Breast Cancer-associated Genes Research Articles

Identification of novel breast cancer‐associated transcripts by uniGene database mining and gene expression analysis in normal and malignant cells

Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation.

Mouse models of BRCA1 and their application to breast cancer research

Germline mutations of human breast cancer-associated gene 1 (BRCA1) predispose women to breast and ovarian cancers. In mice, over 20 distinct mutations, including null, hypomorphic, isoform, conditional, and point mutations, have been created to study functions of Brca1 in mammary development and tumorigenesis. Analyses using these mutant mice have yielded an enormous amount of information that greatly facilitates our understanding of the gender- and tissue-specific tumor suppressor functions of BRCA1, as well as enriches our insights into applying these preclinical models of disease to breast cancer research. Here, we review features of these mutant mice and their applications to cancer prevention and therapeutic treatment.

Chromosomal instability induced by mammography X-rays in primary human fibroblasts from BRCA1 and BRCA2 mutation carriers

Purpose: Mammography X-rays are known to induce DNA double-strand breaks (DSB) whose error-free recombinational repair requires the function of the tumour repressor genes BRCA1 (breast-cancer-associated gene 1) and BRCA2 (breast-cancer-associated gene 2). Since un- or misrepaired DSB lead to chromosomal anomalies which may promote the development of breast cancer, we have studied the potential of mammography X-rays for immediate and delayed induction of chromosomal anomalies in human primary fibroblasts from BRCA1 and BRCA2 mutation carriers.Materials and methods: Primary human fibroblasts from three BRCA1, three BRCA2 mutation carriers, one BRCA2-deficient fanconi anemia (FA) patient and three normal individuals were exposed to various doses of mammography X-rays. Chromosomal anomalies at first mitosis and at several population doublings post-irradiation were assayed (Giemsa staining and Fish [fluorescence in situ hybridization]).Results: No effect of the BRCA mutation status was observed on survival curves after exposure to mammography X-rays and on the dose-dependent increase of chromosomal anomalies at first mitosis post-irradiation. In contrast, several population doublings after exposure to a low dose of only 0.5 Gy chromosomal instability, manifested as gross chromosomal rearrangements and aneuploidy, had developed in BRCA2-deficient FA fibroblasts and in some – but not all – BRCA heterozygous fibroblasts.Conclusions: Low doses of mammography X-rays have the potential to induce chromosomal instability in fibroblasts from BRCA mutation carriers: Cells exhibit gross chromosomal rearrangements and aneuploidy similar to those observed in breast cancer cells. These results suggest that for women carrying a BRCA mutation early and frequent screening with mammography X-rays may not be the method of choice to detect breast cancer.

Heterozygous Mutations in DNA Repair Genes and Hereditary Breast Cancer: A Question of Power

Heterozygous Mutations in DNA Repair Genes and Hereditary Breast Cancer: A Question of Power

Differential Regulation of Breast Cancer-Associated Genes by Progesterone Receptor Isoforms PRA and PRB in a New Bi-Inducible Breast Cancer Cell Line

Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (AREG) playing important role in cancer. Interestingly, unliganded PRA increases AREG expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB.

Transcriptional activation of breast cancer-associated gene 2 by estrogen receptor

RNF115, or Breast Cancer-Associated Gene 2 (BCA2), encodes a RING-finger ubiquitin E3 ligase, expression of which was associated with estrogen receptor (ER)-positive status in human breast tumors. Although the BCA2 promoter contains several estrogen response element (ERE) half-sites, the role of ER in the regulation of BCA2 transcription has not been reported. The aim of this study is to investigate the molecular mechanism by which estrogen regulates BCA2 transcription. BCA2 mRNA and protein levels were examined by RT-PCR and Western blot analysis, respectively, and localization was assessed by immunofluorescence. BCA2 promoter activity in response to E(2) was tested by a dual luciferase reporter assay and ER binding to the BCA2 promoter was examined by chromatin immunoprecipitation assay. We found that BCA2 mRNA and protein levels are regulated by estrogen in ER-positive MCF7 breast cancer cells and MDA MB 231 cells stably transfected with ER. Estrogen treatment in hormonal depleted MCF7 and MDA MB 231/ER stably transfected cells resulted in increased nuclear ER and cytoplasmic and nuclear BCA2 staining. Cycloheximide is not able to inhibit BCA2 mRNA levels, suggesting potential BCA2 regulation at the transcriptional level. Anti-estrogens like tamoxifen and ICI 182 178 counteracted E(2)-induced BCA2 protein and knockdown of ER by ER siRNA resulted in a significant decrease in BCA2 protein and a lower nuclear expression pattern. Estrogen treatment lead to a significant increase in BCA2 promoter response, associated with increased binding of ER to the ERE region of the BCA2 promoter. BCA2 is therefore a newly identified transcriptional target of estrogen receptor.

Differential expression of breast cancer-associated genes between stage- and age-matched tumor specimens from African- and Caucasian-American Women diagnosed with breast cancer

BackgroundRecent studies suggest that the poorer breast cancer outcome observed in African-American women (AAW) may, in part, result from underlying molecular factors. The purpose of this study was to investigate gene expression differences between Caucasian-American women (CAW) and AAW that may contribute to this poorer prognosis.MethodsThe expression of 84 genes involved in breast carcinoma prognosis, response to therapy, estrogen signaling, and tumor aggressiveness was assessed in age- and stage-matched CAW and AAW paraffin-embedded breast cancer specimens. The Wilcoxon–Mann–Whitney Test was used to identify genes with a significant difference in expression between CAW and AAW. To determine if the differentially expressed genes could segregate between the CAW and AAW, we performed semi-supervised principal component analysis (SSPCA).ResultsTwenty genes were differentially expressed between AAW and CAW. SSPCA incorporating these 20 genes segregated AAW and CAW into two distinct groups. AAW were significantly (p < 0.05) more likely to display aberrations in G1/S cell-cycle regulatory genes, decreased expression of cell-adhesion genes, and low to no expression of ESR1, PGR, ERBB2 and estrogen pathway targets.ConclusionsThe gene expression differences identified between AAW and CAW may contribute to more aggressive disease, resistance to therapy, enhanced metastatic potential and poor clinical outcome. These findings support the hypothesis that breast cancer specimens collected from AAW display distinct gene expression differences compared to similar tissues obtained from CAW. Additional population-based studies are necessary to determine if these gene expression variations contribute to the highly aggressive and treatment-resistant breast cancer phenotype frequently observed in AAW.

Abstract 693: Development of a protein-based functional assay for the determination of BRCA1 carrier status

Abstract While most breast cancers are sporadic, 5-10% are hereditary and attributable to the presence of a mutation in the breast cancer associated gene 1 (BRCA1). The BRCA1 gene encodes a tumour suppressor with several well described roles in maintaining genomic integrity, such as cell cycle checkpoint control and DNA repair. BRCA1 is also known to play a role in the differentiation of breast epithelial cells. Given the high risk of cancer development associated with BRCA1 mutation carriers, it is important that they be identified early and accurately. Previous work in our lab developed a novel functional assay to predict BRCA1 status based on gene expression profiles. That study analyzed EBV-transformed lymphocyte cell lines (LCLs) of BRCA1 mutation carriers (BRCA1+/−) and controls (BRCA1+/+) and found that lymphocytes from BRCA1 carriers could be distinguished with high fidelity (∼90% accuracy) from individuals with two wild type copies of the gene. Interestingly, it was found that many of the genes used to distinguish BRCA1 mutation carriers were also markers of differentiation in blood cells. We are now using this information to develop a flow cytometric protein-based biomarker assay. To do this, our transcriptome microarray data was analyzed to identify genes with strong edictability of BRCA1 status, including genes with known roles in lymphocyte differentiation such as TBX21 and CXCR3. These genes were then used to model the assay in-silico using a training set of data comprised of 53 LCLs. The model was designed by selecting genes both up- and down-regulated in BRCA1 carriers, placing them in pairs and looking at the ratio of their expression values. This model was subsequently validated using a test data set comprising 16 LCLs. Overall accuracy of the model was 94%, correctly predicting 54/59 LCLs. Based on in-silico modelling, we chose six antibodies to develop the assay. To identify other candidate proteins, LCL lysates were analyzed by mass spectrometry (MS). The results identified several other candidate proteins for assay development including IgG1, IgD, BCR and MX1. Interestingly, as was the case in our transcriptome analysis, these MS identified proteins also suggested that BRCA1 mutation carriers displayed defects in cellular differentiation. This was indicated by the higher expression of surface markers in responding to less differentiated B-lymphocytes in comparison with controls. Ultimately, the assay will be applied to fresh patient blood samples with the expectation that it will become an assay that can be implemented in a clinical setting to provide accurate identification of BRCA1 mutation carriers and aid in disease management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 693. doi:1538-7445.AM2012-693

Abstract 2744: Identification of synergistic effect of autophagy inhibitors and resveratrol on BRCA1 mutant cancer cells

Abstract Over 200,000 new breast cancer cases are diagnosed in the US each year, with 5% of cases caused by mutations in BRCA1 (Breast cancer associated gene 1). Mutation carries have a lifetime risk of up to 80% for developing breast cancer. Current treatment options are limited and often do not lead to cure. We showed previously that treatment with resveratrol, although initially reduced more the tumor size in BRCA1 mutant (mt) tumors, with respect to BRCA1 wild-type (wt) in mice, tumors continued to grow after prolonged treatment. To identify small molecules that can be repurposed and synergize the killing effect of resveratrol in BRCA1-mt tumors, we screened the NCGC Pharmaceutical Collection in which, more than 52% are drugs approved for human or animal use by FDA, and the rest are drugs approved in other countries or in clinical trial. Using a high-throughput cell viability assay by measuring intracellular ATP content, we identified the inhibitory effect of 43 compounds that exhibited synergy with resveratrol (Res) with efficacy&amp;gt;50% and potency &amp;lt;35uM. We further analyzed 4 of them, cantharidin, chloroquine, amodiaquin and colchicine for in vivo study. Utilizing BRCA1-mt breast tumor implant models in aythymic mice reveal that mice treated with combination therapy (Drug + Res) exhibit significantly reduced tumor volumes and increased survival rate compared with mice on monotreatment and this effect was even more significant when compared to wt-BRCA1. Also, some mammary tumors developed in the BRCA1 mammary tissue specific mutant mice exhibited a total regression with combination therapy. In order to identify a shared molecular and biochemical change which make BRCA1-mt cancer cells more susceptible to the combination treatment, we found that these compounds inhibit autophagy at different levels, targeting the mTOR/AKT pathway at different levels and/or inhibiting the fusion between the lysosome and the phagosome producing strong vacuolization in the cytoplasm during treatment, increasing LC3-II in a Beclin independent manner, followed by a faster induction of apoptosis on the BRCA mutant cells. This approach offers the potential to combat breast cancer cells and sensitize BRCA1 deficient cells to more effective treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2744. doi:1538-7445.AM2012-2744

The In Vivo Dynamic Organization of BRCA1‐A Complex Proteins at DNA Damage‐Induced Nuclear Foci

The breast cancer associated gene 1 (BRCA1)-A protein complex assembles at DNA damage-induced nuclear foci to facilitate repair of double-stranded breaks. Here, we describe the first systematic comparison of the dynamics, copy number and organization of its core components at foci. We show that the protein pools at individual foci generally comprise a small immobile fraction (∼20%) and larger mobile fraction (∼80%), which together occupy the same focal space but exist at different densities. In the mobile fraction, Abraxas (CCDC98) and the heterodimer BARD1-BRCA1 share similar rates of dynamic exchange (complete turnover in ∼500 seconds). In contrast, RAP80, which is required for initial foci assembly, was more dynamic with 25-fold faster turnover at mature foci. In addition, Abraxas, BARD1, BRCA1 and Merit40 (NBA1) were stably retained in the immobile fraction of foci under conditions causing loss of BRCC36 and RAP80, suggesting a shift to RAP80-independent localization after foci formation. These results, combined with our finding that RAP80 (∼1200 copies per focus) is twofold more abundant than Abraxas/BARD1/BRCA1 at foci, suggest new models defining the dynamic organization of BRCA1-A complex at mature foci, wherein the unusually fast turnover of RAP80 may contribute to its regulation of BRCA1-dependent DNA repair.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of ∼20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located ∼2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

CDDO-Methyl Ester Delays Breast Cancer Development in Brca1-Mutated Mice

The breast cancer-associated gene 1 (BRCA1) is the most frequently mutated tumor suppressor gene in familial breast cancers. Mutations in BRCA1 also predispose to other types of cancers, pointing to a fundamental role of this pathway in tumor suppression and emphasizing the need for effective chemoprevention in these high-risk patients. Because the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) is a potent chemopreventive agent, we tested its efficacy in a highly relevant mouse model of BRCA1-mutated breast cancer. Beginning at 12 weeks of age, Brca1(Co/Co); MMTV-Cre;p53(+/-) mice were fed powdered control diet or diet containing CDDO-Me (50 mg/kg diet). CDDO-Me significantly (P < 0.05) delayed tumor development in the Brca1-mutated mice by an average of 5.2 weeks. We also observed that levels of ErbB2, p-ErbB2, and cyclin D1 increased in a time-dependent manner in the mammary glands in Brca1-deficient mice, and CDDO-Me inhibited the constitutive phosphorylation of ErbB2 in tumor tissues from these mice. In BRCA1-deficient cell lines, the triterpenoids directly interacted with ErbB2, decreased constitutive phosphorylation of ErbB2, inhibited proliferation, and induced G(0)-G(1) arrest. These results suggest that CDDO-Me has the potential to prevent BRCA1-mutated breast cancer.

A genome-wide RNAi screen identifies novel targets of neratinib resistance leading to identification of potential drug resistant genetic markers

Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.

P2-06-03: Differential Expression of Breast Cancer-Associated Genes between Stage- and Age-Matched Tumor Specimens from African- and Caucasian-American Women Diagnosed with Breast Cancer.

Abstract Background: Recent studies suggest that the poorer outcome of breast cancer patients observed in African-American women (AAW) may, in part, result from underlying genetic factors. The purpose of this study was to investigate gene expression differences between Caucasian-American women (CAW) and AAW that may contribute to this poorer prognosis. Methods: The expression of genes involved in breast carcinoma prognosis, response to therapy, estrogen signaling, and tumor aggressiveness was assessed in age- and stage-matched CAW and AAW paraffin-embedded breast cancer specimens. The Wilcoxon-Mann-Whitney Test was used to identify genes with a significant difference in expression between CAW and AAW. To determine if the differentially expressed genes could segregate between the CAW and AAW, we performed semi-supervised principle component analysis (SSPCA). Results: Twenty genes were differentially expressed between AAW and CAW. SSPCA incorporating these 20 genes separated AAW and CAW into two distinct groups. AAW were significantly (p &amp;lt; 0.05) more likely to display aberrations in G1/S cell-cycle regulatory genes, decreased expression of cell-adhesion genes, and low to no expression of ESR1, PGR, ERBB2 and estrogen pathway targets. Conclusions: The gene expression differences identified between AAW and CAW may contribute to more aggressive disease, resistance to therapy, enhanced metastatic potential and poor clinical outcome. Impact: These findings support the hypothesis that breast cancer specimens collected from AAW display distinct genetic differences compared to similar tissues obtained from CAW. Additional population-based studies are necessary to determine if these genetic variations contribute to the highly aggressive and treatment-resistant breast cancer phenotype frequently observed in AAW. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-06-03.

Abstract CN04-01: New drugs for the chemoprevention of experimental breast cancer

Abstract Tamoxifen, raloxifene and exemestane are all effective for prevention of estrogen-receptor positive (ER+) breast cancer in women. Despite these significant advances, novel drugs are needed for the chemoprevention of estrogen-receptor negative (ER−) breast cancer, especially for women with hereditary breast cancers who frequently harbor BRCA (breast cancer-associated gene) mutations. Currently, “watchful waiting” or bilateral prophylactic mastectomy is offered to these patients, although these high risk women are ideal candidates for an effective, safe chemopreventive regimen. Two relevant mouse models were used to study drugs that are used to treat human breast cancer or drugs with known chemopreventive activity in other animal models. When conditional knockout (co) of BRCA1 is coupled with a mutation in p53, Brca1Co/Co;MMTV-Cre;p53+/− mice develop ER− mammary tumors within 6–10 months of age. In the PyMT mouse model, the polyomamiddle T oncoprotein drives carcinogenesis by inducing significant infiltration of tumor-associated macrophages (TAM). The efficacy of synthetic triterpenoids, rexinoids, histone deacetylase inhibitors, and PARP (Poly ADP ribose polymerase) inhibitors as well as potential biomarkers and mechanisms for these drugs will be discussed. Supported by the Breast Cancer Research Foundation and Komen for the Cure Citation Information: Cancer Prev Res 2011;4(10 Suppl):CN04-01.

Yin Yang 1 positively regulates BRCA1 and inhibits mammary cancer formation

Expression of the breast cancer-associated gene 1 (BRCA1) in sporadic breast cancers is usually reduced, yet the underlying mechanisms remains elusive. To identify factors that are responsible for reduced BRCA1 expression, we screened 92 known transcription factors for their ability to regulate expression of BRCA1. Among several potential regulators, the Gli-Krueppel-related transcription factor Yin Yang 1 (YY1) showed the most dramatic transactivation of the BRCA1 promoter. YY1 binds to the promoter of BRCA1, and its overexpression resulted in increased expression of BRCA1 and a number of BRCA1 downstream genes. We further showed that overexpression of YY1 in cancer cells inhibited cell proliferation, foci formation and tumor growth in nude mice. To assess the clinical relevance between YY1 and BRCA1, we studied expression of YY1 and BRCA1 from human breast cancer samples and tissue arrays, and detected a significant positive correlation between the level of YY1 and BRCA1 expression in these cancers. Taken together, these findings suggest that YY1 is a key regulator of BRCA1 expression and may be causally linked to the molecular etiology of human breast cancer.

Abstract 4035: The breast cancer associated gene 2 (BCA2) binds to Ubc9 and is involved in sumoylation

Abstract SUMOylation is a common post-translational modification in which small ubiquitin-like modifier (SUMO) protein moiety is attached to a lysine residue of the target protein and alters its stability, protein-protein interactions, protein-DNA interactions and subcellular localization. Recently, there is an increased recognition for this process as SUMO conjugation plays a critical role in basic cellular processes like proliferation, intracellular signaling and stabilization of transcriptional factors and chromatin remodeling factors. We are studying the Breast Cancer Associated Gene 2 (BCA2), a well characterized ubiquitin E3 ligase which we discovered by subtractive hybridization cloning. In order to identify substrates for the E3 ligase, we performed bacterial two-hybrid screening and found that the SUMO conjugating enzyme Ubc9 as a binding partner. In this study, we examined, whether the BCA2 ligase might be involved in sumoylation.Bioinformatic analyses of BCA2 sequences by SUMOplotTM prediction revealed Lysine 32 as the possible SUMO modification site. To establish direct protein-protein interactions, we performed pull down assays using GST- tagged Ubc9 expressed by the pGEX4T3 expression vector and purified recombinant wild-type BCA2. Results were analyzed by SDS-PAGE Western blotting. To assess if BCA2 and Ubc9 co-localize, we transfected Hek293T cells with BCA2 and Ubc9 constructs and analyzed them using Immunoflorescence staining. We found that BCA2 interacts with Ubc9 and they are co-localized in both the cytoplasm and nucleus. To investigate whether BCA2 is sumoylated by SUMO 1 or SUMO 2/3 we performed in vitro sumoylation assays. SUMO E1 and E2 were incubated along with recombinant BCA2, SUMO1 or SUMO 2/3 respectively for 2 hours at 37°C. The Western blots were probed with SUMO1, SUMO 2/3 and BCA2 antibodies. We also performed in vivo sumoylation with exogenous BCA2 by transfecting pCMV BCA2-Flag vector into Hek293T cells by using Fugene transfection reagent and after 48 hours of transfection, cells were lysed with denaturing lysis buffer and pull downs were performed using anti Flag beads followed by Western blots. These in vitro and in vivo sumoylation studies reveal that BCA2 is sumoylated by SUMO 2/3, but not SUMO1. In the recent past it has become more clear that both the ubiquitination and sumoylation systems communicate and jointly affect the properties of common substrate proteins and there is an increasing evidence in breast cancer development that deregulation of these processes play a vital role. Our findings of a possible involvement of BCA2 in both of these processes opens up avenues that should be further exploited in order to understand the regulatory role of BCA2 in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4035. doi:10.1158/1538-7445.AM2011-4035

Abstract 3341: TGFβ/ TNFα-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype

Abstract Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2011-3341

Hereditary Breast Cancer and the BRCA1-Associated FANCJ/BACH1/BRIP1

It is clear that FANCJ, also known as BACH1 or BRIP1, is an essential tumor suppressor gene based on the identification of clinically relevant mutations not only in breast cancer, but also the childhood cancer syndrome, Fanconi anemia. This conclusion is further supported by the direct and functional interaction between FANCJ and the hereditary breast cancer-associated gene product BRCA1. In the absence of the FANCJ DNA helicase or its interaction with BRCA1, cells have defects in several aspects of the DNA damage response. In particular, the BRCA1-FANCJ interaction is essential for promoting error-free repair, checkpoint control and for limiting DNA damage tolerance. As the number of FANCJ clinical mutations and affected patients accumulate, it will be critical to understand whether the associated tumors resemble BRCA-associated tumors. If so, FANCJ patients could also benefit from new therapies that selectively sensitize DNA repair-defective tumors and spare healthy cells. In this article, we summarize the breast cancer-associated FANCJ mutations and discuss functional outcomes for DNA repair and tumor suppression.

High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer

Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.