Serine-Phosphorylated STAT3 Promotes Tumorigenesis Via Modulation of RNA Polymerase Transcriptional Activity

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

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

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.

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).

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 Il11r1, 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 functional validation, we reveal RuvB- like AAA ATPase 1 (RUVBL1/Pontin) and enhancer of rudimentary homolog (ERH) proteins as interacting partners of pS-STAT3 essential for maximal transcriptional efficiency of RNA polymerase on STAT3-regulated genes in GC.

Materials and Methods

Human biopsies

Antral gastric biopsies (tumor/matched non-tumor) were collected by surgical resection from

GC patients at Monash Medical Centre (MMC; Melbourne, Australia) and Xin Hua Hospital

(Shanghai, China) (Supplementary Table S1). Non-cancerous (normal control) antral gastric tissue was collected from cancer-free individuals undergoing endoscopy (MMC). Biopsies were snap-frozen in liquid nitrogen or stored in 10% formalin, the latter for histopathology and H. pylori status (21). Written informed patient consent was obtained, and collections were approved by Monash Health Human Research Ethics Committee and Xin Hua Hospital Ethics

Committee. Patient studies were conducted in accordance with the World Medical

Association Declaration of Helsinki statement on the ethical principles for medical research involving human subjects.

GC mouse models and treatments

The gp130F/F, gp130F/F:Stat3+/-, gp130:Il11ra-/- and Stat3SA/SA mice (13, 18, 19, 22), along with genetically matched (129Sv×C57BL/6) wild-type mice as littermate controls, were housed under specific pathogen–free conditions. All experiments were approved by the MMC

“B” Animal Ethics Committee.

Recombinant human IL-11 (5μg; PeproTech) or PBS was intraperitoneal injected into

6-8-week-old mice. For xenografts, MKN-28 STAT3-WT, STAT3-SD or STAT3-SA cells

(1.75×106) resuspended in 50% v/v PBS and Matrigel (Cultrex) were subcutaneously injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ/Arc (NSG) mice (Animal Resources Centre, Australia), and tumor size was measured weekly using electronic callipers to calculate tumor volume

(2×width×length)/2=Vmm3).

Bone marrow chimeras

6-8-week-old gp130F/F mice were lethally irradiated with a single 9.5Gy dose, following which mice were reconstituted via intravenous injection with 107 cells from unfractionated bone marrow of donor mice. Recipient mice were culled at 10-12 weeks post-transplant.

Isolation of nuclear and cytoplasmic tissue fractions, and gastric epithelial glands

Nuclear and cytoplasmic fractions were isolated from fresh antral tissues using the NE-

PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (ThermoScientific). For epithelial gland isolation, gastric antral tissue was collected in ice-cold Hank’s Balanced Salt Solution

(HBSS; Gibco), and tissue was incubated in HBSS containing 30mM EDTA for 15min at

37oC. Tissue was repeatedly flushed using a 1mL pipette to liberate glands from the basement membrane. Glands were collected by centrifugation and lysed in RIPA buffer.

Histology and immunohistochemistry

Following formalin fixation and paraffin embedding (FFPE), histological assessment of mouse stomachs and MKN-28 xenografts was performed on 4-6m hematoxylin and eosin

(H&E)-stained tissue sections. For immunohistochemistry, sections were deparaffinised in xylene and re-hydrated by sequential submersions in 100%, 70% and 0% v/v ethanol/dH2O.

Antigen retrieval for pY-STAT3 immunohistochemistry was performed in heated 1mM

EDTA, pH8.0 solution, and for other immunohistochemistry in heated 10mM sodium citrate acid, pH6.0 solution. Endogenous peroxidase was blocked in 3% v/v hydrogen peroxidase/methanol, and sections were blocked against non-specific binding by incubating in 10% v/v serum/Tris-buffered saline, matched to the species of secondary antibody.

Immunohistochemistry was performed with primary antibodies against pY-STAT3 (1:1,000 dilution), total STAT3 (1:1,000 dilution) and PCNA (1:5,000 dilution) (Cell Signaling

Technology), pS-STAT3 (1:1,000 dilution) (Santa Cruz Biotechnology), and CD45 (1:100 dilution) (BD Biosciences), along with concentration matched rabbit-IgG control (Vector

Labs). Following overnight 4oC incubations, sections were incubated at room temperature with biotinylated anti-rabbit IgG antibodies (Vector Labs), biotin labelled with HRP

(Vectastain ABC HRP Kit; Vector Labs), and developed using a liquid diaminobenzidine chromogen substrate system (Dako). PCNA staining was performed using the Mouse on

Mouse (M.O.M™) detection kit (Vector Labs). Sections were counterstained with hematoxylin, images acquired using CellSens software (Olympus). PCNA and STAT3 (total, pY- and pS-) positivity was determined using the positive pixel count algorithm v9.0 (Leica

Biosystems) on whole slide scans of stained sections; positivity was calculated as the total number of positive pixels/total number of pixels.

For mucosal thickness measurements, whole H&E-stained slides were imaged using an

Aperio Scanscope AT Turbo digital pathology scanner (Leica Biosystems). Mucosal thickness was measured at 10 random points using the linear measurement tool of Aperio

Image Scope software (Leica Biosystems), and the average taken. For PAS/Alcian blue staining, sections were incubated in 1% w/v Alcian blue (in 3% v/v acetic acid/dH2O, pH2.5) followed by oxidation in 1% v/v periodic acid/dH2O. Slides were immersed in Schiff’s reagent and counterstained with haemotoxylin prior to mounting.

Immunofluorescence

FFPE human and mouse gastric sections were deparaffinised and subjected to antigen retrieval, along with endogenous peroxidase blockade, as with immunohistochemistry.

Following blocking of non-specific binding with CAS-Block (Invitrogen), immunofluorescence was performed on sections with antibodies against pS-STAT3 (Santa

Cruz Biotechnology), ERH (Abcam) and Pontin (Cell Signaling Technology), each at 1:100

dilution. Alexa Fluor conjugates (Invitrogen) were used as secondary antibodies. Nuclear staining was achieved using 4’,6-diamidino-2-phenylindole (DAPI). Sections that underwent the above staining protocol, but in the absence of primary antibodies, served as negative controls. Images were acquired using a Nikon C1 confocal microscope. To quantify cellular staining, digital images of photomicrographs (60× high power fields) were viewed using

Image J software. Positive-staining cells were counted manually (n=8 fields).

Cell lines

Human GC cell lines MKN-28 and MKN-1 (Japanese Collection of Research Bioresources

Cell Bank) were maintained in Roswell Park Memorial Institute 1640 media (Gibco) containing 10% fetal calf serum (FCS; Bovogen). Cell line identification was authenticated by short-tandem repeat profiling (PowerPlex HS16 System KIT, Promega) in our laboratory after receipt in 2013, and cells were passaged during experiments for under 12 weeks at a time between freeze/thaw cycles. Lenti-XTM 293T cells (Clontech) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS. Cells were routinely tested for mycoplasma contamination (MycoAlertTM PLUS Mycoplasma Detection Kit, Lonza). Cells were incubated at 37ºC supplemented with 5% CO2 in a humidified chamber.

Isolation of murine primary gastric epithelial cells

Murine primary gastric epithelial cells were isolated from 4-5-week-old mouse gastric antral tissues as before (23).

Engineered cell lines, and CRISPR RNA (crRNA) screen

MKN-28 STAT3-WT, STAT3-SD or STAT3-SA cells were generated by reconstituting

STAT3-null cells (24) with STAT3-WT, STAT3-SD or STAT3-SA (18, 25, 26) lentiviral

expression constructs (pLVX-IRES-mCherry; Clontech). After viral transduction, STAT3-

WT, STAT3-SD or STAT3-SA cells were sorted for mCherry by flow cytometry to select for cell populations with equivalent STAT3 expression levels that approximated endogenous

STAT3 expression in parental MKN-28 cells.

For the crRNA screen, Alt-R® CRISPR/Cas9 crRNA and trans-activating crRNA

(tracrRNA) (Integrated DNA Technologies) were duplexed (single guide RNA) and transfected into MKN-28 cells stably expressing Cas9 (generated using lentiviral-encoded pLentiCas9-Blast vector). For transfection control, tracrRNA alone was used. The effect of crRNA target gene knockdown on SOCS3 mRNA induction was determined by qPCR. All vectors were sequenced for validation, and sequences for crRNAs and tracrRNAs, along with cloning primers, are available upon request.

RNA isolation and gene expression analyses

Total RNA was isolated from human GC cell lines and human/mouse gastric tissues using

TRI Reagent® Solution (Sigma), followed by on-column RNeasy® Mini Kit RNA clean-up and DNase treatment (Qiagen). RNA was transcribed using the Superscript® III First-Strand

Synthesis System (InvitrogenTM) and Transcriptor High Fidelity cDNA Synthesis Kit

(Roche), respectively. Quantitative real-time PCR (qPCR) was performed on samples in technical triplicates using the 7900HT Fast RT-PCR System (Applied BiosystemsTM), and data acquired and analysed (27). Mouse and human primer sequences are in Supplementary

Table S2.

Mitochondrial DNA (mtDNA) copy number

Genomic DNA was isolated from snap-frozen tissue using the ISOLATE II Genomic DNA

Kit (Bioline), and subjected to RNAse-A treatment (Qiagen). The ratio of mtDNA copy

number to the amount of nuclear DNA was calculated by qPCR (7900HT Fast RT-PCR

System using SensiMix™ SYBR® Hi-ROX (Bioline)) as previously described (28).

Microarray analysis

Gene expression profiling of Cyanine-3-labelled cRNA from 4-week-old mouse gastric antral tissues (n=6/genotype) was performed on Agilent SurePrint G3 Mouse Gene

Expression 8×60K chips. Scanned images of array slides were analysed with Feature

Extraction Software 11.0.1.1 (Agilent) using default parameters (GE1-1100_Jul11 and Grid:

028005_D_F_20120201) to obtain processed signal intensities. Data was imported and integrated using Genespring 13.0 (Agilent).

Gene set enrichment analysis (GSEA)

JavaGSEA Desktop Application v2.2.2 was employed on microarray datasets comprising

45578 native features (Agilent probes) which were collapsed to 9300 genes, and h.all.v5.2.symbols.gmt, c2.all.v5.symbols.gmt and c5.all.v5.2.symbols.gmt gene sets

(https://software.broadinstitute.org/gsea/msigdb/) were used. 1000 ‘gene_set’ permutations were used to test statistics. Significant, positive enrichments were set at normalised enrichment score >0 and false discovery rate q-value of <0.05. All basic and advanced fields were set to default.

Human GC survival datasets

Overall survival analyses were performed on the “Gastric Cancer Project '08 Singapore”

(GSE15459) intestinal-type GC patient cohort (29) using low (bottom third) versus high (top third) median expression values for ERH, RUVBL1 or SOCS3. Treatment among the patients

(n=99) comprised surgery alone (n=68) or adjuvant 5-Fluorouracil therapy (n=12), with the remainder unknown (n=19).

Immunoblotting

Antibodies against pY-STAT3, pS-STAT3, STAT3, Vimentin and E-Cadherin (Cell

Signaling Technology), Tubulin and OXPHOS antibody cocktail (Abcam), and Actin and

FLAG-M2 (Sigma-Aldrich), were used. Membranes were analysed using the Odyssey® CLx

Imaging System (Li-Cor). Protein expression was quantified using ImageJ (National Institutes of Health).

Quantitative chromatin immunoprecipitation (ChIP)

Detailed methodology for in vitro ChIP on MKN-28 cells and in vivo ChIP on mouse gastric tissue is provided in the “Supplementary Methods”. For quantitative in vitro and in vivo ChIP data analyses, % input and fold enrichment were calculated using the 2-ΔΔCT method (30).

Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME)

In vitro RIME on MKN-28 cells and in vivo RIME on mouse gastric tissue was performed using modified published protocols (31, 32), which are extensively detailed in the

“Supplementary Methods”.

Proximity ligation assay (PLA)

Cells were seeded in µ-Slide chambers (Ibidi), cultured and IL-11-treated as per ChIP and

RIME experiments. Cells were fixed in 4% formaldehyde/PBS and permeabilised with 100% ice-cold methanol. PLA was performed using the Duolink® In Situ Red Starter Kit

(Mouse/Rabbit) (Sigma). Antibody combinations included STAT3 (Cell Signaling

Technology) with either ERH (Abcam) or Pontin (Cell Signaling Technology). STAT3- deficient cells were employed to determine assay background. Images were acquired on a

Nikon C1 confocal microscope, and analysis and quantification were performed using

CellProfiler software version 3.0.0 (Broad Institute Imaging Platform).

Statistics

Statistical analyses were performed using GraphPad Prism V7.0 software or R package.

Statistical significance (P<0.05) between the means of two groups was determined using unpaired t-tests or Mann-Whitney U tests, and for matched datasets involved Wilcoxon signed-rank tests. Statistical significance between the means of multiple groups were determined using ordinary one-way ANOVA or Kruskal-Wallis tests. Data are presented as the mean ± standard error of the mean (SEM). The log-rank test was used to calculate the statistical significance of the difference in survival between two groups.

Results

Genetic ablation of constitutive pS-STAT3 in gp130F/F mice suppresses gastric tumorigenesis

In gp130F/F mice, gastric adenomatous hyperplasia occurs by 6-weeks of age with 100% penetrance, followed by formation of adenomas (tumors) by 12-weeks of age which continue to grow until a maximal tumor size is reached by 52 weeks (18, 19). In wild-type gp130+/+ gastric tissues, pY-STAT3 levels are low, while pY-STAT3 levels are elevated in 4-week-old pre-tumor bearing and 12-week-old tumor-bearing gp130F/F gastric tissues (Fig. 1A;

Supplementary Fig. S1A and B). By contrast, comparable pS-STAT3 levels were detected in gastric tissues from 4-week-old and 12-week-old gp130+/+ and gp130F/F mice (Fig. 1A;

Supplementary Fig. S1A and B).

To define whether constitutive pS-STAT3 expression is required for gastric tumorigenesis, we crossed gp130F/F mice with pS-STAT3-deficient Stat3SA/SA mice (13). At 4- weeks of age, the increased gastric mucosal thickness, stomach mass and immunohistochemical staining for the PCNA cell proliferation marker, each indicative of the hyper-proliferative response within the gastric epithelium of gp130F/F mice, were markedly reduced in gp130F/F:Stat3SA/SA mice (Supplementary Fig. S1C-G). Also, stomachs of 12-week- old gp130F/F:Stat3SA/SA mice were free of tumors, and characterised by significant reductions

(compared to age-matched gp130F/F mice) in stomach mass, gastric mucosal thickness and

PCNA-positive cell numbers, comparable to those in gp130+/+ controls (Fig. 1B-F). From 26- weeks of age onwards, one-third of gp130F/F:Stat3SA/SA mice developed small gastric adenomas (Supplementary Fig. S2A and B). Furthermore, gp130F/F:Stat3+/SA mice containing only one Stat3SA allele displayed a marked reduction in tumorigenesis at 12- and 26-weeks of age, indicating a gastric gene dosage effect of pS-STAT3 (Fig. 1B, D and E; Supplementary

Fig. S2A and B). Indeed, the magnitude of gastric tumor suppression in gp130F/F:Stat3SA/SA

mice was comparable to that in gp130F/F:Stat3+/- mice, where the total signaling capacity of

STAT3 has been genetically reduced to wild-type levels by heterozygous ablation of one endogenous Stat3 allele (Supplementary Fig. S2A-H) (18, 19). The comparable rescue in gastric tumorigenesis of gp130F/F:Stat3SA/SA and gp130F/F:Stat3+/- mice aligned with a significant survival benefit, with both genotypes displaying 71% and 76% survival at 1-year, respectively, compared to 13% of gp130F/F mice (Supplementary Fig. S2H and I). Therefore, these data reveal an obligate requirement for constitutive pS727 by STAT3 for its pro- tumorigenic actions in the stomach.

Constitutive pS-STAT3 promotes the proliferative potential of human GC cells in vivo

In support of our in vivo findings, immunohistochemistry revealed strong and comparable pS-

STAT3 levels in GC patient biopsies (62% and 50% positive staining in non-tumor and tumor tissue sections, respectively) and cancer-free controls (57% positive staining in normal tissue sections) (Supplementary Fig. S3A-C, Supplementary Table S1). By contrast, pY-STAT3 was barely detectable in cancer-free controls (4% positive staining), and increased pY-STAT3 staining intensity was observed in GC patient non-tumor (11% positive staining) and tumor

(18% positive staining) tissues, albeit at levels markedly below those for pS-STAT3

(Supplementary Fig. S3A-C). Furthermore, immunoblotting of a panel of human GC cell lines indicated that pS-STAT3 expression was detected in all cell lines, while pY-STAT3 levels were only detected in 4 out of 7 cell lines (Supplementary Fig. S3D). These observations demonstrate that pS-STAT3, similar to the gp130F/F mouse model, is constitutively expressed in both malignant and non-malignant human gastric tissues.

We next evaluated whether constitutive pS-STAT3 was required for the growth of established human GC cell line-derived xenografts. Upon CRISPR/Cas9-mediated knock-out of STAT3 expression in human MKN-28 GC cells (24), cells were reconstituted with either

wild-type STAT3 (STAT3-WT), a STAT3 mutant (serine to aspartic acid substitution at site

727; STAT3-SD) that mimics constitutive S727 phosphorylation (26), or S727 phosphorylation-defective STAT3 (STAT3-SA), via lentiviral transduction. The growth of

STAT3-SA-expressing human MKN-28 xenografts in NSG mice was significantly impaired compared to STAT3-WT and STAT3-SD MKN-28 xenografts (displaying a comparable growth rate), and was associated with reduced numbers of PCNA-positive proliferating cells

(Supplementary Fig. S4A-D). These data support a key role for constitutive pS-STAT3 in promoting the proliferative potential of the gastric epithelium during GC.

Suppressed gastric tumorigenesis in gp130F/F:Stat3SA/SA mice is characterised by abrogated tumor inflammation and angiogenesis, yet is independent of the bone- marrow-derived myeloid lineage

We next evaluated whether the suppression of gastric tumorigenesis in gp130F/F:Stat3SA/SA mice coincided with changes to inflammation. Histopathological scoring of H&E-stained gastric antrum sections from 12-week-old gp130F/F and gp130F/F:Stat3SA/SA mice, along with wild-type littermates, demonstrated that the marked gastric inflammation observed in gp130F/F mice was significantly attenuated in gp130F/F:Stat3SA/SA mice (Fig. 2A). Similarly, immunohistochemistry revealed that CD45-positive infiltrating immune cells present throughout the gastric compartment of gp130F/F mice were largely absent in gp130F/F:Stat3SA/SA stomachs, the latter of which were comparable to wild-type controls (Fig.

2B).

To address whether pS-STAT3 expression in immune cells contributed to gastric tumorigenesis, we initially performed immunohistochemistry for pS-STAT3 on gastric tumor sections from 12-week-old gp130F/F mice. This indicated that pS-STAT3 was widely expressed in inflammatory cell aggregates distributed within the submucosal, muscularis and

serosal layers, as well as throughout the tumor mucosal epithelium (Fig. 2C). Immunoblotting also demonstrated that pS-STAT3 expression levels were comparable among protein extracts isolated from the glandular epithelium and stroma (containing immune cells) of 12-week-old gp130F/F mouse tumors (Fig. 2D). Next, we assessed whether immune cells expressing pS-

STAT3 contributed to gastric tumorigenesis by reconstituting bone marrow of irradiated gp130F/F recipient mice with donor bone marrow from gp130F/F:Stat3SA/SA mice. However, the appearance of stomachs of gp130F/F recipients reconstituted with either gp130F/F:Stat3SA/SA or control gp130F/F bone marrow, along with their stomach mass and tumor burden, were comparable (Fig. 2E-G).

We next determined whether additional features of the intestinal-type gastric tumor phenotype of gp130F/F mice, namely augmented angiogenesis and production of acidic mucins, were also dependent on pS-STAT3. Expression profiling by qPCR revealed a significant reduction in mRNA levels of angiogenesis-related genes in gastric tissues of 12- week-old gp130F/F:Stat3SA/SA versus gp130F/F mice (Fig. 2H). In addition, Alcian blue-

Periodic Acid Schiff staining demonstrated that stomachs of gp130F/F:Stat3SA/SA (and wild- type) mice were devoid of acidic mucins that are indicative of the intestinal-type gastric tumor pathology of gp130F/F mice (Fig. 2I). Collectively, these data support the notion that pS727 plays a critical role in facilitating the oncogenic potential of STAT3 – via cell proliferation, inflammation and angiogenesis – during the initiation and establishment of GC in a cell intrinsic manner.

pS727 modulates the transcriptional programming of STAT3 independent of mitochondrial or metabolic gene networks during gastric tumorigenesis

We next examined the molecular basis by which pS-STAT3 promotes the onset of GC by

DNA microarray profiling the transcriptome of pre-tumorigenic (and non-inflamed) gastric

antrum tissue from 4-week-old gp130F/F mice, and age-matched wild-type and gp130F/F:Stat3SA/SA mice. A total of 987 genes were significantly differentially expressed (>2- fold, P<0.05) in gastric antrum of gp130F/F compared to wild-type mice, with 477 and 510 genes up-regulated and down-regulated, respectively (Fig. 3A; Supplementary Table S3).

Strikingly, the gastric antrum transcriptome of gp130F/F:Stat3SA/SA mice resembled that of wild-type mice, with a 3-fold reduction in the number of significantly differentially-expressed genes (322; 185 up-regulated, 137 down-regulated) compared to gp130F/F gastric antrum (Fig.

3A).

Since pS-STAT3 is implicated in mitochondrial function and associated metabolic reprogramming, a hallmark of cancer (33-35), we investigated whether gene networks associated with these processes were differentially modulated by pS-STAT3 in gp130F/F gastric antrum. Heat map analyses of DNA microarrays indicated that mitochondrial-encoded genes exhibited a comparable expression profile among the gastric antrum genotypes (Fig.

3B). In addition, qPCR and immunoblotting demonstrated that mRNA and protein levels of mitochondrial-encoded and nuclear-encoded mitochondrial genes were similar among gastric antrum from wild-type, gp130F/F and gp130F/F:Stat3SA/SA mice aged 4-weeks and 12-weeks, the latter including tumor and matched non-tumor tissues from gp130F/F mice (Fig. 3C-E;

Supplementary Fig. S5A). Similarly, the mitochondrial DNA copy number was not affected by altered gp130-dependent signaling in gp130F/F and gp130F/F:Stat3SA/SA mice

(Supplementary Fig. S5B and C). Notably, the expression profile of metabolic genes for glycolysis and the citric acid (TCA) cycle were also unchanged (Fig. 3F and G). By contrast,

GSEA illustrated an enrichment of gene signatures for inflammatory response, regulation of innate immunity, epithelial wound healing (migration) and JAK-STAT3 pathways in gp130F/F

(versus wild-type) compared to gp130F/F:Stat3SA/SA gastric antrum (Fig. 3H). Further interrogation of a signature comprising 30 STAT3 target genes (e.g. Socs3, Il11, Myc, Mmp9,

Vegfa) (18, 19) indicated that pS-STAT3 deficiency restored their expression to wild-type levels in gp130F/F:Stat3SA/SA gastric antrum (Fig. 3I; Supplementary Fig. S5D and E).

Collectively, these data show that pS727 plays a central role in modulating the transcriptional activity of STAT3 in gastric tumorigenesis.

pS-STAT3 is independent of IL-11-mediated pY-STAT3

Since the IL-11-gp130-JAK-STAT3 signaling axis promotes gastric tumorigenesis in gp130F/F mice (18), we reasoned that reduced gastric IL-11 expression levels in gp130F/F:Stat3SA/SA mice may dampen the overall STAT3 oncogenic signal output, leading to suppressed tumorigenesis (Fig. 3I; Supplementary Fig. S5D and E). Indeed, immunoblotting and/or immunohistochemistry showed that pY-STAT3 and IL-11 protein levels were reduced in the gastric antrum of gp130F/F:Stat3SA/SA versus gp130F/F mice (Fig. 1A and 4A-E; Supplementary

Fig. S1A), and gastric pY-STAT3 levels were similarly lower in both nuclear and cytoplasmic subcellular fractions from gp130F/F:Stat3SA/SA mice (Fig. 4F). Furthermore, we demonstrated a rescue effect on gastric pY-STAT3 levels in gp130F/F:Stat3SA/SA mice upon the administration of IL-11 over 6 hours, coincident with augmented protein levels of c-Myc (a representative pro-tumorigenic STAT3-regulated target) (Fig. 4G).

Considering these observations, we investigated whether IL-11 upregulated pS-STAT3 levels in addition to pY-STAT3 during gastric tumorigenesis. However, in 4-week old gp130F/F:Il11r-/- mice deficient in IL-11 signaling (18), gastric pS-STAT3 levels remained unaltered, whereas pY-STAT3 levels were substantially reduced compared to gp130F/F mice

(Fig. 4H). Similarly, pY-STAT3 levels were robustly increased in gastric antrum tissues of wild-type mice or human MKN-1 GC cell lines treated with IL-11, while pS-STAT3 levels remained unchanged (Fig. 4I and J). Furthermore, treatment of these human GC cell lines with JAK inhibitors abrogated pY-STAT3 whilst having no effect on pS-STAT3 (Fig. 4K).

Collectively, these data suggest that the upstream pathways leading to pY-STAT3 and pS-

STAT3 signaling are not interdependent, but rather co-operate to drive GC. Moreover, the reduction in pY-STAT3 levels in gp130F/F:Stat3SA/SA mice are likely caused by reduced IL-11 expression, which is the major driver of pY-STAT3 in this GC model.

pS727 modulates the transcriptional activity of RNA polymerase on STAT3 target genes

To investigate whether pS727 plays a critical role in modulating the transcriptional output of

STAT3 by influencing its recruitment to target gene promoters, we performed ChIP. The

Socs3 gene promoter was used as a representative STAT3-regulated target since Socs3 gene expression levels strongly align with STAT3 activity in GC (18, 24, 27). To avoid any bias conferred by different gastric pY-STAT3 expression levels (observed between gp130F/F and gp130F/F:Stat3SA/SA mice), we used gastric tissues from IL-11-treated wild-type and Stat3SA/SA mice, which results in comparable pY-STAT3 induction in the stomach (Fig. 5A). Whilst equivalent gastric pY-STAT3 protein levels bound to the Socs3 promoter region in IL-11- treated wild-type and Stat3SA/SA mice, there was a significant reduction in Socs3 mRNA levels in the Stat3SA/SA gastric antrum (Fig. 5B and C). This finding was confirmed by ChIP in IL-

11-stimulated human MKN-28 GC cells expressing either STAT3-WT or STAT3-SA (Fig.

5D-F).

The above observations dissociate pY-STAT3 promoter recruitment from the transcriptional induction of STAT3-regulated genes, and suggest that the requirement of pS727 for maximal transcriptional capacity of STAT3 is independent of its loading onto target promoters. We therefore hypothesised that the impaired transcriptional response in the absence of pS-STAT3 could be due to inefficient promoter recruitment of RNA polymerase II

(Pol II), the initiation of transcription, or elongation of nascent RNA transcripts. To investigate this, we performed ChIP on IL-11-stimulated human MKN-28 GC cells

expressing STAT3-WT and STAT3-SA for the binding of distinct serine phosphorylation sites within Pol II that are associated with transcription initiation and 5’-end mRNA processing (RNA Pol II pSer5 CTD) or transcription elongation (RNA Pol II pSer2 CTD)

(Fig. 5G) at the SOCS3 promoter. The accumulation of pSer5-RNA Pol II on the active

SOCS3 promoter was comparable in IL-11-stimulated cells expressing STAT3-WT (44% increase) and STAT3-SA (33% increase), which suggests that pS-STAT3 does not have a major influence on the recruitment of RNA Pol II during transcription (Fig. 5H). By contrast, in IL-11-stimulated STAT3-SA cells, pSer2-RNA Pol II levels bound to the SOCS3 gene were significantly impaired (by 90%) compared to IL-11-stimulated STAT3-WT cells

(STAT3-WT, 270% increase versus STAT3-SA, 25% increase) (Fig. 5H). Notably, in

STAT3-deficient cells compared to STAT3-WT and STAT3-SA cells, SOCS3 promoter loading by pSer5-RNA Pol II was equivalent in the absence of IL-11 stimulation, yet was not increased following IL-11 stimulation (Fig. 5H). Collectively, these data indicate that STAT3 is required for increased promoter recruitment of pSer5-RNA Pol II following stimulation, yet this is independent of pS727. Rather, pS-STAT3 primarily regulates transcription by augmenting RNA Pol II-mediated elongation, but not initiation.

pS-STAT3 interacts with ERH and Pontin to augment transcription of STAT3 target genes

The ability of pS-STAT3 to regulate RNA Pol II elongation but not promoter recruitment of either STAT3 or RNA Pol II suggests that additional transcriptional co-regulators may interact with pS-STAT3. To identify interacting partners specific for pS-STAT3, we undertook a combinatorial in vitro and in vivo proteomics RIME approach using gastric tissues of 4-week-old gp130F/F and gp130F/F:Stat3SA/SA mice together with IL-11-treated human MNK-28 GC cells expressing STAT3-WT and STAT3-SA (Fig. 6A; Supplementary

Fig. S6A and B). Using this unbiased strategy, 14 candidate serine phosphorylation-dependent

STAT3-interacting proteins were identified (Fig. 6A).

To validate the functional requirement of the 14 candidate pS-STAT3-binding proteins for the transcriptional capacity of STAT3, we established a CRISPR/Cas9 (36) screen to knock-out each of these 14 genes in human MKN-28 GC cells, along with that of STAT3 as a control, and assess the effect on SOCS3 gene transcription in response to IL-11 stimulation.

While transfection of tracrRNA alone had no impact on SOCS3 induction, in contrast, transfection with the tracrRNA-crRNA duplex targeting STAT3 reduced SOCS3 transcript levels (by 56%) to that in unstimulated cells. Among these 14 genes, genetic ablation of either

RUVBL1 (encoding Pontin) or ERH markedly impaired the capacity of IL-11 to upregulate mRNA levels of SOCS3 and the additional STAT3 target gene, MMP9 (Fig. 6B-D;

Supplementary Fig. S6C). The requirement of Pontin and ERH for IL-11-dependent transcription was specific for STAT3-regulated genes (e.g. SOCS3), but not genes regulated by the related IL-11/STAT1 axis (e.g. SOCS1) (18) (Fig. 6E).

To confirm the physical interaction of pS-STAT3 with Pontin and ERH, we employed in situ PLA on IL-11-treated human MKN-28 STAT3-WT and STAT3-SA cells using antibodies against STAT3 and either ERH or Pontin. The abundance of puncta denoting

STAT3-Pontin or STAT3-ERH interactions was significantly diminished in STAT3-SA expressing cells, confirming the requirement for pS-STAT3 to form complexes with Pontin and ERH (Fig. 6F and G). STAT3-ERH interactions were predominantly localised to the nucleus of MKN-28 STAT3-WT cells (Fig. 6F), consistent with an exclusive nuclear functional role for ERH (37). By contrast, Pontin is localised to both the nucleus and cytoplasm (38) which likely accounts for the nuclear and cytoplasmic STAT3-Pontin interactions observed in MKN-28 STAT3-WT cells (Fig. 6F). These observations support

ERH and Pontin as in vivo interacting partners of pS-STAT3 to augment the transcriptional induction of STAT3 target genes in GC.

The translational relevance of our findings was validated in human GC patients, whereby ERH, RUVBL1 and SOCS3 expression was significantly upregulated in 85% (17/20),

70% (14/20) and 70% (14/20) of tumor versus matched non-tumor tissues, respectively, and increased expression correlated with worse overall patient survival, albeit not significant for

ERH (Fig. 7A-C, Supplementary Fig. S7A). Furthermore, mRNA levels for ERH and

RUVBL1 demonstrated significant positive correlations with those for SOCS3, as well as with pS-STAT3 protein levels (Fig. 7D and E). Interestingly, the increased expression of these genes was independent of disease stage (Supplementary Fig. S7B). Importantly, immunofluorescence staining revealed a pronounced co-localisation of pS-STAT3 with ERH and Pontin in human GC tumor and matched non-tumor tissues, with dual pS-STAT3/ERH or pS-STAT3/Pontin staining higher in tumors (Fig. 7F-H). The co-localisation of pS-STAT3 with ERH and Pontin was also confirmed in tumors of the gp130F/F GC model

(Supplementary Fig. S7C and D), thus supporting ERH and Pontin as in vivo interacting partners of pS-STAT3 in GC.

Discussion

In cancer, emerging studies have suggested an essential requirement for pS-STAT3 by

oncogenic Ras-addicted malignancies that is independent of its canonical nuclear (i.e. gene

transcription) role, but rather is dependent on mitochondrial function via augmenting the

mitochondrial electron transport chain to support aerobic glycolysis over oxidative

phosphorylation for the production of cellular ATP (the ‘Warburg effect’) (8, 11, 27).

However, in contrast to Ras-driven oncogenesis, the relative role of pS-STAT3 nuclear and

mitochondrial activities to the molecular pathogenesis of many epithelial malignancies driven

by excessive cytokine secretion or tyrosine kinase activity, such as GC, is unknown.

Here, we define an obligatory and unprecedented role for pS-STAT3 in the initiation

and maintenance of GC. Specifically, in the STAT3-driven gp130F/F GC model, we

demonstrate that pS-STAT3 deficiency suppresses gastric tumorigenesis and is associated

with the attenuation of proliferative, inflammatory, angiogenic and metaplastic processes.

Furthermore, our bone marrow reconstitution studies, in concert with human GC (epithelial)

cell line xenografts, imply that the requirement for pS-STAT3 is intrinsic to the non-

hematopoietic (i.e. epithelial) compartment, consistent with our previous data for STAT3-

dependency by the gastric tumor epithelium (18, 27). Notably, the marked tumor suppressive

effect, along with improved survival rate, of ablating pS-STAT3 in gp130F/F:Stat3SA/SA mice

was comparable to that achieved by monoallelic deletion of Stat3 (gp130F/F:Stat3+/-), which

also phenocopies gp130F/F mice lacking the capacity to transduce IL-11 signals (18). In this

regard, microarray-based transcriptomic analyses assigned the pro-tumorigenic actions of pS-

STAT3 to the transcriptional modulation of nuclear encoded STAT3 target genes – implicated in numerous oncogenic cellular processes, independent of mitochondrial function and metabolism – among which includes IL-11, the primary upstream activator of STAT3 in

GC (18, 27).

SOCS3 is a negative regulator of STAT3 transcriptional function, and epigenetic

silencing of SOCS3 is observed in some cancer types (e.g. endometrial). However, in GC, the

transcriptional upregulation of SOCS3 is likely reflecting elevated STAT3 activation (18, 19,

27), and high SOCS3 mRNA levels significantly correlate with poor survival outcomes (Fig.

7C). Therefore, SOCS3 was employed in our current study as a robust molecular read-out of

STAT3 transcriptional activity in GC. In this regard, we demonstrate that the magnitude of

oncogenic STAT3 transcriptional activity is enhanced by the capacity of pS727 to augment

RNA Pol II-mediated transcript elongation of STAT3-target genes (e.g. SOCS3), rather than

facilitate RNA Pol II (or STAT3) promoter recruitment and transcription initiation. This is

supported by the report that pS727 did not influence the capacity of STAT3 to accumulate on

the SOCS3 promoter (14). Moreover, our demonstration that RNA Pol II occupies the SOCS3

promoter in the absence IL-11-stimulated STAT3 signaling is consistent with the rapid

transcriptional induction of many primary response genes associated with oncogenesis,

including those upregulated by STAT3 (e.g. Myc), whose promoters are preloaded with a

proximally paused RNA Pol II that is primed for the transition from RNA Pol II transcript

initiation to elongation (39).

Another key finding of our study was the identification of Pontin and ERH as pS-

STAT3-dependent interacting partners that augment the transcriptional activation of STAT3

target genes in GC. Pontin is a highly conserved AAA+ ATPase family member, and while

STAT3 can interact with other ATPases, in particular BRG1 which is critical for Ser2 phosphorylation of RNA Pol II and transcriptional elongation, unlike our current study the role of phosphorylated STAT3 in the interaction with BRG was not investigated (40). Pontin has been shown to directly modulate gene transcription, as well as participate in protein super complexes to regulate diverse cellular processes including chromatin remodelling (INO80 complex), histone modification (NuA4/TIP60 complex), snoRNP biogenesis and RNA Pol II

assembly (R2TP complex) (38, 41-44). Interestingly, Pontin can interact with the

transcription activation domain of STAT2 to facilitate maximal RNA Pol II recruitment and

initiation at the promoters of interferon-stimulated genes (45, 46). This contrasts our current

finding supporting a role for Pontin (via its interaction with pS-STAT3) in the RNA Pol II-

driven transcription elongation phase through STAT3 target genes, suggesting that the role

for Pontin in augmenting STAT-driven gene transcription may be dependent on cellular

context, the upstream STAT-activating stimuli and/or the specific STAT family member.

Indeed, we show here that the genetic ablation of Pontin (and ERH) in human GC cells had no effect on the transcription of the STAT1-regulated gene SOCS1. With respect to ERH, its

diverse molecular functions include regulation of transcription, cell cycle, RNA splicing

and pyrimidine metabolism (47). Notably, ERH can interact with SPT5 and FCP1, essential

factors that modulate the processing capacity of RNA Pol II, and thus transcriptional

elongation rates (48-50). Therefore, the actions of Pontin and ERH in promoting transcription

elongation of RNA Pol II are consistent with our data assigning such a transcriptional role for

pS-STAT3. Furthermore, since STAT3 target genes affect numerous oncogenic cellular

processes, such as proliferation, apoptosis, invasion, and angiogenesis (2, 4), the increased

expression of RUVBL1 and ERH that we observed in human GC would likely further

augment the oncogenic transcriptional capacity of STAT3, thus favouring a more aggressive

tumor phenotype in patients.

Hyper-activation of IL-11/gp130/STAT3 signaling in GC recapitulates what is

observed in many human tumors where oversupply of IL-6 family cytokines converge,

primarily via JAKs, to maintain pY-STAT3 levels (4, 18, 19, 51). However, we reveal here

that pS-STAT3 is independent from IL-11-driven upregulation of pY-STAT3, as evidenced

by pS-STAT3 levels remaining unaltered in in vitro and in vivo GC models following either

IL-11 treatment, IL-11R ablation or JAK inhibition. Rather, constitutive pS-STAT3 is

observed at comparable levels in mouse/human gastric non-tumor/tumor tissues, and among

human GC cell lines, which may be a consequence of at least one of the diverse upstream

kinases that drive serine phosphorylation of STAT3, including ERK, p38 and JNK MAPKs,

protein kinase C delta, glycogen synthase kinase 3α/β and cyclin-dependent kinase-1, being

engaged at all times (12, 52-56). We therefore speculate that STAT3 tyrosine and serine phosphorylation events may have coevolved to confer a broader spectrum of transcriptional potentials capable of graded regulation. Moreover, we hypothesise, at least in GC, that IL-11 signaling within the gastric epithelium represents a response to tissue injury, infection and/or inflammation, and engages JAK-bound gp130 receptor complexes to induce pY-STAT3. This tightly controlled molecular switch then facilitates STAT3 nuclear translocation and DNA binding affinity, thus licencing STAT3 with a broad transcriptional capacity. Upon entering the nucleus, basal pS-STAT3 then recruits and exploits various co-activators, such as Pontin

and ERH, to augment the transcriptional elongation of STAT3 target genes by RNA Pol II.

Accordingly, as evidenced by gp130F/F and human GC cell xenograft models, in the absence

of pS-STAT3, the induction of pY-STAT3 alone is insufficient to attain the threshold of

STAT3 transcriptional output that is necessary for gastric tumorigenesis.

In summary, our current study demonstrates that STAT3-driven GC is dependent upon

the capacity of pS727 to maximise RNA Pol II transcription rates of STAT3-target genes by

facilitating interaction of STAT3 with Pontin and ERH, identified here as hitherto unknown

transcriptional co-activators. Therefore, identifying key sites governing the interaction

between STAT3 and ERH or Pontin, as well as the mechanism(s) by which ERH and Pontin

regulate STAT3-driven gene transcription, provides opportunities for pharmacological

targeting of STAT3 in GC, and other STAT3-driven cancers (e.g. colorectal, pancreatic), which thus far has been problematic (1, 6). While our data (Fig. 7F) suggest that the targeted disruption of pS-STAT3/Pontin and pS-STAT3/ERH interactions might occur in normal (i.e.

non-tumor) and tumor tissues, nonetheless, the specific blockade of S727 independently of

Y705 in STAT3 represents an alternative approach to selectively suppress the oncogenic

transcriptional activity of pS-STAT3 in GC, while simultaneously preserving the homeostatic

functions (e.g. mucosal wound healing) of pY-STAT3. An advantage of such a therapeutic strategy is to maintain overall gastric function, and thus minimise non-specific toxicities. In the absence of current pS-STAT3-specific inhibitors, the identification of the kinase(s) responsible for gastric STAT3 serine phosphorylation will foster the evaluation of existing drugs targeting candidate kinases (e.g. MEK/MAPK inhibitors, Trametinib and Binimetinib)

as effective indirect pS-STAT3 inhibitors, and thus anti-cancer agents, in GC.

Acknowledgements

We thank N. Williamson and the Bio21 Mass Spectrometry and Proteomics Facility, the

Monash Health Translational Precinct Medical Genomics Facility, and the Monash Histology

Platform for providing core and technical services.

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Figure Legends

Figure 1.

Suppressed gastric tumorigenesis in gp130F/F mice lacking pS-STAT3. A, Representative immunoblots of 12-week-old gp130+/+ wild-type (WT), gp130F/F gastric tumor (T; F/FT) and non-tumor (NT; F/FNT), and tumor-free gp130F/F:Stat3SA/SA (F/F:SA/SA), gastric tissue lysates. Each lane represents an individual mouse. B, Scatter plot depicting total mass (grams) of stomachs from 12-week-old WT, SA/SA, F/F, gp130F/F:Stat3+/SA (F/F:+/SA) and

F/F:SA/SA mice. n=12 mice/genotype. C, Representative low power (left images, solid line) and high power (right images, dotted-line) photomicrographs of PCNA-immunostained gastric cross-sections of 12-week-old mice. Scale bars: 50m. Arrows depict the PCNA- positive proliferative zone. D and E, Scatter plots depicting (D) total mass (grams) of gastric tumors (n=12 mice/genotype), and (E) antral mucosal thickness (m) (n=4 mice/genotype), of

12-week-old mice. F, Representative appearance of stomachs (left images), H&E-stained whole stomach longitudinal cross-sections (middle images), and magnification of the antral mucosa region depicted by dotted insets in middle images (right images), from the indicated genotypes. Scale bars: 10mm (left images), 1mm (middle images) and 100m (right images).

Arrows depict macroscopically visible tumors. Left panel: F, fundus; C, corpus A, antrum;

S.I, small intestine. Right panel: M, mucosa; MM, muscularis mucosa; SM, submucosa; ME, muscularis external; S, serosa.

In (B), (D) and (E), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Kruskal-Wallis test.

Figure 2. pS-STAT3 promotes gastric tumorigenesis associated with inflammation and angiogenesis, yet is independent of bone marrow-derived inflammatory cells. A, Inflammatory scores (0,

none; 1, mild; 2, moderate; 3, severe) from 12-week-old gp130+/+ wild-type (WT), gp130F/F

(F/F) and gp130F/F:Stat3SA/SA (F/F:SA/SA) mouse stomachs. n=6 mice/genotype. B,

Representative photomicrographs of CD45-immunostained gastric cross-sections from 12- week-old mice (3 stomachs/genotype). Scale bars; 50m. C, Representative photomicrographs of pS-STAT3-immunostained antral tumor cross-sections from 12-week- old F/F mice. Magnification panels (right) depict high power images of positively stained epithelial cells and inflammatory cell aggregates. Scale bars; 200m. D, Representative immunoblots of lysates from 12-week-old tumor-bearing F/F whole antrum, as well as epithelial and stromal enrichments. E, Representative appearance of stomachs (left), and

H&E-stained whole stomach longitudinal cross-sections of the antral mucosa (right), from 16-

18-week-old recipient F/F mice reconstituted with either F/F (F/FF/F) or F/F:SA/SA

(F/FF/F:SA/SA) donor bone marrow. Scale bars: 10mm (left images) and 1mm (right images).

Arrows depict macroscopically visible tumors. F, fundus; C, corpus A, antrum; S.I, small intestine. F and G, Scatter plots depicting total mass (grams) of (F) stomachs and (G) antral tumors from F/FF/F and F/FF/F:SA/SA chimeric mice. n=8 mice/genotype. H, qPCR of angiogenesis genes in gastric antral tissue from 12-week-old WT, F/F gastric tumor (F/FT) and non-tumor (F/FNT), and tumor-free F/F:SA/SA gastric tissues (n=6 mice/genotype).

Expression data are normalised to 18srRNA. I, Representative low power (left) and high power (right) photomicrographs showing combined Alcian Blue/PAS-stained cross-sections through the antral stomach region of the indicated 12-week-old mice. Neutral mucins stain light purple and acid mucins stain dark purple. Scale bars: 50m.

In (A) and (F-H), *P<0.05, **P<0.01, ***P<0.001; Kruskal-Wallis test (A, H) and unpaired t-test (F, G).

Figure 3.

pS727 is required for the gastric transcriptional regulation of STAT3-dependent gene networks, independent of metabolic and mitochondrial pathways, in gp130F/F mice. A, Line graph (left image) of gene microarray expression data showing significantly upregulated

(green) and downregulated (red) genes (fold change >2, P<0.05) in the indicated gastric tumor-free 4-week-old tissues (n=6 mice/genotype). Bar graph (right image) depicting the number of genes differentially expressed (fold change >2, P<0.05) in gp130F/F (F/F) and gp130F/F:Stat3SA/SA (F/F:SA/SA) samples compared to gp130+/+ wild-type (WT) (x-axis) samples. B, Heat map of gene microarray analysis of representative samples from (A) depicting expression levels of mitochondrial (mt)-encoded genes. Each column represents an individual mouse. C, qPCR of mt-encoded genes in the indicated tumor-free 4-week-old gastric tissues. Expression data (n=6 mice/genotype) are normalised to 18srRNA. D,

Representative immunoblots of 12-week-old tumor-free WT, F/FNT and F/F:SA/SA gastric tissue lysates. Each lane represents an individual mouse. E, Densitometry quantification of immunoblots from (D) with each protein normalised to Actin. n=3 mice/genotype. F, G and I,

Representative heat maps of gene microarray data depicting expression levels of gene-sets for

(F) glycolysis and (G) the citric acid (TCA) cycle pathways, and (I) STAT3-regulated genes.

H, Enrichment plots generated by GSEA of ranked gene microarray expression data from (A) comparing F/F versus F/F:SA/SA 4-week-old tumor-free gastric samples.

In (B), (F), (G) and (I), coloured side scales depict Log2-fold change (same scale used for (F) and (G)). In (C) and (E), Kruskal-Wallis test.

Figure 4.

Gastric pS-STAT3 is independent of IL-11-mediated pY-STAT3, which is reduced in gp130F/F:Stat3SA/SA mice. A, Representative photomicrographs of low power (solid line, left images) and high power (dotted line, right images) pY-STAT3- and total STAT3-

immunostained gastric cross-sections from the indicated 12-week-old mice. Scale bars; 50m.

B and D, Representative immunoblots of lysates from (B) tumor-free 4-week-old gp130F/F

(F/F) and gp130F/F:Stat3SA/SA (F/F:SA/SA) gastric tissues, and (D) 12-week-old F/F non- tumor (F/FNT), F/F tumor (F/FT) and F/F:SA/SA tumor-free gastric tissues, with the indicated antibodies. C and E, Densitometry quantification of immunoblots from (B) and (D), respectively, with IL-11 normalised to Tubulin. F-K, Representative immunoblots on (F) nuclear and cytoplasmic enrichments of gastric tissue lysates from tumor-free 4-week-old F/F and F/F:SA/SA mice, (G) gastric tissue lysates from 4-week-old FF and F/F:SA/SA mice intraperitoneal injected with IL-11 for the indicated time points, (H) gastric tissue lysates from tumor-free 4-week-old wild-type (WT), Il11r1-/- (IL11R), F/F and gp130F/F:Il11r1-/-

(F/F:IL11R) mice, (I) gastric tissue lysates from 4-week-old WT mice after IL-11 injections for the indicated time points, (J) lysates from human GC MKN-1 cells treated with IL-11 for the indicated time points, and (K) lysates from MKN-1 cells either untreated (-) or treated with the specified JAK inhibitors for 60min. Tof, tofacitinib; Rux, ruxolitinib,

In (B), (D), (F) and (G), each lane represents an individual mouse. In (C) and (E), data are from n=3 mice/genotype. *P<0.05; unpaired t-test (C) and Kruskal-Wallis test (E).

Figure 5. pS-STAT3 modulates the transcriptional activity of RNA Polymerase II on STAT3 target genes. A, Representative immunoblots with the indicated antibodies on lysates from 4-6- week-old wild-type (WT) and Stat3SA/SA (SA/SA) mice administered for 30min with PBS (-) or IL-11 (+). Each lane represents an individual mouse. B, Nuclear enrichments from antral tissues of mice from (A) were subjected to ChIP analysis with antibodies against either pY-

STAT3 or IgG isotype control. qPCR (n=4 mice/genotype) was performed with primers against the STAT3 binding region of the Socs3 promoter (-64 TSS) or control primers against

the Socs3 3’-UTR (+2192 TSS). TSS, transcription start site; UTR, untranslated region. C, qPCR of Socs3 (normalised to 18srRNA) from gastric tissues of mice from (A). n=4 mice/group. D, Representative immunoblots on lysates from MKN-28 STAT3 knock-out

(KO), STAT3-WT and STAT3-SA expressing cells either untreated (-) or treated with IL-11

(+) for 30min. E, Nuclear enrichments from MKN-28 cells in (D) were subjected to ChIP with an anti-pY-STAT3 antibody, and the STAT3 binding region of the human SOCS3 promoter (-61 TSS) or the SOCS3 3’-UTR (+2174 TSS) was amplified by qPCR. Expression data are from 4 independent experiments. F, qPCR of SOCS3 (normalised to 18SrRNA) from

MKN-28 cells in (D). Expression data from 4 independent experiments. G, Promoter and gene structure of the human SOCS3 gene. Primer pairs to detect RNA Polymerase II (Pol II) loaded on the promoter, and its progress through the gene body, are highlighted. pSer5 of the

C-terminal domain (CTD) of Pol II marks transcription initiation, and pSer2 CTD marks Pol

II elongation. H, Nuclear enrichments from MKN-28 cells in (D) were subjected to ChIP with antibodies against pSer5 CTD RNA Pol II, pSer2 CTD Pol II, or IgG isotype control, and proximal promoter (-61 TSS) or distal gene (+2815) regions of SOCS3 were qPCR amplified.

Expression data are from 4 independent experiments.

In (B), (C), (E), (F) and (H), *P<0.05, **P<0.01, ****P<0.0001; ordinary one-way

ANOVA.

Figure 6.

ERH and Pontin proteins interact with pS-STAT3 to augment transcription of STAT3 target genes. A, Workflow of in vitro and in vivo RIME experiments to identify STAT3-interacting proteins, and the CRISPR-based gene editing screen to validate candidate regulators (upon their deletion) of STAT3 target gene (e.g. SOCS3) expression. LC-MS/MS, liquid chromatography tandem mass spectrometry; PLA, proximity ligation assay. B, qPCR of

SOCS3 expression (normalised to 18SrRNA) in MKN-28-Cas9 cells transfected with the indicated crRNAs, and left untreated (-) or treated with IL-11 (+) for 30min. Percentages of

SOCS3 upregulation relative to transfected, IL-11-stimulated MKN-28-Cas9 (-) cells (grey bar) are indicated. C, Immunoblots of lysates from MKN-28-Cas9 cells transfected with the indicated crRNAs. D and E, qPCR analysis (4 independent experiments) of SOCS3 (D) and

SOCS1 (E) expression (normalised to 18SrRNA) in MKN-28-Cas9 cells (-) transfected with tracrRNA control, or crRNAs against RUVBL1, ERH or STAT3 and treated with IL-11 (+) for

30min. F, Representative in situ PLA images of MKN-28-WT or -SA cells treated will IL-11 for 30min and probed with antibody combinations against STAT3 with either ERH or Pontin.

PLA foci, red; DAPI, blue. Scale bar: 10m. G, Quantification of PLA dots using Broad

Institute’s CellProfiler. Data are presented from 4 independent experiments.

In (D), (E) and (G), *P<0.05, **P<0.01, ****P<0.0001; ordinary one-way ANOVA

(D, E), and unpaired t-test (G). In (D) and (E), P values for comparisons versus non-targeted cells stimulated with IL-11 (“-”) are identical to those versus control “+tracRNA” cells stimulated with IL-11.

Figure 7.

Co-localisation of ERH and Pontin with pS-STAT3, and correlations of elevated expression of ERH and RUVBL1 with SOCS3 and impaired survival, in human GC. A and B, qPCR of

ERH, RUVBL1 and SOCS3 (normalised to 18SrRNA) in (A) paired gastric tumor (T) and adjacent non-tumor (NT) tissue, and (B) in T tissue relative to paired NT tissue, in GC patients (n=20). C, Kaplan-Meier 5-year survival analysis of the “Gastric Cancer Project '08

Singapore” patient cohort stratified into 2 groups based on low (n=33) or high (n=33) ERH,

RUVBL1 and SOCS3 expression. D, qPCR-based correlation analyses of gene expression of

ERH and RUVBL1 with SOCS3 (normalised to 18SrRNA) in gastric tissues (paired T and NT)

from GC patients (n=20). E, Correlation of ERH and RUVBL1 mRNA with pS-STAT3 levels in representative GC patient tissue lysates (n=8 paired T and NT) from (D) that were immunoblotted. Densitometry on pS-STAT3 and total STAT3 immunoblots to enumerate expression values for pS-STAT3 (normalised against total STAT3) for correlations with ERH and RUVBL1. F, Quantification of pS-STAT3/ERH or pS-STAT3/Pontin positive stained cells (as a percentage of total pS-STAT3 positive cells) from GC patient (n=5) paired T and

NT sections. G and H, Immunofluorescence images of T sections from a representative GC patient (F) co-stained with antibodies against pS-STAT3 (green) and either ERH (G, red) or

Pontin (H, red). DAPI nuclear staining is blue. Scale bars: 50m. Arrowheads in merged images indicate representative dual positive pS-STAT3-expressing cells (white/yellow).

In (A) and (F), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Mann-Whitney U test

(A) and (F), Wilcoxon signed rank test (B), log-rank test (C). In (D) and (E), r is the Pearson correlation coefficient.

Serine-phosphorylated STAT3 promotes tumorigenesis via modulation of RNA polymerase transcriptional activity

Jesse Balic, Daniel J Garama, Mohamed Saad, et al.

Cancer Res Published OnlineFirst September 3, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0974

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/08/31/0008-5472.CAN-19-0974.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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