A major mechanism of DNA repair related to homologous recombination is the Fanconi anemia (FA) pathway. FA genes collaborate with BRCA genes to form foci of DNA repair on chromatin after DNA damage or during the S phase of the cell cycle. Our goal was to develop a method capable of evaluating the functional status of the pathway in patients' tumor tissue, which could also be practically incorporated into large-scale screening. To develop this method, we first used Western immunoblot to detect FANCD2 protein monoubiquitination in fresh tumor specimens of patients with ovarian cancer undergoing surgery and stained formalin-fixed paraffin-embedded tumor tissue simultaneously with 4',6-diamidino-2-phenylindole, FANCD2, and Ki67 antibodies, eventually extending this method to other solid tumors. This triple stain permitted evaluation of the presence, or lack thereof, of FANCD2 subnuclear repair foci in proliferating cells by immunofluorescence microscopy. Overall, we evaluated 156 formalin-fixed paraffin-embedded tumor samples using the FA triple-staining immunofluorescence method. The ratios of FANCD2 foci-negative tumors in ovarian, lung, and breast tumor samples were 21%, 20%, and 29.4%, respectively. Our studies have led to the development of a suitable method for screening, capable of identifying tumors with somatic functional defects in the FA pathway. The use of paraffin-embedded tissues renders the reported method suitable for large-scale screening to select patients for treatment with DNA interstrand crosslinking agents, poly ADP-ribose polymerase inhibitors, or their combination.
[Show abstract][Hide abstract] ABSTRACT: Gynecologic malignancies annually account for over 91,000 new cancer cases and approximately 28,000 deaths in the United States. Although there have been advancements in cytotoxic chemotherapies, there has not been significant improvement in overall survival in these patients. While targeted therapies have shown some benefit in many solid tumors, further development of these agents is needed for the treatment of gynecologic malignancies. Poly(ADP-ribose) polymerase (PARP) catalyzes the polyADP-ribosylation of proteins involved in DNA repair. Inhibitors of PARP were originally developed for cancers with homologous recombination deficiencies, such as those harboring mutations in BRCA1 or BRCA2 genes. However, pre-clinical research and clinical trials have suggested that the activity of PARP inhibitors is not limited to those with BRCA mutations. PARP inhibitors may have activity in cancers deficient in other DNA repair genes, signaling pathways that mitigate DNA repair, or in combination with DNA-damaging agents independent of DNA repair dysfunction. Currently there are seven different PARP inhibitors in clinical development for cancer. While there has been promising clinical activity for some of these agents, there are still significant unanswered questions regarding their use. Going forward, specific questions that must be answered include timing of therapy, use in combination with cytotoxic agents or as single-agent maintenance therapy, and whether there is a predictive biomarker that can be used with PARP inhibition. Even with large strides in the treatment of many gynecologic malignancies in recent years, it is imperative that we develop newer agents and methods to identify patients that may benefit from these compounds. The focus of this review will be on pre-clinical data, current clinical trials, and the future of PARP inhibitors in the treatment of ovarian, endometrial, and cervical cancer.
Frontiers in Oncology 10/2013; 3:237. DOI:10.3389/fonc.2013.00237
[Show abstract][Hide abstract] ABSTRACT: Alveolar rhabdomyosarcoma that harbors the PAX3/FOXO1 fusion gene (t-ARMS) is a common and lethal subtype of this childhood malignancy. Improvement in clinical outcomes in this disease is predicated upon the identification of novel therapeutic targets.
Robust mouse models were used for in vivo analysis, and molecular studies were performed on xenografts treated in parallel. Two independent patient sets (n=101 and 124) of clinically-annotated tumor specimens were used for analysis of FANCD2 levels and its association with clinical and molecular characteristics and outcomes.
Our xenograft studies reveal a selective suppression of FANCD2 by m-TOR kinase inhibition and radiosensitization of the t-ARMS line only. In the initial patient set, we showFANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the co-presence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and non-random enrichment of mTOR-associated genes that correlate with FANCD2 gene expression within the t-ARMS samples, but not within other cases. In the second patient set, we show that, on a protein level, FANCD2 expression correlates with PAX3/FOXO1 fusion gene and is strongly associated with phospho-p70S6K expression in cases with the fusion gene.
Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS. Indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy.
Clinical Cancer Research 04/2014; 20(14). DOI:10.1158/1078-0432.CCR-13-0556 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin embedded (FFPE) tumor samples. DNA repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA damaging drugs. We screened 139 non-small cell lung cancer (NSCL) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel targeted agents in the background of FA deficiency we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down non-small cell lung cancer cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5µM) and BMN673 (0.5µM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5M. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL2/XL inhibitor ABT263 at dose of 2 M. The treated cells were harvested at 24, 48 and 72 hours post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 hours post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathw
Frontiers in Oncology 12/2014; 4:368. DOI:10.3389/fonc.2014.00368
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