Alshareeda, Alaa
(2014)
Assessment of DNA-damage repair in breast cancer.
PhD thesis, University of Nottingham.
Abstract
Background: Current evidence indicates that DNA damage response (DDR) is a highly complex process that involves various pathways working in an orchestrated and interwoven manner in response to different types of damage to DNA. Although specific defects of DDR remain to be deciphered in cancer as a general, there is certainly an undeniable relationship between a particular dysfunction of DDR and the phenotype of tumour [1, 2]. It has been demonstrated that familial forms of breast and ovarian cancer are characterised by defects in one of the main mechanisms of DDR homologous recombination (HR) as a result of germline loss-of-function mutations in one of HR modifying genes, such as BRCA1 and BRCA2 [1, 3, 4]. Defects of genes involved in other DDR pathways are also associated with specific types of cancers; for instance hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. Several previous studies have demonstrated that impaired DDR play a fundamental role in the pathogenesis and behaviour of breast cancer (BC). However, characterisation of this complex process, the expression and co-expression of the key proteins involved in the various DDR pathways and their prognostic significance in BC remain to be defined. In BC, it is reported that genes involved in DNA double strand breaks (DSB) repair are the most important. Two main pathways are involved in the repair of DNA-DSB; HR and Non Homologous End Joining (NHEJ) [3]. The common characteristics of global DDR are multiple genes induction directly associated with sensing and repair of DNA, arrest of cell cycle, and cell division inhibition. As a result DDR process does not only include genes activation involved in damage sensing as well as repair but additionally genes involved in control of cell-cycle [5]. Despite the fact that DDR may possibly involve activation of several pathways (such as SUMOylation (SUMO)) [6, 7] and many genes are engaged in different overlapping mechanisms, each pathway is characterised by activation and expression of a unique set of genes. This could allow discovering the active or aberrant pathway in a given tumour [1, 4, 5].
This study explores the hypothesis that investigation of alterations in the different pathways of DNA-DSB, may contribute to the characteristics of BC. Therefore, the aim was to perform a comprehensive profiling of key proteins involved in the different DNA-DSB repair pathways in the different molecular classes of BC. This approach aims to address the inherent problems arising from the complexity of DDR mechanism in BC with the potential of discovering a key pathway that is active or inactive in specific forms of BC that can be helpful to identify DNA repair status in individual BC patients.
Method: The study cohort comprises three BC groups: A) Large series of unselected primary sporadic operable invasive tumours (n=1904) in addition to B) 386 cases of oestrogen receptor (ER) negative tumours and C) a well-characterised series of BC from patients with known BRCA1 germline mutations (n=24). The proteins investigated in this study are known to participate in different DNA-DSB repair pathways including, DNA damage sensors (ATM and ATR), HR repair (BRCA1, BARD1, Rad51, γH2AX and SMC6L1), DNA damage checkpoint signalling protein (CHK1 and CHK2), NHEJ repair (KU70/KU80, and DNA-PK), and SUMO (PIAS1, PIAS4, and UBC9). Because subcellular localisation of DDR proteins may affect their function, two markers that have role in nuclear transport in the cell were examined (NPM and KPNA2). The expression of these proteins was assessed using the well-established immunohistochemical technique utilising tissue microarray technology. The expression of proteins was further evaluated in various cell lines; BRCA1 deficient HeLaSilenciX® cells, and control BRCA1 proficient HeLaSilenciX®, MDA-MB-436 (BRCA1 deficient), and MCF-7 (BRCA1 proficient and ER+) using Reverse Phase Protein Microarray (RPPA).
Results: Both cytoplasmic and nuclear expression was observed for expression of Rad51, SMC6L1, BRCA1, BARD1; (HR markers), PIAS1, UBC9 (SUMO markers), γH2AX (DNA-DSB marker) and CHK1 (checkpoint signalling protein). In contrast, both NHEJ markers and most of the DNA damage sensors (ATM and ATR), CHK2 and PIAS4 were mainly expressed in the nucleus. Generally, tumours that showed positive cytoplasmic/negative nuclear expression such as CHK1, PIAS1, Rad51, and BRCA1, and positive nuclear NHEJ markers showed an association with a poor outcome and adverse prognostic characteristics including high histologic grade, high mitotic frequency, high nuclear pleomorphism and larger tumour size in addition to ER negativity, and triple negative breast cancer (TNBC). Conversely, nuclear+/cytoplasmic- expression showed an association the better outcome. Interestingly, ATM protein expression showed no association with the expression of the two NHEJ markers, whereas ATR showed an association with cytoplasmic expression of BRCA1 and BARD1 and was positively associated with NHEJ markers. In non-TNBC, tumours showing BRCA1-/KU70/KU80- phenotype had worse breast cancer specific survival (BCSS) than positive expression (P<0.0001), whereas in the TN cohort,complex of KU70/KU80-&DNA-PK+ had the worst BCSS (P=0.001), and both are independent prognostic markers for BC.
KPNA2, but not NPM was highly associated with poor BCSS (P<0.0001). At least one of nucleocytoplasmic transport markers (NPM or KPNA2) was significantly associated with the subcellular localisation of the most of the markers that showed cytoplasmic expression including SMC6L1, γH2AX, BRCA1, BARD1, UBC9, PIAS1 ,Rad51 and CHK1. RPPA was used to investigate the protein expression in different cell lines, although the correlation between RPPA and IHC was not significant, the results of RPPA were consistent with that demonstrated by IHC further supporting the finding of the current study.
Conclusion: This study highlight the complexity of DDR related proteins and the overlap between different pathways involved in DDR. The finding of this study may help in the classification of BC and therefore, targeting active pathways in the development of drugs would enhance better patients’ outcomes. Major prognostic and predictive variables can be very important in choosing suitable treatment plans, identifying the risk of recurrence and classifying patients for clinical trials. Our results show that the HR- repair marker Rad51, complex of HR and NHEJ repair markers (BRCA1&KU70/KU80) in non-TNBC, and a complex of NHEJ markers (KU70/KU80&DNA-PK) are all independent prognostic markers for BC. In addition to expression, subcellular localisation of DDR proteins appeared to be a major factor in their role. Particularly, HR repair markers (but not NHEJ) showed worse features of cytoplasmic location of expression, whereas nuclear expression was associated with more favourable features. Finally, the results of this study provide further evidence to support combined use of IHC with the parallel analytic capability of protein microarray RPPA to investigate protein alterations in human tumours.
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