Laki Buluwela

Histone Deacetylase Inhibitor Trichostatin A Represses Estrogen Receptor α-Dependent Transcription and Promotes Proteasomal Degradation of Cyclin D1 in Human Breast Carcinoma Cell Lines

Purpose: Estrogen receptor α (ERα)-positive breast cancer cell lines are up to 10 times more sensitive than ERα-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response. Experimental Design and Results: In the ERα-positive MCF-7 cell line, TSA repressed ERα and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G1-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERα-negative MDA-MB-231 cell line, which arrested in G2-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA. Conclusions: Tamoxifen has been shown to inhibit ERα-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERα-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERα-dependent and ERα-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

Microarray coupled to quantitative RT–PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells

The androgen receptor (AR) mediates the growth-stimulatory effects of androgens in prostate cancer cells. Identification of androgen-regulated genes in prostate cancer cells is therefore of considerable importance for defining the mechanisms of prostate-cancer development and progression. Although several studies have used microarrays to identify AR-regulated genes in prostate cancer cell lines and in prostate tumours, we present here the results of gene expression microarray profiling of the androgen-responsive LNCaP prostate-cancer cell line treated with R1881 for the identification of androgen-regulated genes. We show that the expression of 319 genes is stimulated by 24 h after R1881 addition, with a similar number (300) of genes being significantly repressed. Expression of the upregulated genes, as well as of 60 of the most robustly downregulated genes, was carried out using quantitative RT–PCR (Q-RT–PCR) over a time-course of R1881 treatment from 0 to 72 h. Q-RT–PCR was also carried out following treatment with other AR agonists (dihydrotestosterone, estradiol and medroxyprogesterone) and antagonists (cyproterone acetate, hydroxyflutamide and bicalutamide). This study provides a comprehensive analysis of androgen-regulated gene expression in the LNCaP prostate cancer cell line, and identifies a number of androgen-regulated genes, not described previously, as candidates for mediating androgen responses in prostate cancer cells.

Antiestrogens and Their Therapeutic Applications in Breast Cancer and Other Diseases

The identification of the link between breast cancer and estrogens has led to the development of antiestrogens, in particular tamoxifen, to inhibit the activities of estrogen receptors (ERs) in breast cancer cells. The clinical use of tamoxifen has played a major part in decreasing breast cancer mortality over the past 30 years. Though antiestrogenic in the breast, some antiestrogens have estrogen-like actions in other tissues, acting to promote bone density and protect against cardiovascular disease, thus raising the possibility of their use in counteracting the effects of estrogen loss following menopause. Moreover, antiestrogens show efficacy as chemopreventive agents in women at high risk of developing breast cancer. Thus, antiestrogens define an important and well-understood class of cancer drug, which continue to be a mainstay in breast cancer treatment.

The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells

Estrogen receptor-α (ER) is expressed in the great majority of breast cancers, and the inhibition of ER action is a key part of breast cancer treatment. The inhibition of ER action is achieved using anti-estrogens, primarily tamoxifen, and with aromatase inhibitors that inhibit estrogen biosynthesis, thereby preventing ER activation. However, resistance to these therapies is common. With the aim of identifying new molecular targets for breast cancer therapy, we have identified the liver receptor homolog-1 (LRH-1) as an estrogen-regulated gene. RNA interference and over-expression studies were used to investigate the role of the LRH-1 in regulating breast cancer growth and to identify the targets of an LRH-1 action. Promoter recruitment was determined using reporter gene and chromatin immunoprecipitation (ChIP) assays. We show that LRH-1 regulates breast cancer cell growth by regulating the ER expression. Reporter gene and in vitro DNA-binding assays identified an LRH-1-binding site in the ER gene promoter, and ChIP assays have demonstrated in vivo binding at this site. We also provide evidence for new LRH-1 variants in breast cancer cells arising from the use of alternative promoters. Previous studies have shown that LRH-1 functions in estrogen biosynthesis by regulating aromatase expression. Our findings extend this by highlighting LRH-1 as a key regulator of the estrogen response in breast cancer cells through the regulation of ER expression. Hence, inhibition of LRH-1 could provide a powerful new approach for the treatment of endocrine-resistant breast cancer.

APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer

Summary Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.

Discovery of a New Class of Liver Receptor Homolog-1 (LRH-1) Antagonists: Virtual Screening, Synthesis and Biological Evaluation

Targeting LRH-1: Virtual screening and molecular modeling were used to identify novel antagonists of liver receptor homolog-1 (LRH-1), an emerging therapeutic target for breast cancer. Hit compounds were synthesized and biologically assayed, and the preliminary results suggest that raloxifene-based analogues, substituted at the position C-7 of the benzothiophene ring, might generate an inactive protein conformation through binding and thus antagonize this nuclear receptor.

Capsaicin-sensitive primary sensory neurons in the mouse express N-Acyl phosphatidylethanolamine phospholipase D

Our previous finding, that the capsaicin- and KCl-induced Ca2+-dependent production of the intra- and intercellular signaling molecule N-arachidonoyl ethanolamine (anandamide) in cultured primary sensory neurons could be abolished and reduced by ∼2/3 by capsaicin-induced degeneration of capsaicin-sensitive neurons, respectively suggests that a major sub-population of capsaicin-sensitive cells together with a group of non-capsaicin-sensitive cells should express enzymes involved in Ca2+-dependent anandamide synthesis. N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is known to be involved in Ca2+-dependent anandamide production. Hence, here, we used reverse transcriptase and quantitative real time polymerase chain reaction to study NAPE-PLD expression in dorsal root ganglia and to clarify the sub-population of cells expressing this enzyme. Cultures prepared from mouse dorsal root ganglia were grown either in the absence or presence of the neurotoxin, capsaicin (10 μM) overnight. We report, that NAPE-PLD is expressed both in dorsal root ganglia and cultures prepared from dorsal root ganglia and grown in the absence of capsaicin. Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia. These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia. These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.