Research ArticleInterferon-gamma-induced dephosphorylation of STAT3 and apoptosis are dependent on the mTOR pathway
Introduction
Interferon-gamma (IFN-γ), in addition to its well-known anti-viral activities, also exhibits anti-proliferative, proapoptotic effects on various tumor cells [1], including prostate cancer cells [2]. The anti-proliferative, proapoptotic effects of IFN-γ have been largely attributed to the activation of the Janus kinase (JAK) and Signal Transducer and Activator of Transcription-1 (STAT1) signaling pathway [3], [4], [5]. The cascade of signal transduction is initiated upon the binding of dimeric IFN-γ to its receptor, followed by the activation of the receptor-associated Jak1 and Jak2, which in turn phosphorylate tyrosines on the receptor. The phospho-tyrosines serve as the docking sites for multiple cytosolic proteins, including STAT1. Subsequent tyrosine-phosphorylation of STAT1 by the Jaks leads to homodimerization of phosphorylated STAT1 and translocation into the nucleus, where STAT1 functions as a transcription factor.
In addition to the JAK/STAT pathway, IFN-γ can also activate the MAPK pathway and the PI-3K/mTOR pathway (reviewed recently in [6]). The role of these pathways in IFN-γ-induced biological effects, however, has been less clearly defined. The mTOR pathway, critically involved in cell proliferation, cell cycle progression, and cell survival, regulates ribosome biogenesis and protein synthesis in response to the availability of nutrients and stimulation from growth factors and cytokines [7]. The upstream signaling pathways affecting the activity of mTOR include the PI-3K and MAPK/ERK pathways, both of which have been shown to regulate the mTOR pathway through modulating the activity of the Tuberous Sclerosis tumor suppressor (TSC1/2) complex [7], [8], [9]. The direct targets activated by mTOR include p70 S6K, 4EBP1, and eIF4G [7]. The activation of the mTOR pathway by IFN-γ appears to be mediated through the PI-3K pathway, while STAT1 activation and gene transcription via GAS elements are mTOR-independent [10]. The majority of evidence indicates that an activated mTOR pathway positively regulates cell growth and protects cells from apoptosis [7]. However, one recent study suggests that, on the contrary, an intact PI3-K/mTOR pathway was required by IFN-α to induce apoptosis in multiple myeloma cells, although the underlying mechanism(s) remains to be established [11].
In normal cells, STAT activation is transient and tightly controlled, whereas in a large number of primary tumors and cancer-derived cell lines, including prostate cancer cells, STAT3, in particular, is deregulated and remains constitutively activated [12], [13], [14]. STAT3 has been considered a potential oncogene because of its ability to induce tumorigenic transformation and block apoptosis [15]. Direct inhibition of constitutively tyrosine-phosphorylated STAT3 can elicit growth inhibition and apoptosis in various cancer cells, including prostate cancer cells [16], [17]. In this study, we demonstrate that, in M12 cells, a human metastatic prostate cancer cell line, the IFN-γ-induced apoptosis, and inhibition of cell proliferation are associated with persistent suppression of constitutively tyrosine-phosphorylated STAT3, and furthermore, an intact mTOR pathway is required for the IFN-γ-induced apoptosis and suppression of STAT3.
Section snippets
Cell culture
M12 cells are a highly metastatic derivative of a SV40-T antigen transformed, low tumorigenic human prostate cancer cell line [18]. M12 cells were cultured in defined RPMI 1640 supplemented with 5 μg/ml insulin–transferrin–sodium selenite (ITS, Sigma, Aldrich, St. Louis, MO), 0.2 μM dexamethasone, 10 ng/ml epidermal growth factor (EGF) at 37°C and 5% CO2. For experiments detecting changes in signaling pathways in response to various treatments, M12 cells were grown to 60–70% confluence on
IFN-γ inhibited cell proliferation and induced apoptosis in M12 cells
M12 cells were treated with either type I interferon (IFN-α or IFN-β) or type II interferon, IFN-γ, at a concentration of 20 U/ml, 100 U/ml, 500 U/ml, or 2500 U/ml. Proliferation of M12 cells was dose-dependently inhibited by IFN-γ, with 100 U/ml inducing greater than 70% growth inhibition, relative to untreated cells, 96 h post-treatment (Fig. 1A). Higher doses of IFN-γ suppressed growth by more than 85%. In contrast, IFN-α or IFN-β, at a high concentration of 2500 U/ml, only modestly
Discussion
In this study, we have demonstrated that the potent inhibitory effect on cell growth and the induction of apoptosis exhibited by IFN-γ are associated with the persistent suppression of constitutive pY-STAT3 in M12 cells, a derived human metastatic prostate cancer cell line. IFN-γ primarily activates STAT1 [4], although STAT3 and 5 can also be activated [19], [23]. In M12 cells, IFN-γ could induce phosphorylation of STAT1 (Fig. 3A) and STAT5 (data not shown). The immediate effect of IFN-γ on
Acknowledgments
This work was supported by National Institutes of Health Grant CA58110 (to RGR), and by Department of Defense Grant W81XWH-04-1-0107 (to PF).
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