Co-expression of HER2 and HER3 receptor tyrosine kinases enhances invasion of breast cells via

12 Pages·2012·0.63 MB·English

by  Nicola Aceto

UCSF
UC San Francisco Previously Published Works
Title
Co-expression of HER2 and HER3 receptor tyrosine kinases enhances invasion of breast
cells via stimulation of interleukin-8 autocrine secretion
Permalink
https://escholarship.org/uc/item/6106p69m
Journal
Breast Cancer Research, 14(5)
ISSN
1465-5411
Authors
Aceto, Nicola
Duss, Stephan
MacDonald, Gwen
et al.
Publication Date
2012-10-12
DOI
http://dx.doi.org/10.1186/bcr3329

Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California
Acetoetal.BreastCancerResearch2012,14:R131
http://breast-cancer-research.com/content/14/5/R131
RESEARCH ARTICLE Open Access
Co-expression of HER2 and HER3 receptor tyrosine
kinases enhances invasion of breast cells via
stimulation of interleukin-8 autocrine secretion
Nicola Aceto1, Stephan Duss1, Gwen MacDonald1, Dominique S Meyer1,2, Tim-C Roloff1, Nancy E Hynes1 and
Mohamed Bentires-Alj1*
Abstract
Introduction: The tyrosine kinase receptors HER2 and HER3 play an important role in breast cancer. The HER2/
HER3 heterodimer is a critical oncogenic unit associated with reduced relapse-free and decreased overall survival.
While signaling cascades downstream of HER2 and HER3 have been studied extensively at the level of post-
translational modification, little is known about the effects of HER2/HER3 overexpression and activation on gene
expression in breast cancer. We have now defined the genetic landscape induced by activation of the HER2/HER3
unit in mammary cells, and have identified interleukin (IL)8 and CXCR1 as potential therapeutic targets for the
treatment of HER2/HER3-overexpressing breast cancers.
Methods: Three-dimensional (3D) cultures, invasion and migration assays were used to determine the effects of
HER2 and HER3 co-expression and activation. Gene expression analysis was performed to identify the gene
network induced by HER2/HER3 in 3D cultures. Bioinformatic analysis and neutralizing antibodies were used to
identify key mediators of HER2/HER3-evoked invasion.
Results: Co-expression of the tyrosine kinase receptors HER2 and HER3 induced migration and invasion of MCF10A
cells. Microarray analysis of these cells revealed a specific “HER2/HER3 signature” comprising 80 upregulated
transcripts, with IL8 being the highest (11-fold upregulation). Notably, examination of public datasets revealed high
levels of IL8 transcripts in HER2-enriched as well as basal-like primary breast tumors, two subtypes characterized by
a particularly poor prognosis. Moreover, IL8 expression correlated with high tumor grade and ER-negative status.
Importantly, treatment with IL8-neutralizing antibodies prevented invasion of MCF10A-HER2/HER3 and BT474 cells
in 3D cultures, highlighting the importance of IL8 autocrine signaling upon HER2/HER3 activation.
Conclusions: Our findings demonstrate that HER2 and HER3 co-expression induces IL8 autocrine signaling, leading
to the invasion of mammary cells. Agents targeting IL8 or its receptor CXCR1 may be useful for the treatment of
HER2/HER3/IL8-positive breast cancers with invasive traits.
Introduction The importance of HER2 in cancer is highlighted by the
HER2 (ErbB2) and HER3 (ErbB3) are members of a clinical efficacy of the anti-HER2 humanized monoclo-
family of four receptor tyrosine kinases that also nal antibody trastuzumab (Herceptin), especially when
includes the epidermal growth factor receptor (HER1/ combined with chemotherapy, for the treatment of
EGFR) and HER4 (ErbB4) [1]. HER2 overexpression HER2-overexpressing breast cancers [3,4]. HER3 has
accounts for approximately 20% of all breast cancers been strongly implicated as a dimerization partner of
and is commonly associated with a poor prognosis [2]. HER2 in promoting malignant transformation [5],
mainly via activation of the PI3K pathway [6,7]. The
HER2/HER3 heterodimer, stabilized by heregulin
*Correspondence:[email protected] (HRG), has been identified as a strong oncogenic unit in
1MechanismsofCancerDepartment,FriedrichMiescherInstitutefor
BiomedicalResearch,Maulbeerstrasse66,Basel,CH-4058Switzerland
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Acetoetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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breast cancer [5,8] and is associated with reduced inserts at a concentration of 10,000 cells in 200 μl
relapse-free and decreased overall survival [9]. DMEM/F12 containing 0.1% BSA. The lower chamber
In HER2-dependent breast cancer cells, loss of HER3 was filled with 750 μl DMEM/F12 containing 0.1% BSA
reduces cell proliferation and decreases PI3K activity and 100 ng/ml heregulin. The cells were incubated for
[10,11]. Moreover, inhibition of HER2 phosphorylation 24 hat37°C in5% CO2, at which point, cellsremaining
by tyrosine kinase inhibitors (TKIs) targeting EGFR and intheupperchamberwereremovedusingacottonswab.
HER2 triggers feedback overexpression and activation of Cells on the lower surface of the membrane were fixed
HER3 [12], thereby limiting the inhibitory effect of with 4% paraformaldehyde (PFA), stained with crystal
HER-targeting TKIs. These studies highlight a central violet and imaged. The same protocol was followed for
role for HER3 in HER2-mediated breast tumorigenesis the invasion assays, using BD BioCoat growth factor
and response to therapy. reduced Matrigel invasion chambers (BD Biosciences,
The signaling pathways activated by HER2 and HER3 San Jose, CA, USA), however the cells were not starved
have been studied extensively [13]. For example, while andwereseededintheupperchamberat25,000perwell.
HER2 generally activates the Ras-MAPK, PLCg-PKC,
SHP2, STATs, PI3K/AKT and Src-dependent pathways Reagents
[7], HER3 contains six binding sites for the p85 subunit Heregulin (Sigma, St. Louis, MO, USA) 10 ng/ml and
ofPI3K,whichallowpotent activationofthe PI3K/AKT growth factor reduced Matrigel basement membrane
pathway [7]. In contrast, little is known about the effect matrix (BD Biosciences, San Jose, CA, USA) were used
oftheHER2/HER3oncogenicunitongeneexpressionin for three-dimensional cultures, and TRIzol reagent (Invi-
breastcancer. trogen, Carlsbad, CA, USA) for RNA extraction. The
Inthisstudy,usingthree-dimensionalcultures,migra- neutralizing antibodies anti-CXCR1, anti-CXCR2 and
tionand invasionassays, genetictoolsandbioinformatic anti-IL8 (R&D Systems, Minneapolis, MN, USA) were
analysis, we have defined the genetic landscape induced used according to the manufacturer’s instructions. Anti-
bysimultaneousexpressionofHER2andHER3inmam- bodies anti-Ki67 used for assessment of proliferation of
mary cells. These results reveal IL8 and its receptor MCF10A cells grown in two- and three-dimensional cul-
CXCR1aspotentialtherapeutictargetsforthetreatment tures were purchased from Thermo Scientific, Waltham,
ofHER2/HER3-overexpressingbreastcancers. MA, USA. Human recombinant IL8 was purchased
from Invitrogen, Carlsbad, CA, USA.
Materials and methods
Monolayer and three-dimensional cultures Retroviral infection
MCF10A cells were obtained from J Brugge (Harvard MCF10A cells were infected overnight with retroviral
Medical School, Boston, MA, USA) and propagated in particles containing pWZL-Hygro-hHER2 or pBabe-
DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) Neo-hHER3 at multiplicity of infection (MOI) = 10 in
supplemented with 5% horse serum (Hyclone, Logan, the presence of 8 μg/ml polybrene (Sigma, St. Louis,
UT, USA), 20 ng/ml EGF (Peprotech, Rocky Hill, NJ, MO, USA), and selected with 250 μg/ml G418 (Gibco,
USA), 0.5 μg/ml hydrocortisone (Sigma, St. Louis, MO, Carlsbad, CA, USA) and/or 100 μg/ml hygromycin
USA), 100 ng/ml cholera toxin (Sigma, St. Louis, MO, (Invivogen, San Diego, CA, USA).
USA),10μg/mlinsulin(Sigma,St.Louis,MO,USA),100
IU/ml penicillin and 100 μg/ml streptomycin. MCF10A Microarray analysis
cells were grown in three-dimensional cultures as Total RNA was extracted from tumors with TRIzol
described previously [14]. BT474 cells were obtained reagent (Invitrogen, Carlsbad, CA, USA). Total RNA
from the American Type Culture Collection (ATCC, (300 ng) was processed with GeneChip WT cDNA
Manassas,VA,USA)andgrowninDMEMmediumsup- Synthesis Kit and the GeneChip WT Terminal Labeling
plementedwith10%fetalbovineserum(Sigma,St.Louis, Kit (Affymetrix, Santa Clara, CA, USA), hybridized for
MO, USA). For three-dimensional cultures, BT474 cells 16 h to GeneChip Human Gene 1.0 ST arrays (Affyme-
were grown in DMEM medium containing 1% fetal trix, Santa Clara, CA, USA) and washed and scanned on
bovine serum and 10 ng/ml heregulin (Sigma, St. Louis, a GeneChip Scanner 3000 with autoloader according to
MO,USA). the manufacturer’s instructions. Expression values were
normalized and probeset-level values calculated with
Migration and invasion assays robust multi-array average (RMA) as implemented in
MigrationassayswereperformedusingBDChambersas the R/Bioconductor package, affy (R version 2.8). Con-
per the manufacturer’s instructions. Briefly, cells were trasts between MCF10A-HER2/HER3 and the other
starved overnight in DMEM/F12 containing 0.1% BSA groups were statistically analyzed in Genedata Expres-
andthenseededintheupperchamberofcollagen-coated sionist (version 5.1). Venn diagrams were created for
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probesets with an absolute linear fold change > 1.5 and HER2/HER3 heterodimer [5,6]. When grown in three-
a P-value < 0.05 to find probesets commonly up- or dimensional cultures, MCF10A cells expressing the
downregulated in all contrasts. CEL files have been emptyvectorformedhollowsphereswithpolarizedcells,
deposited in the GEO repository (GSE37009). Lists of mimicking the morphology of normal mammary acinar
differential genes including fold-changes and P-values structures (Figure S1 in Additional file 1). Interestingly,
were uploaded to Ingenuity Pathway analysis to study HER2expressioninducedtheformationoflargerspheres
biological network enrichment. thatwerepolarizedandcharacterizedbyahollowlumen.
Of note, HER3 expression alone had no major effect on
Analysis of IL8 expression in publicly available breast MCF10Acellsinthree-dimensionalcultures,withonlya
cancer datasets minorproportionofstructuresbeingmulti-acinarwitha
Weperformedgenesetanalysis(GSA)ofIL8onprimary filled lumen. In contrast, consistent with our previous
breasttumorswiththeGeneExpression-basedOutcome observations [18], simultaneous expression of HER2/
of Breast Cancer Online (GOBO) platform as described HER3 in MCF10A cells in the presence of heregulin
previously [15]. Examination of IL8 expression and its inducedtheformationofhighlyinvasiveandunpolarized
associationwithclinical parameterswasconductedona structures containing a filled lumen, while HER2/HER3
merged dataset comprising 1,881 primary breast tumor overexpression in the absence of heregulin did not
samples obtained from multiple experiments and classi- (Figure 1A; Figure S1 in Additional file 1; data not
fied into breast cancer subtypes by two different classi- shown).Thesedataindicatethatheregulinisrequiredfor
fiers[16,17]. HER2/HER3-induced invasion, loss of polarization and
luminal filling in three-dimensional cultures of human
Real-time PCR mammaryepithelialcells.
TotalRNAwasextractedwithTRIzolreagentandusedas In addition, we performed a migration assay using the
atemplateforproductionofcDNA.ThecDNAwasused Boyden chamber as well as an invasion assay using
inSYBR-basedquantitativereal-timePCRforquantifica- Matrigel-coated chambers. In both cases, co-expression
tion of IL8, E-cadherin, N-cadherin, vimentin and fibro- of HER2 and HER3 increased migration and invasion
nectin1 transcript levels. IL8 forward primer: 5’- towards a heregulin-containing medium (Figure 1B and
AAGCTGGCCGTGGCTCTCTT-3’ and reverse primer: Figure S2 in Additional file 1). Of note, HER2 expres-
5’-TGGTGGCGCAGTGTGGTCCA-3’. E-cadherin for- sion alone increased migration of MCF10A cells but had
ward primer:5’-TGCCCAGAAAATGAAAAAGG-3’and no effect on invasion (Figure 1B).
reverseprimer:5’-GTGTATGTGGCAATGCGTTC-3’.N- Moreover,we grewMCF10Acellsexpressingacontrol
cadherin forward primer: 5’-ACAGTGGCCACCTA- vector, HER2, HER3 or HER2/HER3 in two- or three-
CAAAGG-3’andreverseprimer:5’-CCGAGATGGGGTT dimensionalculturesfor15daysandassessedproliferation
GATAATG-3’.Vimentinforwardprimer: 5’-GAGAACT bystainingwithanti-Ki67antibodies.Whilewefoundno
TTGCCGTTGAAGC-3’andreverseprimer:5’-GCTTCC differenceinproliferationamongcelllineswhengrownin
TGTAGGTGGCAATC-3’. Fibronectin1forwardprimer: two-dimensionalcultures,weobservedanincreaseinthe
5’-CAGTGGGAGACCTCGAGAAG-3’ and reverse pri- percentageofKi67-positivecellsinMCF10A-HER2/HER3
mer: 5’-TCCCTCGGAACATCAGAAAC-3’. Glyceralde- grown in three-dimensional culture (Figure 1C and 1D).
hyde-3-phosphate dehydrogenase (GAPDH) levels were Moreover,weextractedRNAfromMCF10Acellsexpres-
usedtonormalizethedata.TheGAPDHforwardprimer sing an empty vector, HER2, HER3 or HER2/HER3 that
was 5’-ACCCAGAAGACTGTGGATGG-3’ and the weregrowninthree-dimensionalculturesfor15daysand
GAPDH reverse primer 5’-TCTAGACGGCAGGT- assessed expression levels of epithelial-to-mesenchymal
CAGGTC-3’. transition(EMT)markers. While we found no change in
expression ofE-cadherinand vimentin, we observed sig-
Results nificantupregulationofthemesenchymalmarkersN-cad-
Co-expression of HER2 and HER3 induces mammary cell herin and fibronectin1 (Figure 1E). Altogether, these
invasion, migration and proliferation in the presence of results indicate that co-expression of HER2 and HER3
heregulin inducesmigration,invasionandproliferationofmammary
Weexpressedthereceptortyrosinekinases(RTKs)HER2 cellsinthepresenceofheregulin.
and HER3 into the immortalized but non-transformed
mammary epithelial cells MCF10A. We then seeded Gene expression analysis of MCF10A-HER2/HER3 cells
MCF10Acellsexpressingemptyvector,HER2,HER3,or grown in three-dimensional cultures reveals the presence
HER2 plus HER3 (HER2/HER3) in three-dimensional of a HER2/HER3-driven gene signature
culturesfor15daysinthepresenceorabsenceofheregu- The gene expression profile induced by HER2/HER3 co-
lin,aknownHER3-ligandthatstabilizesandactivatesthe expression in breast epithelial cells has not been
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A 3D cultures B
es 100 - heregulin * 6 Migration **
ur + heregulin Invasion
uct 80 nt 5
asive str 60 zed cou 34 * **
ge of inv 2400 Normali 12
a
nt
e
c
Per Empty vector HER2 HER3 HER2/HER3 Empty vector HER2 HER3 HER2/HER3
C
Empty vector HER2 HER3 HER2/HER3
Nuclei
Ki-67
s
e
ur
ult
c
D
2
200(cid:80)m
s
e
ur
ult
c
D
3
50(cid:80)m
D 7-positive cells 1680000 2D cultures E 700 E cadherin *
Ki6 40 n 600 Vimentin
Percentage of 20 Evemcptotyr HER2 HER3 HER2/HER3 ntage of expressio 345000000 NFi bcraodnheecrtiinn 1 *
67-positive cells 22330505 3D cultures * Perce 120000 Empty vector HER2 HER3 HER2/HER3
of Ki 15
e 10
g
nta 5
Perce Empty HER2 HER3 HER2/HER3
vector
Figure1HER2andHER3co-expressioninducesmammarycellinvasion,migrationandproliferation.(A)Percentagesofinvasivestructures
inthepresenceorabsenceofheregulin10ng/ml.Resultsrepresentmeans±standarderrorofthemean(SEM)(n=3,*P<0.05).(B)Normalized
cellcountforthemigrationandinvasionassays.Resultsrepresentmeans±SEM(n=3,*P<0.003,**P<0.002).(C)Representativeconfocalimages
ofequatorialcross-sectionsofMCF10Acellsexpressingemptyvector,HER2,HER3orHER2/HER3grownintwo-orthree-dimensionalculturesfor15
daysinthepresenceof10ng/mlheregulinandstainedwithTO-PRO(blue)andanti-Ki67antibodies(red).(D)PercentageofKi67-positivecells
whengrownintwo-orthree-dimensionalcultures.Resultsrepresentmeans±SEM(n=3,*P<0.03).(E)Quantitativereal-timePCRonRNAextracts
ofMCF10Acellsexpressingemptyvector,HER2,HER3orHER2/HER3grownintwo-orthree-dimensionalculturesfor15daysinthepresenceof10
ng/mlheregulin.Quantitativereal-timePCRwasperformedusingprimersagainsttheepithelial-to-mesenchymaltransition(EMT)markersE-
cadherin,N-cadherin,vimentinandfibronectin1.Resultsrepresentmeans±SEM(n=3,*P<0.05).
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examined previously. To this end, we extracted RNA In addition, to test whether IL8 signaling contributes
from MCF10A cells expressing an empty vector, HER2, to HER2/HER3-evoked invasion, we grew MCF10A-
HER3, or HER2/HER3 grown in three-dimensional cul- HER2/3 and BT474 cells in three-dimensional cultures
tures for 15 days in the presence of heregulin and per- in the presence of an IL8-neutralizing antibody. Remark-
formed microarray analysis on the RNA obtained from ably, blockade of IL8 autocrine signaling reduced the
the different groups to obtain gene expression contrasts invasive behavior of these cells (Figure 2C, D). We also
of MCF10A-HER2/HER3 versus the other conditions. tested whether inhibition of the IL8 receptors CXCR1
We identified a set of 157 Affymetrix probe set IDs dif- and CXCR2 affected invasion. While there was no dif-
ferentially regulated in MCF10A-HER2/HER3 cells (fold ference in invasion upon CXCR2 blockade, the
change > 1.5-fold, P < 0.05) compared with all the other MCF10A-HER2/3 and BT474 cells-invasive/aberrant
groups (Figure S3 in Additional file 1). Characterization phenotype was significantly reduced upon neutralization
of these IDs led to the identification of 137 differentially of CXCR1 (Figure 2C, D). These results indicate that
regulated genes, of which 80 were upregulated specifi- IL8 autocrine signaling acts predominantly via CXCR1.
cally in MCF10A-HER2/HER3 cells, and referred to as
the HER2/HER3 signature (Figure S4 and Table S1 in IL8 is highly expressed in HER2-enriched and basal-like
Additional file 1). Gene ontology analysis of the HER2/ primary breast cancers
®
HER3 signature with the Ingenuity Systems resource To assess whether IL8 is overexpressed in human
revealed enrichment in pathways involved in cellular tumors, we analyzed IL8 expression levels in a public
movement, cell-to-cell signaling and interaction, cell dataset containing gene expression data from, 1,881 pri-
death, cellular development and cellular growth and mary breast tumors [15], classified by two independent
proliferation (Figure S5 in Additional file 1). These classification methods [16,17]. Unequivocally, we found
results indicate that the co-expression and activation of high expression of IL8 in HER2-enriched and basal
HER2/HER3 induces a specific gene signature associated breast cancers (Figure 3A, B), two subtypes associated
with invasiveness. Among these genes, IL8 was upregu- with poor prognosis.
lated approximately 11-fold and was the most upregu- Next, we examined whether IL8 expression correlates
lated gene in MCF10A-HER2/HER3 cells relative to all with clinicopathological parameters such as estrogen
other conditions (Table S1 in Additional file 1). Further- receptor (ER) status and tumor grade in breast cancer
more, IL8 was found to be involved in all pathways patients.Tothisend,primarybreasttumorsweredivided
identified by the Ingenuity resource (Figure S5 in Addi- intoER-negativeandER-positivegroupsandIL8expres-
tional file 1; data not shown). Thus, IL8 may play an sion was analyzed in each group. IL8 was significantly
important role downstream of the HER2/HER3 hetero- upregulatedinER-negativetumors(Figure 4A).Inaddi-
dimer in breast cells. tion, primary breast tumors were divided into three
groups according to tumor grade 1, 2, or 3, and IL8
IL8 contributes to HER2/HER3-induced invasion in three- expression was analyzed in each group. In line with our
dimensional cultures findingscorrelatingIL8withtheaggressivenessofHER2/
Our results suggestthatHER2/HER3-inducedIL8auto- HER3cells,weobservedsignificantoverexpressionofIL8
crinesecretioncontributestotheinvasionoftransformed inhigh-gradeprimarytumors(Figure4B).
breastcells.Givenourfindingthatco-activationofHER2 In summary, our data show that activation of the
and HER3 induces IL8 production, we compared IL8 HER2/HER3 oncogenic unit upregulates several genes
expression levels in BT474 cells, a HER2- and HER3- including IL8, which in turn acts via CXCR1 to induce
positivebreastcancercell line,toMCF10A cellsexpres- invasion ofmammary epithelialcells(Figure 5).Wealso
sing an empty vector, HER2, HER3 or HER2/3. The IL8 show that high IL8 expression correlates with ER-nega-
transcriptwashighlyexpressedinMCF10A-HER2/HER3 tive status and high tumor grade. These results provide
aswellasinBT474cells(Figure2A). new insights into the changes in gene expression upon
We then asked whether IL8 stimulation perse induces activation of the HER2/HER3 oncogenic unit and point
invasion of MCF10A cells grown in three-dimensional to IL8 and its receptor CXCR1 as possible therapeutic
cultures. We seeded MCF10A cells inthree-dimensional targets for the treatment of invasive HER2/HER3/IL8-
cultures and found that stimulation with increasing positivebreastcancers.
concentrations of IL8 induced invasion (Figure 2B and
Figure S6 in Additional file 1), although not to the same Discussion
extentastheco-expressionofHER2andHER3inthepre- The signaling cascades activated by the tyrosine kinase
senceofheregulin(Figure1A).Theseresultssuggestthat receptors HER2 and HER3 have been studied extensively
additional pathways downstream of HER2/HER3 are in the neoplastic breast [7]. In contrast, little is known
neededtoachievetheirhigherinvasionphenotype. about the effects of HER2/HER3 co-activation on gene
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A B
s
e 9 * ure 100
n fold chang 5678 * vasive struct 6800 * *
o n
L8 expressi 1234 NS NS entage of i 2400 *
I erc
CF10ecAt-or A-HER2 A-HER3 2/HER3 BT474 P - 10 20 50 IL8 (ng/ml)
mMpty v MCF10 MCF10 A-HER
E 10
F
C
M D
C
CTRL IL8 NAb CTRL IL8 NAb
50(cid:80)m 50(cid:80)m
CXCR1 NAb CXCR2 NAb CXCR1 NAb CXCR2 NAb
es 100 NS es 100
ur ur
Percentage of invasive struct 24680000 CTRL 8 NA*b 1 NA*b 2 NAb Percentage of aberrant struct 24680000 CTRL 8 NA*b 1 NA*b 2 NNAbS
L R R L R R
I C C I C C
X X X X
C C C C
Figure2IL8orCXCR1blockadereducesHER2/HER3-evokedinvasionofmammarycells.(A)Quantitativereal-timePCRofIL8.Thebar
graphshowsthepercentagesofIL8expressioninMCF10Acellsexpressingemptyvector,HER2,HER3orHER2/HER3,aswellasintheHER2/
HER3-positivebreastcancercelllineBT474.Resultsrepresentmeans±standarderrorofthemean(SEM)(n=3,*P<0.018;NS,notsignificant).
(B)PercentageofinvasivestructuresuponstimulationofMCF10AcellswithIL8.Resultsrepresentmeans±SEM(n=3,*P<0.05).(C)
Representativephase-contrastpicturesofMCF10A-HER2/HER3cellsgrowninthree-dimensionalculturesfor15daysinthepresenceofheregulin
andtheindicatedneutralizingantibodies.Thebargraphshowsthepercentagesofinvasivestructures.Resultsrepresentmeans±SEM(n=3,*P
<0.0004;NS,notsignificant).(D)Representativephase-contrastpicturesofBT474cellsgrowninthree-dimensionalculturesfor15daysinthe
presenceofheregulinandneutralizingantibodies.Thebargraphshowsthepercentagesofaberrantstructures.Resultsrepresentmeans±SEM
(n=3;*P<0.05;NS,notsignificant).CTRL,control.
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A Hu et al. subtype classification
n =357 152 482 289 257 344
6
8
L
f I 4
o
n
o 2
si
s
e
pr 0
x
E
2 −2
g
o
L
−4
Basal HER2 LumA LumB mal-like assified
r cl
o n
N U
B
Parker et al. subtype classification
n = 304 240 465 471 304 97
6
8
L
f I 4
o
n
o 2
si
s
e
pr 0
x
E
2 −2
g
o
L
−4
Basal nriched LumA LumB mal-like assified
e r cl
2 o n
R N U
E
H
Figure3IL8expressioncorrelateswithHER2-enrichedandbasalbreastcancers.Genesetanalysis(GSA)ofIL8expressioninadataset
comprising1,881primarybreasttumorshttp://co.bmc.lu.se/gobo.(A)BoxplotofIL8expressioninbreasttumorsamplesclassifiedintosix
molecularsubtypesaccordingtotheclassifiersofHuetal.[16];P<0.0001byanalysisofvariance(ANOVA).(B)BoxplotofIL8expressionin
breasttumorsamplesclassifiedintosixmolecularsubtypesaccordingtotheclassifiersofParkeretal.[17];P<0.0001byANOVA.Numbers
abovethechartsreportthenumberofpatientsineachsubtypegroup.
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A
395 1225
6
8
L
f I 4
o
n
o 2
si
s
e
pr 0
x
E
2 −2
g
o
L
−4
ER negative ER positive
B
239 677 495
8
L 4
f I
o
n 2
o
si
s
e 0
r
p
x
E
2 −2
g
o
L
−4
Tumor grade: 1 2 3
Figure4IL8expressioncorrelateswithER-negativeandhigh-gradeprimarybreasttumors.Genesetanalysis(GSA)ofIL8inadataset
comprising1,881primarybreasttumorshttp://co.bmc.lu.se/gobo.(A).IL8expressioncorrelateswithestrogenreceptor(ER)-negativetumors.Box
plotofIL8expressioninER-negativeandER-positiveprimarybreastcancers;P<0.0001byanalysisofvariance(ANOVA).B.IL8expression
correlateswithhightumorgrade.BoxplotofIL8expressioningrade1,2and3primarybreastcancers,respectively;P<0.0001byANOVA.
Numbersabovethechartsshowthenumberofpatientsineachsubtypegroup.
expression. We have used three-dimensional cultures of invasion downstream of HER2/HER3, we showed pre-
mammary cells and bioinformatic analysis to specifically viously that knockdown of another of these upregulated
investigate this aspect. We found that activation of the genes, the zinc finger E-box binding homeobox 1
HER2/HER3 oncogenic unit causes upregulation of sev- (ZEB1), also blocks invasion in MCF10A-HER2/HER3
eral genes including the chemokine IL8, which was dis- cells [18]. These results highlight the fact that several
covered originally as a chemotactic factor for leukocytes genes of the HER2/HER3 signature may contribute to
[19] and shown later to contribute to human cancer the invasive phenotype induced by co-activation of
progression through its potential mitogenic, motogenic HER2/HER3 receptors. Our study reveals novel potential
and angiogenic functions [20]. targets for the treatment of HER2- and HER3-positive
Following HER2/HER3 co-activation in mammary human breast tumors.
cells, we identified 80 upregulated genes. While in this We show here that IL8 upregulation is a consequence
study we found IL8 to be an important factor mediating of HER2/HER3 co-activation in human mammary cells.
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IL-8
Heregulin
HER2/3 CXCR1
heterodimer
IL-8
IL-8 gene
Invasion
Figure 5 Model of the HER2/HER3/IL8 signaling cascade. Heregulin-induced heterodimerization and activation of HER2/HER3 causes
upregulationoftheIL8transcript,whichinturnmediatesinvasionviaCXCR1.
Elevated IL8 levels have previously been described in expressionhave remainedobscure. For example, stimuli
breast tumor specimens relative to normal breast tissue suchaslipopolysaccharide,phorbol12-myristate13-acet-
[21]. We analyzed public datasets and found high IL8 ate, IL1 and TNF can induce IL8 production [20], and
expression specifically in HER2-positive and basal-like stress factors such as hypoxia, acidosis, nitric oxide and
tumors. It is conceivable that HER2/HER3-independent cell density have also been shown to influence IL8
mechanisms may be responsible for high IL8 expression expressioninparticularcelltypes[20].Here,wedemon-
in some triple negative breast cancers (TNBCs) [20]. strate a direct relationship between HER2/HER3activa-
Interestingly, otherstudies have demonstratedthat the tionandIL8expressioninbreastcancer.Itisconceivable
IL8/CXCR1 signaling is important for breast cancer cell that the activity of the HER2/HER3 heterodimer also
invasion [22] and predominantly active in cells with the promotesIL8expressionincancersofdifferentorigins.
cancer stem cell (CSC) phenotype when compared to
non-CSCs[23,24].Moreover,blockadeofCXCR1signifi- Conclusions
cantly reduces the CSC population, leading to decreased Our study provides evidence for a link between HER2/
tumorigenicity and metastasis [23,24]. In line with these HER3 overexpression and IL8 production in mammary
findings, we have previously reported that co-expression cells. First, we show that co-activation of HER2 and
ofHER2andHER3inMCF10Acellsincreasesthepropor- HER3 causes upregulation of IL8, which in turn contri-
tion of cells with the CSC phenotype [18]. Altogether, butes to HER2/HER3-evoked cellular invasion. More-
theseresultshighlightanimportantroleforHER2/HER3/ over, we show that the IL8 transcript is highly expressed
IL8intheregulationofinvasionandtheCSCphenotype. in HER2-enriched and basal-like human primary breast
WeobservedacorrelationbetweenhighIL8expression cancers, and that high IL8 levels correlate with ER-nega-
levelsandnegativeERstatusaswellashightumorgrade. tive status and high tumor grade. Finally, treatment with
Inadditiontobreast cancer[21],IL8isoverexpressedin IL8- or CXCR1-neutralizing antibodies reduced invasive
othertypes of cancers,includingcolon[25],gastric[26], behavior of HER2/HER3-driven malignant breast cells,
melanoma [27,28], ovarian [29,30], pancreatic [31] and highlighting a role for IL8 downstream of activated
prostate cancer[32].Generally, IL8 expression hasbeen HER2/HER3. Our results identify IL8 and its receptor
shown to contribute to human cancer progression CXCR1 as potential targets for the treatment of breast
[20,33]; however, the mechanisms regulating IL8 cancers with active HER2/HER3/IL8 signaling.