STAT3, but not ERKs, mediates the IL-6-induced proliferation of renal cancer cells, ACHN and 769P.
BACKGROUND
Although interleukin-6 (IL-6) has been suggested to function as an autocrine growth factor in renal cell carcinoma (RCC), the underlying mechanism responsible for the IL-6-induced proliferation of RCC has not been defined. The aim of this study was to characterize the signaling cascades mediating IL-6-induced proliferation and to investigate the use of effective novel interventions to block the IL-6-induced autocrine growth of renal cancer cells.


METHODS
IL-6-induced proliferation and intracellular signaling cascades were analyzed in four human renal cancer cell lines Caki-1, ACHN, 769P and A498. IL-6-induced activation of STAT3 (signal transducer and activator of transcription-1) and extracellular signal-regulated kinases (ERKs), and the effects of anti-IL-6 neutralizing antibody, Jak inhibitor AG 490, and MEK1 inhibitor PD 98059 were analyzed by Western blotting using phospho-specific antibodies. The DNA-binding activities of STATs were analyzed by EMSA. Apoptosis was determined by using nuclear staining and the TUNEL assay. Changes in the apoptosis-related proteins, bcl-2, bcl-xL, and bax were analyzed by Western blotting.


RESULTS
IL-6 induced tyrosine phosphorylation and increased the DNA binding activity of STAT3 and, to a lesser extent, STAT1 in all cell lines except for Caki-1, which did not express the IL-6 receptor subunit gp130. ERKs were constitutively activated in all cell lines and the activation level was not up-regulated further by exogenously added IL-6 nor down-regulated by anti-IL-6 neutralizing antibody. IL-6-induced STAT3 tyrosine phosphorylation and DNA binding activity was inhibited by treatment with Jak specific inhibitor AG 490; however, it was not affected by the MEK1 inhibitor PD 98059. Moreover, treatment with AG 490 inhibited IL-6-induced proliferation of ACHN and 769P cells and induced apoptosis with the down-regulation of bcl-2 and the up-regulation of bax.


CONCLUSIONS
This study identified STAT3, but not ERKs, to be a major mediator of IL-6-induced proliferation of renal cancer cells. Although ERKs were constitutively activated, ERKs were not found to be essential for the IL-6-induced proliferation and modulation of the STAT3 activity. Because the Jak specific inhibitor AG 490 effectively inhibited the IL-6-induced STAT3 activity and induced apoptosis, the blockade of the STAT3 signaling pathways is considered to be potentially useful as a novel therapeutic approach for RCC.
Horiguchi
Akio
A
Department of Urology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Oya
Mototsugu
M
Marumo
Ken
K
Murai
Masaru
M
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D015850:Interleukin-6 / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D007425:Intracellular Membranes / Q000502:physiology; D007680:Kidney Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D019947:Receptors, Interleukin-6 / Q000378:metabolism; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D014443:Tyrosine; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one