il-6-stat-3-hif
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Cancer Therapy: Clinical
IL6-STAT3-HIF Signaling and Therapeutic Response to theAngiogenesis Inhibitor Sunitinib in Ovarian Clear Cell Cancer
Michael S. Anglesio1,5, Joshy George1,2, Hagen Kulbe7, Michael Friedlander3, Danny Rischin1, Charlotte Lemech3,
Jeremy Power1, Jermaine Coward7, Prue A. Cowin1, Colin M. House1, Probir Chakravarty7, Kylie L. Gorringe1,Ian G. Campbell1, Australian Ovarian Cancer Study Group, Aikou Okamoto8, Michael J. Birrer9, David G. Huntsman5,
Anna de Fazio4, Steve E. Kalloger6, Frances Balkwill7, C. Blake Gilks5,6, and David D. Bowtell1,2
Abstract
Purpose: Ovarian clear cell adenocarcinoma (OCCA) is an uncommon histotype that is generally
refractory to platinum-based chemotherapy. We analyzehere the mostcomprehensivegene expression and
copy number data sets, to date, to identify potential therapeutic targets of OCCA.
Experimental Design:Gene expression and DNA copy number were carried out using primary human
OCCA tumor samples, and findings were confirmed by immunohistochemistry on tissue microarrays.
Circulatinginterleukin (IL) 6 levels were measured in serum from patients with OCCA or high-grade serous
cancers and related to progression-free and overall survival. Two patients were treated with sunitinib, and
their therapeutic responses were measured clinically and by positron emission tomography.Results: We find specific overexpression of the IL6-STAT3-HIF(interleukin 6-signal transducer and
activator of transcription 3-hypoxia induced factor) pathway in OCCA tumors compared with high-grade
serous cancers. Expression ofPTHLHand high levels of circulating IL6 in OCCA patients may explain
the frequent occurrence of hypercalcemia of malignancy and thromboembolic events in OCCA. We
describe amplification of several receptor tyrosine kinases, most notablyMET, suggesting other potential
therapeutic targets. We report sustained clinical and functional imaging responses in two OCCA patients
with chemotherapy-resistant disease who were treated with sunitinib, thus showing significant parallels
with renal clear cell cancer.
Conclusions: Our findings highlight important therapeutic targets in OCCA, suggest that more
extensive clinical trials with sunitinib in OCCA are warranted, and provide significant impetus to the
growing realization that OCCA is molecularly and clinically distinct to other forms of ovarian cancer.Clin
Cancer Res; 17(8); 253848. 2011 AACR.
Introduction
Ovarian clear cell adenocarcinoma (OCCA) is a histo-logic subtype of ovarian cancer that is characterized by a
particularly poor response rate to current chemotherapyregimens. The occurrence of OCCA is associated withcoexistent endometriosis (1). OCCAs are generally refrac-tory to platinum-based therapy, with a response rate ofonly 11% to 15% (2). Given the poor response rates, thereis a need todevelop novel clinical approaches toOCCA andthis rests on first gaining insight into the biology of thedisease. Investigation of OCCA has been neglected in favorof the more common high-grade serous carcinoma(HGSC), and most studies include only a small numberofOCCA samples as part ofa larger series of ovarian tumors(36).
Relatively little is known about the signaling pathways
that drive OCCA. Hepatocyte nuclear factor 1 beta(HNF1b) was discovered as a biomarker of OCCA (7).It seems to be a lineage-specific markerthat is expressed inpreneoplastic lesions and may play a role in apoptoticescape. A gene expression pattern typical of OCCA hasrecently been derived from OCCA cell lines and involves
genes associated with oxidative stress, glyconeogenesis,and mitogen activated protein kinase and cytokine acti-vation (8). Independent karyotypic and array-based gen-
ome-wide measures of DNA copy number have identified
Authors' Affiliations: 1Peter MacCallum Cancer Centre, East Melbourne,Victoria; 2Department of Biochemistryand MolecularBiology, University ofMelbourne, Parkville, Victoria; 3Department of Medical Oncology, RoyalHospital for Women; 4Westmead Institute for Cancer Research, Universityof Sydney at Westmead Millennium Institute, Westmead Hospital, Sydney,Australia; 5Department of Pathology, University of British Columbia;6Genetic Pathology Evaluation Centre of the Prostate Research Centreand Department of Pathology, Vancouver General Hospital and Universityof British Columbia, Vancouver, British Columbia, Canada; 7Centre forCancer and Inflammation,Barts Cancer Institute, Queen MaryUniversity of
London, London, United Kingdom;8
Departments of Obstetrics and Gyne-cology and Gene Therapy, Institute of DNA Medicine, The Jikei UniversitySchool of Medicine, Tokyo, Japan; and 9Massachusetts General Hospital,Boston, Massachusetts
Note:Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: DavidD. Bowtell, PeterMacCallumCancer Centre,LockedBag1 ABeckettSt., Melbourne8006, Victoria, Australia. Phone:61 396561356; Fax: 61 3 96561414; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-10-3314
2011 American Association for Cancer Research.
Clinical
Cancer
Research
Clin Cancer Res; 17(8) April 15, 20112538
American Association for Cancer ResearchCopyright 2011on December 3, 2011clincancerres.aacrjournals.orgDownloaded from
Published OnlineFirst February 22, 2011; DOI:10.1158/1078-0432.CCR-10-3314
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gain at 17q23-25 (911) as being associated with over-expression ofPPM1D, a protein phosphatase that regu-lates the stress-induced extracellular signal-regulatedkinase p38.
To date, no published genomic studies have involvedcharacterization of a large number of tumors by using high-resolution contemporary gene expression and DNA copynumber platforms. Here we explore the genomic features of
a cohort of 59 OCCAs, showing amplification and over-expression of multiple cytokine and growth factor receptorsand signaling components.
Methods
Patient cohortsOCCA samples were drawn from the AOCS, a popula-
tion-based cohort of more than 1,800 women with ovariancancer recruited between 2002 and 2006 (12). Patients forthis project were ascertained from AOCS initially by usingabstracted pathology reports, followed by review of diag-nostic hematoxylin and eosinstained sections from tissuecollected at primary surgery by a panel of anatomic pathol-ogists to confirm an OCCA diagnosis. Samples from a
cohort of primary OCCA patients were subjected to par-tially overlapping genomic, IHC, and serum IL6 measure-ments. Summary clinical characteristics of this OCCA
cohort and a control set of HGSC samples for IHC areprovided in Table 1. Details of the analyses carried outwith individual AOCS samples are provided in Supple-mentary Table S1. For validation studies, single nucleo-
tide polymorphism (SNP) microarray data were obtainedfrom 18 Japanese OCCA samples (Supplementary
Table 1. Sample cohort used for genomic and IHC analyses
OCCA HGSC
Overall cohort
(n 59)
SNP 6.0
(n 39)
HG-U133 Plus
2.0 (n 31)
TMA
(n 40)
TMA
(n 16)
Gradea
1 0
2 7
3 59 39 31 40 9
Stage
I 26 19 13 19 2
II 9 6 6 5 1
III 18 10 10 13 11
IV 1 1 0 1 2
Unknown 5 3 2 2 0
Age
Median 56 58 56 56 60
Range 3178 3178 3378 3278 4373
Primary treatment
Plt/Tx 39 24 23 32 13
Plt only 7 4 4 5 1
Other/unknown 13 12 4 3 2
Abbreviations: Plt/Tx, platinum-taxane; Plt, platinum.aAll OCCA tumors are considered "high-grade" by convention.
Translational Relevance
Advanced stage ovarian clear cell adenocarcinoma
(OCCA) is associated with poor patient outcome, with
low response rates to platinum-taxanebased regimens.We observed distinctly different gene expression and
copy number patterns in OCCA compared with high-grade serous carcinoma (HGSC). Collectively these find-ings argue for novel therapeutic approaches for OCCA.Our findings highlightMETand IL6. Although METis
gainedinbothHGSCandOCCA,thefocalnatureofMETgain and consistent overexpression in OCCA are sugges-tive of a driver mutation. Our findings indicate thatOCCA patients shouldbe specificallyincluded in currentclinical trials of anti- interleukin (IL) 6 antibodies andthat overexpression of IL6and PTHLH maycontribute tothrombosis and hypercalcemia of malignancy com-monly seen in OCCA. The impressive clinical responsesof 2 patients to sunitinib are consistent with molecular
parallels between renal clear cell cancer and OCCA andthe overexpression of EPAS1 (HIF2A) and HIF1A inOCCA observed in this study.
IL6-STAT3-HIF Signaling in Ovarian Clear Cell Cancer
www.aacrjournals.org Clin Cancer Res; 17(8) April 15, 2011 2539
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Table S2). Validat ion of IHC data was done using 246ovarian tumor samples (29 OCCAs, 217 HGSCs)obtained from the British Columbia Cancer Agency (Sup-plementary Table S2). Two patients with platinum-resis-
tant OCCA were treated with the small molecule inhibitorsunitinib, which was obtained from Pfizer. Patientconsent, sample collection, and project design wereapproved by Institutional Review Boards at the contribut-
ing institutions.
Sample processingDNA and RNA were isolated from serial sections(912
100 mm) of snap-frozen tissue embedded in optimal cut-ting temperature (OCT) compound and sectioned using acryomicrotome. Flanking 5-mm sections stained withhematoxylin and eosin were used to estimate the propor-tion of tumor cells present in the specimen. Whole tumorsections were used for all gene expression studies, and forDNA copy number analysis, all sections were microdis-
sected (except for 5 sections wherein percentage tumornuclei exceeded 90%) prior to DNA extraction. RNA andDNA extraction and quantification were done as describedpreviously (13).
Microarray analysesAffymetrix U133 Plus 2.0 and SNP 6.0 oligonucleotide
arrays were used to measure global changes in gene expres-sion and DNA copy number. Gene expression and DNAcopy number data, along with data analysis package insweave format, are available at http://www.petermac.org/research/supplements.html. Analysis of gene expressiondata was essentially as described previously (12, 13).DNA copy number analysis was essentially as describedpreviously (14). Additional information on normalization,
filtering, hierarchical clustering, differential gene expres-sion statistical analysis, gene ontology, and copy numberanalyses can be found in Supplementary Methods.
Tissue microarray and immunohistochemistryAn advanced tissue arrayer (Chemicon International)
was used to obtain 0.6-mm cores from representativeformalin-fixed, paraffin-embedded tissues and constructtissue microarrays(TMA) in an agarose matrix as previouslydescribed (12). Immunohistochemistry (IHC) was carriedout using 3-mm sections that were dewaxed, rehydrated,and stained using a Dako autostainer with Envisionamplification (Dako). Staining was visualized with diami-nobenzadine. Antibodies were HIF1A (Novus; NB100-479
at 1:1,500), pSTAT3 (Cell Signaling Technologies; D3A7 at1:50), LYN (Cell Signaling Technologies; C13F9 at 1:200),and IL6. Two sample sets were used to generate TMAs forIHC analysis: One set of 40 OCCA specimens was obtainedfrom AOCS, of which 22 specimens were used for geneexpression analysis and 29 specimens were used for DNAcopy number analysis (Supplementary Table S1). A secondset of samples was obtained from the OvCaRe group atthe British Columbia Cancer Agency and was completelyindependent of the genomic analysis (Supplementary
Table S2). Results for both TMAs were scored by an ana-tomic pathologist (C.B.G). Statistical comparisons of pro-tein expression in OCCA and serous cancers were madeusing the AOCS and Canadian samples independently. In
both cases, scoring systems were binarized into high andlow groups during statistical analysis (see figure legends).
Cell cultureOvarian cell lines were obtained from public repositories
(American Type Culture Collection: TOV112D, TOV21G,and ES2; National Cancer Institute: SKOV3 and IGROV1;Human Science Research Resource Bank: RMG1; RIKEN:JHOC5, JHOC7, and JHOC9). HAC2 was kindly providedby Dr. Aikou Okamoto (Japan) and OVHS1 from Dr. Ian
Campbell (Australia). All cell lines were maintained inRPMI 1640 with 10% FBS in a humidified incubator at37C with 5% CO2. Experiments were conducted withsubconfluent cells in log-phase growth unless otherwisenoted.
Measurement of circulating IL6 levels in conditionedmedia and serum
Cells were plated at 3 105 cells/well in a 6-well plate,cell culture supernatants were removed after 48 hours ofculture, and cytokine concentrations were measured usingQuantikine ELISA kits following manufacturers instruc-tions (R&D Systems). OCCA and HGSC serum sampleswere obtained from the AOCS cohort, relying only onpatients wherever serum samples were collected immedi-ately presurgically. Clotted blood samples were collected inplain tubes, allowed to clot, serum collected by centrifuga-tion at 2,500 rpmor 1,300gfor 10minutes, and stored in1 mL aliquots at80C until processed. Serum IL6 con-centrations were estimated using electrochemilumines-
cence detection with Meso Scale Discovery (MSD) assaysand done according to the manufacturers protocol. Afterdefrosting at 4C, samples were centrifuged briefly at 2,000gfor 1 to 2 minutes at 4C. The calibrator standards andsamples from patients and normal healthy controls wereincubated on MSD single-plex IL6 microplates (MSD catno. K151AKB-2). Plates were washed and read usingSECTOR Imager 2400 software (MSD). Statistical signifi-cance of difference between OCCA and HGSC serumsamples was calculated using a Wilcoxon rank-sum test.A KaplanMeier survival curve was plotted, and log-rankstatistic was computed to test the significance of serum IL6levels versus patient outcome.
Results
Clear cell ovarian cancer accounts for less than 10% ofinvasive ovarian cancer diagnoses in Western countries andit was therefore necessary to access a large patient cohort toobtain substantial numbers of samples. Patients wereobtained from the Australian Ovarian Cancer Study(AOCS), a population-based cohort of more than 1,800women accrued from 2002 to 2006 (12). Details of the
clinical characteristics of the AOCS OCCA patient cohort
Anglesio et al.
Clin Cancer Res; 17(8) April 15, 2011 Clinical Cancer Research2540
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and genomic analyses done are provided in Table 1 andSupplementary Tables S1 and S2.
Overexpression and amplification of cytokine and
receptor tyrosine kinase signalingAffymetrix U133 2.0 GeneChip arrays were used toobtain comprehensive gene expression signatures for 31OCCA samples, which were compared with data generated
previously by our group on more than 200 HGSC andendometrioid cancers (12) and human ovarian surfaceepithelium (HOSE; ref. 15). Unsupervised clustering of
the top 9,500 most variant genes easily segregated OCCAfrom other ovarian cancer histotypes (Fig. 1A). A largecoregulated cluster of genes was highly expressed in OCCAthat included the immunohistochemical (IHC) biomarkerof OCCAHNF1b(TCF2; ref. 9), the transcriptional targetsofHNF1b,PKHD1(16) and P450 xenobiotic metabolism
genes CYP2C9, CYP2C19, and CYP2C18 (17), the prolactinreceptor (PRLR), and UDP glycosyl transferases involved infatty acid and steroid metabolism (Fig. 1B). IHC studieshave shown that immune infiltration is less prevalent in
OCCA than in HGSC (18), and this was reflected in thelow-level expression of immune markers (Fig. 1B).We noted highly specific upregulation of PTHLH
(PTHRP; Fig. 1C; Supplementary Fig. S1), a gene whoseexpression is commonly associated with hypercalcemia ofmalignancy (19), and stanniocalcin-1 (STC1; Fig. 1C), aprotein hormone that regulates calcium/phosphate home-
ostasis and is upregulated by interleukin (IL) 6 in responseto hypoxia (20). Strong and specific overexpression ofIL6was observed (P < 0.001; Fig. 1C and Supplementary
Fig. S1). IL6 is known to regulate PTHLH expression(21) and to be strongly proangiogenic in ovarian cancer(22). IL6 expression was tightly correlated with both
A
serous/endometrioidmolecular subtypes
B
Clea
r
Cell
C Serous Clear Cell
Stromal
IL6EPAS1 (Hif2a)
LYN
Xenobiotic metabolismUDP-GlucosyltransferasesReceptors
HNF1BTransmembrane transporters
MET
Immune
Mesenchymal
Proliferation
IL6HIF2AMETSTC1PTHLHCYP2C9
HNF1BPRLR
Legend
HOSELMP/LMP-like (C3)
Low GradeEndometrioid (C6)Clear Cell (OCCA)
HGSC Subtypes:High stroma (C1)High Lymphocyte (C2)Low Stroma (C4)Mesenchymal (C5)
Figure 1.Gene expression of OCCA. A, dendrogram of agglomerative unsupervised clustering including OCCA samples, HOSE, serous and endometrioid
ovarian carcinomas ("Tothill molecularsubtypes" of HGSC; ref.12), low-grade/borderline serous carcinomas, andlow-grade endometrioidcarcinomas. OCCA
samples are distinctly separatedfrom theother groups ofovarian tumors.B, an expression heatmap ofHOSE, molecularsubtypes of serous andendometrioid
carcinomas, and OCCA. Samples are ordered by subtype class, showing discrete clusters of coregulated genes. A coregulated gene cluster highly specific to
OCCA samples including specific biomarkers of OCCA such as HNF1b and a number of its transcriptional targets is clearly visible. Molecular subtypes
ofserous andendometrioidcarcinomas definedby Tothilland colleagues (NCBI GEO accessionGSE12172) are labeled and usedfor comparison in (A) and(B),
asper colorlegend, asare HOSE samples from Bonome andcolleagues(datadonated byDr. Michael Birrer; ref. 15). C, zoomed imageof selected genes from
the larger heatmap whose expression is tightly correlated with the OCCA subtype. Serous and clear cell histologic types are shown.
IL6-STAT3-HIF Signaling in Ovarian Clear Cell Cancer
www.aacrjournals.org Clin Cancer Res; 17(8) April 15, 2011 2541
American Association for Cancer ResearchCopyright 2011on December 3, 2011clincancerres.aacrjournals.orgDownloaded from
Published OnlineFirst February 22, 2011; DOI:10.1158/1078-0432.CCR-10-3314
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PTHLH (PTHRP; P < 0.0001) and EPAS1 (HIF2A; P