SIPAR negatively regulates STAT3 signaling and inhibits progression of melanoma

Cellular Signalling 25 (2013) 2272–2280

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SIPAR negatively regulates STAT3 signaling and inhibits progression of melanoma Fangli Ren a, Fuqin Su a, Hongxiu Ning a,b, Yangmeng Wang a, Yongtao Geng c, Yarui Feng a, Yinyin Wang a, Yanquan Zhang a, Zhe Jin a, Yi Li c, Baoqing Jia d,e, Zhijie Chang a,⁎ a

State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Medicine, School of Life Sciences, Tsinghua University, Beijing 100084, China Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143, USA College of Life Sciences, Peking University, Beijing 100871, China d Department of General Surgery, Chinese PLA General Hospital, Beijing 100853, China e Department of Pathology, Chinese PLA General Hospital, Beijing 100853, China b c

a r t i c l e

i n f o

Article history: Received 19 April 2013 Received in revised form 8 July 2013 Accepted 26 July 2013 Available online 31 July 2013 Keywords: SIPAR STAT3 Dephosphorylation Melanoma

a b s t r a c t Persistently activated STAT3 is important for tumorigenesis in a variety of cancers, including melanoma. Although many co-factors in the regulation of STAT3 activity have been identified, it remains unclear how STAT3 phosphorylation is negatively regulated. Here, we report that SIPAR (STAT3-Interacting Protein As a Repressor) inhibits STAT3 activity by accelerating its dephosphorylation. We observed that SIPAR directly interacted with STAT3 upon IL-6 stimulation. Moreover, over-expression of SIPAR reduced, whereas depletion enhanced, the level of phosphorylated STAT3. We further demonstrated that SIPAR inhibited the growth of melanoma cells by decreasing the level of phosphorylated STAT3 and the expression of its target genes. These results suggest that SIPAR, functioning as a new negative regulator, inhibits STAT3 activity by enhancing its dephosphorylation and represses melanoma progression. © 2013 Elsevier Inc. All rights reserved.

1. Introduction STATs (signal transducers and activators of transcription) are a family of latent cytoplasmic transcription factors and are activated by cytokine receptors and subsequent phosphorylation of the receptorassociated Janus kinases (JAKs). STAT proteins form homo- or heterodimers by reciprocal interaction between SH2 domains after phosphorylation of tyrosine residues, translocate into the nucleus, bind to DNA and regulate the expression of their target genes [1]. In normal cells, the level and duration of STAT activation are controlled by mechanisms including phosphorylation of the receptor complex, dimerization of STATs, interaction with co-factors and dephosphorylation by protein tyrosine phosphatases (PTPases) [2–4]. STAT3, a member of the STAT family, plays an indispensable role in cell growth, differentiation, and survival [5–7]. Ablation of the STAT3 gene leads to embryonic lethality [8]. Conditional removal of STAT3 in specific tissues revealed its important effects on the regulation of the immune response and tolerance [9,10], obesity [11], pluripotent stem cell maintenance [12,13], wound healing [14], and mammary gland development [15]. In contrast to normal cells, persistently activated STAT3 has been reported at a very high frequency in diverse cancer cell lines and tumor ⁎ Corresponding author at: School of Medicine, School of Life Sciences, Tsinghua University, Beijing 100084, China. Tel.: +86 10 62785076; fax: +86 10 62773624. E-mail address: [email protected] (Z. Chang). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.07.023

tissues, including breast cancer, head and neck squamous cell carcinoma, prostate cancer, various leukemias, multiple myeloma and melanoma [16–24]. STAT3 promotes the expression of genes regulating tumor cell proliferation (e.g. Cyclin D1, p21waf1, c-Myc), apoptosis (e.g. Bcl2 and Bcl-xL) and transformation (E-cadherin, Snail and LIV-1) [25]. Recent studies demonstrated that STAT3 is also required for the proliferation and maintenance of tumor stem cells [26,27]. STAT3 has been suggested to be an oncoprotein involved in tumorigenesis [19,28–31]. Several studies indicated that STAT3 could be used as a target for tumor therapy [32,33]. For instance, targeting STAT3 in the murine B16 melanoma resulted in tumor regression, accompanied by apoptosis [34], and targeting STAT3 in tumors inhibited tumor growth in vivo [35,36]. Interference of STAT3 activity by over-expressing STAT3 (Y705F), a dominant-negative STAT3, or by introducing a small interfering RNA (siRNA), repressed tumor cell proliferation and tumor growth, leading to apoptosis of the tumor cells inoculated into nude mice [34,37]. To further understand the regulation of STAT3, we have identified a novel gene encoding a protein named SIPAR (STAT3 Interacting Protein As a Repressor) (GenBank accession number NM_026050). We found that SIPAR interacted with STAT3 by a yeast two hybrid experiment and SIPAR inhibited the transcriptional activity of STAT3 in a luciferase reporter experiment [38]. SIPAR was shown to affect zebrafish development in our previous study [38]. Recently, SIPAR (with another name: Acpin1) was reported to be highly expressed in the testis, in particular in the acrosomal region of sperm, and different alternative splicing

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forms of the transcript were found [39]. It appears that SIPAR may play a role in spermiogenesis or spermatogenesis [39], however, the biological and pathological functions of SIPAR in STAT3 signaling in mammalian cells remain unknown. In this study, we report that SIPAR inhibits the phosphorylation of STAT3 and suppresses the progression of melanoma by repressing STAT3 targeted gene expression. 2. Materials and methods 2.1. Plasmids and reagents Myc-tagged mouse SIPAR expression vector (pCMV/Myc-SIPAR) and GST-tagged SIPAR (pGEX-4T1/SIPAR) were constructed by PCR based on the sequence of the mouse SIPAR we had previously cloned into a pACT2 vector (pACT2/SIPAR) [38]. Mammalian expression vectors, including pXJ40/Flag-STAT3, pXJ40/GST, pXJ40/GST-STAT3, pXJ40/Flag-STAT3 and pXJ40/GST-STAT3 deletions, were provided by Dr. Xinmin Cao from National University of Singapore. Luciferase reporter vector pGL3/(APRE)4-luc was a gift from Dr. Sachiko Ezoe from Osaka University. pRc-CMV/Flag-STAT3, Flag-STAT3-CA and FlagSTAT3 (Y705F) were provided by James E. Darnell Jr. from the Rockefeller University. A plasmid with siRNA against mouse SIPAR was constructed by annealing two single-stranded DNA fragments, 5′-GAT CCCTCTCAAGTTCTTCAGATGTTCAAGAGACATCTGAAGAACTTGAGAGTTA-3′ and 5′-AGCTTAACTCTCAAGTTCTTCAGATGTCTCTTGAACATCTGAA GAACTTGAGAGG-3′ (the underlined sequences indicate the siRNA target of the mouse SIPAR gene), which were then inserted between the BamHI and HindIII sites of pSilencer 4.1/CMV vector (a gift from Dr. Xun Shen, Chinese Academy of Science, Beijing). Human recombinant interleukin-6 (IL-6) and IL-6 soluble receptor were purchased from R&D Biotechnology. Anti-STAT3 rabbit polyclonal antibodies (C-20), anti-Cyclin D1 monoclonal antibody (A-12), anti-Bcl-2 monoclonal antibody (H-5) and anti-actin monoclonal antibody (C-2) were purchased from Santa Cruz Biotechnology. Anti-p-STAT3 polyclonal antibodies (9131L) were purchased from Cell Signaling Technology.

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beads. Recombinant GST-STAT3 protein was purified from the lysates of HEK293T cells transfected with pXJ40/GST-STAT3. GST fusion proteins were eluted with elution buffer (50 mM Tris–HCl, 10 mM reduced glutathione). For cytoplasmic and nuclear protein fraction, cell pellets were first re-suspended in 1 ml of ice cold Buffer I (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 25 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and protease inhibitors, pH 8.0) and incubated on ice for 15 min. Nonidet P-40 (10%) was then added to a final concentration of 1%. After a 10 s vortex the samples were centrifuged at top speed for 1 min. The supernatants were collected as the cytoplasmic fraction. The nuclear pellets were re-suspended in 200 μl of ice-cold Buffer II (20 mM HEPES, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 25 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and protease inhibitors, pH 8.0) and rotated vigorously at 4 °C for 30 min. Samples were centrifuged at 4 °C at top speed for 10 min, with the supernatant being the nuclear fraction. Lysate, 180 μl, was mixed with the indicated antibodies and incubated at 4 °C for 2 h or overnight, followed by the addition of protein G-agarose beads to pellet the immune complexes. The immunoprecipitants and 5% of the lysates were analyzed by immunoblotting for the indicated proteins. 2.4. Immunostaining and confocal microscopy Cells growing on glass cover slips in 6 well clusters were washed with PBS, fixed for 20 min at room temperature with 4% paraformaldehyde in PBS, and permeabilized for 10 min with 0.2% Triton X-100 in PBS. Cells were blocked with 10% normal goat serum for 1 h at room temperature. Cover slips were incubated with primary antibodies, diluted in 3% BSA–PBS, overnight at 4 °C and bound antibodies were detected with FITC- or TRITC-conjugated goat anti-rabbit or anti-mouse IgG. Cover slips were mounted in a glycerol-based anti-fade mounting medium and analyzed with a laser scanning confocal microscopy with a 60× oil-immersion objective. 2.5. Luciferase assay

2.2. Mammalian cell lines and transfection HEK293T and MCF7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37 °C in a 5% CO2 containing atmosphere. B16 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. The NIH3T3/v-src cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. Lipofectamine 2000 (Invitrogen) was used for transient transfection for HEK293T, MCF7 and B16 cells. For the establishment of the SIPAR depletion cell line, an RNAi vector (pSilence4.1/SIPARi) was transfected into B16 cells using Lipofectamine 2000 according to the manufacturer's instructions. Cells were transfected with the respective plasmids and single clones were selected in culture medium containing 1 mg/ml neomycin (G418). 2.3. Immunoprecipitation, immunoblotting and GST pull-down assay For co-immunoprecipitation assays, HEK293T cells in 60 mm dishes were transfected with the indicated plasmids. Cells were lysed 36 h after transfection in 800 μl of lysis buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, and 1 mM sodium orthovanadate in the presence of protease inhibitors. An aliquot of 500 μl of whole-cell lysate was incubated with 2 μg of the indicated monoclonal antibody and 30 μl protein G Sepharose beads at 4 °C for 4 h. Beads were washed 4 times with cell lysis buffer. Precipitants were eluted with 2× SDS PAGE sample buffer and analyzed by Western blotting. GST-SIPAR or His-SIPAR fusion protein was induced with 0.1 mM IPTG for 12 h at 16 °C and purified with glutathione agarose beads (Amersham Pharmacia Biotech) or Ni-nitrilotriacetic acid (NTA)

Luciferase assays were performed with the indicated plasmid mixtures using HEK293T cells. The reporter was pGL3/(APRE)4-luc. Data were normalized with an internal control (pRL-TK vector from Promega). STAT3 luciferase activity was measured using a luciferase assay system (Promega). Experiments were done in triplicate and the means with SEs were calculated. 2.6. Tumor xenografts For tumor formation, 5 male five-week-old athymic Balb/C nu/nu mice were injected with 2 × 105 B16 cells infected with adenovirus expressing GFP (Ad/GFP) or SIPAR (Ad/SIPAR). Tumors appeared at day 5 after injection. Tumor volume was measured every 5 days and calculated as width2 × length × 0.5. Tumor growth curves were drawn according to average of tumor volumes (mm3). Tumors were weighed when the mice were sacrificed 20 days after injection. All mice experiments were handled according to a protocol approved by the Animal Research Committee of Beijing Administration of Laboratory Animals. 2.7. Immunohistochemistry Paraffin-embedded melanoma specimens from B16 mice infected with Ad-GFP or Ad-SIPAR were fixed on glass slides for immunohistochemical analyses. Rabbit anti-SIPAR antibodies at a dilution of 1:2000, anti-p-STAT3 at a dilution of 1:50, and anti-STAT3, anti-Cyclin D1 and anti-Bcl2 antibodies at a dilution of 1:500 were used in the immunohistochemistry using an immunohistochemistry polymer double detection kit (Zhongshan Golden Bridge Bio, China) according to the manufacturer's protocol.

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3. Results

(Fig. 1F). The SIPAR protein is hard to detect in many tissues, including the lung, liver, stomach, spleen, kidney and colon after the birth, although it is observed in the thymus and heart only at postnatal day 5 (Fig. 1F). All these results suggest that SIPAR is predominantly expressed in the brain, both in the embryo and after birth.

3.1. Sequence information and the expression pattern of SIPAR family proteins SIPAR was originally cloned in a yeast two-hybrid screen using mouse STAT3 as bait [38]. The full-length SIPAR protein is composed of 260 amino acids in mouse and 259 in human (Fig. 1A). A Northern blot analysis demonstrated that SIPAR is abundantly expressed in the heart, spleen, brain, lung and muscle, and hard to detect in the intestine, liver, stomach and kidney, of the adult mouse (Fig. 1B). A Western analysis using antibodies against mouse SIPAR indicated that SIPAR remains at a high level in the cerebra and cerebellum, two sections of the brain tissues in mouse (Fig. 1C), consistent with our previous observation that SIPAR is mainly distributed in the brain from an in situ hybridization experiment in mouse embryos [40]. A high level of SIPAR protein is also observed in the ovary (Fig. 1C, last lane). While the mRNA level for mSIPAR is high in the spleen (Fig. 1B, lane 4) the protein level is low (Fig. 1C, lane 9). We further confirmed that SIPAR is highly expressed during embryonic development from day 11.5 (Fig. 1D) and it appeared that the protein remains at high levels in the head and whole body in different stages of mouse embryos (Fig. 1E). Interestingly, the level of SIPAR protein remains at a high level in the brain (cerebra and cerebellum) of the mouse at the different postnatal days examined

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3.2. SIPAR interacts with STAT3 in vitro and in vivo Our previous study indicated that SIPAR interacts with STAT3 in a yeast two-hybrid system [38]. To further examine whether the interaction of SIPAR and STAT3 occurs in mammalian cells, we co-expressed Flag-tagged STAT3 (Flag-STAT3) and Myc-tagged SIPAR (Myc-SIPAR) in HEK293T cells. Whole-cell lysates were immunoprecipitated with an anti-Myc or anti-Flag antibody. Western blot analyses demonstrated that Myc-SIPAR precipitated down Flag-STAT3 (Fig. 2A, left panel), and reciprocally Flag-STAT3 precipitated down the Myc-SIPAR protein (Fig. 2A, right panel), suggesting that SIPAR interacts with STAT3 in these cells. Furthermore, we validated the interaction in a GST pull down experiment using GST tagged SIPAR (GST-SIPAR) or STAT3 (GST-STAT3) in HEK293T cells. Indeed, GST-SIPAR pulled down FlagSTAT3 in these cells (Fig. 2B). To demonstrate a direct interaction of the two proteins, we performed an in vitro GST pull down experiment using GST-STAT3 purified from HEK293T cells and His-SIPAR proteins purified from Escherichia coli (BL21). The result showed that GST-

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KGSVGRSSAPAASLGKAVALSPAPSEEQLAGMSHGIGDALG-SDWPGREPRATD-NRGQYLKGESWVSGRPGHPKLREVGFLRGEPPSAG RESVRRNPASAATPSTAVGLFPAPT-ECFARVSCSGVEALGRRDWLGGGPRATDGHRGQCPKGEPRVSRLPRHQKVPEMGSFQDDPPSAF

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Fig. 1. Sequences and expression of SIPAR genes. (A) Alignment of the human and mouse SIPAR protein sequences. Identical residues are enclosed in black boxes. (B) mRNA levels of mouse SIPAR in different tissues. A Northern blot was performed using the indicated mouse tissues. (C) Protein levels of SIPAR in adult mouse tissues. A Western blot was performed with the indicated mouse tissues. (D). Expression of SIPAR during mouse embryogenesis. (E) SIPAR protein levels in the head of the mouse compared with the whole body of the embryo. (F) Expression of SIPAR in different tissues at different ages after birth. Rabbit polyclonal antibodies against SIPAR (anti-SIPAR) were used for the Western blot analyses. β-Actin was used as an internal control.

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Fig. 2. Identification of STAT3 as a SIPAR-interacting protein. (A) Flag-STAT3 and Myc-SIPAR interact in mammalian cells. HEK293T cells were transfected for expression of Flag-tagged STAT3 and/or Myc-tagged SIPAR. Cell lysates were immunoprecipitated with either an anti-Myc or an anti-Flag antibody as indicated. Precipitates were blotted with an anti-Flag or anti-Myc antibody. (B) GST-SIPAR interacts with Flag-STAT3 in vivo. A GST-pull-down assay was performed to demonstrate the in vivo interaction of GST-SIPAR and Flag-STAT3. GSTSIPAR fusion protein was used to pull down Flag-STAT3 from HEK293T cell lysate. (C) SIPAR interacts with STAT3 directly. An in vitro GST pull-down assay using purified His-SIPAR combined with GST or GST-STAT3 was performed in vitro. (D) Endogenous SIPAR interacts with STAT3 in vivo. The whole cell lysates from adult mouse brain and liver were used for an immunoprecipitation with an anti-STAT3 antibody and blotted with anti-SIPAR antibodies. (E) The C-terminus, SH2, LD and DB domains of STAT3 responded to the interaction with SIPAR. Schematic representation of the wild-type (WT) and truncations of STAT3. An immunoprecipitation assay showed the interaction of Myc-SIPAR and Flag tagged truncations of STAT3. (F) SIPAR binds to the DB and SH3 domains of STAT3. Schematic representation of domains of STAT3. Immunoprecipitation and immunoblot of cell lysates from HEK293T cells expressing Myc-SIPAR and domains of the Flag-STAT3 are shown.

STAT3 and His-SIPAR strongly interacted in the test tube, suggesting a direct interaction (Fig. 2C). All these results indicated that SIPAR interacts with STAT3 directly both in vitro and in vivo. To examine whether the endogenous STAT3 protein interacts with SIPAR, we used cell lysates from mouse brain tissue, where SIPAR is highly abundant, and mouse liver, where it is less abundant (see Fig. 1A and 2D). Immunoprecipitation experiments using antibodies against mouse SIPAR demonstrated that SIPAR interacted with STAT3 in the brain but that no interaction occurred in the liver (Fig. 2D) and suggested that SIPAR interacts with STAT3 under physiological conditions. To characterize the domains responsible for the interaction between SIPAR and STAT3, we employed a variety of mutants with different domains of STAT3 to map the interaction sites. An immunoprecipitation experiment indicated that both the region encoding the DNA binding domain (DB) and linker domain (LD), designed as DB-LD, and the region encoding the Src homologue 2 domain (SH2) and C-terminal domain (CT), assigned as SH2-CT, are associated with Myc-SIPAR (Fig. 2E, last two lanes). More detailed domain mapping experiments revealed that the DB and SH2 domains are associated with SIPAR very strongly, while the other domains showed weak or no interaction (Fig. 2F).

These analyses indicated that SIPAR interacts with STAT3 via the DNA binding and SH2 domains. 3.3. IL-6 treatment enhances the interaction of SIPAR with STAT3 The interaction of SIPAR with the SH2 domain of STAT3 prompted us to examine whether the interaction is regulated by the treatment of IL-6, a cytokine that triggers a signaling cascade and leads to the phosphorylation of STAT3 [41]. For this purpose, we co-expressed FlagSTAT3 and Myc-SIPAR in HEK293T cells treated with 10 ng/ml of IL-6 for 30 min. An immunoprecipitation assay using an anti-Flag antibody demonstrated that IL-6 treatment enhanced the interaction of SIPAR with STAT3 (Fig. 3A). A quantitative analysis of the Flag-STAT3 and Myc-SIPAR complex showed that IL-6 induced the interaction of STAT3 and SIPAR (Fig. 3B). Furthermore, co-expression of GST-STAT3 with Myc-SIPAR confirmed the enhanced interaction of STAT3 with SIPAR in MCF7 cells treated with IL-6 (Fig. 3C). These results implied that SIPAR might associate preferably with phosphorylated STAT3 or nucleus-localized STAT3 since IL-6 stimulates STAT3 phosphorylation and nuclear translocation [1,42]. To examine whether IL-6 enhances the interaction of endogenous STAT3 and SIPAR protein in cells, we

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Myc-SIPAR Fig. 3. IL-6 treatment enhances SIPAR-STAT3 interaction in the nucleus. (A) IL-6 induces the interaction of Flag-STAT3 and Myc-SIPAR. HEK293T cells were transfected for expression of Flag-STAT3 and/or Myc-SIPAR with/without IL-6 stimulation. Cell lysates were immunoprecipitated with an anti-Flag antibody and blotted with an anti-Myc antibody as indicated. p-STAT3 was shown as an indicator of activation of IL-6 (10 ng/ml). (B) A quantitative analysis of the Flag-STAT3 and Myc-SIPAR complex. The interaction of Flag-STAT3 and Myc-SIPAR was quantified and expressed as the relative density from three independently repeated experiments. The bar indicates the standard deviation. (C) IL-6 enhances the interaction of GSTSIPAR and Flag-STAT3. GST-SIPAR protein was used to pull down Flag-STAT3 from the cell lysates of HEK293T treated with or without IL-6. (D) IL-6 promotes the interaction of endogenous STAT3 and SIPAR in vivo in the intact cell. B16 cells were treated with/without IL-6 and the cell lysates were immunoprecipitated with anti-SIPAR antibodies and blotted with an anti-STAT3 antibody as indicated. IgG was used as a control in the immunoprecipitation. (E) The interaction of STAT3 and SIPAR induced by IL-6 is dependent on the phosphorylation of STAT3. An immunoprecipitation was performed for the cytoplasmic or nuclear component from HEK293T cells after IL-6 treatment for 30 min. β-Tubulin or Jun-B was used as the cytoplasmic or nuclear marker. (F) SIPAR prefers to interact with p-STAT3 in the nucleus. A quantitative analysis of the Flag-STAT3 and Myc-SIPAR complex in the nucleus is shown. The experiments for the interaction of STAT3 and SIPAR were repeated three times. (G) STAT3 (Y705F), a mutant form of STAT3, abolished the interaction with SIPAR. HEK293T cells were transfected for the expression of wild-type Flag-STAT3 (WT) or mutant STAT3 (Y705F) in the presence or absence of Myc-SIPAR with/without IL-6 stimulation. Cell lysates were immunoprecipitated with an anti-Flag antibody and blotted with an anti-Myc antibody as indicated. p-STAT3 was shown as an indicator of IL-6 stimulation. (H) GFP-SIPAR colocalized with Flag-STAT3 in the nucleus. MCF7 cells transfected with Flag-STAT3 and/or GFP-SIPAR were treated with or without IL-6 for 30 min. Cells were immunostained with an anti-Flag antibody (TRITC). DAPI was used for nucleus staining. Merged image is shown for the demonstration of co-localization of Flag-STAT3 and GFP-SIPAR. Scale, 10 μm.

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performed an immunoprecipitation experiment using antibodies against STAT3 and SIPAR. The result showed that antibodies against SIAPR strongly precipitated down endogenous STAT3 in the presence of IL-6 (Fig. 3D). These results suggested that SIPAR interacts with STAT3 in the intact cells and it appeared that SIPAR prefers to interact with phosphorylated STAT3 in the nucleus. STAT3 is distributed in the cytoplasm and translocates into the nucleus upon cytokine stimulation [1,42]. In our previous study, we observed that SIPAR is mainly located in the nucleus and is present in lower levels in the cytoplasm [38]. To determine the location where the interaction of STAT3 and SIPAR occurs, we performed an immunoprecipitation experiment using separated nuclear and cytoplasmic proteins from cells co-expressing Flag-STAT3 and Myc-SIPAR under IL-6 treatment. The results showed that Myc-STAT3 interacts with Flag-SIPAR both in the cytoplasm and the nucleus upon treatment with IL-6 (Fig. 3E). Interestingly, we observed almost no detectable interaction of STAT3 and SIPAR occurred in the absence of IL-6 in the nucleus while a significant interaction remained in the cytoplasmic fraction (Fig. 3E, top panel). The level of the Flag-STAT3 and Myc-SIPAR complex increased in the nucleus upon IL-6 stimulation, concomitant with the increased level of phosphorylated STAT3 and Flag-STAT3 protein (Fig. 3E, see Flag-STAT3 level in the nucleus). This result suggested that SIPAR interacts with p-STAT3 in the nucleus. In parallel, the SIPAR/ STAT3 complex was increased in the cytoplasm after stimulation with IL-6, accompanied by the presence of a p-STAT3 and SIPAR complex (Fig. 3E, see the interaction in the cytoplasmic fraction). However, we observed that the complex of SIPAR with p-STAT3 remained at a very high level in the nucleus in comparison with that seen in the cytoplasm (Fig. 3E, second panel). A quantitative analysis of these results indicated that IL-6 treatment significantly induced the level of the nuclear complex of SIPAR and STAT3 in the nucleus (Fig. 3F). Furthermore, we demonstrated that STAT3 (Y705), a mutant that has lost the ability to be phosphorylated, failed to interact with Myc-SIPAR (Fig. 3G), suggesting that the phosphorylation of STAT3 is required in the interaction with SIPAR. These results suggest that SIPAR interacts with both phosphorylated and unphosphorylated STAT3 but prefers to interact with p-STAT3 in the nucleus. To further examine whether STAT3 and SIPAR co-localize in mammalian cells, we performed an immunostaining assay in MCF7 cells. The results demonstrated that GFP-SIPAR was mainly localized to the nucleus, which is consistent with our previous observation [38]. Significantly, STAT3 translocated into the nucleus where it interacted with SIPAR after IL-6 treatment (Fig. 3H). Taken together, these data suggest that SIPAR interacts with STAT3 not only in the cytoplasm but also in the nucleus. 3.4. SIPAR negatively regulates STAT3 transcription activity To reveal the biological function of SIPAR, we examined STAT3 signaling based on our observations that SIPAR interacted with STAT3. We employed a luciferase reporter linked with a STAT3 binding element to examine the transcriptional activity of STAT3. Examination of the luciferase activity indicated that over-expression of SIPAR inhibited the transcriptional activity of STAT3 in response to IL-6 stimulation in HEK293T cells (Fig. 4A). This result is consistent with our previous study [38]. Since the activity of STAT3 corresponds to its phosphorylation status, we examined the level of phosphorylated STAT3 (p-STAT3) in the presence of SIPAR. For this purpose, we generated an adenovirus vector to over-express Myc-SIPAR (Fig. 4B). Western blot analyses indicated that the level of p-STAT3 decreased dramatically when Myc-SIPAR was over-expressed in either B16 (Fig. 4C) or MCF7 (Fig. 4D) cells. These results indicated that SIPAR impairs the phosphorylation of STAT3. Since STAT3 is constitutively activated in v-Src-transformed cells [29,43], we sought to determine the role of SIPAR in the regulation of v-Src activated STAT3. A Western blot analysis demonstrated that

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adenovirus-expressed SIPAR (Ad/SIPARs) significantly decreased the level of p-STAT3 in v-Src transformed cells (Fig. 4E). To address whether the endogenous SIPAR protein is relevant to the phosphorylation of STAT3, we generated two DNA-vector-based small interference RNAs (siRNAs) to knock-down endogenous SIPAR expression. A Western blot analysis revealed that the SIPAR protein was efficiently depleted by these two specific siRNAs targeting mouse SIPAR in HEK293T cells (Fig. 4F). For simplicity, we used a mixture of the two siRNAs to examine the effect of the siRNAs on p-STAT3 levels in v-Src-transformed cells (3T3/v-Src) (Fig. 4F, left) and B16 cells (Fig. 4F, right) as STAT3 was reported to be active in both cell lines [24,29,43]. As expected, depletion of SIPAR resulted in significantly elevated p-STAT3 levels without changing the level of total STAT3 in both cell lines (Fig. 4F). These data consistently suggested that SIPAR negatively regulates the phosphorylation level of STAT3. 3.5. SIPAR enhances STAT3 dephosphorylation As the level of p-STAT3 is maintained by phosphorylation, induced by kinases, and dephosphorylation, by phosphatases, we sought to clarify the role of SIPAR on the maintenance of STAT3 phosphorylation. In a preliminary study, we found that the over-expression of SIPAR had no significant affect on the level of p-STAT3 immediately after stimulation by IL-6 (data not shown). We therefore questioned whether the decreased levels of p-STAT3 at longer time points after IL-6 stimulation were due to the dephosphorylation process. To examine this hypothesis, we employed an adenovirus to over-express SIPAR in MCF7 cells. Cells were stimulated with IL-6 for 30 min and then the cytokine was withdrawn with the cells placed in serum-free medium for different lengths of time. A Western blot analysis indicated that p-STAT3 remained at a high level in the control cells up to 90 min after withdrawal of the cytokine, while its level was dramatically decreased in cells that were infected with the Ad/SIPAR as early as 30 min after the withdrawal of the cytokine (Fig. 4G). This result suggested that SIPAR promotes the dephosphorylation of STAT3. 3.6. SIPAR inhibits the progression of melanoma caused by constitutively active STAT3 As SIPAR decreases the phosphorylation of STAT3 and negatively regulates STAT3 transcriptional activity, we sought to determine whether SIPAR regulates the expression of STAT3 targeted genes in response to IL-6 stimulation. For this purpose, we infected MCF-7 cells with an adenovirus vector that over-expresses SIPAR. A Western blot analysis indicated that over-expression of SIPAR not only significantly decreased p-STAT3 levels upon IL-6 stimulation, but also dramatically inhibited the expression of IL-6 inducible genes including Cyclin D1, c-Myc and Bcl-2 (Fig. 5A). Since expression of these genes is highly correlated with cell proliferation, we speculated that the over-expression of SIPAR might affect the progression of tumors induced by persistently activated STAT3. To address this hypothesis, we over-expressed SIPAR using an adenovirus (Ad/SIPAR) in B16 cells where p-STAT3 remains at a high level [24]. We examined the effect of SIPAR on tumor formation as this cell line shows a strong ability for tumorigenesis in nude mice [24]. Adenoviruses, of the same titers, expressing GFP or SIPAR were used to infect cells which were injected into the right (GFP) and left (SIPAR) flanks of nude mice subcutaneously. Tumor growth was measured as tumor volumes at different days after inoculation with the tumor cells and was analyzed statistically. The results showed that tumors formed 10 days after inoculation and gradually grew larger (Fig. 5B). Tumors formed from cells infected with SIPAR grew slowly in comparison with the control cells infected with GFP (Fig. 5B). Finally, mice were sacrificed and the size and weights of the tumors were examined. Results showed that both tumor size (Fig. 5C, top panel) and tumor weight (Fig. 5C, bottom panel) were reduced in B16 cells infected with SIPAR compared to the same cells infected with the control GFP

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Fig. 4. SIPAR inhibits STAT3 transcriptional activity by promoting the dephosphorylation of STAT3. (A) The effect of SIPAR on STAT3-mediated transcription. HEK293T cells were transfected with plasmids encoding Flag-STAT3 as indicated together with APRE-Luc, which responds to the STAT3/STAT3 complex binding. Twenty-four hours after transfection, cells were starved with serum-free medium for another 24 h followed by stimulation with cytokines (IL-6, 10 ng/ml) for 8 h. Cells were extracted and measured for luciferase activity according to the manufacture's protocol (Promega). Data were normalized by co-transfection with Renilla luciferase vector and expressed as mean (n = 3) plus SD (bars). All the assays were carried out in triplicate. (B) A microscopic analysis of GFP expression 2 days after adenoviral infection shows equally efficient transduction (up to 85%) of B16 melanoma cells. GFP is expressed from the same viral vector as Ad/SIPAR. (C, D, E) SIPAR decreases the level of p-STAT3. Phosphorylated STAT3 levels were examined from B16 (C) and MCF7 (D) and NIH3T3/v-src (E) cells that were infected with Ad/GFP or Ad/SIPAR in the presence or absence of IL-6 for 30 min. The total STAT3 level was used as a loading control and the GFP level was used as an equal titer control. (F) Depletion of SIPAR facilitates STAT3 phosphorylation. Levels of p-STAT3 were examined in stable NIH3T3/v-src (left) and B16 (right) cells under depletion of SIPAR by two different siRNAs (noted as SIPAR RNAi) and pSilence4.1 vector (noted as Mock). Total STAT3 was used as a loading control. (G) SIPAR enhances dephosphorylation of STAT3. MCF7 cells infected with Ad/SIPAR or Ad/GFP were stimulated with IL-6 (10 ng/ml) for 30 min, followed by starvation (noted as withdrawal) for the times shown. p-STAT3 and total STAT3 were examined by an immunoblot. GFP was used as an equal titer control and total STAT3 and β-actin were used as loading controls.

adenovirus. A histopathological study indicated that there were fewer p-STAT3 positive tumor cells in the Ad-SIPAR infected tumors than in control tumors (Fig. 5D, a vs a′), while the numbers of STAT3 positive cells remained unchanged (Fig. 5D, c vs c′). Moreover, the numbers of Cyclin D1 and Bcl-2 positive cells were also dramatically reduced in the tumors formed from B16 cells infected with Ad-SIPAR in comparison with cells infected with the GFP adenovirus (Fig. 5C, d vs d′, e vs e′). These results were consistent with the Western blot analysis that showed that the levels of Cyclin D1 and Blc-2 were reduced in SIAPR1 over-expressing cells (Fig. 5A). Taken together, these data suggested that SIPAR inhibits tumor growth by reducing levels of STAT3 phosphorylation. 4. Discussion STAT3 plays very important roles in cell survival, differentiation and proliferation. STAT3 is activated by phosphorylation in response to many different cytokines. Under normal physiological conditions, activated STAT3 is transient and is subject to rapid turn-over through a dephosphorylation process. The level of active STAT3 is strictly maintained through phosphorylation and dephosphorylation. In tumors, however, STAT3 activity has been found to be persistently elevated, which is either the result of constitutive phosphorylation by positive regulators or impaired dephosphorylation by negative regulators [33]. In this study, we found that SIPAR, a novel negative regulator, represses the activation of STAT3. We have shown that SIPAR physically interacts with STAT3 in vitro and in vivo, and that the interaction occurs not only in the cytoplasm but also in the nucleus of the cell. It appears that SIPAR

preferentially associates with phosphorylated STAT3, which might be due to the interaction of SIPAR at the DNA-binding and SH2 domains of STAT3 (see Fig. 2E–F), as the SH2 domain is critical for phosphorylation [41]. Intriguingly, we observed that the interaction of SIPAR with STAT3 resulted in an inhibition of the STAT3 transcriptional activity. We reasoned that the inhibitory role of SIPAR on the activity of STAT3 is because of the decreased phosphorylation of STAT3. We demonstrated that SIPAR leads to the dephosphorylation of STAT3. All these data are consistent with the observations that SIPAR is an intrinsic inhibitor for STAT3. As the activation of STAT3 is an important event for different physiological processes, including cell proliferation, differentiation and apoptosis, we envisioned that there must be different regulators to control the activity of STAT3. During the last decade, several positive and negative regulators of STAT3 have been identified [4]. In this study, we revealed that SIPAR is a novel repressor that promotes the dephosphorylation of STAT3. Our study provided new information in the understanding of the regulation of STAT3 activity. While we provided evidence that SIPAR promoted the dephosphorylation process of STAT3, we did not find any phosphatase activity (data not shown). To date, several phosphatases have been reported that regulate the de-phosphorylation of STAT3 [2–4]. The amino acid sequence of SIPAR is not related to phosphatases (Fig. 1A), therefore, we envision that SIPAR may associate with a phosphatase, which then mediates the dephosphorylation of STAT3. We are currently attempting to clarify the identity of this phosphatase. We have shown that SIPAR was mainly localized to the nucleus (Fig. 3H), however, we also observed cytoplasmic SIPAR (see Fig. 3E).

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Fig. 5. SIPAR represses the progression of melanoma and is correlated with decreased phosphorylation of STAT3. (A) SIPAR decreases the expression of STAT3 target genes. The protein levels of STAT3 target genes were examined from MCF7 cells that were infected with Ad/GFP or Ad/SIPAR in the presence or absence of IL-6 for 30 min. Total STAT3 level was used as a loading control. (B–C) SIPAR inhibits tumor growth. 2 × 105 of B16 cells infected with Ad/GFP or Ad/SIPAR was injected into both flanks of nude mice (n = 6). Tumor sizes (B) were monitored during a period of 20 days. Xenograft tumors from both flanks in the same animals are shown (C, top). Tumor weights were measured at 20 days after injection (C, bottom). (D) Correlated expression of p-STAT3 and STAT3 target genes in the tumors formed by B16 cells. Cells were infected with a control (Ad/GFP) or an adenovirus expressing SIPAR (Ad/SIPAR). Immunohistochemistry was performed on the tumors with anti-SIPAR, p-STAT3, Cyclin D1 and Bcl-2 antibodies. Total STAT3 was used as a control. Scale: 100 μm.

SIPAR, thus, is associated with STAT3 both in the nucleus and cytoplasm. Since STAT3 translocates from the cytoplasm into the nucleus after cytokine stimulation, we observed that the interaction of SIPAR with STAT3 increased upon IL-6 stimulation (Fig. 3E). These results suggest that SIPAR associates with phosphorylated STAT3 both in the cytoplasm and the nucleus, indeed, when we used STAT3 (Y705F), a mutant that has lost the ability to be phosphorylated at tyrosine 705, we found no interaction of STAT3 and SIPAR (Fig. 3G).

Importantly, we observed that the over-expression of SIPAR in B16 melanoma, which has persistently active STAT3, led to the regression of the tumor growth. We demonstrated the role of SIPAR in B16 cells since this cell line was reported to form tumors in nude mice [24,34]. Our data showed that over-expression of SIPAR not only reduced tumor volume and weight (Fig. 5B–C) but also the expression of tumor related genes (Fig. 5A). These results are consistent with the observation that SIPAR repressed the phosphorylation and transcriptional

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activity of STAT3 (Fig. 4). Furthermore, in the tumors that formed, we observed that the level of p-STAT3 was decreased dramatically in the cells over-expressing SIPAR (Fig. 5D). In conclusion, we demonstrated that SIPAR enhances the dephosphorylation of STAT3 and negatively regulates STAT3 activity. The next step should be to study whether SIPAR suppresses STAT3-mediated cell transformation, and the mechanisms SIPAR involved in the regulation of STAT3 phosphorylation. Furthermore the role of SIPAR in physiological processes can be addressed as its expression is restricted to specific tissues (mainly the brain) in adults and is highly expressed in the whole embryo. Acknowledgments This work was supported by grants from the 973 Project (2011CB910502), NSFC (30871286, 31071225, 31030040), Tsinghua Science Foundation (20121080018), and the 863 Project (2012AA021703) in China. We thank Dr. David M Irwin from the University of Toronto for his editing of the manuscript. References [1] J.E. Darnell Jr., I.M. Kerr, G.R. Stark, Science 264 (5164) (1994) 1415–1421. [2] P.C. Heinrich, I. Behrmann, S. Haan, H.M. Hermanns, G. Muller-Newen, F. Schaper, The Biochemical Journal 374 (Pt 1) (2003) 1–20. [3] M. Kubo, T. Hanada, A. Yoshimura, Nature Immunology 4 (12) (2003) 1169–1176. [4] F. Su, F. Ren, Y. Rong, Y. Wang, Y. Geng, M. Feng, Y. Ju, Y. Li, Z.J. Zhao, K. Meng, Z. Chang, Breast Cancer Research 14 (2) (2012) R38. [5] J.E. Darnell Jr., Science 277 (5332) (1997) 1630–1635. [6] D.E. Levy, C.K. Lee, The Journal of Clinical Investigation 109 (9) (2002) 1143–1148. [7] J.G. Williams, Current Opinion in Genetics & Development 10 (5) (2000) 503–507. [8] K. Takeda, K. Noguchi, W. Shi, T. Tanaka, M. Matsumoto, N. Yoshida, T. Kishimoto, S. Akira, Proceedings of the National Academy of Sciences of the United States of America 94 (8) (1997) 3801–3804. [9] F. Cheng, H.W. Wang, A. Cuenca, M. Huang, T. Ghansah, J. Brayer, W.G. Kerr, K. Takeda, S. Akira, S.P. Schoenberger, H. Yu, R. Jove, E.M. Sotomayor, Immunity 19 (3) (2003) 425–436. [10] T. Wang, G. Niu, M. Kortylewski, L. Burdelya, K. Shain, S. Zhang, R. Bhattacharya, D. Gabrilovich, R. Heller, D. Coppola, W. Dalton, R. Jove, D. Pardoll, H. Yu, Nature Medicine 10 (1) (2004) 48–54. [11] S.H. Bates, W.H. Stearns, T.A. Dundon, M. Schubert, A.W. Tso, Y. Wang, A.S. Banks, H.J. Lavery, A.K. Haq, E. Maratos-Flier, B.G. Neel, M.W. Schwartz, M.G. Myers Jr., Nature 421 (6925) (2003) 856–859. [12] T. Matsuda, T. Nakamura, K. Nakao, T. Arai, M. Katsuki, T. Heike, T. Yokota, The EMBO Journal 18 (15) (1999) 4261–4269. [13] R. Raz, C.K. Lee, L.A. Cannizzaro, P. d'Eustachio, D.E. Levy, Proceedings of the National Academy of Sciences of the United States of America 96 (6) (1999) 2846–2851.

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