CTRP9 Promotes Brown Adipose Tissue Lipolysis in Mice Fed a High-Fat Diet.

BACKGROUND: This study aimed to elucidate the molecular mechanism through which C1q/tumor necrosis factor (TNF)-related protein 9 (CTRP9) acts in the formation and differentiation of brown adipose tissue (BAT). METHODS: Adenovirus particles encoding CTRP9 and green fluorescent protein were inoculated into the scapula of C57BL/6J mice and fed a high-fat diet for 8 weeks; the body weight, lipid droplet morphology, glucose tolerance, insulin tolerance, and protein expression levels were analyzed. In addition, CTRP9 adenovirus was transfected into brown preadipocytes, and differentiation was induced to identify the effect of CTRP9 overexpression on adipocyte differentiation. RESULTS: CTRP9 overexpression significantly increased the weight gain of mice. Additionally, the CTRP9 overexpression group exhibited significantly increased adipose tissue weight and glucose clearance rates and decreased insulin sensitivity and serum triglyceride levels compared to the control group. Furthermore, CTRP9 overexpression significantly upregulated the adipose triglyceride lipase (ATGL) and perilipin 1 protein expression levels in BAT. The cell experiment results confirmed that CTRP9 overexpression significantly inhibited the adipogenesis of brown adipocytes as evidenced by the downregulation of uncoupling protein 1, beta-3 adrenergic receptor, ATGL, and hormone-sensitive lipase mRNA levels and the significant suppression of uncoupling protein 1, ATGL, and perilipin 1 protein levels in brown adipocytes. CONCLUSIONS: The finding of this study demonstrated that CTRP9 promotes lipolysis by upregulating ATGL expression in vivo and inhibits the differentiation of brown preadipocytes in vitro.

CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis.

The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.

Promotional effects of HIF1α and KDM3A interaction on vascular smooth muscle cells in thoracic aortic dissection.

Aim: The current study was performed to define the role of KDM3A in thoracic aortic dissection (TAD). Methods: The binding of HIF1α and KDM3A in HES1 was detected by ChIP and dual-luciferase reporter gene assay. Loss and gain-of function assays of HIF1α, KDM3A and HES1 were further performed in Ang-II-induced mouse aortic smooth muscle cell line (MOVAS) cells. Lastly, in vivo TAD models were established. Results: HIF1α was highly expressed in TAD. KDM3A promoted the transcription activation of HES1. HIF1α enhanced the proliferation and migration of Ang-II-induced MOVAS cells, in addition to increasing thoracic aorta dilation to induce TAD formation in vivo. Silencing of HES1 reversed the effects of HIF1α in vivo and in vitro. Conclusion: The findings indicated that interaction between HIF1α and KDM3A enhances the proliferation and migration of MOVAS cells to induce TAD.

The current study aimed to clarify the role of the KDM3A gene, which is involved in thoracic aortic dissection (TAD; a sudden tear in the inner layer of the aortic wall) as well as its underlying mechanism. The findings revealed that overexpression of HIF1α increased the formation and movement of Ang-II-induced mouse aortic smooth muscle cell line cells, whereas HIF1α silencing caused the opposite results. The KDM3A gene supported the transcriptional activation of HES1 by interacting with HIF1α. HIF1α increased TAD formation in vivo and the silencing of the HES1 transcription factor reversed the effects of HIF1α in vivo and in vitro. These discoveries deepen our understanding of the causes of TAD and highlight novel therapeutic targets for the development of effective targeted therapy against TAD.

Analysis of MCM Proteins' Role as a Potential Target of Statins in Patients with Acute Type A Aortic Dissection through Bioinformatics.

Acute aortic dissection is one of the most severe vascular diseases. The molecular mechanisms of aortic expansion and dissection are unclear. Clinical studies have found that statins play a protective role in aortic dissection development and therapy; however, the mechanism of statins' effects on the aorta is unknown. The Gene Expression Omnibus (GEO) dataset GSE52093, GSE2450and GSE8686 were analyzed, and genes expressed differentially between aortic dissection samples and normal samples were determined using the Networkanalyst and iDEP tools. Weight gene correlation network analysis (WGCNA), functional annotation, pathway enrichment analysis, and the analysis of the regional variations of genomic features were then performed. We found that the minichromosome maintenance proteins (MCMs), a family of proteins targeted by statins, were upregulated in dissected aortic wall tissues and play a central role in cell-cycle and mitosis regulation in aortic dissection patients. Our results indicate a potential molecular target and mechanism for statins' effects in patients with acute type A aortic dissection.

Functional aspects of primary cilium in signaling, assembly and microenvironment in cancer.

The primary cilium is an antennae-like structure extent outside the cell surface. It has an important role in regulating cell-signaling transduction to affect proliferation, differentiation and migration. Evidence is accumulating that ciliary defects lead to ciliopathies and ciliary deregulation also play crucial roles in cancer formation and progression. Interestingly, restoring the cilia can suppress proliferation in some cancer cell. However, t he role of primary cilia in cancer still be debated. In this article, we review the role of the primary cilium in cancer through architecture, signaling pathways, cilia assembly and disassembly regulators, and summarized the new findings of the primary cilium in tumor microenvironments and different cancers, highlighting novel possibilities for therapeutic target in cancer.

Simvastatin and Atorvastatin inhibit DNA replication licensing factor MCM7 and effectively suppress RB-deficient tumors growth.

Loss or dysfunction of tumor suppressor retinoblastoma (RB) is a common feature in various tumors, and contributes to cancer cell stemness and drug resistance to cancer therapy. However, the strategy to suppress or eliminate Rb-deficient tumor cells remains unclear. In the present study, we accidentally found that reduction of DNA replication licensing factor MCM7 induced more apoptosis in RB-deficient tumor cells than in control tumor cells. Moreover, after a drug screening and further studies, we demonstrated that statin drug Simvastatin and Atorvastatin were able to inhibit MCM7 and RB expressions. Further study showed that Simvastatin and Atorvastatin induced more chromosome breaks and gaps of Rb-deficient tumor cells than control tumor cells. In vivo results showed that Simvastatin and Atorvastatin significantly suppressed Rb-deficient tumor growth than control in xenograft mouse models. The present work demonstrates that 'old' lipid-lowering drugs statins are novel weapons against RB-deficient tumors due to their effects on suppressing MCM7 protein levels.

Simvastatin suppresses the DNA replication licensing factor MCM7 and inhibits the growth of tamoxifen-resistant breast cancer cells.

Acquired tamoxifen resistance (TamR) remains a major challenge in breast cancer endocrine therapy. The mechanism of acquiring tamoxifen resistance remains elusive, and no effective drugs are available. In this investigation, we determined that the expression of the DNA damage marker γH2AX is upregulated under minichromosome maintenance protein 7 (MCM7) knockdown in phospho Ser807/811-retinoblastoma protein (p-Rb) defect cells. In addition, the expression of p-Rb was lower in TamR cells than in parental cells, and the expression of γH2AX was significantly upregulated when MCM7 was knocked down in TamR cells. Simvastatin, an agent for hypercholesterolemia treatment, activated the MCM7/p-RB/γH2AX axis and induced DNA damage in TamR cells, especially when combined with tamoxifen. Finally, in vitro and in vivo experiments demonstrated that simvastatin combined with tamoxifen increased TamR cell apoptosis and inhibited xenograft growth. In conclusion, simvastatin may suppress TamR cell growth by inhibiting MCM7 and Rb and subsequently inducing DNA damage.

Loss of DLG5 promotes breast cancer malignancy by inhibiting the Hippo signaling pathway.

Discs Large Homolog 5 (DLG5) plays an important role in the maintenance of epithelial cell polarity. Recent research showed that DLG5 is decreased in Yes-associated protein (YAP)-overexpressing cells. However, the exact relationship between DLG5 and YAP is not clear. In this study, we showed that loss of DLG5 promoted breast cancer cell proliferation by inhibiting the Hippo signaling pathway and increasing nuclear YAP expression. Furthermore, depletion of DLG5 induced epithelial-mesenchymal transition (EMT) and disrupted epithelial cell polarity, which was associated with altered expression of Scribble, ZO1, E-cadherin and N-cadherin and their mislocalization. Interestingly, we first reported that loss of DLG5 inhibited the interaction of Mst1 and Lats1 with Scribble, which was crucial for YAP activation and the transcription of TEA domain (TEAD) family members. In summary, loss of DLG5 expression promoted breast cancer malignancy by inactivating the Hippo signaling pathway and increasing nuclear YAP.

MCM7 promotes cancer progression through cyclin D1-dependent signaling and serves as a prognostic marker for patients with hepatocellular carcinoma.

DNA replication is a central procedure of cell proliferation, whereas aberrant DNA replication is indicated to be a driving force of oncogenesis. Minichromosome maintenance complex component 7 (MCM7) plays an essential role in initiating DNA replication. To investigate the potential oncogenic properties and prognostic value of MCM7 in hepatocellular carcinoma (HCC), we conducted immunohistochemistry staining of MCM7 in 153 HCC samples and found that MCM7 high expression level was associated with worse overall survival (OS) of HCC patients. Mechanistically, knockdown of MCM7 significantly inhibited cellular proliferation in vitro and HCC tumorigenicity in vivo. Cyclin D1 was proved to be regulated by MCM7-MAPK signaling pathway. Clinically, high expression of both MCM7 and cyclin D1 exhibited a relatively high sensitivity and specificity to predict worse outcome of HCC patients. Taken together, our results suggest that MCM7-cyclin D1 pathway may participate in cancer progression and serve as a biomarker for prognosis in HCC.

CRB3 regulates contact inhibition by activating the Hippo pathway in mammary epithelial cells.

The loss of contact inhibition is a hallmark of cancer cells. The Hippo pathway has recently been shown to be an important regulator of contact inhibition, and the cell apical polarity determinant protein CRB3 has been suggested to be involved in Hippo signalling. However, whether CRB3 regulates contact inhibition in mammary cells remains unclear, and the underlying mechanisms have not been elucidated. As shown in the present study, CRB3 decreases cell proliferation, promotes apoptosis, and enhances the formation of tight and adherens junctions. Furthermore, we report for the first time that CRB3 acts as an upstream regulator of the Hippo pathway to regulate contact inhibition by recruiting other Hippo molecules, such as Kibra and/or FRMD6, in mammary epithelial cells. In addition, CRB3 inhibits tumour growth in vivo. Collectively, the present study increases our understanding of the Hippo pathway and provides an important theoretical basis for exploring new avenues for breast cancer treatment.

Simvastatin exerts anti-hepatitis B virus activity by inhibiting expression of minichromosome maintenance protein 7 in HepG2.2.15 cells.

Simvastatin (SIM), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been reported to inhibit the activity of hepatitis B virus (HBV), however, the mechanism underlying its antiviral function remains unknown. Minichromosome maintenance (MCM) 7, a component of the MCM complex, has been reported to act as an important host factor aiding virus genome replication in host cells. The present study demonstrated that downregulation of MCM7 inhibited the expression of proteins transferred by adenoviral vectors. This suggests an association between MCM7 and viral DNA expression. Thus, the current study aimed to investigate whether SIM affected MCM7 expression. Notably, the results of the present study indicated that following exposure to SIM the protein expression levels of MCM7 in HepG2.2.15, a human HBV­transfected liver cell line, was decreased. In addition, the HBV DNA replication in the cell line was suppressed. As quantitative polymerase chain reaction experiments demonstrated that SIM did not downregulate the mRNA expression level of MCM7, the current study further investigated whether SIM affects the translation of MCM7. Western blot experiments indicated that SIM improved the activation of eukaryotic initiation factor­2α (eIF2α), a protein synthesis initiation factor, and upregulated the upstream factors of eIF2α, protein kinase RNA­like endoplasmic reticulum kinase, which is regulated by the liver kinase B1 (LKB1)­AMP­activated protein kinase (AMPK) signaling pathway. These results indicated that SIM induced HBV downregulation via an MCM­dependent mechanism, and SIM may inhibit MCM7 expression by increasing the phosphorylation of eIF2α, which is mediated by the LKB1-AMPK signaling pathway.

Metastasis-associated in colon cancer-1 is associated with poor prognosis in hepatocellular carcinoma, partly by promoting proliferation through enhanced glucose metabolism.

Metastasis-associated in colon cancer-1 (MACC1) is a newly identified gene that is involved in the development and progression of hepatocellular carcinoma (HCC), however its investigation has not been comprehensive. In the present study, in vitro techniques, including immunohistochemistry, western blotting, reverse transcription quantitative polymerase chain reaction, metabolic assay, MTT assay, colony formation assay and prognostic analysis were used to confirm the involvement of MACC1 in HCC. Histological examination confirmed that the protein expression of MACC1 was upregulated in HCC and was associated with the hexokinase-2 (HK2) protein, which also indicates a poor prognosis. Knockdown of MACC1 induced the reduction of glycogen consumption and lactate production, which then lead to a marked reduction of proliferation in the MHCC-97H cells. However, the overexpression of MACC1 produced the opposite results in the HepG2 cells. These results suggested that MACC1 leads to a poor prognosis in HCC, partly by promoting proliferation via enhancement in glucose metabolism by HK2. Therefore, this pathway has the potential to become an important therapeutic target in HCC.

MACC1 expression correlates with PFKFB2 and survival in hepatocellular carcinoma.

OBJECTIVE: To validate the relationship between MACC1 and 6-phosphofructo-2-kinase/fructose 2, 6 bisphosphatase (PFKFB2) expression as well as its clinicopathological features and prognostic significance in hepatocellular carcinoma. METHODS: By using immunohistochemistry, we investigated the MACC1 and PFKFB2 protein expression in 60 pairs of hepatocellular carcinoma and corresponding non-tumor tissues. Using the Mann-Whitney U test, the Chi-square test, Kaplan-Meier survival analysis, Cox proportional hazard regression analysis and Spearman analysis, we studied the relationship between MACC1 and PFKFB2 protein expression and postoperative overall survival (OS) of the HCC patients. RESULTS: MACC1 and PFKFB2 positive staining rates were significantly higher in hepatocellular carcinoma than in the corresponding nontumor tissues (P=0.012 and 0.04, respectively). The clinicopathological features evaluation revealed that positive expression of MACC1 was associated with a high Edmondson classification (P=0.007) and advanced TNM stage (P=0.027). Similar findings were evident for PFKFB2 expression (P=0.002 and P=0.027). MACC1 and PFKFB2 positive expression was associated with a lower OS rate (P=0.004 and 0.03, respectively). Kaplan-Meier survival and Cox proportional hazard regression analyses revealed MACC1 positive expression to be a prognostic factor for postoperative OS, but PFKFB was not. CONCLUSION: Highly expressed MACC1 and PFKFB2 protein were associated with TNM stage, Edmondson -Steier classification and overall survival. MACC1 may affect tumor metabolism partly through expression and phophorylation of PFKFB2.

Caveolin-1 is up-regulated by GLI1 and contributes to GLI1-driven EMT in hepatocellular carcinoma.

Caveolin-1 (Cav-1) has been recently identified to be over-expressed in hepatocellular carcinoma (HCC) and promote HCC cell motility and invasion ability via inducing epithelial-mesenchymal transition (EMT). However, the mechanism of aberrant overexpression of Cav-1 remains vague. Here, we observed that Cav-1 expression was positively associated with GLI1 expression in HCC tissues. Forced expression of GLI1 up-regulated Cav-1 in Huh7 cells, while knockdown of GLI1 decreased expression of Cav-1 in SNU449 cells. Additionally, silencing Cav-1 abolished GLI1-induced EMT of Huh7 cells. The correlation between GLI1 and Cav-1 was confirmed in tumor specimens from HCC patients and Cav-1 was found to be associated with poor prognosis after hepatic resection. The relationship between protein expression of GLI1 and Cav-1 was also established in HCC xenografts of nude mice. These results suggest that GLI1 may be attributed to Cav-1 up-regulation which plays an important role in GLI1-driven EMT phenotype in HCC.

Role of the Hedgehog pathway in hepatocellular carcinoma (review).

The Hedgehog (Hh) pathway is an evolutionarily conserved signaling mechanism that controls many aspects of cell differentiation and the development of tissues and organs during embryogenesis. Early investigations have focused on the effects of Hh activity on the development of organs including skin, gut, the nervous system and bone. However, in addition to normal developmental processes, these investigations also found that Hh signaling is involved in aberrant proliferation and malignant transformation. Consequently, the role of Hh in cancer pathology, and its modulation by environmental factors is the subject of many investigations. Numerous environmental toxins, alcohol, and hepatitis viruses can cause hepatocellular carcinoma (HCC), which is the most common form of liver cancer. Significant hyperactivation of Hh signaling has been observed in liver injury and cirrhosis which often leads to the development of HCC lesions. Moreover, Hh activity plays an important role in the progression of HCC. Here, we review findings relevant to our understanding of the role of Hh signaling in HCC pathogenesis.