UBE2T-mediated Akt ubiquitination and Akt/ß-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism.

The oncogene protein ubiquitin-conjugating enzyme E2T (UBE2T) is reported to be upregulated in hepatocellular carcinoma (HCC) and correlated with poor clinical outcomes of HCC patients. However, the underlying mechanism by which UBE2T exerts its oncogenic function in HCC remains largely unexplored. In this study, in vitro and in vivo experiments suggested that UBE2T promoted HCC development including proliferation and metastasis. GSEA analysis indicated that UBE2T was positively correlated with pyrimidine metabolism, and LC/MS-MS metabolomics profiling revealed that the key products of pyrimidine metabolism were significantly increased in UBE2T-overexpressing cells. UBE2T overexpression led to the upregulation of several key enzymes catalyzing de novo pyrimidine synthesis, including CAD, DHODH, and UMPS. Moreover, the utilization of leflunomide, a clinically approved DHODH inhibitor, blocked the effect of UBE2T in promoting HCC progression. Mechanistically, UBE2T increased Akt K63-mediated ubiquitination and Akt/ß-catenin signaling pathway activation. The disruption of UBE2T-mediated ubiquitination on Akt, including E2-enzyme-deficient mutation (C86A) of UBE2T and ubiquitination-site-deficient mutation (K8/14 R) of Akt impaired UBE2T's effect in upregulating CAD, DHODH, and UMPS. Importantly, we demonstrated that UBE2T was positively correlated with p-Akt, ß-catenin, CAD, DHODH, and UMPS in HCC tumor tissues. In summary, our study indicates that UBE2T increases pyrimidine metabolism by promoting Akt K63-linked ubiquitination, thus contributing to HCC development. This work provides a novel insight into HCC development and a potential therapeutic strategy for HCC patients.

Wnt/ß-catenin inhibitor ICG-001 enhances the antitumor efficacy of radiotherapy by increasing radiation-induced DNA damage and improving tumor immune microenvironment in hepatocellular carcinoma.

BACKGROUND AND PURPOSE: Radiotherapy (RT) has a promising anti-tumor effect depending on its effects on both cancer cells and tumor immune microenvironment (TIME). As one of the most common alterations in hepatocellular carcinoma (HCC), wnt/ß-catenin pathway activation, has been reported to induce radioresistance and suppressive TIME. In this study, we aim to explore the effect of wnt/ß-catenin inhibitor ICG-001 on radiosensitivity and RT-related TIME of HCC and the underlying mechanism. MATERIALS AND METHODS: C57BL/6 and nude mouse tumor models were used to evaluate the efficacy of different treatments on tumor growth, recurrence and mice survival. Flow cytometry was performed to assess tumor infiltrating lymphocytes (TILs). DNA damage response (DDR) and radioresistance was investigated by colony formation assays, γ-H2AX and micronuclei measurements. RESULTS: The addition of ICG-001 to RT exhibited better anti-tumor and survival-prolong efficacy in C57BL/6 than nude mice. TILs analysis revealed that ICG-001 plus RT boosted the infiltration and IFN-γ production of TIL CD8+ T cells, meanwhile reduced the number of Tregs. Moreover, mechanistic study demonstrated that ICG-001 increased the radiation-induced DDR of HCC cells by suppressing p53, thus leading to stronger activation of cGAS/STING pathway. Utilization of cGAS/STING pathway inhibitors impaired the therapeutic effect of combination therapy. Furthermore, combination therapy led to stronger immunologic memory and tumor relapse prevention. CONCLUSIONS: Our findings showed that ICG-001 displayed both local and systematic effects by increasing radiosensitivity and improving immunity in HCC, which indicated that ICG-001 might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in HCC patients.

Upregulated long non­coding RNA LincIN promotes tumor progression via the regulation of nuclear factor 90/microRNA­7/HOXB13 in esophageal squamous cell carcinoma.

Long non­coding RNA LincIN has been reported to be overexpressed and to be involved in the metastasis of breast cancer. However, the expression and role of LincIN in esophageal squamous cell carcinoma (ESCC) remain unsolved. In the present study, LincIN expression was examined in ESCC by RT­qPCR, and the roles of LincIN in ESCC were determined using cell growth, migration and invasion assays. In addition, the effects of LincIN on nuclear factor 90 (NF90) and microRNA/miR (miR)­7 were examined by RNA immunoprecipitation assay, RT­qPCR, dual­luciferase reporter assay and western blot analysis. The results revealed that LincIN expression was significantly increased in ESCC tissues and cell lines. The increased expression of LincIN was positively associated with invasion depth, lymph node metastasis, TNM stage and a poor prognosis. Functional assays revealed that the overexpression of LincIN promoted ESCC cell growth, migration and invasion. Mechanistic analysis revealed that LincIN physically bound to NF90, enhanced the binding between NF90 and primary miR­7 (pri­miR­7), and further enhanced the inhibitory effects of NF90 on miR­7 biogenesis. Therefore, LincIN downregulated miR­7 expression in ESCC. The expression of miR­7 inversely correlated with that of LincIN in ESCC tissues. By downregulating miR­7, LincIN increased the expression of HOXB13, a target of miR­7. The overexpression of miR­7 or the depletion of HOXB13 both attenuated the tumor­promoting roles of LincIN in ESCC cell growth, migration and invasion. On the whole, the findings of the present study suggest that LincIN is overexpressed and plays an oncogenic role in ESCC via the regulation of the NF90/miR­7/HOXB13 axis. Thus, LincIN may prove to be a promising prognostic biomarker and therapeutic target for ESCC.

UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation.

BACKGROUND: Radioresistance is the major obstacle in radiation therapy (RT) for hepatocellular carcinoma (HCC). Dysregulation of DNA damage response (DDR), which includes DNA repair and cell cycle checkpoints activation, leads to radioresistance and limits radiotherapy efficacy in HCC patients. However, the underlying mechanism have not been clearly understood. METHODS: We obtained 7 pairs of HCC tissues and corresponding non-tumor tissues, and UBE2T was identified as one of the most upregulated genes. The radioresistant role of UBE2T was examined by colony formation assays in vitro and xenograft tumor models in vivo. Comet assay, cell cycle flow cytometry and γH2AX foci measurement were used to investigate the mechanism by which UBE2T mediating DDR. Chromatin fractionation and immunofluorescence staining were used to assess cell cycle checkpoint kinase 1(CHK1) activation. Finally, we analyzed clinical data from HCC patients to verify the function of UBE2T. RESULTS: Here, we found that ubiquitin-conjugating enzyme E2T (UBE2T) was upregulated in HCC tissues, and the HCC patients with higher UBE2T levels exhibited poorer outcomes. Functional studies indicated that UBE2T increased HCC radioresistance in vitro and in vivo. Mechanistically, UBE2T-RNF8, was identified as the E2-E3 pair, physically bonded with and monoubiquitinated histone variant H2AX/γH2AX upon radiation exposure. UBE2T-regulated H2AX/γH2AX monoubiquitination facilitated phosphorylation of CHK1 for activation and CHK1 release from the chromatin to cytosol for degradation. The interruption of UBE2T-mediated monoubiquitination on H2AX/γH2AX, including E2-enzyme-deficient mutation (C86A) of UBE2T and monoubiquitination-site-deficient mutation (K119/120R) of H2AX, cannot effectively activate CHK1. Moreover, genetical and pharmacological inhibition of CHK1 impaired the radioresistant role of UBE2T in HCC. Furthermore, clinical data suggested that the HCC patients with higher UBE2T levels exhibited worse response to radiotherapy. CONCLUSION: Our results revealed a novel role of UBE2T-mediated H2AX/γH2AX monoubiquitination on facilitating cell cycle arrest activation to provide sufficient time for radiation-induced DNA repair, thus conferring HCC radioresistance. This study indicated that disrupting UBE2T-H2AX-CHK1 pathway maybe a promising potential strategy to overcome HCC radioresistance.

ATR inhibitor AZD6738 enhances the antitumor activity of radiotherapy and immune checkpoint inhibitors by potentiating the tumor immune microenvironment in hepatocellular carcinoma.

BACKGROUND: Radioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation leads to the inhibition of immune microenvironment, thus impairing the antitumor effect of radioimmunotherapy. However, it is unclear whether inhibition of the DDR pathway can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of DDR inhibitor AZD6738 on the combination of radiotherapy and immune checkpoint inhibitors (ICIs) in HCC. METHODS: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control and tumor recurrence inhibition. Effects of each treatment regimen on the alterations of immunophenotypes including the quantification, activation, proliferating ability, exhaustion marker expression, and memory status were assessed by flow cytometry. RESULTS: AZD6738 further increased radiotherapy-stimulated CD8+ T cell infiltration and activation and reverted the immunosuppressive effect of radiation on the number of Tregs in mice xenografts. Moreover, compared with radioimmunotherapy (radiotherapy plus anti-PD-L1 (Programmed death ligand 1)), the addition of AZD6738 boosted the infiltration, increased cell proliferation, enhanced interferon (IFN)-γ production ability of TIL (tumor-infiltrating lymphocyte) CD8+ T cells, and caused a decreasing trend in the number of TIL Tregs and exhausted T cells in mice xenografts. Thus, the tumor immune microenvironment was significantly improved. Meanwhile, triple therapy (AZD6738 plus radiotherapy plus anti-PD-L1) also induced a better immunophenotype than radioimmunotherapy in mice spleens. As a consequence, triple therapy displayed greater benefit in antitumor efficacy and mice survival than radioimmunotherapy. Mechanism study revealed that the synergistic antitumor effect of AZD6738 with radioimmunotherapy relied on the activation of cyclic GMP-AMP synthase /stimulator of interferon genes (cGAS/STING) signaling pathway. Furthermore, triple therapy led to stronger immunologic memory and lasting antitumor immunity than radioimmunotherapy, thus preventing tumor recurrence in mouse models. CONCLUSIONS: Our findings indicate that AZD6738 might be a potential synergistic treatment for radioimmunotherapy to control the proliferation of HCC cells, prolong survival, and prevent tumor recurrence in patients with HCC by improving the immune microenvironment.

Knockdown of UACA inhibitsproliferation and invasion and promotes senescence of hepatocellular carcinoma cells.

Uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA/Nucling), has been reported to be upregulated in various cancers. However, its expression and function have not been studied in hepatocellular carcinoma (HCC). In the present study, expression of UACA was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the results revealed that UACA was upregulated in 23 cases of HCC compared with paired corresponding non-tumor liver tissues. In addition, the upregulation of UACA in HCC was further validated by analyzing the datasets from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) and GSE36376. Furthermore, knockdown of UACA suppressed the proliferative and invasive ability as well as inducing senescence of HCC cells. Besides, the expression level of UACA was positively associated with Hif1α (hypoxia-inducible factor 1α) in HCC datasets from TCGA-LIHC and GSE54236. Moreover, treatment with CoCl2 led to the increased expression and the localization alteration of UACA in HCC cells. In summary, UACA is upregulated in HCC and knockdown of UACA ameliorated malignant behaviors of HCC cells, and UACA was correlated with and under control of Hif1α.