Yoshio Miki

Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage

BRCA1 (BReast‐CAncer susceptibility gene 1) and BRCA2 are tumor suppressor genes, the mutant phenotypes of which predispose to breast and ovarian cancers. Intensive research has shown that BRCA proteins are involved in a multitude of pivotal cellular processes. In particular, both genes contribute to DNA repair and transcriptional regulation in response to DNA damage. Recent studies suggest that BRCA proteins are required for maintenance of chromosomal stability, thereby protecting the genome from damage. New data also show that BRCAs transcriptionally regulate some genes involved in DNA repair, the cell cycle, and apo ptosis. Many of these functions are mediated by a large number of cellular proteins that interact with BRCAs. The functions of BRCA proteins are also linked to distinct and specific phosphory‐lation events; however, the extent to which phosphorylation‐acti‐vated molecular pathways contribute to tumor suppressor activity remains unclear. Finally, the reasons why mutations in BRCA genes lead to the development of breast and ovarian cancers are not clearly understood. Elucidation of the precise molecular functions of BRCAs is expected to improve our understanding of hereditary as well as sporadic mammary carcinogenesis.

Protein kinase Cδ is responsible for constitutive and DNA damage‐induced phosphorylation of Rad9

The mammalian homolog of the Schizosaccharomyces pombe Rad9 is involved in checkpoint signaling and the induction of apoptosis. While the mechanisms responsible for the regulation of human Rad9 (hRad9) are not known, hRad9 is subject to hyperphosphorylation in the response of cells to DNA damage. The present results demonstrate that protein kinase Cδ (PKCδ) associates with Rad9 and that DNA damage induces this interaction. PKCδ phosphorylates hRad9 in vitro and in cells exposed to genotoxic agents. The functional significance of the interaction between hRad9 and PKCδ is supported by the finding that activation of PKCδ is necessary for formation of the Rad9–Hus1–Rad1 complex. We also show that PKCδ is required for binding of hRad9 to Bcl‐2. In concert with these results, inhibition of PKCδ attenuates Rad9‐mediated apoptosis. These findings demonstrate that PKCδ is responsible for the regulation of Rad9 in the Hus1–Rad1 complex and in the apoptotic response to DNA damage.

Identification of Rad51 alteration in patients with bilateral breast cancer

AbstractThe human Rad51 gene, HsRAD51, is a homolog of RecA of Escherichia coli and functions in recombination and DNA repair. BRCA1 and BRCA2 proteins form a complex with Rad51, and these genes are thought to participate in a common DNA damage response path-way associated with the activation of homologous recombination and double-strand break repair. Additionally, we have shown that the pattern of northern blot analysis of the Rad51 gene is closely similar to those of the BRCA1 and BRCA2 genes. It is therefore possible that alterations of the Rad51 gene may be involved in the development of hereditary breast cancer. To investigate this possibility, we screened Japanese patients with hereditary breast cancer for Rad51 mutations and found a single alteration in exon 6. This was determined to be present in the germline in two patients with bilateral breast cancer, one with synchronous bilateral breast cancer and the other with synchronous bilateral multiple breast cancer. In both patients, blood DNAs showed a G-to-A transition in the second nucleotide of codon 150, which results in the substitution of glutamine for arginine. As this alteration was not present in any patients with breast or colon cancer examined, we assume that this missense alteration is likely to be a disease-causing mutation.

Expression profiling to predict postoperative prognosis for estrogen receptor-negative breast cancers by analysis of 25,344 genes on a cDNA microarray

Estrogen receptor (ER) status is an essential determinant of clinical and biological behavior of human breast cancers. While ER‐positive breast cancers respond well to adjuvant hormone therapy, ER‐negative tumors are generally resistant. To date, no attempts have succeeded in finding molecular markers for classifying ER‐negative breast cancers with respect to postoperative prognosis. To identify a set of prognostic markers for this type of cancer, we used a cDNA microarray consisting of 25,344 human genes to investigate expression profiles of ten primary breast cancers from patients who had died of breast cancer within 5 years after surgery (5y‐D) and 10 from patients who had survived disease‐free for more than 5 years (5y‐S). Sets of genes characterizing each group were identified by Mann‐Whitney and random‐permutation tests. We documented 71 genes with higher expression in the 5y‐D group than in the 5y‐S group, and 15 with higher expression in the 5y‐S group than in the 5y‐D group. Semi‐quantitative RT‐PCR experiments were carried out to confirm the results of the microarray analysis. We established a scoring system for predicting postoperative prognosis of ER‐negative breast cancers on the basis of aberrant gene expression. The list of genes reported here provides valuable information with regard to progression of breast cancer and is a source of possible target molecules for development of novel drugs to treat patients with ER‐negative breast cancers.

Familial breast and ovarian cancers.

About 60% of familial breast and ovarian cancers in Japan involve germline mutations of the BRCA1 or BRCA2 (BRCA1/2) genes. These genes contribute to genetic stability and DNA repair and act as tumor suppressor genes. Mutation analysis of the BRCA1/2 genes has improved our understanding of both common mutation patterns in Japanese patients and the clinicopathological features of BRCA1/2-related cancers. BRCA1-related breast cancers are characterized by poor prognosis, a low rate of estrogen receptor positivity, and histological predominance of solid-tubular carcinoma. BRCA1-related ovarian cancers are associated with a high frequency of serous adenocarcinoma and a good outcome. Further large-scale studies are needed to delineate genotype-phenotype relations and penetrance in BRCA1/2-related breast and ovarian cancers in Japan. The development of systems for clinical genetics in Japan, including genetic counseling, has led to the increased use of genetic testing for the clinical management of BRCA1/2-related cancers. Three options are available for carriers of BRCA1/2 mutations: intensive surveillance, chemoprevention, and prophylactic surgery. Studies done in other countries indicate that prophylactic surgery effectively prevents the development of breast and ovarian cancers in carriers of BRCA1/2 mutations. However, prophylactic mastectomy remains controversial in Japan, and now systematic intensive surveillance is generally performed for the prevention of breast cancer in women at high risk. Early detection of ovarian cancer remains challenging, resulting in increased acceptance of the need for prophylactic oophorectomy in women at risk. This review summarizes experimental and clinical findings about familial breast and ovarian cancers, including data on Japanese patients.

Genotyping of single nucleotide polymorphism using model-based clustering

MOTIVATIONnSingle nucleotide polymorphisms have been investigated as biological markers and the representative high-throughput genotyping method is a combination of the Invader assay and a statistical clustering method. A typical statistical clustering method is the k-means method, but it often fails because of the lack of flexibility. An alternative fast and reliable method is therefore desirable.nnnRESULTSnThis paper proposes a model-based clustering method using a normal mixture model and a well-conceived penalized likelihood. The proposed method can judge unclear genotypings to be re-examined and also work well even when the number of clusters is unknown. Some results are illustrated and then satisfactory genotypings are shown. Even when the conventional maximum likelihood method and the typical k-means clustering method failed, the proposed method succeeded.

Gene expression patterns as marker for 5-year postoperative prognosis of primary breast cancers

Purpose To establish the novel prognostic markers for breast cancer, gene expression profile was examined genome-wide.Methods We used cDNA microarray consisting of 18,432 human genes to compare genome-wide expression profiles of eight primary breast cancers, four from patients who died of breast cancer within 5xa0years after surgery (5D group) and four who survived disease-free for more than 5xa0years (5S group).Results We identified 21 genes whose expression was greater in tumors from the 5D group than in 5S tumors, and 23 with higher expression in the 5S group than in the 5D group. We established a Prognostic Index (PI) for prediction of postoperative prognosis, based on the aberrant expression profiles of ten of those genes. Among 20 additional cases chosen blindly, ten presented with high prognostic scores (>7, good) according to the PI; the remaining ten cases revealed scores <7 (poor). The PI predicted the actual 5-year clinical outcomes of these 20 cases with 100% accuracy.Conclusion Our PI system is reliable in clinical settings for predicting postoperative risk for breast cancer. The extensive list of genes provides valuable information about progression of breast cancer and suggests potential target molecules for treating this disease.