ERK is a negative feedback regulator for IFN-γ/STAT1 signaling by promoting STAT1 ubiquitination

Human ESCC cell lines EC1 and KYSE150, were used in this study. EC1 cell lines was purchased from Hongkong University in 2011 and KYSE150 was purchased from DSMZ in 2012. They were maintained in RPMI 1640 or Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). All cells were verified to be free of mycoplasma contamination.

STAT1 siRNA and scramble RNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transfection of siRNA was performed using Lipofectamine RNAimax (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Plasmids including eGFP-STAT1, eGFP-STAT1Y701F, eGFP-STAT1S727A, Flag-STAT1β were purchased from Addgene (Cambridge, MA, USA). Flag-tagged, constitutive-active STAT1 (or STAT1C) cloned into the backbone of pcDNA3.1 was a gift from Dr. Toru Ouchi (Roswell Park Cancer Institute, University at Buffalo, NY, USA). The constitutively active MEK-1 (HA-ca-MEK) vector was a gift from Dr. Nathalie Rivard (Université de Sherbrooke, Québec, CA). The proteasome inhibitor N-carbobenzoxyl-L-leucinyl-L-norleucinal (MG132) was purchased from Calbiochem (La Jolla, CA, USA) and 5-Aza-2′-deoxycytidine (5-Aza) was purchased from Sigma (St Louis, MO, USA). U0126, a Mitogen-activated protein kinase 1 (MEK1) inhibitor was purchased from Sigma (St Louis, MO, USA).

Co-immunoprecipitation, immunoprecipitation and Western blot analysis were performed as described previously [5]. Antibodies reactive with human β-actin, caspase-9, caspase-3, Bcl-2, Bcl-xL, STAT1, phospho-STAT1 serine 727 (or p-STAT1 S727), phospho-STAT1 tyrosine 701 (or p-STAT1 Y701), phospho-ERK (or p-ERK), ERK, Ubiquitin (or Ub), phospho-JAK2 (or p-JAK2), JAK2, Human influenza hemagglutinin (or HA) and Poly ADP ribose polymerase (PARP) were purchased from Cell Signaling.

To evaluate the effect of MG132 on the growth of ESCC cell lines, cell viability was determined by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI) and trypan blue assay (sigma, St Louis, MO, USA) according to the manufacturer’s protocol. Colony formation assay was performed as described previously [5].

Using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), total cellular RNA was extracted from cells following the manufacturer’s protocol. Primer sequence for IFNγ is: Forward: 5′- TGACCAGAGCATCCAAAAGA-3′, Reverse: 5′-CTCTTCGACCTCGAAACAGC-3′. IFNγ receptor: Forward: 5′-TCTTTGGGTCAGAGTTAAAGCCA-3′, Reverse: 5′-TTCCATCTCGGCATACAGCAA-3′. Human GAPDH was used as control.

Immunofluorescence was performed as previously described [5]. Cells were visualized with a Zeiss LSM 710 confocal microscope at the Core Cell Imaging Facility, Cross Cancer Institute, Alberta, Canada.

Apoptosis was measured by using the FITC-conjugated Annexin V/PI assay kit (Invitrogen, Carlsbad, CA, USA) and flow cytometry. After treatment with scramble RNA or siRNA STAT1 for 24 h, cells were treated with 10 μM MG132 for 24 h and then collected according to the manufacturer’s protocol.

The supernatant of cell suspension from harvested cells were collected, the level of IFNγ was analyzed by ELISA using the commercially available ELISA kits in a 96-well microplate (eBioscience, San Diego, CA, USA) at an optical density of 450 nm, according to the manufacturer’s protocol.

The cBioPortal for Cancer Genomics is an open-access downloaded bio-database, providing visualization and analyzing tool for large-scale cancer genomics data sets (www.​cbioportal.​org). This portal collected records that were derived from 147 individual cancer studies, in which 31 types of cancer were analyzed, which included over 21,000 samples. Analysis of the 185 esophageal cancer samples from this database was performed in silico.

To investigate the mechanism(s) by which STAT1 is down-regulated in ESCC, we questioned if the STAT1 gene is silenced via gene methylation. Thus, we treated two ESCC cell lines EC1 and KYSE150 with 1–10 μM 5-Aza for 0–48 h. By Western blots and quantitative RT-PCR, we did not find any appreciable change in STAT1 nor phospho(p)-STAT1 expression, suggesting that gene methylation does not play a role in suppressing STAT1 expression in ESCC (Additional file 1: Figure S1).

In view of one previous report that STAT1 can be degraded via the ubiquitin-proteosome pathway in mouse embryonic fibroblasts [11], we tested if this mechanism contributes to the low expression level of STAT1 in ESCC. Thus, we treated EC1 and KYSE150 with varying concentrations (1–10 μM) of MG132 for 24 h. By Western blots, we found that the STAT1 protein level in all cell lines was dramatically up-regulated in a dose-dependent manner, and this STAT1 up-regulation was detectable at an MG132 concentration as low as 1 μM (Fig. 1a). Furthermore, MG132 induced an increase in STAT1 in time-dependent manner (Fig. 1b). With the exception of EC1, a cell line that did not express p-STAT1^S727, the STAT1 phosphorylation level at Y701 and S727 generally increased in parallel with the total protein level of STAT1. These findings suggest that there are mechanism(s) that constitutively activate STAT1 in ESCC cells at the steady state.

We performed immunoprecipitation and Western blots to detect STAT1 ubiquitination in EC1 and KYSE150 cells. As shown in Fig. 2a, STAT1 ubiquitination was decreased in the presence of U0126 compared to the negative controls. Moreover, transfection of the constitutively-activated MEK/ERK plasmid increased STAT1 polyubiquitination, compared with the empty vector (Fig. 2b). Taken together, these results support the concept that ERK activation promotes polyubiquitination and proteasomal degradation of STAT1.

As mentioned, one previous study using mouse embryonic fibroblasts has shown that ERK phosphorylates STAT1 at S727 and targets it for proteasomal degradation [11]. To determine whether the two phosphorylation sites of STAT1 (Y701 and S727) contribute to STAT1 polyubiquitination, we transfected plasmids encoding GFP-tagged STAT1 or the two STAT1 mutants (S727A and Y701F) into the EC1 cells, and the results are illustrated in Fig. 2c. As expected, treatment of U0126 effectively decreased the amount of p-ERK, and this change correlated with a reciprocal increase in GFP and STAT1 proteins. The levels of both STAT1^S727A and STAT1^Y701F increased in response to the U0126 treatment. In addition, silencing of ERK by inhibitor U0126 decreased the polyubiquitination of STAT1, either in the form of wild-type and mutants (Fig. 2d). These findings suggest that the degradation of STAT1 mediated by ERK is independent of the phosphorylation at Y701 or S727. STAT1β is a C-terminal-truncated version of STAT1 that lacks S727, but retains Y701 [12] . To further support the concept that phosphorylation of STAT1^S727 is not required for ERK-mediated STAT1 proteasomal degradation, we transfected Flag-tagged STAT1α and STAT1β plasmids into EC1 cells with or without U0126. As shown in Fig. 2e, we found that U0126 can increase both exogenous STAT1α and STAT1β in both cell lines; in addition, ERK can promote the polyubiquitination of both STAT1α and STAT1β (Fig. 2f). These results further support that active ERK can mediate STAT1 degradation that is independent of the phosphorylation of S727.

To further delineate the mechanisms underlying ERK-mediated STAT1 proteasomal degradation, we examined whether ERK binds to STAT1. Co-immunoprecipitation experiments were performed using EC1 and KYSE150 cells, with or without MG132 treatment. As shown in Fig. 3a, immunoprecipitation of ERK pulled down STAT1 in both cell lines, and MG132 treatment appreciably increased the amount of STAT1 bound to ERK, probably due to the fact that the total amount of STAT1 increased in response to proteasomal inhibition by MG132. Reciprocal pull-down experiments showed essentially the same results, except that we did not see a MG132-induced increase in the amount of ERK bound to STAT1, probably due to the fact that the protein level of ‘bioavailable’ STAT1 was substantially lower than that of ERK. Moreover, the co-localization of ERK and STAT1 was confirmed by confocal microscopy (Fig. 3b). To determine whether the phosphorylation status of STAT1 is required for the ERK-STAT1 interaction in ESCC cells, we transfected EC1 cells with wild-type STAT1, STAT1^Y701F or STAT1^S727A. All of the plasmids used were GFP-tagged. Results are illustrated in Fig. 3c. ERK was pulled down along with STAT1 as well as STAT1 mutants. These results indicated that STAT1 can bind to ERK independent of the phosphorylation status of STAT1 at the Y701 or S727, two sites known to be important for the function of STAT1. These findings are in parallel with the previous experiments in which we showed that ERK can mediate STAT1 degradation independent of the two STAT1 phosphorylation sites mutants.

The ERK signaling pathway has been reported to play a crucial role in IFNγ/STAT1 signaling in human macrophages [13]. We hypothesized that, in addition to decreasing STAT1 expression, ERK may further down-regulate STAT1 signaling by suppressing IFNγ production. Our data is in support of this concept. Using quantitative RT-PCR, we found that inhibition of ERK by U0126 increased the expression of IFNγ and IFNγ receptor in both ESCC cell lines (Fig. 4a). As shown in Fig. 4b, similar results were found when we performed the ELISA to detect the active form of IFNγ. As shown in Fig. 4c, we also found evidence that IFNγ up-regulated p-ERK, which appears to serve as a gatekeeper to prevent over-stimulation of STAT1, which is known to provide potent pro-apoptotic signal in ESCC cells [9]. To demonstrate the effectiveness of ERK in blocking IFNγ-mediated activation of STAT1, we transfected EC1 with the constitutively active ERK vector before the addition of IFNγ to the cell culture. As shown in Fig. 4c, the expression of constitutively active ERK substantially dampened the up-regulation of p-STAT1 induced by IFNγ. Moreover, we performed the colony formation to evaluate the biological effect of ERK on STAT1 in both cell lines. As shown in Fig. 4d, U0126 significantly diminished the clonogenic ability of ESCC cell with STAT1 knockdown by siRNA; at the same time, constitutive-active MEK significantly increased the clonogenic ability of ESCC cells transfected with STAT1C (p < 0.05). Taken together, these findings suggest that ERK is a key regulator of STAT1 function in ESCC cells.

The observation that MG132 can induce effective apoptosis in various types of cancer, including ESCC, has been widely published [10, 14]. In view of our findings that STAT1 can induce effective apoptosis in ESCC [5], and our current observation that proteasome degradation is a key pathway to down-regulate STAT1, we hypothesized that MG132 also induces apoptosis in ESCC via a STAT1-dependent pathway. First, we confirmed that MG132 effectively induced apoptosis in ESCC cells, as evidenced by the reduction of viable cells as well as enhanced the cleavage of caspase 3 and PARP expression in dose and time dependent manner of MG132 treatment (Fig. 5a and b). Second, we found that siRNA knockdown of STAT1 significantly attenuated MG132-induced cell inhibition (at 5 μM) in EC1 and KYSE150 cells (Fig. 5c), and these findings correlated with the the lack of change in caspase 3, PARP and Annexin-V/propodium iodide (Fig. 5d-e). Taken together, these observations suggest that MG132-induced apoptosis in ESCC is highly STAT1-dependent.

In the previous published papers, we demonstrated the expression of STAT1 and ERK and its correlation with the clinical significance of ESCC patients [7]. Then we further investigate STAT1 and MAPK1(ERK) expression in ESCC. The bioinformatics analysis was performed to detect these gene alterations and expression in ESCC, using cBioportal Web resource online (cBioportal for Cancer Genomic)(Fig. 6a). However, we didn’t find the significance correlation between patients survival and STAT1/ERK alteration (Fig. 6b). As shown in Fig. 6c, the mutual exclusivity and co-occurrence analysis by Cbioportal revealed that STAT1 and ERK have a tendency to occur together. The results from the database supports our previous finding that ERK inhibit STAT1 expression and activation in ESCC and consistent with our previous results that ERK inhibited STAT1 expression [7].