Background Bone metastasis is a main cause of morbidity in breast cancer. Since breast cancer is a heterogeneous disease, the interactions of cancer cells with the skeletal host cells might also be diverse. We hypothesized that gene expression signatures induced by heterotypic interaction of breast cancer cells and osteoblasts might be of clinical relevance. Methodology/Principal Findings We established an ex vivo co-culture model using benign breast epithelial cells or a panel of 5 malignant breast epithelial cells in combination with primary human osteoblasts and determined associated gene expression changes with HEEBO microarrays. Pretreatment gene expression profiles of 295 early stage breast cancers published from the Netherlands Cancer Institute with a median follow up of 12.6 years allowed evaluating in vitro effects in the in vivo situation.The effects of the interaction between osteoblasts and breast cancer cell lines of different origin were very heterogeneous. Hs578T cells started to proliferate in co-culture with osteoblasts, SKBR-3 induced a TGF-β response and MDA-MB231 cells showed two distinct sets of up-regulated genes: A set of interferon response genes associated with an up-regulation of STAT1 was in vivo remarkably coherent providing a basis for segregation of tumors into two groups. In a uni-variate analysis, early stage tumors with high expression levels (n = 136) of this gene set had a significantly lower overall survival rate (p = 0.005) (63% at 10 years) than tumors with low expression levels (n = 159) (overall survival: 77% at 10 years). The second gene set was associated with IL-6 and did not significantly change the overall survival rate (p = 0.165), but was significantly associated with a shorter time to bone metastasis (p = 0.049; 74% vs. 83% at 10 years). Conclusion/Significance An IL-6 gene expression pattern induced by heterotypic interaction of breast cancer cells with osteoblasts in vitro is associated with a higher rate of bone metastasis in vivo.
References
[1]
Roodman G (2004) Mechanisms of bone metastasis. The New England journal of medicine 350: 1655.
[2]
Coleman R, Rubens R (1987) The clinical course of bone metastases from breast cancer. British journal of cancer 55: 61.
[3]
Paget S (1889) The distribution of secondary growths in cancer of the breast. Cancer Metastasis Rev 8: 98–101.
[4]
Thomas R, Guise T, Yin J, Elliott J, Horwood N, et al. (1999) Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 140: 4451.
[5]
Kingsley L, Fournier P, Chirgwin J, Guise T (2007) Molecular biology of bone metastasis. Molecular Cancer Therapeutics 6: 2609.
[6]
Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, et al. (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences of the United States of America 98: 10869–10874.
[7]
Egeblad M, Littlepage L, Werb ZThe fibroblastic coconspirator in cancer progression; 2005. 383 p. NIH Public Access.
[8]
Zumsteg A, Christofori G (2009) Corrupt policemen: inflammatory cells promote tumor angiogenesis. Current Opinion in Oncology 21: 60.
[9]
Iiizumi M, Mohinta S, Bandyopadhyay S, Watabe K (2007) Tumor-endothelial cell interactions: therapeutic potential. Microvascular research 74: 114–120.
[10]
Buess M, Nuyten DS, Hastie T, Nielsen T, Pesich R, et al. (2007) Characterization of heterotypic interaction effects in vitro to deconvolute global gene expression profiles in cancer. Genome Biol 8: R191.
[11]
Harada S, Rodan G (2003) Control of osteoblast function and regulation of bone mass. Nature 423: 349–355.
[12]
Jones D, Nakashima T, Sanchez O, Kozieradzki I, Komarova S, et al. (2006) Regulation of cancer cell migration and bone metastasis by RANKL. Nature 440: 692–696.
[13]
Li L, Neaves WB (2006) Normal stem cells and cancer stem cells: the niche matters. Cancer Res 66: 4553–4557.
[14]
Karrison T, Ferguson D, Meier P (1999) Dormancy of mammary carcinoma after mastectomy. JNCI Journal of the National Cancer Institute 91: 80.
[15]
Karnoub A, Dash A, Vo A, Sullivan A, Brooks M, et al. (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449: 557–563.
Mauri D, Valachis A, Polyzos N, Tsali L, Mavroudis D, et al. (2010) Does Adjuvant Bisphosphonate in Early Breast Cancer Modify the Natural Course of the Disease? A Meta-Analysis of Randomized Controlled Trials. Journal of the National Comprehensive Cancer Network 8: 279.
[18]
Buess M, Rajski M, Vogel-Durrer B, Herrmann R, Rochlitz C (2009) Tumor-endothelial interaction links the CD44+/CD24？phenotype with poor prognosis in early-stage breast cancer. Neoplasia 11: 987–1002.
[19]
Spandidos A, Wang X, Wang H, Seed B (2010) PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res 38: D792–799.
[20]
Demeter J, Beauheim C, Gollub J, Hernandez-Boussard T, Jin H, et al. (2007) The Stanford Microarray Database: implementation of new analysis tools and open source release of software. Nucleic Acids Res 35: D766–770.
[21]
Edgar R, Domrachev M, Lash A (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic acids research 30: 207.
[22]
Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences of the United States of America 95: 14863–14868.
[23]
Boyle EI, Weng S, Gollub J, Jin H, Botstein D, et al. (2004) GO::TermFinder–open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20: 3710–3715.
[24]
van de Vijver MJ, He YD, van't Veer LJ, Dai H, Hart AA, et al. (2002) A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347: 1999–2009.
[25]
Bos P, Xiang H, Nadal C, Shu W, Gomis R, et al. (2009) Genes that mediate breast cancer metastasis to the brain. Nature 459: 1005–1009.
[26]
Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, et al. (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the National Academy of Sciences of the United States of America 100: 8418–8423.
[27]
Rajski M, Zanetti-D？llenbach R, Vogel B, Herrmann R, Rochlitz C, et al. (2010) IGF-I induced genes in stromal fibroblasts predict the clinical outcome of breast and lung cancer patients. BMC medicine 8: 1.
[28]
R Development Core Team (2011) A language and environment for statistical computing. Available: http://www.R-project.org via the Internet. Accessed 2011 Nov 24.
[29]
Mourskaia A, Dong Z, Ng S, Banville M, Zwaagstra J, et al. (2008) Transforming growth factor- 1 is the predominant isoform required for breast cancer cell outgrowth in bone. Oncogene 28: 1005–1015.
[30]
Safran M, Dalah I, Alexander J, Rosen N, Iny Stein T, et al. (2010) GeneCards Version 3: the human gene integrator. Availble: http://www.genecards.org via the Internet. Accessed 2011 Nov 24.
[31]
Coussens L, Werb Z (2002) Inflammation and cancer. Nature 420: 860–867.
[32]
Diehn M, Sherlock G, Binkley G, Jin H, Matese J, et al. (2003) SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data. Nucleic acids research 31: 219.
[33]
van 't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, et al. (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415: 530–536.
[34]
Chang HY, Nuyten DS, Sneddon JB, Hastie T, Tibshirani R, et al. (2005) Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci U S A 102: 3738–3743.
[35]
Mundy GR (2002) Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2: 584–593.
[36]
Smid M, Wang Y, Zhang Y, Sieuwerts A, Yu J, et al. (2008) Subtypes of breast cancer show preferential site of relapse. Cancer research 68: 3108.
[37]
Smid M, Wang Y, Klijn J, Sieuwerts A, Zhang Y, et al. (2006) Genes associated with breast cancer metastatic to bone. Journal of Clinical Oncology 24: 2261.
[38]
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100: 3983–3988.
[39]
Fillmore C, Kuperwasser C (2008) Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res 10: R25.
[40]
Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, et al. (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer research 69: 1302.
[41]
Sansone P, Storci G, Tavolari S, Guarnieri T, Giovannini C, et al. (2007) IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. Journal of Clinical Investigation 117: 3988–4002.
[42]
Rusciano D, Burger M (2000) In vivo cancer metastasis assays. Cancer metastasis: experimental approaches. Amsterdam, The Netherland: Elsevier. pp. 207–242.
[43]
Dhurjati R, Krishnan V, Shuman L, Mastro A, Vogler E (2008) Metastatic breast cancer cells colonize and degrade three-dimensional osteoblastic tissue in vitro. Clinical and Experimental Metastasis 25: 741–752.
[44]
Udagawa N, Takahashi N, Katagiri T, Tamura T, Wada S, et al. (1995) Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors. The Journal of experimental medicine 182: 1461.
[45]
Hoyland J, Freemont A, Sharpe P (1994) Interleukin 6, IL 6 receptor, and IL 6 nuclear factor gene expression in paget's disease. Journal of Bone and Mineral Research 9: 75–80.
[46]
Devlin R, Bone H 3rd, Roodman G (1996) Interleukin-6: a potential mediator of the massive osteolysis in patients with Gorham-Stout disease. Journal of Clinical Endocrinology & Metabolism 81: 1893.
[47]
Bataille R, Jourdan M, Zhang X, Klein B (1989) Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias. Journal of Clinical Investigation 84: 2008.
[48]
Bachelot T, Ray-Coquard I, Menetrier-Caux C, Rastkha M, Duc A, et al. (2003) Prognostic value of serum levels of interleukin 6 and of serum and plasma levels of vascular endothelial growth factor in hormone-refractory metastatic breast cancer patients. British journal of cancer 88: 1721–1726.
[49]
Nikiteas N, Tzanakis N, Gazouli M, Rallis G, Daniilidis K, et al. (2005) Serum IL-6, TNFalpha and CRP levels in Greek colorectal cancer patients: prognostic implications. World J Gastroenterol 11: 1639–1643.
[50]
Berishaj M, Gao SP, Ahmed S, Leslie K, Al-Ahmadie H, et al. (2007) Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer. Breast Cancer Res 9: R32.
[51]
Sheridan C, Kishimoto H, Fuchs R, Mehrotra S, Bhat-Nakshatri P, et al. (2006) CD44+/CD24？breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 8: R59.
[52]
Kennecke H, Yerushalmi R, Woods R, Cheang M, Voduc D, et al. (2010) Metastatic Behavior of Breast Cancer Subtypes. Journal of clinical oncology 28: 3271.
[53]
Chang HY, Sneddon JB, Alizadeh AA, Sood R, West RB, et al. (2004) Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS Biol 2: E7.
