Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including ␥-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of ␥-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate ␥-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of ␥-tubulin in vitro, and expression in cells of ␥-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.Cancer cells frequently have unstable numbers of chromosomes (reviewed in reference 20). One mechanism for chromosomal instability is improper centrosome duplication, since the centrosome is the organelle that organizes the spindle for separation of chromosomes during mitosis. The presence of more than two centrosomes in a cell can result in lost or fragmented chromosomes after cell division. Human tumors derived from breast and other tissues have abnormal centrosome numbers in early-stage lesions. As an example, abnormal centrosome numbers have been detected in ductal carcinoma in situ, the first stage of breast cancer (21, 33), and BRCA1 has been shown to have a role in regulating centrosome number (reviewed in reference 9).BRCA1 is a tumor suppressor that is mutated in inherited breast and ovarian cancer cases, and it is also epigenetically down-regulated in sporadic breast cancers. Strikingly, BRCA1 function is required for nearly all cell types to grow; it has many roles in the cell. These functions include the regulation of DNA damage repair, transcription, and X-chromosome inactivation (reviewed in references 37 and 41). All of these processes could be important in protecting mammary cells from uncontrolled growth, but it is not clear why loss of BRCA1 specifically results in breast and ovarian cancer.There is growing evidence that BRCA1 functions as a regulator of centrosome number. First, BRCA1 is localized to the centrosome in mitotic cells (17,23). Second, interference with BRCA1 function by various methods can cause an increased centrosome number. For example, mouse fibroblasts derived from BRCA1 exon 11 knockouts have ...

In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of ␥-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of ␥-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of ␥-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.BRCA1 is a breast-and ovary-specific tumor suppressor, and mutations in this gene have been found in approximately 40% of familial breast cancer cases and most of combined familial breast and ovarian cancers (1,8,43). BRCA1 is a large phosphoprotein consisting of 1,863 amino acids in humans, with a number of domains that interact directly or indirectly with many proteins with diverse functions such as transcription control, cell cycle regulation, chromatin remodeling, and DNA repair (30,40). BRCA1 has a RING domain at its amino terminus, and in association with BARD1, the heterodimer is an E3 ubiquitin ligase (16,46). Identifying the critical role for the BRCA1-dependent ubiquitin ligase activity in breast cell biology has been a major focus of research. In this study, we find that the BRCA1-associated E3 ubiquitin ligase directly regulates centrosome function.Centrosomes are the major microtubule (MT)-organizing centers of animal cells. Centrosomes control the number, polarity, and distribution of MTs, which are important in regulating cell polarity, shape, motility, intracellular transport, and cell division (13). In a normal cell, centrosomes start duplicating at early-S phase, and by M phase the cell has two mature centrosomes that form the bipolar spindle and ensure proper segregation of chromosomes to the two daughter cells. Currently more than 150 proteins have been shown to localize to centrosomes (3).The cells in many tumor types, including breast cancer, display numerical and structural centrosome aberrations, which have been collectively termed centrosomal hypertrophy. Structural abnormalities include increased centrosomal volu...

In India, socioeconomic inequality limiting access to treatment is a major factor towards increased cancer burden; therefore, incorporation of a cost-effective and comprehensive multi-gene test will be helpful in ensuring widespread implementation of genetic screening in the clinical practice for hereditary breast and/or ovarian cancers.

Breast and/or ovarian cancer (BOC) are among the most frequently diagnosed forms of hereditary cancers and leading cause of death in India. This emphasizes on the need for a cost-effective method for early detection of these cancers. We sequenced 141 unrelated patients and families with BOC using the TruSight Cancer panel, which includes 13 genes strongly associated with risk of inherited BOC. Multi-gene sequencing was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect pathogenic mutations in 51 (36.2%) cases, out of which 19 were novel mutations. When we considered familial breast cancer cases only, the detection rate increased to 52%. When cases were stratified based on age of diagnosis into three categories, ⩽40 years, 40-50 years and >50 years, the detection rates were higher in the first two categories (44.4% and 53.4%, respectively) as compared with the third category, in which it was 26.9%. Our study suggests that next-generation sequencing-based multi-gene panels increase the sensitivity of mutation detection and help in identifying patients with a high risk of developing cancer as compared with sequential tests of individual genes.

The breast and ovarian cancer specific tumor suppressor BRCA1, bound to BARD1, has multiple functions aimed at maintaining genomic stability in the cell. We have shown earlier that the BRCA1/BARD1 E3 ubiquitin ligase activity regulates centrosome-dependent microtubule nucleation. In this study, we tested which domains of BRCA1 and BARD1 were required to control the centrosome function. In the present study, (a) we confirmed that the ubiquitination activity of BRCA1 regulates centrosome number and function in Hs578T breast cancer cells; (b) we observed that both the amino and carboxyl termini of BRCA1 are required for regulation of centrosome function in vitro; (c) an internal domain (770-1,290) is dispensable for centrosome regulation; (d) BARD1 is required for regulation of centrosome function and protein sequences within the terminal 485 amino acids are necessary for activity; and (e) BARD1 is localized at the centrosome throughout the cell cycle. We conclude that the BRCA1-dependent E3 ubiquitin ligase functions to restrain centrosomes in mammary cells, and loss of BRCA1 in the precancerous breast cell leads to centrosomal hypertrophy, a phenotype commonly observed in incipient breast cancer.