Nicole Dalla Venezia

BRCA1 interacts with acetyl-CoA carboxylase through its tandem of BRCT domains.

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 is a 220-kDa protein that contains a tandem of two BRCA1 C-Terminal (BRCT) domains. Among missense and nonsense BRCA1 mutations responsible for family breast cancer, some are located into the BRCT tandem of BRCA1 coding sequence. In an attempt to understand how BRCT is critical for BRCA1 function, we search for partners of this BRCT tandem of BRCA1. Using a glutathione-S-transferase (GST) pull-down assay with murine cells, we isolated only one major BRCA1-interacting protein, further identified as Acetyl Coenzyme A (CoA) Carboxylase α (ACCA). We showed that this interaction is conserved through murine and human species. We also delineated the minimum interacting region as being the whole tandem of BRCT domains. We demonstrated that BRCA1 interacts in vitro and in vivo with ACCA. This interaction is completely abolished by five distinct germline BRCA1 deleterious mutations affecting the BRCT tandem of BRCA1. Interestingly, ACCA originally known as a rate-limiting enzyme for fatty acids biosynthesis, has been recently shown to be over-expressed in breast cancers and considered as a potential target for anti-neoplastic therapy. Furthermore, our observation is making a bridge between the genetic factors involved in susceptibility to breast and ovarian cancers, and environmental factors such as nutrition considered as key elements in the etiology of those cancers.

BRCA1 Affects Lipid Synthesis through Its Interaction with Acetyl-CoA Carboxylase

Germ line alterations in BRCA1 (breast cancer susceptibility gene 1) are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 acts as a scaffold protein implicated in multiple cellular functions, such as transcription, DNA repair, and ubiquitination. However, the molecular mechanisms responsible for tumorigenesis are not yet fully understood. We have recently demonstrated that BRCA1 interacts in vivo with acetyl coenzyme A carboxylase α (ACCA) through its tandem of BRCA1 C terminus (BRCT) domains. To understand the biological function of the BRCA1·ACCA complex, we sought to determine whether BRCA1 is a regulator of lipogenesis through its interaction with ACCA. We showed here that RNA inhibition-mediated down-regulation of BRCA1 expression induced a marked increase in the fatty acid synthesis. We then delineated the biochemical characteristics of the complex and found that BRCA1 interacts solely with the phosphorylated and inactive form of ACCA (P-ACCA). Finally, we demonstrated that BRCA1 affects lipid synthesis by preventing P-ACCA dephosphorylation. These results suggest that BRCA1 affects lipogenesis through binding to P-ACCA, providing a new mechanism by which BRCA1 may exert a tumor suppressor function.

BRCA1 expression during prenatal development of the human mammary gland.

Germ-line alterations of BRCA1 are associated with elevated risk of breast cancer. Evidence for the involvement of Brca1 in cellular differentiation and morphogenesis has been obtained in mouse models during embryogenesis. Although the presence of well-conserved functional domains might suggest a similar function for both human and mouse genes, very few data on BRCA1 expression in human fetal tissues are available. We have, therefore, investigated the expression of BRCA1 in the mammary gland from human female fetuses aged between 15 and 33 weeks. Quantification of BRCA1 transcripts, using a competitive reverse transcriptase PCR method, indicates a progressive decrease in BRCA1 expression with increasing fetal age between the 15th and 30th week of gestation. Subsequently, the amount of BRCA1 transcripts becomes similar to that found in adult mammary gland. Analysis of BRCA1 protein revealed, in fetal samples, a 220u2009kDa band corresponding to the 220u2009kDa BRCA1 protein described in human cell lines. These later experiments confirm that the relative level of the 220u2009kDa BRCA1 protein is highest in the early stages of mammary gland development. The temporal patterns of BRCA1 expression in human fetuses suggest a role for BRCA1 in the morphogenesis and differentiation of the human mammary gland.

BRCA1 Interacts with Poly(A)-binding Protein IMPLICATION OF BRCA1 IN TRANSLATION REGULATION

BRCA1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair, cell cycle control, and apoptosis. We identified poly(A)-binding protein 1 (PABP) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by in vitro assays and coimmunoprecipitation in mammalian cells. Endogenous interaction between BRCA1 and PABP was also observed. This interaction was abolished by BRCA1 cancer-associated mutations, suggesting that it may be physiologically relevant. Deletion mapping demonstrated that the RNA recognition motifs 1–4 region of PABP is required to mediate the interaction with BRCA1. To understand the biological function of the BRCA1-PABP complex, we sought to determine whether BRCA1 is a modulator of translation. We showed here that inhibition of endogenous BRCA1 using a small interfering RNA-based approach decreased protein synthesis. Conversely, overexpression of BRCA1 activated translation. Using a RNA transfection approach, we clearly showed that BRCA1 modulates translation, independently of any transcriptional activity. The data presented here suggest that BRCA1 modulates protein synthesis via its interaction with PABP, providing a novel mechanism by which BRCA1 may exert its tumor suppressor function.

BRCA1-Dependent Translational Regulation in Breast Cancer Cells.

BRCA1 (Breast Cancer 1) has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair and protein ubiquitination. We previously demonstrated that BRCA1 interacts with PABP1 (Poly(A)-Binding Protein 1) and that BRCA1 modulates protein synthesis through this interaction. To identify the mRNAs that are translationally regulated by BRCA1, we used a microarray analysis of polysome-bound mRNAs in BRCA1-depleted and non-depleted MCF7 cells. Our findings show that BRCA1 modifies the translational efficiency of approximately 7% of the mRNAs expressed in these cells. Further analysis revealed that several processes contributing to cell surveillance such as cell cycle arrest, cell death, cellular growth and proliferation, DNA repair and gene expression, are largely enriched for the mRNAs whose translation is impacted by BRCA1. The BRCA1-dependent translation of these species of mRNAs therefore uncovers a novel mechanism through which BRCA1 exerts its onco-suppressive role. In addition, the BRCA1-dependent translation of mRNAs participating in unexpected functions such as cellular movement, nucleic acid metabolism or protein trafficking is indicative of novel functions for BRCA1. Finally, this study contributes to the identification of several markers associated with BRCA1 deficiency and to the discovery of new potential anti-neoplastic therapeutic targets.

Differential expression and subcellular localization of murine BRCA1 and BRCA1-δ11 isoforms in murine and human cell lines

BRCA1 mutations are involved in breast and ovarian cancer predisposition in humans. The biological functions of the murine BRCA1 gene have been extensively studied but little is known about murine BRCA1 proteins. To better characterize these proteins, we have cloned the full‐length murine BRCA1 cDNA and a splice variant deleted of exon 11, BRCA1‐Δ11, by RT‐PCR method. Three polyclonal antibodies raised against various parts of murine BRCA1 were used in our study: D16, M20 and 5MO, which were generated in our laboratory. This allowed us to analyze the expression and subcellular localization of both isoforms in murine and human cell lines by immunoblotting, immunoprecipitation, cell fractionation and immunofluorescence. Endogenous BRCA1 was detected in murine cell lines but not splice variant BRCA1‐Δ11, whereas both ectopically expressed murine isoforms were detected in transfected human Bosc 23 cells. Subcellular fractionation and immunofluorescence results showed that the BRCA1 protein was mainly located in the nucleus, whereas BRCA1‐Δ11 was preferentially cytoplasmic. The conservation of exon 11 splicing and the differential subcellular localization of BRCA1 and BRCA1‐Δ11 in human and mouse suggest that these proteins could play distinct roles and that they could differentially act in the pathological mechanisms leading to the development of breast and ovarian cancer. The characterization of the murine BRCA1 proteins and antibodies will be useful to further study BRCA1 functions in murine models. Int. J. Cancer 88:519–524, 2000.

Caspase-dependent BRCA1 cleavage facilitates chemotherapy-induced apoptosis

BRCA1 acts as a tumor suppressor gene, and germ-line mutations in this gene are found in a large proportion of families with breast and ovarian cancers. The BRCA1 protein has been implicated in several cellular processes, such as transcription regulation, DNA responses to DNA damage signals, cell cycle control, and apoptosis. Apoptosis plays a critical role in radiation- and chemotherapy-induced cytotoxicity, and its impairment contributes to resistance to tumor treatments. In an attempt to elucidate the role of BRCA1 in apoptosis, we examined the response to chemotherapeutic drugs of cells expressing physiological levels of BRCA1 protein. We showed that chemotherapy-induced apoptosis leads to a caspase-mediated cleavage of BRCA1. We then showed that the BRCA1-p90 cleavage product is mainly localized in the cytoplasm. Finally, we demonstrated that cancer-associated mutations affecting the BRCT tandem repeat abolish its pro-apoptotic function. The data presented here provide new insight into the role of endogenous BRCA1 as a mediator of apoptosis and show that BRCA1 functions as a molecular determinant of response to a range of cytotoxic chemotherapeutic agents.

Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism

5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes. However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA. Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.

Low glucose microenvironment of normal kidney cells stabilizes a subset of messengers involved in angiogenesis.

As glucose is a mandatory nutrient for cell proliferation and renewal, it is suspected that glucose microenvironment is sensed by all cell types to regulate angiogenesis. Several glucose‐sensing components have been partially described to respond to high glucose levels. However, little is known about the response to low glucose. Here, we used well‐differentiated isolated normal rat renal tubules under normal oxygenation conditions to assess the angiogenic response to low glucose. In apparent paradox, but confirming observations made separately in other models, high glucose but also low glucose increased mRNA level of vascular endothelial growth factor A (VEGFA). A subset of mRNAs including hypoxia‐inducible factor 1A (HIF1A), angiopoietin receptor (TIE‐2), and VEGF receptor 2 (FLK1) were similarly glucose‐sensitive and responded to low glucose by increased stability independently of HIF1A and HIF2A proteins. These results contribute to gain some insights as to how normal cells response to low glucose may play a role in the tumor microenvironment.