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Title: The role of IGF-1R signaling in the DNA damage response of prostate cancer
Author: Lodhia, Kunal
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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The type 1 insulin like growth factor receptor (IGF-1R) is a cell surface receptor that mediates proliferation and cell survival. The aim of this project was to explore the contribution of the IGF-1R to the DNA damage response in human prostate cancer cells. Previous work from our group showed IGF-1R to be over expressed in prostate cancer. Furthermore, IGF-1R depletion enhances sensitivity of prostate cancer cells to DNA damaging agents, and IGF-1R depletion or inhibition delays DSB repair. Prior work in melanoma cells indicated that IGF-1R down-regulation enhances radiosensitivity and impairs the function of ATM, a key protein in the response to DNA DSB. This led to the initial hypothesis that IGF-1R signalling can influence ATM function, possibly via the PI3K-AKT pathway. Two putative AKT phosphorylation sites were identified on ATM and initial experiments detected a 350 kDa protein equivalent to the size of ATM, that was phosphorylated on RXRXXS/T motif(s) in response to IGF-1. However, it did not prove possible to identify this immunoreactivity in ATM immunoprecipitates, and AKT inhibition, although radiosensitizing, did not induce features of defective ATM function. Therefore this project was halted and two new projects exploring the role of IGF signalling in DNA repair were undertaken. The first project continues from work published from our laboratory showing that IGF-1R undergoes nuclear translocation in prostate cancer cells. The function of nuclear IGF-1R is unclear, and mass spectrometry (MS) was utilized to identify potential nuclear binding partners. Nuclear extraction and IGF-1R immunoprecipitation methods were modified in order to achieve optimal conditions for subsequent MS analysis. Three independent MS analyses have been performed aiming to identify binding partners of nuclear IGF-1R in cells grown in complete medium, cells that were serum-starved and IGF-1 treated, or exposed to irradiation-induced DNA damage. Candidate binding partners included transcription factors such as Bruton’s tyrosine kinase associated protein 135 (BAP-135) also known as general transcription factor II-I (TF II-I), suggesting possible involvement of nuclear IGF-1R in the regulation of transcription. Initial validation experiments suggested IGF-1 induced complex formation between IGF-1R and TF II-I, and also tyrosine phosphorylation of TF II-I in response to IGF-1, raising the possibility that IGF-1 influences TF II-I function. The second project is based on a high throughput screen completed by another member of our laboratory, that sought to identify DNA repair-related proteins that when depleted increased the sensitivity of prostate cancer cells to IGF-1R inhibition. One of the potential hits is RAD51, a recombinase central to the strand invasion step of homologous recombination (HR). Initial experiments validated RAD51 knockdown by the screen siRNAs, and showed that RAD51 depletion did indeed enhance sensitivity to a novel IGF-1R inhibitor in PTEN wild type prostate cancer cells. Loading of RAD51 onto single stranded DNA requires BRCA2, and in a PTEN wild type colorectal cell line model, lack of BRCA2 was shown to sensitize colorectal cancer cells to IGF-1R inhibition, compared to isogenic cells that expressed functional BRCA2. Finally, experiments also sought to mimic effects of RAD51 depletion using a small molecule CDK1inhibitor, R0-3306, recently shown to impair HR by inhibiting BRCA1 function. In prostate cancer cells, R0-3306 at the determined GI50 value (1 μM) suppressed formation of RAD51 foci, consistent with HR attenuation. Furthermore, CDK1 inhibition phenocopied the effects of RAD51 depletion, sensitizing prostate cancer cells to IGF-1R inhibition with a 2.4 fold reduction in GI50 value and 13 fold reduction in the GI80. These data suggest that the sensitivity of human prostate cancer cells to IGF-1R inhibition can be enhanced by genetic and chemical approaches to suppress HR, and that BRCA mutant cancers may be intrinsically sensitive to IGF-1R inhibition. The ultimate aim of this work is to understand how IGF-1R biology influences DNA damage repair, in order to guide the development of new treatments for prostate cancer.
Supervisor: Macaulay, Val; Aleksic, Tamara Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available