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Supplemental materials and methods, supplemental figure legends

Tumor-suppressor genes (TSG) are often deleted or transcriptionally suppressed in cancer. PGR codes for progesterone receptor (PR), a transcription factor whose function depends on its ligand. Although PR expression is often undetectable in cervical cancer, its relevance to the endocrine-related etiology of this prevalent gynecological disease remains unclear. In this study, we show that the deletion of one Pgr allele in cervical epithelium promoted spontaneous cervical cancer in human papilloma viral oncogene-expressing transgenic mice as efficiently as the ablation of both Pgr alleles. We also show that tumors arising in the transgenic mice with one or both Pgr alleles did not express PR or expressed at the reduced levels compared with the normal epithelium. PR status correlated with estrogen receptor α (ERα) status in the mouse model and the Cancer Genome Atlas (TCGA) dataset. TCGA data analyses revealed that PGR expression significantly decreased in cervical cancer and that the biallelic deletion of PGR was rare. Furthermore, low PGR expression was associated with poor prognosis in young patients with cervical cancer. These discoveries point to PGR as a haploinsufficient TSG in the uterine cervix. They also raise the possibility that the restoration of PGR expression may improve the survival rate.Implications:The decreased expression of PR may increase the risk of cervical cancer in human papillomavirus–infected women.Visual Overview:http://mcr.aacrjournals.org/content/molcanres/19/1/42/F1.large.jpg.

IHC results for p16 and Mcm7.

IHC conditions and information for antibodies and chemicals.

Supplementary Table 1 from Positive Cross-Talk between Estrogen Receptor and NF-κB in Breast Cancer

Supplementary Tables 1-3 from Gene Expression Preferentially Regulated by Tamoxifen in Breast Cancer Cells and Correlations with Clinical Outcome

Estrogen receptors (ER) and nuclear factor-κB (NF-κB) are known to play important roles in breast cancer, but these factors are generally thought to repress each other's activity. However, we have recently found that ER and NF-κB can also act together in a positive manner to synergistically increase gene transcription. To examine the extent of cross-talk between ER and NF-κB, a microarray study was conducted in which MCF-7 breast cancer cells were treated with 17β-estradiol (E2), tumor necrosis factor α (TNFα), or both. Follow-up studies with an ER antagonist and NF-κB inhibitors show that cross-talk between E2 and TNFα is mediated by these two factors. We find that although transrepression between ER and NF-κB does occur, positive cross-talk is more prominent with three gene-specific patterns of regulation: (a) TNFα enhances E2 action on ∼30% of E2-upregulated genes; (b) E2 enhances TNFα activity on ∼15% of TNFα-upregulated genes; and (c) E2 + TNFα causes a more than additive upregulation of ∼60 genes. Consistent with their prosurvival roles, ER and NF-κB and their target gene, BIRC3, are involved in protecting breast cancer cells against apoptosis. Furthermore, genes positively regulated by E2 + TNFα are clinically relevant because they are enriched in luminal B breast tumors and their expression profiles can distinguish a cohort of patients with poor outcome following endocrine treatment. Taken together, our findings suggest that positive cross-talk between ER and NF-κB is more extensive than anticipated and that these factors may act together to promote survival of breast cancer cells and progression to a more aggressive phenotype. [Cancer Res 2009;69(23):8918–25]

The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERα-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERα and reversed by E2 or ICI 182,780. Introduction of ERβ into MCF-7 cells reverses tamoxifen action on ∼75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy. (Cancer Res 2006; 66(14): 7334-40)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high mortality rate and few effective treatments. A growing area of cancer therapeutics seeks to exploit the metabolic dysregulation of cancer cells, such as glucose, amino acid, and fatty acid metabolism, to selectively target malignant cells. As ligand-dependent transcription factors and critical regulators of metabolism, liver X receptors (LXRs) are amenable to small-molecule targeting for such purposes. We have profiled the transcriptomic, metabolomic, and cytotoxic effects of a newly discovered small-molecule LXR modulator, GAC0003A4 (3A4), in PDAC cell lines. On the transcriptomic level, marked changes in gene expression were observed, including downregulation of LXR target genes and pathways. Gene set enrichment analysis determined downregulation of several metabolic pathways, such as fatty acid and cholesterol metabolism, while upregulated pathways involved TNFα/NF-κB and other stress-induced processes. Metabolomic analyses revealed altered metabolites in several pathways, the most enriched categories being lipids and amino acid metabolites, while phospholipids and sphingolipids, including ceramides, were also found to be significantly altered. Insights from transcriptomic and metabolomic studies helped guide the determination of alterations in cholesterol and ceramides as integral to the antiproliferative mechanisms of 3A4. Additionally, a concurrent programmed cell death mechanism involving apoptosis and necroptosis was shown to be activated. These studies provide novel insights into the effects of LXR modulation on gene expression, metabolism, and cell death induction in PDAC cells. The metabolic and cytotoxic effects of LXR modulation on the PDAC cell lines used in this study could also aid in the design and application of drugs to target other refractory cancers.

Background Alcohol consumption is a risk factor for breast cancer, contributing up to nearly 23,000 new cases each year. Mechanistic studies show that alcohol increases aggressiveness and metastatic potential, promotes angiogenesis, induces chronic-inflammation, and deregulates RNA polymerase III related genes. Alcohol has also been shown to affect estrogen signaling in breast cancer, including in our previously published study on the transcriptomic effects of alcohol in breast cancer cells. Methods To elucidate mechanisms of action of alcohol in breast cancer, we carried out secondary analyses of our alcohol responsive transcriptome data using gene ontology and pathway databases and analysis tools and cistromic data analysis of candidate transcription factors which may mediate the transcriptomic alterations. Predicted alcohol-responsive pathways and mechanisms were perturbed and examined experimentally in breast cancer cells. Clinical relevance of identified genes were determined by expression profiles in patient samples and correlation with disease outcomes and alcohol consumption in previously published study cohorts. Results Gene ontology analysis showed that alcohol alters the expression of many metabolism related genes, and cistromic data of differentially expressed genes revealed the potential involvement of nuclear factor of activated T-cells 3 (NFATC3) in mediating the transcriptomic effects of alcohol. Pathway analysis further predicts regulation of calcium signaling by alcohol in breast cancer cells. Chemical perturbation of this pathway reversed the effect of alcohol on breast cancer cell growth and reduced the elevated cytosolic Ca2+ levels induced by alcohol. Expression levels of alcohol-responsive genes in tumor samples from breast cancer patients are associated with poor disease outcomes. Moreover, expression of some of these genes were altered in breast cancer patients who consumed alcohol previously as compared to those who did not drink. Conclusion Alcohol alters expression of genes that regulate intracellular calcium levels and downstream signaling pathways which drive breast cancer cell proliferation and disease progression.

Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer. PDACs harbor oncogenic mutations in the KRAS gene, and ongoing efforts to directly target its mutant protein product to inhibit tumor growth are a priority not only in pancreatic cancer but in other malignancies such as lung and colorectal cancers where KRAS is also commonly mutated. An alternative strategy to directly targeting KRAS is to identify and target druggable receptors involved in dysregulated cancer hallmarks downstream of KRAS dysregulation. Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-modulated transcription factors and are involved in the regulation of genes which function in key cancer-related processes, including cholesterol transport, lipid and glucose metabolism, and inflammatory and immune responses. Modulation of LXRs via small molecule ligands has emerged as a promising approach for directly targeting tumor cells or the stromal and immune cells within the tumor microenvironment. We have previously shown that only one of the two LXR subtypes (LXRβ) is expressed in pancreatic cancer cells, and targeting LXR with available synthetic ligands blocked the proliferation of PDAC cells and tumor formation. In a screen of a focused library of drug-like small molecules predicted to dock in the ligand-binding pocket of LXRβ, we identified two novel LXR ligands with more potent antitumor activity than current LXR agonists used in our published studies. Characterization of the two lead compounds (GAC0001E5 and GAC0003A4) indicates that they function as LXR inverse agonists which inhibit their transcriptional activity. Prolonged treatments with novel ligands further revealed their function as LXR "degraders" which significantly reduced LXR protein levels in all three PDAC cell lines tested. These findings support the utility of these novel inhibitors in basic research on ligand design, allosteric mechanisms, and LXR functions and their potential application as treatments for advanced pancreatic cancer and other recalcitrant malignancies.

The response of the uterus to 17β-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous studies from our laboratory using high (C57BL6/J; B6) and low uterine responders (C3H/HeJ; C3H) to E2led to the identification ofEstq1-Estq5, five quantitative trait loci (QTL) controlling phenotypic variation in uterine growth and eosinophilic leukocyte infiltration. Transcriptional profiling subsequently identified coronin actin binding protein 2A (Coro2a) as a candidate for an expression quantitative trait gene (eQTG) underlyingEstq1. Here we show that like mRNA expression, allele specific expression of CORO2A in both the uterine epithelium and stroma similarly co-segregates with uterine responsiveness. Utilizing B6.C3H congenic mice carrying theCoro2aC3Hallele, we confirm the genetic linkage betweenCoro2aandEstq1. Lastly, we show that B6-Coro2a-/-and B6-Coro2aB6/-mice phenocopy uterine responsiveness of mice carrying theCoro2aC3H/C3HandCoro2aB6/C3Halleles respectively, consistent with a haploinsufficiency model of genetic control. Together, these results identifyCoro2aas the eQTG underlyingEstq1, and establish a critical role forCoro2ain E2-regulated responses.

Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer with a high mortality rate due to the lack of early detection and effective treatment options for advanced diseases. Metabolic reprogramming, a common hallmark of malignant transformation in pancreatic cancer, is critical for the growth and survival of cancer cells and a potential target mechanism for the treatment of pancreatic cancer. PDAC cells have upregulated glutamine metabolism to meet their biosynthetic and oxidative demands. Liver X receptors (LXRs) are ligand-dependent transcription factors involved in maintaining metabolic homeostasis. LXRs regulate critical cancer-related processes and pathways, including cholesterol, glucose and lipid metabolism, and inflammatory and immune responses. Analysis of transcriptomic data from PDAC clinical samples reveals overexpression of LXRs and their target genes in tumors as compared to normal tissue controls. Targeting LXRs with the novel LXR inverse agonist and degrader GAC0001E5 inhibited PDAC cell proliferation. Using a metabolomics approach, we discovered that 1E5 inhibits glutamine anaplerosis and induces oxidative stress, which are detrimental to PDAC cells. These findings highlight a novel role for LXR in regulating cancer metabolism and the potential application of LXR modulators in targeting cancer metabolism in pancreatic cancer and other malignancies.

Tumor-suppressor genes (TSG) are often deleted or transcriptionally suppressed in cancer. PGR codes for progesterone receptor (PR), a transcription factor whose function depends on its ligand. Although PR expression is often undetectable in cervical cancer, its relevance to the endocrine-related etiology of this prevalent gynecological disease remains unclear. In this study, we show that the deletion of one Pgr allele in cervical epithelium promoted spontaneous cervical cancer in human papilloma viral oncogene-expressing transgenic mice as efficiently as the ablation of both Pgr alleles. We also show that tumors arising in the transgenic mice with one or both Pgr alleles did not express PR or expressed at the reduced levels compared with the normal epithelium. PR status correlated with estrogen receptor α (ERα) status in the mouse model and the Cancer Genome Atlas (TCGA) dataset. TCGA data analyses revealed that PGR expression significantly decreased in cervical cancer and that the biallelic deletion of PGR was rare. Furthermore, low PGR expression was associated with poor prognosis in young patients with cervical cancer. These discoveries point to PGR as a haploinsufficient TSG in the uterine cervix. They also raise the possibility that the restoration of PGR expression may improve the survival rate. Implications: The decreased expression of PR may increase the risk of cervical cancer in human papillomavirus–infected women. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/1/42/F1.large.jpg.

Estrogen receptor β (ERβ) was first identified in the rodent prostate and is abundantly expressed in human and rodent prostate epithelium, stroma, immune cells and endothelium of the blood vessels. In the prostates of mice with inactivated ERβ, mutant phenotypes include epithelial hyperplasia and increased expression of androgen receptor (AR)-regulated genes, most of which are also upregulated in prostate cancer (PCa). ERβ is expressed in both basal and luminal cells in the prostate while AR is expressed in luminal but not in the basal cell layer which harbors the prostate stem cells. To investigate the mechanisms of action of ERβ and its potential cross-talk with AR, we used RNA-seq to study the effects of estradiol or the synthetic ligand, LY3201, in AR-positive LNCaP PCa cells which had been engineered to express ERβ. Transcriptomic analysis indicated relatively few changes in gene expression with ERβ overexpression, but robust responses following ligand treatments. There is significant overlap of responsive genes between the two ligands, as well as ligand-specific alterations. Gene set analysis of down-regulated genes identified an enrichment of androgen-responsive genes, such as FKBP5, CAMKK2, and TBC1D4. Consistently, AR transcript, protein levels, and transcriptional activity were down-regulated following ERβ activation. In agreement with this, we find that the phosphorylation of the CAMKK2 target, AMPK, was repressed by ligand-activated ERβ. Down-regulation of TBC1D4, a major regulator of glucose uptake in prostate, indicates that ERβ is changing glucose metabolism in the prostate. These findings suggest that ERβ-mediated signaling pathways are involved in the negative regulation of AR expression and activity, thus supporting a tumor suppressive role for ERβ in PCa.

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV+ than HPV− cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV+ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.

Regulation of gene expression in immune cells is known to be under genetic control, and likely contributes to susceptibility to autoimmune diseases such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice, more accurately reflecting genetically diverse human populations, we provide an extensive characterization of the genetic regulation of gene expression in five different naive immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control, exhibiting diverse patterns: global, cell-specific and sex-specific. Bioinformatic analysis of the genetically controlled transcript networks reveals reduced cell type specificity and inflammatory activity in wild-derived PWD/PhJ mice, compared with the conventional laboratory strain C57BL/6J. Additionally, candidate MS-GWAS (genome-wide association study candidate genes for MS susceptibility) genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared with PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis, the principal model of MS, and skewing of the encephalitogenic T-cell responses. Taken together, our results provide functional insights into the genetic regulation of the immune transcriptome, and shed light on how this in turn contributes to susceptibility to autoimmune disease.

Members of the nuclear receptor superfamily of ligand-dependent transcription factors carry out vital cellular functions and are highly druggable therapeutic targets. Liver X receptors (LXRs) are nuclear receptor family members that function in cholesterol transport, glucose metabolism and the modulation of inflammatory responses. There is now accumulating evidence to support the involvement of LXRs in a variety of malignancies and the potential efficacy of their ligands in these diseases. This Review summarizes the discovery and characterization of LXRs and their ligands, their effects and mechanisms in preclinical cancer models, and the future directions of basic and translational LXR research in cancer therapeutics.

The estrogens are female sex hormones that are involved in a variety of physiological processes, including reproductive development and function, wound healing, and bone growth. They are mainly known for their roles in reproductive tissues—specifically, 17β-estradiol (E2), the primary estrogen, which is secreted by the ovaries and induces cellular proliferation and growth of the uterus and mammary glands. In addition to the role of estrogens in promoting tissue growth and development during normal physiological states, they have a well-established role in determining susceptibility to disease, particularly cancer, in reproductive tissues. The responsiveness of various tissues to estrogen is genetically controlled, with marked quantitative variation observed across multiple species, including humans. This variation presents both researchers and clinicians with a veritable physiological puzzle, the pieces of which—many of them unknown—are complex and difficult to fit together. Although genetics is known to play a major role in determining sensitivity to estrogens, there are other factors, including parent of origin and the maternal environment, that are intimately linked to heritable phenotypes but do not represent genotype, per se. The objectives of this review article were to summarize the current knowledge of the role of genotype, and uterine and neonatal environments, in phenotypic variation in the response to estrogens; to discuss recent findings and the potential mechanisms involved; and to highlight exciting research opportunities for the future.—Wall, E. H., Hewitt, S. C., Case, L. K, Lin, C.-Y., Korach, K. S., Teuscher, C. The role of genetics in estrogen responses: a critical piece of an intricate puzzle.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer. It is predicted to become the third leading cause of cancer-related deaths by 2030. PDAC has a poor survival rate of 5 years with a 7% success rate. Current treatments include nontargeted highly toxic chemotherapeutic agents. More than 95% of PDAC patients have activating mutations of Kristen RAt Sarcoma virus (KRAS) gene, and it is known to drive metabolic reprogramming that increases cancer cell survival and resistance against the chemotherapeutic drugs. KRAS promotes noncanonical consumption of glutamine by upregulating the expression of glutaminase 1 (GLS1) enzyme. This, in turn, supports PDAC growth and survival by maintaining redox homeostasis and also by replenishing citric acid cycle intermediates. Liver X receptor beta (LXRβ), a nuclear receptor, is a ligand-dependent transcription factor that is ubiquitously expressed in all tissues. It plays an important role in maintaining cholesterol, lipid, and glucose homeostasis under normal physiologic conditions. Agonist-bound LXRβ activates the transcription of genes involved in lipogenesis, reverse cholesterol transport, and glucose metabolism. Our group has previously shown that LXRβ is expressed in PDAC tissues, and treatment with GW3965, an LXRβ agonist, reduced PDAC proliferation. However, LXRβ agonists have adverse side effects of increasing liver and circulating triglycerides. This study identified a novel ligand of LXRβ 1E5, which significantly reduced PDAC cell proliferation. 1E5 decreased the basal expression of LXRβ target genes such as sterol regulatory element-binding protein 1 (SREBP1c) and ATP binding cassette subfamily A member 1 (ABCA1). It also increased the binding of co-repressor peptides such as nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the LXRβ ligand binding domain, whereas it decreased the binding of the co-activator peptide as demonstrated by the time-resolved fluorescence energy transfer (TR-FRET) assay, thereby inhibiting the transcriptional activity of LXRβ. Therefore, 1E5 acts as an LXRβ inverse agonist. Furthermore, the metabolomics study revealed that 1E5 disrupts glutamine metabolism in PDAC cells by downregulating the glutamine, aspartate, and alpha-ketoglutarate levels in cells. Gene expression analysis showed that it significantly downregulated GLS1 expression. These results demonstrate that the novel inverse agonist is a candidate agent in the treatment of PDAC by targeting glutamine metabolism. Citation Format: Shivangi Srivastava, Scott Widmann, Chin-Yo Lin. Liver X receptor ligand targets glutamine metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C53.

Estrogen receptor alpha (ERα) is a cellular receptor for the female sex hormone estrogen and other natural and synthetic ligands and play critical roles in normal development and physiology and in the etiology and treatment of endocrine-related diseases. ERα is a member of the nuclear receptor superfamily of transcription factors and regulates target gene expression in a ligand-dependent manner. It has also been shown to interact with G-protein coupled receptors and associated signaling molecules in the cytoplasm. Transcriptionally, ERα either binds DNA directly through conserved estrogen response element sequence motifs or indirectly by tethering to other interacting transcription factors and nucleate transcriptional regulatory complexes which include an array of co-regulator proteins. Genome-scale studies of ERα transcriptional activity and localization have revealed mechanistic complexity and insights including novel interactions with several transcription factors, including FOXA1, AP-2g, GATA3, and RUNX1, which function as pioneering, collaborative, or tethering factors. The major challenge and exciting prospect moving forward is the comprehensive definition and integration of ERα complexes and mechanisms and their tissue-specific roles in normal physiology and in human diseases.

The uterotropic response of the uterus to 17β-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high [C57BL/6J (B6)] or low [C3H/HeJ (C3H)] responders to E2 led to the identification of quantitative trait (QT) loci associated with phenotypic variation in uterine growth and leukocyte infiltration. The mechanisms underlying differential responsiveness to E2, and the genes involved, are unknown. Therefore, we used a microarray approach to show association of distinct E2-regulated transcriptional signatures with genetically controlled high and low responses to E2 and their segregation in (C57BL/6J×C3H/HeJ) F1 hybrids. Among the 6664 E2-regulated transcripts, analysis of cellular functions of those that were strain specific indicated C3H-selective enrichment of apoptosis, consistent with a 7-fold increase in the apoptosis indicator CASP3, and a 2.4-fold decrease in the apoptosis inhibitor Naip1 (Birc1a) in C3H vs. B6 following treatment with E2. In addition, several differentially expressed transcripts reside within our previously identified QT loci, including the ERα-tethering factor Runx1, demonstrated to enhance E2-mediated transcript regulation. The level of RUNX1 in uterine epithelial cells was shown to be 3.5-fold greater in B6 compared to C3H. Our novel insights into the mechanisms underlying the genetic control of tissue sensitivity to estrogen have great potential to advance understanding of individualized effects in physiological and disease states.—Wall, E. H., Hewitt, S. C., Liu, L., del Rio, R., Case, L. K., Lin, C.-Y., Korach, K. S., Teuscher, C. Genetic control of estrogen-regulated transcriptional and cellular responses in mouse uterus.

Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumorderived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.

Liver X receptors (LXRs) are nuclear receptors with well-known functions in cholesterol transport, fatty acid and glucose metabolism, and modulation of immune responses. Natural and synthetic ligands have been identified and are under development for the treatment of metabolic and inflammatory conditions and diseases. There is mounting evidence pointing to functional roles for LXRs in a variety of malignancies and the potential therapeutic efficacy of their ligands.This review summarizes the discovery and characterization of LXRs and their ligands, surveys their effects and mechanisms of action in cell-based and animal models of cancer, and proposes the future direction of basic and translational studies of LXRs and their ligands in cancer research and therapeutics.Targeting LXRs is a promising strategy for cancer treatment, particularly for those cancers which do not have effective treatment options. Key questions remain, however, regarding the specific mechanisms of action, effects on other target cells within the tumor microenvironment, and receptor status in patient populations. Moreover, LXR ligands optimized for disease-specific functions and cancer-related endpoints are currently not available. These issues represent both challenges and significant opportunities for future research and development efforts.

List of custom primers and catalog primers used amplify lncRNAs by TaqMan Q-RTPCR

Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics.

Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics.

Expression, regulation, and evolution of 26 lncRNAs.

SiRNA knockdown validations and post-knockdown phenotypes after RNAi of estrogen-induced lncRNAs in MCF7 cells.

Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics.

MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

The central dogma of molecular biology relegates RNAs to the role of "messengers" of genetic information, with proteins as the end products that perform key roles as regulators and effectors of biological processes. Notable exceptions include non-protein-coding RNAs, which function as adaptors (tRNAs) and ribosomal components (rRNAs) during translation, as well as in splicing (snRNAs) and RNA maturation including editing (snoRNAs). Genome and transcriptome projects have revealed, however, a significant number, rivaling the protein-coding transcripts, of non-protein-coding RNAs not related to these previously characterized transcript classes. Non-protein-coding RNA research has primarily focused on microRNAs, a small subclass of non-protein-coding RNAs, and their regulatory roles in gene expression, and these findings have been reviewed extensively. Here, we turn our attention to the larger, in number and size, long non-coding RNAs (lncRNAs), and review their evolutionary complexity and the growing evidence for their diverse mechanisms of action and functional roles in basic molecular and cellular biology and in human disease. In contrast to the focus on in-silico and expression studies in existing lncRNA literature, we emphasize direct evidence for lncRNA function, presenting experimental approaches and strategies for systematic characterization of lncRNA activities, with applications to known gene regulatory networks and diseases.

Most commonly reported non-commercial protein isolation methods require the use of detergents and/or chaotropes for better yield, and they all need additional Purification or clean-up steps for subsequent mass spectrometry analysis. Moreover, there is no simple procedure available for obtaining both soluble and membrane proteins from the same sample. Here we describe a simple and detergent-free chloroform-assisted protein isolation (ChlAPI) method for mammalian cells and tissues, and demonstrated its suitability to mass spectrometry based proteome analysis. In this single-step method, cultured cells or grounded tissue were mixed in 10% chloroform in ammonium bicarbonate buffer to separate whole cell proteome into biphasic layers. Total number of aqueous phase proteins, as assessed by 2DE, was comparable to the proteins isolated with commonly used detergent containing buffer. Shotgun proteomics analysis of the aqueous and organic phase proteome fractions of MCF7 cells by LC-MS/MS resulted in Identification of a total of 752 and 593 proteins, respectively from IPI human protein database. Among the total of 1134 distinct and nonredundant proteins, 29.5% were predicted to be membrane localized; 78% of them wereidentified from organic phase fraction. Application of this new procedure to estrogen-treated MCF cells lead to the Identification of previously known as well as unknown estrogen-responsive gene products. These fi ndings suggest that the simple and inexpensive ChlAPI method described here is suitable for protein isolation from mammalian samples, and is readily compatible with 2DE and LC-MS/MS analyses.

Estrogen receptors (ER) and nuclear factor-kappaB (NF-kappaB) are known to play important roles in breast cancer, but these factors are generally thought to repress each other's activity. However, we have recently found that ER and NF-kappaB can also act together in a positive manner to synergistically increase gene transcription. To examine the extent of cross-talk between ER and NF-kappaB, a microarray study was conducted in which MCF-7 breast cancer cells were treated with 17beta-estradiol (E(2)), tumor necrosis factor alpha (TNFalpha), or both. Follow-up studies with an ER antagonist and NF-kappaB inhibitors show that cross-talk between E(2) and TNFalpha is mediated by these two factors. We find that although transrepression between ER and NF-kappaB does occur, positive cross-talk is more prominent with three gene-specific patterns of regulation: (a) TNFalpha enhances E(2) action on approximately 30% of E(2)-upregulated genes; (b) E(2) enhances TNFalpha activity on approximately 15% of TNFalpha-upregulated genes; and (c) E(2) + TNFalpha causes a more than additive upregulation of approximately 60 genes. Consistent with their prosurvival roles, ER and NF-kappaB and their target gene, BIRC3, are involved in protecting breast cancer cells against apoptosis. Furthermore, genes positively regulated by E(2) + TNFalpha are clinically relevant because they are enriched in luminal B breast tumors and their expression profiles can distinguish a cohort of patients with poor outcome following endocrine treatment. Taken together, our findings suggest that positive cross-talk between ER and NF-kappaB is more extensive than anticipated and that these factors may act together to promote survival of breast cancer cells and progression to a more aggressive phenotype.

We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.

Regulation of hairy and enhancer of split homologue-1 (HES-1) by estradiol and all-trans retinoic acid affects proliferation of human breast cancer cells. Here, we identify and characterize cis-regulatory elements involved in HES-1 regulation. In the distal 5′ promoter of the HES-1 gene, we found a retinoic acid response element and in the distal 3′ region, an estrogen receptor α(ER)α binding site. The ERα binding site, composed of an estrogen response element (ERE) and an ERE half-site, is important for both ERα binding and transcriptional regulation. Chromatin immunoprecipitation assays revealed that ERα is recruited to the ERE and associates with the HES-1 promoter. We also show recruitment of nuclear receptor co-regulators to the ERE in response to estradiol, followed by a decrease in histone acetylation and RNA polymerase II docking in the HES-1 promoter region. Our findings are consistent with a novel type of repressive estrogen response element in the distal 3′ region of the HES-1 gene.

The expression of carbonic anhydrase XII (CA12), a gene that encodes a zinc metalloenzyme responsible for acidification of the microenvironment of cancer cells, is highly correlated with estrogen receptor α (ERα) in human breast tumors. Here, we show that CA12 is robustly regulated by estrogen via ERα in breast cancer cells, and that this regulation involves a distal estrogen-responsive enhancer region. Upon the addition of estradiol, ERα binds directly to this distal enhancer in vivo, resulting in the recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3, and changes in histone acetylation. Mutagenesis of an imperfect estrogen-responsive element within this enhancer region abolishes estrogen-dependent activity, and chromosome conformation capture and chromatin immunoprecipitation assays show that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intrachromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homologue, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in the regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers. [Cancer Res 2008;68(9):3505–15]

The lactose operon and its control is a fundamental transcriptional regulatory concept presented in introductory and many advanced molecular biology courses. Much is known about the positive and negative control mechanisms that govern levels of expression of this operon. One basic principle that is taught about the lac operon is that it is "leaky," meaning that the transcriptional control of the operon is not 100% efficient and that in wild-type cells, transcription from the promoter is never completely "off," but there is always some basal transcription. In contrast, the arabinose operon is often used as an example of a tightly controlled operon, and transcription from the ara promoter is very low in the absence of inducer. The relative levels of control of these two operons can be illustrated using Western blots of proteins expressed in the presence and absence of the appropriate inducers and antibodies against the gene products. Different times of growth and the addition of inducer can also be examined. The results are very dramatic and help to reinforce the principles of promoter control.