Dsp-0187 Structure

[Crisoforo Schuhmacher]

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Lung cancer is a leading cause of cancer mortality worldwide. A variety of factors, including genetic alterations and tumor environment, contribute to lung cancer development and drug resistance. High interleukin-6 (IL-6) expression is common in patients with lung cancer and is indicative of poor prognosis1,2. IL-6 is released from tumor cells and immune cells and is involved in tumor initiation, progression, and metastasis via activation of multiple intracellular signaling pathways, including the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), phosphoinositide-3 kinase/AKT, RAS/mitogen-activated protein kinase (MAPK), MEK/extracellular signal-regulated kinase 5 (ERK5), and p38/JNK pathways3,4,5. Binding of IL-6 to IL-6R activates the JAK tyrosine kinases to induce phosphorylation of STAT3 at Y705, thereby activating it. This activated STAT3 plays crucial roles in proliferation, survival, metastasis, and self-renewal of cancer cells6,7,8. In lung cancer cells, STAT3 is activated by tyrosine kinases such as epidermal growth factor (EGF) receptor, JAK2, Src, and IL-6R3,5,9,10. Phosphorylated STAT3 molecules form homodimers, which enter the nucleus and transcribe target genes, such as survivin, Bcl-2, Mcl-1, c-Myc, cyclin D1, slug, and matrix metalloproteinase-2, involved in tumorigenesis, survival, and metastasis7,8,11,12,13,14. Aberrant activation of STAT3 is associated with malignant cancers with poor clinical prognosis, and STAT3 inhibition causes cancer cell death, suggesting STAT3 as a potential target for cancer therapy7,8,15.

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 transcription25,26. SOCS3 also negatively regulates JAK-STAT3, p44/P42 MAPK, and p53 in prostate and hepatocellular cancer27,28,29. STAT3-interacting 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 cancers30. 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 cancer31. 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 STAT332.

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 DDIAS-knockdown-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.

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.

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.

a DDIAS binds to STAT3. Flag-DDIAS and HA-STAT3 were co-transfected into HEK293T cells in the indicated combinations. The cell lysates were subjected to an immunoprecipitation assay using anti-Flag agarose or anti-HA beads, and the immunoprecipitates were probed with anti-HA or anti-Flag antibody. b Binding of endogenous STAT3 and DDIAS. Immunoprecipitation was performed using anti-STAT3 or anti-DDIAS in NCI-H1703 cells. c Mapping of STAT3-binding region on DDIAS. Flag-DDIAS and HA-STAT3 deletion constructs were co-transfected into HEK293T cells in the indicated combinations, and their interactions were analyzed by co-immunoprecipitation. d Mapping of STAT3-binding region on PTPRM. HA-STAT3 deletion constructs and Myc-PTPRM were co-transfected into HEK293T cells in the indicated combinations. The cell lysates were subjected to an immunoprecipitation assay using anti-HA antibody, and the immunoprecipitates were probed with anti-Myc or anti-HA antibody. e Effect of Flag-DDIAS on the interaction between STAT3 and PTPRM. HEK293T cells co-transfected with FLAG-DDIAS and/or Myc-PTPRM and HA-STAT3. Co-immunoprecipitation was performed using anti-HA antibody.

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 activity21,22,44. However, it is believed that the D2 domain of RPTP is catalytically inactive and functions as a substrate-binding domain that modifies the RPTP structure53. It has been shown that PTPRK directly binds to STAT3 and dephosphorylates STAT3 Y7051. In contrast, PTPRM dephosphorylates catenin p120ctn by binding to the N-terminus of catenin p120ctn, which is important for tyrosine phosphorylation54,55,56. 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 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.

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