A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate.

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse.

Study of intracellular anabolism of 5-fluorouracil and incorporation in nucleic acids based on an LC-HRMS method.

5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.

Uncovering the Translational Regulatory Activity of the Tumor Suppressor BRCA1.

BRCA1 inactivation is a hallmark of familial breast cancer, often associated with aggressive triple negative breast cancers. BRCA1 is a tumor suppressor with known functions in DNA repair, transcription regulation, cell cycle control, and apoptosis. In the present study, we demonstrate that BRCA1 is also a translational regulator. We previously showed that BRCA1 was implicated in translation regulation. Here, we asked whether translational control could be a novel function of BRCA1 that contributes to its tumor suppressive activity. A combination of RNA-binding protein immunoprecipitation, microarray analysis, and polysome profiling, was used to identify the mRNAs that were specifically deregulated under BRCA1 deficiency. Western blot analysis allowed us to confirm at the protein level the deregulated translation of a subset of mRNAs. A unique and dedicated cohort of patients with documented germ-line BRCA1 pathogenic variant statues was set up, and tissue microarrays with the biopsies of these patients were constructed and analyzed by immunohistochemistry for their content in each candidate protein. Here, we show that BRCA1 translationally regulates a subset of mRNAs with which it associates. These mRNAs code for proteins involved in major programs in cancer. Accordingly, the level of these key proteins is correlated with BRCA1 status in breast cancer cell lines and in patient breast tumors. ADAT2, one of these key proteins, is proposed as a predictive biomarker of efficacy of treatments recently recommended to patients with BRCA1 deficiency. This study proposes that translational control may represent a novel molecular mechanism with potential clinical impact through which BRCA1 is a tumor suppressor.

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.

Cell cycle-dependent conjugation of endogenous BRCA1 protein with SUMO-2/3.

BACKGROUND: BRCA1, the main breast and ovarian cancer susceptibility gene, has a key role in maintenance of genome stability, cell cycle and transcription regulation. Interestingly, some of the numerous proteins which interact with BRCA1 protein undergo conjugation with small ubiquitin-like modifiers (SUMO). This post-translational modification is related to transcription, DNA repair, nuclear transport, signal transduction, and to cell cycle stress response. METHODS AND RESULTS: Protein sequence analysis suggests that sumoylation target sites belong to the RING finger and BRCT domains (BRCA1 C-terminus), two crucial regions for BRCA1 function. Moreover putative SUMO interacting motifs are present in the sequence of many proteins of BRCA1 network. Using immunoprecipitations and western blotting, we show the conjugation of endogenous nuclear BRCA1 protein with SUMO-2/3. BRCA1 conjugation with SUMO-2/3 is linked to the cell cycle in a cell line dependent manner since no cell cycle dependence of sumoylation is observed in MCF7 breast cancer cells. In contrast, BRCA1 conjugation with SUMO-2/3 is linked to the oxidative stress independently to the cell line, in DU145, MCF7 and 293 T cells. CONCLUSION AND GENERAL SIGNIFICANCE: Our data reveal a new BRCA1 regulation pathway implying sumoylation in response to cell cycle progression and oxidative stress, providing a possible mechanism for the involvement of BRCA1 gene in tumorigenesis.

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

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 alpha (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.

Acetyl-CoA carboxylase alpha gene and breast cancer susceptibility.

The identification of an interaction between BRCA1 and acetyl-CoA carboxylase alpha (ACCalpha), a key enzyme in lipid synthesis, led us to investigate the role of ACCalpha in breast cancer development, where it might contribute to the energy-sensing mechanisms of malignant transformation. In order to investigate if certain ACCalpha alleles may be high-risk breast cancer susceptibility alleles, 37 extended breast and breast/ovarian cancer families negative for BRCA1 and BRCA2 mutations were exhaustively screened for sequence variations in the entire coding sequence, intron-exon junctions, 5'UTR, 3'UTR (untranslated regions) and the promoter regions of the ACCalpha gene. Two possibly disease-associated ACCalpha variants were each identified in a single family and were not present in 137 controls. Multiple polymorphisms were detected in breast cancer families, including 12 single nucleotide polymorphisms where the frequency of the rare allele estimated in controls was >0.10. The observed lack of variation in the ACCalpha coding region along with the presence of extended areas of linkage disequilibrium and low haplotype diversity indicates an overall high preservation of this gene. The prevalence of the ACCalpha haplotypes composed of common polymorphisms was determined in 453 breast cancer cases and 469 female controls. One haplotype was found to be associated with a substantial and highly significant increase in breast cancer risk (odds ratio = 3.10, 95% confidence interval 1.87-5.14, P < 0.0001), whereas three other haplotypes were found to have a protective effect. Our results indicate that mutations in the ACCalpha gene are unlikely to be a major cause of high-risk breast cancer susceptibility; however, certain common ACCalpha alleles may influence breast cancer risk. This study provides the first insight into the involvement of the ACCalpha gene in breast cancer predisposition and calls for further, large-scale studies that will be needed to understand the role of ACCalpha in tumour susceptibility and development.

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 alpha (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.

Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation.

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.