RNA binding motif protein 45-mediated phosphorylation enhances protein stability of ASCT2 to promote hepatocellular carcinoma progression.

Targeting metabolic remodeling represents a potentially promising strategy for hepatocellular carcinoma (HCC) therapy. In-depth understanding on the regulation of the glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2) contributes to the development of novel promising therapeutics. As a developmentally regulated RNA binding protein, RBM45 is capable to shuttle between nucleus and cytoplasm, and directly interacts with proteins. By bioinformatics analysis, we screened out that RBM45 was elevated in the HCC patient specimens and positively correlated with poor prognosis. RBM45 promoted cell proliferation, boosted xenograft tumorigenicity and accelerated HCC progression. Using untargeted metabolomics, it was found that RBM45 interfered with glutamine metabolism. Further results demonstrated that RBM45 positively associated with ASCT2 in human and mouse specimens. Moreover, RBM45 enhanced ASCT2 protein stability by counteracting autophagy-independent lysosomal degradation. Significantly, wild-type ASCT2, instead of phospho-defective mutants, rescued siRBM45-suppressed HCC cell proliferation. Using molecular docking approaches, we found AG-221, a mutant isocitrate dehydrogenase 2 (mIDH2) inhibitor for acute myeloid leukemia therapy, pharmacologically perturbed RBM45-ASCT2 interaction, decreased ASCT2 stability and suppressed HCC progression. These findings provide evidence that RBM45 plays a crucial role in HCC progression via interacting with and counteracting the degradation of ASCT2. Our findings suggest a novel alternative structural sites for the design of ASCT2 inhibitors and the agents interfering with RBM45-ASCT2 interaction may be a potential direction for HCC drug development.

RHNO1 disruption inhibits cell proliferation and induces mitochondrial apoptosis via PI3K/Akt pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) represents one of the primary liver malignancies with poor prognosis. RHNO1, which implicated in the ATR-CHK1 signaling pathway thus functions in the DNA replication stress response. However, the role and molecular mechanisms of RHNO1 in HCC remain largely elusive. Here, we imply that RHNO1 is elevated in HCC tumor tissues and that high expression of RHNO1 predicts poor prognosis of HCC patients. Moreover, RHNO1 mRNA, especially protein levels were significantly increased in most HCC cells. Knockdown of RHNO1 through small interfering RNAs (siRNAs) inhibited the proliferation and triggered cell apoptosis of HCC cells both in vitro and in vivo. Specifically, we find that RHNO1 deficiency confers apoptosis via mitochondrial-mediated pathway. Mechanistically, silencing of RHNO1 impeded HCC proliferation and induced apoptosis by inactivating the PI3K/Akt pathway. Overall, these findings unravel that RHNO1 functions as an oncogene in HCC, and involved in regulating mitochondrial apoptosis to promote HCC thus may serve as a therapeutic and diagnostic target for HCC.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.

GPR27 Regulates Hepatocellular Carcinoma Progression via MAPK/ERK Pathway.

Purpose: Orphan GPCRs (GPRs) play important roles in the malignant progression of cancer and have the potential to develop into anti-tumor drug targets. However, the biological processes and molecular mechanisms of GPR27 have not been properly assessed in cancer. Our objective was to reveal the effect of GPR27 on the progression of hepatocellular carcinoma (HCC). Methods: GPR27 levels were detected in HCC cell lines using quantitative reverse transcriptase-polymerase chain reaction and Western blot analysis. Next, the changes of phenotypes after GPR27 knockdown or overexpression were evaluated using in vitro methods. Finally, the mechanism of GPR27 in HCC was tested using RNA-seq and in vivo mouse xenograft model. Results: In the present study, we reported that suppression of GPR27 expression inhibited proliferation, colony formation, cell viability, and induced cell S phase arrest of HCC cells, whereas GPR27 overexpression led to the opposite outcomes. Moreover, suppression of GPR27 expression resulted in blocking MAPK/ERK signal pathway which indicated the inhibition of HCC cells proliferation. Further study in vivo confirmed that GPR27 can affect the proliferation of HCC cells through the MAPK/ERK pathway. Conclusion: Taken together, the findings of the present study uncover biological functions of GPR27 in HCC cells, and delineate preliminary molecular mechanisms of GPR27 in modulating HCC development and progression.

Preclinical efficacy against acute myeloid leukaemia of SH1573, a novel mutant IDH2 inhibitor approved for clinical trials in China.

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults, with increasing incidence with age and a generally poor prognosis. Almost 20% of AML patients express mutant isocitrate dehydrogenase 2 (mIDH2), which leads to the accumulation of the carcinogenic metabolite 2-hydroxyglutarate (2-HG), resulting in poor prognosis. Thus, global institutions have been working to develop mIDH2 inhibitors. SH1573 is a novel mIDH2 inhibitor that we independently designed and synthesised. We have conducted a comprehensive study on its pharmacodynamics, pharmacokinetics and safety. First, SH1573 exhibited a strong selective inhibition of mIDH2 R140Q protein, which could effectively reduce the production of 2-HG in cell lines, serum and tumors of an animal model. It could also promote the differentiation of mutant AML cell lines and granulocytes in PDX models. Then, it was confirmed that SH1573 possessed characteristics of high bioavailability, good metabolic stability and wide tissue distribution. Finally, toxicological data showed that SH1573 had no effects on the respiratory system, cardiovascular system and nervous system, and was genetically safe. This research successfully promoted the approval of SH1573 for clinical trials (CTR20200247). All experiments demonstrated that, as a potential drug against mIDH2 R140Q acute myeloid leukaemia, SH1573 was effective and safe.

C118P, a novel microtubule inhibitor with anti-angiogenic and vascular disrupting activities, exerts anti-tumor effects against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a hypervascular solid tumor, is the most leading cause of cancer mortality worldwide. Microtubule binding agents targeting tumor vasculature have been investigated and employed clinically. C118P is a newly synthesized analog of CA4 with improved water solubility and extended half-life. The current studies investigated the pharmacological effects of C118P and its active metabolite C118. Here, we first confirmed by in vitro assays that C118 exerts microtubule depolymerization activity and by molecular docking revealed that it fits to the colchicine binding site of tubulin. In addition, we found that C118P and C118 altered microtubule dynamics and cytoskeleton in human umbilical vein endothelial cells. Accordingly, we observed that C118P and C118 inhibited angiogenesis and disrupted established vascular networks using tube formation assays and chick chorioallantoic membrane angiogenesis assays. In addition, our data showed that C118P and C118 exhibited board anti-proliferative effect on various cancer cells, including HCC cell lines, in MTT assays or Sulforhodamine B assays. Moreover, we found that C118P induced G2/M phase cell cycle arrest and apoptosis in HCC cell lines BEL7402 and SMMC7721 using flow cytometry analysis and immunoblotting assays. Finally, we confirmed that C118P suppressed HCC growth via targeting tumor vasculature and inducing apoptosis in the SMMC7721 xenograft mouse model. In conclusion, our studies revealed that C118P, as a potent microtubule destabilizing agent, exerts its multiple pharmacological effects against HCC by inducing cell cycle arrest and apoptosis, as well as targeting tumor vasculature. Thus, C118P might be a promising drug candidate for liver cancer treatment.

The comprehensive landscape of miR-34a in cancer research.

MicroRNA-34 (miR-34) plays central roles in human diseases, especially cancers. Inactivation of miR-34 is detected in cancer cell lines and tumor tissues versus normal controls, implying its potential tumor-suppressive effect. Clinically, miR-34 has been identified as promising prognostic indicators for various cancers. In fact, members of the miR-34 family, especially miR-34a, have been convincingly proved to affect almost the whole cancer progression process. Here, a total of 512 (miR-34a, 10/21), 85 (miR-34b, 10/16), and 114 (miR-34c, 10/14) putative targets of miR-34a/b/c are predicted by at least ten miRNA databases, respectively. These targets are further analyzed in gene ontology (GO), KEGG pathway, and the Reactome pathway dataset. The results suggest their involvement in the regulation of signal transduction, macromolecule metabolism, and protein modification. Also, the targets are implicated in critical signaling pathways, such as MAPK, Notch, Wnt, PI3K/AKT, p53, and Ras, as well as apoptosis, cell cycle, and EMT-related pathways. Moreover, the upstream regulators of miR-34a, mainly including transcription factors (TFs), lncRNAs, and DNA methylation, will be summarized. Meanwhile, the potential TF upstream of miR-34a/b/c will be predicted by PROMO, JASPAR, Animal TFDB 3.0, and GeneCard databases. Notably, miR-34a is an attractive target for certain cancers. In fact, miR-34a-based systemic delivery combined with chemotherapy or radiotherapy can more effectively control tumor progression. Collectively, this review will provide a panorama for miR-34a in cancer research.

Sepiapterin reductase promotes hepatocellular carcinoma progression via FoxO3a/Bim signaling in a nonenzymatic manner.

Sepiapterin reductase plays an enzymatic role in the biosynthesis of tetrahydrobiopterin, which is reported in limited studies to regulate the progression of several tumors. However, the role of sepiapterin reductase in hepatocellular carcinoma remains largely unknown. Here, we found that sepiapterin reductase was frequently highly expressed in human hepatocellular carcinoma, which was significantly associated with higher T stage, higher tumor node metastasis stage, and even shorter survival of hepatocellular carcinoma patients. Furthermore, cell and animal experiments showed that sepiapterin reductase depletion inhibited cancer cell proliferation and promoted cancer cell apoptosis. Importantly, the results suggested that sepiapterin reductase enzymatic activity was not necessary for the progression of hepatocellular carcinoma, based on the comparison between SMMC-7721 and SMMC-7721 containing sepiapterin reductase mutant. Moreover, we showed that sepiapterin reductase regulated the development of hepatocellular carcinoma via the FoxO3a/Bim-signaling pathway. Collectively, our study suggests that sepiapterin reductase controls hepatocellular carcinoma progression via FoxO3a/Bim signaling in a nonenzymatic manner, which provides a potential prognostic factor and therapeutic strategy for hepatocellular carcinoma.

The structure and regulation of the E3 ubiquitin ligase HUWE1 and its biological functions in cancer.

E3 ligases are a class of critical enzymes that can catalyse the transfer of ubiquitin (Ub) from an E2 enzyme to the substrate and are essential to cellular processes. The E3 ligase HUWE1 (also known as ARF-BP1, HECTH9, HSPC272, Ib772, LASU1, MULE, URE-B1, UREB1, and HECT, UBA and WWE domain-containing E3 ubiquitin protein ligase 1) is encoded by the huwe1 gene. HUWE1 is a key regulator of the DNA damage response, transcription, autophagy, apoptosis and metabolism in a variety of cancers. Due to its pivotal role in conferring substrate specificity, HUWE1 has attracted enormous attention as a promising anticancer drug target. In this review, we indicate the specific molecular structure of HUWE1 and its role in various cellular signalling pathways and highlight new insights into HUWE1 in cancer. Finally, we discuss outstanding questions regarding HUWE1 in oncology and highlight its limitations in drug development and clinical guidance to better define the role of HUWE1 in multiple cancers.

Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop.

Aberrant energy metabolism is critical for cancer progression. Tumor-associated macrophages (TAMs) can stimulate tumor angiogenesis and enhance cancer metastasis; however, the metabolic interaction between cancer cells and macrophages characterized by lactate shuttles remains unclear. Here, we showed that lactate activated human macrophages to a TAM-like phenotype and stimulated the secretion of CCL5 by activation of Notch signaling in macrophages. Reciprocally, CCL5 increased cell migration, induced cancer cell EMT, and promoted aerobic glycolysis in breast cancer cells, suggesting a positive metabolic feedback loop in the co-culture system. Inhibition of CCR5, the cognate receptor of CCL5, or neutralization of CCL5, broke the metabolic loop and decreased cancer cell migration and EMT. Inhibition of aerobic glycolysis significantly reduced breast cancer cell EMT, indicated that aerobic glycolysis was necessary for the invasive phenotype of cancer cells. We further showed that TGF-ß signaling regulated the expression of CCR5 in the co-culture system, and CCL5 induced glycolysis by mediation of AMPK signaling. The expression of CCL5-CCR5 axis was highly associated with macrophage infiltration, TGF-ß and p-AMPK in clinical samples. CCL5-CCR5 axis promoted breast cancer metastasis in vivo. Our findings suggested a pivotal role of CCL5-CCR5 axis in the metabolic communication between cancer cells and macrophages.

MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-ß signaling in human breast cancer cells.

BACKGROUND: Cordyceps has long been used to treat cancer. However, its pharmacologically active components as well as the molecular mechanisms underlying its effects are still unclear. PURPOSE: To investigate the effect of MHP-1, a newly isolated polysaccharide from Mortierella hepialid (the asexual structure of C. sinensis), on breast cancer metastasis. STUDY DESIGN: The effect of MHP-1 on breast cancer cell migration, epithelial-mesenchymal transition (EMT) and TGF-ß signaling were investigated in vitro and in vivo. The effect of MHP-1 against topotecan-resistant MCF-7 cells that developed an EMT-like phenotype was also examined. METHODS: The in vitro effect of MHP-1 on breast cancer cell proliferation and migration was evaluated by CCK8 and transwell assay. Morphological changes were observed and EMT markers were detected by western blot. The production of MMPs was measured by quantitative PCR and ELISA assay. To further investigate the mechanism that MHP-1 inhibited breast cancer EMT, western blot, ELISA, luciferase reporter gene assay, siRNA, quantitative PCR, immunohistochemistry, and xenograft tumor model were performed. RESULTS: MHP-1 inhibited breast cancer cell migration but did not cause any cytotoxicity. MHP-1 significantly surpressed breast cancer EMT, and slightly decreased MMP-9 secretion. TGF-ß signaling was selectively inhibited after MHP-1 treatment, and other EMT-related pathways, like Wnt and Notch, were not affected. MHP-1 reduced the secretion of TGF-ß1, but rarely affected other EMT-induced cytokines. Dual luciferase assay and Smad2/3 phosphorylation analysis indicated that MHP-1 suppressed TGF-ß signaling. We further showed that MHP-1 restored sensitivity in topotecan (TPT)-resistant MCF-7 cells that developed an EMT-like phenotype. Similarly, the effect of TPT on resistant MCF-7 cells was also increased either by ALK5 (TGFßRI) siRNA or by a small molecular inhibitor of ALK5, SB-431542. MHP-1 inhibited breast cancer metastasis in the MDA-MB-231 xenograft model, and the immunohistochemical staining showed dramatically decreased expression of ALK5 and vimentin, and increased expression of E-cadherin. CONCLUSION: MHP-1 significantly inhibited breast cancer metastasis and restored drug sensitivity in TPT-resistant cells via down-regulation of TGF-ß signaling and EMT program. The combination of non-toxic agents like MHP-1 and current anti-cancer drugs should be considered in the future treatment of cancer.

[Effects and mechanisms of Xiao-Ai-Ping injection on angiogenesis].

This study was designed to investigate the effect of Xiao-Ai-Ping injection on cancer angiogenesis. CCK8 assay and Brd U incorporation immunofluorescence assay were used to detect the effect of Xiao-Ai-Ping injection on HUVECs proliferation; wound healing assay and transwell assay were employed to test the effect of Xiao-Ai-Ping injection on HUVECs migration. The anti-angiogenic effect of Xiao-Ai-Ping injection was examined by tube formation assay, rat aortic ring assay and chicken chorioallantoic membrane(CAM) assay. ELISA assay was used to measure the secretion of vascular endothelial growth factor(VEGF); and the activation of vascular endothelial growth factor receptor 2(VEGFR2) protein and its downstream signaling pathways were examined by Western blot. Our data demonstrated that Xiao-Ai-Ping injection inhibited HUVECs proliferation in a time- and dose-dependent manner, and the IC(50) (mg·m L(-1)) values for 24, 48 and 72 h were 48.7 ± 7.14, 29.1 ±2.25 and 22.0 ± 4.53, individually. Xiao-Ai-Ping injection inhibited HUVECs DNA synthesis and migration. Xiao-Ai-Ping injection suppressed HUVECs tube formation, and reduced microvessel sprouting from rat aortic rings and vessel growth in CAMs. Furthermore, Xiao-Ai-Ping injection attenuated the secretion of VEGF, and inhibited the expression of p-VEGFR2 and phosphorylation of protein kinase B(p-AKT). We conclude that Xiao-Ai-Ping injection inhibits angiogenesis by down-regulation of VEGF signaling and AKT pathway.