Cell type specificity of progesterone action in the normal human breast and in breast cancer

Bent, F. van der (Fleur) (2014) Cell type specificity of progesterone action in the normal human breast and in breast cancer. thesis, Medicine.

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Abstract

At the end of the 19th century it was recognized that the ovarian hormones, progesterone (P) and estrogen, play a critical role in breast cancer, as removal of the ovaries resulted in effective treatment of patients with advanced breast cancer. Although the role of estrogen in the development of breast cancer has been well described, the role of P is still poorly understood. Only recently more evidence has accumulated which links P to the differentiation and proliferation of the epithelial cell compartment in the human breast.The exact molecular mechanisms by which P increases proliferation in the breast are not yet fully established, but growing evidence suggests that the DNA replication licensing machinery, which ensures the cell replicates DNA only once per cell cycle, and therefore is a caretaker of genomic stability, is influenced by. The tumor suppressor protein, BRCA1, has an important role in DNA repair, but also plays a role in proliferation and differentiation of the mammary epithelial compartment, as several studies have shown that knockdown of BRCA1 exaggerates the proliferative potential of the mammary gland in mice. Normally, an exaggerated proliferative response must be counteracted by the process of programmed cell death, apoptosis, to prevent uncontrollable tissue growth and maintain tissue homeostasis, and both BRCA1 and P are able to induce this process. In addition, both P and BRCA1 can influence cell proliferation. This is important as both proliferation and differentiation play a role in tumorigenesis, and dysregulation of these processes could lead to an increased risk of breast cancer. Growing evidence now suggests that BRCA1 and P can interact and potentially contribute to carcinogenesis in a number of different ways. Firstly, repression of carcinogenesis by BRCA1 has been attributed to its regulation of both the estrogen receptor (ER) and progesterone receptor (PR). Furthermore, BRCA1 and P can exert their functions on overlapping targets of the DNA replication licensing machinery. From the information described above, we hypothesised that BRCA1 inhibits proliferation of the mammary epithelial cell compartment, and that in BRCA1 mutation carriers this repression of proliferation induced by P is lost, leading to an increased cancer risk in carriers of this genetic predisposition. To test this hypothesis we compared the transcriptomes of P-treated wild type BRCA1 T47D breast cancer cells with that of T47D cells where BRCA1 expression was knocked down. To establish BRCA1 knockdown, we constructed a lentiviral vector with two different short hairpin RNA sequences and confirmed its repression by quantitative PCR. We then performed microarray analysis and genes which were significantly regulated by P, with or without BRCA1 knockdown, were identified using Genome Studio software and Excel, and clustered using the database for annotation, visualization, and integrated discovery (DAVID) tool. We identified that many of the genes upregulated by P in the BRCA1 knockdown cells were associated with apoptosis. From a list of those targets which displayed the highest fold upregulation by P, we observed that interestingly, many appeared to be further upregulated by P when BRCA1 was knocked down. We identified that most of these genes were involved in inhibition of the apoptotic process, thus promoting cell survival. Furthermore, we found a number of targets which were involved in cell motility and migration. We selected 9 of these targets for validation. From the results obtained in this study we can conclude that BRCA1 can regulate the expression of P-induced anti-apoptotic genes, as knockdown of BRCA1 caused significantly higher expression of P regulated genes involved in the anti-apoptotic process, thus promoting cell survival. In addition, knockdown of BRCA1 caused higher expression of a number of P regulated genes involved in cell motility. These two features together, that is, resistance of tumour cells to cell death and a higher metastatic potential, could possibly account for the aggressive phenotype of breast cancers seen in carriers of the mutant BRCA1 gene.

Item Type:	Thesis (Thesis)
Supervisor name:	Kallenberg, Prof. dr. C.G.M.
Supervisor name:	Hilton, Dr. H.N. and Clarke, Prof. C.L. and Westmead Institute for Cancer Research, Sydney and University of Sydney at Westmead Millennium Institute
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 11:05
Last Modified:	25 Jun 2020 11:05
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/2499

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