The Fanconi Anemia–BRCA Pathway
Defects of the DNA repair and DNA damage response have at least two important implications in cancer biology. First, these defects cause genomic instability, which promotes malignant transformation of cells. Second, they lead to cellular hypersensitivity to DNA-damaging agents, which can contribute to effective chemo/radio-therapy. To achieve greater understanding of cancer and to develop new diagnostic and therapeutic strategies, it is important to elucidate the cause of genomic instability and the mechanisms surrounding the DNA damage response/DNA repair pathway. The recently emerged concept of the Fanconi anemia–BRCA pathway, which is a signaling pathway that is activated by DNA damage and regulates DNA repair, provides a good model for studying these implications.
The study of rare genetic diseases with cancer susceptibility has produced insights into the pathogenesis of cancer in the general population. For example, mutations in p53, Rb, and ATM genes are, respectively, responsible for the genetic diseases Li-Fraumeni syndrome, familial retinoblastoma, and ataxia telangiectasia. Similarly, Fanconi anemia, a rare cancer susceptibility syndrome, has more recently emerged in the DNA repair and signaling field. Understanding this genetic disorder may greatly enhance our knowledge of the pathogenesis and progression of human cancers.
Fanconi anemia, an autosomal-recessive (or X-linked) cancer susceptibility syndrome, is characterized by chromosomal instability and cellular hypersensitivity to DNA-crosslinking agents, such as cisplatin and mitomycin C. Fanconi anemia comprises at least 13 complementation groups; all of the 13 Fanconi anemia genes (FANCA, B, C, D1 [BRCA2], D2, E, F, G, I, J [BACH1/BRIP1], L, M, and N [PALB2]) have been identified. The breast/ovarian cancer susceptibility gene products (BRCA1 and BRCA2 proteins) and all of the Fanconi anemia proteins cooperate in the Fanconi anemia–BRCA pathway, a common pathway required for the cellular resistance to DNA-crosslinking agents.
The key event in the Fanconi anemia–BRCA pathway is the monoubiquitination of one of the Fanconi anemia proteins, FANCD2. Eight Fanconi anemia proteins (A, B, C, E, F, G, L, and M) are components of a multisubunit ubiquitin ligase complex (FA core complex) required for the monoubiquitination of FANCD2. FANCI and FANCD2 form another protein complex, the ID complex.
This pathway is activated in response to DNA damage. After DNA damage, FANCD2 is monoubiquitinated and targeted to BRCA1/BRCA2/RAD51-containing nuclear foci at the sites of DNA damage. The FA core complex, BRCA1, and a DNA damage-signaling kinase called ATR are required for this process. After ionizing radiation (IR) exposure, FANCD2 is directly phosphorylated by ATM, another DNA damage-signaling kinase; this phosphorylation is required for the establishment of an IR-inducible S-phase checkpoint.
The Fanconi anemia–BRCA pathway is inactivated in a wide variety of human cancers, for example, by methylation of one of the Fanconi anemia genes, FANCF. This inactivation can cause cisplatin sensitivity of cancer cells.
Our long-term objective is to elucidate molecular mechanisms of DNA damage response/DNA repair pathways (such as the Fanconi anemia–BRCA pathway) and their involvement in carcinogenesis and to use such information to refine diagnosis and therapy of patients with cancer or with Fanconi anemia.
Our current research focuses on analysis of the involvement of the Fanconi anemiaBRCA pathway in resistance of human cancer to DNA-crosslinking agents. Our projects include (1) elucidation of the roles of microRNAs in the regulation of the Fanconi anemia–BRCA pathway, (2) elucidation of the role of BRCA1/2 in platinum resistance of cancer cells, and (3) identification and characterization of small molecules that inhibit the Fanconi anemia–BRCA pathway and sensitize cancer cells to DNA crosslinking agents.
MicroRNA and the Fanconi Anemia–BRCA Pathway
MicroRNAs (miRNAs) are 20- to 24-nucleotide noncoding RNA molecules that post-transcriptionally regulate gene expression. MiRNAs are involved in biological processes such as cell proliferation, differentiation, and apoptosis, and are deregulated in cancer. However, their roles in DNA repair, especially the Fanconi anemia–BRCA pathway, have not been elucidated. We hypothesized that the Fanconi anemia–BRCA pathway is regulated by miRNAs, and we used a cell-based assay to screen human microRNA mimic and inhibitor libraries. This assay, which uses decrease of DNA damage-induced FANCD2/RAD51 foci as a readout, identified several candidate microRNAs that affect FANCD2/RAD51 foci formation. Currently we are analyzing these miRNAs. This study will lead to the discovery of novel factors (miRNAs and their targets) that regulate the pathway, are potential targets for chemosensitization, and may be novel Fanconi anemia genes or breast/ovarian cancer susceptibility genes, as are many other factors involved in the pathway.
BRCA1/2 and Platinum Resistance of Cancer Cells
Platinum compounds, such as cisplatin and carboplatin, are key drugs for the treatment of ovarian cancer, and primary and acquired resistance to platinum compounds is a serious problem clinically. The breast/ovarian cancer susceptibility genes, BRCA1 and BRCA2 (BRCA1/2), play a critical role in repairing DNA damaged by platinum compounds, and BRCA1/2-deficient cells are hypersensitive to platinum compounds. Therefore, cisplatin or its derivative, carboplatin, is a logical choice for the treatment of BRCA1/2-deficient tumors, and women with BRCA1/2-mutated ovarian carcinoma have a better prognosis than those without the BRCA1/2 mutation if they receive platinum-based therapy. However, even patients with BRCA1/2-mutated ovarian cancer sometimes experience primary or acquired platinum resistance, and mechanisms of this drug resistance have been largely unknown.
Recently, we found that cisplatin resistance of BRCA1/2-mutated cancer can be mediated by secondary intragenic mutations in BRCA1/2 that restore the wild-type BRCA1/2 reading frame in some cell line models. We also found several cases of BRCA1/2-mutated platinum-resistant ovarian cancer with secondary mutations of BRCA1/2 that restored the wild-type BRCA1/2 reading frame. We plan to determine the clinical relevance of this phenomenon and to elucidate mechanisms of inactivation and reactivation of BRCA1/2 in cancer.
Small Molecules That Inhibit the Fanconi Anemia–BRCA Pathway
We have demonstrated that inactivation of the Fanconi anemia–BRCA pathway causes hypersensitivity of cancer cells to DNA-crosslinking agents and that reactivation of the pathway causes acquired resistance to the drugs. These observations led us to hypothesize that small molecules that inhibit the pathway sensitize tumor cells to DNA-crosslinking agents. Because these agents are widely used in cancer chemotherapy, the Fanconi anemia–BRCA pathway inhibitors may be beneficial for many patients as chemosensitizers.
We have developed a high-throughput cell-based screening assay for small-molecule inhibitors of the pathway utilizing DNA damage–induced FANCD2 foci formation as a readout, have screened thousands of chemicals, and have identified several chemicals that inhibit the pathway. We are determining whether these candidate Fanconi anemia–BRCA pathway inhibitors are useful as chemosensitizers. We also plan to elucidate mechanisms of action of these chemicals. These studies should provide insights about the signaling of the pathway and are expected to lead to the discovery of drugs that can be used as chemosensitizers in the treatment of cancer.
Grants from the National Institutes of Health and the Fanconi Anemia Research Fund provided partial support for these projects.
As of May 30, 2012