Evaluating homologous recombination activity in tissues to predict the risk of hereditary breast and ovarian cancer and olaparib sensitivity.

Homologous recombination (HR) repairs DNA damage including DNA double-stranded breaks and alterations in HR-related genes results in HR deficiency. Germline alteration of HR-related genes, such as BRCA1 and BRCA2, causes hereditary breast and ovarian cancer (HBOC). Cancer cells with HR deficiency are sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors and DNA-damaging agents. Thus, accurately evaluating HR activity is useful for diagnosing HBOC and predicting the therapeutic effects of anti-cancer agents. Previously, we developed an assay for site-specific HR activity (ASHRA) that can quantitatively evaluate HR activity and detect moderate HR deficiency. HR activity in cells measured by ASHRA correlates with sensitivity to the PARP inhibitor, olaparib. In this study, we applied ASHRA to lymphoblastoid cells and xenograft tumor tissues, which simulate peripheral blood lymphocytes and tumor tissues, respectively, as clinically available samples. We showed that ASHRA could be used to detect HR deficiency in lymphoblastoid cells derived from a BRCA1 pathogenic variant carrier. Furthermore, ASHRA could quantitatively measure the HR activity in xenograft tumor tissues with HR activity that was gradually suppressed by inducible BRCA1 knockdown. The HR activity of xenograft tumor tissues quantitatively correlated with the effect of olaparib. Our data suggest that ASHRA could be a useful assay for diagnosing HBOC and predicting the efficacy of PARP inhibitors.

The centrosome: a critical hub for cell cycle regulation.

Cyclins and cyclin-dependent kinases (CDKs) localize to the centrosome, but their significance in the cell cycle is unclear. Recently, Roberts et al. revealed that centrosomal cyclin B-CDK is required for mitotic entry and phosphorylation of substrates. This suggests that the centrosome acts as a signaling hub controlling the cell cycle.

Protocol to detect OLA1 polyubiquitination by Aurora A in vivo and in vitro.

Aurora A is a critical kinase that functions in centrosome maturation and bipolar spindle assembly. On the other hand, Aurora A has E3 ubiquitin ligase activity and polyubiquitinates Breast cancer gene 1 (BRCA1)-interacting protein Obg-like ATPase 1 (OLA1), targeting it for proteasomal degradation. Here, we present a protocol to detect OLA1 ubiquitination. We describe steps for recovering frozen cells and protein purification. We then detail assays for both in vivo and in vitro ubiquitination of OLA1 by Aurora A. For complete details on the use and execution of this protocol, please refer to Fang et al.1.

Knockout of Brca1-interacting factor Ola1 in female mice induces tumors with estrogen suppressible centrosome amplification.

Obg-like ATPase 1 (OLA1) is a binding protein of Breast cancer gene 1 (BRCA1), germline pathogenic variants of which cause hereditary breast cancer. Cancer-associated variants of BRCA1 and OLA1 are deficient in the regulation of centrosome number. Although OLA1 might function as a tumor suppressor, the relevance of OLA1 deficiency to carcinogenesis is unclear. Here, we generated Ola1 knockout mice. Aged female Ola1+/- mice developed lymphoproliferative diseases, including malignant lymphoma. The lymphoma tissues had low expression of Ola1 and an increase in the number of cells with centrosome amplification. Interestingly, the proportion of cells with centrosome amplification in normal spleen from Ola1+/- mice was higher in male mice than in female mice. In human cells, estrogen stimulation attenuated centrosome amplification induced by OLA1 knockdown. Previous reports indicate that prominent centrosome amplification causes cell death but does not promote tumorigenesis. Thus, in the current study, the mild centrosome amplification observed under estrogen stimulation in Ola1+/- female mice is likely more tumorigenic than the prominent centrosome amplification observed in Ola1+/- male mice. Our findings provide a possible sex-dependent mechanism of the tumor suppressor function of OLA1.

Bevacizumab increases the sensitivity of olaparib to homologous recombination-proficient ovarian cancer by suppressing CRY1 via PI3K/AKT pathway.

PARP inhibitors have changed the management of advanced high-grade epithelial ovarian cancer (EOC), especially homologous recombinant (HR)-deficient advanced high-grade EOC. However, the effect of PARP inhibitors on HR-proficient (HRP) EOC is limited. Thus, new therapeutic strategy for HRP EOC is desired. In recent clinical study, the combination of PARP inhibitors with anti-angiogenic agents improved therapeutic efficacy, even in HRP cases. These data suggested that anti-angiogenic agents might potentiate the response to PARP inhibitors in EOC cells. Here, we demonstrated that anti-angiogenic agents, bevacizumab and cediranib, increased the sensitivity of olaparib in HRP EOC cells by suppressing HR activity. Most of the γ-H2AX foci were co-localized with RAD51 foci in control cells. However, most of the RAD51 were decreased in the bevacizumab-treated cells. RNA sequencing showed that bevacizumab decreased the expression of CRY1 under DNA damage stress. CRY1 is one of the transcriptional coregulators associated with circadian rhythm and has recently been reported to regulate the expression of genes required for HR in cancer cells. We found that the anti-angiogenic agents suppressed the increase of CRY1 expression by inhibiting VEGF/VEGFR/PI3K pathway. The suppression of CRY1 expression resulted in decrease of HR activity. In addition, CRY1 inhibition also sensitized EOC cells to olaparib. These data suggested that anti-angiogenic agents and CRY1 inhibitors will be the promising candidate in the combination therapy with PARP inhibitors in HR-proficient EOC.

Functional evaluation of BRCA1/2 variants of unknown significance with homologous recombination assay and integrative in silico prediction model.

Numerous variants of unknown significance (VUSs) exist in hereditary breast and ovarian cancers. Although multiple methods have been developed to assess the significance of BRCA1/2 variants, functional discrepancies among these approaches remain. Therefore, a comprehensive functional evaluation system for these variants should be established. We performed conventional homologous recombination (HR) assays for 50 BRCA1 and 108 BRCA2 VUSs and complementarily predicted VUSs using a statistical logistic regression prediction model that integrated six in silico functional prediction tools. BRCA1/2 VUSs were classified according to the results of the integrative in vitro and in silico analyses. Using HR assays, we identified 10 BRCA1 and 4 BRCA2 VUSs as low-functional pathogenic variants. For in silico prediction, the statistical prediction model showed high accuracy for both BRCA1 and BRCA2 compared with each in silico prediction tool individually and predicted nine BRCA1 and seven BRCA2 variants to be pathogenic. Integrative functional evaluation in this study and the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines strongly suggested that seven BRCA1 variants (p.Glu272Gly, p.Lys1095Glu, p.Val1653Leu, p.Thr1681Pro, p.Phe1761Val, p.Thr1773Ile, and p.Gly1803Ser) and four BRCA2 variants (p.Trp31Gly, p.Ser2616Phe, p.Tyr2660Cys, and p.Leu2792Arg) were pathogenic. This study demonstrates that integrative evaluation using conventional HR assays and optimized in silico prediction comprehensively classified the significance of BRCA VUSs for future clinical applications.

Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation.

The BRCA1-interacting protein Obg-like ATPase 1 (OLA1) functions in centriole duplication. In this study, we show the role of the mitotic kinase Aurora A in the reduction of centrosomal OLA1. Aurora A binds to and polyubiquitinates OLA1, targeting it for proteasomal degradation. NIMA-related kinase 2 (NEK2) phosphorylates the T124 residue of OLA1, increases binding of OLA1 to Aurora A and OLA1 polyubiquitination by Aurora A, and reduces centrosomal OLA1 in G2 phase. The kinase activity of Aurora A suppresses OLA1 polyubiquitination. The decrease in centrosomal OLA1 caused by Aurora A-mediated polyubiquitination promotes the recruitment of pericentriolar material proteins in G2 phase. The E3 ligase activity of Aurora A is critical for centrosome amplification induced by its overexpression. The results suggest a dual function of Aurora A as an E3 ubiquitin ligase and a kinase in the regulation of centrosomal OLA1, which is essential for proper centrosome maturation in G2 phase.

Phase II study of biweekly cetuximab plus mFOLFOX6 or mFOLFIRI as second-line treatment for metastatic colorectal cancer and exploratory analysis of associations between DNA methylation status and the efficacy of the anti-EGFR antibody: T-CORE1201.

Background: Little is known about the biweekly combined use of cetuximab and chemotherapy as second-line treatment of metastatic colorectal cancer (mCRC). Recently, DNA methylation status has been reported to be a new possible predictor of the efficacy from the anti-epidermal growth factor receptor (EGFR) antibody treatment. The purpose of this study was to examine the efficacy and safety of biweekly cetuximab plus mFOLFOX6 or mFOLFIRI as a second-line treatment for KRAS exon 2 wild-type mCRC. We also investigated the predictability of DNA methylation status on the efficacy of the EGFR antibody-containing treatment. Methods: Patients who were refractory or intolerant to the first-line chemotherapy were enrolled and received biweekly cetuximab plus mFOLFOX6 or mFOLFIRI. The primary endpoint was progression-free survival (PFS). Tumor evaluations were performed every 2 months using Response Evaluation Criteria in Solid Tumor (RECIST) version 1.1. Adverse events (AEs) were evaluated according to the Common Terminology Criteria for Adverse Events version 4.0. DNA methylation status of colorectal cancer cells was defined by a modified MethyLight assay. Results: Sixty-six cases were enrolled. The median PFS (mPFS) was 5.1 [95% confidence interval (CI), 3.8-7.6] months. The median overall survival (mOS) was 12.7 (95% CI, 7.5-15.3) months. Grade 3 or higher neutropenia occurred in 53.0% of patients, whereas skin disorders with a grade 3 or higher occurred in <15% of patients. In multivariate analysis, DNA methylation status could not be an independent predictor of PFS [hazard ratio (HR), 1.43; P=0.39] and OS (HR, 2.13; P=0.086). However, in RAS/BRAF wild-type patients, the mPFS and mOS in the low-methylated colorectal cancer (LMCC) group was numerically better than those in the highly-methylated colorectal cancer (HMCC) group, although the difference was not statistically significant [mPFS: 8.5 (95% CI, 6.1-10.9) vs. 3.3 (95% CI, 1.2-not reached) months, P=0.79; ΔmPFS, 5.2 months; mOS: 15.3 (95% CI, 11.9-23.5) vs. 6.5 (95% CI, 3.1-not reached) months, P=0.53; ΔmOS, 8.8 months]. Conclusions: Biweekly cetuximab plus mFOLFOX6 or mFOLFIRI is a useful second-line therapy for mCRC. DNA methylation status warrants further exploration as a predictive biomarker for anti-EGFR efficacy in mCRC.

Paclitaxel sensitizes homologous recombination-proficient ovarian cancer cells to PARP inhibitor via the CDK1/BRCA1 pathway.

OBJECTIVE: An effective treatment strategy for epithelial ovarian cancer (EOC) with homologous recombination (HR)-proficient (HRP) phenotype has not been established, although poly (ADP-ribose) polymerase inhibitors (PARPi) impact the disease course with HR-deficient (HRD) phenotype. Here, we aimed to clarify the cellular effects of paclitaxel (PTX) on the DNA damage response and the therapeutic application of PTX with PARPi in HRP ovarian cancer. METHODS: Two models with different PTX dosing schedules were established in HRP ovarian cancer OVISE cells. Growth inhibition and HR activity were analyzed in these models with or without PARPi. BRCA1 phosphorylation status was examined in OVISE cells by inhibiting CDK1, which was reduced by PTX treatment. CDK1 expression was evaluated in EOC patients treated with PTX-based neoadjuvant chemotherapy. RESULTS: PTX suppressed CDK1 expression resulting in impaired BRCA1 phosphorylation in OVISE cells. The reduced CDK1 activity by PTX could decrease HR activity in response to DNA damage and therefore increase the sensitivity to PARPi. Immunohistochemistry showed that CDK1 expression was attenuated in samples collected after PTX-based chemotherapy compared to those collected before chemotherapy. The decrease in CDK1 expression was greater with dose-dense PTX schedule than with the conventional PTX schedule. CONCULSIONS: PTX could act synergistically with PARPi in HRP ovarian cancer cells, suggesting that the combination of PTX with PARPi may be a novel treatment strategy extending the utility of PARPi to EOC. Our findings provide cules for future translational clinical trials evaluating the efficacy of PTX in combination with PARPi in HRP ovarian cancer.

BRCA1 transports the DNA damage signal for CDDP-induced centrosome amplification through the centrosomal Aurora A.

Breast cancer gene 1 (BRCA1) plays roles in DNA repair and centrosome regulation and is involved in DNA damage-induced centrosome amplification (DDICA). Here, the centrosomal localization of BRCA1 and the kinases involved in centrosome duplication were analyzed in each cell cycle phase after treatment with DNA crosslinker cisplatin (CDDP). CDDP treatment increased the centrosomal localization of BRCA1 in early S-G2 phase. BRCA1 contributed to the increased centrosomal localization of Aurora A in S phase and that of phosphorylated Polo-like kinase 1 (PLK1) in late S phase after CDDP treatment, resulting in centriole disengagement and overduplication. The increased centrosomal localization of BRCA1 and Aurora A induced by CDDP treatment involved the nuclear export of BRCA1 and BRCA1 phosphorylation by ataxia telangiectasia mutated (ATM). Patient-derived variants and mutations at phosphorylated residues of BRCA1 suppressed the interaction between BRCA1 and Aurora A, as well as the CDDP-induced increase in the centrosomal localization of BRCA1 and Aurora A. These results suggest that CDDP induces the phosphorylation of BRCA1 by ATM in the nucleus and its transport to the cytoplasm, thereby promoting the centrosomal localization Aurora A, which phosphorylates PLK1. The function of BRCA1 in the translocation of the DNA damage signal from the nucleus to the centrosome to induce centrosome amplification after CDDP treatment might support its role as a tumor suppressor.

Cisplatin Plus Capecitabine After Adjuvant S-1 in Metastatic Gastric Cancer: A Phase II T-CORE1102 Trial.

BACKGROUND/AIM: This phase II study assessed the efficacy of capecitabine plus cisplatin in patients with advanced gastric cancer refractory to adjuvant S-1. PATIENTS AND METHODS: This single-arm, open-label, multicenter, phase II study was conducted by Tohoku Clinical Oncology Research and Education Society (T-CORE) in Japan. Patients aged ≥20 years with advanced HER2-negative gastric cancer that was refractory to S-1 were enrolled. Patients received 80 mg/m2 cisplatin on day 1 intravenously and 1,000 mg/m2 capecitabine twice daily from day 1 to day 14, in 3-week cycles. The primary endpoint was progression-free survival (PFS). The threshold overall response rate (ORR) was estimated to be 15%. The secondary endpoints were overall survival (OS), time to treatment failure, ORR, and toxicities. RESULTS: In total, 21 patients were enrolled from seven hospitals. The median patient age was 63 years. Nineteen patients received the protocol treatment. Median PFS was 3.7 months [90% confidence interval (CI)=2.7-5.6 months], which did not reach the predefined threshold of 4.0 months. ORR was 5.9% (95%CI=0.0-17.1%). Median OS was 11.9 months (95% CI 6.3-19.4 months). Febrile neutropenia was observed in 5.3% of patients. The most frequently observed grade 3 non-hematologic toxicities were nausea (15.8%) and hyponatremia (15.8%). CONCLUSION: The addition of a fluoropyrimidine to a platinum agent after adjuvant therapy is not suitable for gastric cancer.

Roles of RACK1 in centrosome regulation and carcinogenesis.

Receptor for activated C kinase 1 (RACK1) regulates various cellular functions and signaling pathways by interacting with different proteins. Recently, we showed that RACK1 interacts with breast cancer gene 1 (BRCA1), which regulates centrosome duplication. RACK1 localizes to centrosomes and spindle poles and is involved in the proper centrosomal localization of BRCA1. The interaction between RACK1 and BRCA1 is critical for the regulation of centrosome number. In addition, RACK1 contributes to centriole duplication by regulating polo-like kinase 1 (PLK1) activity in S phase. RACK1 binds directly to PLK1 and Aurora A, promoting the phosphorylation of PLK1 and activating the Aurora A/PLK1 signaling axis. Overexpression of RACK1 causes centrosome amplification, especially in mammary gland epithelial cells, inducing overactivation of PLK1 followed by premature centriole disengagement and centriole re-duplication. Other proteins, including hypoxia-inducible factor α, von Hippel-Lindau protein, heat-shock protein 90, ß-catenin, and glycogen synthase kinase-3ß, interact with RACK1 and play roles in centrosome regulation. In this review, we focus on the roles and underlying molecular mechanisms of RACK1 in centrosome regulation mediated by its interaction with different proteins and the modulation of their functions.

Dysregulation of the centrosome induced by BRCA1 deficiency contributes to tissue-specific carcinogenesis.

Alterations in breast cancer gene 1 (BRCA1), a tumor suppressor gene, increase the risk of breast and ovarian cancers. BRCA1 forms a heterodimer with BRCA1-associated RING domain protein 1 (BARD1) and functions in multiple cellular processes, including DNA repair and centrosome regulation. BRCA1 acts as a tumor suppressor by promoting homologous recombination (HR) repair, and alterations in BRCA1 cause HR deficiency, not only in breast and ovarian tissues but also in other tissues. The molecular mechanisms underlying BRCA1 alteration-induced carcinogenesis remain unclear. Centrosomes are the major microtubule-organizing centers and function in bipolar spindle formation. The regulation of centrosome number is critical for chromosome segregation in mitosis, which maintains genomic stability. BRCA1/BARD1 function in centrosome regulation together with Obg-like ATPase (OLA1) and receptor for activating protein C kinase 1 (RACK1). Cancer-derived variants of BRCA1, BARD1, OLA1, and RACK1 do not interact, and aberrant expression of these proteins results in abnormal centrosome duplication in mammary-derived cells, and rarely in other cell types. RACK1 is involved in centriole duplication in the S phase by promoting polo-like kinase 1 activation by Aurora A, which is critical for centrosome duplication. Centriole number is higher in cells derived from mammary tissues compared with in those derived from other tissues, suggesting that tissue-specific centrosome characterization may shed light on the tissue specificity of BRCA1-associated carcinogenesis. Here, we explored the role of the BRCA1-containing complex in centrosome regulation and the effect of its deficiency on tissue-specific carcinogenesis.

BRCA1/ATF1-Mediated Transactivation is Involved in Resistance to PARP Inhibitors and Cisplatin.

Homologous recombination (HR)-deficient cells are sensitive to PARP inhibitors through a synthetic lethal effect. We previously developed an HR activity assay named Assay of Site-Specific HR Activity (ASHRA). Here, we evaluated the HR activity of 30 missense variants of BRCA1 by ASHRA and found that several BRCA1 variants showed intermediate HR activity, which was not clearly discerned by our previous analyses using a conventional method. HR activity measured by ASHRA was significantly correlated with sensitivity to olaparib. However, cells expressing the severely HR-deficient BRCA1-C61G variant were resistant to olaparib, and resistance was dependent on high expression of activating transcription factor 1 (ATF1), which binds to BRCA1 and activates the transcription of target genes to regulate cell proliferation. The BRCA1-C61G variant bound to ATF1 and stimulated ATF1-mediated transactivation similar to wild-type BRCA1. High expression of ATF1 conferred resistance to olaparib and cisplatin activating BRCA1/ATF1-mediated transcription without affecting HR activity in BRCA2-knockdown or RAD51-knockdown cells, but not in BRCA1-knockdown cells. These results suggest that ASHRA is a useful method to evaluate HR activity in cells and to predict the sensitivity to PARP inhibitors. The expression level of ATF1 might be an important biomarker of the effect of PARP inhibitors and platinum agents on HR-deficient tumors with the BRCA1-C61G variant or alteration of non-BRCA1 HR factors such as BRCA2 and RAD51. Significance: ASHRA could evaluate HR activity in cells and predict the sensitivity to PARP inhibitors. High expression level of ATF1 may predict the resistance of BRCAness tumors with alterations of non-BRCA1 HR factors to PARP inhibitors and platinum agents.

RACK1 regulates centriole duplication through promoting the activation of polo-like kinase 1 by Aurora A.

Breast cancer gene 1 (BRCA1) contributes to the regulation of centrosome number. We previously identified receptor for activated C kinase 1 (RACK1) as a BRCA1-interacting partner. RACK1, a scaffold protein that interacts with multiple proteins through its seven WD40 domains, directly binds to BRCA1 and localizes to centrosomes. RACK1 knockdown suppresses centriole duplication, whereas RACK1 overexpression causes centriole overduplication in a subset of mammary gland-derived cells. In this study, we showed that RACK1 binds directly to polo-like kinase 1 (PLK1) and Aurora A, and promotes the Aurora A-PLK1 interaction. RACK1 knockdown decreased phosphorylated PLK1 (p-PLK1) levels and the centrosomal localization of Aurora A and p-PLK1 in S phase, whereas RACK1 overexpression increased p-PLK1 level and the centrosomal localization of Aurora A and p-PLK1 in interphase, resulting in an increase of cells with abnormal centriole disengagement. Overexpression of cancer-derived RACK1 variants failed to enhance the Aurora A-PLK1 interaction, PLK1 phosphorylation and the centrosomal localization of p-PLK1. These results suggest that RACK1 functions as a scaffold protein that promotes the activation of PLK1 by Aurora A in order to promote centriole duplication.This article has an associated First Person interview with the first author of the paper.

The Function of BARD1 in Centrosome Regulation in Cooperation with BRCA1/OLA1/RACK1.

Breast cancer gene 1 (BRCA1)-associated RING domain protein 1 (BARD1) forms a heterodimer with BRCA1, a tumor suppressor associated with hereditary breast and ovarian cancer. BRCA1/BARD1 functions in multiple cellular processes including DNA repair and centrosome regulation. Centrosomes are the major microtubule-organizing centers in animal cells and are critical for the formation of a bipolar mitotic spindle. BRCA1 and BARD1 localize to the centrosome during the cell cycle, and the BRCA1/BARD1 dimer ubiquitinates centrosomal proteins to regulate centrosome function. We identified Obg-like ATPase 1 (OLA1) and receptor for activated C kinase (RACK1) as BRCA1/BARD1-interating proteins that bind to BARD1 and BRCA1 and localize the centrosomes during the cell cycle. Cancer-derived variants of BRCA1, BARD1, OLA1, and RACK1 failed to interact, and aberrant expression of these proteins caused centrosome amplification due to centriole overduplication only in mammary tissue-derived cells. In S-G2 phase, the number of centrioles was higher in mammary tissue-derived cells than in cells from other tissues, suggesting their involvement in tissue-specific carcinogenesis by BRCA1 and BARD1 germline mutations. We described the function of BARD1 in centrosome regulation in cooperation with BRCA1/OLA1/RACK1, as well as the effect of their dysfunction on carcinogenesis.

Relationship among DNA double-strand break (DSB), DSB repair, and transcription prevents genome instability and cancer.

DNA double-strand break (DSB) is a serious type of DNA damage and is known to trigger multiple responses within cells. In these responses, novel relationships among DSB, DSB repair, and transcription machineries are created. First, transcription is repressed if DSB occurs near or at the transcription site, termed DSB-induced transcriptional repression, which contributes to DSB repair with the aid of DNA damage-signaling pathways, ATM- or DNA-PKcs-signaling pathways. DSB-induced transcriptional repression is also regulated by transcriptional factors TLP1, NELF, and ENL, as well as chromatin remodeling and organizing factors ZMYND8, CDYL1, PBAF, and cohesin. Second, transcription and RNA promote DSB repair for genome integrity. Transcription factors such as LEDGF, SETD2, and transcriptionally active histone modification, H3K36, facilitate homologous recombination to overcome DSB. At transcriptional active sites, DNA:RNA hybrids, termed R-loops, which are formed by DSB, are processed by RAD52 and XPG leading to an activation of the homologous recombination pathway. Even in a transcriptionally inactive non-genic sites, noncoding RNAs that are produced by RNA polymerase II, DICER, and DROSHA, help to recruit DSB repair proteins at the DSB sites. Third, transcriptional activation itself, however, can induce DSB. Transcriptional activation often generates specific DNA structures such as R-loops and topoisomerase-induced DSBs, which cause genotoxic stress and may lead to genome instability and consequently to cancer. Thus, transcription and DSB repair machineries interact and cooperate to prevent genome instability and cancer.

Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing efficiency.

Homologous recombination (HR) contributes to the repair of DNA double-strand breaks (DSBs) and inter-strand crosslinks. The HR activity in cancer cells can be used to predict their sensitivity to DNA-damaging agents that cause these damages. To evaluate HR activity, we developed a system called Assay for Site-specific HR Activity (ASHRA), in which cells are transiently transfected with an expression vector for CRISPR/Cas9 and a HR donor sequence containing a marker gene. DSBs are created by Cas9 and then repaired by HR using donor vector sequences homologous to the target gene. The level of genomic integration of the marker gene is quantified by Western blotting, flowcytometry, or quantitative PCR (qPCR). ASHRA detected HR deficiency caused by BRCA1, BARD1, or RAD51 knockdown or introduction of BRCA1 variants. The influence of BRCA1 variants on HR, as determined by qPCR, was consistent with the chemosensitivities of the transfected cells. The qPCR format of ASHRA could measure HR activity in both transcribed and un-transcribed regions. Knockdown of BRCA1 nor BARD1 did not affect HR activity in a transcriptionally inactive site. ASHRA can evaluate HR activity and will be useful for predicting sensitivity to chemotherapy, screening drugs that affect HR, and investigating the mechanisms of HR.

RACK1 regulates centriole duplication by controlling localization of BRCA1 to the centrosome in mammary tissue-derived cells.

Breast cancer gene 1 (BRCA1) is a tumor suppressor that is associated with hereditary breast and ovarian cancer. BRCA1 functions in DNA repair and centrosome regulation together with BRCA1-associated RING domain protein (BARD1), a heterodimer partner of BRCA1. Obg-like ATPase 1 (OLA1) was identified as a protein that interacts with BARD1. OLA1 regulates the centrosome by binding to and collaborating with BRCA1 and BARD1. We identified receptor for activated C kinase (RACK1) as a protein that interacts with OLA1. RACK1 directly bound to OLA1, the N-terminal region of BRCA1, and γ-tubulin, associated with BARD1, and localized the centrosomes throughout the cell cycle. Knockdown of RACK1 caused abnormal centrosomal localization of BRCA1 and abrogated centriole duplication. Overexpression of RACK1 increased the centrosomal localization of BRCA1 and caused centrosome amplification due to centriole overduplication. The number of centrioles in cells with two γ-tubulin spots was higher in cell lines derived from mammary tissue compared to those derived from other tissues. The effects of aberrant RACK1 expression level on centriole duplication were observed in cell lines derived from mammary tissue, but not in those derived from other tissues. Two BRCA1 variants, R133H and E143K, and a RACK1 variant, K280E, associated with cancer, which weakened the BRCA1-RACK1 interaction, interfered with the centrosomal localization of BRCA1 and reduced centrosome amplification induced by overexpression of RACK1. These results suggest that RACK1 regulates centriole duplication by controlling the centrosomal localization of BRCA1 in mammary tissue-derived cells and that this is dependent on the BRCA1-RACK1 interaction.

Identification of KLF9 and BCL3 as transcription factors that enhance reprogramming of primordial germ cells.

Primordial germ cells (PGCs) are precursors of eggs and sperm. Although PGCs are unipotent cells in vivo, they are reprogrammed into pluripotent stem cells (PSCs), also known as embryonic germ cells (EGCs), in the presence of leukemia inhibitory factor and basic fibroblast growth factor (bFGF) in vitro. However, the molecular mechanisms responsible for their reprogramming are not fully understood. Here we show identification of transcription factors that mediate PGC reprogramming. We selected genes encoding transcription factors or epigenetic regulatory factors whose expression was significantly different between PGCs and PSCs with in silico analysis and RT-qPCR. Among the candidate genes, over-expression (OE) of Bcl3 or Klf9 significantly enhanced PGC reprogramming. Notably, EGC formation was stimulated by Klf9-OE even without bFGF. G-protein-coupled receptor signaling-related pathways, which are involved in PGC reprogramming, were enriched among genes down-regulated by Klf9-OE, and forskolin which activate adenylate cyclase, rescued repressed EGC formation by knock-down of Klf9, suggesting a molecular linkage between KLF9 and such signaling.