Interleukin-6 is a potential therapeutic target in interleukin-6 dependent, estrogen receptor-α-positive breast cancer

Abstract

Introduction

Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis.

Methods

The role of the phosphorylated signal transducer and activator of transcription 3 pathway was investigated in ERα-positive breast cancer. A panel of cell lines was treated with exogenous IL-6. An IL-6 specific gene signature was generated by profiling ten ERα-positive breast cancer cell lines alone or following treatment with 10 ng/mL recombinant IL-6 or human marrow stromal cell-conditioned media, with or without siltuximab (a neutralizing anti-IL-6 antibody) and grown in three-dimensional tumor microenvironment-aligned cultures for 4 days, 5 days, or 6 days. The established IL-6 signature was validated against 36 human ERα-positive breast tumor samples with matched serum. A comparative MCF-7 xenograft murine model was utilized to determine the role of IL-6 in estrogen-supplemented ERα-positive breast cancer to assess the efficacy of anti-IL-6 therapy in vivo.

Results

In eight of nine ERα-positive breast cancer cell lines, recombinant IL-6 increased phosphorylation of tyrosine 705 of STAT3. Differential gene expression analysis identified 17 genes that could be used to determine IL-6 pathway activation by combining their expression intensity into a pathway activation score. The gene signature included a variety of genes involved in immune cell function and migration, cell growth and apoptosis, and the tumor microenvironment. Validation of the IL-6 gene signature in 36 matched human serum and ERα-positive breast tumor samples showed that patients with a high IL-6 pathway activation score were also enriched for elevated serum IL-6 (≥10 pg/mL). When human IL-6 was provided in vivo, MCF-7 cells engrafted without the need for estrogen supplementation, and addition of estrogen to IL-6 did not further enhance engraftment. Subsequently, we prophylactically treated mice at MCF-7 engraftment with siltuximab, fulvestrant, or combination therapy. Siltuximab alone was able to blunt MCF-7 engraftment. Similarly, siltuximab alone induced regressions in 90% (9/10) of tumors, which were established in the presence which were established in the presence of hMSC expressing human IL-6 and estrogen.

Conclusion

Given the established role for IL-6 in ERα-positive breast cancer, these data demonstrate the potential for anti-IL-6 therapeutics in breast cancer.

Keywords:

• breast cancer
• estrogen receptor
• gene signature
• paracrine IL-6
• siltuximab

Supplementary material

Summary of role of gene signature components in breast cancer

The expression of some of the genes within the IL-6 signature is known to be altered in breast cancer. The two progesterone-metabolizing enzyme genes in the signature, aldoketone reductases 1C1 and 1C2, may be down-regulated in some breast tumors. This can lead to reduced progesterone catabolism and ultimately persistent activation of progesterone receptors.¹ CCAAT/enhancer binding protein, delta (CEBPδ), a transcription factor, is known to exhibit tumor-suppressing properties;² however, it is inactivated in many cases of estrogen receptor (ER)-positive breast cancer by methylation, and this is associated with relapse, metastasis, and reduced survival.^3,4 Conversely, high expression of interferon-inducible guanylate binding protein 2 (GBP2) is associated with a more favorable prognosis in ER-positive breast cancer and may be predictive of response to anthracycline-based chemotherapy.⁵

The signature also contains three members of the calcium binding S100 family, S100A7 (psoriasin), S100A8 (calgranulin B), and S100A9; the expression of psoriasin has been observed in ERα-positive breast cancer, but more strongly in ERα-negative breast cancer.⁶ Psoriasin may enhance metastasis in ERα-negative breast cancer through regulation of matrix metalloproteinase −9 secretion and the actin cytoskeleton.⁷ Interestingly, expression of psoriasin is induced by epidermal growth factor in breast cancer cells.⁸ Furthermore, overexpression of calgranulin B and SA100A9 has been observed in ~50% of invasive ductal breast carcinomas, and this was associated with poor tumor differentiation, higher pathologic stage, and node metastasis.⁹ Lipocalin 2 (LCN2) is also overexpressed in some breast cancers, and it has been suggested that this may promote breast cancer progression by promoting angiogenesis and may indicate poor prognosis^10–12; similar findings have been reported for β-tubulin isotype III (TUBB3).¹³ It has also been shown that in ER-positive breast tumors that exhibited poor response to the aromatase inhibitor letrozole, α-1-antichymotrypsin, a serine proteinase inhibitor (SerpinA3), expression was significantly reduced.¹⁴

The expression of complement factor B is upregulated in breast cancer relative to normal breast tissue,¹⁵ and has previously been identified within a 76-gene signature of ER-positive primary node-negative breast tumors that may be predictive of metastasis.¹⁶ Transmembrane channel-like protein 5 is also upregulated in breast cancer,¹⁷ most notably in breast tumors with mutations in PI3K, which is associated with IL-6 downstream signaling.¹⁸ To date, no association between transmembrane channel-like protein 5 and outcomes in breast cancer has been reported.

Interferon-inducible transmembrane protein (IFITM) genes 1, 2, and 3 are ubiquitously expressed in humans and are induced by the Jak-STAT pathway signaling downstream of IL-6 and also oncostatin. While IFITM3 is upregulated following viral infection,¹⁹IFITM1, IFITM2, and IFITM3 are also upregulated specifically in colorectal tumors.²⁰ No studies have been published to date that have explored the functions of IFITM genes in breast cancer.

References

• JiQAoyamaCNienYDSelective loss of AKR1C1 and AKR1C2 in breast cancer and their potential effect on progesterone signalingCancer Res200464207610761715492289
   PubMed Web of Science ®Google Scholar
• GerySTanosakiSHofmannWKKoppelAKoefflerHPC/EBPdelta expression in a BCR-ABL-positive cell line induces growth arrest and myeloid differentiationOncogene20052491589159715674331
   PubMed Web of Science ®Google Scholar
• PalmieriCMonteverdeMLattanzioLSite-specific CpG methylation in the CCAAT/enhancer binding protein delta (CEBPdelta) CpG island in breast cancer is associated with metastatic relapseBr J Cancer2012107473273822782348
   PubMed Web of Science ®Google Scholar
• NaderiATeschendorffAEBarbosa-MoraisNLA gene-expression signature to predict survival in breast cancer across independent data setsOncogene200726101507151616936776
   PubMed Web of Science ®Google Scholar
• GodoyPCadenasCHellwigBInterferon-inducible guanylate binding protein (GBP2) is associated with better prognosis in breast cancer and indicates an efficient T cell responseBreast Cancer201421449149923001506
   PubMed Web of Science ®Google Scholar
• WolfRVoscopoulosCWinstonJHighly homologous hS100A15 and hS100A7 proteins are distinctly expressed in normal breast tissue and breast cancerCancer Lett2009277110110719136201
   PubMed Web of Science ®Google Scholar
• SnehADeolYSGanjuADifferential role of psoriasin (S100A7) in estrogen receptor alpha positive and negative breast cancer cells occur through actin remodelingBreast Cancer Res Treat2013138372773923535840
   PubMed Web of Science ®Google Scholar
• ParuchuriVPrasadAMcHughKBhatHKPolyakKGanjuRKS100A7-downregulation inhibits epidermal growth factor-induced signaling in breast cancer cells and blocks osteoclast formationPLoS One200833e174118320059
   PubMed Web of Science ®Google Scholar
• AraiKTakanoSTerataniTItoYYamadaTNozawaRS100A8 and S100A9 overexpression is associated with poor pathological parameters in invasive ductal carcinoma of the breastCurr Cancer Drug Targets20088424325218537548
   PubMed Web of Science ®Google Scholar
• NachtMFergusonATZhangWCombining serial analysis of gene expression and array technologies to identify genes differentially expressed in breast cancerCancer Res199959215464547010554019
   PubMed Web of Science ®Google Scholar
• YangJMcNeishBButterfieldCMosesMALipocalin 2 is a novel regulator of angiogenesis in human breast cancerFASEB J2013271455022982376
   PubMed Web of Science ®Google Scholar
• BauerMEickhoffJCGouldMNMundhenkeCMaassNFriedlANeutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancerBreast Cancer Res Treat2008108338939717554627
   PubMed Web of Science ®Google Scholar
• PentheroudakisGBatistatouAKalogerasKTPrognostic utility of beta-tubulin isotype III and correlations with other molecular and clinicopathological variables in patients with early breast cancer: a translational Hellenic Cooperative Oncology Group (HeCOG) studyBreast Cancer Res Treat2011127117919321390496
   PubMed Web of Science ®Google Scholar
• MillerWRLarionovAChanges in expression of oestrogen regulated and proliferation genes with neoadjuvant treatment highlight heterogeneity of clinical resistance to the aromatase inhibitor, letrozoleBreast Cancer Res2010124R5220646288
   PubMed Web of Science ®Google Scholar
• DoustjalaliSRYusofRYipCHLooiLMPillayBHashimOHAberrant expression of acute-phase reactant proteins in sera and breast lesions of patients with malignant and benign breast tumorsElectrophoresis200425142392240115274022
   PubMed Web of Science ®Google Scholar
• WangYKlijnJGZhangYGene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancerLancet2005365946067167915721472
   PubMed Web of Science ®Google Scholar
• CizkovaMCizeron-ClairacGVacherSGene expression profiling reveals new aspects of PIK3CA mutation in ERalpha-positive breast cancer: major implication of the Wnt signaling pathwayPLoS One2010512e1564721209903
   PubMed Web of Science ®Google Scholar
• WegielBBjartellACuligZPerssonJLInterleukin-6 activates PI3K/Akt pathway and regulates cyclin A1 to promote prostate cancer cell survivalInt J Cancer200812271521152918027847
   PubMed Web of Science ®Google Scholar
• DiamondMSFarzanMThe broad-spectrum antiviral functions of IFIT and IFITM proteinsNat Rev Immunol2013131465723237964
   PubMed Web of Science ®Google Scholar
• AndreuPColnotSGodardCIdentification of the IFITM family as a new molecular marker in human colorectal tumorsCancer Res20066641949195516488993
   PubMed Web of Science ®Google Scholar

Acknowledgments

The authors thank the following for their work in performing the experiments and analyses described in this paper: panels of Western blots to address alternate IL-6 pathways were performed by Genway Biotech, Inc; Western blots to assess pSTAT3 signaling were performed by Dr Nick Sullivan at Dr Tania Oberyszyn’s laboratory, Ohio State University; pSTAT3 digital image analyses were performed by Flagship Bioscience LLC. Brenda Hertzog performed pSTAT3 IHC work. Editorial assistance was provided by Christopher J Jones, PhD, of MedErgy and was funded by Janssen Research and Development, LLC. This study was supported by Janssen Research and Development.

Author contributions

Tineke Casneuf, Brett Hall, and Kate Sasser conceived and designed the study. Karin Verstraeten and Amy Axel performed the gene expression experiments. Tineke Casneuf

Disclosure

JLW was a consultant of Janssen Research and Development. TC, AEA, PK, JDA, TV, KV, AKS are employees of Janssen Research and Development. BMH is a former employee of Janssen Research and Development. No other potential conflicts of interest were disclosed by the other authors.