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. 2011 May 19;30(20):2345-55.
doi: 10.1038/onc.2010.605. Epub 2011 Jan 24.

IL-6 promotes prostate tumorigenesis and progression through autocrine cross-activation of IGF-IR

A Rojas  1 , G Liu, I Coleman, P S Nelson, M Zhang, R Dash, P B Fisher, S R Plymate, J D Wu
Affiliations

IL-6 promotes prostate tumorigenesis and progression through autocrine cross-activation of IGF-IR

A Rojas et al. Oncogene. .

Abstract

As an established mediator of inflammation, interleukin-6 (IL-6) is implicated to facilitate prostate cancer progression to androgen independence through transactivation of the androgen receptor. However, whether IL-6 has a causative role in de novo prostate tumorigenesis was never investigated. We now provide the first evidence that IL-6 can induce tumorigenic conversion and further progression to an invasive phenotype of non-tumorigenic benign prostate epithelial cells. Moreover, we find that paracrine IL-6 stimulates the autocrine IL-6 loop and autocrine activation of insulin-like type I growth factor receptor (IGF-IR) to confer the tumorigenic property and also that activation of signal transducer and activator of transcription 3 (STAT3) is critical in these processes. Inhibition of STAT3 activation or IGF-IR signaling suppresses IL-6-mediated malignant conversion and the associated invasive phenotype. Inhibition of STAT3 activation suppresses IL-6-induced upregulation of IGF-IR and its ligands, namely IGF-I and IGF-II. These findings indicate that IL-6 signaling cooperates with IGF-IR signaling in the prostate microenvironment to promote prostate tumorigenesis and progression to aggressiveness. Our findings suggest that STAT3 and IGF-IR may represent potential effective _targets for prevention or treatment of prostate cancer.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
IL-6 induces EMT in SV40T immortalized benign prostate epithelia P69 cells. A, Microscopic images demonstrating depolarized morphology and loss of E-cadherin and gain of Vimentin expression in P69 cells after exposure to IL-6. Scale bar, 100 μm. B, Western blotting confirming reduction in E-cadherin and increase in Vimentin expression in P69 cells after exposure to IL-6. GAPDH was used as the loading control. C, Real-time RT-PCR demonstrating the changes in expression of EMT-associated genes in 69 cells after exposure to IL-6 (50 ng/ml), including E-cadherin, Vimentin, N-Cadherin, and the transcriptional factor Snail. RQ, relative quantity. D, Representative micrographs (top panel) and statistical analysis (bottom panel) of wounding assay demonstrate increased migration of P69 cells in the presence of IL-6. Scale bar, 250 μm. Data expressed as mean + s.d. *, P <0.05. Data shown are experiments with 50 ng/ml of IL-6.
Figure 2
Figure 2
IL-6 induces EMT through activation of STAT3 and IGF-IR. A, Western blotting shows that phosphorylation of STAT3 is required for IL-6-induced loss of E-Cadherin in P69 cells. Blocking STAT3 phosphorylation with AG594 inhibits the change of E-Cadherin in P69 cells. B, Western blotting shows that IGF-IR signaling is involved in IL-6 induced changes of Vimentin and E-Cadherin in P69 and BPH-1 cells. C, immunofluorescence staining demonstrates that IGF-IR signaling is critical for IL-6 induced-EMT in P69 and BPH-1 cells. Scale bar, 50μm. Blocking IGF-IR signaling with the monoclonal antibody (mAb) IMC-A12 inhibits loss of E-cadherin and gain of Vimentin.
Figure 3
Figure 3
IL-6 induces changes in three-dimensional (3-D) acini formation and malignant phenotypes of benign prostate epithelial P69 (A) and BPH-1 (B) cells in association with EMT. Top panel, micrographs demonstrate the morphology of acini spheres formed in 3-D Matrigel. Middle Panel, immunofluorescence staining demonstrates IL-6-induced changes in E-Cadherin (Green) and Vimentin (Red) in 3-D Matrigel culture. Bottom Panel, representative sections observed under confocal microscopy. Scale bar, 50μm.
Figure 4
Figure 4
IL-6 induces transformation of benign P69 and BPH-1 cells through activation of IGF-IR. A, MTT assay (mean +s.d.) showing that IL-6 (50 ng/ml) stimulates proliferation of P69 and BPH-1 cells and the mitogenic effect is blocked by the anti-IGF-IR mAb IMC-A12. B and C, IL-6 facilitates colony formation of P69 cells in soft agar. B, numbers of colonies formed by P69 cells in the presence of IL-6 at indicated time points. C, representative size of colonies formed by P69 cells in the presence of IL-6 at indicated time points. Scale bar, 5mm. D. Reporter assay showing markedly increased transcriptional activation of the cancer-specific promoter PEG-3 in P69 and PBH-1 cells in the presence of IL-6. Data also show that inhibition of STAT3 activation with AG490 or blocking IGF-IR signaling with the mAb IMC-A12 inhibits the effect of IL-6. *, P < 0.01. #, not significantly different from the control.
Figure 5
Figure 5
IL-6 promotes progressive tumorigenesis and metastatic progression of benign immortalized prostate epithelial cells in vivo. Human IL-6 was stably expressed in benign P69 and BPH-1cells (designated as P69IL-6 and BPH-1IL-6 respectively). 2 × 106 /mouse P69IL-6 or BPH-1IL-6, or parental P69 or BPH-1 cells were s.c. implanted into groups of nude mice. A, Tumor incidence in animals that were implanted with P69IL-6 or BPH-1IL-6 stable genetically modified cells or parental P69 or BPH-1 cells. Data show that all the animals that were implanted with P69IL-6 or BPH-1IL-6 cells developed tumors within 10 days, whereas no tumor was formed in animals that were implanted with parental P69 or BPH-1 cells. B, growth and volume of tumors (mean ± s.e.) formed by P69IL-6 and BPH-1IL-6 cells. C, IHC staining of SV40T-Ag and Vimentin showing that tumors formed by P69IL-6 cells metastasized to the lymph node and that Vimentin was abundantly expressed in these tumors. Scale bar, 100 μm.
Figure 6
Figure 6
IL-6 induces autocrine activation of IGF-IR and autocrine loops of IL-6 signaling in P69 cells. A, Western blotting showing that IL-6 (50 ng/ml) induces markedly increased phosphorylation of IGF-IR in P69 cells in serum-free cultures and the effect was blocked by the STAT3 inhibitor AG490. β-tubulin was used as the loading control. B–D, Real-time RT-PCR showing that IL-6 induces increased expression of the ligands IGF-I and IGF-II and the receptor IGF-IR in P69 cells. Data also show that the effect of IL-6 was blocked by the STAT3 inhibitor AG490. E–G, Real-time RT-PCR showing that IL-6 stimulates autocrine IL-6 loop through upregulation of expression of IL-6 and its signaling components IL-6R and gp130. Data shown as mean +s.d. RQ, relative quantity. *, P < 0.001. *, P < 0.05. #, not significantly different from the control.
Figure 7
Figure 7
Transcriptional re-programming induced by IL-6 to facilitate tumorigenesis. A, Microarray expression profile showing clusters of genes that are upregulated by IL-6. A list of significantly upregulated genes with known function is shown in Supplemental Table 1. B, Real-time RT-PCR validating the upregulated expression of the two malignancy-associated genes, K-Ras and LCN2. Data also show that IL-6-induced upregulation of K-Ras and LCN2 was inhibited by blocking IGF-IR signaling with antibody IMC-A12 or STAT3 inhibitor AG490. *, P < 0.05.
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