Oncogenic functions of IGF1R and INSR in prostate cancer include enhanced tumor growth, cell migration and angiogenesis

Abstract

We scrutinized the effect of insulin receptor (INSR) in addition to IGF1R in PCa using in vitro and in vivo models. In-vitro overexpression of IGF1R and INSRA, but not INSRB increased cell proliferation, colony formation, migration, invasion and resistance to apoptosis in prostate cancer cells (DU145, LNCaP, PC3). Opposite effects were induced by downregulation of IGF1R and total INSR, but not INSRB. In contrast to tumor cells, non-cancerous epithelial cells of the prostate (EP156T, RWPE-1) were inhibited on overexpression and stimulated by knockdown of receptors. In-vivo analyses using the chicken allantoic membrane assay confirmed the tumorigenic effects of IGF1R and INSR. Apart of promoting tumor growth, IGF1R and INSR overexpression also enhanced angiogenesis indicated by higher vessel density and increased number of desmin-immunoreactive pericytes. Our study underscores the oncogenic impact of IGF1R including significant effects on tumor growth, cell migration, sensitivity to apoptotic/chemotherapeutic agents and angiogenesis, and characterizes the INSR, in particular the isoform INSRA, as additional cancer-promoting receptor in prostate cancer. Both, the insulin-like growth factor receptor 1 and the insulin receptor exert oncogenic functions, thus proposing that both receptors need to be considered in therapeutic settings.

Figure 1. IGF1R/INSRA expression levels influence PCa cell proliferation and colony formation potential but have minor effects on cancer stem/progenitor cell marker levels

A) IGF1R/INSRA impact on PCa cell proliferation. New DNA synthesis determined by thymidine incorporation assay was measured to assess cell proliferation in PCa cells (DU145, DuCaP, LNCaP and PC3) and non-cancerous prostate cells (EP156T and RWPE-1) following IGF1R, INSRA or INSRB overexpression using overexpression plasmids as well as IGF1R, INSR or INSRB downregulation applying specific siRNAs. B) IGF1R/INSRA modulate the colony formation potential of PCa cells. Relative number of colonies of PCa cells (DU145, DuCaP, LNCaP and PC3) and non-cancerous prostate cells (EP156T and RWPE-1) following IGF1R/INSR overexpression and downregulation was determined by 2D colony formation assay. Not only colony sizes, but also colony numbers were strongly influenced by cellular IGF1R/INSR expression levels. C) Identification of the cancer stem/progenitor cell marker panel CD24^low/CD44^high/CD49b^high in PCa cells overexpressing IGF1R, INSRA and INSRB (data shown for PC3). Cells transfected with IGF1R/INSRA/INSRB overexpression plasmids were analyzed for CD24, CD44 and CD49b expression and compared to cells transfected with ctrl plasmid. On the right representative dot blots of CD49b positiv control cells and cells overexpressing IGF1R, INSRA and INSRB, respectively, analyzed for CD44 and CD24 expression are shown. D) ALDH activity in PCa cells overexpressing IGF1R/INSR (data shown for PC3 cells). ALDH activity was analyzed by flow cytometry and compared to control cells. A specific ALDH inhibitor (DEAB) was used as a control for each sample to define and subtract background fluorescence. E) ALDH1 mRNA levels in PCa cells following IGF1R/INSR overexpression (data shown for PC3 cells). Data are presented as mean ± SD of a minimum of four independent experiments. Statistics, Student's t-test.

Figure 2. IGF1R/INSRA expression levels influence PCa cell migration, invasion and resistance to apoptosis

A) IGF1R/INSA influence PCa cell migration. Migrated PCa (DU145, DuCaP, LNCaP and PC3) and non-cancerous prostate cells (EP156T and RWPE-1) normalized to total cell numbers were analyzed in a Boyden chamber assay following IGF1R/INSR overexpression or downregulation using overexpression plasmids (IGF1R, INSRA, INSRB) and specific siRNAs (IGF1R, INSR, INSRB), respectively. B) IGF1R/INSA influence PCa cell invasion. Invaded cells were analyzed in a Matrigel-coated Boyden chamber assay and normalized to total cell numbers. C-D) IGF1R/INSRA levels impact on PCa sensitivity to apoptotic stimuli such as treatment with docetaxel or cylcohexamide. PCa cells (PC3 cells shown) were transfected with overexpression plasmids or siRNAs specific for IGF1R/INSR prior exposure to apoptotic stimuli. Apoptosis was measured by determining caspase 3/7 activity (Caspase activity ELISA, C) or propidium iodide staining (flow cytometry analysis, determination of sub-G1 fraction, D). Data are presented as mean ± SD of a minimum of four independent experiments. Statistics, Student's t-test.

Figure 3. IGF1R/INSR expression levels influence tumor growth and tumor-infiltrating blood vessel density in vivo.

Data were achieved using the chicken chorioallontoic membrane (CAM) assay and the PCa model system PC3. A) Successful target overexpression (IGF1R, INSRA, INSRB) or downregulation (IGF1R, INSR, INSRB) was confirmed by qPCR after harvesting of the onplant tumors B) IGF1R/INSR expression levels influence tumor size in vivo. C) Tumor IGF1R/INSR levels impact on the amount of tumor-infiltrating vessels determined by visualization of desmin-immunoreactive pericytes by immunohistochemistry. D) Representative pictures of tumor onplants and anti-desmin staining to visualize tumor-infiltrating blood vessels. Data are presented as mean ± SD of three independent experiments analyzing four different onplants per CAM and treatment. Statistics, Student's t-test.