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Suppression of STAT3 NH2 -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

ABSTRACT: Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients.

SUBMITTER: Ray S

PROVIDER: S-EPMC6132241 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Suppression of STAT3 NH<sub>2</sub> -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

Ray Sutapa S Coulter Don W DW Gray Shawn D SD Sughroue Jason A JA Roychoudhury Shrabasti S McIntyre Erin M EM Chaturvedi Nagendra K NK Bhakat Kishor K KK Joshi Shantaram S SS McGuire Timothy R TR Sharp John G JG

Molecular carcinogenesis 20180125 4

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH<sub>2</sub> terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several b ...[more]

PMID: 29280516

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Project description:Signal Transducers and Activator of Transcription-3 (STAT3) are latent transcription factors that are regulated by post-translational modifications (PTMs) in response to cellular activation by the IL-6 superfamily of cytokines to regulate cell cycle progression and/or apoptosis. Here we observe that STAT3 is inducibly mono-ubiquitinated and investigate its consequences. Using domain mapping and highly specific selected reaction monitoring-mass spectrometric assays, we identify lysine (K) 97 in its NH2-terminal domain as the major mono-ubiquitin conjugation site. We constructed a mono-ubiquitinated mimic consisting of a deubiquitinase-resistant monomeric ubiquitin fused to the NH2 terminus of STAT3 (ubiquitinated-STAT3 FP). In complex assays of ectopically expressed ubi-STAT3-FP, we observed enhanced complex formation with bromodomain-containing protein 4 (BRD4), a component of the activated positive transcriptional elongation factor (P-TEFb) complex. Chromatin immunoprecipitation experiments in STAT3(+/-) and STAT3(-/-) MEFs showed BRD4 recruitment to STAT3-dependent suppressor of cytokine signaling-3 gene (SOCS3). The effect of a selective small molecule inhibitor of BRD4, JQ1, to inhibit SOCS3 expression demonstrated the functional role of BRD4 for STAT3-dependent transcription. Additionally, ectopic ubiquitinated-STAT3 FP expression upregulated BCL2, BCL2L1, APEX1, SOD2, CCND1 and MYC expression indicating the role of ubiquitinated STAT3 in anti-apoptosis and cellular proliferation. Finally we observed that ubiquitinated-STAT3 FP suppressed TNFα-induced apoptotic cell death, indicating the functional importance of mono-ubiquitinated STAT3 in antiapoptotic gene expression. We conclude that STAT3 mono-ubiquitination is a key trigger in BRD4-dependent antiapoptotic and pro-proliferative gene expression programs. Thus, inhibiting the STAT3 mono-ubiquitination-BRD4 pathway may be a novel therapeutic target for the treatment of STAT3-dependent proliferative diseases.

Dataset Information

Suppression of STAT3 NH2 -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

Publications

Suppression of STAT3 NH<sub>2</sub> -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

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