OBJECTIVE—Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS—We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP, PEPCK, and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes, and metformin–and adenovirus SHP (Ad-SHP)–mediated hepatic glucose production was measured in B6-Lepob/ob mice. RESULTS—Hepatic SHP gene expression was induced by metformin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK), as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4α–or FoxA2-mediated promoter activity of PEPCK and G6Pase, and the inhibition was blocked with siRNA SHP. Additionally, SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore, oral administration of metformin increased SHP mRNA levels in B6-Lepob/ob mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lepob/ob mice. CONCLUSIONS—We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP.
RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.
The human POK family members are transcription factors with a POZ domain and zinc fingers that act primarily as transcriptional repressors. Several members of this family are involved in oncogenesis and this prompted us to assess whether expression levels of individual POK family members are associated with clinical outcomes in cancer. We have observed that ZBTB4 is downregulated in breast cancer patients, and that its expression is significantly correlated with relapse-free survival. Further integrative analysis of mRNA and microRNA (miR) expression data from the NCI-60 cell lines revealed an inverse correlation between ZBTB4 and oncogenic miRs derived from the miR-17-92 cluster and its paralogues. The experimental results using MDA-MB-231 and MCF-7 human breast cancer cells confirm that miRNAs derived from these clusters, containing miR-17-5p, miR-20a, miR-106a, miR-106b and miR-93, negatively regulate ZBTB4 expression. Overexpression of ZBTB4 or restoration of ZBTB4 by using an antagomir inhibit growth and invasion of breast cancer cells, and this effect is due, in part, to ZBTB4-dependent repression of the specificity protein 1 (Sp1), Sp3, and Sp4 genes, and subsequent downregulation of several Sp-dependent oncogenes, in part, through competition between ZBTB4 and Sp transcription factors for GC-rich promoter sequences. These results confirm that ZBTB4 functions as a novel tumor suppressor gene with prognostic significance for breast cancer survival, and the oncogenic miR-17-92/ZBTB4/Sp axis may be a potential therapeutic target.
Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor and acts as a coregulator of various nuclear receptors. Herein, we examined a novel cross talk between SHP and a forkhead transcription factor HNF3 (hepatocyte nuclear factor 3/Foxa. Transient transfection assay demonstrated that SHP inhibited the transcriptional activity of all three isoforms of HNF3, HNF3alpha, beta, and gamma. In vivo and in vitro protein interaction studies showed that SHP physically interacted with HNF3. Adenovirus-mediated overexpression of SHP significantly decreased the mRNA levels of glucose-6-phosphase (G6Pase), cholesterol 7-alpha-hydroxylase (CYP7A1), and phosphoenolpyruvate carboxykinase (PEPCK) in HepG2 cells and rat primary hepatocytes. Moreover, the mRNA level of G6Pase was notably increased by down-regulation of SHP with small interfering RNA. Interestingly, HNF3 transactivity was still repressed by SHPDelta128-139 that fails to repress nuclear receptors. Mapping of interaction domain revealed that SHP interacted with forkhead DNA binding domain of HNF3alpha. Gel mobility shift and chromatin immunoprecipitation assays demonstrated that SHP inhibits DNA binding of HNF3. These results suggest that SHP is involved in the regulation of G6Pase, CYP7A1, and PEPCK gene expression via novel mechanism of inhibition of HNF3 activity and expand the role of SHP as a coregulator of other family of transcription factors in addition to nuclear receptors.
Graphical AbstractHighlights d LKB1-deficient T reg cells produce Th1 and Th17 cytokines d The loss of LKB1 compromises the mevalonate pathway in T reg cells d Mevalonate or GGPP treatment restores function and stability in LKB1-deficient T reg cells d LKB1-mediated regulation of T reg cells is independent of AMPK SUMMARY The function of regulatory T (T reg ) cells depends on lipid oxidation. However, the molecular mechanism by which T reg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in T reg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for T reg cell functional competency and stability by inducing T reg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of T reg cells and enhanced survival of LKB1-deficient T reg mice. Thus, LKB1 is a key regulator of lipid metabolism in T reg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.
Purpose Resistance to vascular endothelial growth factor receptor (VEGFR) inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors. Experimental Design We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes. Results Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In cancer patients receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival. Conclusions HGF/c-MET pathway mediates VEGFR inhibitor-resistance and vascular remodeling in NSCLC.
In vitro three-dimensional (3D) tumour models mimic natural cancer tissue in vivo, bridging the gap between conventional 2D in vitro testing and animal models. Stromal and cancer tissues with extracellular matrix (ECM) can provide a tumour microenvironment (TME) with cell-to-cell and cell-to-ECM interactions. These interactions induce the exchange of biophysical factors, contributing to the progression, metastasis, and drug resistance of cancer. Here, we describe a 3D in vitro lung cancer model cultured in a microfluidic channel that is able to confirm the role and function of various stromal cells in tumourigenesis, thereby representing an in vivo-like TME. We founded that biophysical factors contribute to the role of fibroblast cells in tumour formation, especially, producing a nascent vessel-like tubular structure, resulting in the formation of vascularized tumour tissue. Fibroblast cells altered the gene expression of the cancer cells to enhance metastasis, survival, and angiogenesis. The device could be used for developing and screening anti-cancer drugs through the formation of the same multicellular tumour spheroids under TME interactions. We believe this microfluidic system provides interaction of TME for cancer research by culturing stromal tissue.
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