Investigating the biology of nuclear IGF-1R in prostate cancer

Insulin-like growth factor 1 receptor (IGF-1R) is a transmembrane receptor tyrosine kinase comprised of two extracellular α- and two transmembrane β-subunits in a heterotetrameric structure. IGF-axis signalling is implicated in cancer progression, metastasis and resistance to treatment including radiotherapy. Recent work by our lab and others has shown the presence and activity of IGF-1R in the nucleus of a range of cancer cell types, interacting with nuclear proteins and undergoing recruitment to chromatin at gene promoters. Nuclear IGF-1R has been detected in malignant but rarely in benign epithelia, and higher levels of nuclear IGF-1R have been shown to be associated with adverse clinical outcomes. Responses in specific subsets of patients have also suggested nuclear IGF-1R may be a potential biomarker for tumours that may be more responsive to anti-IGF-1R targeted treatment. Therefore, nuclear IGF-1R may be a tumour selective property of the IGF-axis that could act as a novel target for treatment. This thesis aimed to further investigate the biological role of nuclear IGF-1R including screening of potential novel inhibitors specific for nuclear IGF-1R, identification of IGF-1R chromatin binding sites in vivo, and identification of novel nuclear interacting proteins to further elucidate the role of nuclear IGF-1R in cancer progression and as a treatment target.
The first aim of this work was to investigate the role of the extracellular α-subunit of IGF-1R in the nucleus, as previous work focussed on the β-subunit only. IGF-1Rα-subunit was detected in the nucleus of two prostate cancer cell lines, DU145 and 22Rv1, by co-immunoprecipitation, immunofluorescence microscopy and chromatin immunoprecipitation (ChIP). These experiments showed for the first time that IGF-1Rα- and β-subunits interacted in the nucleus, levels of nuclear IGF-1Rα-subunit were IGF-responsive, and IGF-1Rα-subunit was recruited to chromatin at β-subunit binding sites identified previously by our group. These data suggest that α-subunit contributes to the function of nuclear IGF-1R.
The second objective, based on potential tumour selective properties of nuclear IGF-1R, aimed to develop a screening platform for small molecule inhibitors of nuclear IGF-1R. Despite multiple attempts to optimise scaled-down immunofluorescence and generate stable IGF-responsive luciferase reporters incorporating IGF-1R DNA binding sites, this screen could not be undertaken. However, these luciferase reporter plasmids can be used in future investigation of the transcriptional function of nuclear IGF-1R.
The third aim was to assess the clinical relevance of recruitment of IGF-1R to chromatin in vivo. To assess this, ChIP for IGF-1R was performed from fresh frozen tissue samples obtained from patients undergoing radical prostatectomy. Sequencing analysis detected genome wide recruitment of IGF-1R to chromatin in vivo for the first time. IGF-1R peaks were shown to cluster proximal to gene transcription start sites (TSS) and 38% of these peaks were coincident with H3K4me1, a histone mark for active enhancer regions. This suggests IGF-1R is recruited to regulatory DNA regions at these sites, and therefore may play a role in influencing gene expression. Further work is now needed to determine whether there is a link between IGF-1R recruitment and gene expression at the transcriptional and/or epigenetic level and therefore the contribution of IGF-1R recruitment to chromatin to cancer growth and progression in vivo.
The final aim was to investigate the nuclear interactome of IGF-1R using 22Rv1 clones expressing WT IGF-1R or IGF-1R null, generated by another student and shown to be radiosensitive. Proteins co-precipitating with IGF-1Rβ in 22Rv1 WT cells were analysed by mass spectrometry. Protein hits were ranked by peptide numbers detected in WT vs IGF-1R null cells, and the top hit was the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), required for DNA repair by non-homologous end-joining (NHEJ). Co-precipitation validated the interaction of nuclear IGF-1R with DNA-PKcs and with phospho-S2056 DNA-PKcs, an auto-phosphorylation that occurs upon recruitment to chromatin. Consistent with a requirement for IGF-1R, irradiation-induced DNA-PKcs chromatin recruitment and S2056 phosphorylation were reduced in IGF-1R null cells. Further work is needed to investigate whether direct interaction with nuclear IGF-1R and/or downstream IGF-1R signalling contribute to DNA-PKcs function and radioresistance.
In summary, this work has shown a biological role in prostate cancer for the IGF-1Rα-subunit in the nucleus. For the first time, this work detected recruitment of IGF-1R to chromatin binding sites in vivo in malignant prostate cancer tissue at sites proximal to gene TSS and in some cases coincident with histone marker for active enhancer regions. Further in vitro work also showed an interaction between nuclear IGF-1R and DNA-PKcs that may play an important role in DNA-PKcs recruitment to chromatin. These factors may have significance for the biological role of nuclear IGF-1R in cancer progression and may contribute to the clinical relevance of nuclear IGF-1R in prostate cancer.

Keywords: ChIP-qPCR
co-immunoprecipitation
prostate cancer
mass spectrometry
tissue ChIP-seq
nuclear receptor
nuclear interactome
nuclear translocation
insulin-like growth factor-1 receptor
luciferase assays
Advisor: Macaulay, V
Mills, I