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Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

Nature Cell Biology volume 18, pages 1078–1089 (2016)Cite this article

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An Erratum to this article was published on 27 October 2016

This article has been updated

Abstract

Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.

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Figure 1: LIFR:STAT3 signalling is downregulated in patients with bone metastases and repressed by hypoxia.
Figure 2: Hypoxic regulation of LIFR:STAT3 signalling is independent of HIF1α or HIF2α.
Figure 3: The LIFR:STAT3 signalling pathway is intact in breast cancer cells with low metastatic potential and not intact in cells with high metastatic potential.
Figure 4: Inhibition of LIFR signalling alters dormancy-, quiescence- and cancer stem cell-associated genes.
Figure 5: Elevated Parathyroid hormone-related protein (PTHrP) signalling blocks LIFR signalling and reduces dormancy-, quiescence- and cancer stem cell-associated genes.
Figure 6: LIFR knockdown results in greater bone destruction via increased osteoclastogenesis and proliferation in vivo.
Figure 7: Loss of STAT3 signalling results in greater bone destruction in vivo.
Figure 8: Targeting STAT3 or SOCS3 mimics effects of loss of LIFR signalling on dormancy genes.

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Change history

  • 30 September 2016

    In the version of this Article originally published, Fig. 3b was duplicated in Fig. 3c. This has been corrected in the online versions of the Article.

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Acknowledgements

The authors wish to acknowledge A. Diep, G. Nelson and the Stanford Veterinary Service Center for technical assistance, and N. Sims and J. Martin for thoughtful discussion of LIFR signalling. Cell sorting/flow cytometry analysis for this project was performed on instruments in the Stanford Shared FACS Facility. The results published here are in whole or in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov and the ENCODE consortium (see Methods). R.W.J. received fellowship support from the Stanford Cancer Institute. This work was funded by NIH grant K99CA194198 (R.W.J.). J.A.S. was supported by VA grant 1I01BX001957 and NIH grant CA163499. A.J.G. was supported by NIH grants CA67166, CA197713 and CA198291, the Silicon Valley Foundation, The Kimmelman Fund, and the Skippy Frank Foundation.

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Authors and Affiliations

  1. Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University, Stanford, California 94305, USA

    Rachelle W. Johnson, Elizabeth C. Finger, Monica M. Olcina, Marta Vilalta, Todd Aguilera, Yu Miao & Amato J. Giaccia

  2. Department of Veterans Affairs: Tennessee Valley Healthcare System (VISN 9), Nashville, Tennessee 37212, USA

    Alyssa R. Merkel & Julie A. Sterling

  3. Department of Medicine, Division of Clinical Pharmacology, Nashville, Tennessee 37232, USA

    Alyssa R. Merkel & Julie A. Sterling

  4. Vanderbilt Center for Bone Biology, Nashville, Tennessee 37232, USA

    Alyssa R. Merkel & Julie A. Sterling

  5. Department of Medicine, Division of Endocrinology, Stanford University, Stanford, California 94305, USA

    Joshua R. Johnson & Joy Y. Wu

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Contributions

R.W.J. directed the project, developed the hypothesis, performed experiments, analysed data, and wrote the manuscript. E.C.F. designed and developed the LIFR promoter construct, performed experiments and edited the manuscript. M.M.O., M.V., T.A., Y.M., A.R.M. and J.R.J. performed experiments and edited the manuscript. J.A.S. performed experiments, conceptualized and interpreted data. J.Y.W. conceptualized and interpreted data. A.J.G. conceived the hypothesis, interpreted data and edited the manuscript.

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Correspondence to Amato J. Giaccia.

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Integrated supplementary information

Supplementary Figure 1 LIFR, STAT3 and SOCS3 alterations frequently co-occur.

(a–c) The Cancer Genome Atlas (TCGA) analysis of invasive breast carcinoma (Nature 2012 dataset) patient samples for mRNA down-regulation of (a) LIFR alone (n = 682 patients with no mRNA down-regulation, n = 17 patients with mRNA down-regulation), (b) STAT3 alone (n = 682 patients with no mRNA down-regulation, n = 22 patients with mRNA down-regulation), and (c) SOCS3 alone (n = 682 patients with no mRNA down-regulation, n = 6 patients with mRNA down-regulation). Logrank test. (d) Tendency for co-occurrence of alterations in LIFR/STAT3 (top line) and STAT3/SOCS3 (bottom line) in patient samples from invasive breast carcinoma (Nature 2012 dataset). Fisher exact test. n = 825 patients. Graphs represent the mean/group and error bars represent standard error of the mean (s.e.m.). ∗P < 0.05 and ∗∗∗P < 0.001.

Supplementary Figure 2 LIFR is regulated via DNA methylation in breast cancer patients likely to recur.

(a) Western blot for LIFR in MCF7 whole cell lysates following lentiviral knockdown. MCF7 non-silencing control (MCF7NSC) and MCF7shRNA #3 cells were used in all remaining experiments. Vinculin = loading control. (b) PGK1 mRNA levels for MCF7NSC cells cultured in normoxia (Nx) or hypoxia (Hx; 0.5% oxygen) for 24 h. 3 technical replicates from a single experiment representative of 2 independent experiments. (c) LIFR mRNA levels in SUM159 human breast cancer cells following 24 h culture in normoxia (Nx) or hypoxia (Hx; 0.5% oxygen). 3 technical replicates from a single experiment. (d) Correlation of LIFR mRNA levels (y-axis) or (e) STAT3 mRNA levels (y-axis) and LIFR DNA methylation (x-axis) in patients with invasive breast carcinoma (TCGA Provisional dataset, n = 1,104 patients). Pearson Correlation and Spearman Correlation. (f) LIFR mRNA levels (n = 850 patients disease free; n = 68 patients recurred/progressed) and (g) LIFR DNA methylation (n = 575 patients disease free; n = 51 patients recurred/progressed) in patients with invasive breast carcinoma (TCGA Provisional dataset). Student’s unpaired t-test. Source data for Suppl. 2b available in Supplementary Table 1. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001.

Supplementary Figure 3 LIF inhibits proliferation in breast cancer cells with low metastatic potential.

(a–c) XTT assay to determine proliferation of MCF7, MDA-MB-231b, and 4T1BM2 cancer cells in response to recombinant LIF (50 ng ml−1) or TGF-β (5 ng ml−1) over 3 days. Two-Way ANOVA with Tukey’s multiple comparisons test. n = 3 biological replicates/day. (d,e) SOCS3 mRNA levels in MCF7 and MDA-MB-231b cells following 1 or 6 h treatment with recombinant LIF (0–100 ng ml−1). One-Way ANOVA with Sidak’s multiple comparisons test. n = 3 biological replicates, each being an average from 3 independent experiments. Source data for Suppl. 3a–c available in Supplementary Table 1. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001.

Supplementary Figure 4 LIF stimulates STAT3:SOCS3 induction in breast cancer cells with low metastatic potential.

(a,c,e) Western blot for LIFR, pSTAT3 (Y705), total Stat3, and β-actin or vinculin (loading control) after 15 or 30 min treatment with PBS (vehicle control), recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1) in (a) PyMT and (c) D2.0R cells with low metastatic potential and (e) D2A1 cells with high metastatic potential. Blots represent 3 independent biological replicates/group. (b,d,f) SOCS3 mRNA levels after 1 or 6 h treatment with PBS, recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1). h: Mann-Whitney test. j,l: Student’s unpaired t-test.n = 3 biological replicates, each being an average from 3 independent experiments. (g,i) Western blot for LIFR, pSTAT3 (Y705), total Stat3, and vinculin (loading control) after 15 or 30 min treatment with PBS (vehicle control), recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1) in (g) 4T1 parental cells and (i) MDA-MB-231 parental cells. Blots represent 3 independent biological replicates/group. (h,j) SOCS3 mRNA levels after 1 or 6 h treatment with PBS, recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1) in (h) 4T1 parental cells and (j) MDA-MB-231 parental cells. One-Way ANOVA. n = 3 biological replicates, each being an average from 3 independent experiments. Unprocessed blots in Supplementary Fig. 9. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Supplementary Figure 5 LIFR knockdown does not alter proliferation but confers an invasive phenotype in vitro.

(a) Western blot for pSTAT3 (Y705), total Stat3, and β-actin (loading control) in MCF7NSC and MCF7shLIFR (#3 from Supplementary Fig. 1g) following 15 min treatment with PBS (vehicle control), recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1). (b) SOCS3 mRNA levels in MCF7NSC and MCF7shLIFR cells following 1 h treatment with PBS, recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1). 3 technical replicates from a single experiment representative of 2 independent experiments. (c) Cell counting assay to assess changes in proliferation in MCF7NSC and MCF7shLIFR cells over 3 days. 2 biological replicates/point. (d) Average tumor volume for MCF7NSC (n = 4 mice) or MCF7shLIFR (n = 3 mice) tumors inoculated into the mammary fat pad (500K cells/fat pad). Student’s unpaired t-test. (e) Images (left) and quantification (right) of scratch wound assay in MCF7NSC and MCF7shLIFR cells in vitro. Scale bar = 200 μm. Student’s unpaired t-test. 2 biological replicates, one each from 2 independent experiments. (f) 3D culture of MCF7NSC and MCF7shLIFR cells in type I rat collagen. Images taken at day 5 (scale bar = 200 μm) and day 9 (scale bar = 50 μm) after tumor cell seeding. Images representative of 3 independent experiments. Source data for Suppl. 5b-e available in Supplementary Table 1 and unprocessed blots in Supplementary Fig. 9. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001.##P < 0.01 versus MCF7NSC + LIF treatment.

Supplementary Figure 6 Quiescence and cancer stem cell-associated genes implicated in tumor cell dormancy.

(a) Table of genes identified as validated dormancy genes in ER + ve breast cancer cells and quiescence-related genes. (b) LIFR mRNA levels in SUM159 cells with LIFR lentiviral knockdown. 3 technical replicates from a single experiment. Source data for Suppl. 6b available in Supplementary Table 1. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗∗∗P < 0.001.

Supplementary Figure 7 The effects of LIFR signaling on dormancy are mediated via STAT3 versus PI3K:mTOR or MAPK signaling.

(a) TCGA analysis of invasive breast carcinoma (Nature 2012 dataset) patient samples for mRNA down-regulation of LIFR, PIK3CA, and mTOR genes (n = 543 patients with no down-regulation, n = 32 patients with mRNA down-regulation). Logrank test. (b) TCGA analysis of invasive breast carcinoma (Nature 2012 dataset) patient samples for mRNA down-regulation of LIFR, MAPK3, and MAPK1 genes (n = 666 patients with no down-regulation, n = 52 patients with mRNA down-regulation). Logrank test. (c) Western blot for phospho-Akt, Akt, and loading control Actin protein levels in MCF7 cells treated with vehicle (water), 10 nM, 100 nM, or 1,000 nM BEZ235 (PI3K inhibitor) for 24 h. (d) mRNA levels for dormancy and quiescence-associated genes in MCF7 cells after 24 h treatment with BEZ235. Multiple t-tests with Holm-Sidak post-test. n = 3 biological replicates, each being an average from 3 independent experiments. (e) Western blot for phospho-Erk1/2, Erk1/2, and loading control Actin protein levels in MCF7 cells treated with vehicle (DMSO), 0.1 μM, 1 μM, or 10 μM AZD6244 (ERK1/2 inhibitor) for 24 h. (f) mRNA levels for dormancy and quiescence-associated genes in MCF7 cells after 24 h treatment with AZD6244. Multiple t-tests with Holm-Sidak post-test. n = 3 biological replicates, each being an average from 3 independent experiments. (g,h) mRNA levels for cancer stem cell-associated genes in MCF7 cells after 24 h treatment with (g) BEZ235 or (h) AZD6244. Multiple t-tests with Holm-Sidak post-test. n = 3 biological replicates, each being an average from 3 independent experiments. Unprocessed blots in Supplementary Fig. 9. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Supplementary Figure 8 Loss of LIFR in MCF7 cells does not alter lung colonization.

(a) Negative controls for Ki67 staining in MCF7NSC and MCF7shLIFR tumor-bearing tibiae (8 mice/group). Scale bar = 100 μm. (b) Representative PIMO staining at the tumor-bone interface for 2 mice/group (of a total 3 mice/group where this was observed) in MCF7NSC and MCF7shLIFR tumor-bearing bones. Scale bar = 100 μm. (c) Tumor burden/total area histomorphometric analysis of lung sections from MCF7NSC and MCF7shLIFR tumor-inoculated mice. 3/10 MCF7NSC and 1/10 MCF7shLIFR tumor-inoculated mice had tumor detectable by H&E. Representative H&E images on right. Student’s unpaired t-test.n = 10 mice/group. Scale bar = 100 μm. (d) Human β-2microglobulin mRNA levels in homogenized lungs from MCF7NSC and MCF7shLIFR inoculated mice. 6/10 MCF7NSC and 5/10 MCF7shLIFR tumor-inoculated mice had detectable human mRNA in the lungs. HMBS = housekeeping gene. Student’s unpaired t-test.n = 10 mice/group. (e) Analysis of Minn et al. dataset for LIFR mRNA levels in breast cancer patients with a poor prognosis based on Van ’t Veer signature (n = 32 patients with no lung metastasis, n = 13 patients with lung metastasis). Student’s unpaired t-test with Welch’s Correction. (f) Structure of the Stat3 inhibitor ML116 (Stat3i). (g) 3D model of Stat3i docking in predicted DNA binding site of a Stat3 monomer. (h) SOCS3 mRNA levels in MCF7 cells following 1 h treatment with LIF (50 ng ml−1) ± 1 h Stat3i pre-treatment (1–50 μM). 3 technical replicates from a single experiment. (i) mRNA levels of LIFR-independent dormancy/quiescence-associated genes in MCF7 cells following 24 h treatment with 5 μM or 50 μM of Stat3 inhibitor ML116 (Stat3i). Multiple t-tests with Holm-Sidak post-test. n = 3 biological replicates, one each from 3 independent experiments. (j) SOCS3 mRNA levels in MCF7 cells transfected with siRNA against SOCS3. Student’s unpaired t-test.n = 3 biological replicates, each being an average from 3 independent experiments. (k,l) mRNA levels for (k) dormancy and quiescence and (l) cancer stem cell-associated genes in SUM159 cells transfected with siRNA against SOCS3 for 48 h. Student’s unpaired t-test.n = 3 biological replicates, each being an average from 3 independent experiments. Source data for Suppl. 8c,d,h, available in Supplementary Table 1. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Supplementary Figure 9 Unprocessed Western Blots.

Unprocessed Western blots developed on a Chemidoc XRS are provided for figures as indicated.

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Johnson, R., Finger, E., Olcina, M. et al. Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow. Nat Cell Biol 18, 1078–1089 (2016). https://doi.org/10.1038/ncb3408

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