Student Theses, Dissertations and Capstones

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD) in Pharmacy

Copyright Statement

All rights reserved. This publication is intended for use solely by faculty, students, and staff of Nova Southeastern University. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

First Advisor

Jean J Latimer

Second Advisor

Luigi Cubeddu

Third Advisor

Rais Ansari

Publication Date / Copyright Date

2018

Publisher

Nova Southeastern University

Abstract

NER plays an important role in remediating DNA damage. Latimer et al., have previously shown nucleotideexcision repair (NER) to be intrinsically deficient in stage I sporadic BC by function and gene expression. Previous work performed on 12 isogenically matched stage I tumors and non-tumor adjacent (NTA) samples identified 2 types of NTA tissue with regard to functional NER. One type that represented 75% ofthe samples, had lower NER capacity, similar to the tumor (Low: Low pair). The other type had high NER relative to the tumor (High: Low pair). Two isogenic NTA/tumor cell line pairs representing these 2 types ofNTA were identified by the UDS assay for downstream molecular analyses. Expression of the 20 canonical NER genes using microarray analysis was consistent with functional NER of the Low: Low and High: Low cell lines. Findings from expression microarray were validated using RNA sequencing, where 16/20 genes were significantly higher in the NTA line of the High: Low pair compared to the tumor line, but none of the 20 genes were significantly different in expression among the Low: Low pair. Protein expression was also evaluated for RPA3, XPC and RAD23B, however only RAD23B showed promising trends. We believe the mechanism of downregulation of NER genes was epigenetic based on downregulation in multiple genes and multiple patients. DNA methylation was explored as the mechanism of this phenomenon. Using MethylationEPIC array, analysis of promoter level, gene level and CpG island level methylation no correlation between methylation and gene expression in our cell line pairs. DDB1 showed differential methylation among the both High: Low and Low: Low pairs but in a direction opposite to gene expression, indicating possible inhibition of a repressor at its promoter region. RNA sequencing allowed us to explore the presence of single nucleotide variants in the 20 NER genes along with other BC genes. We discovered notable variants inERCC2, ERCC5, ERCC6 as well as in BRCA1, CHK1 and ATR, which warrant further investigation using The Cancer Genome Atlas. Finally, we were able to construct person-specific maps or our own “Vogelsteinograms” for breast carcinogenesis.

Disciplines

Pharmacy and Pharmaceutical Sciences

Keywords

Breast cancer, DNA repair, Gene expression, Next generation sequencing, Nucleotide excision repair, Personalized medicine

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