Interleukin-6 is a potential therapeutic target in interleukin-6 dependent, estrogen receptor-α-positive breast cancer - PubMed (original) (raw)

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

Tineke Casneuf et al. Breast Cancer (Dove Med Press). 2016.

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.

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Figures

Figure 1

Figure 1

Effect of recombinant IL-6 on STAT3-Tyr705 phosphorylation in ERα-positive and ERα-negative cell lines. Notes: Cell lines were treated with IL-6 and analyzed at baseline and 24 hours posttreatment. Each blot represents independent experiments and bands were digitally excised without modification and rearranged for presentation. Abbreviations: IL-6, interleukin-6; pSTAT3, phosphorylated signal transducer and activator of transcription 3; ER, estrogen receptor.

Figure 2

Figure 2

Effect of recombinant IL-6 on Akt, MEK 1/2, and ERK 1/2 phosphorylation in ERα-positive and ERα-negative cell lines. Notes: Cell lines were treated with IL-6 and analyzed at baseline and 24 hours posttreatment. Each blot represents independent experiments and bands were digitally excised without modification and rearranged for presentation. Abbreviations: IL-6, interleukin-6; Akt, serine-threonine kinase; MEK, MAPK/ERK kinase; ERK, extracellular signal regulated kinase; ER, estrogen receptor.

Figure 3

Figure 3

Strength of the IL-6 signature in ERα-positive breast cancer cell lines at different time points and under different treatment conditions. Notes: Bar graphs represent each sampling time point (days 4–6) following administration of additional treatment(s) at either day 0 or day 1. aD4–D1: IL-6: cell lines treated with IL-6 on day 1, harvested on day 4. bD5–D1: IL-6: cell lines treated with IL-6 on day 1, harvested on day 5. cD6–D1: IL-6: cell lines treated with IL-6 on day 1, harvested on day 6. dD4–D0: siltuximab: cell lines treated with siltuximab on day 0, harvested on day 4. eD4–D0: IL-6 + D1: siltuximab: cell lines treated with IL-6 on day 0, with siltuximab on day 1, harvested on day 4. fD4–D1: IL6 + D1: siltuximab: cell lines treated with IL-6 and siltuximab on day 1, harvested on day 4. gD4: hMSC-CM: hMSC-CM harvested on day 4. hD4: hMSC-CM + D0: siltuximab: hMSC-CM grown in presence of siltuximab as of day 0, harvested on day 4. Abbreviations: D, day; IL-6, interleukin-6; ER, estrogen receptor; hMSC-CM, human marrow stromal cell-conditioned media.

Figure 4

Figure 4

The relationship between elevated serum IL-6 and increased intratumoral phosphorylated STAT3-Y705 in human breast cancer samples. Notes: (A) Elevated IL-6 versus nonelevated IL-6 serum levels were analyzed using the Wilcoxon rank sum test. pSTAT3 was analyzed using immunohistochemistry (IHC); serum IL-6 was analyzed using a panoptic IL-6 meso scale detection (MSD) assay, _P_=0.04. Seven of 36 breast cancer samples were not profiled for pSTAT3 immunohistochemistry; and therefore, only 29 samples are represented in the figure. (B) Waterfall plot of IL-6 pathway activation score versus serum IL-6 concentration in human breast cancer samples. Abbreviations: IL-6, interleukin-6; pSTAT3, phosphorylated signal transducer and activator of transcription 3.

Figure 5

Figure 5

Summary of findings from MCF-7 murine tumor xenograft model. Notes: (A) 5×106 MCF-7 cells were coinjected into the murine mammary fat pad with either estrogen pellets (E2), human mesenchymal stromal cells (hMSCs, 0.5×106), or both. Top left, neither hMSC nor E2; top right, E2 only; bottom left, hMSC only; bottom right, hMSC + E2. (B) Percentages of animals demonstrating tumor volume ≥500 mm3.

Figure 6

Figure 6

Changes in tumor volume in the MCF-7 murine tumor xenograft model over a 6-week period. Notes: (A) Prophylactic treatment with 1) control (vehicle, □); 2) siltuximab 20 mg/kg bodyweight twice weekly (▲); 3) fulvestrant 200 mg/kg bodyweight once weekly (▼); or 4) siltuximab + fulvestrant (♦). Each treatment group contained ten mice. (B) Treatment of established tumors (volume 100–150 mm3) with 1) control (vehicle, □); 2) siltuximab 20 mg/kg bodyweight twice weekly (▲); 3) fulvestrant 200 mg/kg bodyweight once weekly (▼); or 4) siltuximab + fulvestrant (♦). Each treatment group contained ten mice. Abbreviation: SEM, standard error of the mean.

Figure 7

Figure 7

Functional interpretation of the IL-6 gene signature and its relationship to the three main downstream pathways of IL-6. Notes: The 17 genes are part of the Jak-STAT IL-6 downstream pathway. Members of the gene signature are represented by green boxes, and upstream pathway members are represented by white boxes. Abbreviations: IL-6, interleukin-6; MEK, MAPK/ERK kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidyl-inositol-3-kinase; Akt, serine-threonine kinase; STAT3, signal transducer and activator of transcription 3; CEBPD, CCAAT/enhancer binding protein; MMP9, matrix metalloproteinase 9; TUBB3, β-tubulin isotype III; CFB, complement factor B; TMC-5, transmembrane channel-like protein 5; GBP2, guanylate binding protein 2; AKR, aldoketone reductase; LCN2, lipocalin 2; IFITM, interferon-inducible transmembrane protein.

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