BackgroundG-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer.MethodsThe expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed.ResultsAnalysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue.ConclusionThe results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.

Background: is an oncomiR in human hepatocellular carcinoma and is highly expressed in liver, but its regulation is uncharacterized. Results: Dehydroepiandrosterone (DHEA) rapidly increases miR-21 transcription in HepG2 cells by activating G-proteincoupled estrogen receptor (GPER). Conclusion: miR-21 transcription is regulated by DHEA through GPER. Significance: GPER may be among the activators of miR-21 expression in human hepatocellular carcinoma.

Epidemiological evidence indicates that cadmium and arsenic exposure increase lung cancer risk. Cadmium and arsenic are environmental contaminants that act as endocrine disruptors (EDs) by activating estrogen receptors (ERs) in breast and other cancer cell lines but their activity as EDs in lung cancer is untested. Here, we examined the effect of cadmium chloride (CdCl2) and sodium arsenite (NaAsO2) on the proliferation of human lung adenocarcinoma cell lines. Results demonstrated that both CdCl2 and NaAsO2 stimulated cell proliferation at environmentally relevant nM concentrations in a similar manner to 17β-estradiol (E2) in H1793, H2073, and H1944 cells but not in H1792 or H1299 cells. Further studies in H1793 cells showed that 100 nM CdCl2 and NaAsO2 rapidly stimulated mitogen-activated protein kinase (MAPK, extracellular-signal-regulated kinases) phosphorylation with a peak detected at 15 min. Inhibitor studies suggest that rapid MAPK phosphorylation by NaAsO2, CdCl2, and E2 involves ER, Src, epidermal growth factor receptor, and G-protein coupled ER (GPER) in a pertussis toxin-sensitive pathway. CdCl2 and E2 activation of MAPK may also involve ERβ. This study supports the involvement of membrane ER and GPER signaling in mediating cellular responses to environmentally relevant nM concentrations of CdCl2 and NaAsO2 in lung adenocarcinoma cells.

Oestrogen receptor α (ERα+) breast tumours rely on mitochondria (mt) to generate ATP. The goal of the present study was to determine how oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete compared with dextran-coated charcoal (DCC)-stripped foetal bovine serum (FBS)-containing medium ('serum-starved'). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respiratory stateapparent was unchanged by serum deprivation. 4-OHT reduced OCR independent of serum status. Despite having a higher mt DNA/nuclear DNA ratio than MCF-7 cells, T47D cells have a lower OCR and ATP levels and higher proton leak. T47D express higher nuclear respiratory factor-1 (NRF-1) and NRF-1-regulated, nuclear-encoded mitochondrial transcription factor TFAM and cytochrome c, but lower levels of cytochrome c oxidase, subunit IV, isoform 1 (COX4, COX4I1). Mitochondrial reserve capacity, reflecting tolerance to cellular stress, was higher in serum-starved T47D cells and was increased by 4-OHT, but was decreased by 4-OHT in MCF-7 cells. These data demonstrate critical differences in cellular energetics and responses to 4-OHT in these two ERα+ cell lines, likely reflecting cancer cell avoidance of apoptosis.

Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα) + breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress.

Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis.

Mucin 1 (MUC1) is a diagnostic factor and therapy target in lung adenocarcinoma. MUC1 C-terminal intracellular domain (CD) interacts with estrogen receptor α (ERα) and increases gene transcription in breast cancer cells. Because lung adenocarcinoma cells express functional ERα and estrogen receptor β (ERβ) we examined MUC1 expression and MUC1-ER interaction. Since blocking MUC1 CD with an inhibitory peptide (PMIP) inhibited breast tumor growth, we tested whether PMIP would inhibit lung adenocarcinoma cell proliferation. We report that MUC1 interacts with ERα and ERβ within the nucleus of H1793 lung adenocarcinoma cells in accordance with MUC1 expression. PMIP was taken up by H23 and H1793 cells and inhibited the proliferation of H1793, but not H23 cells, concordant with higher MUC1 in H1793 cells. Lower MUC1 protein in H23 does not correspond to miR-125b and miR-145 that have been reported to reduce MUC1 expression. PMIP had no effect on the viability of normal human bronchial epithelial cells, which lack MUC1 expression. PMIP inhibited estradiol (E2) –activated reporter gene transcription and endogenous cyclin D1 and nuclear respiratory factor-1 (NRF-1) gene transcription in H1793 cells. These results indicate MUC1-ER functional interaction in lung adenocarcinoma cells and that inhibiting MUC1 inhibits lung adenocarcinoma cell viability.

The frequency of a splice variant of mucin 1 (MUC1), MUC1/A was lower in dry eye disease patients compared to normal controls, suggesting a link between the absence of MUC1/A and the development of dry eye disease which is characterized by chronic inflammation. The objectives of the present study were to clone and characterize the phenotype of cells expressing solely MUC1/A versus MUC1/B or a variant lacking the extracellular domain (ΔEX) and to determine whether MUC1/A and MUC1/B differentially modulate inflammatory responses in transfected cells. The additional 27 bp and SNP present in the N-terminus of MUC1/A were cloned into a FLAG-MUC1/B expression vector. Transient transfection of MUC1/A and MUC1/B plasmids into MUC1-null COS-7 cells resulted in similar protein expression and plasma membrane localization. MUC1/B and MUC1/A differed in their ability to modulate tumor necrosis α (TNFα)-induced transcription of IL-1β and IL-8. MUC1/B and MUC1/A inhibited IL-8 induction by TNFα at 4 h. However with 24 h TNFα, MUC1/A increased IL-1β and IL-8 whereas MUC1/B had no effect on cytokine expression. MUC1/B inhibited TNFα-induced luciferase activity from an NF-κB reporter whereas MUC1/A either inhibited or increased this luciferase activity depending on the time of TNFα treatment. MUC1/A, but not MUC1/B, increased the basal TGFβ expression. Both MUC1/B and MUC1/A blocked TNFα-induced miR-21 expression. These data demonstrate that MUC1/A and MUC1/B have different inflammatory activities and support the hypothesis that MUC1 genotypic differences may affect susceptibility to ocular surface damage in dry eye disease.