Arora, Arvind
(2019)
Exploring RECQ Helicase landscape in breast cancer.
DM thesis, University of Nottingham.
Abstract
Background
Breast cancer is the second most common cause of cancer death in females in the UK. Approximately 20 percent of breast cancers diagnosed are triple negative (TNBC) intrinsic subtype, which are aggressive high-grade tumours carrying a poor prognosis. About 20% of TNBC harbour a mutation in breast cancer gene (BRCA). BRCA plays important role in homologous recombination. There is evidence to suggest that non-familial breast cancers harbour homologous recombination repair defects that may be useful targets for therapy.
The RECQ protein family is a highly conserved group of DNA helicases, which play diverse roles in various DNA metabolic processes such as DNA replication and recombination repair. In humans, five RECQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) have been identified and three of them namely, WRN, BLM, and RECQL4 have been associated with autosomal recessive disorders which are characterized by genomic instability, premature aging, and predisposition to various cancers. The role of RECQ helicase expression in sporadic breast cancer pathogenesis is unknown. We hypothesized that dysregulation in RECQ helicase expression may have an impact on breast cancer pathogenesis and survival. The aim of this thesis was to have a comprehensive assessment of the prognostic, predictive and clinicopathological significance of five RECQ helicases at the transcriptome and protein level.
Methods
Investigation of the protein expression of RECQ helicases in breast cancer was performed in two patient cohorts. The first cohort was a series of 1650 primary invasive breast cancer patients who were diagnosed between 1986 and 1999 and entered into the Nottingham tenovus series. The second cohort used for evaluation was an independent series of 252 ER-negative invasive breast cancers diagnosed and managed at Nottingham University Hospitals between 1999 and 2007. Standard immunohistochemistry technique was used for staining the breast TMA (tissue microarray) and assessment of staining was done using H-scoring. Evaluation of the mRNA expression was done in the METABRIC dataset (1977 cases). Data analysis was performed using SPSS (SPSS, version 22 Chicago, IL). Frequency histogram distributions and X tile (Version 3.6.1) were used to establish the cut-offs for expression values such that the resulting subgroups have significantly different survival courses. Cumulative survival probabilities were estimated using the Kaplan–Meier method. Statistical significant differences between survival outcomes were tested using the log-rank test. P values for each test were adjusted with (Benjamini & Hochberg, 1995) multiple P-value adjustments and an adjusted p-value of <0.05 was considered significant. Multivariate analysis with survival Cox proportional hazard model was performed where a statistically significant survival outcome was seen in the univariate analysis. Cytotoxicity of Cisplatin in BLM Ctr and BLM KD Hela cells was assessed using clonogenic and MTS cell survival assays. Functional consequences of Cytotoxicity in BLM KD Hela cell lines was determined using flow cytometry technique. Weasel (Victoria, Australia) flow cytometry analysis software was utilized for data analysis. Graphical visualisation and statistical analysis were carried out in GraphPad Prism 6 (GraphPad, La Jolla, USA). Two way ANOVA was used to determine the statistically significant differences between the variables.
Results
Low RECQL1 expression showed a statistically significant association with aggressive breast cancer phenotypes. Furthermore, low RECQL1 was linked to poor breast cancer survival (p<0.05) in the whole cohort and ER-positive patients including those who had adjuvant endocrine therapy, suggesting the prognostic and predictive significance of RECQL1 in sporadic breast cancers.
High RECQL5 mRNA expression and high RECQL5/low RAD51 protein expression revealed a statistically significant association with aggressive breast cancer phenotypes and poor survival which was more pronounced in ER-positive subgroup. This suggests the predictive and prognostic significance of RECQL5 in breast cancer. RECQL5 remained an independent predictor of survival on multivariate analysis.
RECQL4 amplification/gain in copy numbers, high RECQL4 mRNA, expression and low nuclear/high cytoplasmic RECQL4 protein expression were associated with aggressive breast cancer phenotypes, and poor survival (p<0.05). In tumours with high nuclear RECQL4 protein expression, c-MYC could stratify patients into prognostic groups (p<0.05).This confirms the clinicopathological, predictive and prognostic significance of RECQL4 in breast cancer.
Low WRN mRNA levels were significantly associated with poor breast cancer-specific survival in the whole cohort and ER-positive patients including those who had adjuvant endocrine therapy, informing the prognostic and predictive significance. At the protein level, WRN protein expression remained an independent predictor of survival on multivariate analysis. ER-positive tumours, including those who had endocrine therapy, with high TOPO1/high WRN expression showed poor breast cancer-specific survival (p<0.05).
High BLM mRNA levels were significantly associated with aggressive breast cancer phenotypes and poor breast cancer-specific survival in the whole cohort and ER-positive tumour including those who had endocrine therapy, showing predictive and prognostic significance. Low nuclear BLM/low nuclear RAD51 protein levels were associated with poor survival(p<0.05) in the whole cohort and ER-negative patients including those who had chemotherapy showing resistance to chemotherapy. BLM mRNA expression remained an independent predictor of survival on multivariate analysis. Moreover, preliminary cell line work showed that BLM-KD HeLa cells were sensitive to platinum chemotherapy.
Conclusion
RECQ helicases expression in breast cancer clearly has predictive, prognostic, and clinicopathological significance. RECQ helicases could have a synthetic lethality relationship with PARP inhibitors or other DNA repair inhibitors. It is important to do further cell line work in breast cancer cell lines through BLM knockdown with siRNA and Knock out with CRISPR cas-9.
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