Both IGF1R and INSR Knockdown Exert Antitumorigenic Effects in Prostate Cancer In Vitro and In Vivo

Abstract

The IGF network with its main receptors IGF receptor 1 (IGF1R) and insulin receptor (INSR) is of major importance for cancer initiation and progression. To date, clinical studies targeting this network were disappointing and call for thorough analysis of the IGF network in cancer models. We highlight the oncogenic effects controlled by IGF1R and INSR in prostate cancer cells and show similarities as well as differences after receptor knockdown (KD). In PC3 prostate cancer cells stably transduced with inducible short hairpin RNAs, targeting IGF1R or INSR attenuated cell growth and proliferation ultimately driving cells into apoptosis. IGF1R KD triggered rapid and strong antiproliferative and proapoptotic responses, whereas these effects were less pronounced and delayed after INSR KD. Down-regulation of the antiapoptotic proteins myeloid cell leukemia-1 and survivin was observed in both KDs, whereas IGF1R KD also attenuated expression of prosurvival proteins B cell lymphoma-2 and B cell lymphoma-xL. Receptor KD induced cell death involved autophagy in particular upon IGF1R KD; however, no difference in mitochondrial energy metabolism was observed. In a mouse xenograft model, induction of IGF1R or INSR KD after tumor establishment eradicated most of the tumors. After 20 days of receptor KD, tumor cells were found only in 1/14 IGF1R and 3/14 INSR KD tumor remnants. Collectively, our data underline the oncogenic functions of IGF1R and INSR in prostate cancer namely growth, proliferation, and survival in vitro as well as in vivo and identify myeloid cell leukemia-1 and survivin as important mediators of inhibitory and apoptotic effects.

Figure 1.

IGF1R and INSR KD attenuated growth of prostate cancer cells. Efficiency of IGF1R and INSR KD by dox induction of shRNAs for 4 days was confirmed at mRNA (A) and at protein (B) level by qRT-PCR and Western blot analysis in shPC3 cells (shLUC control, shIGF1R, and shINSR). C, Representative images of shPC3 cells ± dox incubation for 4 and 12 days. D, Growth of shLUC control, shIGF1R, and shINSR cells ± dox stimulation for 4 and 12 days. E, Analysis of DNA synthesis assessed by measurement of [methyl-³H]-thymidine incorporation ± dox for 4 and 12 days. All data represent mean ± SD from 4 independent experiments (*, P < .05; **, P < .01; ***, P < .001, ANOVA).

Figure 2.

IGF1R and INSR KD eradicated xenograft tumors in nude mice. Seven BALB/c nu/nu mice per group were injected with shIGF1R, shINSR, or shLUC control cells in both flanks. After tumor establishment for 1 week, KD treatment was started by the addition of dox to the drinking water. A, Tumor volume was measured using a Caliper twice a week. Data represent mean ± SEM of 7 mice (14 tumors) (*, P < .05; **, P < .01; ***, P < .001, ANOVA). B, After 20 days, the mice were killed, and tumor weight was measured. C, Representative tumor images of the different treatment groups after preparation. Scale bar, 1 cm. D, Formalin-fixed and paraffin-embedded sections of shLUC, shIGF1R, and shINSR tumors were stained with hematoxylin/eosin (H&E) and scanned for tumor cells. Scale bar, 200 μm.

Figure 3.

Induction of apoptosis upon IGF1R and INSR KD. A, Activity of effector caspases 3/7 was measured in shPC3 cells 4 and 12 days after incubation ± dox (RLU, relative luminescence units). B, Cleaved PARP (cPARP) protein level was analyzed by Western blotting after 4 and 12 days ± dox (representative Western blotting). C, Sub-G₁ phase apoptotic cells were analyzed by flow cytometry after 4 and 12 days ± dox and subsequent staining with propidium iodide. D, Analysis of cell viability after 4 and 12 days ± dox using the water-soluble tetrazolium reagent. Data represent mean ± SD from 4 independent experiments (*, P < .05; **, P < .01; ***, P < .001, ANOVA). Differences between cells without dox treatment were statistically insignificant.

Figure 4.

Mechanisms of tumor cell inhibition in response to IGF1R and INSR KD. A, Alteration of antiapoptotic Bcl-2 family proteins Mcl-1, Bcl-2, and Bcl-xL by Western blot analysis 4 and 12 days after IGF1R and INSR KD. Representative Western blotting images together with mean relative band intensities of 3 independent experiments calculated relative to GAPDH are shown (top). Histograms represent analysis of Bcl-2 protein band intensities after 12 days ± dox. Analysis of DNA synthesis (B), DNA fragmentation (C), and caspase 3/7 activity (D) in shPC3 cells after Mcl-1 KD using siRNAs in the absence of dox. E, Survivin protein levels after IGF1R/INSR KD for 12 days determined by Western blotting. A representative Western blotting image and quantitative analysis of 3 independent experiments are shown. Analysis of DNA synthesis (F) and DNA fragmentation (G) after treatment of shPC3 cells with the survivin inhibitor YM155 (3.5nM [F] and 25nM [G], respectively) for 72 hours in the presence or absence of dox. H, Inhibition of cell growth in response to survivin inhibitor YM155 (3.5nM, 72 h) and Mcl-1 inhibitor UMI-77 (5μM, 72 h). I, Rescue from apoptosis by simultaneous overexpression of survivin and Mcl-1. DNA fragmentation of shIGF1R/INSR cells was measured 72 hours after overexpression of survivin and Mcl-1 in the presence or absence of dox. Histograms represent mean ± SD (*, P < .05; **, P < .01; ***, P < .001, ANOVA).

Figure 5.

Induction of autophagy upon IGF1R and INSR KD. A and B, Activation of autophagy after induction of receptor KD was detected by Western blot analysis of the autophagy marker LC3-II. Representative Western blotting image (A) and determination of protein band intensities (B). C, IF staining of the autophagy marker LC3-II in cells treated for 48 hours ± dox and in addition treated with autophagy modulators ± CQ (25μM), ± 3-MA (5mM), and ± Mf (10mM). Scale bar, 40 μm. D, IF images of dox treated cells with higher magnification. Scale bar, 20 μm. E, Detection of autophagy marker proteins using Western blotting. F, DNA synthesis of shPC3 cells after 4 days ± dox treated with autophagic modulators for 48 hours. G, Measurement of mitochondrial respiratory O₂ consumption in intact cells (basal state), in cells permeabilized by digitonin and stimulated with metabolites fueling electrons into OXPHOS complexes CI and II (malate, pyruvate, glutamate, and succinate) and maximal ETS capacity in uncoupled cells. H, Mitochondrial mass analyzed by determination of the mitochondrial marker protein cytochrome c oxidase (COX IV) using Western blotting.

Figure 6.

Effect of IGF1R and INSR KD in AR-positive LNCaP and DuCaP cells. Down-regulation of antiapoptotic proteins after receptor KD using siRNA targeting was investigated in AR-positive LNCaP and DuCaP cells in comparison with AR-negative PC3 cells. Western blot images (A) and quantitative protein band analysis (B) of apoptotic proteins 72 hours after siRNA transfection. DNA synthesis 72 hours after siRNA Mcl-1 KD (C) and after survivin KD with YM155 (PC3, 3.5nM and 25nM; LNCaP and DuCaP, 50nM and 100nM) (D). Data represent mean ± SD from 3 experiments (*, P < .05; **, P < .01; ***, P < .001, ANOVA and t test [C], respectively).