Interferon-gamma-induced dephosphorylation of STAT3 and apoptosis are dependent on the mTOR pathway

Abstract

Interferon-gamma (IFN-γ) exhibits diverse biological activities, including control of cell growth and tumor suppression. Here, we report that the treatment of M12 cells, a human metastatic prostate cancer cell line, with IFN-γ, resulted in marked inhibition of cell proliferation and induced apoptosis. These effects were not seen with either IFN-α or IFN-β. M12 cells, like many other human cancer cells, contain constitutively activated signal transducer and activator of transcription 3 (STAT3). The basal levels of both Akt and ERK1/2 phosphorylation are also markedly elevated in M12 cells. Strikingly, IFN-γ-induced apoptosis and growth inhibition of M12 cells were associated with persistent suppression of the constitutive tyrosine-phosphorylated STAT3 (pY-STAT3). The IFN-γ-induced dephosphorylation of pY-STAT3, however, was inhibited when the mTOR pathway was specifically blocked by rapamycin. Inhibition of PI-3K with low-dose LY294002, or MAPK with PD98059 also suppressed the mTOR/p70 S6k pathway, and correlated with the blockage of IFN-γ-induced dephosphorylation of pY-STAT3. Simultaneously, treatment with LY294002, PD98059, or rapamycin abolished IFN-γ-induced apoptosis in M12 cells. The inhibition of the mTOR pathway, however, did not affect IFN-γ-induced activation of STAT1 pathway, and suppression of STAT1 expression by siRNA had no effect on IFN-γ-induced dephosphorylation of pY-STAT3. Taken together, these results demonstrate that an intact mTOR pathway is critical for IFN-γ-induced suppression of pY-STAT3 and apoptosis. Our study thus provides novel insights into the contributions of signaling pathways other than the classical JAK/STAT1 pathway in the anti-proliferative, proapoptotic actions of IFN-γ.

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.