Serine-Phosphorylated STAT3 Promotes Tumorigenesis Via Modulation of RNA Polymerase Transcriptional Activity
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Author Manuscript Published OnlineFirst on September 3, 2019; DOI: 10.1158/0008-5472.CAN-19-0974 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Serine-phosphorylated STAT3 promotes tumorigenesis via modulation of RNA polymerase transcriptional activity Jesse J. Balic1,2, Daniel J. Garama2,3,#, Mohamed I. Saad1,2,#, Liang Yu1,2, Alison C. West1,2, Alice J. West1,2, Thaleia Livis1,2, Prithi S. Bhathal2, Daniel J. Gough2,3,* and Brendan J. Jenkins1,2,* 1Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, Victoria, 3168, Australia. 2Department of Molecular and Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, 3800, Australia. 3Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia. #Daniel J. Garama and Mohamed I. Saad contributed equally. *To whom correspondence should be addressed: Brendan J. Jenkins, Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, Victoria 3168, Australia. Tel +61 3 8572 2740; E-mail [email protected]. Daniel J. Gough, Centre for Cancer Research, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, Victoria 3168, Australia. Tel +61 3 8572 2710; E-mail [email protected] Running title: STAT3 serine phosphorylation promotes gastric cancer 1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 3, 2019; DOI: 10.1158/0008-5472.CAN-19-0974 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Key words: ERH, gastric cancer, rapid immunoprecipitation mass spectrometry of endogenous protein, RNA polymerase II, RUVBL1/Pontin, STAT3, transcriptional regulation. Financial support: This work is supported by a research grant awarded by the National Health and Medical Research Council (NHMRC) of Australia to B.J. Jenkins, as well as the Operational Infrastructure Support Program by the Victorian Government of Australia. J.J. Balic is supported by an Australian Postgraduate Awards scholarship from the Australian Government. A.C. West is supported by an NHMRC Early Career Fellowship, and D.J. Gough by an NHMRC Career Development Fellowship and a grant from the United States Department of Defence. B.J. Jenkins is supported by an NHMRC Senior Medical Research Fellowship. Disclosures No conflicts of interest, financial or otherwise, are declared by all authors. Significance: This study reveals that constitutive STAT3 serine phosphorylation is essential for gastric tumorigenesis via interaction with co-factors Pontin and ERH to augment RNA polymerase-driven transcription. Word count: 8,092 Figures: 7 Tables: 0 2 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 3, 2019; DOI: 10.1158/0008-5472.CAN-19-0974 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Deregulated activation of the latent oncogenic transcription factor signal transducer and activator of transcription (STAT)3 in many human epithelial malignancies, including gastric cancer (GC), has invariably been associated with its canonical tyrosine phosphorylation and enhanced transcriptional activity. By contrast, serine phosphorylation (pS) of STAT3 can augment its nuclear transcriptional activity and promote essential mitochondrial functions, yet the role of pS-STAT3 among epithelial cancers is ill-defined. Here, we reveal that genetic ablation of pS-STAT3 in the gp130F/F spontaneous GC mouse model and human GC cell line xenografts abrogated tumor growth that coincided with reduced proliferative potential of the tumor epithelium. Microarray gene expression profiling demonstrated that the suppressed gastric tumorigenesis in pS-STAT3-deficient gp130F/F mice associated with reduced transcriptional activity of STAT3-regulated gene networks implicated in cell proliferation and migration, inflammation and angiogenesis, but not mitochondrial function or metabolism. Notably, the pro-tumorigenic activity of pS-STAT3 aligned with its capacity to primarily augment RNA polymerase II-mediated transcriptional elongation, but not initiation, of STAT3 target genes. Furthermore, by employing a combinatorial in vitro and in vivo proteomics approach based on the rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) assay, we identified RuvB-like AAA ATPase 1 (RUVBL1/Pontin) and enhancer of rudimentary homolog (ERH) as interacting partners of pS- STAT3 that are pivotal for its transcriptional activity on STAT3 target genes. Collectively, these findings uncover a hitherto unknown transcriptional role and obligate requirement for pS-STAT3 in GC that could be extrapolated to other STAT3-driven cancers. 3 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 3, 2019; DOI: 10.1158/0008-5472.CAN-19-0974 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Elevated tyrosine phosphorylation at residue 705 (pY705) of STAT3 is an indirect indicator of its transcriptional activity on diverse gene networks which promote many cancer- associated cellular processes, such as proliferation and survival, inflammation, angiogenesis, metastasis and immunosuppression (1, 2). The interleukin (IL)-6 cytokine family (IL-6, IL- 11, IL-27, among others) is a prominent activator of STAT3 via dimerization of the gp130 signal-transducing co-receptor. Here, activation of gp130-associated Janus kinases (JAKs) tyrosine phosphorylate STAT3, enabling formation of STAT3 homodimers, or heterodimers with additional transcription factors (e.g. STAT1, NANOG, c-Jun/c-Fos, OCT-1) and co- activators (e.g. p300/CBP), which then translocate to the nucleus (3, 4). Also, pY-STAT3 can indirectly influence gene transcriptional programming by inducing the expression of other transcription factors (e.g. MYC) (4). In a non-phosphorylated state, STAT3 can also associate with NF-B to drive a distinct transcriptional signature comprising genes implicated in oncogenic and immune responses (5). It has also emerged that post-translational modifications of STAT3, namely acetylation, methylation, S-glutathionylation, ubiquitination and SUMOylation, can collectively modulate its dimerization, nuclear retention and DNA binding capacity, and thus influence transcriptional outputs (6, 7). Notably, the tumor promoting potential of STAT3 extends to its role in the mitochondria, where it acts as a central regulator of cellular metabolism. While nuclear STAT3 can reprogram metabolism in cancer cells by directing the transcription of MYC and HIF1A, both of which are master regulators of the ‘Warburg effect’, STAT3 can also enter the mitochondria and directly regulate the activity of the electron transport chain that is dependent on serine phosphorylation at residue 727 (pS727) (8-10). Mitochondrial pS- STAT3 appears to be a requisite for mutant Ras-driven tumors, whereas the extent of its role in the pathogenesis of other cancer types remains unknown (6-8, 11). 4 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 3, 2019; DOI: 10.1158/0008-5472.CAN-19-0974 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. There are contrasting reports on the requirement of pS727 for STAT3 DNA binding and transcriptional activities, and modulation of Y705 phosphorylation (12-17). Among these, embryonic fibroblasts derived from Stat3SA/SA mice harbouring an S727A knock-in substitution in the endogenous Stat3 locus (SA allele) exhibited a 50% reduction in the IL-6- dependent transcriptional response compared to wild-type cells (13). Furthermore, Stat3SA/SA mice employed in a model of angiotensin II-induced hypertension demonstrate reduced expression of STAT3-dependent cardiac remodelling genes (17). These observations suggest a role for pS-STAT3 in transcriptional regulation, along with its potential contribution to physiological and pathological states, albeit ill defined. We have previously generated a STAT3-driven genetic mouse model (gp130F/F) of spontaneous intestinal-type gastric cancer (GC) (18-20). These mice are homozygous for a phenylalanine (F) knock-in substitution of the cytoplasmic Y757 residue in endogenous gp130, which blocks binding of the negative regulator suppressor of cytokine signaling (SOCS)3, leading to exaggerated IL-11-driven pY-STAT3 levels in the gastric compartment (18, 19). The causal role for dysregulated IL-11/gp130-dependent STAT3 activation in the development of gastric tumors in gp130F/F mice was shown by the suppressed tumorigenesis observed upon either heterozygous or homozygous ablation of Stat3 or Il11r1, respectively (18, 19). However, whether pS-STAT3 plays any role in GC is unknown. Here, we employ a genetic strategy to demonstrate that constitutive serine phosphorylation is essential for the oncogenic activity of STAT3 in the gastric epithelium. Moreover, by combining a proteomics-based screen with CRISPR/Cas9-mediated