Zino, Samer M. Walid
Investigations into the expression of sirtuins in breast cancer: in vivo and in vitro studies.
PhD thesis, University of Glasgow.
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Breast cancer remains the most common malignancy in women and a major cause of morbidity and mortality in the western World, despite the advances in diagnosis and treatment. The main challenge remains to identify new, and improve existing treatment modalities. Understanding the mechanism by which tumours grow and metastasise is key to developing new therapeutic targets
Similar to most cancers, the incidence of breast cancer increases with age. Therefore, genes involved in biological ageing and factors affecting genomic integrity, considered critical to cellular senescence and organismal life span, are also relevant to neoplastic transformation and tumour growth. Thus exploring factors associated with biological ageing in cancer may improve our understanding of the disease as an aberration of normal biological ageing and result in new prognostic markers or therapeutic targets. There is increasing evidence for the involvement of sirtuins in biological ageing, along with other essential cellular processes including cell cycle control, DNA damage repair and differentiation. This is suggestive of a possible role for sirtuins in cancer. Therefore, this study was conducted to investigate a potential role for sirtuins in breast cancer disease (including anti-tumour treatment).
Firstly, Real time PCR was used to compare the transcriptional expression level of individual sirtuin genes in vivo. The experimental result showed that only SIRT1 and SIRT4 showed an association with age in “normal patients” (normal and non malignant patient grouped together), with decreasing levels of SIRT1 and increasing levels of SIRT4 being associated with increasing chronological age. All sirtuin levels were significantly decreased in malignant tumours, when compared to either normal and/ or non-malignant biopsies. Decreased relative transcriptional expression of SIRT1, SIRT2, SIRT3, SIRT6 and SIRT7 showed significant association with higher tumour grade, when breast cancer patients were divided according to the known histopathological markers.
The Kaplan-Meier analysis for cancer specific survival and tumour recurrence was preformed on entire patient cohorts and in patient subgroups selected to have moderate prognosis (ER+ve and NPI between 3.4 and 5.4). The Kaplan-Meier survival analysis showed that higher levels of SIRT6 and SRT7 were associated with a longer survival period in all patient cohorts. Furthermore, higher levels of SIRT6 remained significantly associated with better survival, when breast cancer patients were selected to have intermediate prognosis (ER+ve and NPI between 3.4 and 5.4). Whereas, higher levels of SIRT7 remained significantly associated with longer survival period only in patients with ER+ve tumours. The Kaplan-Meier analysis showed that lower levels of SIRT1 gene expression were associated with longer patient survival and lower tumour recurrence in a patient group, selected by NPI, to have an intermediate clinical prognosis. Multivariate Cox-regression analysis demonstrated that the relative transcriptional level of the SIRT6 gene was independent of tumour size, grade, nodal status, oestrogen receptor status, lymphovascular invasion status, and the NPI in influencing survival.
The decreased sirtuin gene expression observed in this study is consistent with an anti-cancer effect and suggests that sirtuins might be implicated in breast cancer pathogenesis. For example, decreased levels of SIRT2 might assist DNA-damaged tumour cells, as indicated by the decreased expression of another sirtuin involved in DNA damage responses, SIRT6, in escaping cell cycle arrest during tumour initiation and progression. Furthermore, The associations between sirtuins and survival period suggest that these sirtuins (especially SIRT6) might be used as an additional prognostic marker in breast cancer patients, especially in those individuals who have equivocal prognostic pathological markers. Therefore, the level of expression of sirtuin genes (SIRT6) might help explaining those breast cancer cases, which behave unexpectedly, according to the known pathological prognostic markers.
Secondly, The changes in the relative transcriptional expression levels of the sirtuin genes were investigated in response to adjuvant chemotherapy therapies, commonly used in breast cancer (Tamoxifen and Docetaxel), in breast caner cell lines. The first experiment determining sirtuins changes in response to Docetaxel treatment for 72 hours in ER-ve breast cancer cell line (MDA-MB-231) showed significant increase in the relative transcriptional expression levels of all sirtuins after Docetaxel treatment. These data were consistent with the pro-apoptotic role for SIRT1, SIRT3 and SIRT7, and suggestive of DNA damage involvement at higher doses of Docetaxel, as indicated by increased SIRT6 and XRCC5. Finally, increased SIRT2 levels are suggestive of SIRT2 involvement in the mitotic arrest caused by Docetaxel, through its contribution to microtubule dysfunction.
The second experiment, determining sirtuin changes in response to Tamoxifen treatment in ER+ve (MCF-7) and ER-ve (MDA-MB-453) breast cancer cell lines, showed significant increase in the relative transcriptional expression levels of all sirtuins after Tamoxifen treatment. These data were consistent with the pro-apoptotic role for sirtuins. Furthermore, the observed increased levels of SIRT6 are suggestive of DNA damage involvement at higher doses of Tamoxifen. Another noteworthy result of this experiment is the increased levels of SIRT2 in response to Tamoxifen treatment. This might explain the failure of a TAM-treated cell to proceed through the cell cycle, in spite of the increases in transcription factors that promote cell cycle after Tamoxifen treatment. There was no significant difference in sirtuin changes after Tamoxifen treatment between these two cell lines to indicate that sirtuin changes were ER-dependent.
In total, the data accumulated from this study demonstrated the involvement of sirtuins in breast cancer disease (pathogenesis and anti-tumour treatment) and suggest the possible use of SIRT6 as a novel, additional and biological prognostic marker. Finally, this study suggests that sirtuins activators, rather than inhibitors, might be beneficial in breast cancer disease and enhance the response to adjuvant chemotherapy.
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