Amanda McCann

Publications of Amanda McCann

• Commentary on paper: 5-Aza-2'-deoxycytidine increases sialyl Lewis X on MUC1 by stimulating β-galactoside:α2,3-sialyltransferase 6 gene (Chachadi et al.).

  Authors: Radka Saldova, Amanda McCann, Pauline M Rudd

  The international journal of biochemistry & cell biology. 02/2012; 44(5):737.

• Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer.

  Authors: Fiona Furlong, Patricia Fitzpatrick, Sharon O'Toole, Sine Phelan, Barbara McGrogan, Aoife Maguire, Anthony O'Grady, Michael Gallagher, Maria Prencipe, Aloysius McGoldrick, Paul McGettigan, Donal Brennan, Orla Sheils, Cara Martin, Elaine W Kay, John O'Leary, Amanda McCann

  The Journal of pathology. 11/2011;

  Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol(®) ) in combination withEpithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol(®) ) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high-grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression-free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel-induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR-433 binding domain in the MAD2 3' UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down-regulated in pre-miR-433 transfected A2780 cells. Secondly, pre-miR-433 suppressed the activity of a reporter construct containing the 3'-UTR of MAD2. Thirdly, blocking miR-433 binding to the MAD2 3' UTR protected MAD2 from miR-433 induced protein down-regulation. Importantly, reduced MAD2 protein expression in pre-miR-433-transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high-grade serous EOC. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

• 5-AZA-2'-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer.

  Authors: Radka Saldova, Eugene Dempsey, Marta Pérez-Garay, Karina Mariño, Jenny A Watson, Alfonso Blanco-Fernández, Weston B Struwe, David J Harvey, Stephen F Madden, Rosa Peracaula, Amanda McCann, Pauline M Rudd

  Epigenetics : official journal of the DNA Methylation Society. 11/2011; 6(11).

  Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stablyGlycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation patterning are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells (OVCAR3) resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of GMDS and FX, key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes such as MGAT5 reflect the changes seen in the branching and sialylation of secreted glycans. Overall, our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.

• Alpha T-catenin (CTNNA3): a gene in the hand is worth two in the nest.

  Authors: James D Smith, Maria H Meehan, John Crean, Amanda McCann

  Cellular and molecular life sciences : CMLS. 05/2011; 68(15):2493-8.

  Alpha-T-Catenin (CTNNA3) is a key protein of the adherens junctional complex in epithelial cells playing a crucial role in cellular adherence. What makes this gene particularly interesting is that itAlpha-T-Catenin (CTNNA3) is a key protein of the adherens junctional complex in epithelial cells playing a crucial role in cellular adherence. What makes this gene particularly interesting is that it is located within a common fragile site, is epigenetically regulated, is transcribed through multiple promoters, and generates a variety of alternate transcripts. Finally, CTNNA3 has a nested gene (LRTMM3) embedded within its genomic context transcribed in the opposite direction. Apart from the complexity of its regulation, alterations in both CTNNA3 and LRTMM3 are implicated in human disease.

• Homocysteine levels impact directly on epigenetic reprogramming in astrocytes.

  Authors: Ying Jin, Ana Amaral, Amanda McCann, Lorraine Brennan

  Neurochemistry international. 03/2011;

  Although the neurotoxic effects of homocysteine have been well elucidated, the effects of homocysteine in astrocytes have received little attention until recently. Previously we have demonstratedAlthough the neurotoxic effects of homocysteine have been well elucidated, the effects of homocysteine in astrocytes have received little attention until recently. Previously we have demonstrated that elevated levels of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. However, the mechanisms behind such alterations remain unclear. As homocysteine is a key metabolite in one-carbon metabolism the present study examined if the effects of homocysteine on astrocyte function are mediated through an epigenetic mechanism. Following exposure to homocysteine for 72h, global DNA methylation and H3K9 acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity and protein levels of DNA methyltransferase 3B were measured. Exposure to homocysteine resulted in global DNA hypomethylation (p<0.05) and histone hyperacetylation (p<0.05). Total DNA methyltransferase activity significantly decreased following exposure to homocysteine (from 11.5±3.9 to 6.0±1.7OD/h/mg protein, p<0.01) which was accompanied by a significant reduction in protein levels of DNA methyltransferase 3B (p<0.05). Treatment of astrocytes with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, mimicked the functional changes induced by homocysteine. In conclusion, the results demonstrate significant epigenetic modifications following exposure to homocysteine in astrocytes and these changes seem to mediate functional alterations.

• MAD2 downregulation in hypoxia is independent of promoter hypermethylation.

  Authors: Maria Prencipe, Aloysius McGoldrick, Antoinette S Perry, Anthony O'Grady, Sine Phelan, Barbara McGrogan, Patricia Fitzpatrick, Jenny A Watson, Fiona Furlong, Donal J Brennan, Mark Lawler, Elaine Kay, Amanda McCann

  Cell cycle (Georgetown, Tex.). 07/2010; 9(14):2856-65.

  Aberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancerAberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancer cell lines, the mechanism(s) underlying its downregulation remain elusive. The objective of this study was to establish the impact of hypoxia on MAD2 expression and to investigate the potential role of aberrant promoter methylation as a possible mechanism of MAD2 downregulation. For this purpose, three ovarian cancer cell lines, displaying differing levels of MAD2, were treated with chromatin modifying drugs, pre and post-hypoxia exposure and a DHPLC analysis of DNA promoter methylation carried out. We show that hypoxia induces downregulation of MAD2 expression, independently of MAD2 promoter methylation. We also show no evidence of MAD2 promoter methylation in breast and prostate cancer cells or in breast cancer clinical material. While our findings provide no evidence for MAD2 promoter methylation, we show a concomitant upregulation of p21 with downregulation of MAD2 in hypoxia. Our in vitro results were also confirmed in an ovarian cancer tissue microarray (TMA), where a reciprocal staining of MAD2 and CAIX was found in 21/60 (35%) of tumours. In summary, MAD2 downregulation may be a crucial mechanism by which hypoxic cells become chemorefractory. This stems from our previous work where we demonstrated that MAD2 downregulation induces cellular senescence, a viable cellular fate, with resultant cellular resistance to paclitaxel. Moreover, MAD2 downregulation could play a central role in the induction of chemoresistance in hypoxia, a key tumour microenvironment associated with chemoresistance.

• Epigenetics, the epicenter of the hypoxic response.

  Authors: Jenny A Watson, Chris J Watson, Amanda McCann, John Baugh

  Epigenetics : official journal of the DNA Methylation Society. 05/2010; 5(4):293-6.

  It is becoming increasingly apparent that epigenetics plays a crucial role in the cellular response to hypoxia. Such epigenetic regulation may work hand in hand with the hypoxia-induced transcriptionIt is becoming increasingly apparent that epigenetics plays a crucial role in the cellular response to hypoxia. Such epigenetic regulation may work hand in hand with the hypoxia-induced transcription factor (HIF) family or may contribute in a more substantial way to the maintenance of a hypoxia-adapted cellular phenotype long after HIF has initiated the immediate response pathways. In this article we discuss the current research implicating epigenetic mechanisms in the cellular response to hypoxic environments. This includes; the role of epigenetics in both the stabilization and binding of HIF to its transcriptional targets, the role of histone demethylase enzymes following direct HIF transactivation, and finally, the impact of hypoxic environments on global patterns of histone modifications and DNA methylation.

• Generation of an epigenetic signature by chronic hypoxia in prostate cells.

  Authors: Jenny A Watson, Chris J Watson, Ann Maria McCrohan, Kathryn Woodfine, Miriam Tosetto, Jennifer McDaid, Emma Gallagher, David Betts, John Baugh, Jacintha O'Sullivan, Adele Murrell, R William G Watson, Amanda McCann

  Human molecular genetics. 08/2009;

  Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study we have usedIncreasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterised by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL1b, IL6, IL8 and TNF cytokines. In association with these phenotypic changes and the absence of HIF-1alpha protein expression we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

• Recurrence of urothelial carcinoma of the bladder: a role for insulin-like growth factor-II loss of imprinting and cytoplasmic E-cadherin immunolocalization.

  Authors: Emma M Gallagher, Deirdre M O'Shea, Patricia Fitzpatrick, Michèle Harrison, Breege Gilmartin, Jenny A Watson, Trevor Clarke, Martin O Leonard, Aloysius McGoldrick, Maria Meehan, Chanel Watson, Fiona Furlong, Patrick O'Kelly, John M Fitzpatrick, Peter A Dervan, Anthony O'Grady, Elaine W Kay, Amanda McCann

  Clinical cancer research : an official journal of the American Association for Cancer Research. 12/2008; 14(21):6829-38.

  PURPOSE: This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was alsoPURPOSE: This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II. EXPERIMENTAL DESIGN: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1 cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pT(a) noninvasive and 44 pT(1) lamina propria invasive) using the commercially available Novocastra antibody. RESULTS: IGF-II LOI was evident in 7 of 17 (41%) UCB tumors and 4 of 11 (36%) tumor-associated normal urothelial samples. Two of four pT(1) grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1 cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-II maintenance of imprinting tumors had concomitant CDH1 cytoplasmic localization. UCB cell lines displaying cytoplasmic CDH1 immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1 staining. Finally, cytoplasmic CDH1 staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage. CONCLUSIONS: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery.

• Urinary insulin-like growth factor 2 identifies the presence of urothelial carcinoma of the bladder.

  Authors: Jenny A Watson, Keith Burling, Patricia Fitzpatrick, Elaine Kay, John Kelly, John M Fitzpatrick, Peter A Dervan, Amanda McCann

  BJU international. 12/2008;

  OBJECTIVE To examine urinary insulin-like growth factor 2 (IGF-2) levels in patient urine samples and determine the potential of IGF-2 as a marker for the presence of urothelial carcinoma of theOBJECTIVE To examine urinary insulin-like growth factor 2 (IGF-2) levels in patient urine samples and determine the potential of IGF-2 as a marker for the presence of urothelial carcinoma of the bladder (UCB). PATIENTS AND METHODS The current gold standard for diagnosis of UCB is cystoscopy and cytological analysis. The identification of an accurate urine marker for UCB with the potential to replace unnecessary cystoscopy would benefit patients with UCB and others investigated after detecting haematuria. In the present study, we analysed 65 urine samples, and optimized an enzyme-linked immunosorbent assay-based approach to measure urinary levels of IGF-2. RESULTS Based on a threshold of 5.4 ng/mL, patients with UCB have significantly elevated levels of urinary IGF-2 (P = 0.009) and this difference remained significant after adjustment for age and sex (P = 0.04). Sensitivity and specificity values of 80% and 52%, respectively, were determined for urinary IGF-2 alone and when combined with nuclear matrix protein 22 (NMP22; an approved biomarker for detection of UCB). There was a positive correlation between urinary IGF-2 levels and NMP22 levels in patient urine samples and the combined assay improved the detection of UCB (sensitivity 85% and specificity 52%). CONCLUSION Substantiated evidence has identified IGF-2 as a valuable marker for UCB. In addition, the novel observations of the present study have shown that aberrant levels of IGF-2 occurring in the presence of UCB, can now be determined through a simple and inexpensive urine assay.

• Novel image analysis approach for quantifying expression of nuclear proteins assessed by immunohistochemistry: application to measurement of estrogen and progesterone receptor levels in breast cancer.

  Authors: Elton Rexhepaj, Donal Brennan, Peter Holloway, Elaine Kay, Amanda McCann, Goran Landberg, Michael Duffy, Karin Jirstrom, William Gallagher

  Breast cancer research : BCR. 10/2008; 10(5):R89.

  ABSTRACT: INTRODUCTION: Manual interpretation of immunohistochemistry (IHC) is a subjective, time consuming and variable process, with an inherent intra- and inter-observer variability. AutomatedABSTRACT: INTRODUCTION: Manual interpretation of immunohistochemistry (IHC) is a subjective, time consuming and variable process, with an inherent intra- and inter-observer variability. Automated image analysis approaches offer the possibility of developing rapid, uniform indicators of IHC staining. Here, we describe the development of a novel approach for automatically quantifying estrogen receptor (ER) and progesterone receptor (PR) protein expression assessed by IHC in primary breast cancer. METHODS: Two cohorts of breast cancer patients (n = 179 and n = 564) were used in the study. Digital images of breast cancer tissue microarrays were captured using the Aperio ScanScope XT slide scanner (Aperio Technologies, Vista, CA). Image analysis algorithms were developed using MatLab 7 (MathWorks, Apple Hill Drive, MA). A fully automated nuclear algorithm was developed to discriminate tumour from normal tissue, as well as to quantify ER and PR expression, and applied to both cohorts. Random forest clustering was employed to identify optimum thresholds for survival analysis. RESULTS: The accuracy of the nuclear algorithm was initially confirmed by a histopathologist, who validated the output in 18 representative images. For these 18 samples, an excellent correlation was evident between the results obtained by manual and automated analyses (Spearman's Rho = 0.9, p-value, <0.001). Optimum thresholds for survival analysis were identified using random forest clustering. This revealed that 7% positive tumor cells was the optimum threshold for ER and 5% positive tumor cells for PR. Moreover, a 7% cut-off for ER predicted a better response to tamoxifen than the currently used 10% threshold. Finally, the application of linear regression to duplicate cores demonstrated a more homogenous pattern of expression for ER (R = 0.860) than for PR (R = 0.681). CONCLUSIONS: In summary, we present data on the automated quantification of ER and PR in 743 primary breast tumours using a novel unsupervised image analysis algorithm. This novel approach provides a useful tool for the quantification of biomarkers on tissue specimens, as well as objective identification of appropriate cut-offs for biomarker positivity.

• Progesterone receptor B (PRB) promoter hypermethylation in sporadic breast cancer : Progesterone receptor B hypermethylation in breast cancer.

  Authors: Orla Mc Cormack, Wen Y Chung, Patricia Fitzpatrick, Fiachra Cooke, Barbara Flynn, Michele Harrison, Edward Fox, Emma Gallagher, Aloysius McGoldrick, Peter A Dervan, Amanda McCann, Michael J Kerin

  Breast cancer research and treatment. 09/2008; 111(1):45-53.

  Introduction Oestrogen receptor alpha (ER alpha) is traditionally measured on all breast tumour specimens to identify those patients more likely to respond to anti-oestrogens. Progesterone receptorIntroduction Oestrogen receptor alpha (ER alpha) is traditionally measured on all breast tumour specimens to identify those patients more likely to respond to anti-oestrogens. Progesterone receptor (PR) status has contributed useful information in defining more responsive subgroups. PR negativity may be a marker for increased signalling through growth factor receptor tyrosine kinase pathways. Progesterone acts through two PRs, PRA and PRB. PRB, the functionally active PR, can be silenced by promoter hypermethylation. Methods Following DNA and RNA extraction from 94 breast carcinomas, the methylation status of the PRB promoter was assessed by sodium bisulphite modification and methylation sensitive PCR (MSP). A quantitative realtime PCR analysis (QRTPCR) was used to determine the levels of PRB mRNA expression. Protein expression was evaluated immunohistochemically with a commercially available PRB antibody. Results 76% of the primary breast carcinoma samples demonstrated a methylated band for PRB. PRB methylation significantly compromised total PR immunohistochemistry (IHC) expression (P = 0.03). PRB mRNA correlated positively with total PR IHC (r = 0.58, P = 0.04), ER alpha IHC (P = 0.02), and tumour grade (P = 0.01). PRB protein expression was significantly associated with a number of favourable prognostic variables including smaller (P = 0.004) lower grade (P = 0.007), ER alpha IHC positive tumours (P < 0.001), and tumours with a low Nottingham Prognostic Index (NPI) (P = 0.0008). PRB mRNA levels were significantly associated with better overall survival (P = 0.04) in a univariate analysis. Conclusion The majority of tumours were methylated for PRB. This did not directly compromise PRB expression suggesting that other factors may down regulate the PR gene. When PRB was expressed, it correlated with good prognostic markers and better overall survival.

• Small interfering RNAs induce macrophage migration inhibitory factor production and proliferation in breast cancer cells via a double-stranded RNA-dependent protein kinase-dependent mechanism.

  Authors: Michelle E Armstrong, Michael Gantier, Lili Li, Wen Y Chung, Amanda McCann, John A Baugh, Seamas C Donnelly

  Journal of immunology (Baltimore, Md. : 1950). 06/2008; 180(11):7125-33.

  Small interfering RNAs (siRNAs) represent a novel tool to induce gene silencing in mammalian cells and clinical trials are currently ongoing to assess the therapeutic efficacy of siRNAs in variousSmall interfering RNAs (siRNAs) represent a novel tool to induce gene silencing in mammalian cells and clinical trials are currently ongoing to assess the therapeutic efficacy of siRNAs in various human diseases, including age-related macular degeneration and respiratory syncytial virus infection. However, previously reported off-target, nonspecific effects of siRNAs, including activation of type I IFNs and proinflammatory cytokines, remain an outstanding concern regarding use of these agents in vivo. Macrophage-migration inhibitory factor (MIF) is a pleiotropic cytokine with well-described roles in cell proliferation, tumorigenesis, and angiogenesis and represents a target gene for siRNA-based therapy in the treatment of breast cancer. However, in this study we describe an increase in MIF production from mammary adenocarcinoma (MCF-7) cells following transfection with MIF siRNA and various control siRNAs. This effect was shown to be dose-dependent and was attenuated in the presence of a double-stranded RNA-dependent protein kinase inhibitor, 2-aminopurine. Furthermore, treatment of MCF-7 cells with poly(I:C) also stimulated a PKR-dependent increase in MIF production from MCF-7 cells. The biological consequence of the siRNA-induced increase in MIF production from MCF-7 cells was a PKR-dependent increase in proliferation of breast cancer cells. Furthermore, in cDNAs prepared from a primary human breast cancer cohort, we demonstrated a significant correlation (Spearman rank correlation coefficient, r = 0.50, p < 0.0001, n = 63) between PKR- and MIF-mRNA expression. In conclusion, this study highlights the potential biological consequences of off-target, nonspecific effects of siRNAs and underlines the safety concerns regarding the use of siRNAs in the treatment of human diseases, such as cancer.

• Taxanes, microtubules and chemoresistant breast cancer.

  Authors: Barbara T McGrogan, Breege Gilmartin, Desmond N Carney, Amanda McCann

  Biochimica et biophysica acta. 05/2008; 1785(2):96-132.

  The taxanes, paclitaxel and docetaxel are microtubule-stabilizing agents that function primarily by interfering with spindle microtubule dynamics causing cell cycle arrest and apoptosis. However, theThe taxanes, paclitaxel and docetaxel are microtubule-stabilizing agents that function primarily by interfering with spindle microtubule dynamics causing cell cycle arrest and apoptosis. However, the mechanisms underlying their action have yet to be fully elucidated. These agents have become widely recognized as active chemotherapeutic agents in the treatment of metastatic breast cancer and early-stage breast cancer with benefits gained in terms of overall survival (OS) and disease-free survival (DFS). However, even with response to taxane treatment the time to progression (TTP) is relatively short, prolonging life for a matter of months, with studies showing that patients treated with taxanes eventually relapse. This review focuses on chemoresistance to taxane treatment particularly in relation to the spindle assembly checkpoint (SAC) and dysfunctional regulation of apoptotic signaling. Since spindle microtubules are the primary drug targets for taxanes, important SAC proteins such as MAD2, BUBR1, Synuclein-gamma and Aurora A have emerged as potentially important predictive markers of taxane resistance, as have specific checkpoint proteins such as BRCA1. Moreover, overexpression of the drug efflux pump MDR-1/P-gp, altered expression of microtubule-associated proteins (MAPs) including tau, stathmin and MAP4 may help to identify those patients who are most at risk of recurrence and those patients most likely to benefit from taxane treatment.

• Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer.

  Authors: Alison M Hosey, Julia J Gorski, Margaret M Murray, Jennifer E Quinn, Wen Y Chung, Gail E Stewart, Colin R James, Susan M Farragher, Jude M Mulligan, Alistair N Scott, Peter A Dervan, Patrick G Johnston, Fergus J Couch, Peter A Daly, Elaine Kay, Amanda McCann, Paul B Mullan, D Paul Harkin

  Journal of the National Cancer Institute. 11/2007; 99(22):1683-94.

  BACKGROUND: BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined aBACKGROUND: BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. METHODS: We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. RESULTS: Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). CONCLUSIONS: BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

• Role of the Progesterone Receptor (PR) and the PR Isoforms in Breast Cancer.

  Authors: Orla Mc Cormack, Michele Harrison, Michael J Kerin, Amanda McCann

  Critical reviews in oncogenesis. 02/2007; 13(4):283-302.

  Known risk factors for breast cancer include overexposure to exogenous or endogenous hormones, namely, estrogen and progesterone. The effects of progesterone acts via two isoforms of the progesteroneKnown risk factors for breast cancer include overexposure to exogenous or endogenous hormones, namely, estrogen and progesterone. The effects of progesterone acts via two isoforms of the progesterone receptor (PR) termed A (PRA) and B (PRB). There is a single human PR gene with two distinct promoter regions in exon 1 encoding the two isoforms. Studies have shown that in poor prognostic tumors, the ratio between PRA and PRB is altered, with a predominance of PRA and loss of PRB. PRA and PRB regulate different subsets of genes involved in particular functional pathways. Breast tumors that express PR are associated with slow growth, better differentiation, and better overall prognosis in the short term. Both estrogen receptor alpha (ERalpha) and total progesterone receptor (PR) are immunohistochemically measured on breast cancer specimens to help determine those patients who would benefit from hormonal treatment. Depending on the hormone receptor status and the menopausal status of the patient, different treatments are available. Although the value of ERalpha in predicting response to hormone therapy in early breast cancer patients is undisputed, the value of PR is currently under debate. Historically, PR was thought to be a surrogate marker for ER expression; however, it is now known that lack of PR expression can indicate underlying epidermal growth factor receptor signaling or promoter methylation. This has implications for and can dictate hormone therapy responsiveness. Further studies investigating the specific isoforms and their pathways will help to reveal the underlying mechanisms of PR-induced breast tumori-genesis and help in assigning the most appropriate patient-tailored treatment.

• A multi-centre investigation towards reaching a consensus on the immunohistochemical detection of ERbeta in archival formalin-fixed paraffin embedded human breast tissue.

  Authors: Pauline J Carder, Claire E Murphy, Peter Dervan, Maria Kennedy, Amanda McCann, Philippa T K Saunders, Abeer M Shaaban, Christopher S Foster, Caroline J Witton, John M S Bartlett, Rosemary A Walker, Valerie Speirs

  Breast cancer research and treatment. 09/2005; 92(3):287-93.

  Estrogen receptor (ER) alpha is a well-established independent prognostic factor in breast cancer whose presence determines the clinical implications of adjuvant endocrine therapy. A second receptor,Estrogen receptor (ER) alpha is a well-established independent prognostic factor in breast cancer whose presence determines the clinical implications of adjuvant endocrine therapy. A second receptor, ERb has been described, and a number of studies have examined its expression in breast tissue. However elucidation of the role played by ERb has been hampered by published immunohistochemical studies employing a variety of protocols and scoring systems such that inter-laboratory comparisons are difficult. Here we present a multi-centre study designed to critically evaluate inter-laboratory differences in methodology. Six UK and Irish centres participated in this study. A small series of breast cancers were stained using centre-specific laboratory protocols and scored using both centrespecific and standard scoring protocols. There was generally poor agreement as to what constituted a positive or negative case when centre-specific scoring systems were used with less than half of all cases in agreement. Concordance was improved when a standard scoring system was used but varied according to threshold for positivity employed and primary antibody. Our results emphasise the need for further studies addressing the role of ERb to be based on a wider consensus on criteria for positivity. Ideally this should be based on calibration against clinical outcome.

• Promoter switch: a novel mechanism causing biallelic PEG1/MEST expression in invasive breast cancer.

  Authors: Inge Søkilde Pedersen, Peter Dervan, Alo McGoldrick, Michèle Harrison, Frederique Ponchel, Valerie Speirs, John D Isaacs, Thomas Gorey, Amanda McCann

  Human molecular genetics. 07/2002; 11(12):1449-53.

  We have previously reported on the biallelic expression of the imprinted PEG1/MEST gene in infiltrating carcinomas of the breast. Putative loss of imprinting (LOI) of PEG1/MEST has subsequently alsoWe have previously reported on the biallelic expression of the imprinted PEG1/MEST gene in infiltrating carcinomas of the breast. Putative loss of imprinting (LOI) of PEG1/MEST has subsequently also been implicated in the aetiology of lung adenocarcinomas and colon cancer. Taking advantage of our previous study, identifying seven infiltrating carcinomas of the breast, displaying biallelic PEG1/MEST expression, we have analysed the allelic usage of the two alternative PEG1/MEST transcripts encoding isoforms 1 and 2, separately. In addition, expression levels of the two transcripts have been measured by real-time RT-PCR, in order to elucidate the mechanism behind the switch from monoallelic transcription in normal breast tissue to biallelic expression in invasive cancer. The isoform 1 transcript is imprinted in both the paired normal tissue and the breast carcinomas. In contrast, the isoform 2 transcript is biallelically expressed, or in one case expressed from the opposite allele to isoform 1, raising the possibility that isoform 2 is polymorphically imprinted in normal breast tissue. In all the paired normal samples, isoform 1 is predominantly expressed, explaining the monoallelic profiles of these samples. However, in four of the seven biallelic carcinomas, isoform 2 is expressed at higher levels than isoform 1, indicating that a switch in expression from isoform 1 to isoform 2 is responsible for the biallelic profiles in these samples. Our results not only suggest a novel mechanism leading to biallelic expression detected when analysing the common 3'-UTR of the PEG1/MEST transcriptional unit, they are also indicative of the existence of further alternative PEG1/MEST transcripts.

• Taxanes, microtubules and chemoresistant breast cancer

  Authors: Barbara T. McGrogan, Breege Gilmartin, Desmond N. Carney, Amanda McCann

  Biochimica et Biophysica Acta (BBA) - Reviews on Cancer.

  The taxanes, paclitaxel and docetaxel are microtubule-stabilizing agents that function primarily by interfering with spindle microtubule dynamics causing cell cycle arrest and apoptosis. However, theThe taxanes, paclitaxel and docetaxel are microtubule-stabilizing agents that function primarily by interfering with spindle microtubule dynamics causing cell cycle arrest and apoptosis. However, the mechanisms underlying their action have yet to be fully elucidated. These agents have become widely recognized as active chemotherapeutic agents in the treatment of metastatic breast cancer and early-stage breast cancer with benefits gained in terms of overall survival (OS) and disease-free survival (DFS). However, even with response to taxane treatment the time to progression (TTP) is relatively short, prolonging life for a matter of months, with studies showing that patients treated with taxanes eventually relapse. This review focuses on chemoresistance to taxane treatment particularly in relation to the spindle assembly checkpoint (SAC) and dysfunctional regulation of apoptotic signaling. Since spindle microtubules are the primary drug targets for taxanes, important SAC proteins such as MAD2, BUBR1, Synuclein-gamma and Aurora A have emerged as potentially important predictive markers of taxane resistance, as have specific checkpoint proteins such as BRCA1. Moreover, overexpression of the drug efflux pump MDR-1/P-gp, altered expression of microtubule-associated proteins (MAPs) including tau, stathmin and MAP4 may help to identify those patients who are most at risk of recurrence and those patients most likely to benefit from taxane treatment.

• Imprinted expression of the canine IGF2R, in the absence of an anti-sense transcript or promoter methylation.

  Authors: Fiona M O'Sullivan, Susan K Murphy, Lauren R Simel, Amanda McCann, John J Callanan, Catherine M Nolan

  Evolution & development. 9(6):579-89.

  Imprinted genes are epigenetically modified in a parent of origin-dependent manner, and as a consequence, are differentially expressed. Although the evolution of genomic imprinting is a subject ofImprinted genes are epigenetically modified in a parent of origin-dependent manner, and as a consequence, are differentially expressed. Although the evolution of genomic imprinting is a subject of intense debate, imprinted genes have been studied primarily in mice and humans and in a small number of marsupial mammals. Comparative studies involving rodents and primates are of limited value, as they belong to the same superordinal group of eutherian mammals (Euarchontoglires). On the other hand, comparisons involving marsupials may not be informative, due to phylogenetic distance. Canis familiaris belongs to Laurasiatheria, a sister-group of Euarchontoglires, and should prove useful in comparative studies of imprinted genes. Using RT-PCR we demonstrate monoallelic expression of the canine IGF2R in several tissues, including uterus and umbilical cord. In the case of umbilical cord, we identify the expressed allele as maternally derived. The canine IGF2R is thus an imprinted gene. Using bisulfite sequencing, we show that the canine IGF2R resembles the imprinted mouse Igf2r in having a CpG island in intron 2 that is hemi-methylated. However, it differs from the mouse gene in that maintenance of the monoallelic expression of canine IGF2R does not require expression of an anti-sense transcript from the paternally derived allele, or methylation of the repressed IGF2R promoter. In these two important features, the imprinted canine gene resembles the imprinted opossum IGF2R. Our data suggest that these features were properties of the ancestral imprinted IGF2R and that the anti-sense transcript (Air) and promoter methylation observed in mouse are derived features of the mouse Igf2r locus.