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    Molecular Mechanism of Nucleotide Excision Repair Deficiency in Novel Breast Tumor Cell Lines

    Scientific Abstract:
    Molecular Mechanism of Nucleotide Excision Repair Deficiency in Novel Breast Tumor Cell Lines Background: Although many studies have identified critical genetic and epigenetic changes that mark transformation of cells in tissues such as colon, pancreas, and lung, similar studies in breast cancer have met with limited success. Markers such as c-erb, cyclin D1, and others have all been shown to only be overexpressed in only 30% of breast tumors examined, indicating that few molecular alterations common to the majority of breast cancers have been found. In striking contrast to this, we have shown that 100% of stage 1 breast cancers (17 samples) manifest a significant (p < 0.001) loss in Nucleotide Excision Repair (NER) relative to breast reduction mammoplasty cultures (22 samples). This is also the first functional assay to show such a global phenomenon in breast cancer. In order to determine the genes involved in this loss of function, we have performed RNase Protection Assays (RPA) on both breast tumor explants and breast reduction explants. We have identified 5 candidate genes with decreased mRNA expression in the tumor relative to breast reductions, including members of the TFIIH complex. Objective/Hypothesis: We hypothesize that the decrease in mRNA expression levels in the candidate genes is due to an epigenetic mechanism, i.e. methylation of the 5’ elements of the XP-B, XP-D, TFIIHp56, TFIIH p44, and TFIIH p34 genes. Specific Aims: In this study we will: (1) compare the results of our RPAs with protein levels of these gene products using Western analysis. If the Western results are consistent with the mRNA expression results, then we will address the molecular mechanisms. We will look at epigenetic regulation via sodium bisulfite sequencing of the 5’ regions of the candidate genes. In explants where reduced repair capacity has been shown, we will restore function by treatment with 5’-deoxy-azacytidine to remove methylation. (3) If epigenetic regulation is not the mechanism of decreased expression, we will analyze the cells for loss of genetic material using array Comparative Genomic Hybridization (CGH) and standard Loss of Heterozygosity (LOH) techniques at the NER gene loci. In explants where genetic loss is the mechanism of decreased expression, we will restore function using exogenous expression of the lost NER gene. Study Design: The existence of novel explant cultures and cell lines in this lab provides a unique opportunity to perform these assays. From these we can generate the large numbers of cells required for molecular techniques. In order to confirm that the protein levels of our 5 candidate genes are also decreased in tumor explants relative to breast reduction explants, we will perform Western blot analysis using commercially available antibodies. Once we have confirmed these genes are our candidates, we will then explore the molecular mechanisms behind their decreased expression. We will first look at an epigenetic regulation mechanism by undertaking sodium bisulfite sequencing of the candidate genes, paying particular attention to their 5’ regulatory regions’ CpG islands to determine their methylation status. We will then employ the use of 5’-deoxy-azacytidine to remove this methylation and restore gene expression. If methylation is not the mechanism of decrease for these genes, we will look for loss of genetic material first with array CGH to determine gross deletions. In addition we will analyze LOH using flanking sets of highly polymorphic microsatellite markers around the NER genes (including the candidate genes) to determine more subtle losses of genetic material. Lastly, we will restore NER function using exogenous expression of the lost genetic material under the control of an inducible promoter. Relevance: Unlike most markers that have been shown to be associated with 30% of breast cancers, we now show a marker associated with 100% of 17 early stage tumors. Based on this data and additional unpublished data we have shown that loss of NER is intrinsic in sporadic breast cancer. BRCA-1 is also beginning to be linked functionally to NER the recent literature. This would suggest that our data on sporadic breast cancer and the data on genetic breast cancer cases are converging on a critical mechanism relevant to breast cancer etiology. The extended explant cultures created in this laboratory are an innovative tool that have allowed our laboratory to examine the loss of NER at the molecular level using assays where larger numbers of live cells are necessary. Elucidation of the genes involved in tumorigenesis will aid in both prognosis and therapy by suggesting more timely and targeted treatment since the patients can be classified by tumor etiology and in vitro vulnerability to DNA damaging drugs.

    Lay Abstract:
    Molecular Mechanism of Nucleotide Excision Repair Deficiency in Novel Breast Tumor Cell Lines Breast cancer is a common disease, affecting 182,000 American women each year and killing 46,000. At 10 years, there is only a 50% survival rate. Breast cancer is also a heterogeneous disease. Just as no two women are alike, no two tumors are alike. No two patients respond the same way to the same treatment, and yet historically scientists have studied only a handful of stage IV, drug-resistant cell lines due to the limitation of cell culture. Fortunately, tissue culture and tissue engineering methods have improved and it is now possible to study many more tumor types and patients. Our laboratory has developed 109 explants representing 109 different women. Because molecular biology requires large numbers of cells for analysis, we can now use these explants (and provide them to others) to analyze potential causes of breast cancer. The main focus of research in this laboratory is to determine the mechanism of DNA repair deficiencies in the etiology of breast tumors. The formation of tumors in the human body includes the accumulation of mutations. Some of these mutations involve tumor suppressor genes, proto-oncogenes, and “mutator” genes also known as DNA repair genes. We have shown that the loss of Nucleotide Excision Repair (NER) is intrinsic in sporadic breast cancer etiology. We have analyzed the global genomic repair of primary cultures derived from 17 stage 1 tumors relative to 22 breast reduction mammoplasties and found a significant loss of NER in 100% these early stage tumors (p<0.001). Although many studies have identified critical genetic and epigenetic changes that mark transformation of cells in tissues such as colon, pancreas, and lung, similar studies in breast cancer have met with limited success. Markers such as c-erb, cyclin D1, and others have all been shown to only be overexpressed in only 30% of breast tumors examined, indicating that few molecular alterations common to the majority of breast cancers have been found. In striking contrast, we have seen this effect in 100% of the samples tested. This is also the first functional assay to show such a global phenomenon in breast cancer. Using an RNAse protection assay, we have identified 5 candidate genes with decreased mRNA expression levels in tumor explants relative to breast reduction explants, which we believe are responsible for the observed functional decrease. We will study in more detail the molecular mechanism responsible for the decrease in their expression. We hypothesize that epigenetic regulation mechanism are responsible for the decreased expression of our 5 candidate genes: XP-B, XP-D, TFIIHp56, TFIIHp44, and TFIIHp34. The technique of Western blot analysis will first be utilized to confirm our identification of these 5 genes. We will then investigate the possibility of an epigenetic regulation mechanism being behind their decreased expression through the use of sodium bisulfite sequencing. Ultimately, we will restore function within these cells using a chemical called 5’-deoxy-azacytidine to remove the methylation. Should methylation not be the mechanism of decreased expression we will look for large losses in genetic material with array Comparative Genomic Hybridization (CGH), and subtle losses of genetic material with Loss of Heterozygosity (LOH) techniques as an alternative explanation. If this is the case, we will restore function by reinserting the lost NER gene into the cells. The explants and cell lines created in this laboratory are an innovative tool that have allowed and will allow this laboratory to examine the loss of NER in early stage tumors and puts this lab at a unique position for the study of breast cancer etiology. Elucidation of the genes involved in the formation of tumors will aid in both prognosis and therapy by suggesting more timely and targeted treatment since the patients can be classified by tumor etiology and its in vitro vulnerability to DNA damaging drugs. With continuing studies on NER in breast cancer and the identification of the remaining NER genes, it is hoped that a simpler and faster assay can and will be developed so that breast cancer treatment can be tailored to each individual based upon her, and her tumor’s, DNA repair capabilities. By accurately tailoring treatment for each woman, at the very least the morbidity associated with breast cancer can significantly be reduced.