Therapeutic inhibition of Jak activity inhibits ...€¦ · mice Emma Stuart1,2 , Michael Buchert1,2 , Tracy Putoczki1,2, Stefan Thiem1,2, ... observation that the intestine specific

Stuart et al., Page 1

Therapeutic inhibition of Jak activity inhibits progression of gastrointestinal tumors in

mice

Emma Stuart1,2 , Michael Buchert1,2 , Tracy Putoczki1,2, Stefan Thiem1,2, Ryan Farid1,2, Joachim

Elzer1,5, Dennis Huszar3, Paul M. Waring4, Toby J. Phesse1,2 and Matthias Ernst1,2,*

1 The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia

2 Ludwig Institute for Cancer Research Melbourne, Australia.

3 AstraZeneca, Oncology iMed, Waltham, MA02451, USA

4 Dept of Pathology, The University of Melbourne, VIC 3010, Australia

5 present address: GABA GmbH, D-79539 Lörrach, Germany * Corresponding author

Running title: Jak inhibition limits gastrointestinal tumor growth

Key words: Jak/Stat3, gastrointestinal cancer, inflammation-associated cancer

on August 17, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 7, 2014; DOI: 10.1158/1535-7163.MCT-13-0583-T

http://mct.aacrjournals.org/


Financial Support: This work was generously supported by the Ludwig Institute for Cancer Research, the Victorian State Government Operational Infrastructure Support, the IRIISS scheme of the National Health and Medical Research Council Australia (NHMRC), and NHMRC grants #487922 (M. Ernst, M. Buchert), #433617 (M. Ernst), #603122 (M. Ernst) and #603127 (T.J. Phesse, M. Buchert). M. Ernst is a NHMRC Senior Research Fellow (#331004).

Corresponding Author: Matthias Ernst, The Walter and Eliza Hall Institute of Medical Research 1G Royal Parade Parkville, VIC 3052, Australia Phone +61 3 9345 2555 Fax +61 3 9347 0852 [email protected]

Disclosures: The authors declare no conflicting interests

Word Count (excl abstract): 2654

Total number of figures: 4 and 4 Supplementary





ABSTRACT

Aberrant activation of the latent transcription factor STAT3 and its downstream targets is a

common feature of epithelial-derived human cancers, including those of the gastrointestinal tract.

Mouse models of gastrointestinal malignancy implicate Stat3 as a key mediator of inflammatory

driven-tumorigenesis, where its cytokine/gp130/Jak-dependent activation provides a functional link

through which the microenvironment sustains tumor promotion. Although therapeutic targeting of

STAT3 is highly desirable, such molecules are not available for immediate clinical assessment.

Here we investigated whether the small molecule Jak1/2 inhibitor AZD1480 confers therapeutic

benefits in two mouse models of inflammation-associated gastrointestinal cancer, which are strictly

dependent of excessive Stat3 activation. We confirm genetically that Cre-mediated, tumor cell-

specific reduction of Stat3 expression arrests the growth of intestinal-type gastric tumors in

gp130F/F mice. We find that systemic administration of AZD1480 readily replicates this effect,

which is associated with reduced Stat3 activation and correlates with diminished tumor cell

proliferation and increased apoptosis. Likewise, AZD1480 therapy also conferred a cytostatic effect

on established tumors in a colitis-associated colon cancer model in wild-type mice. As predicted

from our genetic observations in gp130F/F mice, the therapeutic effect of AZD1480 remains fully

reversible upon cessation of compound administration. Collectively, our results provide the first

evidence that pharmacological targeting of excessively activated wild-type Jak kinases affords

therapeutic suppression of inflammation-associated gastrointestinal cancers progression in vivo.





INTRODUCTION

Aberrant activation of the latent transcription factor Signal Transducers and Activators of

Transcription (STAT) 3 is a common molecular characteristic of many epithelial-derived human

cancers (1). STAT3 plays a critical role in controlling transcriptional programs that fuel many

processes necessary for tumor promotion (2). Although STAT3 activation is observed in response to

various growth factors and cytokines, the IL-6 family of cytokines, defined by their shared use of

the GP130 receptor β-chain, arguably play the most important role during tumor promotion (3).

Upon ligand engagement of the receptor, the GP130-associated Janus family kinases (JAK) JAK1,

JAK2 and TYK2 undergo activation before recruiting and phosphorylating cytoplasmic STAT3 to

promote its dimerization, nuclear translocation and transcriptional activity (4).

Since oncogenic mutations in the GP130/JAK/STAT3 pathway occur infrequently in human

cancers, aberrant STAT3 signaling predominantly results from excessive autocrine and paracrine

signaling by IL-6 family cytokines, making this pathway a prime candidate for therapeutic

interference (2). Although STAT3 is a common signaling node for all of the redundantly acting IL6-

family cytokines, small molecules that disrupt STAT3’s protein-protein or protein-DNA

interactions with sufficient specificity have not yet been developed for clinical use (5). An

alternative, and immediately available approach exists in the inhibition of upstream activators

including JAK kinases, for which several small molecule inhibitors undergo advanced clinical

testing for diseases associated with constitutively activating JAK mutations (6). However, since

these JAK inhibitors are competitive antagonists of the enzyme’s ATP-binding sites, they also

inhibit activity of the corresponding wild-type kinases.

Here, we pursue this notion with the Jak1/2-specific inhibitor AZD1480 (7) in two models of

inflammation-associated and Stat3-driven gastrointestinal cancers in mice. We show that





therapeutic Jak inhibition confers an unexpected cytostatic benefit in situations where tumor

promotion depends on excessive activation of the gp130-associated wild-type forms of these

kinases.





MATERIALS and METHODS

Mice

All mice were on a mixed C57BL/6 x 129Sv background and maintained under specific pathogen-

free conditions. Construction of the transgenic gpa33-CreERT2 strain will be described elsewhere

(M. Buchert and M. Ernst, unpublished observations), and gp130F/F (8), R26LacZ (9) and Stat3fl/fl1

(10) mice have been described before. For experiments, littermates were randomly assigned to

individual treatment cohorts. Tumor-bearing mice received either AZD1480 (30 mg/kg,

AstraZeneca; diluted in 0.5% hydroxypropyl methylcellulose (Dow, #009004-65-3)/0.1%Tween-80

(Fisher, #BP338-500) daily by oral gavage for 6 weeks or diluent alone. Colitis-associated colon

cancer was induced and monitored by endoscopy as described previously (11,12,13) with the

exception that we administered dextran sodium sulfate at a concentration of 2% (w/v) in drinking

water. Collection and processing of stomach and intestine was conducted as described (12,13). All

animal experimentation was approved by the Ludwig Institute for Cancer Research/Department of

Surgery, Royal Melbourne Hospital Animal Ethics Committee.

Immunohistochemistry

Immunohistochemical analysis of gastric and colonic tissue was performed as described (12,13).

Apoptosis was detected with the ApopTag® Peroxidase detection kit (Millipore) according to the

manufacturer’s protocol.

Q-PCR analysis

Tissue was disrupted in Trizol (Invitrogen) using a Qiagen TissueLyser and RNA prepared as

described2. cDNA was synthesized using an Applied Biosystems high-capacity cDNA reverse

transcription kit, where each reaction contained 2 µg of total RNA. We performed Q-PCR on a

Corbett Research Rotogene RG3000 (San Francisco, CA), and for expression of Il6 and Il11 with





an Applied Biosystems 7300 Real Time PCR system using TaqMan probes (Applied Biosystems)

as described (12,13). Expression was normalized to GAPDH and we used the 2-ΔΔCT method to

calculate fold-change. Mann-Whitney U tests to determine statistical differences between ΔCT

values with values p≤0.05 being considered as significant. Primer sequences are detailed in

Supplementary Table 1.

Protein analysis

Protein lysates from whole tissue was prepared as described using RIPA buffer, and 40 µg were

separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes as

described (12,13). Membranes were blocked with 5% BSA, incubated with primary antibodies

(detailed in Supplementary Table 2) overnight and the appropriate secondary antibodies. Signals

were visualized with a Chemidoc XRS+ (BioRad, Hercules, CA) chemiluminescence detection

system.

Statistical analyses

All data are expressed as Mean±SEM. Differences between treatment groups were analyzed by two-

sided Student’s t-test.





RESULTS

Genetic limitation of epithelial Stat3 expression inhibits gastric tumor promotion in gp130F/F

mice

To test the therapeutic potential of Jak inhibition in an inflammation-associated epithelial cancer

model, we used the gp130F/F mutant mouse as a validated preclinical model of intestinal-type

gastric cancer that shares molecular and histological features with the human disease (12).

Although systemic reduction of Stat3 expression in gp130F/F;Stat3+/- mice delays the onset and

reduces the growth of tumors (8), it remains unclear whether this is directly attributable to excessive

Stat3 activity in the metaplastic epithelium of tumors gp130F/F mice. We therefore made use of our

observation that the intestine specific glycoprotein gpA33 (14) is expressed in the metaplastic

epithelium of gp130F/F mice, but not in the surrounding non-neoplastic glandular epithelium of the

antrum (Fig S1a,b). Therefore, we generated a novel transgenic mouse strain based on a bacterial

artificial chromosome (BAC) that includes all of the cis-regulatory elements of the gpa33 gene

locus to drive expression of a tamoxifen (Tmx)-dependent CreERT2 DNA recombinase. We then

generated corresponding compound gpa33-CreERT2;gp130F/F;R26LacZ/+ mutant mice, and following

Tmx administration we detected LacZ reporter activity only in the tumor epithelium but not in the

adjacent normal glandular epithelium of the antrum (Fig S1c). As expected, we also detected

recombination in the intestinal epithelium including the duodenum (data not shown).

We next induced Cre activity in 8 week-old gpa33-CreERT2;gp130F/F;Stat3fl/fl mice with

established tumors (Fig 1a), and as anticipated from the LacZ staining pattern, we observed partial

recombination of the Stat3fl/fl alleles and associated reduction in Stat3 expression in the tumors (Fig

S1d,e). Importantly, 8 weeks later we observed reduced tumor burden in the Tmx-treated cohort

when compared to the vehicle control group, which coincided with a reduction in tumor size, but

not in tumor number (Fig 1b). Furthermore, in Tmx-treated gpa33-CreERT2;gp130F/F;Stat3fl/fl mice





we observed reduced staining for the proliferation marker PCNA in the dysplastic epithelium of the

branching, tortuos gastric glands in the tumors that often develop cystic dilations (15). These

histopathological observations correlated with reduced expression of the Stat3 target gene Ccnd1

encoding the cell cycle regulator cyclin D1 (Fig 1c,d). Collectively, this data suggests that genetic

suppression of Stat3 expression in neoplastic epithelium reduces tumor growth in the antrum of

gp130F/F mice.

AZD1480 treatment reduces gastric tumor burden in gp130F/F mice

In order to test whether pharmacological inhibition of Jak kinase activity would confer a therapeutic

benefit in gp130F/F mice, we treated 8 week-old mice with AZD1480 for 6 weeks (Fig 2a). In mice

collected immediately after the end of this treatment period (referred to as “Acute cohort”), we

observed a reduced tumor burden (Fig 2b and FigS2A). Histological analysis of lesions from the

AZD1480-treated cohort revealed that they were entirely intramucosal and lacked the signs of

hemorrhage and inflammatory cell infiltrates observed in tumors of untreated mice. The latter

showed the previously observed mucinus metaplasia with signs of epithelial invasion into

muscularis and chronic abrasion of the epithelium facing the lumen of the stomach (FigS2A and

Ref 15). Likewise, mice that we aged for 6 additional weeks after AZD1480-treatment (“Follow-up

cohort”) also had reduced tumor burden when compared to their age-matched vehicle cohort (Fig

2b and FigS2). However, the tumor burden in 20 week-old mice of the “Follow-up” cohort was

larger than in the 14 week-old AZD1480-treated “Acute” cohort, indicating that the growth

inhibitory effect of AZD1480 was reversible. The reduced number of both, emerging small (<25

mg) as well as established large tumors (25-50 mg), suggests that AZD1480 treatment suppressed

early steps of tumorigenesis as well as subsequent tumor growth (Fig 2c). Accordingly, in the

“Follow-up” cohort we observed an increase in larger tumors, presumably arising from the small

(25-50 mg) tumors, as well as a “replenishment” of newly arising tumor in the latter group.





AZD1480 reduces gastric tumor burden in a Stat3-dependent manner

We next confirmed that AZD1480 inhibited Jak1/2 activity in the tumors by reduced abundance of

phosphorylated Jak1 (pJak1) and Jak2 (pJak2), and this difference was no longer observed in the

“Follow-up” cohort (Fig 3a). We also performed immunohistochemical analysis for phosphorylated

Stat3 (pStat3) to confirm that AZD1480-treatment suppressed Stat3 activity, and observed a marked

reduction in pStat3 expression in tumors from the “Acute” cohort (Fig 3a and FigS3a), which

completely reverted in the “Follow-up” cohort. As predicted from the original application of

AZD1480 to inhibit Jak2-mediated Stat5 activation in hemopoietic cells (7), we also detected

reversible inhibition of Stat5 in these tumors most likely arising from tumor infiltrating cells (13).

We have previously described co-activation of the Jak/Stat and PI3K/mTor pathways in these

tumors in a gp130-dependent manner, and found that pharmacological blockade of mTor alone was

sufficient to suppress tumor burden in gp130F/F mice despite persistent Stat3 activity (12).

Therefore, we investigated whether AZD1480 treatment also affected PI3K pathway activation.

Surprisingly, we observed similar staining for the mTor target prpS6 in tumors of AZD1480 and of

vehicle-treated mice consistent with pAkt expression in tumors remaining unaffected by AZD1480

treatment (Fig 3a and FigS3b). These findings suggest that gp130-dependent activation of the

PI3K/mTor pathway may occur independent of Jak1/2 activity. Consistent with AZD1480 treatment

suppressing Stat3 activity, we found that the reduced expression of the bona fide Stat3-target gene

Socs3 in the “Acute” cohort reverted in the “Follow-up” cohort to levels observed in the vehicle

control cohorts (Fig 3b). Since tumor development in gp130F/F mice requires Il-11, but not Il-6

signaling (13), we next investigated expression of these two cytokines within the tumors. While Il6

expression was unaffected, we observed a marked raise of Il11 expression following AZD1480

treatment (Fig 3c). Since Il11 expression levels were again normalized in the tumors of the

“Follow-up” cohort, we interpret this finding as part of a compensatory mechanism by which tumor

cells adapt to an environment of impaired Jak/Stat signaling.





AZD1480 treatment induces apoptosis and reduces proliferation in gp130F/F tumors

Since our data suggested that AZD1480 inhibited the growth of established tumors, we next

investigated whether it affected cell survival and proliferation. We observed more ApopTag-

positive cells in tumors of the “Acute” cohort than in tumors from control mice, but this effect was

not sustained at the end of the treatment-free “Follow-up” period (FigS3c). This finding was

consistent with Western blot analysis that revealed reduced expression of the Stat3 target genes

encoding the pro-survival proteins Bcl-XL and Bcl-2 in the “Acute”, but not in the “Follow-up”

cohort (Fig 3a). Likewise, AZD1480 also reduced the number of PCNA-positive epithelial cells in

tumors of the “Acute”, but not of the “Follow-up” cohort (FigS3d) and this observation coincided

with reduced expression of the cell cycle regulators ccnd1 and cdc25 (Fig 3d). Collectively, our

data indicates that AZD1480 treatment reduces Stat3 activity and limits the growth of gastric

tumors in gp130F/F mice by simultaneously suppressing cell survival and proliferation.

AZD1480 suppresses tumor growth in inflammation-associated colon cancer in wild-type mice

To investigate whether Jak1/2 inhibition could also confer therapeutic benefits in an inflammation-

associated tumor model where all gp130-signaling components remained wild-type, we explored

the efficacy of AZD1480-treatment in the colitis-associated colon cancer (CAC) model in wild-type

mice. The CAC model depends on administration of the somatic mutagen azoxymethane (AOM) to

induced mutations in exon 3 of the ctnnb1 gene to confer constitutive activation of the canonical

WNT signaling pathway (3,13). Subsequent repeated administration of the luminal toxin dextran

sodium sulfate (DSS) meanwhile triggers flares of colonic inflammation and the development of

colitis (3,12,13) (FigS4a). We, and others, have shown that in the CAC-model, IL-6 family

cytokines promote survival and proliferation of AOM-mutagenized cells to form tubular adenomas

in the distal colon, which is suppressed in the absence of Stat3 expression in the intestinal

epithelium (3). We therefore randomly assigned CAC challenged mice after the second cycle of

DSS mice to AZD1480 or control treatments and we employed serial endoscopy prior to and at the





end of 40 day treatment period to monitor tumor progression in individual mice. Using established

scoring parameters for the endoscopic exams (11,13), we detected the expected increase in disease

in the vehicle cohort. By contrast, the disease scores remained stable or decreased in all mice of the

AZD1480-treated cohort (Fig 4a and FigS4b). These observations were confirmed upon autopsy,

when we observed fewer and smaller macroscopic tumors in mice of the AZD1480-treated cohort

(Fig 4b). Furthermore, histological analyses of colonic tissue revealed a reduction in PCNA

positive epithelial staining in colons of AZD1480-treated mice, which coincided with a reduction of

inflammatory cell aggregates (FigS4c). Western blot analysis revealed that the colonic epithelium

of AZD1480-treated mice contained less pJak1 and pJak2, which correlated with suppressed pStat3

(and pStat5) levels when compared to the colonic epithelium of the control cohort. Likewise, we

also observed reduced abundance of Bcl-2 and Bcl-XL proteins in colonic epithelium of AZD1480-

treated mice (Fig 4c) although the abundance of IL11 protein in these tumors remained unaffected.

Collectively, this data indicates that AZD1480 treatment effectively suppressed tumor growth in

wild-type mice and that the underlying molecular mechanism is likely to relate to impairment of

Stat3-driven proliferation and survival of neoplastic intestinal epithelium.





DISCUSSION

The tumor microenvironment is a rich source of inflammatory cytokines, among which the IL-6

family is most widely implicated in conferring tumor promoting activities in breast, prostate, liver

and gastrointestinal cancers. In turn, persistent and aberrant activation of Stat3 in neoplastic tissue

promotes expression of many hallmarks of cancer (16) and is associated with a poor prognosis in

many solid cancers including those arising from gastric (17) and colonic epithelium (18). In human

cancers, somatic mutations have been identified in that confer constitutive activation, in GP130,

JAK1, JAK2 and STAT3 alongside or epigenetic silencing of the negative regulator SOCS3, in the

majority of cases excessive signaling results from an overabundance of IL-6 family ligands (2).

Here we show in two independent models of inflammation-associated cancer that the dual Jak1/2

inhibitor AZD1480 confers therapeutic benefits.

In both models the inhibition of tumorigenesis coincided with reduced Stat3 phosphorylation,

reduced expression of the anti-apoptotic proteins Bcl-XL and Bcl-2, and the cell cycle regulators

Ccnd1 and Cdc25. These observations are consistent with findings in xenograft models where

Jak1/2 inhibition reduces the growth of breast, prostate and ovarian cancers and coincided with

reduced pStat3 expression in glioblastoma (19), thyroid carcinoma (20) and myeloma (21). We

have previously shown that epithelial-specific Stat3 deletion in the intestine confers a prophylactic

benefit to CAC-challenged mice3. Here we observe that mosaic lacZ activation by gpa33-CreERT2

and the corresponding reduction of Stat3 expression by approximately 30% is sufficient to reduce

overall tumour growth. It is therefore likely that at least some of the effects conferred by systemic

AZD1480 administration occurred at the level of the neoplastic epithelium. These findings support

an exquisite sensitivity of tumor cells to excessive Stat3 signaling where, for example, genetic

ablation of one Stat3 allele is already sufficient to block gastric tumorigenesis in gp130F/F;Stat3+/-

mice (8). By the same token, collectively our above observations argue strongly against a

significant contribution of Stat5 inhibition, which we also observed in AZD1480-tretade mice.





Conversely, normal tissue homeostasis remains unaffected in Stat3+/- mice and is also not affected

after a 40 day treatment with AZD1480 (E. Stuart, T.J. Phesse and M. Ernst; unpublished

observations). However, systemic Jak-inhibition may also confer additional therapeutic benefits,

including suppression of tumor angiogenesis and possibly restore the anti-tumour response

frequently impaired as a consequence of excessive Stat3 activation in immune cells in response to

tumor-derived production of cytokines such as IL-10 (22).

Developing therapeutic strategies for selective Stat3 inhibition has proven challenging and therefore

targeting of upstream components provides a pharmacologically viable alternative including

monoclonal antibodies directed towards IL-6 or its α-chain receptor subunit or small molecule

inhibitors for Jak family of kinases. Although originally developed to inhibit the constitutive active

JAK2(V617F) mutant associated with myeloproliferative disorders, here we show surprising

efficacy of AZD1480 on excessively activated wild-type Jak1 and Jak2 to confer a cytostatic effect.

Since recent evidence suggests that combinations of JAK inhibitors with HDAC and mTOR

antagonists provide an effective strategy to induce killing of tumor cells harboring the

JAK2(V617F) mutations (23) similar combinations may result in the desired killing of cancerous

gastrointestinal epithelium.





REFERENCES

1. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 2002,109:1139-42. 2. Jarnicki A, Putoczki T and Ernst M. Stat3: linking inflammation to epithelial cancer - more

than a "gut" feeling? Cell Division 2010,5:14-28. 3. Bollrath J, Phesse TJ, von Burstin VA, Putoczki T, Bennecke M, Bateman T, et al. gp130-

mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell 2009,15:91-102.

4. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F. Principles

of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 2003,374:1-20. 5. Haftchenary S, Avadisian M and Gunning PT. Inhibiting aberrant Stat3 function with

molecular therapeutics: a progress report. Anti-cancer Drugs 2011,22:115-27. 6. Noor SM, Bell R and Ward AC. Shooting the messenger: targeting signal transduction

pathways in leukemia and related disorders. Crit Rev Oncology/Hematology 2011,78:33-44. 7. Hedvat M, Huszar D, Herrmann A, Gozgit JM, Schroeder A, Sheehy A, et al. The JAK2

inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis in solid tumors. Cancer Cell 2009,16:487-97.

8. Jenkins BJ, Grail D, Nheu T, Najdovska M, Wang B, Waring P, et al. Hyperactivation of

Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-beta signaling. Nat Med 2005,11:845-52.

9. Sorriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet

1999,21:70-71. 10. Alonzi T, Maritano D, Gorgoni B, Rizzuto G, Libert C, Poli V. Essential role of STAT3 in

the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver. Mol Cell Biol 2001,21:1621-32.

11. Becker C, Fantini MC and Neurath MF. High resolution colonoscopy in live mice. Nat

Protocols 2006,1:2900-04. 12. Thiem S, Pierce TP, Palmieri M, Putoczki TL, Buchert M, Preaudet A, et al. mTORC1

inhibition restricts inflammation-associated gastrointestinal tumorigenesis in mice. J Clin Invest 2013,123:767-81.

13. Putoczki TL, Thiem S, Loving A, Busuttil RA, Wilson NJ, Ziegler PK, et al. Interleukin-11

is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis and can be targeted therapeutically. Cancer Cell 2013,24:257-71.

14. Johnstone CN, White SJ, Tebbutt NC, Clay FJ, Ernst M, Biggs WH, et al. Analysis of the

regulation of the A33 antigen gene reveals intestine-specific mechanisms of gene expression. J Biol Chem 2002,277:34531-39.





15. Judd LM, Alderman BM, Howlett M, Shulkes A, Dow C, Moverley J, et al. Gastric cancer development in mice lacking the SHP2 biding site on the IL-6 family co-receptor gp130. Gastroenterology 2004,126:196-207.

16. Hanahan D. and Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011,144:

646-74. 17. Kim DY, Cha ST, Ahn DH, Kang HY, Kwon CI, Ko KH, et al. STAT3 expression in gastric

cancer indicates a poor prognosis. J Gastro Hepatol 2009,24:646-51. 18. Morikawa T, Baba Y, Yamauchi M, Kuchiba A, Nosho K, Shima K, et al. STAT3

expression, molecular features, inflammation patterns, and prognosis in a database of 724 colorectal cancers. Clin Cancer Res 2011,17:1452-62.

19. McFarland BC, Ma JY, Langford CP, Gillespie GY, Yu H, Zheng Y, et al. Therapeutic

potential of AZD1480 for the treatment of human glioblastoma. Mol Cancer Therap 2011,10:2384-93.

20. Couto JP, Almeida A, Daly L, Sobrinho-Simões M, Bromberg JF, Soares P. AZD1480

Blocks Growth and Tumorigenesis of RET- Activated Thyroid Cancer Cell Lines. PloS one 2012,7:e46869.

21. Scuto A, Krejci P, Popplewell L, Wu J, Wang Y, Kujawski M. The novel JAK inhibitor

AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival. Leukemia 2011,25:538-50.

22. Yu H, Pardoll D and Jove R. STATs in cancer inflammation and immunity: a leading role

for STAT3. Nat Rev Cancer 2009,9:798-809. 23. Britschgi A, Andraos R, Brinkhaus H, Klebba I, Romanet V, Müller U, et al. JAK2/STAT5

inhibition circumvents resistance to PI3K/mTOR blockade: a rationale for cotargeting these pathways in metastatic breast cancer. Cancer Cell 2012,22:796-811.





LEGENDS

FIG 1 Epithelial Stat3 ablation reduces established gastric tumor burden in

BAC(gpA33:CreERT2);gp130F/F;Stat3fl/fl mice.

(A) Schematic representation of experimental design. Eight week old

gpA33:CreERT2;gp130F/F;Stat3fl/fl mice were treated for 5 consecutive days with daily

injections of 2mg tamoxifen (i.p., in 10% ethanol 90% sunflower oil as vehicle) or vehicle

alone and aged for 8 weeks when all analysis was carried out.

(B) Combined weight of all resected tumors from each mouse was determined, and individual

tumors were classified according to their weight (n>5 mice per cohort). Mice were

randomly assigned to the cohort receiving tamoxifen (+Tmx) or vehicle (-Tmx).

(C) Representative immunohistochemical stains for PCNA from gastric tumors of mice from

the indicated treatment cohort. Boxed areas are also shown at higher magnifications. Scale

bar is 100 µM (25 µM for insets).

(D) Quantitative PCR analysis for Ccnd1 encoding cyclinD1 of pooled tumors from >5

individual mice per treatment cohort.

Data are Mean±SEM; with * p<0.05, *** p<0.001.





FIG 2 Systemic administration of AZD1480 reduces established gastric tumor burden in

gp130F/F mice.

(A) Schematic representation of experimental design. All mice were treated with AZD1480 (30

mg/kg, p.o. once daily for 5 days per week) and analysed either immediately after

completion of their treatment (“Acute”-cohort) or after an additional 6 week treatment-free

follow-up period (“Follow-up” cohort).

(B) Combined weight of all resected tumors from each mouse was determined, and individual

tumors were classified according to their weight (n>5 mice per cohort). At the end of the

treatment period, mice were randomly assigned to the Acute and Follow-up cohorts.

Significances indicate differences between age-matched cohorts only.

Data are Mean±SEM; with * p<0.05, ** p<0.01, *** p<0.001.





FIG 3 Biochemical analysis of gastric tumors from AZD1480-treated gp130F/F mice.

(A) Representative Western blot analyses for the indicated proteins and their phosphorylated

(p) isoforms of cell lysates prepared from pooled tumors of individual mice and collected

as described in Figure 2.

(B) Quantitative PCR analysis for the bona fide Stat3 target genes Socs3 in pooled tumors for

>5 individual mice.

(C) Quantitative TaqMan PCR analysis for expression of Il6 and Il11 in pooled tumors for >5

individual mice.

(D) Quantitative PCR analysis for the cell cycle genes ccnd1 and cdc25 in pooled tumors for

>5 individual mice.

Data are Mean±SEM; with * p<0.05, ** p<0.01.





FIG 4 Systemic administration of AZD1480 reduces established colitis-associated colon

cancer burden in wild-type mice.

(A) Tumor burden of individual mice was serially assessed by endoscopy as described in

Becker et al. 4, immediately prior to and at the end of a 40 day treatment of AZD1480 (30

mg/kg, p.o. once daily for 5 days per week) or vehicle. Mice were randomly assigned to

the AZD1480 and vehicle-treatment control cohorts. Resulting endoscopy scores for each

cohort scores were statistically assessed.

(B) Total number of macroscopically visible colon tumors per mouse at time of autopsy. The

combined tumor area was estimated from assuming a circular shape of the resulting

adenomas and determining their diameter. n>3 mice per cohort.

(C) Representative Western blot analysis for the indicated proteins and their phosphorylated

(p) isoforms of cell lysates prepared from pooled colonic tumors of individual mice

collected at the end of a 40 day treatment of AZD1480 (30 mg/kg, p.o. once daily for 5

days per week) or vehicle.

Data are Mean±SEM; with * p<0.05




A250 ***

B >25 mg 25-50 mg <50 mg

Figure 1

-Tmx +Tmx0

50

100

150

200

250

Tu

mo

r b

urd

en (m

g) ***

Tu

mo

r n

um

ber

C

0.03

0.04

ssio

n

*D

0.00

0.01

0.02

CycD1

exp

res

- Tmx + Tmx

CreERT2 negativeplus Tmx

CreERT2 positiveplus Tmx




Figure 2

ATumor burden

0 4 8 12 16 20AZD1480 (30 mg/kg)

Age (Weeks)

Acute

Follow up

A

A

250 ***

B **

AZD1480 (30 mg/kg) VehicleFollow-up

A =Time point of analysis

<25 mg 25-50 mg >50 mg

50

100

150

200

250

um

or

bu

rde

n (m

g) **

***T

um

or

nu

mb

er

** *

0

50Tu

Acute Follow upVehicle AZD1480 Vehicle AZD1480

T

**




Figure 3

8

10

pre

ssio

n

***C

pJak1Jak1

A Acute Follow-upVehicle AZD1480 Vehicle AZD1480

Vehic

le

ZD1480

Vehic

le

ZD1480

Vehic

le

ZD1480

Vehic

le

ZD1480

0

2

4

6

Tra

nsc

rip

t exp2

pStat5Stat5

pJak2Jak2

pStat3Stat3

VeAZD Ve

AZD VeAZD Ve

AZD

Acute Follow-up Acute Follow-up

Il6 Il11

Stat5pAktAkt

prpS6rpS6

BclxL

2.5 2.0*** **12 ***B D

Bcl2Bax

Actin

Vehic

le

D1480

Vehic

le

D1480

0.0

0.5

1.0

1.5

2.0

Ccn

d1 e

xp

res

sio

n

ehic

le

D1480

ehic

le

D1480

0.0

0.5

1.0

1.5

Cdc

25 e

xp

res

sio

n *

Vehic

le

ZD1480

Vehic

le

ZD1480

0

2

4

6

8

10

SOC

S3 e

xp

res

sio

n

VeAZD Ve

AZD Veh

AZD1Veh

AZD1

Acute Follow-up Acute Follow-up

VeAZD Ve

AZD

Acute Follow-up




Figure 4

40 VehicleA AZD1480

+ +- -C

10

20

30

En

do

sco

py

sc

ore

VehicleAZD1480

* pJak2

Jak2

pJak1 Jak1

+ + - -

pJak2

pJak1Jak1

Jak2

40 days 80 days0

B

pStat3

Stat3

pStat5

pStat3Stat3

Stat5

IL 119

12

ber

80

100

mm

2)

* *

Actin Bax

Bcl-xL

Bcl-2

IL-11Bcl-xL

Bcl-2

ActinBax

Vehicle AZD14800

3

6

Tu

mo

r n

um

Control AZD14800

20

40

60

Tu

mo

r a

rea

(m

Vehicle AZD1480 Control AZD1480




Published OnlineFirst January 7, 2014.Mol Cancer Ther Emma C Stuart, Michael Buchert, Tracy Putoczki, et al. gastrointestinal tumors in miceTherapeutic inhibition of Jak activity inhibits progression of

Updated version

10.1158/1535-7163.MCT-13-0583-Tdoi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2014/01/09/1535-7163.MCT-13-0583-T.DC1

Access the most recent supplemental material at:

Manuscript

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

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2014/01/07/1535-7163.MCT-13-0583-TTo request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-13-0583-T

http://mct.aacrjournals.org/content/suppl/2014/01/09/1535-7163.MCT-13-0583-T.DC1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2014/01/07/1535-7163.MCT-13-0583-T


Documents

Therapeutic inhibition of Jak activity inhibits ...€¦ · mice Emma Stuart1,2 , Michael Buchert1,2 , Tracy Putoczki1,2, Stefan Thiem1,2, ... observation that the intestine specific