Title: Autocrine CCL5 Effect Mediates Trastuzumab Resistance by ERK Pathway

Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 TITLE PAGE

2 Title: Autocrine CCL5 effect mediates trastuzumab resistance by ERK pathway

3 activation in HER2-positive breast cancer

4 Authors: Sandra Zazo1, Paula González-Alonso1, Ester Martín-Aparicio1, Cristina

5 Chamizo1, Melani Luque1, Marta Sanz-Álvarez1, Pablo Mínguez2, Gonzalo Gómez-

6 López3, Ion Cristóbal4, Cristina Caramés4, Jesús García-Foncillas4, Pilar Eroles5, Ana

7 Lluch5,6, Oriol Arpí7, Ana Rovira7,8, Joan Albanell7,8,9, Juan Madoz-Gúrpide1§, Federico

8 Rojo1§

9 Affiliations: 1Pathology, Fundación Jiménez Díaz University Hospital Health Research

10 Institute (IIS—FJD, UAM)—CIBERONC, Madrid 28040, Spain; 2Genetics

11 Department, Health Research Institute-Fundación Jiménez Díaz (IIS-FJD, UAM),

12 Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid

13 28040, Spain; 3Bioinformatics Unit, Spanish National Cancer Research Centre (CNIO),

14 Madrid 28029, Spain; 4Translational Oncology Division, OncoHealth Institute, Health

15 Research Institute-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain;

16 5Institute of Health Research INCLIVA-CIBERONC, Valencia 46010, Spain;

17 6Medicine Department, University of Valencia, Valencia 46010, Spain; 7Cancer

18 Research Program, IMIM (Hospital del Mar Research Institute), Barcelona 08003,

19 Spain; 8Medical Oncology Department, Hospital del Mar-CIBERONC, Barcelona

20 08003, Spain; 9Universitat Pompeu Fabra, Barcelona 08002, Spain; §these authors

21 contributed equally to this work.

22 Running title: Trastuzumab resistance by CCL5/ERK axis activation

23 Keywords: breast cancer, HER2-positive, anti-receptor therapy, trastuzumab,

24 resistance, cytokines, CCL5, cell lines

Downloaded from mct.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Financial support: The present work was supported by grants from the Spanish

2 Ministry of Health, Consumer Affairs and Social Welfare (AES Program, grants

3 PI15/00934; PI18/00382 and PI18/00006); the Biomedical Research Networking Centre

4 for Cancer (CIBERONC); the Biobanks Platform, PT13/0010/0012; the Community of

5 Madrid (S2010/BMD-2344); and ProteoRed (PRB2-ISCIII, PT13/0001). PGA was

6 supported by a Fundación Conchita Rábago de Jiménez Díaz grant. PM was supported

7 by the ISCIII Miguel Servet Program (CP16/00116).

8 Corresponding authors: §Dr. Juan Madoz-Gúrpide, Ph.D., Pathology Department, IIS-

9 Fundación Jiménez Díaz, UAM, Avda. Reyes Católicos 2, E-28040 Madrid, Spain. E-

10 mail: [email protected]. Phone: +34-915504800.

11 §Dr. Federico Rojo, M.D. Ph.D., Pathology Department, University Hospital Fundación

12 Jiménez Díaz, Avda. Reyes Católicos 2, E-28040 Madrid, Spain. E-mail: [email protected].

13 Phone: +34-915504800.

14 Conflict of interest: AL has a consulting or advisory role in Novartis, Pfizer,

15 Roche/Genentech, Eisai, Celgene (recipient herself) and research funding from Roche

16 Pharma AG, AstraZeneca, Merck, PharmaMar, Boehringer Ingelheim, Amgen,

17 GlaxoSmithKline, Novartis, Pfizer, Eisai, Celgene, Pierre Fabre. The rest of the authors

18 declare no competing financial interests.

1 ABSTRACT

2 HER2-positive breast cancer is currently managed with chemotherapy in combination

3 with specific anti-HER2 therapies, including trastuzumab. However, a high percentage

4 of patients with HER2-positive tumors do not respond to trastuzumab (primary

5 resistance) or either recur (acquired resistance), mostly due to molecular alterations in

6 the tumor that are either unknown or undetermined in clinical practice. Those alterations

7 may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor

8 proliferation and metastasis.

9 Using continued exposure of a HER2-positive cell line to trastuzumab we

10 generated a model of acquired resistance characterized by increased expression of

11 several cytokines. Differential gene expression analysis indicated an overexpression of

12 15 genes, including 5 different chemokines, and highlighting CCL5/RANTES as the

13 most overexpressed one. Functional studies, either by in vitro gene silencing or by in

14 vitro and in vivo pharmacological inhibition of the CCL5/CCR5 interaction with

15 maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance

16 to trastuzumab, which was mediated by ERK activation. In patient samples, increased

17 CCL5 expression significantly correlated with lower rates of complete response after

18 neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA.

19 Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was

20 statistically associated with worse disease-free survival and overall cancer survival in

21 patients with early HER2-positive breast cancer.

1 INTRODUCTION

2 Breast cancer accounts for 20-25% of all cancer cases worldwide and is the most

3 prevalent in women(1). Breast cancer comprises many biologically different diseases

4 with distinct pathological features and clinical implications, thus making accurate

5 grouping of clinically relevant subtypes of importance(2). Among these subtypes,

6 HER2-positive breast cancer, which accounts for 20% of all breast cancers(3), is

7 characterized by gene overexpression, high rates of cell proliferation and metastasis,

8 poor prognosis, low overall survival (OS), and variable chemotherapy response with

9 poor outcome(4). Together with this prognostic value, the HER2 receptor is currently

10 considered part of the standard assessment protocol as a predictor of response to

11 treatment(5).

12 During the past decade, systemic therapeutic management of breast cancer has

13 undergone a significant transformation, leading to the emergence of targeted therapy.

14 For HER2-positive breast cancer patients, targeting HER2 has become an attractive

15 therapeutic approach. Trastuzumab (Herceptin), a humanized IgG1 monoclonal

16 antibody that selectively targets the HER2 receptor, became the first FDA-approved

17 targeted therapy for metastatic breast cancer in 1998(6). Since then, therapies such as

18 trastuzumab combined with chemotherapy have been considered the standard of care for

19 HER2-positive breast cancer patients(7). However, about 25% of HER2-positive breast

20 cancers do not respond initially to trastuzumab(8), and 70% of the trastuzumab-

21 responsive metastatic cancers progress to therapy within the first year due to acquisition

22 of trastuzumab resistance(9). Several potential resistance mechanisms to trastuzumab

23 have been reported during the last decade and their details have been described in

24 numerous reviews(10-12).

1 Nowadays, it is widely accepted that many pathways may be involved in the

2 development of resistance to antibody-based treatments. Specifically, there is a

3 widespread belief that a tumor must be examined in the context of its

4 microenvironment, and therefore should be considered as an entity with a heterogeneous

5 cellular origin in continuous interaction with the stroma, non-tumor cells, and the

6 immune system(13), highly modulated by the inflammatory cells found in the

7 tumor(14). This interaction of the tumor with the microenvironment is mainly regulated

8 through cytokines, which signal the participation of distinct pathways in processes of

9 cell proliferation and differentiation(15).

10 Many chemokines and their receptors are expressed by tumor cells. The

11 chemokine CCL5/ RANTES, is well-recognized for its activities in the immune context,

12 where it induces leukocyte-directed motility. CCL5 has affinity for the G protein-

13 coupled receptors (GPCR) CCR1, CCR3, and, especially, CCR5; and a lesser binding

14 capacity with other receptors. Recently, CCL5/CCR5 have been implicated in

15 proliferation and metastasis in breast cancer(16, 17) and have been recognized as

16 potential therapeutic targets. Moreover, a recent study showed that CCL5 signaling

17 promotes breast cancer recurrence following HER2 inhibition through the recruitment

18 of macrophages(18). In the context of HIV/AIDS studies, potent antagonist inhibitors

19 have developed, which prevent binding of ligands to the receptors; the only CCR5

20 antagonist currently approved by the FDA and the EMA for the treatment of infected

21 patients is maraviroc (Pfizer, USA)(19, 20). The use of this drug in breast cancer could

22 prevent the activation of the CCR5 receptor mediated by the autocrine action of

23 CCL5(21). Preliminary studies on cell lines and murine models have shown that

24 maraviroc prevents binding of CCL5 to CCR5 by decreasing proliferation and

25 development of metastases(22).

1 In the present study, we aimed to identify, quantify, and functionally evaluate

2 potential biomarkers that might be involved in trastuzumab resistance in breast cancer.

3 We examined differential gene expression in HER2-positive trastuzumab sensitive and

4 acquired resistant human breast cancer cell models(23). Differential gene expression

5 analysis indicated an overexpression of 16 genes, highlighting CCL5 as the most

6 overexpressed chemokine with involvement in breast cancer. Because of the emerging

7 role of these proteins as mediators of normal proliferation, migration, and metastasis(16,

8 24, 25), we explored the CCL5 implication in acquired resistance to trastuzumab by

9 functional studies, which were validated in clinical samples from HER2-positive breast

10 cancer. Notably, our results reveal the previously undescribed involvement of CCL5 in

11 the development of trastuzumab resistance.

1 MATERIALS AND METHODS

2 Cells and treatments

3 BT-474, HCC1569, and HCC1954 cell lines were purchased from ATCC (USA) and

4 authenticated (LGC Standards, UK; tracking no: 710259498). JIMT1 cell line was

5 purchased from DSMZ (Germany). Trastuzumab-resistant (BT-474.rT) cell line was

6 generated by continuous exposure to trastuzumab, cultured, authenticated, and tested for

7 Mycoplasma as previously described(23). Cell growth and proliferation assays were

8 performed as previously reported(23). Trastuzumab (Genentech, USA) was supplied by

9 the pharmacy of our hospital. Maraviroc and selumetinib were obtained from

10 Selleckchem (USA). Recombinant human CCL5r was from R&D Systems (USA).

11 Transwell migration assay

12 Migration assays were performed using 24-well plates with transwell permeable

13 supports of 6.5 mm insert and a polycarbonate membrane with an 8-µm pore size

14 (Costar 3422, Corning, USA). Cells were seeded in the upper chamber at 2×104

15 cells/mL in 0.1 mL of serum-free RPMI-1640 media. A volume of 0.8 mL of media

16 supplemented with 10% FBS was placed in the bottom well as a chemo-attractant. After

17 incubation for 24 h at 37 ºC in an atmosphere containing 5% CO2, migrated cells on the

18 lower surface were stained using crystal violet and counted under a light microscope.

19 Clonogenic assay

20 Experiments were performed in 6-well plates coated with 3 mL of 0.6% soft agarose

21 (Sigma-Aldrich, USA) in medium. A total of 5×103 cells were suspended in 0.3%

22 agarose in medium and plated in triplicates over the pre-coated wells. Fresh medium

23 was supplied twice a week. After 21 days, colonies were stained with MTT (M-565,

24 Sigma-Aldrich) for 4 h at 37 ºC. Then, colonies were fixed by adding dimethyl

25 sulfoxide (DMSO) overnight at 37 ºC. Colony numbers were determined from

1 triplicates and three independent experiments were carried out for each condition and

2 cell line.

3 Quantitative real-time RT-PCR

4 Total RNA extracts were isolated using RNeasy Mini kit (Qiagen, Netherlands), their

5 quality was assessed by NanoDrop determination, and RNA was transcribed to cDNA

6 using Universal Transcriptor cDNA kit (Roche, Switzerland). qPCR amplification was

7 performed in a LightCycler-480 system (Roche) using assays specific for ATP5E,

8 CCL5, CCR5, CXCL10, CXCL11, IFNλ1, and IFNλ2 (Table S1). Relative gene

9 expression was calculated according to the comparative method(26), using ATP5E

10 expression for normalization.

11 Western blotting (WB) analysis

12 Total protein lysates were prepared with RIPA buffer containing protease and

13 phosphatase inhibitors. Nuclear and cytosolic protein fractions were isolated using the

14 K266-25 Nuclear/Cytosol Fractionation Kit (BioVision, USA). Protein extracts were

15 clarified, denatured, and subjected to SDS-PAGE and WB. The primary antibodies

16 were: AKT, pAKT-Thr308, pAKT-Ser473, ERK1/2, pERK1/2-Thr202/Tyr204, HER2

17 (Cell Signaling Technology, USA); CCL5 (R&D Systems); and GAPDH (Sigma-

18 Aldrich). Secondary antibodies were conjugated to alkaline phosphatase (Sigma-

19 Aldrich).

20 ELISA

21 The CCL5/RANTES Immunoassay (R&D Systems) was used in cell protein extracts,

22 cell culture supernatants, or patient sera, in duplicate. Absorbance was measured at

23 450 nm.

24 Gene expression analysis

1 Total RNA from BT-474 and BT-474.rT cells either untreated or treated with 15 µg/ml

2 trastuzumab for 48 h from independent biological duplicates was used for gene

3 expression profiling with the Genechip Human Gene 2.0 ST (Affymetrix, USA). Data

4 were processed following the methodology previously described(27). Cutoff value was

5 set so that genes with >2-fold change (sensitive/resistant) in expression levels were

6 considered significantly altered. Further identification of prospective biomarkers of

7 resistance to trastuzumab was performed using a filtering process to determine

8 candidates that were differentially regulated between BT-474 and BT-474.rT, both at

9 basal conditions and trastuzumab-exposure conditions. Data are available through Gene

10 Expression Omnibus with dataset identifier GSE89216.

11 Gene set enrichment analysis (GSEA)

12 GSEA(28) was applied using annotations from MsigDB, Reactome, KEGG, and NCI

13 databases. Genes were ranked based on limma moderated t-statistic. After Kolmogorov-

14 Smirnoff testing, those gene sets showing FDR<0.05 were considered enriched between

15 classes under comparison.

16 siRNA silencing

17 BT-474.rT cells were transfected with siRNAs targeting CCL5 (Smart-pool of 4

18 siRNAs: on-target-plus CCL5 siRNA L-007844-00-0005, 5 nmol) and CCR5 (Smart-

19 pool of 4 siRNAs: on-target-plus CCR5 siRNA L-004855-00-0005, 5 nmol), and

20 scrambled siRNA as a control (Dharmacon, USA), dissolved in a mixture of Opti-MEM

21 and Lipofectamine 2000 (Invitrogen, USA). After transfection, BT-474.rT cells were

22 treated with 15 µg/ml trastuzumab for 7 days, and cell growth was assessed.

23 Transfection was repeated after 72 h to maintain silencing. For gene- and protein-

24 expression analysis, cells were subjected to siRNA silencing for 48 h, lysed, and

25 subjected to qPCR and WB assays as described above.

1 Murine model

2 We developed an in vivo xenograft subcutaneous breast cancer model at the facilities of

3 the Barcelona Biomedical Research Park (PRBB). All experiments were performed in

4 accordance with the 2010/63/EU Directive on the protection of animals and approved

5 by the Ethical Committee for Animal Research of the Barcelona Biomedical Research

6 Park (EEA-PRBB). Six-week-old female mice, with severe combined

7 immunodeficiency/beige (SCID/Beige, Charles River, USA), were selected for

8 inoculation. A 17β-estradiol pellet, 0.72 mg, 60-day release (Innovative Research of

9 America, USA) was implanted subcutaneously into each mouse 48 h before cell

10 injection. Twenty mice were subcutaneously inoculated in their right flank with 2.5x106

11 BT-474.rT cells mixed with 1:1 Matrigel (BD Biosciences, USA) in PBS as previously

12 described(29). Tumor diameters were serially measured with digital calipers and tumor

13 volumes were calculated by the equation: volume = (width2 x length)/2. When the

14 average volume of tumors reached 100 mm3, mice were randomly allocated into four

15 groups of five mice each. For therapeutic studies, the concentration and time of

16 treatments were based on previous reports, and administered as follows: Group 1, mice

17 received control treatment with human IgG1ĸ (10 mg/kg); Group 2, trastuzumab (10

18 mg/kg); Group 3, maraviroc (10 mg/kg); Group 4, mice received the combination of

19 trastuzumab and maraviroc (10 mg/kg and 10 mg/kg, respectively). All treatments were

20 freshly prepared in PBS, allowing for an injection volume of 100 µl/20 g mouse

21 intraperitoneally every other day for three weeks. After three weeks, tumor xenografts

22 obtained from BT-474.rT cells were excised and measured.

23 Immunohistochemistry (IHC)

24 FFPE sections (2-3 µm) were obtained from cell pellets of patient samples and human

25 tumor xenografts in mice, and IHC was performed as previously described(30). Primary

1 antibodies against pERK, cleaved-caspase3, pHistone3 (Cell Signaling Technology),

2 and CCL5 (R&D Systems) were used, and HER2 status was assessed by HercepTest

3 (Agilent Technologies). A semiquantitative histoscore (Hscore) was calculated by

4 estimation of the percentage of tumor cells positively stained with low, medium, or high

5 staining intensity, and the results ranged from 0 to 300(31). All immunohistochemical

6 staining was performed on a Dako Autostainer platform (Agilent Technologies).

7 Analysis of TCGA samples

8 We downloaded read count data for 1097 primary breast tumors from TCGA(32, 33)

9 (http://cancergenome.nih.gov, January, 2015) using the R package in TCGA-Assembler.

10 We obtained clinical data for the breast-cancer samples from the original clinical dataset

11 (clinical_patient_piblic_brca.txt) as described in the previous studies.

12 Patients and tumor samples

13 One hundred and forty-six specimens from primary breast tumors were obtained from

14 the Fundación Jiménez Díaz Biobank. Tumor specimens from FFPE blocks were

15 retrospectively selected from consecutive breast cancer patients diagnosed between

16 2000 and 2014, following these criteria: infiltrating carcinomas, operable, neoadjuvant

17 (N=64), or adjuvant therapy (N=82) containing trastuzumab, enough available tissue

18 and clinical follow-up. In addition, 17 cases from which serum samples were available

19 prior to treatment were selected from the Biobank of the Hospital del Mar. For all cases,

20 clinical data were collected from medical clinical records by oncologists, following

21 written informed consent from the patients. The studies were conducted in accordance

22 with ethical guidelines from the Declaration of Helsinki. The ethical committee and

23 institutional review boards from our hospitals approved the project. Clinical tumor

24 response to primary chemotherapy was evaluated for pathological response according to

25 the International Union against Cancer Criteria (UICC/AJCC) staging system(34).

1 Statistical analysis

2 Receiver operating characteristic (ROC) analysis was used to determine the optimal

3 cutoff point based on relapse end point for CCL5 expression as previously

4 described(35). Survival was analyzed by the Kaplan-Meier method using the log-rank

5 test. OS was defined as the time from diagnosis to the date of death from any cause or

6 last follow-up. DFS was defined as the time from diagnosis until the first event, in

7 which relapse at any location, death, or end of follow-up were considered events.

8 Multivariate analyses were carried out using the Cox proportional hazards model.

9 Analysis of experimental conditions was done by paired t-test. Statistical significance

10 was analyzed by a two-tailed Student’s t-test (*: p-value<0.05; **: p-value<0.01; ***:

11 p-value<0.001). This work was carried out in accordance with REMARK

12 guidelines(36).

1 RESULTS

2 The gene expression profiles of HER2-positive breast-cancer lines with acquired

3 resistance to trastuzumab revealed overexpression of cytokines

4 After generation of trastuzumab resistant lines(23), we decided to search for differences

5 at the gene level between these cells and their corresponding parental sensitive lines, in

6 the absence or presence of 15 μg/ml trastuzumab. Changes in gene expression were

7 considered as significant for -2≥logFC≥2 and a p-value<0.05. Principal component

8 analysis revealed separate clusters corresponding to the sensitive BT-474 and the

9 resistant BT-474.rT cells and proved that the differences in their gene expression pattern

10 were independent of trastuzumab exposure in culture. Expression-level data were

11 obtained for 10,508 genes, and we identified 25 genes with differential expression

12 between BT-474 and BT-474.rT. Of these, 15 genes were overexpressed in BT-474.rT

13 compared to BT-474 (Figure S1).

14 Gene set enrichment analysis (GSEA) demonstrated that the ERBB2/HER2 gene

15 set is enriched in BT-474.rT, and that the genes with higher overexpression in this line

16 relative to the parental contributed to the enrichment of this gene set. The most

17 significant contributing genes are interferon-inducing proteins IFI44, IFIT1, IFI44L, all

18 members of the superfamily of cytokines (Figure S2). Given their involvement in

19 proliferation and metastasis processes in breast cancer, 5 genes from the cytokine family

20 (CXCL10, CCL5, CXCL11, INFL1, and INFL2) were selected for assessment of their

21 transcript expression profiles by RT-qPCR. All 5 exhibited elevated expression levels in

22 the BT-474.rT line compared to the sensitive BT-474 (Figure 1A). In addition, their

23 expression levels were assessed on lines with primary resistance to trastuzumab

24 (JIMT11, HCC1569, and HCC1954): CCL5, CXCL10, and CXCL11 were found to be

25 overexpressed in all three lines, though to a lesser degree than BT-474.rT, whereas

1 IFNL1 showed levels of intermediate expression between BT-474.rT and BT-474

2 (Figure S3).

3 Cells with acquired resistance produced and secreted more CCL5 protein

4 Immunodetection by WB showed a marked increase in CCL5 protein in BT-474.rT

5 relative to its parental line, which had very similar protein levels to the HCC1569 line

6 (Figure 1B). This protein overexpression was also verified by IHC from cell pellets

7 (Figure 1C): no expression of CCL5 was detected in BT-474, whereas a heterogeneous

8 CCL5 overexpression was observed in BT-474.rT cells. In the primary resistance line,

9 HCC1569, a homogeneous CCL5 intermediate expression was identified in almost all

10 cells. As cytokines are known for their abilities to induce cellular migration, and CCL5

11 overexpressing cells are specifically reported to acquire invasion and migration

12 abilities(37), cell invasion assays were also performed to detect the invasive capacity of

13 these breast cancer cells. Interestingly, we observed significantly increased migration in

14 BT-474.rT cells in comparison with BT-474 control cells (Figure 1D), thereby

15 evidencing that resistance acquisition plays a relevant role in regulating the migration of

16 the cells. Additionally, clonogenic assays were performed to analyze whether

17 mechanistic changes due to acquired resistance could alter the malignancy of BT-474.rT

18 cells. We detected that treatment with trastuzumab for 21 days did not significantly

19 impair the colony-forming ability of cells with acquired resistance, in comparison with

20 sensitive BT-474 cells, proving that it was a stable resistance (Figure S4).

21 Finally, we assessed whether the CCL5 protein produced by these cell lines was

22 secreted into the medium, as happens in physiological conditions with chemokines. The

23 increase in CCL5 synthesis in BT-474.rT cells was determined by an ELISA assay in

24 the secretion to the medium (Figure 1E), with a concentration of 7,504 pg/ml CCL5 in

25 BT-474.rT compared to 144 pg/ml in BT-474.

1 Increased endogenous CCL5 levels were implicated in acquired resistance to

2 trastuzumab

3 The silencing of CCL5 by siRNA in BT-474.rT reversed trastuzumab resistance

4 significantly (p-value=0.001) in 7-day cell proliferation experiment with respect to the

5 control (54% vs. 82%), with a ΔGR value of 1.3 being sensitive according to the

6 algorithm defined by O'Brien(38) (Figure 2A). However, the HCC1569 line transfected

7 with siCCL5 presents a growth in the presence of trastuzumab similar to the control

8 condition (siC), indicating that this line remains resistant to trastuzumab independently

9 of the reduction of CCL5 levels (Figure 2A). The silencing of its receptor CCR5 and the

10 dual silencing of CCL5/CCR5 reversed trastuzumab resistance even more intensely,

11 adding more evidence to the implication of CCL5 in the acquisition of resistance

12 (Figure 2B). The efficiency of CCL5 and CCR5 silencing was confirmed by qPCR and

13 ELISA in both lines (Figure S5).

14 Next, we evaluated whether sensitization to trastuzumab in the BT-474.rT line

15 caused by the silencing of CCL5 was compensated by the addition of exogenous CCL5

16 (CCL5r) (Figure S6). The addition of CCL5r under CCL5 silencing conditions

17 significantly increased cell growth (p-value=0.003) in the presence of trastuzumab up to

18 resistance levels (ΔGR=1.1). These data support the argument that an increase in levels

19 of CCL5 is implicated in acquired resistance to trastuzumab.

20 Pharmacological inhibition of CCL5 activity with maraviroc resensitized cells to

21 treatment with trastuzumab

22 Maraviroc is a negative allosteric modulator antagonist of the CCR5 receptor and

23 blocks its activation by preventing the binding of CCL5 to the receptor(22). The cell

24 lines BT-474 and BT-474.rT were exposed to different concentrations of maraviroc (5-

25 200 μM) to assess its effect in cell growth. A count after 7 days revealed that exposure

1 to maraviroc at high concentrations did not result in a significant inhibition of cell

2 proliferation in any of the lines tested. When BT-474.rT cells were treated with

3 15 μg/ml trastuzumab—either with 10 μM maraviroc or with the combination of both

4 drugs—it was observed that treatments individually did not provoke inhibition of

5 proliferation; however, the combination of trastuzumab plus maraviroc caused a

6 significant decrease in cell proliferation and reversed acquired resistance (47%, p-

7 value<0.001) (Figure 2C).

8 In treatment conditions with trastuzumab, a marked decrease in pERK was

9 observed in BT-474 (70% decrease on average, by densitometric analysis), while ERK

10 phosphorylation levels are less affected by trastuzumab treatment in BT-474.rT (20%

11 decrease). This fact indicates that, on the BT-474.rT cell line, treatment with

12 trastuzumab is not able to block the activation of ERK (Figure 2D). At the molecular

13 level, the combination of trastuzumab and maraviroc caused a significant decrease in

14 ERK activation levels in the resistant cell line that was not observed in the single

15 treatment with maraviroc or trastuzumab (Figure 2E). Treatment with maraviroc in

16 combination with trastuzumab causes re-sensitization to trastuzumab in the resistant

17 line, due to decreased levels of pERK, suggesting that increased CCL5 levels favor

18 activation of ERK (Figure 2E). In addition, treatment with maraviroc alone or in

19 combination with trastuzumab did not result in a decrease in HER2 levels. Similarly, a

20 decrease in pAKT levels (both in Thr308 and Ser473 levels) was attributed to

21 trastuzumab treatment (alone or in combination) but not to maraviroc alone (Figure S7).

22 The confirmation of our hypothesis that high CCL5 levels cause the activation of ERK

23 in the resistant cells was revealed with the dual treatment of the BT-474.rT cell line with

24 trastuzumab plus selumetinib (Figure 2F). When the activation of ERK was repressed

25 with the MEK specific inhibitor selumetinib (Figure 2G), cell proliferation of resistant

1 cells treated with the drug combination significantly decreased as compared to

2 trastuzumab-treatment alone (Figure 2F), therefore improving its therapeutic response.

3 Effect of combined therapy with trastuzumab and maraviroc on xenograft tumor

4 growth

5 The assessment of maraviroc-mediated restoration of sensitivity to trastuzumab was

6 performed with a BT-474.rT cell line-derived xenograft model to investigate the role of

7 CCL5 in tumor growth. After tumors reached a minimum volume of 100 mm3, mice

8 were allocated at random to one of four treatment groups: control group (10 mg/kg

9 IgG1ĸ) and treatment groups (trastuzumab 10 mg/kg, maraviroc 10 mg/kg, and

10 trastuzumab 10 mg/kg plus maraviroc 10 mg/kg in combination). Similar to the in vitro

11 results, mice treated with the combination of trastuzumab and maraviroc displayed

12 significantly less tumor growth (10%, compared with day 0; p=0.004) than those of the

13 control group treated with IgG ĸ and those of the groups receiving trastuzumab or

14 maraviroc treatments alone (170%, 120% and 60%, respectively, compared with day 0,

15 Figure 3).

16 CCL5 expression analysis in human HER2-positive breast cancer

17 The validation of CCL5 detection was performed according to the Rimm algorithm for

18 IHC validation(39), and the optimal dilution of the anti-CCL5 antibody was determined

19 to be 1:40. The CCL5 overexpression threshold was determined by the ROC curve

20 based on the endpoint of relapse, calculated as the area under the curve (AUC) (Figure

21 S8). Samples with values of H-score>150 (sensitivity=75%, specificity=100%) were

22 considered as having high CCL5 overexpression. Tumor tissue sections showed a CCL5

23 cytoplasmic expression in patches or homogeneous staining in neoplastic cells, with

24 variable intensity ranging between weak and strong. In normal mammary epithelial

25 tissue adjacent to the tumor, CCL5 expression could not be observed. Consistently, and

1 as expected, CCL5 expression was detected in lymphocytes and plasma cells present in

2 the tumor stroma and adjacent mammary parenchyma. This expression of CCL5 in

3 mononuclear inflammatory cells was used as positive internal control in the cases

4 evaluated in the different study cohorts (Figure 4A). For pERK1/2, nuclear staining was

5 required for considering a tumor cell as positive (Figure 4A). A tumor was scored as

6 positive when any proportion of tumor cells was stained. Endothelial cells and

7 lymphocytes were considered as internal positive and negative controls for each slide.

8 CCL5 expression in tumor cells predicted benefit to trastuzumab therapy in early

9 HER2-positive breast cancer

10 Clinical assessment of CCL5 overexpression in resistance to trastuzumab was studied in

11 samples from a clinical cohort of 146 cases of early HER2-positive breast carcinoma

12 treated with trastuzumab in different regimens. Sixty-four FFPE samples of neoadjuvant

13 treatment of trastuzumab plus chemotherapy were included in this cohort (Table S2).

14 One-third of these cases showed high levels of CCL5 expression in tumor cells.

15 Expression of CCL5 was not significantly correlated with hormonal status, histological

16 type and grade, hormone receptors, proliferation, or stage. However, CCL5 expression

17 did correlate significantly with complete pathological response to treatment (Table S2,

18 Figure 4B). Seventy-one percent of the tumors with no evidence of pathological

19 response (Miller & Payne grade G1-G3) had a high expression of CCL5, while only

20 14% of tumors with either almost complete tumor response (G4) or complete response

21 (G5) had high expression of CCL5 (p-value<0.001). In fact, none of the tumors with

22 complete tumor response (G5) had high expression of CCL5. Considering the degree of

23 lymph node response, CCL5 overexpression was detected in 52% of the cases without

24 evidence of response to treatment (B and C) (Figure 4C). On the other hand, high levels

1 of CCL5 were detected in 5 cases with negative lymph nodes (A) and 5 cases with

2 lymph nodes without residual neoplastic infiltration (D) (p-value=0.013).

3 Taking together the pathological response in the breast and in the axilla, 24 cases

4 with complete pathological response were identified, all with low CCL5 expression

5 levels. Conversely, most of the 40 cases that did not present complete pathological

6 response showed high levels of CCL5 (p-value<0.001) (Table S2). Finally, CCL5

7 overexpression correlated with more frequent disease relapse: 73% of cases with

8 increased expression suffered relapse, compared to 27% of those with low expression

9 levels (p-value=0.002) (Table S2). These findings indicated that an increase in CCL5

10 expression levels in the tumor component predicts a worse response to trastuzumab

11 treatment.

12 The adjuvant cohort consisted of 82 samples taken prior to therapy from patients

13 with early breast cancer treated with trastuzumab plus chemotherapy (Table S3). In this

14 cohort, CCL5 overexpression was detected in 22% of the cases. CCL5 expression was

15 not significantly associated with stage, histology, tumor grade, hormonal receptors, or

16 proliferation. Relapse of the disease (35% of the patients) was significantly correlated

17 with the expression of CCL5 (p-value<0.001), with high levels of CCL5 being

18 identified in 72% of cases with relapse (Table S3). As we had evidence, from the

19 molecular assays in cell lines, of the potential role of ERK activation as a mediator of

20 the resistance elicited by CCL5, we also determined the expression levels of pERK in

21 this cohort. We found a strong correlation of the overexpression of both markers (p-

22 value=0.001, Figure S9).

23 Finally, we correlated CCL5 expression levels with evolution of patients in the

24 entire cohort of early breast cancer. Increased CCL5 expression in the tumor component

25 was significantly associated with lower DFS (median 29 vs. 42 months, p-value<0.01,

1 Figure 5A), and a lower OS (p-value<0.001, Figure 5B). The DFS analysis separately in

2 neoadjuvant and adjuvant series was also significant (median 54 vs. 104 months, p-

3 value=0.001 in neoadjuvant; 44 vs. 93 months, p-value<0.001 in adjuvant setting;

4 Figure S10). A multivariate Cox analysis including all the significant clinical-

5 pathological factors from the univariate study, as well as estrogen receptors and

6 chemotherapy regimen, revealed that CCL5 overexpression behaved as an independent

7 factor of poor prognosis in patients with early HER2-positive breast cancer (HR: 13.6;

8 95% CI: 3.4-54.8; p-value<0.001) (Table S4).

9 Serum CCL5 levels in early HER2-positive breast cancer predicted pathological

10 complete response in neoadjuvant trastuzumab containing therapy

11 A small cohort of 14 sera samples from patients with HER2-positive breast cancer

12 collected prior to initiation of trastuzumab treatment in a neoadjuvant regimen was also

13 analyzed. CCL5 concentration in sera was determined by ELISA assay, and the

14 optimum cutoff point was calculated using a ROC curve, which was established at a

15 concentration of CCL5≥90 ng/ml in serum (sensitivity=71%, specificity=86%). Forty-

16 three percent of serum samples displayed high CCL5 concentrations, correlating

17 significantly with the degree of pathological response: 75% of the patients with low

18 CCL5 concentration in serum presented complete response, whereas only 17% of

19 patients with a high concentration of CCL5 showed this complete response (Table S5).

20 The expression of CCL5 is modulated in the natural history of the disease

21 We decided to evaluate the modulation of CCL5 expression in response to trastuzumab

22 exposure in a cohort of 44 patients with paired samples (pre- and post-treatment

23 samples). Of these, 25 pairs were obtained from patients with early HER2-positive

24 breast cancer treated in a neoadjuvant regimen (the pre-treatment sample corresponded

25 to the diagnostic biopsy and the post-treatment sample to the surgical specimen).

1 Continued exposure to trastuzumab in those patients caused a significant increase in

2 CCL5 expression: 40% of post-treatment samples showed increased CCL5 expression

3 in their tumor component as compared to their pre-treatment samples (p-value=0.022),

4 whereas 28% of them exhibited lower CCL5 levels, and the remainder did not vary

5 (Figure 5C).

6 Moreover, 19 additional cases were selected from patients in whom progression of

7 the disease had been detected during the conventional treatment of chemotherapy plus

8 trastuzumab, and for whom both a pre-treatment diagnostic sample and a metastatic

9 after-treatment sample were available. Analysis of CCL5 expression revealed that 58%

10 of the post-treatment samples presented a statistically significant increase in CCL5

11 expression (p-value=0.012), while 16% of samples showed a decrease in CCL5

12 expression after treatment (and 26% of paired samples did not change their CCL5

13 expression levels) (Figure 5D).

14 Overexpression of CCL5 in patients with HER2-positive breast cancer associated

15 with lower OS

16 We analyzed mRNA expression levels of CCL5 in a series of 182 HER2-positive breast

17 cancer patients from available data on TCGA and correlated with OS. A tendency

18 toward association of CCL5 overexpression with worse OS was demonstrated, although

19 this was non-significant (p-value=0.069) (Figure S11). On the other hand, expression of

20 CCR5 (the most common receptor for CCL5) showed a non-significant tendency to be

21 associated with overall poorer prognosis (p-value=0.086). The correlation study with

22 the CCL5 remaining receptors (CCR1, CCR2, CCR3, and CCR4) showed that

23 overexpression of CCR3 correlated significantly with a lower OS in HER2-positive

24 breast cancer patients (p-value=0.038) (Figure S11).

1 DISCUSSION

2 HER2-positive tumors account for 20-25% of all cases of breast cancer. With the

3 introduction of anti-HER2 therapies in clinical practice, however, the prognosis of these

4 tumors is now favorable, and DFS and OS are increasing. However, a high percentage

5 of patients with HER2-positive tumors do not respond to therapy with trastuzumab,

6 mostly due to the presence of other genetic alterations in the tumor that are either

7 unknown or undetermined in clinical practice. These additional alterations may cause

8 the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation

9 and metastasis.

10 Our results suggest that continued exposure to trastuzumab in cellular models

11 leads to drug resistance. Differential gene expression analysis identified 16 significantly

12 overexpressed genes on the BT-474.rT line compared to the BT-474 line. Most

13 strikingly, 13/16 genes belong to the cytokine superfamily (CXCL10, CCL5, CXCL11,

14 IFNL1, and IFNL2) or are involved in the activation of members of this family (IFIT3,

15 IFI44, IFI6, IFIT1, IFI44L, IFIT2, OAS1, and OASL). Cytokines are the main proteins

16 secreted into the extracellular domain, and although their main role is the recruitment

17 and activation of the immune response, their implication becomes increasingly relevant

18 in neoplastic processes of invasion, metastasis, and immune response evasion(40, 41).

19 Indeed, it has been reported that the cytokine profile secreted from the tumor site varies

20 between different breast cancer subtypes. In the HER2-positive breast cancer subtype,

21 production of cytokines such as IL6 and CCL5 are predominant and implicated in cell

22 proliferation(42). Notoriously, CCL5 was one of the top genes differentially

23 overexpressed in the expression analysis. The main function of the CCL5 chemokine is

24 its chemotactic activity in the immune system; however, it has an important autocrine

25 function in the tumoral component. In addition, reports suggest that CCL5 is frequently

1 overexpressed in basal-phenotype breast cancer, HER2-positive, as well as in advanced

2 disease(42). In all these settings, high levels of CCL5 in breast lines are associated with

3 increased proliferation and migration(43), stem phenotype(44), metastasis(17), and

4 immune cell infiltration(18). Although the other chemokines detected in the differential

5 expression array made in resistant and sensitive cells are also described in breast cancer,

6 their role is more controversial, and is mainly associated with luminal type breast

7 tumors.

8 CCL5 overexpression induction in tumor cells is modulated by the transcription

9 factor AP-1, activated by the binding of the also transcription factor c-Jun, which in turn

10 is activated by the cellular signaling path JNK. The activation of AP-1 for CCL5

11 synthesis is mainly by NF-κB via AKT, and the MAPKs pathway(44). Notably, the

12 overexpression of CCL5 prompted cell proliferation and migration through the

13 activation of mTOR(25). Recently, CCL5 has been associated with resistance to

14 different treatments in breast cancer: in cellular models of luminal subtype,

15 overexpression of CCL5 caused phosphorylation and activation of STAT3, and it was

16 postulated as a possible mechanism of resistance to tamoxifen(45). Another study

17 showed that the activation of an IL6-inflammatory loop including CCL5 mediated

18 trastuzumab resistance in HER2-positive breast cancer cellular models by expanding the

19 cancer stem-cell population(46): an IL6-mediated increase resulted in activation of

20 AKT, STAT3, NF-κB, and the subsequent rise of CCL5 levels, ultimately responsible

21 for the resistance. Recently, a study linked the inhibition of HER2 with an increased

22 CCL5 signaling, which promoted macrophage recruitment and ultimately resulted in

23 tumor recurrence(18). One might speculate that CCL5 plays a role in acquired

24 trastuzumab resistance by attracting macrophages in the stroma, which supply residual

25 cancer cells with collagen and ultimately promoting tumor growth. Other reports,

1 however, suggest that overexpression of CCL5 is associated with a better response to

2 treatment with trastuzumab(42). The acquisition of resistance to specific anti-HER2

3 therapies by autocrine production of ligands that activate compensatory pathways has

4 also been described in HER2-positive breast cancer models. In particular, acquired

5 therapeutic resistance was reported when incomplete inhibition of EGFR by lapatinib

6 resulted in selection of an heregulin-driven feedback that activated the HER3-EGFR-

7 PI3K-PDK1 signaling axis(47). Similar, insulin growth factor-I (IGF-I) activation of

8 receptor IGF-IR signaling has been associated with trastuzumab resistance in HER2-

9 positive breast cancer models(48).

10 Our functional studies indicate that CCL5 is involved in acquired resistance to

11 trastuzumab. Both the silencing of CCL5 and its pharmacological inhibition by

12 maraviroc provoke a reversal of resistance to trastuzumab in BT-474.rT cells. Our

13 results further suggest that such resistance could be produced by the constitutive

14 activation of ERK: unlike that which occurs in the sensitive model, treatment with

15 trastuzumab does not affect ERK activation in resistant cells, indicating that this

16 pathway is being activated by other mechanisms. In addition, treatment with maraviroc

17 plus trastuzumab results in a decrease in ERK activation, suggesting that the reversal of

18 resistance to trastuzumab caused by CCL5 blockade is mediated by a reduction in ERK

19 activation levels. These results agree with recent studies describing that the interaction

20 of CCL5 with its receptor CCR5 causes direct activation of ERK, thus promoting an

21 increase in cell migration(49) and providing antiapoptotic signals for cell survival(50).

22 In addition, the interaction of CCL5 with other receptors such as CCR1 in different

23 cancer types also causes an increase in ERK phosphorylation that promotes the

24 expression of MMP2 and MMP9 in taxane resistance models(51, 52). In addition, the

25 decrease in cell proliferation of resistant cells treated with trastuzumab in combination

1 with selumetinib, a specific MEK repressor, confirmed the role of ERK activation in

2 CCL5-mediated acquisition of trastuzumab resistance. Our results also indicate that the

3 resistance was not mediated by pAKT, since the combined treatment of trastuzumab

4 with maraviroc modulates AKT in a way that resembles trastuzumab monotherapy. This

5 agrees with previous reports indicating that CCR5 chemokines did not induce any

6 significant AKT phosphorylation(53).

7 Our proposal of this role of CCL5 as a mediator of resistance acquisition to

8 trastuzumab was not applicable to primary resistant cell models. As it was proved for

9 the HCC1569 line, blocking of CCL5 (either by RNA silencing or by treatment with

10 maraviroc) did not significantly decrease cell proliferation. Additionally, the evidence

11 of hyperactivation of the PI3K/AKT/mTOR pathway in HCC1569, mediated by the loss

12 of PTEN expression(38), suggested that CCL5 was not responsible of the primary

13 resistance in those cells.

14 On the other hand, the addition of exogenous CCL5 (CCL5r) to the sensitive line

15 does not lead to an increase in resistance to trastuzumab, suggesting that the acquisition

16 of resistance is a complex process that may require additional alterations to the

17 overexpression of CCL5. Previous studies identified that the interaction between CCL5

18 and CCR5 was required to observe proliferation and migration processes, which in

19 addition required overexpression of both CCL5 and its receptor(22, 25). In summary,

20 our work suggests that CCL5 overexpression causes resistance to trastuzumab treatment

21 through ERK activation, and that the resistance process could be mediated by different

22 CCL5 receptors. Notably, there are other cytokines overexpressed in our resistance cell

23 model that have not been assessed in this study and might also contribute to acquired

24 resistance to trastuzumab.

1 Finally, the tumorigenic potential of the BT-474.rT cell line and the trastuzumab-

2 resistant phenotype of the derived tumors were both confirmed in a BT-474.rT

3 xenograft model in mice. Although maraviroc administration as a single treatment was

4 ineffective, it showed significant antitumor effects when it was administered in

5 combination with trastuzumab, in terms of size reduction. This supported our previous

6 in vitro observations, and therefore suggested that acquired trastuzumab resistance may

7 be mediated by CCL5 activity in vivo. Collectively, these results indicate that the

8 combined therapy with verteporfin overcomes acquired trastuzumab resistance in vivo

9 by blocking tumor growth and inducing tumor reversion.

10 Overexpression of CCL5 has also been described in breast cancer patients,

11 correlated with an advanced disease and presence of a greater number of metastases,

12 and associated with worse prognosis(17, 54). The increase in plasma CCL5

13 concentration has also been associated with poor prognosis of the disease(43). In

14 addition, both the basal and the HER2-positive molecular subtypes exhibit a higher

15 CCL5 expression in the tumor component(42). Our analyses demonstrate that CCL5

16 overexpression is an independent factor of poor prognosis that is significantly

17 associated with lower DFS and OS in early HER2-positive breast cancer, and to lower

18 OS in advanced HER2-positive breast cancer.

19 In addition, overexpression of this chemokine could be implicated in resistance to

20 trastuzumab in early HER2-positive breast cancer. Patients with high levels of CCL5

21 have significantly poorer response rates to trastuzumab when treated with neoadjuvant

22 anti-HER2 trastuzumab, and no pathological complete response has been observed in

23 these cases. The analysis of CCL5 in serum samples prior to treatment showed that a

24 high CCL5 concentration significantly correlated with a poorer pathological response to

25 neoadjuvant treatment. Furthermore, the analysis of the evolution of CCL5 levels over

1 the course of the disease (by comparing pre- and post-treatment samples) significantly

2 demonstrated that continued exposure to trastuzumab resulted in increased CCL5

3 expression, which correlated with a higher rate of relapse.

4 Previous reports have proposed CCL5 as a good predictor of response to

5 neoadjuvant trastuzumab therapy in HER2-positive breast cancer because of its primary

6 function as chemoattractant of lymphocytes and immune cells(55). The CCL5 transcript

7 levels determined in those works corresponded, however, to global expression levels

8 that do not discriminate between expression in the stroma, lymphocytes, or tumor

9 component; they also value the paracrine effect of CCL5, which increases lymphocyte

10 recruitment. Our study, on the other hand, emphasizes the importance of CCL5

11 expression in the tumor component, revealing a lower pCR to trastuzumab when CCL5

12 expression is increased. Survival analysis from results from the TCGA database showed

13 that patients with CCL5 overexpression exhibited a tendency toward a worse OS. In

14 addition, the correlation analysis with the CCL5 receptors showed that the increase of

15 CCR3 is significantly associated with a lower OS, and that there is a trend in the same

16 direction for increased expression of CCR1 and CCR5. These data support the

17 hypothesis that an increase in CCL5 levels and its implication in resistance to

18 trastuzumab or its incidence in survival must be associated with a corresponding

19 increase of its receptors (CCR1, CCR3, and CCR5). Accordingly, the scientific

20 literature confirms that in HER2-positive breast tumors, CCL5 overexpression

21 correlates with CCR5 overexpression as well as an increase in CCR1 expression in the

22 basal and HER2-positive subtypes(22, 42).

23 Our findings in samples of patients with HER2-positive breast cancer treated with

24 trastuzumab indicate that CCL5 overexpression in the infiltrating tumor component

25 behaves as an independent predictive factor of lower response to treatment and is

1 associated with a poorer prognosis of the disease. In addition, we suggest that the

2 activation or increase in the expression rate of some CCL5 receptor might be implicated

3 in the acquired resistance to trastuzumab. In any case, determination of CCL5/CCL5-

4 receptor expression levels in the tumor component in patients with early HER2-positive

5 breast cancer who are candidates for treatment with trastuzumab could be used as a

6 predictor of response to treatment.

1 AUTHORS’ CONTRIBUTIONS

2 Conception and design: JMG, FR. Development of methodology: SZ, PGA, EMA, PM,

3 JMG, FR. Acquisition of data: SZ, PGA, EMA, CCh, ML, MSA, CC, PE, OA. Analysis

4 and interpretation of data: SZ, PGA, CCh, GGL, PM, IC, AR, JMG, FR. Writing,

5 review, and/or revision of the manuscript: SZ, JMG, FR. Administrative, technical, or

6 material support: PGA, SZ, EMA, CC, IC, AR. Study supervision: JGF, AL, JA, JMG,

7 FR.

1 ACKNOWLEDGMENTS

2 We thank Oliver Shaw for linguistic correction of the article.

3 The present work was supported by grants from the Spanish Ministry of Health,

4 Consumer Affairs and Social Welfare (AES Program, grants PI15/00934; PI18/00382

5 and PI18/00006); the Biomedical Research Networking Centre for Cancer

6 (CIBERONC); the Biobanks Platform, PT13/0010/0012; the Community of Madrid

7 (S2010/BMD-2344); and ProteoRed (PRB2-ISCIII, PT13/0001). PGA was supported

8 by a Fundación Conchita Rábago de Jiménez Díaz grant. PM was supported by the

9 ISCIII Miguel Servet Program (CP16/00116).

1 REFERENCES

2 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: a cancer journal for

3 clinicians. 2016;66:7-30.

4 2. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al.

5 Molecular portraits of human breast tumours. Nature. 2000;406:747-52.

6 3. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human

7 breast cancer: correlation of relapse and survival with amplification of the HER-2/neu

8 oncogene. Science. 1987;235:177-82.

9 4. Nguyen PL, Taghian AG, Katz MS, Niemierko A, Abi Raad RF, Boon WL, et

10 al. Breast cancer subtype approximated by estrogen receptor, progesterone receptor, and

11 HER-2 is associated with local and distant recurrence after breast-conserving therapy. J

12 Clin Oncol. 2008;26:2373-8.

13 5. Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, et al. American

14 Society of Clinical Oncology 2007 update of recommendations for the use of tumor

15 markers in breast cancer. J Clin Oncol. 2007;25:5287-312.

16 6. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al.

17 Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast

18 cancer that overexpresses HER2. The New England journal of medicine. 2001;344:783-

19 92.

20 7. Hudis CA. Trastuzumab--mechanism of action and use in clinical practice. The

21 New England journal of medicine. 2007;357:39-51.

22 8. de Melo Gagliato D, Jardim DL, Marchesi MS, Hortobagyi GN. Mechanisms of

23 resistance and sensitivity to anti-HER2 therapies in HER2+ breast cancer. Oncotarget.

24 2016;7:64431-46.

1 9. Gajria D, Chandarlapaty S. HER2-amplified breast cancer: mechanisms of

2 trastuzumab resistance and novel targeted therapies. Expert review of anticancer

3 therapy. 2011;11:263-75.

4 10. Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ. Mechanisms of disease:

5 understanding resistance to HER2-targeted therapy in human breast cancer. Nature

6 clinical practice. 2006;3:269-80.

7 11. Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L.

8 Treatment of HER2-positive breast cancer: current status and future perspectives.

9 Nature reviews Clinical oncology. 2012;9:16-32.

10 12. Pohlmann PR, Mayer IA, Mernaugh R. Resistance to Trastuzumab in Breast

11 Cancer. Clin Cancer Res. 2009;15:7479-91.

12 13. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell.

13 2011;144:646-74.

14 14. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet.

15 2001;357:539-45.

16 15. Taub DD, Oppenheim JJ. Chemokines, inflammation and the immune system.

17 Therapeutic immunology. 1994;1:229-46.

18 16. Azenshtein E, Luboshits G, Shina S, Neumark E, Shahbazian D, Weil M, et al.

19 The CC chemokine RANTES in breast carcinoma progression: Regulation of

20 expression and potential mechanisms of promalignant activity. Cancer Research.

21 2002;62:1093-102.

22 17. Stormes KA, Lemken CA, Lepre JV, Marinucci MN, Kurt RA. Inhibition of

23 metastasis by inhibition of tumor-derived CCL5. Breast Cancer Research and

24 Treatment. 2005;89:209-12.

1 18. Walens A, DiMarco AV, Lupo R, Kroger BR, Damrauer JS, Alvarez JV. CCL5

2 promotes breast cancer recurrence through macrophage recruitment in residual tumors.

3 eLife. 2019;8:e43653.

4 19. Pfizer. Celsentri/Selzentry® (Maraviroc). Available from:

5 https://wwwviivhealthcarecom/media/press-releases/2011/september/regulatory-update-

6 celsentriselzentry-maravirocaspx. Accessed May 31, 2017.

7 20. Woollard SM, Kanmogne GD. Maraviroc: a review of its use in HIV infection

8 and beyond. Drug design, development and therapy. 2015;9:5447-68.

9 21. Abel S, Back DJ, Vourvahis M. Maraviroc: pharmacokinetics and drug

10 interactions. Antiviral Therapy. 2009;14:607-18.

11 22. Velasco-Velazquez M, Jiao X, De La Fuente M, Pestell TG, Ertel A, Lisanti MP,

12 et al. CCR5 antagonist blocks metastasis of basal breast cancer cells. Cancer Res.

13 2012;72:3839-50.

14 23. Zazo S, Gonzalez-Alonso P, Martin-Aparicio E, Chamizo C, Cristobal I, Arpi O,

15 et al. Generation, characterization, and maintenance of trastuzumab-resistant HER2+

16 breast cancer cell lines. American journal of cancer research. 2016;6:2661-78.

17 24. Soria G, Ben-Baruch A. The inflammatory chemokines CCL2 and CCL5 in

18 breast cancer. Cancer letters. 2008;267:271-85.

19 25. Murooka TT, Rahbar R, Fish EN. CCL5 promotes proliferation of MCF-7 cells

20 through mTOR-dependent mRNA translation. Biochemical and biophysical research

21 communications. 2009;387:381-6.

22 26. Pfaffl MW. A new mathematical model for relative quantification in real-time

23 RT-PCR. Nucleic Acids Res. 2001;29:e45.

1 27. Vassena R, Montserrat N, Carrasco Canal B, Aran B, de Onate L, Veiga A, et al.

2 Accumulation of instability in serial differentiation and reprogramming of

3 parthenogenetic human cells. Hum Mol Genet. 2012;21:3366-73.

4 28. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA,

5 et al. Gene set enrichment analysis: a knowledge-based approach for interpreting

6 genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545-50.

7 29. Garcia-Parra J, Dalmases A, Morancho B, Arpi O, Menendez S, Sabbaghi M, et

8 al. Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity

9 in HER2 overexpressing breast cancer. Eur J Cancer. 2014;50:2725-34.

10 30. Madoz-Gurpide J, Zazo S, Chamizo C, Casado V, Carames C, Gavin E, et al.

11 Activation of MET pathway predicts poor outcome to cetuximab in patients with

12 recurrent or metastatic head and neck cancer. Journal of translational medicine.

13 2015;13:282.

14 31. Rincon R, Cristobal I, Zazo S, Arpi O, Menendez S, Manso R, et al. PP2A

15 inhibition determines poor outcome and doxorubicin resistance in early breast cancer

16 and its activation shows promising therapeutic effects. Oncotarget. 2015;6:4299-314.

17 32. Cancer Genome Atlas N. Comprehensive molecular portraits of human breast

18 tumours. Nature. 2012;490:61-70.

19 33. Ciriello G, Gatza ML, Beck AH, Wilkerson MD, Rhie SK, Pastore A, et al.

20 Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer. Cell.

21 2015;163:506-19.

22 34. AJCC. AJCC Cancer Staging Handbook From the AJCC Cancer Staging

23 Manual: Springer-Verlag New York; 2010.

24 35. Generali D, Buffa FM, Berruti A, Brizzi MP, Campo L, Bonardi S, et al.

25 Phosphorylated ERalpha, HIF-1alpha, and MAPK signaling as predictors of primary

1 endocrine treatment response and resistance in patients with breast cancer. J Clin Oncol.

2 2009;27:227-34.

3 36. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM, et al.

4 Reporting recommendations for tumor marker prognostic studies. J Clin Oncol.

5 2005;23:9067-72.

6 37. Zhang Y, Yao F, Yao X, Yi C, Tan C, Wei L, et al. Role of CCL5 in invasion,

7 proliferation and proportion of CD44+/CD24- phenotype of MCF-7 cells and

8 correlation of CCL5 and CCR5 expression with breast cancer progression. Oncol Rep.

9 2009;21:1113-21.

10 38. O'Brien NA, Browne BC, Chow L, Wang Y, Ginther C, Arboleda J, et al.

11 Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab

12 but not lapatinib. Molecular cancer therapeutics. 2010;9:1489-502.

13 39. Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J, et al.

14 Antibody validation. BioTechniques. 2010;48:197-209.

15 40. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer. 2004;4:540-50.

16 41. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al. Involvement

17 of chemokine receptors in breast cancer metastasis. Nature. 2001;410:50-6.

18 42. Fertig EJ, Lee E, Pandey NB, Popel AS. Analysis of gene expression of secreted

19 factors associated with breast cancer metastases in breast cancer subtypes. Scientific

20 reports. 2015;5:12133.

21 43. Luboshits G, Shina S, Kaplan O, Engelberg S, Nass D, Lifshitz-Mercer B, et al.

22 Elevated expression of the CC chemokine regulated on activation, normal T cell

23 expressed and secreted (RANTES) in advanced breast carcinoma. Cancer Res.

24 1999;59:4681-7.

1 44. Jiao X, Katiyar S, Willmarth NE, Liu M, Ma X, Flomenberg N, et al. c-Jun

2 induces mammary epithelial cellular invasion and breast cancer stem cell expansion. J

3 Biol Chem. 2010;285:8218-26.

4 45. Yi EH, Lee CS, Lee JK, Lee YJ, Shin MK, Cho CH, et al. STAT3-RANTES

5 autocrine signaling is essential for tamoxifen resistance in human breast cancer cells.

6 Mol Cancer Res. 2013;11:31-42.

7 46. Korkaya H, Kim GI, Davis A, Malik F, Henry NL, Ithimakin S, et al. Activation

8 of an IL6 inflammatory loop mediates trastuzumab resistance in HER2+ breast cancer

9 by expanding the cancer stem cell population. Mol Cell. 2012;47:570-84.

10 47. Xia W, Petricoin EF, 3rd, Zhao S, Liu L, Osada T, Cheng Q, et al. An heregulin-

11 EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in

12 HER2+ breast cancer models. Breast Cancer Res. 2013;15:R85.

13 48. Lu Y, Zi X, Pollak M. Molecular mechanisms underlying IGF-I-induced

14 attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3

15 breast cancer cells. Int J Cancer. 2004;108:334-41.

16 49. Wang SW, Wu HH, Liu SC, Wang PC, Ou WC, Chou WY, et al. CCL5 and

17 CCR5 interaction promotes cell motility in human osteosarcoma. PLoS ONE.

18 2012;7:e35101.

19 50. Tyner JW, Uchida O, Kajiwara N, Kim EY, Patel AC, O'Sullivan MP, et al.

20 CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during

21 viral infection. Nat Med. 2005;11:1180-7.

22 51. Kato T, Fujita Y, Nakane K, Mizutani K, Terazawa R, Ehara H, et al.

23 CCR1/CCL5 interaction promotes invasion of taxane-resistant PC3 prostate cancer cells

24 by increasing secretion of MMPs 2/9 and by activating ERK and Rac signaling.

25 Cytokine. 2013;64:251-7.

1 52. Chen R, Lee WY, Zhang XH, Zhang JT, Lin S, Xu LL, et al. Epigenetic

2 Modification of the CCL5/CCR1/ERK Axis Enhances Glioma Targeting in

3 Dedifferentiation-Reprogrammed BMSCs. Stem cell reports. 2017;8:743-57.

4 53. Basu S, Broxmeyer HE. CCR5 ligands modulate CXCL12-induced chemotaxis,

5 adhesion, and Akt phosphorylation of human cord blood CD34+ cells. J Immunol.

6 2009;183:7478-88.

7 54. Esquivel-Velazquez M, Ostoa-Saloma P, Palacios-Arreola MI, Nava-Castro KE,

8 Castro JI, Morales-Montor J. The role of cytokines in breast cancer development and

9 progression. J Interferon Cytokine Res. 2015;35:1-16.

10 55. Denkert C, von Minckwitz G, Brase JC, Sinn BV, Gade S, Kronenwett R, et al.

11 Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or

12 without carboplatin in human epidermal growth factor receptor 2-positive and triple-

13 negative primary breast cancers. J Clin Oncol. 2015;33:983-91.

1 FIGURE LEGENDS

2 Figure 1. Acquisition of resistance to trastuzumab increased the expression of

3 CCL5 and other cytokines. A. Validation by qPCR of the results obtained in the gene

4 expression array for the genes CCL5, CXCL10, CXCL11, IFNL1, and IFNL2 in the

5 BT-474 and BT-474.rT cell lines. All genes show an increase in expression in line BT-

6 474.rT vs. the sensitive line. B. Validation of intrinsic CCL5 protein levels in BT-474,

7 BT-474.rT, and HCC1569 cellular lines by immunoblotting from 20 µg total protein

8 extract. C. Representative 100X images of CCL5 expression by IHQ. D. The

9 acquisition of resistance to trastuzumab enhances transwell migration in BT-474.rT

10 cells as compared to parental, sensitive BT-474 cells (* denotes p-value<0.05). E.

11 CCL5 abundance levels from cell culture as determined by ELISA.

12 Figure 2. CCL5 mediates acquired resistance to trastuzumab. A. Effect of CCL5

13 silencing on cell proliferation on BT-474.rT and HCC1569 lines treated with 15 µg/ml

14 trastuzumab for 7 days. The silencing on BT-474.rT reversed the resistance significantly

15 (p-value=0.0013) in 7-day counting experiments, with a ΔGR value of 1.3 being

16 sensitive according to the algorithm defined by O' Brien. B. Effect of CCR5 silencing

17 and dual CCL5/CCR5 silencing on cell proliferation on BT-474.rT. C. Effect on cell

18 proliferation of 10 µM maraviroc treatment in combination with 15 µg/ml trastuzumab,

19 on BT-474.rT and HCC1569 cell lines. D. Immunodetection analysis of indicated

20 proteins performed on BT-474, BT-474.rT, and HCC1569 lines in the presence and

21 absence of 15 µg/ml trastuzumab for 24 hours. E. Immunodetection carried out on the

22 BT-474-rT line under conditions of no treatment, treatment with 15 µg/ml trastuzumab,

23 50 µM maraviroc, and a combination of 15 µg/ml trastuzumab plus 50 µM maraviroc

24 for 24 hours. F. The addition of 5 µM selumetinib to the standard treatment of 15 µg/ml

25 trastuzumab significantly enhanced therapeutic response (by decreasing cell

1 proliferation) in cells with acquired resistance to trastuzumab. G. Specific inhibitor

2 selumetinib diminishes the activation signal of ERK, as revealed by immunodetection

3 of its phosphorylated form.

4 Figure 3. Maraviroc-mediated CCL5-blockade inhibits in vivo tumor resistance to

5 trastuzumab. BT-474.rT cells (2.5×106) were injected subcutaneously into the right

6 flank of the mice. Tumor volume was measured every 3 days after injection until the

7 tumor had grown to ≥ 100 mm3. Treatments were injected intraperitoneally every other

8 day for 3 weeks. Treatment with maraviroc restored sensitivity to trastuzumab (n = 5,

9 ***p < 0.001, two-tailed unpaired-samples Anova test).

10 Figure 4. Low expression of CCL5 predicted benefit to trastuzumab in early

11 HER2-positive breast cancer patients. A. IHC representative images (200X) of CCL5

12 and pERK from sections of FFPE samples of HER2-positive breast tumors. B. Box plot

13 of CCL5 expression levels in HER2-positive early breast cancer samples in neoadjuvant

14 treatment according to the degree of tumor pathological response by the Miller & Payne

15 system. C. Id. according to degree of lymph node response.

16 Figure 5. Kaplan-Meier's analysis of DFS and OS in the cohort of 146 HER2-

17 positive early breast cancer patients. A. CCL5 overexpression (gray line) was

18 associated with lower DFS (p-value<0.001) and B. OS (p-value<0.001). C. Modulation

19 of CCL5 expression levels throughout the natural history of the disease. Line chart

20 of CCL5 expression levels in paired pre- and post-treatment samples of 25 cases of

21 HER2-positive early breast cancer in neoadjuvant scheme. Full black lines correspond

22 to tumors showing an increase of CCL5 expression in the post-treatment specimen

23 (10/25); dashed-dotted lines indicate a decrease in CCL5 expression (7/25); and dashed

24 lines indicate no modification of CCL5 expression (8/25). D. Line chart of CCL5

1 expression levels in paired pre- and post-treatment samples of 19 cases that showed

2 progression to a metastatic lesion. Line codes as above.

Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

figure4_mct-19-1172r.tif

Autocrine CCL5 effect mediates trastuzumab resistance by ERK pathway activation in HER2-positive breast cancer

Sandra Zazo, Paula González-Alonso, Ester Martin-Aparicio, et al.

Mol Cancer Ther Published OnlineFirst May 13, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-19-1172

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2020/05/13/1535-7163.MCT-19-1172.DC1

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

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

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2020/05/13/1535-7163.MCT-19-1172. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.