Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis.

BACKGROUND & AIMS: Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. METHODS: HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. RESULTS: HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. CONCLUSIONS: Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

Inhibiting the transcription and replication of Ebola viruses by disrupting the nucleoprotein and VP30 protein interaction with small molecules.

Ebola virus (EBOV) causes hemorrhagic fever in humans with high morbidity and fatality. Although over 45 years have passed since the first EBOV outbreak, small molecule drugs are not yet available. Ebola viral protein VP30 is a unique RNA synthesis cofactor, and the VP30/NP interaction plays a critical role in initiating the transcription and propagation of EBOV. Here, we designed a high-throughput screening technique based on a competitive binding assay to bind VP30 between an NP-derived peptide and a chemical compound. By screening a library of 8004 compounds, we obtained two lead compounds, Embelin and Kobe2602. The binding of these compounds to the VP30-NP interface was validated by dose-dependent competitive binding assay, surface plasmon resonance, and thermal shift assay. Moreover, the compounds were confirmed to inhibit the transcription and replication of the Ebola genome by a minigenome assay. Similar results were obtained for their two respective analogs (8-gingerol and Kobe0065). Interestingly, these two structurally different molecules exhibit synergistic binding to the VP30/NP interface. The antiviral efficacy (EC50) increased from 1 µM by Kobe0065 alone to 351 nM when Kobe0065 and Embelin were combined in a 4:1 ratio. The synergistic anti-EBOV effect provides a strong incentive for further developing these lead compounds in future studies.

[Micro-Raman Spectroscopy of Daughter Minerals in Primary Fluid Inclusions of Rock Salt in Dongying Sag and Their Geological Significances].

Accurate identification of daughter minerals existed in fluid inclusions has a great significance for obtaining the geochemical information on fluids related to processes of sedimentation, diagenesis or mineralization, and using micro-Raman spectra to identify the types of daughter minerals is in-situ, nondestructive, high sensitive, good-stability, quick and convenient, so which has been widely used at present. In this paper, we used micro-Raman spectra, for the first time, to identify the types of daughter minerals in primary inclusions, which trapped in rock salt from the lower submember of Eocene Sha-4 member of Shahejie Formation (Es4x) in Dongying sag. On this basis, the indication significances of these daughter minerals to geochemical characteristics of paleo-brines in Eocene salt lake of Dongying sag were discussed in detail. The results show that anhydrite and starkeyite are the main daughter minerals in primary inclusions in rock salt of Es4x. Among them, anhydrite has strongest Raman band at 1 018 cm-1 and other five additional weaker bands at 500， 611， 629, 676, 1 131 cm-1, respectively. Starkeyite has strongest Raman band at 1 000 cm-1 and other three additional weaker bands at 462, 618, 1 156 cm-1. These two kinds of daughter minerals existed in primary inclusions indicate that salt lake brines contained small amount of compositions of CaSO4 and MgSO4, the brines just reached early stage of evapo-concentrated precipitations at this moment, that is, the end of sulfates deposit and the beginning of early stage of rock salt deposit, and halite was the main product of brines precipitation. Moreover, based on the precipitation temperature of anhydrite and starkeyite, the temperature of paleo-brines of Eocene salt lake during the early stage of evapo-concentrated precipitations was determined, namely, the values of temperature ranged from 37 to 61 ℃ and sedimentary environment was featured by drought.

CCBE1 promotes mitochondrial fusion by inhibiting the TGFß-DRP1 axis to prevent the progression of hepatocellular carcinoma.

The extracellular matrix (ECM), as an important component of the tumor microenvironment, exerts various roles in tumor formation. Mitochondrial dynamic disorder is closely implicated in tumorigenesis, including hyperfission in HCC. We aimed to determine the influence of the ECM-related protein CCBE1 on mitochondrial dynamics in HCC. Here, we found that CCBE1 was capable of promoting mitochondrial fusion in HCC. Initially, CCBE1 expression was found to be significantly downregulated in tumors compared with nontumor tissues, which resulted from hypermethylation of the CCBE1 promoter in HCC. Furthermore, CCBE1 overexpression or treatment with recombinant CCBE1 protein dramatically inhibited HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, CCBE1 functioned as an inhibitor of mitochondrial fission by preventing the location of DRP1 on mitochondria through inhibiting its phosphorylation at Ser616 by directly binding with TGFßR2 to inhibit TGFß signaling activity. In addition, a higher percentage of specimens with higher DRP1 phosphorylation was present in patients with lower CCBE1 expression than in patients with higher CCBE1 expression, which further confirmed the inhibitory effect of CCBE1 on DRP1 phosphorylation at Ser616. Collectively, our study highlights the crucial roles of CCBE1 in mitochondrial homeostasis, suggesting strong evidence for this process as a potential therapeutic strategy for HCC.

MAOA suppresses the growth of gastric cancer by interacting with NDRG1 and regulating the Warburg effect through the PI3K/AKT/mTOR pathway.

OBJECTIVE: Previous studies have indicated that neurotransmitters play important roles in the occurrence and development of gastric cancer. MAOA is an important catecholamine neurotransmitter-degrading enzyme involved in the degradation of norepinephrine, epinephrine and serotonin. To find a potential therapeutic target for the treatment of gastric cancer, the biological functions of MAOA and the underlying mechanism in gastric cancer need to be explored. METHODS: The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) datasets, Kaplan‒Meier (KM) plotter were used to identify the differentially expressed genes, which mainly involved the degradation and synthesis enzymes of neurotransmitters in gastric cancer. We also investigated the expression pattern of MAOA in human and mouse tissues and cell lines by immunohistochemistry and Western blotting analysis. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and a Seahorse experiment were used to identify the molecular mechanism of cancer cell glycolysis. MAOA expression and patient survival were analysed in the Ren Ji cohort, and univariate and multivariate analyses were performed based on the clinicopathological characteristics of the above samples. RESULTS: MAOA expression was significantly downregulated in gastric cancer tissue and associated with poor patient prognosis. Moreover, the expression level of MAOA in gastric cancer tissue had a close negative correlation with the SUXmax value of PET-CT in patients. MAOA suppressed tumour growth and glycolysis and promoted cancer cell apoptosis. We also reported that MAOA can interact with NDRG1 and regulate glycolysis through suppression of the PI3K/Akt/mTOR pathway. MAOA expression may serve as an independent prognostic factor in gastric cancer patients. CONCLUSIONS: MAOA attenuated glycolysis and inhibited the progression of gastric cancer through the PI3K/Akt/mTOR pathway. Loss of function or downregulation of MAOA can facilitate gastric cancer progression. Overexpression of MAOA and inhibition of the PI3K/Akt/mTOR pathway may provide a potential method for gastric cancer treatment in clinical therapeutic regimens.

The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells.

PURPOSE: Gastric cancer (GC) is a malignant tumour with high mortality, and liver metastasis is one of the main causes of poor prognosis. SLIT- and NTRK-like family member 4 (SLITRK4) plays an important role in the nervous system, such as synapse formation. Our study aimed to explore the functional role of SLITRK4 in GC and liver metastasis. METHODS: The mRNA level of SLITRK4 was evaluated using publicly available transcriptome GEO datasets and Renji cohort. The protein level of SLITRK4 in the tissue microarray of GC was observed using immunohistochemistry. Cell Counting Kit-8, colony formation, transwell migration assays in vitro and mouse model of liver metastasis in vivo was performed to investigate the functional roles of SLITRK4 in GC. Bioinformatics predictions and Co-IP experiments were applied to screen and identify SLITRK4-binding proteins. Western blot was performed to detect Tyrosine Kinase receptor B (TrkB)-related signaling molecules. RESULTS: By comparing primary and liver metastases from GC, SLITRK4 was found to be upregulated in tissues of GC with liver metastasis and to be closely related to poor clinical prognosis. SLITRK4 knockdown significantly abrogated the growth, invasion, and metastasis of GC in vitro and in vivo. Further study revealed that SLITRK4 could interact with Canopy FGF Signalling Regulator 3 (CNPY3), thus enhancing TrkB- related signaling by promoting the endocytosis and recycling of the TrkB receptor. CONCLUSION: In conclusion, the CNPY3-SLITRK4 axis contributes to liver metastasis of GC according to the TrkB-related signaling pathway. which may be a therapeutic target for the treatment of GC with liver metastasis.

Diet-Induced Obesity Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma via CX3CL1/CX3CR1 Axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, and the patients are generally diagnosed with distant metastasis. Liver is one of the preferred organs of distant metastasis, and liver metastasis is the leading cause of death in PDAC. Diet-induced obesity (DIO) is a risk factor for PDAC, and it remains unclear whether and how DIO contributes to liver metastasis of PDAC. In our study, we found that DIO significantly promoted PDAC liver metastasis compared with normal diet (ND) in intrasplenic injection mouse model. RNA-seq analysis for liver metastasis nodules showed that the various chemokines and several chemokine receptors were altered between ND and DIO samples. The expression levels of CX3CL1 and CX3CR1 were significantly upregulated in DIO-induced liver metastasis of PDAC compared to ND. Increased CX3CL1 promoted the recruitment of CX3CR1-expressing pancreatic tumor cells. Taken together, our data demonstrated that DIO promoted PDAC liver metastasis via CX3CL1/CX3CR1 axis.

Spatiotemporal quantification of metastatic tumour cell growth and distribution in lymph nodes by whole-mount tissue 3D imaging.

Lymph nodes (LNs) are a common site of metastasis in many solid cancers. Tumour cells can migrate to LNs for further metastatic colonization of distant organs, indicating poor prognosis and requiring different clinical interventions. The histopathological diagnostic methods currently used to detect clinical lymph node metastasis (LNM) have limitations, such as incomplete visualization. To obtain a complete picture of metastatic LNs on the spatial and temporal scales, we used ultimate 3D imaging of solvent-cleared organs (uDISCO) and 3D rapid immunostaining. MC38 cells labelled with EGFP were injected into the left footpads of C57BL/6 mice. Draining lymph nodes (DLNs) harvested from these mice were cleared using the uDISCO method. Metastatic colorectal cancer (CRC) cells in various regions of DLNs from mice at different time points were quantified using 3D imaging of whole-mount tissue. Several stages of tumour cell growth and distribution in LNs were identified: 1) invasion of lymphatic vessels (LVs) and blood vessels (BVs); 2) dispersion outside LVs and BVs for proliferation and expansion; and 3) re-entry into BVs and efferent lymphatic vessels (ELVs) for further distant metastasis. Moreover, these data demonstrated that mouse fibroblast cells (MFCs) could not only promote LNM of tumour cells but also metastasize to LNs together with tumour cells, thus providing a "soil" for tumour cell colonization. In conclusion, 3D imaging of whole-mount tissue and spatiotemporal analysis of LNM may collectively constitute an auxiliary method to improve the accuracy of clinical LNM detection.

Increased Nuclear Transporter KPNA2 Contributes to Tumor Immune Evasion by Enhancing PD-L1 Expression in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies and is known for its high resistance and low response to treatment. Tumor immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Karyopherin alpha 2 (KPNA2), a member of the nuclear transporter family, is elevated in multiple human cancers and accelerates carcinogenesis. However, the specific role of KPNA2 in PDAC remains unclear. In this study, we found that expression of KPNA2 was significantly upregulated in PDAC compared to adjacent nontumor tissue and its high expression was correlated with poor survival outcome by analyzing the GEO datasets. Similar KPNA2 expression pattern was also found in both human patient samples and KPC mouse models through IHC staining. Although KPNA2 knockdown failed to impair the vitality and migration ability of PDAC cells in vitro, the in vivo tumor growth was significantly impeded and the expression of immune checkpoint ligand PD-L1 was reduced by silencing KPNA2. Furthermore, we uncovered that KPNA2 modulated the expression of PD-L1 by mediating nuclear translocation of STAT3. Collectively, our data suggested that KPNA2 has the potential to serve as a promising biomarker for diagnosis in PDAC.

[Cryogenic Raman spectroscopic characteristics of NaCl-H2O, CaCl2-H2O and NaCl-CaCl2-H2O: application to analysis of fluid inclusions].

Accurately diagnosing the types of the salt and calculating the salinity quantitatively are the significant content of fluid inclusions. The traditional method of testing fluid inclusions salinity is cooling. To overcome the difficulty for observing freezing phase transition, the authors tested the spectrum of NaCl-H2O, CaCl2-H2O and NaCl-CaCl2-H2O systems at -180 degrees C by laser Raman spectroscopy. The result demonstrates that the ratio of peak values has linear relationship with salinity. Calibration curves were established by typical ratio of hydro-halite at 3 420 cm(-1) to the ice at 3 092 cm(-1), and the ratio of antarcticite at 3 432 cm(-1) to the ice at 3 092 cm(-1). The calibration curves have very high correlation coefficient. This method is verified by synthetic hydrocarbon-bearing aqueous fluid inclusions and quartz aqueous fluid inclusions of well Fengshen 6 in Dongying sag. The results of the authors' experiments show that cryogenic Raman spectroscopy can not only identify the types of the salts but also determine the salinity effectively in fluid inclusions.