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Title: The role of corticotropin-releasing hormone (CRH) in cellular models of breast cancer
Author: Lal, Suchita K.
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2013
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The role of CRH and CRH related peptides in mediating HPA axis and various pathophysiological processes is well known. Over the last decade the role of CRH in mediating cancer has been widely studied and considered to exert variable functions. This study was undertaken to study the biological role of CRH in ER(+) and ER(-) breast cancer cell lines. Since estrogen is related to normal as well as the neoplastic growth, the CRH effects were studied with and without estrogen in ER (+) MCF7 and ER (-) SKBR3 cell lines. The role of the stress hormone CRH in breast cancer is complex, and its abundance and biological activity may be modulated by estrogen. The signaling mechanisms were investigated by employing phosphokinase assay to identify protein kinases activated by CRH in ER(+) MCF7 cells. CRH activated numerous kinases and downstream effectors, at least some of which were mediated by the CRH receptor type 1 (CRH-R1). MAPK, GSK3β and Akt were further investigated to study downstream effects. The analysis of the spatiotemporal characteristics of MAPK activation suggested that CRH mediated p38 response is strong compared to ERK1/2 which is inhibited by the CRH in MCF7 cells. UCN II also showed a similar response, but the extent of p38 response is not as strong as with CRH. The MAPK effects were studied in SKBR3 cells and interestingly, the CRH and UCN II mediated effect were inhibited by 24 hrs of estrogen treatment. CRH also increased the transcription of many genes that encode effectors, transcriptional targets, or regulators associated with estrogen signaling. CRH mediates its effects by activating two types of CRH receptors, i.e R1 and R2. The tissue sensitivity to agonists is determined by the presence of receptors in the plasma membrane and signal activation. This project was undertaken to investigate cellular expression of CRH-R and CRH-R1 splicing variants (α,β,c,d) and their internalization characteristics. CRH-R has been confirmed in both the cell lines. One of the goals of this project was to identify the expression of CRH-R1 splicing variants in both the cell lines with and without estrogen. CRH-R1α and R1d expression was confirmed in the ER (+) cells. However the CRH-R1d expression was lacking in SKBR3 cells. The CRH mediated splicing of CRH-R1 receptor was dose dependent. This splicing event is regulated by the splicing factors, thus in silico analysis was performed to identify splicing factor, with high affinity binding at exon 12 which is missing in CRH-R1d. It was hypothesized that Estrogen regulated these effect downregulating serine/arginine-rich splicing factor 55 (SRp55) expression resulting in generation of CRH-R1d. The results show that E2 is a driving factor influencing CRH-R1 gene exon 12 splicing and causing an increase in the expression of CRH-R1d. Immunofluorescence demonstrated that CRH treatment initiates the internalization of receptor inside the cytoplasm but this effect is lost when cells were treated for 24 hrs with E2, probably due to generation of CRH-R1d which loses internalization properties. This effect is lost in the absence of E2 receptors (SKBR3 cells).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology ; QP Physiology