We found that Ku70, a known DNA repair factor, has a novel function to bind and inhibit Bax (Bcl-2-associated X protein), a key mediator of apoptosis. Pentapeptides derived from the Bax-binding domain of Ku70 were cell-permeable and protected cells from Bax-mediated apoptosis. These pentapeptides were called BIPs (Bax-inhibiting peptides). BIPs may become a useful therapeutic tool to reduce cellular damage. We also generated BIP mutant pentapeptides that do not inhibit Bax, but retain their cell-penetrating activity. Since both BIPs and BIP mutants are cell-permeable, these peptides were designated CPP5s (cell-penetrating pentapeptides). Among the CPP5s discovered, VPTLK (BIP) and KLPVM (BIP mutant) were confirmed to possess protein transduction activity by examination of the delivery of GFP (green fluorescent protein) into cells by these peptides. The mechanism of cell penetration by CPP5s is not known. CPP5s enter the cell at 0 and 4 degrees C. In preliminary studies, various inhibitors of endocytosis and pinocytosis did not show any significant suppression of CPP5 cell entry. CPP5s have very low toxicity in vitro and in vivo and so may be useful tools in order to develop non-toxic drug-delivery technologies.
Background: There is a strong genetic component to breast cancer, which is the most common neoplasm in women; however current risk markers are few, accounting for less than 10% of breast cancer cases. This means that the majority of familial breast cancers remain unexplained. Our laboratory has studied microsatellite polymorphisms for many years, and we developed techniques to assay these understudied regions of the genome en masse and individually via shot-gun methods to rapidly identify those that are likely to be polymorphic and therefore potentially contribute to phenotype. Material and Methods: Using these novel approaches, we identified a polymorphic AAAG microsatellite repeat in the estrogen-related receptor gamma (ERR-γ) that is expanded in -10% of the general population. We genotyped over 300 individuals and found a longer allelic version (13+ repeat copies) in 14.3% of breast cancer patients, compared to only 4.8% in cancer-free individuals. We computationally identified 22 transcription factors that could bind directly to this AAAG repeat and the surrounding region. We hypothesized that the AAAG-containing region might serve as a promoter for ERR-y. Results: We found a statistically significant number of breast cancer patients with the expanded AAAG allele, and preliminary experiments confirm that the AAAG-containing region of ERR-γ does indeed drive reporter gene expression in estrogen receptor positive (ER+) MCF-7 breast cancer cells. This was not the case for the ER-breast cancer cells (MDA-MB-231) we also examined. Our initial assessments also revealed that SKBR-3 breast cancer cells are naturally heterozygotic for the long and short allelic versions of the AAAG repeat. Our ongoing studies involve knockdown of transcription factors in MCF-7 cells that abrogate the endogenous expression of ERR-γ. Discussion: The role of microsatellites in cancer is well defined for colon cancer, but few studies have been conducted to examine the contribution of these simple sequence repeats in breast cancer. We discovered a polymorphic AAAG repeat in the ERR-γ gene, and our studies indicate that a longer version of this repeat is more prevalent in the genomes of breast cancer patients. Based on our assessment, this allele carries a 2.98 relative risk for the development of breast cancer. The higher incidence of this putative biomarker in breast cancer patients may be a result of altered ERR-γ expression, as the AAAG-containing sequence can drive reporter expression. If the frequency of this potentially predictive marker is sustained in a larger population, and the mechanism by which it confers the cancer phenotype can be identified, it may contribute substantially as a biomarker offering surveillance, prophylactic surgery, and chemoprevention options to patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-03.
#3078 The primary tumors of most breast cancer patients are removed via resection or complete mastectomy. However, years after having their primary tumors removed many patients will need chemotherapy for metastatic disease that arises in distal sites such as lung and brain. It has been reported that many breast cancers overexpress the 2 electron oxidoreducase NQO1, a marker of tumorigenesis for a variety of cancers. Since NQO1 expression levels are low in normal tissue but elevated 10-100 fold in breast cancers, we hypothesized that NQO1 levels could be exploited for chemotherapy with ß-lapachone. ß-lapachone is a naturally derived plant product that is bioactivated by NQO1. In this study we show that ß-lapachone (a DNA repair inhibitor) synergizes with the DNA-PK inhibitor Nu7026 to kill human breast cancer cell lines that express NQO1. Normal human breast tissues and breast cancer cell lines that did not express NQO1 were not affected by ß-lapachone. In addition by using in vivo breast cancer models we demonstrated that ß-lapachone effectively inhibited breast tumor growth in athymic mice. We also showed that ß-lap was efficacious against lung tumors derived from MDA-MB-231 breast cancer cell lines. Finally, our simulated brain metastatic model showed that ß-lap accumulated in brain tumors derived from MDA-MB-2331 cells. Together these data suggest that ß-lap and ß-lap in combination Nu7026 may be a potent combination for treating primary and metastatic breast cancer disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3078.
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