Investigating the role of IGF-1R in radio-sensitivity

The insulin-like growth factor (IGF) axis is an important regulator of cancer cell proliferation, growth, invasion, and can also mediate therapy resistance. Inhibition or depletion of the type 1 IGF receptor (IGF-1R) has been shown by our laboratory and others to enhance sensitivity to ionising radiation (IR) and chemotherapy, and to impair repair of DNA double-strand breaks (DSBs). Our group also reported association of high IGF1R expression with radio-resistance and biochemical recurrence after IR for prostate cancer (PCa). The overall aim of this project was to investigate the molecular basis for IGF-1R-mediated radio-resistance using an androgen receptor-positive (AR+) PCa model. The first aim was to recapitulate in AR+ 22Rv1 PCa cells the radio-sensitisation phenotype observed previously. First, IGF1R gene silencing was shown to radio-sensitise AR- DU145 PCa cells as shown previously in our laboratory, with similar radio-sensitisation in 22Rv1 cells. Secondly, to test whether radio-resistance requires the kinase activity or structural functions of the receptor, CRISPR/Cas9 genome editing was used to generate IGF1R-/- 22Rv1 clones. Attempts were also made to generate cells expressing kinase-dead (KD) IGF-1R; although these efforts were unsuccessful, a hypomorphic IGF1R+/- clone was produced. In 2D clonogenic survival assays, IGF1R+/- and IGF1R-/- displayed enhanced radio-sensitivity, with dose enhancement ratios (DERs) of ≥ 1.46 at 2 Gy, comparable to effects of IGF-1R knockdown. IR-induced DNA damage was quantified using H2AX foci, and IGF1R+/- and IGF1R-/- clones showed significantly increased DNA damage 10-60 minutes post-IR, suggesting increased damage induction. The third aim of this project was to perform unbiased proteomic and phosphoproteomic screens on IGF-1R proficient and deficient 22Rv1 cells before and after IR, to generate information on the role of IGF-1R signalling in the response to IR. Examination of the phosphoproteomic dataset suggested IGF-1R-dependent differences in IR-induced serine 3205 phosphorylation of the major DNA damage response (DDR) protein DNA-dependent protein kinase catalytic subunit (DNA-PKcs), required for classical non-homologous end joining (cNHEJ). This finding prompted exploration of a possible role for IGF-1R in cNHEJ. Indeed, experiments suggested that DNA repair dynamics were altered in IGF1R-/- clones, with increase in repair by microhomology-mediated end joining (MMEJ) and significantly reduced p53-binding protein 1 (53BP1) foci after IR compared to IGF-1R WT cells. Both findings suggested that cNHEJ was compromised. There was further evidence of a role for IGF-1R in cNHEJ, with significantly reduced IR-induced autophosphorylation of DNA-PKcs at serine 2056, and apparent absence of DNA-PKcs recruitment to chromatin in IGF1R-/- clones. These data are consistent with the reported need for DNA-PKcs chromatin recruitment to trigger autophosphorylation. In summary, data presented here support a role for IGF-1R in mediating radio-resistance; a KD clone would be needed to clarify whether this property requires the presence of IGF-1R protein and/or its kinase activity. Finally, a novel role was identified for IGF-1R in radio-resistance and cNHEJ by promoting DNA-PKcs recruitment to chromatin and autophosphorylation. These results have significance both for basic biology and clinical practice. The data shed light on the contribution of IGFs to the DNA damage response, with potential to inform treatment options, selection of patients for radiotherapy, and development of novel approaches to radio-sensitisation.

Advisor: Macaulay, V