No abstract

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

BackgroundRecently, a novel variant of ER-α, ER-α36 was identified and cloned. ER-α36 lacks intrinsic transcription activity and mainly mediates nongenomic estrogen signaling. Here, we studied the role of nongenomic estrogen signaling pathways mediated by ER-α36 in tamoxifen resistance and agonist action.MethodologyThe cellular localization of ER-α36 was examined by immunofluorescence in MCF-7 cells and Hec1A cells. MCF-7 breast cancer cells, MCF-7 cells expressing recombinant ER-α36 (MCF-7/ER36), Hec1A endometrial cancer cells and Hec1A cells with siRNA knockdown of ER-α36 (Hec1A/RNAiER36) were treated with 17β-estradial (E2) and tamoxifen (TAM) in the absence and presence of kinase inhibitor U0126 and LY294002. We examined phosphorylation of signaling molecules and the expression of c-Myc by immunoblotting, and tumor cell growth by MTT assay.ConclusionsER variant ER-α36 enhances TAM agonist activity through activation of the membrane-initiated signaling pathways in endometrial cancer, and that ER-α36 is involved in de novo and acquired TAM resistance in breast cancer.

Icaritin, a prenylflavonoid derivative from Epimedium Genus, regulates many cellular processes. However, the function and the underlying mechanisms of icaritin in breast cancer cell growth have not been well established. Here, we report that icaritin strongly inhibited growth of breast cancer MDA-MB-453 and MCF7 cells. At concentrations of 2–3μM, icaritin induced cell cycle arrest at the G2/M phase accompanied by a down-regulation of the expression levels of the G2/M regulatory proteins such as cyclinB, cdc2 and cdc25C. Icaritin at concentrations of 4–5μM, however, induced apoptotic cell death characterized by accumulation of the annexin V- and propidium iodide-positive cells, cleavage of poly ADP-ribose polymerase (PARP) and down-regulation of the Bcl-2 expression. In addition, icaritin also induced a sustained phosphorylation of extracellular signal regulated kinase (ERK) in these breast cancer cells. U0126, a specific ERK activation inhibitor, abrogated icaritin-induced G2/M cell cycle arrest and cell apoptosis. Icaritin more potently inhibited growth of the breast cancer stem/progenitor cells compared to anti-estrogen tamoxifen. Our results indicate that icaritin is a potent growth inhibitor for breast cancer cells and provide a rational for preclinical and clinical evaluation of icaritin for breast cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a malnourished environment; however, little is known about the mechanisms by which PDAC cells actively promote aerobic glycolysis to maintain their metabolic needs. Gene Expression Omnibus (GEO) was used to identify differentially expressed miRNAs. The expression pattern of miR-30d in normal and PDAC tissues was studied by in situ hybridization. The role of miR-30d/RUNX1 in vitro and in vivo was evaluated by CCK8 assay and clonogenic formation as well as transwell experiment, subcutaneous xenograft model and liver metastasis model, respectively. Glucose uptake, ATP and lactate production were tested to study the regulatory effect of miR-30d/RUNX1 on aerobic glycolysis in PDAC cells. Quantitative real-time PCR, western blot, Chip assay, promoter luciferase activity, RIP, MeRIP, and RNA stability assay were used to explore the molecular mechanism of YTHDC1/miR-30d/RUNX1 in PDAC. Here, we discover that miR-30d expression was remarkably decreased in PDAC tissues and associated with good prognosis, contributed to the suppression of tumor growth and metastasis, and attenuation of Warburg effect. Mechanistically, the m6A reader YTHDC1 facilitated the biogenesis of mature miR-30d via m6A-mediated regulation of mRNA stability. Then, miR-30d inhibited aerobic glycolysis through regulating SLC2A1 and HK1 expression by directly targeting the transcription factor RUNX1, which bound to the promoters of the SLC2A1 and HK1 genes. Moreover, miR-30d was clinically inversely correlated with RUNX1, SLC2A1 and HK1, which function as adverse prognosis factors for overall survival in PDAC tissues. Overall, we demonstrated that miR-30d is a functional and clinical tumor-suppressive gene in PDAC. Our findings further uncover that miR-30d is a novel target for YTHDC1 through m6A modification, and miR-30d represses pancreatic tumorigenesis via suppressing aerobic glycolysis.