Investigating high circulating IGF-1 as a risk factor for development of prostate cancer
Keywords: prostate cancer
IGF-1
IGF

Prostate cancer is the commonest cancer in UK men. Prostate cancers upregulate type 1 insulin-like growth factor receptor (IGF-1R), and men with high serum IGF-1 have an increased risk of prostate cancer and of dying from prostate cancer. Subjects with low circulating IGF-1 are strongly protected from cancer; together with preclinical and bioinformatic data this supports a causative association. Unpublished Macaulay lab immunohistochemistry (IHC) data found association of high serum IGF-1 with IGF-1R upregulation in malignant prostatic epithelium, and a trend to association with nuclear IGF- 1R, which has a transcriptional role. IGFs are also reported to be immunosuppressive, and could therefore influence anti-tumour immune responses. The main aim of this project was to investigate changes in the tumour epithelium and tumour microenvironment (TME) through which high IGF-1 promotes prostate cancer risk and progression. Firstly, IHC in 6 prostate biopsies showed a trend to increased IGF-1R and significant increase in internalised IGF-1R, suggesting receptor activation, in malignant vs benign prostate epithelium. Chromogenic in situ hybridisation in 17 prostatectomies showed IGF1R mRNA upregulation in malignant areas indicating transcriptional regulation. In prostate cancer cell lines, I found that short (24 hrs) and long term (8-12 weeks, to mimic lifelong high IGF supply) culture in IGF-1 downregulated IGF-1R. This may result from feedback of pathway activation and is inconsistent with tissue level findings. IGF-1 did cause persistent increase in nuclear IGF-1R, which has been associated with advanced prostate cancer stage. Secondly, I tested effects of IGF-1 on the immune mediators in prostate cancer cell cultures. IGF-1 upregulated immune associated genes CXCL8, CXCL10, CCL5, IFNB1 and ARG2, reported to be upregulated by the NF-κB and cytosolic DNA sensing cGAS-STING pathways. In DU145 prostate cancer cells IGF-1 caused phosphorylation of cGAS-STING pathway proteins IRF3 and TBK1, however the mechanism of this effect is not yet clear. IGF-1 also resulted in the transcriptional upregulation of CD274, encoding PD-L1, in prostate cancer and colorectal cancer cells. Analysis of TCGA data suggested that prostate cancers with high endogenous IGF1 mRNA had higher expression of CD274, consistent with my cell line data, and of S1P, CXCL12, CCL5 and CCL4, which are associated with increased tumour infiltration of CD4+ T regulatory (Tregs) and immunosuppressive M2 macrophages. Finally, association of IGF axis components with the TME was characterised using multiplex immunofluorescence (M-IF) and RNA sequencing (RNA-seq). In prostatectomies of men with known serum IGF-1, M-IF and RNA-seq were conducted on 32 Formalin-Fixed Paraffin-Embedded tissues. Patients with high circulating IGF-1 had increased tumour infiltration of CD4+ T cells, a trend to increased PD-L1 positivity but no association with CD4+ or FOXP3+ Treg positivity. RNA-seq analysis on tumour areas was challenging due to poor quality of extracted RNAs, and no differentially expressed genes were identified in tumours of patients with high circulating IGF-1. However, dividing cases by high vs low endogenous IGF1 indicated deregulation of focal adhesion, leukocyte transendothelial migration and macrophage associated pathways. In high IGF1 tumours, upregulated genes included IGF pathway genes FOXO1, BCL2, ZEB1, IGFBP5 (a marker of IGF axis activation), and chemokine CXCL12 that promotes chemotaxis of lymphocytes including Tregs. Downregulated genes included TAP1, required for antigen transport and presentation, SOX4 and NF-κB regulator TRAF4. In 9 fresh frozen prostate tumour samples, single cell RNA-seq showed infiltration of M2 macrophages and MDSCs, with enrichment of IGF1 in tumour infiltrating fibroblasts and of IGF-2 scavenger IGF2R in myeloid cells and proliferating CD8 and NK cells. In summary, IGF-1 promoted sustained nuclear IGF- 1R translocation and upregulated PD-L1 and cytokine expression in prostate cancer cells, and endogenous IGF1 associated with CXCL12 upregulation and TAP1 downregulation, supporting an immunosuppressive role for IGF-1 in localised prostate tumours. These findings have potential relevance to prostate cancer risk, immunotherapy and risk of lethal disease.