Heparanase Accelerates Obesity-Associated Breast Cancer … · Tumor Biology and Immunology Heparanase Accelerates Obesity-Associated Breast Cancer Progression Esther Hermano1, Rachel

Tumor Biology and Immunology

Heparanase Accelerates Obesity-AssociatedBreast Cancer ProgressionEsther Hermano1, Rachel Goldberg1, Ariel M. Rubinstein1, Amir Sonnenblick2,Bella Maly3, Daniela Nahmias1, Jin-Ping Li4, Marinka A.H. Bakker5,Johan van der Vlag5, Israel Vlodavsky6, Tamar Peretz1,7, and Michael Elkin1,7

Abstract

Obesewomenhave higher risk of bearing breast tumors thatare highly aggressive and resistant to therapies. Tumor-promoting effects of obesity occur locally via adipose inflam-mation and related alterations to the extracellular matrix(ECM) as well as systemically via circulating metabolicmediators (e.g., free fatty acids, FFA) associated with excessadiposity and implicated in toll-like receptor-mediated acti-vation ofmacrophages—key cellular players in obesity-relatedcancer progression. Although the contribution of macro-phages to proneoplastic effects of obesity is well documented,the role of ECM components and their enzymatic degradationis less appreciated. We show that heparanase, the sole mam-malian endoglucuronidase that cleaves heparan sulfate inECM, is preferentially expressed in clinical/experimentalobesity-associated breast tumors. Heparanase deficiencyabolished obesity-accelerated tumor progression in vivo.Heparanase orchestrated a complex molecular program thatoccurred concurrently in adipose and tumor tissue and sus-tained the cancer-promoting action of obesity. Heparanase

was required for adipose tissue macrophages to produceinflammatory mediators responsible for local induction ofaromatase, a rate-limiting enzyme in estrogen biosynthesis.Estrogen upregulated heparanase in hormone-responsivebreast tumors. In subsequent stages, elevated levels ofheparanase induced acquisition of procancerous phenotypeby tumor-associated macrophages, resulting in activation oftumor-promoting signaling and acceleration of breast tumorgrowth under obese conditions. As techniques to screen forheparanase expression in tumors become available, thesefindings provide rational and amechanistic basis for designingantiheparanase approaches to uncouple obesity and breastcancer in a rapidly growing population of obese patients.

Significance: This study reveals the role of heparanase inpromoting obesity-associated breast cancer and provides amechanistically informed approach to uncouple obesity andbreast cancer in a rapidly growing population of obesepatients.

IntroductionObesity [defined as a bodymass index (BMI) above or equal to

30 kg/m2] is a widespread public health problem that has beenconsistently associatedwith several pathologies, including at least13 different types of cancer (1). In particular, numerous studies

have demonstrated that obese women have a significantly higherrisk of bearing breast tumors, largely estrogen receptor (ER)-positive (2, 3), which are resistant to therapies, more likely torecur, and associatedwith higher death rates (3–5). As the recentlyestimated global prevalence of overweight/obesity in women isapproximately 40% and postmenopausal breast carcinoma hasthe highest incidence among the three obesity-related cancers infemales (i.e., breast, endometrial, and ovarian; ref. 1), elucidationof the precise molecular mechanisms underlying breast tumor—promoting action of obesity is of high importance.

Obesity is widely recognized as a low-grade chronic inflam-matory state, and inflammation is one of the most likely con-tributors to the obesity–cancer link (4–7). Macrophages are keyimmunocytes mediating the tumor promotion action ofobesity (4–7). In the setting of obesity-associated ER-positivebreast carcinoma, the procancerous action is exerted both bytumor-associated macrophages (TAM; ref. 8) and by adiposetissue macrophages (atM; refs. 6, 9–12). Indeed, macrophagemobilization/activation was observed in visceral and mammaryadipose in animal models of obesity, as well as in obesepatients, correlating with both BMI and adipocyte hypertro-phy (4, 5, 9, 11, 13). The atM infiltrating "obese" adipose tissueforms histologically visible crown-like structures (CLS), com-posed of macrophages surrounding dysfunctional adipocytes, ahallmark of white adipose tissue inflammation (3, 9, 10). More-over, unlike in "lean" adipose tissue, atM in "obese" adipose tissue

1Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,Jerusalem, Israel. 2Oncology Division, Tel Aviv Sourasky Medical Center andSackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 3Department ofPathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.4Department of Medical Biochemistry and Microbiology, Uppsala University,Uppsala, Sweden. 5Nephrology Research Laboratory, Department of Nephrol-ogy, Nijmegen Centre for Molecular Life Sciences, Radboud University NijmegenMedical Centre, Nijmegen, the Netherlands. 6Cancer and Vascular BiologyResearch Center, The Rappaport Faculty of Medicine, Technion, Haifa, Israel.7Hebrew University Medical School, Jerusalem, Israel.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

E. Hermano and R. Goldberg contributed equally to this article.

Corresponding Author: Michael Elkin, Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel. Phone: 972-2677-6598; Fax: 972-2642-2794; E-mail: [email protected]

Cancer Res 2019;79:5342–54

doi: 10.1158/0008-5472.CAN-18-4058

�2019 American Association for Cancer Research.

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acquire proinflammatory phenotype, among other factors—dueto activation by fatty acids, derived from the lipid-laden "obese"adipocytes (3, 9, 10, 12). As a result of this activation, reportedlymediated via toll-like receptor 4 (TLR4; refs. 5, 14–16), CLS-residing macrophages secrete inflammatory mediators (i.e.,TNFa; refs. 6, 9, 10), which induce expression of aromatase inadipose stromal cells (i.e.,fibroblasts; refs. 9, 17, 18). Aromatase isthe rate-limiting enzyme in estrogenbiosynthesis, which catalyzesthe conversion of androgens to estrogens. After the cessation ofovarian function, estrogens in postmenopausal women are syn-thesized exclusively in peripheral tissues, with adipose being amajor site of estrogen production. As obesity-associated breasttumors are mostly ER-positive and occur in postmenopausalpatients (2, 3, 11), induction of aromatase expression in "obese"adipose tissue and associated increase in estrogen production,owing to the interplay between "obese" adipocytes, atM, andfibroblasts, are important determinants in breast carcinoma–obesity link (3, 6, 9).

Along with the atM infiltrating excessive adipose tissue, TAMalso contribute to obesity-associated breast cancer. When polar-ized toward tumor-promoting phenotype, TAM supply key obe-sity-associated procancerous cytokines (i.e., IL6, CCL2), growthfactors (e.g., VEGF, EGF), and activating tumor-stimulatingsignaling pathways (e.g., NFkB, STAT3; refs. 4, 7, 19), thuscontributing to breast carcinoma progression (4, 5, 8). Compo-nents of the obese milieu, most notably free fatty acids (FFA),whose circulating levels are often increased in obesity, areamong the candidate agents responsible for adverse TAMactivation (5, 14–16).Nevertheless, given the functional plasticityof macrophages and the notorious ability of TAM to exhibit bothanti- and protumor activities (8), the mechanisms of TAM phe-notypic switch occurringunder obese state are notwell elucidated,and it is not clear whether circulating levels of FFA (20) alone aresufficient to drive protumorigenic TAM polarization. Even less isknown about obesity-associated alterations in the extracellularmatrix (ECM) of breast carcinoma and their role in couplingobese state and breast tumor progression.

Here we provide evidence that heparanase enzyme [the onlyknown mammalian endoglycosidase-degrading heparan sulfate(HS) chains in the ECM] mediates effects of excess adiposity onbreast cancer progression. Upregulation of heparanase is docu-mented in clinical and experimental breast carcinoma (21–24),correlating with larger tumor size, poor survival, and resistance totherapy (21, 25). The enzymatic substrate of heparanase, HS, isubiquitously present at the cell surface and ECM, playing essentialroles in ECM integrity and regulation of receptor–ligand interac-tions that involve a variety of bioactive molecules (26, 27). Inparticular, intact extracellular HS inhibits TLR4 responses andmacrophage activation, whereas its enzymatic removal relievesthis inhibition (28). Moreover, soluble HS fragments generatedby heparanase (29) stimulate TLR4 signaling in vitro (28, 30) andin vivo (31). Hence, heparanase shapes macrophage responses inseveral pathophysiologic conditions (30, 32–35).

Incorporating the aforementioned data with observations thatheparanase deficiency abolishes obesity-accelerated breast tumorprogression in vivo, and that the enzyme is preferentially expressedin both clinical and experimental obesity-associated breasttumors (see "Results" section below), we hypothesized thatheparanase mediates the tumor-promoting effect of obesity bydirecting procancerous action of macrophages at the interface ofbreast carcinoma with excess adiposity and inflammation.

Materials and MethodsClinical data analysis

Breast tumor tissue specimens and clinical data from 123female patients with breast carcinoma were available from theSharett Oncology Institute, Hadassah Medical Center (Jerusalem,Israel). A summary of clinical and pathologic characteristics of thestudy population is shown in Supplementary Table S1. The use ofthese data and formalin-fixed, paraffin-embedded breast carci-noma tissues in research was approved by the Human SubjectsResearch Ethics Committee of the Hadassah Medical Center(Jerusalem, Israel). Tissuemicroarray constructionwas performedas previously (25). Briefly, 5-mm sections stained with hematox-ylin and eosin were obtained to confirm the diagnosis and toidentify representative areas of the specimen. From these definedareas, three tissue cores with a diameter of 0.6 mm were takenfrom the different regions of the tumor and arrayed in triplicateson a recipient paraffin block as described previously (25).Sections of 5 mm of the recipient blocks were cut, deparaffinized,and rehydrated. Tissue was then incubated in 3% H2O2,denatured by boiling (3 minutes) in a microwave oven in citratebuffer (0.01mol/L, pH 6.0) and blocked with 10% goat serum inPBS. Sections were incubated with polyclonal rabbit anti-heparanase antibody (733) directed against a synthetic peptide(158KKFKNSTYRSSSVD171) corresponding to the N-terminus ofthe 50-kDa subunit of the heparanase enzyme (25, 33). Theantibody was diluted 1:100 in 10% goat serum in PBS. Controlslides were incubated with 10% goat serum alone. Color wasdeveloped as described in ref. 25, slides were visualized with aZeiss axioscope microscope, and manually read by an expertpathologist (B. Maly). To define tumor as heparanase-positive,a cut-off point of 25% immunostained tumor cells was chosen onthe basis of an initial overview of the cases, to improve signal-to-noise ratios. Cutoff was chosen before any attempt at correlatingheparanase expression with the obese status of the patients.Immunodetection of ERa was performed as described in ref. 25.

Cell linesHuman ER-positive breast carcinoma cell lines T47D and

MCF7 (authenticated by short tandem repeat profiling at theGenomics Center of the Biomedical Core Facility, TechnionUniversity, Haifa, Israel), mouse breast carcinoma E0771(C57BL/6J syngeneic ER-positive cell line), kindly provided byDr. R. Sharon, Hebrew University Medical School (Jerusalem,Israel), and mouse pancreatic carcinoma Panc02 (C57BL/6J syn-geneic cell line) provided by Dr. M. Dauer (University of Munich,Munich, Germany), were grown in RPMI1640 (T47D, E0771,Panc02) or DMEM (MCF7) supplemented with 1-mmol/L glu-tamine, 50 mg/mL streptomycin, 50 U/mL penicillin, and 10%FCS (Biological Industries) at 37�C and 8% CO2. All cell lineswere tested routinely forMycoplasma by the PCR assay (BiologicalIndustries). Prior to estrogen treatment, cells weremaintained for4 days in phenol red-free medium supplemented with charcoal-stripped FCS (Biological Industries). Then, medium was changedto serum-free medium and cells were treated with estrogen orvehicle alone (ethanol), as indicated in the Results section.

Mouse model of obesity-associated tumor growthTen-week-old, female wt C57BL/6J mice (Harlan Laboratories)

and heparanase-knock out (Hpse-KO) mice (36) on C57BL/6Jbackground (n� 7 per experimental group) were fed high-fat diet(HFD; Teklad TD.06414, 60% of total calories from fat), or

Role of Heparanase in Obesity–Breast Cancer Link

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control diet (CD; Teklad 2018S) for 15 consecutive weeks. By theend of experimental week 12, when both wt and Hpse-KO HFD-fed animals became obese, E0771 cells were injected orthotopi-cally into fourth left mammary fat pads of both HFD-fed (obese)and CD-fed (lean) wt and Hpse-KO mice (5 � 105 cells perinjection). Similarly, HFD-fed (obese) and CD-fed (lean) wt andHpse-KO mice were injected subcutaneously with Panc02 cells(5 � 105 cells per injection). The tumor volume was monitoreduntil experimental week 15. Animals were then sacrificed andtissue samples collected from tumors, collateral (right)mammarygland adipose and visceral fat, and snap-frozen for RNA/proteinextraction or processed for histology. All experiments were per-formed in accordance with the Hebrew University InstitutionalAnimal Care and Use Committee.

Statistical analysisThe results are presented as the mean � SD unless otherwise

stated. P values of� 0.05 were considered statistically significant.Statistical analysis of in vitro and in vivo data was performed byunpaired Student t test. Pearson x2 test was applied to analyze therelationship between heparanase expression and obese status ofpatients with breast carcinoma, using SPSS software (SPSS Inc.).All statistical tests were two-sided.

Study approvalFormalin-fixed, paraffin-embedded human breast carcinoma

tissues and clinical data were obtained in accordance with theethical guidelines of the Declaration of Helsinki. The studieswere approved by the Human Subjects Research Ethics Commit-tee of the Hadassah Medical Center (Jerusalem, Israel). TheCommittee determined that obtaining a written informed con-sent from each subject was unnecessary for this study.

Animal experiments were approved by Institutional AnimalCare Committee of the Hebrew University (Jerusalem, Israel).Additionalmethods are presented in the SupplementaryMethods.

ResultsHeparanase deficiency abolishes obesity-acceleratedorthotopic breast tumor progression

To investigate a role for heparanase in coupling obesity andbreast cancer progression, wild-type (wt) and heparanase-null(Hpse-KO) mice on C57BL6 background were utilized in themodel of HFD-induced obesity, as described in Materials andMethods. HFD-fed C57BL6 mice represent one of the mostreliable and best studied models of diet-induced obesity andrelated pathologic changes, including inflammation (37, 38),fatty acid accumulation (39), and obesity-accelerated tumorgrowth (19, 38). Animals of both genotypes (wt and Hpse-KO)became obese following 12 weeks of HFD, as evidenced by theirsignificantly increased bodyweight and adipocyte hypertrophy inintra-abdominal fat (characteristic feature of diet-inducedobesity, Fig. 1 A and B). On experimental week 12, E0771 cells(syngeneic ER-positive breast cancer cell line; ref. 19) wereinjected orthotopically into the mammary fat pad of bothHFD-fed (obese) and CD-fed (lean) wt and heparanase-KOmice.As expected (19, 38), HFD-induced obesity markedly acceleratedtumor progression in wt mice: 2.3-fold larger tumors wereobserved in wt obese versus wt lean mice on experimental week15, 3 weeks posttumor inoculation (Fig. 1C). Strikingly, hepar-anase deficiency in mouse host abolished tumor-accelerating

effect of obesity, as demonstrated by the lack of statisticallysignificant difference between the volume of tumors growing inlean versus obese Hpse-KO mice (Fig. 1D). Of note, heparanasedeficiency had no inhibitory effect on E0771 tumor growth innonobese conditions, as no statistically significant difference wasdetected between the volume of tumors growing in lean wt versuslean Hpse-KO mice.

Overexpression of heparanase in experimental and humanER-positive breast cancer under obese conditions

We next compared heparanase expression in E0771 breasttumors growing in obese versus lean mice, applying immuno-blotting and IHC. Heparanase expression was barely detected inE0771 tumors derived from the lean host, whereas a markedincrease in heparanase protein levels was detected in tumorsderived from obese wt host (Fig. 2A and B; SupplementaryFig. S1). In agreement with the increased expression of hepar-anase, a significant decrease in the content of HS (enzymaticsubstrate of heparanase) was detected in the tumors of obeseversus lean wt mice, whereas no change in HS content wasdetected in the tumors of obese versus lean heparanase-KO mice(Supplementary Fig. S2A and S2B).

Next, to validate the clinical relevance of these findings, weutilized tissue microarray comprising 123 tumor specimensderived from obese (BMI�30) and nonobese (BMI <30) patientswith breast carcinoma and examined the correlation betweenheparanase expression and obese status of the patients. Becausethe association between obesity and breast cancer is the highestamonghormone-dependent breast tumors (2, 3), ER-positive andER-negative cases were analyzed separately. x2 analysis was thenused to assess the relationship between obese status and upre-gulation of heparanase. In full agreement with the in vivo data(Fig. 2A and B), strong and significant correlation between obesestate of the patient andoverexpression of heparanasewas noted inER-positive breast carcinoma: almost 2-fold higher proportion ofheparanase-overexpressing tumors was detected in obese versusnonobese ER-positive patients with breast carcinoma (63.2% vs.36.8%, x2 test; P ¼ 0.045; Fig. 2C).

Because association between increased adiposity and breastcarcinoma risk/aggressive behavior was repeatedly demonstratedin overweight (BMI �25 kg/m2) patients (1–3), heparanaseexpression in breast carcinoma tumors derived from overweightversus normal weight women (BMI <25 kg/m2) was examined aswell. As shown in Supplementary Fig. S3A, significant correlationbetween overweight condition and heparanase overexpressionwas noted in ER-positive patients with breast carcinoma. Noassociation between heparanase expression and either overweight(Supplementary Fig. S3B) or obesity (Supplementary Fig. S3C)was detected in ER-negative tumors. Of note, the enzyme expres-sion was not detected in any of the 5 control specimens ofnonmalignant breast tissue, included in the array.

Regulation of heparanase expression in ER-positive breastcarcinoma under obese conditions

Both carcinoma cells per se and host-derived stromal cells canpotentially serve as a source of the enzyme in the microenviron-ment of breast carcinoma and other tumor types (40–43). Asshown in Fig. 2A, under nonobese conditions, E0771 carcinomacells express very low or no detectable levels of heparanase. Thisnotion, taken together with the fact that elevated levels of hepar-anase were detected in tumors growing in obese wtmice (but not

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in obese heparanase-KO mice; Fig. 2A, middle), may lead to theassumption that in this experimental setting heparanase ismainlycontributed by the host-derived stromal cells, rather than bycarcinoma cells. However, immunostaining of the mouse tumortissues with heparanase antibody clearly demonstrated thatE0771 carcinoma cells per se represent the main source of theenzyme in tumors growing in obesewtmice (Fig. 2B). In a similarmanner, in patient-derived ER-positive breast carcinoma speci-mens, overexpression of heparanase was noted in breast carcino-ma cells rather than in stromal elements (Fig. 2D).

Estrogen is a potent inducer of the heparanase enzymeexpression in the hormone-responsive cell types (23, 25, 44)including ER-positive breast carcinoma cells of both mouse(i.e., E0771, Fig. 3A) and human (i.e., MCF7, T47D; refs. 23, 25)origins. We therefore next queried whether enhanced estrogensignaling (a known consequence of augmented aromataseproduction by excess adiposity; refs. 3, 4, 9, 11, 13) is respon-sible for the induction of heparanase in E0771 breast carcino-ma cells growing in obese wt mice. Consistent with this modeof action, we detected approximately 6-fold increased expres-sion of progesterone receptor (estrogen-regulated gene com-monly used as an indicator of enhanced estrogen signaling) inE0771 breast tumors growing in obese wtmice but not in obeseHpse-KO mice (Fig. 3B). These data support the notion that

obesity-associated enhanced estrogen signaling occurs in wt butnot in Hpse-KO animals.

As stated above, obese state initiates series of cellular/molecularevents leading to increased production of estrogen in adiposetissue (3, 4, 9, 11, 13), including formation of CLS by atMaggregating around dysfunctional adipocytes (9, 10). These atMundergo abnormal activation by fatty acids released by the lipid-loaded "obese" adipocytes. Notably, female patients store greateramounts of fatty acids (represented by palmitate) in their fatdepots, as comparedwithmale patients (45). Palmitate and othersaturated fatty acids are capable of activating macrophages in"obese" adipose tissue (acting via TLR4; refs. 5, 9, 10, 12, 14–16).Thus, CLS-residing atM are adversely activated and secrete ele-vated levels of cytokines (9, 10, 12), including TNFa, a key inducerof aromatase expression in adipose stromal cells–the fibroblaststhat surround adipocytes (9, 17, 18), eventually leading to extra-gonadal production of estrogen (3, 4, 9, 13, 18).

We, therefore, examined the correlation between obese stateand aromatase expression in visceral adipose tissue of leanand obese mice. As expected (46), no aromatase expression wasdetected in adipose tissue in themajority of leanwtmice (Fig. 3C)In contrast, aromatase expression was easily detected in theadipose tissue in >80% of obese wt mice (Fig. 3C). Statisticalanalysis confirmed correlation between obese state and

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HFD-induced obesity accelerates breast carcinoma progression inwt but not in Hpse-KOmice. Femalewt (A) and Hpse-KO (B) mice were fed HFD (black line) orcontrol (gray line) diet for 15 consecutive weeks. HFD-fed animals of both genotypes (wt and Hpse-KO) became obese, as evidenced by their significantlyincreased body weight. Data are the mean� SE. Two-sided Student t test. � , P < 0.003; �� , P < 0.0001. Inset, adipocyte hypertrophy, characteristic feature ofdiet-induced obesity, was noted in intra-abdominal fat of bothwt (A) and Hpse-KO (B) HFD-fed mice. By experimental week 12 (dashed arrow), E0771 cells wereinjected orthotopically into mammary fat pad of all mice. Volume of E0771 tumors grown in lean (gray line) and obese (black line)wt (C) and Hpse-KO (D) micewas measured until experimental week 15. Data are the mean� SE. Two-sided Student t test. � , P¼ 0.02; �� , P¼ 0.0036; n� 7 mice per condition.






aromatase expression in wt animals (two-sided x2 test;P ¼ 0.003, Fig. 3C). These findings are consistent with theclinically observed correlation between obese status and induc-tion of aromatase in adipose tissue of obese patients withbreast carcinoma, as well as in dietary/genetic models ofobesity (9, 11, 13, 46, 47).

Remarkably, obese state did not result in aromatase inductionin adipose tissue of heparanase-KOmice (Fig. 3C). Given the roleof "obese" adipocyte-derived fatty acids in TLR4-dependent atMactivation (5, 9, 10, 12, 14–16), and the involvement of hepar-anase in sustainingmacrophage stimulationviaTLR4 (32,33, 35),we hypothesized that total lack of heparanase does not allow foradequate atM activation in adipose tissue of obese Hpse-KOanimals. As a result, atM in obese Hpse-KO mice (as opposed toobese wtmice) may not be able to produce sufficient amounts of

inflammatory cytokines (i.e., TNFa) required for the induction ofaromatase expression in adipose tissue fibroblasts. To validatethis hypothesis,wefirst examinedCLS formation in adipose tissuespecimens harvested from lean/obese wt and Hpse-KO mice.Essentially, no CLS were detected in the adipose tissue of bothwt and Hpse-KO lean mice (in agreement with ref. 7), whereas inthe adipose tissue of obese mice (of both genotypes), CLS wereeasily detectable [histologically and by the presence of F4/80-positive macrophages (Fig. 4A, left and middle)]. Of note, therewas no statistically significant difference between the number ofCLS observed in obese wt versus obese Hpse-KO adipose tissue(3.75 vs. 2.67 CLS/500 adipocytes, P ¼ 0.504), suggesting thatheparanase deficiency does not affect the degree of macrophageinfiltration in adipose tissue. Next, we tested the possibilitythat heparanase affects the activation state of CLS-residing

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Expression of heparanase in ER-positive obesity-associated breast carcinoma in experimental (A and B) and clinical (C and D) settings. A and B, Heparanaseprotein (Hpa) levels in the orthotopic E0771 tumors derived from lean and obesewt and Hpse-KOmice. A, Lysates of tumor tissue were analyzed byimmunoblotting (left andmiddle panels). The band intensity was quantified using ImageJ software (right); intensity ratio for Hpa/actin is shown; n� 3 mice percondition. B, Immunostaining (brown) of E0771 tumor tissue sections with antiheparanase antibody. Scale bars, 20 mm. C and D, Heparanase expression in obeseand nonobese ER-positive patients with breast carcinoma. Human breast carcinoma tissue array, comprising 123 tumor specimens derived from obese (BMI >30)and nonobese (BMI <30) patients with breast carcinoma, was processed for IHC with antiheparanase antibody, as described in Supplementary Methods.Obese status was determined from patient history. C,Distribution of heparanase-negative (Hpa�) and heparanase-positive (Hpaþ) breast tumors among obese(n¼ 19) and nonobese (n¼ 57) ER-positive patients with breast carcinoma. Two-sided Pearson x2 test confirmed significant correlation (P¼ 0.045) betweenobese state of the patient and heparanase expression.D, Representative image of positive heparanase immunostaining in breast carcinoma specimens fromtissue microarray. Scale bars, 50 mm.

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macrophages (i.e., level of TNFa production). To this end, weutilized immunostaining for F4/80 (mouse macrophage-specificmarker) and TNFa (a powerful inducer of aromatase expressionin obese adipose tissue; refs. 3, 9, 13, 17, 18) in serial sections ofadipose tissue specimens derived from wt and Hpse-KO mice.TNFa immunostaining revealed markedly lower levels of thecytokine in Hpse-KO mice (Fig. 4A, right), and quantificationstudies confirmed a statistically significant difference in stainingintensity of TNFa in CLS macrophages of adipose specimensharvested from obese wt mice, as compared with obese Hpse-KOmice (Fig. 4B).

We next turned to validate in vitro the decreased ability ofHpse-KOmacrophages to undergo activation in response to obesogenicstimulation (Fig. 4C). For this purpose, we compared expressionof TNFa by macrophages derived from wt and Hpse-KO mice, inresponse to fatty acid, mimicking macrophage activation by fattyacids present in obese adipose tissue (5, 9, 10, 12, 14–16). Pal-mitate (the dominant saturated fatty acid present in fat depots offemale patients; ref. 45) was utilized in our experiments, alongwith other saturated (i.e., stearic) and unsaturated (oleic, linoleic)fatty acids.

Consistent with our hypothesis, stimulation with palmitate(C16:0) in vitro strongly induced expression of TNFa in wtmacrophages but not in heparanase-deficient macrophages(Fig. 4C). Similar results were obtained following stimulationwith stearic acid (C18:0, Fig. 4C). Unsurprisingly, unsaturatedfatty acids [oleic (C18:1) and linoleic (C18:2), Fig. 4C] failed toinduce significant cytokine expression in wt macrophages, con-sistent with reports that attributed TLR-activating/macrophage-stimulating properties mainly to saturated fatty acids (i.e.,palmitate, stearate), whereas unsaturated acids (i.e., oleate)were reported to display only weak macrophage-stimulatingability (14).

Then, to further test our hypothesis that presence of hepar-anase is a prerequisite for the ability of fatty acid–stimulatedmacrophages to undergo activation sufficient to induce aro-matase in adipose stromal cells, we utilized fibroblasts isolatedfrom breast adipose tissue of healthy women undergoingreduction mammoplasty (as described in Supplementary Meth-ods), known as a useful and reliable model to study regulationof aromatase expression in adipose tissue (9, 17, 18). Thefibroblasts were incubated with medium conditioned by wt or

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A, Estrogen induces heparanaseexpression in E0771 breast cancercells. E0771 cells were treated witheither 1� 10�9 M estrogen (E2, blackbar) or vehicle alone (gray bar),as described in SupplementaryMethods. Heparanase mRNAexpression was assessed byqRT-PCR. �� , P¼ 0.002. B,Progesterone receptor (PR)expression in E0771 tumors growinginwt and Hpse-KO lean (gray bars)and obese (black bars) mice,assessed by qRT-PCR (n¼ 3 percondition; �, P¼ 0.048). C,Correlation between obese state andaromatase expression status inmouse adipose tissue. Quantitativereal-time RT-PCR was used todetermine the presence or absenceof aromatase mRNA in visceraladipose tissue samples collectedfromwt and Hpse-KO lean andobese mice. Stacked bar chart isshown, indicating the proportion ofmice positive for aromataseexpression in adipose tissue (black,aromatase positive; white,aromatase negative) in eachexperimental group (n� 6 percondition). Two sided x2 testconfirmed significant correlationbetween obese state and adiposearomatase expression inwt animals(�� , P¼ 0.003). Note that unlike inwtmice, in Hpse-KOmice, obesestate did not confer aromataseexpression in adipose tissue.






heparanase-deficient macrophages, stimulated by palmitate. Inagreement with the hypothesized mode of heparanase action inthis system, medium conditioned by wt macrophages, but notby Hpse-KO macrophages, induced expression of aromatase inthe fibroblasts (Fig. 4D).

These results, showing that heparanase deficiency abolishes themacrophage ability to induce aromatase expression in adiposefibroblasts ex vivo, together with the in vivo findings depictedin Figs. 3C and 4A–C, suggest an explanation for the lack of

aromatase induction in heparanase-deficient obese adipose tissue(Fig. 3C) and therefore the absence of enhanced estrogen signal-ing (Fig. 3B) and lack of heparanase induction in E0771 tumorsgrowing in obese Hpse-KO mice (Fig. 2A and B).

Effect of heparanase on TAM in obesity-associated breastcarcinoma

Although adipose tissue-residing macrophages (atM) are cer-tainly important in obesity-associated ER-positive breast cancer,

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Effect of heparanase deficiency on TNFa expression by macrophages in vivo (A and B) and in vitro (C), as well as on their ability to induce aromatase in adiposestromal cells (fibroblasts) ex vivo (D). A and B, Serial sections of obesewt and Hpse-KOmouse adipose tissue samples (n� 3) were processed for IHC with anti-F4/80 (A, left and middle) and TNFa (A, right) antibodies. A, Photographs are representative ofwt (top) and Hpse-KO (bottom) samples. Scale bar, 20 mm. B,Sections were scored according to TNFa staining intensity (low/no staining¼ 1; medium staining¼ 2; high staining¼ 3). The data shown are the mean� SE ofstaining scores. Two-sided Student t test. � , P¼ 0.005. C, Induction of TNFa expression by fatty acids inwt and Hpse-KOmacrophages in vitro. Primaryperitoneal macrophages, isolated fromwt (dotted bars) or Hpse-KO (empty bars) mice (as described in Supplementary Methods), were either incubated(24 hours, 37�C) with BSA alone (vehicle) or with indicated fatty acids (200 mmol/L; fatty acid:BSA ratio 2:1). Fatty acids used: palmitic (C16:0), stearic (C18:0),oleic (C18:1), and linoleic (C18:2) acids. TNFa expression was evaluated by qRT-PCR. Experiments were repeated twice with similar results. D, Human fibroblasts,isolated from breast adipose tissue of healthy women undergoing reduction mammoplasty, were incubated with dexamethasone (DEX, 250 nmol/L, gray bar)alone, known to trigger aromatase expression in human fibroblasts (17) or with medium conditioned (24 hours, 37�C) by vehicle- or palmitate (C16:0)-stimulatedmacrophages, isolated fromwt or Hpse-KOmice. Quantitative real-time RT-PCR was used to assess the expression of human aromatase mRNA. Error bars,�SE.n.d., nondetectable.

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TAM are undoubtedly the main procancerous immunocyte pop-ulation in general (4, 5, 8) and in obesity-accelerated breastcarcinoma progression in particular (5, 7, 19). Thus, turning backto E0771 tumors, we compared the degree of TAM infiltration intumor tissue specimens derived from lean versus obese mice,applying immunostaining with antibodies directed against F4/80(Fig. 5A) and Mac2 (Supplementary Fig. S4A and S4B)—specificmousemacrophagemarkers (48). Augmented infiltration of TAMwas observed in obesity-associated E0771 breast carcinoma grow-ing inwtmice (2-fold increase, P < 0.02—Fig. 5A and B, consistentwith refs. 7, 19). Importantly, heparanase deficiency abolishedincreased macrophage recruitment in obesity-associated tumors,as no statistically significant difference in TAM infiltration wasdetected in tumors growing in obese versus lean Hpse-KO mice(Fig. 5A and B), mirroring the lack of procancerous action ofobesity in these mice (Fig. 1D).

Macrophage infiltration in breast tumors correlates with highexpression of C-C chemokine ligand 2 (CCL2). Moreover, CCL2,which can be supplied by TAM themselves, acts as a chief inducerof macrophage mobilization in breast carcinoma tissue (7, 49).Furthermore, CCL2-mediated recruitment of macrophages is akey molecular event underlying obesity-associated breast cancerprogression (7). Thus, we compared the expression of CCL2 in

tumors derived from obese versus lean mice. Although no dif-ference in CCL2 levels was noted between tumors derived fromobese versus leanHpse-KOmice, significant increase in bothCCL2mRNA (>2-fold increase, P ¼ 0.048) and protein levels (Fig. 5Cand D) was detected in tumors derived from wt obese, as com-pared with wt lean mice. Given that CCL2 is a well-characterizedHS-binding chemokine, relatively modest increase in CCL2 pro-tein levels detected by immunostaining (Fig. 5C and D) in obesewtmice (vs. >2-fold increase in CCL2mRNA)may reflect elevatedlevels of heparanase enzyme and the resulting decrease in HScontent in the tumors from obese versus leanwtmice (Fig. 2A andB; Supplementary Fig. S2A and S2B). In addition, colocalizationexperiments revealed a 2-fold increase in the percentage of CCL2–positive macrophages infiltrating tumors growing in obese versuslean wt (but not Hpse-KO) mice (Supplementary Fig. S4A andS4B). Collectively, these findings suggest that heparanase over-expression in tumors growing in obese wt mice (Fig. 2A and B)directly affects TAM recruitment and activation.

Heparanase augments cancer-promoting activity of oleate-stimulated macrophages in vitro

The aforementioned findings, along with the emerging roleof heparanase in sustaining macrophage reactivity in several

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Macrophage infiltration and CCL2expression are increased in E0771orthotopic tumors growing in wtobese but not Hpse-KO obesemice. A and C, Tissue specimensderived from E0771 tumorsgrowing in either wt (top) orHpse-KO (bottom); lean (left) andobese (right) mice were stainedwith anti-F4/80 antibody-green(A) or with anti-CCL2 antibody-red (C). Cell nuclei werecounterstained with DRAQ5(blue). Scale bars, 50 mm (A),100 mm (C). B, F4/80-positivemacrophages in tumor tissuespecimens derived from lean(gray bars) and obese (blackbars) mice were quantified per0.04 mm2 microscopic field (allthe fields chosen for analysiswere located �1 mm from thetumor border), based on at leastfour sections from three mice pergroup. Error bars, �SE. Two-sidedStudent t test. � , P ¼ 0.02; �� , P¼ 0.002; n.s, no statisticaldifference. D, CCL2 stainingintensity per microscopic fieldwas quantified using Zensoftware, based on at least foursections from three mice pergroup. Data shown are the meanintensity. Error bars, �SE.Two-sided Student t test.� , P ¼ 0.006; �� , P ¼ 0.03.






cancer-related and nonrelated disorders (32–35, 50), lead tohypothesis that in the setting of obesity, increased intratumorallevels of heparanase (Fig. 2) may direct protumorous actionof TAM stimulated by obesogenic substances present in circula-tion. Specifically, FFAs are among the candidate agentscapable of modulating macrophage responses in the obeseindividuals (5, 9, 10, 12, 14–16). However, unlike within the"obese" adipose tissue, where CLS-residing atM are exposed torelatively high levels of fatty acids due to necrosis of hypertrophicadipocytes (9), obesity-associated circulating levels of FFA (20)alone might not be sufficient to trigger TAM polarization.

To examine in vitro the hypothesized effect of heparanase onphenotypic switch of macrophages exposed to obesogenic sub-stances, we utilized oleic acid, taking advantage of the fact that it isone of themost abundant fatty acids in circulation (51) and in theHFD used in this study (see Materials and Methods). In addition,oleic acid per se was shown to induce only slight effects onmacrophage activation and induction of cytokine expression(i.e., TNFa, IL6; Fig. 4C; ref. 14). We first tested the effect of theenzyme on the ability of oleic acid–stimulated macrophages toproduce protumorous cytokines and to promote breast carcino-ma cell proliferation in vitro. To recapitulate conditions faced bymacrophages infiltrating obesity-associated breast carcinoma tis-sue [i.e., exposure to increased levels of heparanase (Fig. 2) alongwith the presence of elevated levels of circulating FFA], primarymacrophages were treated with oleic acid in the presence ofrecombinant active heparanase; macrophages treated with oleicacid in absence of heparanase or in the presence of heat-inactivated enzyme were used as control. As depictedin Fig. 6A, heparanase strongly and in a dose-dependent manneraugmented productionof IL6 (a keybreast carcinoma–promotingcytokine implicated in the obesity-cancer link; refs. 4–6) by oleicacid–stimulated macrophages. The effect of heparanase on IL6expression/secretion was dose dependent (Fig. 6A; Supplemen-tary Fig. S5A and S5B); IL6 levels reached a plateau and did notcontinue to increase at the enzyme concentrations above 3 mg/mL(Supplementary Fig. S5A and S5B). Importantly, heparanaseeffect was dependent on its enzymatic activity because heat-inactivated heparanase did not affect cytokine levels.

IL6 acts in promotion of cancer through activation of severalsignaling pathways leading to increased tumor cell proliferation,survival, and resistance to therapies (4, 6, 52, 53). In particular,IL6 was shown to enhance tumor cell proliferation in ER-positivebreast cancer (52, 53), and higher IL6 levels in patients withbreast carcinoma correlate with poorer survival and diminish-ed response to therapies (4). One important mechanism bywhich IL6 promotes breast carcinoma is activation of STAT3(4, 52, 53), a critical component of tumor-stimulating signalingin breast carcinoma and other malignancies. Notably, we foundthat medium conditioned by macrophages stimulated by oleicacid in the presence of heparanase markedly enhanced bothSTAT3 signaling (Fig. 6B–D) and proliferation (Fig. 6E) ofE0771 cells in vitro. Proliferation of human breast carcinoma celllines T47DandMCF7was also enhancedbymediumconditionedby macrophages stimulated by oleic acid in the presence ofheparanase (Supplementary Fig. S6).

In contrast, medium conditioned by macrophages stimulatedby oleic acid in the absence of the enzyme had no effect on STAT3signaling (Fig. 6B–D) and cell proliferation (Fig. 6E; Supplemen-tary Fig. S6). In addition, specific STAT3 inhibitor S3I-201 abol-ished the effect of macrophages stimulated by oleic acid in the

presence of heparanase on E0771 cells proliferation (Fig. 6E,dotted bars). Altogether, these findings further support the pro-posed involvement of heparanase in powering procancerousaction of FFA-stimulated TAM in obesity-associated breast carci-noma and role of the IL6–STAT3 axis in this process.

Finally, to place our in vitro findings in the context of obesity-associated breast carcinoma in vivo, we compared the activationstatus of STAT3 in E0771 tumors growing in lean and obese, wtand heparanase-KO mice. As shown in Fig. 6F and G, immunos-taining analysis revealed augmented STAT3 signaling (manifestedby significantly increased levels of phospho-STAT3) in tumorsgrowing in wt obese mice versus tumors growing in their leanlittermates. Importantly, heparanase deficiency prevented obesi-ty-induced STAT3 signaling, as no increase in STAT3 signalingwasdetected in tumors growing in Hpse-KO obese versus lean mice(Fig. 6F and G), echoing failure of obesity to accelerate E0771breast carcinoma growth in Hpse-KO animals. Altogether, thesedata provide an explanation for the lack of tumor-acceleratingeffect of obesity in heparanase-deficient mice and highlight thepreviously unknown role of the enzyme in sustaining obesity–breast cancer link.

DiscussionObesity is a known risk factor in breast cancer (largely ER-

positive; refs. 2–4, 6). Furthermore, once diagnosed, obesepatients with breast carcinoma have worse clinical outcomes thantheir lean counterparts (3–5). Chronic, "smoldering" inflamma-tion, adverse activation of macrophages, and altered productionof estrogens are among key mechanisms through which excessadiposity fosters breast carcinoma progression and therapy resis-tance, acting both locally and systemically (3–6). Heparanaseenzyme was recently linked to modulation of macrophageresponses (32–35, 50) through TLR-dependent mechanism:intact cell surface HS is capable of suppressing TLR4 responses/macrophage activation, its degradation by heparanase abolishesthis suppression (28), and cleavage of cell-surface HS by hepar-anase increases ligand accessibility of TLR4 (33). Moreover,heparanase-generated soluble HS degradation fragments stimu-late TLR4 signaling in vitro (28, 30) and in vivo (31).

Here we show that heparanase is preferentially expressed inclinical and experimental obesity-associated breast carcinoma(Fig. 2) and orchestrates a complex multisite molecular programthat occurs concurrently in adipose (Figs. 3C and 4) and breasttumor (Figs. 5 and 6F and G) tissues; involves tumor cells,hypertrophic "obese" adipocytes, and surrounding macrophages;and sustains obesity-accelerated breast carcinoma growth. Thus,our study not only defines heparanase as a marker of obesity-related, macrophage-driven breast carcinoma progression butalso provides a mechanistic explanation for accelerated growthof estrogen-responsive breast tumors under obese state. In sup-port of this notion, heparanase deficiency abolished obesity-accelerated progression of ER-positive E0771 tumor in vivo.Importantly, we found that heparanase deficiency did not abolishobesity-accelerated growth of estrogen-independent C57BL/6J-syngeneic Panc02 pancreatic carcinoma cell line (SupplementaryFig. S7). Panc02 is awell-establishedmodel of obesity-acceleratedtumor growth in vivo (5, 7, 19) and represents oneof the known12tumor types, other than ER-positive breast carcinoma, associatedwith obesity (1). Thus, our observations that heparanase defi-ciency abolishes obesity-accelerated progression of ER-positive

Hermano et al.





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A–E, Heparanase augments cancer-promoting activity of macrophages stimulated in vitro by oleic acid. A,Mouse peritoneal macrophages were incubated (37�C,24 hours) with oleic acid (C18:1, 200mmol/L; fatty acid: BSA ratio 2:1) or with BSA alone (vehicle) in the absence or presence of active recombinant heparanase atindicated concentrations. Secretion of IL6 was analyzed by ELISA. Error bars,�SD. Two-sided Student t test. � , P� 0.0003. B–D, E0771 cells were seeded inpentaplicates and incubated for 16 hours with medium that was conditioned (24 hours, 37�C) by vehicle-stimulated or oleic acid–stimulated (C18:1) macrophagesin the absence or presence of active recombinant heparanase. B, Left, lysates of E0771 cells were immunoblotted using antibody specific for phospho-STAT3(pSTAT3) and total STAT3. B, Right, the band intensity was quantified using ImageJ software. Error bars,�SE. Two-sided Student t test; � , P¼ 0.043. C, E0771cells were stained with anti-pSTAT3 antibody (red). Cell nuclei were counterstained with DRAQ5 (blue). Overlay (pink) represents cells positive for nuclear-localized pSTAT3. Scale bars, 50 mm. D,Quantification of the degree of association between pSTAT3 and Draq5 staining within E0771 cells in the absence (graybars) or presence (black bars) of active recombinant heparanase was performed using colocalization tool of Zen software (Carl Zeiss). Bar graph shows thePearson correlation coefficient values for pSTAT3/Draq5 colocalization. � , P¼ 0.047; n.s: no statistical difference (P¼ 0.29). E, E0771 cells seeded inpentaplicates in 96-well plate were incubated with medium that has been conditioned (24 hours, 37�C) by vehicle-stimulated or oleic acid–stimulated (C18:1)macrophages in the absence (gray bars) or presence (black bars) of active recombinant heparanase. Specific STAT3 inhibitor S3I-201 (80 mmol/L) was added tosome wells (dotted bars). Bar graph demonstrates the fold change in E0771 cell proliferation, analyzed by MTS assay 48 hours later. � , P¼ 0.04. Error bars,�SD.F and G, Increased pSTAT-3 levels in E0771 tumors growing inwt obese but not in Hpse-KO obese mice. F, Tissue specimens derived from E0771 tumorsgrowing in eitherwt (top) or Hpse-KO (bottom), lean (left), and obese (right) mice were immunostained with anti- pSTAT3 antibody (brown). Scale bars, 50 mm.G, pSTAT3-positive cells in tumor sections from lean (gray bars) and obese (black bars)wt and Hpse-KOmice were quantified (AU, arbitrary units), based onthree sections per mouse (n¼ 4mice per condition), under�200magnification. Error bars,�SE. Two-sided Student t test. � , P¼ 0.02; �� , P¼ 0.013.






E0771, but not of ER-negative Panc02 tumor model, furthercorroborate the mechanistic link between heparanase effect ontumor-promoting action of obesity and estrogen responsiveness.

It seems that macrophage-driven inflammatory events in adi-pose tissue, induction of heparanase in the tumor compartment,and breast carcinoma–promoting action of TAM are inexorablycoupled in obesity-associated ER-positive breast cancer (as sche-matically presented in Fig. 7). The steps occurring in "obese"adipose tissue (Fig. 7, steps i–iv) involve adverse activationof atM,(supported by data depicted in Fig. 4A–C), that is, by saturatedfatty acids locally released from the lipid-overloaded "obese"adipocytes (3, 9, 10), which are known to trigger TLR4. ActivatedatM secrete TNFa (Fig. 4A–C), a powerful trigger of aromataseexpression in adipose fibroblasts (Fig. 7, step iii; refs. 9, 17, 18).Noteworthy, steps ii to iii (Fig. 7) require basic levels of hepar-

anase present in the macrophages themselves, as heparanasedeficiency renders macrophages less responsive to activation bysaturated fatty acids (as in Fig. 4C) and abolishes their ability toinduces aromatase expression in mouse obese adipose tissuein vivo and in human breast adipose fibroblasts ex vivo (asin Figs. 3C and 4D).

Induction of aromatase in adipose tissue eventually results inestrogen-mediated upregulation of heparanase in the breast car-cinoma cells (Fig. 7 steps iv and v, supported by data depictedin Figs. 2A, C, D and 3A and B; also see refs. 23 and 25 for exactmolecular mechanism through which estrogen induces hepara-nase in ER-positive breast carcinoma). During the following stepsoccurring in the breast tumor compartment, breast carcinomacells that succeed in upregulating heparanase can effectivelyrecruit/polarize TAM (Fig. 7, step vi, supported data on Fig. 5

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Amodel of molecular events coupling obesity and breast cancer, orchestrated by heparanase in "obese" adipose tissue (left) and breast tumor (right)compartments. i, CLS composed of atMs (brown pentagons) aggregating around hypertrophic adipocyte (yellow circle), as in Fig. 4A. ii, CLS-residing atM can beadversely activated via TLR4-mediated mechanism, i.e., by locally released FA derived from the lipid-laden "obese" adipocytes (5, 14–16). iii, Activated atMsecrete TNFa (as in Fig. 4A top, B, and C), a key inducer of aromatase expression in adipose stromal cells—fibroblasts (9, 17, 18). iv, Induction of aromatase (asin Figs. 3C and 4D) augments biosynthesis of estrogen (E2) in adipose tissue (3, 4, 9, 13, 18). Note that steps ii to iv require basic levels of heparanase [whichaffect TLR4 signaling through degradation of HS (blue chains); see refs. 28–31 for details], as enzyme deficiency renders macrophages unresponsive to saturatedfatty acids (Fig. 4C) and abolishes their ability to induce aromatase in mouse obese adipose tissue in vivo (Fig. 3C) and in human adipose fibroblasts ex vivo(Fig. 4D). v, Estrogen signaling leads to upregulation of heparanase in ER-positive breast carcinoma cells—supported by results depicted in Figs. 2A, C, D, and 3Aand B; also see refs. 23, 25 for exact molecular mechanism throughwhich estrogen induces heparanase in ER-positive breast carcinoma. vi, Increased levels ofheparanase in obesity-associated breast cancer (BC; as in Fig. 2) and the resulting HS degradation (Supplementary Fig. S2) enhancemobilization of TAM (asin Fig. 5 and Supplementary Fig. S4) and their polarization toward procancerous phenotype (i.e., ability to stimulate breast cancer cell growth, as in Fig. 6 andSupplementary Fig. S5 and S6). This polarization can occur via stimulation by locally circulating FFA (Fig. 6A; Supplementary Fig. S5). Without augmentedheparanase levels, FFA exerts only weakmacrophage-stimulating properties (Fig. 6 and Supplementary Fig. S5, where FFA are exemplified by oleic acid). Inaccordance, in tumors from obese Hpse-KOmice, TAMmobilization is not enhanced (Fig. 5; Supplementary Fig. S4). vii, Increased levels of procancerouscytokines, locally released by TAM, lead to activation of breast cancer-promoting signaling (i.e., along IL6-STAT3 axis, as in Fig. 6B–D, F, and G) and acceleratetumor progression (as in Figs. 1C and 6E). Dashed arrow, increased heparanase levels may also contribute to breast cancer progression via additionalmechanisms (21–24), such as release of ECM-bound growth factors and removal of extracellular barriers for invasion/metastasis (26, 27).

Hermano et al.





and Supplementary Fig. S4), as increased levels of the enzymeenable acquisition of procancerous phenotype by macrophagesstimulated by locally circulating FFA (supported by data depictedin Fig. 6 and in Supplementary Fig. S5 and S6). The above-described scenario results in increased rate of procancerous cyto-kines locally released by TAM within the tumor, activation ofbreast carcinoma-promoting signaling (i.e., STAT3), and acceler-ated tumor growth (Fig. 7, step vii, supported byfindings depictedin Figs. 6B–D, F, G and 1C and 6E, in accordance). Thus, in obesestate, presence of systemic circulating FFA (and perhaps otherobesogenic compounds known to affect macrophage responses),combined with locally induced heparanase in ER-positive breastcarcinoma tissue, creates "opportunity" for abnormal activationof TAM, whereas in the rest of tissues of the same patient (whereheparanase is not expressed), levels of circulating FFA alone arenot sufficient to trigger pathologic macrophage activation.

TAM are well-characterized target for tumor treatment (8) andspecifically for uncoupling obesity–cancer link (5, 7, 19). Yet,translation of TAM-targeting approach into the clinic remainsextremely challenging, in part, due to the highly heterogeneousand dynamic nature of TAM phenotypes (i.e., their ability toexert both pro- and antitumor activity, depending on differenttumor settings; ref. 8). Thus, identification of heparanase as anovel molecular player that dictates the procancerous pattern ofTAM activation in the obesity-related breast carcinoma can pro-vide important information for translation of TAM-targetingapproach into clinical practice and may also help better definepatient populations in which this approach could be particularlybeneficial. It should be noted that the limitation of this study isthe lack of heparanase inhibition in vivo, which will be importantfor further translation of the basic mechanism revealed by ourfindings into therapeutic approach. In light of the above findings,the next question is whether the currently available heparanaseinhibitors could disrupt the obesity–breast cancer linkage. Cur-rently, several heparanase inhibitors, primarily HS-mimickingcompounds such as pixatimod (PG545) and Roneparstat, under-go clinical testing (50). However, due to their structural similarityto soluble HS, the HS-mimicking compoundsmay also act as TLRligands, stimulating macrophages regardless of their heparanase-inhibiting properties (32, 34). In addition, recently identifiedability of heparanase to enhance NK-cell infiltration into tumorsraised concerns that at least in some tumor types the potentialadverse effect of heparanase inhibitors onNK cellsmay limit their

antitumor effect (54). On the other hand, because antitumorfunction of NK cells was reported to be limited/lost under obeseconditions (55), heparanase targeting is still relevant in obesity-associated breast carcinoma. Hence, ongoing development ofheparanase-inhibiting compounds devoid of macrophage-activating properties (such as neutralizing antibodies, small-molecule inhibitors; ref. 50) will likely offer a better strategy toreduce breast carcinoma risk in an increasingly obese populationand to suppress the breast cancer–promoting consequences ofexcess adiposity.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. ElkinDevelopment of methodology: E. Hermano, J.-P. Li, J. van der Vlag, T. Peretz,M. ElkinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Goldberg, A. Sonnenblick, B. Maly,M.A.H. Bakker, I. Vlodavsky, T. Peretz, M. ElkinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Hermano, R. Goldberg, A.M. Rubinstein,A. Sonnenblick, D. Nahmias, M.A.H. Bakker, M. ElkinWriting, review, and/or revision of the manuscript: R. Goldberg,A. Sonnenblick, J. van der Vlag, I. Vlodavsky, T. Peretz, M. ElkinStudy supervision: M. ElkinOther (conduction of the experiments): E. Hermano, R. Goldberg,A.M. Rubinstein, D. NahmiasOther (generation/characterization of clinical databases; review of themanuscript): T. Peretz

AcknowledgmentsThis studywas supported by grants from the Israel Science Foundation (grant

nos. 806/14, 1715/17), the Legacy Heritage Bio-Medical Program (grant no.666/16), and the Mizutani Foundation for Glycoscience Research Grant, allawarded to M. Elkin. It was also supported by grants from the Israel ScienceFoundation (grant nos. 601/14, 2572/16) and the Israel Cancer Research Fund,awarded to I. Vlodavsky.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 2, 2019; revised June 6, 2019; accepted August 26, 2019;published first September 3, 2019.

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2019;79:5342-5354. Published OnlineFirst September 3, 2019.Cancer Res Esther Hermano, Rachel Goldberg, Ariel M. Rubinstein, et al. ProgressionHeparanase Accelerates Obesity-Associated Breast Cancer

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Heparanase Accelerates Obesity-Associated Breast Cancer … · Tumor Biology and Immunology Heparanase Accelerates Obesity-Associated Breast Cancer Progression Esther Hermano1, Rachel