Toshiyasu Taniguchi - US grants

[unreadable] DESCRIPTION (provided by applicant): One of the most difficult problems in the treatment of cancer is acquired resistance to chemotherapy. Defects in DNA repair are likely to play a critical role in the sensitivity of cancer cells to chemotherapeutic drugs, many of which are DNA-damaging agents. Fanconi Anemia (FA) is a genetic disorder characterized by cancer susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin and melphalan. FA proteins and breast/ovarian cancer susceptibility gene products (BRCA1 and BRCA2) cooperate in a common DNA damage-activated signaling pathway (the FA-BRCA pathway), which controls DNA repair. We hypothesize that integrity of the FA-BRCA pathway is a critical determinant of resistance of tumor cells to chemotherapy with DNA crosslinking agents. The goals of our proposed research are 1) to elucidate the role of the FA-BRCA pathway in cisplatin sensitivity/resistance of cancer cells, 2) to determine if the sensitivity of tumor cells to DNA crosslinking agents can be increased by modulating the FA- BRCA pathway using small molecule inhibitors of the pathway, and 3) to further elucidate the mechanism of regulation of the FA-BRCA pathway. We plan to determine the role of one of the FA genes, BRCA2/FANCD1, in acquired resistance to cisplatin by analyzing BRCA2-deficient cancer cell lines and clinical samples of ovarian cancer. We also plan to identify small molecule inhibitors of the FA-BRCA pathway which sensitize tumor cells to DNA crosslinking agents. Several candidate chemicals including cyclin dependent kinase inhibitors and proteasome inhibitors have already emerged from our initial screens. We plan to test if these inhibitors sensitize cancer cells to cisplatin and ultimately to elucidate the mechanism of actions of these drugs. We also plan to elucidate involvement of cyclin-dependent kinases and the proteasome in the regulation of the FA-BRCA pathway using the inhibitors we identified. These studies will lead to discovery of potential drugs that can be used as chemo-sensitizers in the treatment of cancer, provide new insights about the signaling of the FA-BRCA pathway, and may eventually lead to establishment of a strategy to overcome chemo-resistance of cancer. Relevance: Our studies are intended to clarify why some cancers can be effectively treated with drugs that cause DNA damage and others become resistant. They should also lead to a discovery of ways to make cancer chemotherapy more effective. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): DNA-crosslinking agents such as cisplatin, carboplatin, cyclophosphamide, mitomycin C and melphalan are widely-used drugs for treatment of cancers including leukemia, lymphoma, and myeloma. Resistance to these drugs is a major problem for effective cancer therapy. Fanconi anemia is a genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and hypersensitivity to DNA-crosslinking agents. Fanconi anemia proteins and breast cancer susceptibility proteins (BRCA1 and BRCA2) cooperate in a pathway (the Fanconi anemia-BRCA pathway), which controls DNA repair. This pathway is required for cellular resistance to DNA-crosslinking agents. Inhibition of this pathway is therefore an attractive therapeutic strategy to overcome DNA-crosslinker resistance. Key proteins in the pathway, such as FANCD2 and RAD51, accumulate at sites of DNA damage and repair, form nuclear foci and regulate DNA repair. Formation of these nuclear foci is a good marker of the integrity of the pathway. MicroRNAs are non-coding RNA molecules that post-transcriptionally regulate gene expression. MicroRNAs are involved in biological processes such as cell proliferation, differentiation and apoptosis, and are deregulated in cancer. However, regulation of the Fanconi anemia-BRCA pathway by microRNAs has not been investigated. Through screening of microRNA mimics & inhibitors library, we have identified several microRNA mimics and inhibitors which inhibit formation of FANCD2 and RAD51 foci. Therefore, we hypothesize that the Fanconi anemia-BRCA pathway is regulated by microRNAs. Interestingly, some of the miRNAs we identified are known to be deregulated in a subset of human cancers. The goals of our research are 1) to identify microRNAs regulating the Fanconi anemia-BRCA pathway, 2) to elucidate whether the microRNAs affect cellular sensitivity to DNA-crosslinkers and efficiency of DNA repair, and 3) to elucidate the mechanism(s) of regulation of the pathway by the microRNAs. Our study will lead to the discovery of novel factors (microRNAs and their target genes) involved in the Fanconi anemia-BRCA pathway. These factors may be novel cancer/leukemia susceptibility genes or Fanconi anemia genes, as are other factors in the pathway. Deregulation of these factors may determine chemosensitivity. Thus, this project could have an impact on the basic understanding of Fanconi anemia, cancer/leukemia susceptibility and chemosensitivity of cancer cells. PUBLIC HEALTH RELEVANCE Our studies are intended to clarify how DNA repair is regulated by microRNAs. Our studies will provide basic understanding of a blood disease called Fanconi Anemia and also lead to a discovery of ways to make cancer chemotherapy more effective. [unreadable] [unreadable] [unreadable]

Plafinum compounds, such as cisplafin and carboplafin, are key drugs for the treatment of ovarian carcinoma. Both primary and acquired resistance to plafinum compounds are serious clinical problems. The breast/ovarian cancer suscepfibility genes BRCA1 and BRCA2 (BRCA1/2) play a crifical role in repairing the DNA damage caused by plafinum compounds. Consequently, BRCA 7/2-deficient cells are hypersensitive to platinum compounds. Recenfiy, we found that platinum resistance of BRCA V2-mutated cancer can be mediated by secondary intragenic mutations in BRCA1/2 that restore the wild-type BRCA1/2 reading frame. Based on this finding, we hypothesize that restoration of BRCA1/2 is involved in acquired platinum resistance of BRCA1/2-deficient ovarian carcinomas. In this proposal, we focus on determining clinical relevance of restoration of BRCA1/2 function in Bf?CA 7/2-deficient hereditary and sporadic ovarian carcinomas. First, we will determine whether the occurrence of secondary mutations that restore DNA repair function of BRCA1/2 correlates with clinical outcomes of primary and recurrent hereditary ovarian carcinomas occurring in women with inherited BRCA1/2 mutafions. Second, we will evaluate whether restoration of BRCA1 expression is involved in acquired resistance to plafinum in sporadic ovarian carcinomas that inifially have low BRCA1 expression before treatment. We will also determine whether ovarian cancer cells with reduced BRCA1 expression acquire restored BRCA1 function after in vitro selection in the presence of cisplatin and evaluate regulatory mechanisms that lead to restored BRCA1 expression. With these studies, we will assess the clinical significance of restoration of BRCA1/2 function during the treatment of BRCA [unreadable]//2-deficient ovarian carcinoma. RELEVANCE (See instructions): Our study will provide crifical informafion about the mechanisms of plafinum-resistance of BRCA1/2-deficient tumors, which will enable us to predict plafinum resistance of recurrent tumors, and may eventually lead to the establishment of a strategy to overcome platinum resistance.

PROJECT SUMMARY/ABSTRACT Skin cancers are the most common type of cancer in the United States. A primary etiologic agent for skin cancers is ultraviolet (UV) radiation, a carcinogen that induces DNA lesions, such as pyrimidine dimers. The risk of melanoma, a form of skin cancer with a poor prognosis, is also related to sun exposure. Understanding how human cells respond to and repair such DNA lesions is of clinical significance, especially for cancer prevention. As one of the physiologic responses to UV radiation, cells alter the expression of small, non-coding RNAs, known as microRNAs, which negatively regulate gene expression post-transcriptionally. Furthermore, some proteins involved in microRNA biogenesis are required for cellular resistance to UV. However, the molecular mechanisms connecting UV response signaling, the microRNA biogenesis machinery and repair of UV-induced DNA lesions are poorly understood. We hypothesize that UV-induced phosphorylation of human DGCR8, a double-stranded RNA binding protein in the microRNA processing complex, plays a critical role in the cellular response to UV. This is based on our novel finding that a serine residue of DGCR8 is phosphorylated in response to UV, and that this phosphorylation is critical for both cellular survival and efficient removal of pyrimidine dimers after UV irradiation. Surprisingly, the RNA-binding activity of DGCR8 is dispensable for cellular resistance to UV, suggesting an entirely new function of DGCR8 that is independent of microRNA processing. Further, we found that DGCR8 is epistatic with factors involved in transcription coupled-nucleotide excision repair and that recovery of RNA synthesis after UV exposure is delayed in DGCR8-deficient cells, suggesting that DGCR8 is involved in DNA repair of UV-induced lesions by participating in transcription coupled-nucleotide excision repair. We propose to elucidate the mechanisms by which DGCR8 regulates DNA repair of UV-induced lesions and to elucidate how the UV-induced phosphorylation of DGCR8 is regulated. Our studies will identify a novel cellular signaling pathway that connects the microRNA biogenesis machinery and UV-induced DNA damage and repair. We dub this the ?DGCR8-mediated UV response pathway?. Our work will provide mechanistic insights into the cellular UV response and, consequently, the rationale for novel approaches to cancer prevention. Finally, this work will uncover a new biological function of the microRNA biogenesis machinery that is separate from its canonical role.