Nuclear respiratory factor-1 and bioenergetics in tamoxifen-resistant breast cancer cells.

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.

GGNBP2 acts as a tumor suppressor by inhibiting estrogen receptor α activity in breast cancer cells.

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 17ß-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.

Arsenite and Cadmium Activate MAPK/ERK via Membrane Estrogen Receptors and G-Protein Coupled Estrogen Receptor Signaling in Human Lung Adenocarcinoma Cells.

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 17ß-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 ERß. 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.

Anacardic Acid, Salicylic Acid, and Oleic Acid Differentially Alter Cellular Bioenergetic Function in Breast Cancer Cells.

Anacardic acid is a dietary and medicinal phytochemical that inhibits breast cancer cell proliferation and uncouples oxidative phosphorylation (OXPHOS) in isolated rat liver mitochondria. Since mitochondrial-targeted anticancer therapy (mitocans) may be useful in breast cancer, we examined the effect of anacardic acid on cellular bioenergetics and OXPHOS pathway proteins in breast cancer cells modeling progression to endocrine-independence: MCF-7 estrogen receptor α (ERα)+ endocrine-sensitive; LCC9 and LY2 ERα+, endocrine-resistant, and MDA-MB-231 triple negative breast cancer (TNBC) cells. At concentrations similar to cell proliferation IC50 s, anacardic acid reduced ATP-linked oxygen consumption rate (OCR), mitochondrial reserve capacity, and coupling efficiency while increasing proton leak, reflecting mitochondrial toxicity which was greater in MCF-7 compared to endocrine-resistant and TNBC cells. These results suggest tolerance in endocrine-resistant and TNBC cells to mitochondrial stress induced by anacardic acid. Since anacardic acid is an alkylated 2-hydroxybenzoic acid, the effects of salicylic acid (SA, 2-hydroxybenzoic acid moiety) and oleic acid (OA, monounsaturated alkyl moiety) were tested. SA inhibited whereas OA stimulated cell viability. In contrast to stimulation of basal OCR by anacardic acid (uncoupling effect), neither SA nor OA altered basal OCR- except OA inhibited basal and ATP-linked OCR, and increased ECAR, in MDA-MB-231 cells. Changes in OXPHOS proteins correlated with changes in OCR. Overall, neither the 2-hydroxybenzoic acid moiety nor the monounsaturated alky moiety of anacardic acid is solely responsible for the observed mitochondria-targeted anticancer activity in breast cancer cells and hence both moieties are required in the same molecule for the observed effects. J. Cell. Biochem. 117: 2521-2532, 2016. © 2016 Wiley Periodicals, Inc.

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells.

Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3-12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction.

Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by oestradiol and tamoxifen.

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.

5-Aza-2-deoxycytidine and trichostatin A increase COUP-TFII expression in antiestrogen-resistant breast cancer cell lines.

COUP-TFII is reduced in endocrine-resistant breast cancer cells and is negatively associated with tumor grade. Transient re-expression of COUP-TFII restores antiestrogen sensitivity in resistant LCC2 and LCC9 cells and repression of COUP-TFII results in antiestrogen-resistance in MCF-7 endocrine-sensitive cells. We addressed the hypothesis that reduced COUP-TFII expression in endocrine-resistant breast cancer cells results from epigenetic modification. The NR2F2 gene encoding COUP-TFII includes seven CpG islands, including one in the 5' promoter and one in exon 1. Treatment of LCC2 and LCC9 endocrine-resistant breast cancer cells with 5-aza-2'-deoxycytidine (AZA), a DNA methyltransferase (DNMT) inhibitor, +/- trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased COUP-TFII suggesting that the decrease in COUP-TFII is mediated by epigenetic changes. Methylation-specific PCR (MSP) revealed higher methylation of NR2F2 in the first exon in LCC2 and LCC9 cells compared to MCF-7 cells and AZA reduced this methylation. Translational importance is suggested by Cancer Methylome System (CMS) analysis revealing that breast tumors have increased COUP-TFII (NR2F2) promoter and gene methylation versus normal breast.

ß-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression.

Endocrine therapies have been successfully used for breast cancer patients with estrogen receptor α (ERα) positive tumors, but ~40% of patients relapse due to endocrine resistance. ß-glucans are components of plant cell walls that have immunomodulatory and anticancer activity. The objective of this study was to examine the activity of ß-D-glucan, purified from barley, in endocrine-sensitive MCF-7 versus endocrine-resistant LCC9 and LY2 breast cancer cells. ß-D-glucan dissolved in DMSO but not water inhibited MCF-7 cell proliferation in a concentration-dependent manner as measured by BrdU incorporation with an IC50 of ~164 ± 12 µg/ml. ß-D-glucan dissolved in DMSO inhibited tamoxifen/endocrine-resistant LCC9 and LY2 cell proliferation with IC50 values of 4.6 ± 0.3 and 24.2 ± 1.4 µg/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC50 ~464 µg/ml and the proliferation of MDA-MB-231 triple negative breast cancer cells was not inhibited by ß-D-glucan. Concentration-dependent increases in the BAX/BCL2 ratio and cell death with ß-D-glucan were observed in MCF-7 and LCC9 cells. PCR array analysis revealed changes in gene expression in response to 24-h treatment with 10 or 50 µg/ml ß-D-glucan that were different between MCF-7 and LCC9 cells as well as differences in basal gene expression between the two cell lines. Select results were confirmed by quantitative real-time PCR demonstrating that ß-D-glucan increased RASSF1 expression in MCF-7 cells and IGFBP3, CTNNB1 and ERß transcript expression in LCC9 cells. Our data indicate that ß-D-glucan regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation.

Estradiol and tamoxifen regulate NRF-1 and mitochondrial function in mouse mammary gland and uterus.

Nuclear respiratory factor-1 (NRF-1) stimulates the transcription of nuclear-encoded genes that regulate mitochondrial (mt) genome transcription and biogenesis. We reported that estradiol (E2) and 4-hydroxytamoxifen (4-OHT) stimulate NRF-1 transcription in an estrogen receptor α (ERα)- and ERß-dependent manner in human breast cancer cells. The aim of this study was to determine whether E2 and 4-OHT increase NRF-1 in vivo. Here, we report that E2 and 4-OHT increase NRF-1 expression in mammary gland (MG) and uterus of ovariectomized C57BL/6 mice in a time-dependent manner. E2 increased NRF-1 protein in the uterus and MG; however, in MG, 4-OHT increased Nrf1 mRNA but not protein. Chromatin immunoprecipitation assays revealed increased in vivo recruitment of ERα to the Nrf1 promoter and intron 3 in MG and uterus 6 h after E2 and 4-OHT treatment, commensurate with increased NRF-1 expression. E2- and 4-OHT-induced increases in NRF-1 and its target genes Tfam, Tfb1m, and Tfb2m were coordinated in MG but not in uterus due to uterine-selective inhibition of the expression of the NRF-1 coactivators Ppargc1a and Ppargc1b by E2 and 4-OHT. E2 transiently increased NRF-1 and PGC-1α nuclear staining while reducing PGC-1α in uterus. E2, not 4-OHT, activates mt biogenesis in MG and uterus in a time-dependent manner. E2 increased mt outer membrane Tomm40 protein levels in MG and uterus whereas 4-OHT increased Tomm40 only in uterus. These data support the hypothesis of tissue-selective regulation of NRF-1 and its downstream targets by E2 and 4-OHT in vivo.

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer.

BACKGROUND: G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17ß-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 ß (ERα and ERß), 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. METHODS: The 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. RESULTS: Analysis 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. CONCLUSION: The 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.

Targeting the intracellular MUC1 C-terminal domain inhibits proliferation and estrogen receptor transcriptional activity in lung adenocarcinoma cells.

Mucin 1 (MUC1) is a diagnostic factor and therapy target in lung adenocarcinoma. MUC1 C-terminal intracellular domain (CD) interacts with estrogen receptor (ER) α and increases gene transcription in breast cancer cells. Because lung adenocarcinoma cells express functional ERα and ERß, we examined MUC1 expression and MUC1-ER interaction. Because 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 ERß 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 protein expression in H1793 cells. Lower MUC1 protein expression in H23 does not correspond to microRNAs 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-activated reporter gene transcription and endogenous cyclin D1 and nuclear respiratory factor-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.

MUC1/A and MUC1/B splice variants differentially regulate inflammatory cytokine expression.

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-1ß 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-1ß 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 TGFß 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.