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.

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.

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

Our previous finding, that the capsaicin- and KCl-induced Ca(2+)-dependent production of the intra- and intercellular signaling molecule N-arachidonoyl ethanolamine (anandamide) in cultured primary sensory neurons could be abolished and reduced by approximately 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 Ca(2+)-dependent anandamide synthesis. N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is known to be involved in Ca(2+)-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 muM) 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.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF.

Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERalpha plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERalpha and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERalpha is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

Purified malignant mammary epithelial cells maintain hormone responsiveness in culture.

Currently, the therapy for breast cancer is determined by immunohistochemical staining of the primary tumour for oestrogen receptor alpha (ERalpha). However, a proportion of ERalpha-positive patients fail to respond to tamoxifen and a proportion of ERalpha-negative patients show response. Here, we describe a novel procedure for the purification of malignant breast epithelial cells in an attempt to identify these patients at an early stage. Using this procedure, we are able to purify malignant cells to >90% purity as determined by immunohistochemical staining, cytology and fluorescent in situ hybridisation (FISH). While the malignant cells can be maintained in culture they do not proliferate in contrast to purified breast epithelial cells from reduction mammoplasties. Moreover, ERalpha and progesterone receptor (PR) expression is maintained in malignant cells, whereas normal epithelial cells rapidly lose ERalpha and PR. Functional studies were performed on the separated malignant cells in terms of their response to oestradiol and tamoxifen. Four out of the seven ERalpha-positive tumours showed a significant reduction in cell numbers after tamoxifen treatment compared to oestradiol, ERalpha negative tumours failed to show a response. We conclude that (a) it is possible to purify and maintain breast cancer cells for a sufficient period to permit functional studies and (b) ERalpha is retained in culture facilitating the use of these cells in studies of the mechanism of endocrine response and resistance in vitro.

A vector with transcriptional terminators increases efficiency of cloning of an RNA virus by reverse transcription long polymerase chain reaction.

Full-length cDNA clones of RNA viruses are advantageous for maintaining the genomic sequence without the generation of diversity by accumulation of sequence mutations during productive virus replication. They permit in vitro manipulation of the genomic clone to test the effect of sequence changes on the phenotype of reactivated virus. Infectious cDNA clones have been produced by ligation of subgenomic clones but are sometimes difficult to generate in a single cloning operation. We used reverse-transcription to synthesize full-length cDNA from genomic RNA of Coxsackievirus B3 of the Picornavirus family and enzymatically amplified this by long PCR. Five different cloning vectors were used to clone the long PCR product, including the vector Lorist6 which contains transcriptional terminators on either side of the cloning site to prevent transcription of inserts in E. coli. No recombinant colonies were obtained from any of the vectors lacking transcriptional terminators but three full-length clones were obtained using Lorist6. The results suggest that transcriptional terminators increase the recovery of cDNA clones of the 7.4 kb Coxsackie virus genome in this cosmid vector, without resort to phage packaging, representing an advance over previous methods and advantages in the molecular manipulation of these viruses.

Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

Retroviral infection of the FGF2 gene into MCF-7 cells induces branching morphogenesis, retards cell growth and suppresses tumorigenicity in nude mice.

FGF2 (basic fibroblast growth factor) is a multifunctional growth factor and exhibits diverse function in different cell types. In breast, loss of FGF2 expression is associated with malignant progression. In order to understand the role of FGF2 in maintenance of normal breast structures and control of cell growth, we restored FGF2 expression in the breast cancer cell line MCF-7. The FGF2 retrovirally infected MCF-7 cells (MCF-7.F2.5) not only expressed FGF2 in cytoplasm and nuclei, but also released FGF2 into culture medium both on plastic and in Matrigel conditions. The MCF-7.F2.5 cells formed branches in Matrigel and this effect was abolished by the addition of a neutralising anti-FGF2 antibody or function blocking antibodies to alpha2, alpha3 and beta1 integrins. Furthermore, MCF-7.F2.5 cells lost their ability for anchorage-independent growth in soft agar. When MCF-7 and MCF-7.F2.5 cells were injected into nude mice, there was a 1.6- to 3.2-fold reduction of tumour volume with MCF-7.F2.5 cells in comparison with the parental MCF-7 cells. MCF-7.F2.5 cells also demonstrated a reduction in oestrogen receptor-alpha (ERalpha) both in vitro and in vivo. Our results suggest that introduction of the FGF2 gene into MCF-7 cells altered the malignant tumour cells towards a more benign phenotype in vitro and in vivo.

Organization and conservation of the GART/SON/DONSON locus in mouse and human genomes.

The SON gene, which maps to human chromosome 21q22.1-q22.2, encodes a novel regulatory protein. Here we describe the organization of the Son locus in the mouse genome. The mouse Son gene spans a region of approximately 35 kb. The coding region is more than 8 kb in length and has been completely sequenced. The gene is organized into 11 coding exons and 1 noncoding 3'UTR exon, with over 70% of the coding region residing in one 5.7-kb exon. The gene contains at least one alternative exon, N/C exon 1, which can be used, by splicing, to generate a truncated form of the SON protein. Further investigation of the mouse Son locus has identified the genes directly flanking Son. The glycinamide ribonucleotide formyltransferase gene, Gart, is encoded 5' of Son in a head-to-head arrangement, with the start of both genes lying within 899 bp. Sequence comparison with the expressed sequence tagged database identified a novel gene within 65 bp of the 3' end of Son, which we have named Donson. In this unusually compact gene cluster, we have found overlap in the pattern of expression between Gart, Son, and Donson. However, at least two of these genes have very different functions. While GART is involved in purine biosynthesis, we find that SON shows the characteristics of "SR- type" proteins, which are involved in mRNA processing and gene expression.

HGF/SF in mammary epithelial growth and morphogenesis: in vitro and in vivo models.

HGF/SF is a multifunctional cytokine which through binding to its cellular receptor, c-MET, can elicit mitogenic, morphogenic and motogenic responses in target cells. Expression of HGF/SF and c-MET has been shown to be critical in early embryogenesis affecting development of many organs and tissues. The effects of HGF/SF4 on established human and mouse mammary cell lines have previously been reported. This review describes the source and targets for HGF/SF in both human and mouse mammary tissue and discusses the effects of HGF/SF on primary mouse and human mammary epithelial cells in vitro, detailing the individual response of the two epithelial sub-population of cells which comprise this organ. Additionally, the effects of HGF/SF overexpression on mouse mammary gland development in vivo, are summarized.

Endothelial cell transfection with cationic liposomes and herpes simplex-thymidine kinase mediated killing.

Endothelial cells are a promising target for cancer gene therapy because neoangiogenesis is vital for the supply of oxygen and nutrients to solid tumours. However, endothelial cells have been reported to be difficult to transfect. We demonstrate high rates of transfection with the reporter gene pSV40 beta gal using DC-Chol/DOPE cationic liposomes and lower rates with the novel polyamine cationic liposomes ACHx/DC-Chol/DOPE and ACO/DC-Chol/DOPE. Endothelial cells transfected with HSV-thymidine kinase using DC-Chol/DOPE demonstrated 3 log10 increased cytotoxicity compared with controls when exposed to the prodrug ganciclovir, thereby demonstrating significant biological effect.

In vivo effects of hepatocyte growth factor/scatter factor on mouse mammary gland development.

We have recently demonstrated the regulated expression of HGF/SF and its receptor (c-met) during mouse mammary gland development together with the mitogenic, motogenic and morphogenic effects of exogenous HGF/SF on primary mammary epithelial cells in culture. This study was undertaken to analyze the influence of HGF/SF on reconstituted mouse mammary gland development in vivo. Here we report that overexpression of HGF/SF induces a range of alterations in the architecture of virgin mouse mammary gland. These include an enhancement of ductal end bud (mammary gland morphoregulatory control point) size and numbers and hyperplastic branching morphogenesis. These data are the first demonstration of the effects of HGF/SF on mammary epithelium in vivo.

Stable expression of CD1a molecule in human epithelial cell lines shows temperature-dependent expression and affects cell morphology and growth.

The human CD1a molecule is a transmembrane protein which shares structural similarities with HLA class I molecules. It has restricted tissue distribution in normal individuals, and is a useful diagnostic marker for certain disease states such as Langerhans cell histiocytosis. In order to investigate the function of this molecule, a cDNA fragment encoding the CD1a molecule was cloned into several EUKARYOTIC expression vectors which were then used to establish human epithelial cell lines stably expressing the membrane-bound CD1a molecule. Human keratinocytes (HaCaT) and epithelial cells (HeLa) stably expressing CD1a were established by retroviral-mediated gene transfer and DNA transfection, respectively. Expression and localization of the CD1A molecule were then confirmed by Northern blot analysis and immunofluorescence methods. CD1a expression appears to have profound effects on cellular growth and morphology. Both stably CD1a-expressing HeLa and HaCaT cells showed increased doubling times, and up to 20% of CD1a-expressing cells showed altered cell morphology. Clonogenicity experiments demonstrated a reduction in colony size and plating efficiency was augmented in CD1a-positive cells when compared with vector-transfected/infected controls. Our findings suggest that CD1A expression may act as a negative growth regulator in these cells in vitro. Furthermore, lower temperatures greatly enhanced the expression of CD1a at both the protein and mRNA levels in a time-dependent fashion. Since the physiological skin temperatures lie well below the core temperature, this observation may have important implications in the study of Langerhans cells in vitro.

Expression of FGFR2 BEK and K-SAM mRNA variants in normal and malignant human breast.

The expression of mRNA encoding alternative forms of fibroblast growth factor receptor 2 (FGFR2) differing in the carboxy terminal half of their third immunoglobulin-like domain, was investigated in 77 human breast cancer tissues, 12 non-malignant breast biopsies and 29 cell lines, using a reverse transcriptase (RT) polymerase chain reaction (PCR) method. RNA from the two tissue groups yielded PCR product corresponding to both the BEK and the K-SAM form; amounts normalised to glyceraldehyde phosphate dehydrogenase product were similar in both groups. The level of either variant or of the total FGFR2 product was essentially unrelated to prognosis or clinical status except that patients with advanced clinical T staging had a higher proportion of BEK to K-SAM (P = 0.01). RNA from 1/2 normal breast derived and 8/10 breast cancer cell lines expressed exclusively or predominantly the K-SAM form; 2/10 had significant amounts of both BEK and K-SAM mRNA. Of 12 other epithelial lines, seven expressed mainly K-SAM mRNA, four expressed BEK and one was negative. Of five non-epithelial lines, one was negative, two expressed only BEK mRNA and two had significant amounts of both variants. We conclude that tissue levels of FGFR2 mRNA are unaltered in breast cancer extracts and that the splicing mechanism for this exon selection appears not to be significantly disrupted.

Inhibitory effects of activin on the growth and morpholgenesis of primary and transformed mammary epithelial cells.

Activin is a member of the transforming growth factor beta superfamily, which is known to have activities involved in regulating differentiation and development. By using reverse transcription-PCR analysis on immunoaffinity-purified human breast cells, we have found that activin beta a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we have found only four (HBL-100, MCF10-A, PMC-42, and BT 20) to be positive for activin beta a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF- 7 cells (at 2 ng/ml), where it causes an arrest in G(1). Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we demonstrate that activin is an inhibitor of tubule formation by human mammary organoids in vitro. These are the first observations of activin and activin receptor in the normal human breast and in human breast cell lines and suggest a role for activin in mammary cell growth and morphogenesis.

HGF/SF: a potent cytokine for mammary growth, morphogenesis and development.

The mammary gland is a renewing tissue in which morphogenetic processes and differentiation occur cyclically during the menstrual cycle, pregnancy and lactation. These events have been shown to be dependent upon epithelial-mesenchymal interactions. Studies of the effects of individual factors, their cellular source and their target cell populations in the different developmental stages of the mammary gland are greatly facilitated by the accessibility of this organ and the application of new techniques that allow purification of the major epithelial and stromal components of this tissue. Here we demonstrate that HGF/SF and its cellular receptor, c-met, are expressed and regulated temporally during mouse mammary development and differentiation. We show that human and mouse mammary fibroblasts produce HGF/SF and that HGF/SF is not only mitogenic but morphogenic and motogenic for both human and mouse mammary epithelial cells. We have found that human luminal and myoepithelial cells express c-met differentially and that HGF/SF has different effects on these two mammary epithelial cell populations. HGF/SF is mitogenic for luminal cells but not myoepithelial cells, and morphogenic to myoepithelial cells but not luminal cells. This is discussed in the context of the proliferative compartments in the normal mammary gland and the potential role of the myoepithelial cells to act as the skeleton for ductal development.

Conserved gene sequences for species identification: PCR analysis of the 3' UTR of the SON gene distinguishes human and other mammalian DNAs.

In this paper we show that the polymerase chain reaction (PCR) can be used on regions of highly conserved genes, such as the 3' untranslated region (3' UTR) of the SON gene, to identify the mammalian origin of a sample. Using this test, we have been able to distinguish human, monkey, cat, dog, mouse and hamster DNAs. We have also determined the DNA sequence of these different PCR products, which can be used to reinforce species identification. The advantages of this test are that: (i) no prior information is required on the possible species origin of a sample, (ii) the reaction produces a single PCR product which varies in size according to the species of origin, making the test simple to interpret, and (iii) the target region of DNA amplified in these experiments is small and can easily be sequenced and sized using automated techniques. This small size has enabled us to successfully amplify this product from DNA extracted from compromised material (human bone samples) and so demonstrate that the test is valuable for the characterisation of remains in which DNA is degraded.

Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes.

Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no DNA synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen alpha 1(I) and alpha 2(I) chains. It has been suggested that expression of collagen alpha 1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen alpha 1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.