DDIAS promotes STAT3 activation by preventing STAT3 recruitment to PTPRM in lung cancer cells

DNA damage-induced apoptosis suppressor (DDIAS) regulates cancer cell survival. Here we investigated the involvement of DDIAS in IL-6–mediated signaling to understand the mechanism underlying the role of DDIAS in lung cancer malignancy. We showed that DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is constitutively activated in malignant cancers. Interestingly, siRNA protein tyrosine phosphatase (PTP) library screening revealed protein tyrosine phosphatase receptor mu (PTPRM) as a novel STAT3 PTP. PTPRM knockdown rescued the DDIAS-knockdown-mediated decrease in STAT3 Y705 phosphorylation in the presence of IL-6. However, PTPRM overexpression decreased STAT3 Y705 phosphorylation. Moreover, endogenous PTPRM interacted with endogenous STAT3 for dephosphorylation at Y705 following IL-6 treatment. As expected, PTPRM bound to wild-type STAT3 but not the STAT3 Y705F mutant. PTPRM dephosphorylated STAT3 in the absence of DDIAS, suggesting that DDIAS hampers PTPRM/STAT3 interaction. In fact, DDIAS bound to the STAT3 transactivation domain (TAD), which competes with PTPRM to recruit STAT3 for dephosphorylation. Thus we show that DDIAS prevents PTPRM/STAT3 binding and blocks STAT3 Y705 dephosphorylation, thereby sustaining STAT3 activation in lung cancer. DDIAS expression strongly correlates with STAT3 phosphorylation in human lung cancer cell lines and tissues. Thus DDIAS may be considered as a potential biomarker and therapeutic target in malignant lung cancer cells with aberrant STAT3 activation.

Phosphatases (protein tyrosine phosphatases (PTPs)) control the Y705 phosphorylation-mediated activation of STAT3. PTPs are composed of approximately 280 amino acids with a conserved HCX 5 R motif 16,17 . These enzymes are divided into classical PTPs and dual-specificity phosphatases (DSPs). The classical PTPs include 21 receptortype PTPs (RPTPs) or 16 non-transmembrane proteins (NT-PTPs). The intracellular region of RPTPs contains the PTP domain with phosphatase activity, whereas the extracellular region shows functions similar to those of cell adhesion molecules and is involved in cell-cell and cell-matrix contact 16 . The NT-PTPs contain catalytic domains with phosphatase activity and non-catalytic domains that regulate subcellular distribution by restricting access to their particular substrates 16 . STAT3 inactivation occurs via dephosphorylation by PTPs such as SH2-containing phosphatase 2 (SHP2), T cell PTP (TC-PTP), and low molecular weight-DSP2 [18][19][20] . Recent studies reported that STAT3 is dephosphorylated by RPTPs, including PTPRT, PTPRD, and PTPRK in colorectal cancer, glioblastoma multiforme, and nasal-type natural killer/T cell lymphoma, respectively [21][22][23] . PTPRT and PTPRD are tumor suppressors and are frequently inactivated or mutated in human cancers 23,24 .
In addition to PTPs, various negative regulators of STAT3 signaling in cancer cells are known. Protein inhibitor of activated STAT (PIAS1) and suppressor of cytokine-signaling proteins 3 (SOCS3) negatively regulate STAT3 in cancer. PIAS1 can suppress JAK-STAT3 signaling and bind to activated STAT3 dimers to prevent DNA binding for transcription 25,26 . SOCS3 also negatively regulates JAK-STAT3, p44/P42 MAPK, and p53 in prostate and hepatocellular cancer [27][28][29] . STAT3interacting proteins, including chromatin remodeling proteins such as BRG1 and oncogenic transcription factors such as nuclear factor kB (NF-kB) or nuclear factor of activated T cells 1 (NFATc1), have been reported to function as either positive or negative regulators in many types of cancers 30 . Of the positive regulators, annexin A2 binds to STAT3 C-terminal domain containing transactivation domain (TAD) and enhances STAT3 activity, thereby promoting epithelial-to-mesenchymal transition in breast cancer 31 . In contrast, GdX (X-linked gene in G6PD cluster at Xq28) is a negative regulator of STAT3 and promotes STAT3 dephosphorylation by stabilizing the interaction between STAT3 and TC45 (the nuclear isoform of TC-PTP), a nuclear phosphatase of STAT3 32 .
DNA damage-induced apoptosis suppressor (DDIAS), which is abundantly expressed in lung cancer and hepatocellular carcinoma (HCC), regulates the survival of cancer cells via multiple signaling pathways [33][34][35][36] . DDIAS expression is activated by the transcription factor NFATc1 and is induced by ERK5/myocyte enhancer factor 2B pathway 33,34 . Moreover, DDIAS protein stability is negatively regulated by the E3 U-box ubiquitin ligase carboxyl terminus of heat shock protein 70-interacting protein 35 . Furthermore, DDIAS acts as a cofactor for DNA polymerase-primase complex by associating with DNA polymerase to promote tumorigenesis in HCC 37 . DDIAS protects cancer cells from DNA-damaging reagents and tumor necrosis factor-related apoptosisinducing ligand (TRAIL) and positively regulates cancer cell invasion by stabilizing β-catenin 33,34,36,38 . Hence, DDIAS is suggested as a potential therapeutic target for malignant lung cancer 33,35,36 . Although IL-6 is implicated in the malignancy of lung cancers, the association of DDIAS in IL-6-mediated signaling has not been investigated. Therefore, using DDIAS-knockdown experiments, we investigated the mechanisms underlying the role of DDIAS in the IL-6-mediated pathway.
In this study, we demonstrated that DDIAS positively regulated IL-6-mediated STAT3 activation. We also showed that DDIAS physically associated with STAT3 to prevent recruitment of STAT3 to protein tyrosine phosphatase receptor mu (PTPRM), a novel phosphatase for STAT3, thereby sustaining STAT3 Y705 phosphorylation. Our findings suggest a novel pathway for DDIASmediated aberrant activation of STAT3 in malignant lung cancer cells.

DDIAS promotes STAT3 tyrosine phosphorylation in the presence of IL-6
To determine whether IL-6-mediated signaling is controlled by DDIAS, we performed human phosphokinase array using Proteome Profiler TM antibody array in NCI-H1703 cells. Decreased pSTAT3 (Y705) levels were observed in DDIAS-depleted cells in the presence of IL-6 ( Fig. S1). Then we examined the correlation between DDIAS and STAT3 activation in several lung cancer cells. DDIAS expression was strongly correlated with STAT3 expression and STAT3 Y705 phosphorylation (Fig. 1a). To assess whether DDIAS is involved in the IL-6-mediated STAT3 activation, we compared STAT3 phosphorylation in the presence or absence of DDIAS. DDIAS depletion attenuated IL-6-induced STAT3 phosphorylation at Y705 but not at S727 in non-small cell lung cancer (NSCLC) cells, such as NCI-H23 and NCI-H1703 (Fig. 1b). Furthermore, DDIAS knockdown suppressed EGF-induced STAT3 Y705 phosphorylation (Fig. 1c) and inhibited the nuclear translocation of IL-6-mediated pSTAT3 (Fig. 1d). To address whether DDIAS regulates STAT3 transcriptional activity, we performed luciferase assay using a reporter gene (M67-Luc) containing STAT3-binding sites, in the presence or absence of DDIAS. DDIAS depletion significantly repressed the IL-6-induced STAT3 Y705 transcriptional activity in NCI-H1703 (Fig. 1e), whereas Flag-DDIAS overexpression enhanced IL-6-induced STAT3 phosphorylation (Fig. 1f). These results suggest that DDIAS is involved in enhancing STAT3 phosphorylation in the presence of IL-6 or EGF.

PTP inhibition overcomes STAT3 dephosphorylation in DDIAS-knockdown cells
To understand the mechanism underlying STAT3 activation by DDIAS in the presence of IL-6, we investigated the phosphorylation status of JAK1 and JAK2. No significant change was detected in either expression level or phosphorylation degree of both JAK1 and JAK2 in DDIAS-silenced cells (Fig. 2a). Thus the JAK pathway may not be associated with the decrease in STAT3 phosphorylation in the DDIAS-knockdown cells. Therefore, to assess whether DDIAS is involved in the regulation of PTP-mediated dephosphorylation of STAT3, we treated DDIAS-depleted cells with sodium vanadate, a PTP inhibitor, in the presence of IL-6. Sodium vanadate completely recovered STAT3 dephosphorylation in DDIAS-knockdown NCI-H1703 cells (Fig. 2b). Next, we explored the PTPs involved in STAT3 dephosphorylation in the absence of DDIAS. Small interfering RNA (siRNA) screening of validated siRNAs of 36 PTPs was performed to identify PTPs that could recover STAT3 Y705 phosphorylation. Six candidates PTPRM, PTPRN2, DUSP11, PTPRK, PTPRZ1, and DUSP12 overcame the suppression of STAT3 phosphorylation and its transcriptional activity in DDIAS-knockdown cells (Fig. 2c, Fig. S2). The knockdown efficiency of siRNA against the six genes was confirmed using quantitative real-time polymerase chain reaction (qPCR) (Fig. 2d). PTPRK knockdown and PTPRM knockdown completely recovered the DDIASknockdown-induced decrease in STAT3 phosphorylation (Fig. 2e). These results demonstrate that DDIAS is involved in the activation of STAT3 signaling by suppressing that activity of PTPs, such as PTPRM or PTPRK.

PTPRM is a novel phosphatase of STAT3
To confirm that PTPRM regulates STAT3 Y705 dephosphorylation, PTPRM siRNA was transfected into NCI-H23 and NCI-H1703 cells. Compared to the respective controls, PTPRM knockdown cells showed an increased basal level of STAT3 phosphorylation and increased levels of IL-6-induced STAT3 phosphorylation ( Fig. 3a, b), whereas cells with Myc-PTPRM overexpression showed decreased levels of both basal and IL-6-induced STAT3 phosphorylation (Fig. 3c, d). Hence, we next assessed the importance of Y705 for the interaction between PTPRM and STAT3. Co-immunoprecipitation assay revealed that Myc-PTPRM bound to hemagglutinin (HA)-STAT3 wild type (WT) but not HA-STAT3 Y705F (YF; Fig. 3e), indicating that PTPRM is associated with Y705 dephosphorylation of STAT3. Interestingly, PTPRM and STAT3 showed a strong interaction at 60 min of IL-6 treatment (Fig. 3f). These results indicate that PTPRM is a novel phosphatase for STAT3.

DDIAS inhibits PTPRM-STAT3 binding to sustain STAT3 phosphorylation
To understand the association of DDIAS in STAT3 phosphorylation, we investigated the effect of DDIAS on PTPRM activity. First, we examined whether DDIAS depletion affects the mRNA and protein levels of PTPRM. Reverse transcription (RT)-qPCR and western blot analysis revealed that DDIAS did not affect the expression of PTPRM as well as the known STAT3 phosphatases PTPRT, SHP1, and TC-PTP (Fig. 4a, b). Next, we determined the effect of DDIAS on PTP activity using in vitro phosphatase activity assay in DDIAS-knockdown cells. Consistently, DDIAS knockdown did not affect the total PTP activity (Fig. 4c). Therefore, we performed immunoprecipitation analysis using anti-STAT3 antibody in DDIAS-knockdown cells to investigate whether DDIAS is involved in the regulation of PTPRM/STAT3 binding. Notably, in the presence of IL-6, PTPRM and STAT3 showed a stronger interaction in DDIAS-knockdown cells, compared with that in the control cells (Fig. 4d). Next, we performed co-immunoprecipitation analysis to determine whether DDIAS interacts with PTPRM and found that Flag-DDIAS did not interact with Myc-PTPRM in HEK293T cells (Fig. 4e). This result suggests that DDIAS interferes with the interaction between PTPRM and STAT3 without binding to PTPRM. Considering these observations, we investigated the correlation between the expression of PTPRM, STAT3, and DDIAS in different NSCLC cell lines. Interestingly, PTPRM expression in the different NSCLC cell lines was variable and showed no correlation with DDIAS or STAT3 expression in most of the cell lines (Figs. 4f and 1a). To evaluate the correlation between STAT3 activation and expression of DDIAS and PTPRM, we performed immunohistochemistry (IHC) on 40 human lung cancer tissues (Fig. 4g). Consistent with our in vitro human lung cancer cell lines (Fig. 1a), DDIAS expression level was strongly correlated with STAT3 tyrosine phosphorylation. However, overall PTPRM expression level has no correlation with STAT3 phosphorylation in human lung cancer tissues.

DDIAS interacts with STAT3 and interferes with PTPRM/ STAT3 binding
To further understand the mechanism underlying DDIAS-mediated regulation of STAT3 phosphorylation, we assessed whether DDIAS directly interacts with STAT3. Co-immunoprecipitation assay clearly showed that Flag-DDIAS interacted with HA-STAT3 in HEK293T cells (Fig. 5a). Furthermore, endogenous DDIAS bound to STAT3 in NCI-H1703 cells (Fig. 5b). Domain mapping analysis revealed that DDIAS bound to the TAD of STAT3 (Fig. 5c) and that this STAT3 domain was also involved in the interaction with PTPRM (Fig. 5d). Notably, DDIAS overexpression suppressed the interaction between STAT3 and PTPRM (Fig. 5e). These results suggest that DDIAS binds to STAT3 to interfere with PTPRM/STAT3 binding and that PTPRM competes with DDIAS to recruit STAT3 for dephosphorylation.

DDIAS promotes STAT3-mediated malignancy by preventing PTPRM function
We observed that PTPRM overexpression inhibited cell growth in a dose-dependent manner (Fig. S3). Then we examined the effect of PTPRM knockdown on DDIASknockdown-induced suppression of cancer cell growth and invasion (Fig. 6). Clearly, PTPRM knockdown partially rescued the cell growth inhibition induced by DDIAS knockdown (Fig. 6a). Moreover, PTPRM silencing recovered the migration and invasion suppressed by DDIAS knockdown (Fig. 6b, c). As expected, PTPRM depletion increased the expression of STAT3 targets vimentin and slug, which are involved in the migration of DDIAS-knockdown cells (Fig. 6d). Thus PTPRM suppresses STAT3 signaling in DDIAS-knockdown cells, suggesting that DDIAS promotes the IL-6-mediated STAT3 signaling in malignant cancer cells by inhibiting the PTPRM function.
In this study, we found that DDIAS positively regulated IL-6-mediated STAT3 activation. Using siRNA PTP library screening, we first identified PTPRM as a novel PTP that suppresses STAT3 Y705 phosphorylation in NCI-H1703 cells. Furthermore, we found that PTPRK or PTPRT also recovered the STAT3 dephosphorylation induced by DDIAS knockdown (Fig. 2e, Fig. S4). In DDIAS-knockdown cells, PTPRM knockdown recovered the decrease in IL-6-mediated STAT3 Y705 phosphorylation. Similar to PTPRK, PTPRT, and PTPRU, PTPRM belongs to R2B subfamily of RPTPs, which consist of extracellular regions containing one MAM (meprin/A5/μ), one immunoglobulin (Ig)-like, and four fibronectin domains and intracellular regions containing the juxtamembrane domain and tandem intracellular phosphatase domain 17 . PTPRM facilitates hemophilic cell-cell interaction through the extracellular region independent of phosphatase activity in the intracellular domains 47 . Indeed, PTPRM has been shown to inhibit cancer cell proliferation, migration, and invasion [48][49][50] . PTPRM expression is downregulated by genetic and epigenetic alterations such as loss of heterozygosity and hypermethylation in breast cancer, colorectal cancer, and acute lymphoblastic leukemia 48,49,51 . Moreover, PTPRM is downregulated by miRNA-221/-222 in glioblastoma, suggesting its tumorsuppressor function 52 .
Mechanistically, non-receptor PTPs, such as SHP1or SHP2 containing Src homology-2 (SH2) domains, mediate the interaction of PTP with its substrates through SH domains, which function as phospho-tyrosine-binding domains. While receptor-type PTPs such as PTPRK and PTPRT have two intracellular PTP domains (D1 and D2), mutants of the D1 and D2 domain show decreased PTP activity 21,22,44 . However, it is believed that the D2 domain of RPTP is catalytically inactive and functions as a substrate-binding domain that modifies the RPTP structure 53 . It has been shown that PTPRK directly binds to STAT3 and dephosphorylates STAT3 Y705 1 . In contrast, PTPRM dephosphorylates catenin p120 ctn by binding to the N-terminus of catenin p120 ctn , which is important for tyrosine phosphorylation [54][55][56] . Our data show that PTPRM interacted with the STAT3 TAD, which contains the Y705 (Fig. 7). We also observed that DDIAS bound to the STAT3 C-terminal domain containing TAD. These findings suggest that DDIAS competes with PTPRM for STAT3 binding. Thus DDIAS sustains STAT3 phosphorylation by inhibiting the accessibility of PTPRM to STAT3, suggesting a novel mechanism of PTPRM as a tumor suppressor.
We found that DDIAS expression is closely correlated with STAT3 Y705 phosphorylation in NSCLC cell lines and that PTPRM knockdown recovered the reduced STAT3 Y705 phosphorylation in DDIAS-knockdown cells. However, PTPRM expression was variable and showed no correlation with STAT Y705 phosphorylation in lung cancer cell lines and human tissues (Fig. 4f, g); this suggests that mutations in PTPRM or other PTPs are involved in STAT3 dephosphorylation, depending on the cancer cells. The protein levels of PTPRK in the different NSCLC cell lines were also variable and showed no correlation with STAT3 Y705 phosphorylation (Fig. S5a). In NCI-H1703 cells, the expression level of PTPRM was higher than that of PTPRK, PTPRT, or PTPRZ1 in NCI-H1703 cells (Fig. S5b), suggesting that PTPRM is the main phosphatase for STAT3. However, it may be possible that DDIAS prevents STAT3 from recruiting other RPTPs, including PTPRK or PTPRT, by directly binding with STAT3 in other cells. PTPRT expression was very low in most of the NSCLC cell lines (Fig. S5a). Notably, DDIAS affects the involvement of phosphatases in regulation of IL-6-mediated STAT3 phosphorylation.
Previously, we showed that DDIAS promotes proliferation and invasion of cancer cells and is involved in suppression of DNA-damaging-induced apoptosis and TRAIL-mediated extrinsic apoptosis in cancer cells. In this study, we demonstrated that DDIAS promoted IL-6-mediated STAT3 activation in lung cancer cells by competing with PTPRM. DDIAS knockdown caused growth inhibition of lung cancer cells through STAT3 inactivation by PTPRM, suggesting DDIAS as a potential therapeutic target in aberrant STAT3-activated lung cancer. Moreover, IHC analysis demonstrated correlation between the DDIAS expression level and STAT3 phosphorylation in lung cancer patient tissues (Fig. S6). Therefore, we suggest that DDIAS can serve as a biomarker to predict the STAT3 phosphorylation in malignant lung cancer.
We newly identified the DDIAS/PTPRM/STAT3 system in lung cancer cells with aberrant STAT3 expression. We expect multiple, intertwined crosstalks and interactions between DDIAS/PTPRM and other STAT3 regulation system in lung cancer cells. However, we do not have conclusive evidence regarding it. The ability of PTP to compete with DDIAS for STAT3 inactivation depends on its specificity to STAT3, its structural similarity to PTPRM, and its intracellular availability. PTPRM belongs to the same family as PTPRK and PTPRT. Although PTPRT has specificity to STAT3, it is less abundant than PTPRM in the cell. Non-receptor-type PTP, such as SHP1, SHP2, and TC-PTP, are not specific to STAT3. Even though TC-PTP is present at a high level, the dephosphorylation efficiency of STAT3 by TC-PTP is expected to be lower than that by PTPRM. Interestingly, DUSP11 and DUSP12 seem to affect STAT3 inactivation through MAP inhibition.
Using siRNA PTP library screening, we identified PTPRM as an activator of STAT3 Y705 phosphorylation in DDIAS-knockdown cells. However, the PTP siRNA library does not contain all the known PTPs; therefore, it is possible that we may have missed a PTP involved in STAT3 Y705 dephosphorylation. Moreover, a PTP could have been excluded as a false negative in our siRNA screening system, although the experiment using validated siRNA was performed under stringent conditions.  Nevertheless, we demonstrated that DDIAS binds to STAT3 and prevents STAT3 recruitment to PTPRM for Y705 dephosphorylation. We further plan to screen drugs inhibiting the interaction between DDIAS and STAT3 and investigate the effectiveness of this drug in combination with the currently available treatment for lung cancer.
In summary, we provide novel insights into the crucial role of DDIAS in STAT3 signaling in malignant lung cancer. We confirmed that DDIAS is involved in STAT3 activation in malignant lung cancer. We report that PTPRM is a novel STAT3 tyrosine phosphatase and that it negatively regulates STAT3 activation by dephosphorylating Y705 of STAT3 in the presence of IL-6. We also demonstrate that DDIAS promotes STAT3 phosphorylation by blocking PTPRM recruitment of STAT3 via competitive binding with STAT3. Full-length and deletion mutants of Flag-DDIAS were cloned as previously described 35 . 4×M67 pTATA TK-Luc (#8688) and Flag-STAT3 Y705F (#24984) were obtained from Addgene (Cambridge, MA, USA). Myc-DDK-tagged PTPRM (RC226398) was obtained from OriGene (Rockville, MD, USA). Full-length and deletion mutants of STAT3-tagged HA were subcloned from the Flag-STAT3 expression vector (Addgene). STAT3 gene was mutated using the QuickChange Site-Directed Mutagenesis Kit (Enzynomics, Daejeon, Korea). The constructs were verified using sequence analysis.

Luciferase reporter assays
Cells were co-transfected with 300 ng of 4×M67 pTATA TK-Luc and 10 ng of pRL-TK using 1 μl of TurboFect per well in 24-well plates, serum starved for 24 h, and treated with 20 ng/ml of IL-6 for 6 h. Luciferase assay was performed as previously described 33 .

Cell migration and invasion assay
These assays were performed in 24-well Transwell plates (BD Biosciences, San Jose, CA, USA) with 8-μm pore inserts coated with or without Matrigel (Invitrogen). Cells (1 × 10 5 ) were seeded in a culture insert in serumfree media and treated with 20 ng/ml IL-6 into the upper chamber. Complete medium was applied to the lower chamber. Cells were allowed to migrate or invade the layer for 12 or 24 h, respectively. Migrating/invading cells were stained with 0.4% SRB and visualized under a microscope. The experiment was repeated three times.

Immunocytochemistry
Immunofluorescence staining was performed as described previously 34 . Cells were incubated with an anti-pSTAT3 antibody overnight. Fluorescent images were visualized using an LSM 800 microscope (Zeiss, Jena, Germany).

Immunohistochemistry
Human tissue arrays (LC485) were obtained from US Biomax, Inc. (Rockville, MD, USA). IHC staining was performed as previously described 33 . Tissues were incubated with anti-pSTAT3 (Y705), anti-DDIAS, or anti-PTPRM antibodies overnight. The images were visualized using an Olympus BX51 microscope equipped with a DP71 digital camera and DP-B software (Olympus Co, Tokyo, Japan). pSTAT3, DDIAS, and PTPRM positivity was defined as 3+, 2+, 1+, or 0 by IHC. Tissues stained were divided into two high (3+ and 2+) and low (1+ and 0) groups. Scoring of the human tissue array was performed by two independent observers (J.-Y.I. and K.-W.L.), showing high consistency between scores for both pSTAT3 and DDIAS.

Co-immunoprecipitation and western blot analysis
Immunoprecipitation and western blotting were performed as described previously 36 . For endogenous binding, lysates were incubated with anti-STAT3 or anti-DDIAS antibody and subjected to western blot analysis with specific antibodies. Signals were detected using an enhanced chemiluminescence kit (Millipore, Temecula, CA, USA). The densitometry numbers indicated were measured using Image J (NIH).
Tyrosine phosphatase assay PTP activity was measured using a Tyrosine Phosphatase Assay System (Promega, Madison, WI, USA) according to the manufacturer's protocol. NCI-H1703 cells were transfected with siDDIAS for 72 h and lysed with storage buffer. Spin columns were used to remove endogenous free phosphate from the cell lysates. Tyrosine phosphatase activity was measured using the EMax plate reader system (Molecular Devices, San Jose, CA, USA) at 600 nm.

Statistical analysis
Data were obtained from at least three independent experiments with biological triplicates and presented as mean ± SEM. Statistical analyses were performed using two-tailed Student's t test. A value of p < 0.05 was accepted as significant.