Breast Cancer Research and Treatment

, Volume 129, Issue 2, pp 411–419 | Cite as

Inhibition of the c-fms proto-oncogene autocrine loop and tumor phenotype in glucocorticoid stimulated human breast carcinoma cells

  • Eugene P. Toy
  • Tiffany Lamb
  • Masoud Azodi
  • William J. Roy
  • Ho-Hyung Woo
  • Setsuko K. Chambers
Preclinical Study

Abstract

The c-fms proto-oncogene encoded CSF-1 receptor and its ligand represent a feedback loop, which in a paracrine manner, is well known to promote spread of breast cancers. The role of the autocrine feedback loop in promotion of breast tumor behavior, in particular in vitro, is less well understood. The physiologic stimulation of c-fms expression by glucocorticoids (GCs) in vitro and in vivo magnifies the tumor promoting effect seen in these cells from activated c-fms signaling by CSF-1. Targeted molecular therapy against c-fms could therefore abrogate both complementary feedback loops. Using breast cancer cells endogenously co-expressing receptor and ligand, we used complementary approaches to inhibit c-fms expression and function within this autocrine pathway in the context of GC stimulation. Silencing RNA (shRNA), antisense oligonucleotide therapy (AON), and inhibition of c-fms signaling, were all used to quantitate inhibition of GC-stimulated adhesion, motility, and invasion of human breast cancer cells in vitro. shRNA to c-fms downregulated GC-stimulated c-fms mRNA by fourfold over controls, correlating with over twofold reduction in cellular invasiveness. AON therapy was also able to inhibit GC stimulation of c-fms mRNA, and resulted in threefold less invasiveness and 1.5 to 2-fold reductions in adhesion and motility. Finally, the small-molecule c-fms inhibitor Ki20227 was able to decrease in a dose–response manner, breast cancer cell invasion by up to fourfold. Inhibition of this receptor/ligand pair may have clinical utility in inhibition of the autocrine as well as the known paracrine interactions in breast cancer, thus further supporting use of targeted therapies in this disease.

Keywords

Breast cancer Invasion c-fms Proto-oncogene Glucocorticoids 

Abbreviations

Dex

Dexamethasone

FCS

Fetal calf serum

ON

Overnight

AON

Antisense oligonucleotide

GC

Glucocorticoid

qRT-PCR

Quantitative real time reverse transcriptase polymerase chain reaction

siRNA

Silencing RNA

shRNA

Small hairpin RNA

References

  1. 1.
    Kacinski BM, Scata KA, Carter D, Yee LD, Sapi E, King BL, Chambers SK, Jones MA, Pirro MH, Stanley ER, Rohrschneider LR (1991) FMS (CSF-1 receptor) and CSF-1 transcripts and protein are expressed by human breast carcinomas in vivo and in vitro. Oncogene 6:941–952PubMedGoogle Scholar
  2. 2.
    Flick MB, Sapi E, Perrotta PL, Maher MG, Halaban R, Carter D, Kacinski BM (1997) Recognition of activated CSF-1 receptor in breast carcinomas by a tyrosine 723 phosphospecific antibody. Oncogene 14:2553–2561PubMedCrossRefGoogle Scholar
  3. 3.
    Scholl SM, Mosseri V, Tang R, Pouillart P (1993) Expression of colony stimulating factor-1 and its receptor (the protein product of c-fms) in invasive breast tumor cells. Ann NY Acad Sci 698:131–135PubMedCrossRefGoogle Scholar
  4. 4.
    Kluger HM, Dolled-Filhart M, Rodov S, Kacinski BM, Camp RL, Rimm DL (2004) Macrophage colony-stimulating factor-1 receptor expression is associated with poor outcome in breast cancer by large cohort tissue microarray analysis. Clin Cancer Res 10:173–177PubMedCrossRefGoogle Scholar
  5. 5.
    Scholl SM, Pallud C, Beuvon F, Hacene K, Stanley ER, Rohrschneider L, Tang R, Pouillart P, Lidereau R (1994) Anti-colony-stimulating factor-1 antibody staining in primary breast adenocarcinomas correlates with marked inflammatory cell infiltrates and prognosis. J Natl Cancer Inst 86:120–126PubMedCrossRefGoogle Scholar
  6. 6.
    Maher MG, Sapi E, Turner B, Gumbs A, Perrotta PL, Carter D, Kacinski BM, Haffty BG (1998) Prognostic significance of colony-stimulating factor receptor expression in ipsilateral breast cancer recurrence. Clin Cancer Res 4:1851–1856PubMedGoogle Scholar
  7. 7.
    Tang R, Kacinski B, Validire P, Beuvon F, Sastre X, Benoit P, De la Rochefordiere A, Mosseri V, Pouillart P, Scholl S (1990) Oncogene amplification correlates with dense lymphocyte infiltration in human breast cancers: a role for hematopoietic growth factor release by tumor cells? J Cell Biochem 44:189–198PubMedCrossRefGoogle Scholar
  8. 8.
    McDermott RS, Deneux L, Mosseri V, Vedrenne J, Clough K, Fourquet A, Rodriguez J, Cosset JM, Sastre X, Beuzeboc, Pouillart P, Scholl SM (2002) Circulating macrophage colony stimulating factor as a marker of tumour progression. Eur Cytokine Netw 13:121–127PubMedGoogle Scholar
  9. 9.
    Sapi E (2004) The role of CSF-1 in normal physiology of mammary gland and breast cancer: an update. Exp Biol Med 229(1):1–11Google Scholar
  10. 10.
    Aharinejad S, Paulus P, Sioud M, Hofmann M, Zins K, Schafer R (2004) Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice. Cancer Res 64:5378–5384PubMedCrossRefGoogle Scholar
  11. 11.
    Paulus P, Stanley ER, Schafer R, Abraham D, Aharinejad S (2006) Colony-stimulating factor-1 antibody reverses chemoresistance in human MCF-7 breast cancer xenografts. Cancer Res 66:4349–4356PubMedCrossRefGoogle Scholar
  12. 12.
    Lin EY, Nguyen AV, Russell RG, Pollard JW (2001) Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 193(6):727–740PubMedCrossRefGoogle Scholar
  13. 13.
    Pollard JW (2009) Trophic macrophages in development and disease. Nat Rev Immunol 9:259–270PubMedCrossRefGoogle Scholar
  14. 14.
    Hernandez L, Smirnova T, Kedrin D, Wyckoff J, Zhu L, Stanley ER, Cox D, Muller WJ, Pollard JW, Van Rooijen N (2009) Segall JE (2009) The EGF/CSF-1 paracrine invasion loop can be triggered by Heregulin ß1 and CXCL12. Cancer Res 69:3221–3227PubMedCrossRefGoogle Scholar
  15. 15.
    Goswami S, Sahai E, Wyckoff JB, Cammer M, Cox D, Pixley FJ, Stanley ER, Segall JE, Condeelis JS (2005) Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer Res 65:5278–5283PubMedCrossRefGoogle Scholar
  16. 16.
    Wrobel CN, Debnath J, Lin E, Beausoleil S, Roussel MF, Brugge JS (2004) Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture. J Cell Biol 165(2):263–273PubMedCrossRefGoogle Scholar
  17. 17.
    Borycki AG, Smadja F, Stanley R, Leibovitch SA (1995) Colony-stimulating factor 1 (CSF-1) is involved in an autocrine growth control of rat myogenic cells. Exp Cell Res 218:213–222PubMedCrossRefGoogle Scholar
  18. 18.
    Sapi E, Flick MB, Rodov S, Gilmore-Hebert M, Kelley M, Rockwell S, Kacinski BM (1996) Independent regulation of invasion and anchorage-independent growth by different autophosphorylation sites of the macrophage colony-stimulating factor 1 receptor. Cancer Res 56:5704–5712PubMedGoogle Scholar
  19. 19.
    Gunawardane RN, Sgroi DS, Wrobel CN, Koh E, Daley GQ, Brugge JS (2005) Novel role for PDEF in epithelial cell migration and invasion. Cancer Res 65:11572–11580PubMedCrossRefGoogle Scholar
  20. 20.
    Chambers SK, Wang Y, Gertz RE, Kacinski BM (1995) Macrophage colony-stimulating factor mediates invasion of ovarian cancer cells through urokinase. Cancer Res 55:1578–1585PubMedGoogle Scholar
  21. 21.
    Filderman AE, Bruckner A, Kacinski BM, Deng N, Remold HG (1992) Macrophage colony-stimulating factor (CSF-1) enhances invasiveness in CSF-1 receptor-positive carcinoma cell lines. Cancer Res 52:3661–3666PubMedGoogle Scholar
  22. 22.
    Toy EP, Bonafe N, Savlu A, Zeiss C, Zheng W, Flick M, Chambers SK (2005) Correlation of tumor phenotype with c-fms proto-oncogene expression in an in vivo intraperitoneal model for experimental human breast cancer metastasis. Clin Exp Metastasis 22(1):1–9PubMedCrossRefGoogle Scholar
  23. 23.
    Das SK, Stanley ER, Guilbert LJ, Forman LW (1981) Human colony-stimulating factor (CSF-1) radioimmunoassay: resolution of three subclasses of human colony-stimulating factors. Blood 58:630–641PubMedGoogle Scholar
  24. 24.
    Patsialou A, Wyckoff J, Wang Y, Goswami S, Stanley ER, Condeelis JS (2009) Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor. Cancer Res 69:9498–9506PubMedCrossRefGoogle Scholar
  25. 25.
    Sapi E, Flick MB, Gilmore-Hebert M, Rodov S, Kacinski BM (1995) Transcriptional regulation of the c-fms (CSF-1R) proto-oncogene in human breast carcinoma cells by glucocorticoids. Oncogene 10:529–542PubMedGoogle Scholar
  26. 26.
    Chambers SK, Wang Y, Gilmore-Hebert M, Kacinski BM (1994) Post-transcriptional regulation of c-fms proto-oncogene expression by dexamethasone and of CSF-1 in human breast carcinomas in vitro. Steroids 59:514–522PubMedCrossRefGoogle Scholar
  27. 27.
    Kacinski BM, Flick MB, Sapi E (2001) RU-486 can abolish glucocorticoid-induced increases in CSF-1 receptor expression in primary human breast carcinoma specimens. J Soc Gynecol Investig 8:114–116PubMedCrossRefGoogle Scholar
  28. 28.
    Wei S, Nandi S, Chitu V, Yeung YG, Yu W, Huang M, Williams LT, Lin H, Stanley ER (2010) Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells. J Leukoc Biol 88:495–505PubMedCrossRefGoogle Scholar
  29. 29.
    Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW Jr, Ahmed-Ansari A, Sell KW, Pollard JW, Stanley ER (1990) Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA 87:4828–4832PubMedCrossRefGoogle Scholar
  30. 30.
    Toy EP, Azodi M, Folk NL, Zito CM, Zeiss CJ, Chambers SK (2009) Enhanced ovarian cancer tumorigenesis and metastasis by the macrophage colony-stimulating factor (CSF-1). Neoplasia 11(2):136–144PubMedGoogle Scholar
  31. 31.
    Ohno H, Kubo K, Murooka H, Kobayashi Y, Nishitoba T, Shibuya M, Yoneda T, Isoe T (2006) A c-fms tyrosine kinase inhibitor, Ki20227, suppresses osteoclast differentiation and osteolytic bone destruction in a bone metastasis model. Mol Cancer Ther 5(11):2634–2643PubMedCrossRefGoogle Scholar
  32. 32.
    Hiraga T, Nakamura H (2009) Imatinib mesylate suppresses bone metastases of breast cancer by inhibiting osteoclasts through the blockade of c-Fms signals. Int J Cancer 124:215–222PubMedCrossRefGoogle Scholar
  33. 33.
    Chambers SK (2000) In vitro invasion assays. In: Bartlett J (ed) Methods in molecular medicine: ovarian cancer. Humana Press Inc., Totowa, NJ, pp 179–185CrossRefGoogle Scholar
  34. 34.
    Chambers SK, Ivins CM, Kacinski BM, Hochberg RB (2004) An unexpected effect of glucocorticoids on stimulation of c-fms proto-oncogene expression in choriocarcinoma cells expressing little glucocorticoid receptor. Am J Obstet Gynecol 190:974–985PubMedCrossRefGoogle Scholar
  35. 35.
    Chambers SK, Gilmore-Hebert M, Wang Y, Rodov S, Benz EJ, Kacinski BM (1993) Post-transcriptional regulation of CSF-1 and CSF-1 receptor gene expression during inhibition of phorbol-ester induced monocytic differentiation by dexamethasone and cyclosporin A: potential involvement of a destabilizing protein. Exp Hematol 21:1328–1334PubMedGoogle Scholar
  36. 36.
    Woo HH, Zhou Y, Yi X, David CL, Zheng W, Gilmore-Hebert M, Kluger HM, Ulukus EC, Baker T, Stoffer JB, Chambers SK (2009) Regulation of non AU-rich element containing c-fms proto-oncogene expression by HuR in breast cancer. Oncogene 28(9):1176–1186PubMedCrossRefGoogle Scholar
  37. 37.
    Woo HH, Yi X, Lamb T, Menzl I, Baker T, Shapiro DJ, Chambers SK (2010) Post-transcriptional suppression of proto-oncogene c-fms expression by vigilin in breast cancer. Mol Cell Biol. doi:10.1128/MCB.01031-10
  38. 38.
    Lorinz P, Misteli T, Baker BF, Bennett CF, Spector DL (2000) Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 28(2):582CrossRefGoogle Scholar
  39. 39.
    Yang W, Saad M, Pakunlu RI, Khandare JJ, Garbuzenko OB, Vetcher AA, Soldatenkov VA, Pozharov VP, Minko T (2008) Nonviral nanoscale-based delivery of antisense oligonucleotides targeted to hypoxia-inducible factor 1α enhances the efficacy of chemotherapy in drug-resistant tumor. Clin Cancer Res 14(11):3607–3616CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Eugene P. Toy
    • 1
    • 5
  • Tiffany Lamb
    • 4
  • Masoud Azodi
    • 2
  • William J. Roy
    • 3
  • Ho-Hyung Woo
    • 4
  • Setsuko K. Chambers
    • 4
  1. 1.Departments of Obstetrics and Gynecology, Division of Gynecologic OncologyUniversity of RochesterNYUSA
  2. 2.Department of Obstetrics and GynecologyYale University School of MedicineNew HavenUSA
  3. 3.GYN Oncology at the Cancer CenterCredentialed by MD Anderson Physicians NetworkMobileUSA
  4. 4.Arizona Cancer CenterUniversity of ArizonaTucsonUSA
  5. 5.Gynecologic Oncology AssociatesUniversity of RochesterRochesterUSA

Personalised recommendations