antigen receptor T (CAR-T) cell therapy for solid tumors: challenges and opportunities.

Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have been shown to have unprecedented efficacy in B cell malignancies, most notably in B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate using anti-CD19 CAR-T cells. However, CAR T-cell therapy for solid tumors currently is faced with numerous challenges such as physical barriers, the immunosuppressive tumor microenvironment and the specificity and safety. The clinical results in solid tumors have been much less encouraging, with multiple cases of toxicity and a lack of therapeutic response. In this review, we will discuss the current stats and challenges of CAR-T cell therapy for solid tumors, and propose possibl e solutions and future perspectives.