Skip to main content
SpringerLink
Log in

Reelin Deficiency Delays Mammary Tumor Growth and Metastatic Progression

Journal of Mammary Gland Biology and Neoplasia Aims and scope Submit manuscript

Cite this article

Abstract

Reelin is a regulator of cell migration in the nervous system, and has other functions in the development of a number of non-neuronal tissues. In addition, alterations in reelin expression levels have been reported in breast, pancreatic, liver, gastric, and other cancers. Reelin is normally expressed in mammary gland stromal cells, but whether stromal reelin contributes to breast cancer progression is unknown. Herein, we used a syngeneic mouse mammary tumor transplantation model to examine the impact of host-derived reelin on breast cancer progression. We found that transplanted syngeneic tumors grew more slowly in reelin-deficient (rl Orl −/−) mice and had delayed metastatic colonization of the lungs. Immunohistochemistry of primary tumors revealed that tumors grown in rl Orl −/− animals had fewer blood vessels and increased macrophage infiltration. Gene expression studies from tumor tissues indicate that loss of host-derived reelin alters the balance of M1- and M2-associated macrophage markers, suggesting that reelin may influence the polarization of these cells. Consistent with this, rl Orl −/− M1-polarized bone marrow-derived macrophages have heightened levels of the M1-associated cytokines iNOS and IL-6. Based on these observations, we propose a novel function for the reelin protein in breast cancer progression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Fatemi SH. Reelin glycoprotein: structure, biology, and roles in health and disease. New York: Springer; 2008.

    Book  Google Scholar 

  2. Honda T, Kobayashi K, Mikoshiba K, Nakajima K. Regulation of cortical neuron migration by the reelin signaling pathway. Neurochem Res. 2011;36(7):1270–9.

    Article  CAS  PubMed  Google Scholar 

  3. Abadesco AD, Cilluffo M, Yvone GM, Carpenter EM, Howell BW, Phelps PE. Novel disabled-1-expressing neurons identified in adult brain and spinal cord. Eur J Neurosci. 2014;39:579–92.

    Article  PubMed  Google Scholar 

  4. D’Arcangelo G. Reelin in the years: controlling neuronal migration and maturation in the mammalian brain. Adv Neurosci. doi:10.1155/2014/597395.

  5. Herz J, Chen Y. Reelin, lipoprotein receptors and synaptic plasticity. Nat Rev Neurosci. 2006;7(11):850–9.

    Article  CAS  PubMed  Google Scholar 

  6. Bock HH, Herz J. Reelin activates SRC family tyrosine kinases in neurons. Curr Biol. 2003;13(1):18–26.

    Article  CAS  PubMed  Google Scholar 

  7. Kuo G, Arnaud L, Kronstad-O’Brien P, Cooper JA. Absence of Fyn and Src causes a reeler-like phenotype. J Neurosci. 2005;25(37):8578–86.

    Article  CAS  PubMed  Google Scholar 

  8. Diaz-Mendoza MJ, Lorda-Diez CI, Montero JA, Garcia-Porrero JA, Hurle JM. Reelin/DAB-1 signaling in the embryonic limb regulates the chondrogenic differentiation of digit mesodermal progenitors. J Cell Physiol. 2014;229(10):1397–404.

    Article  CAS  PubMed  Google Scholar 

  9. Botella-López A, de Madaria E, Jover R, Bataller R, Sancho-Bru P, Candela A, et al. Reelin is overexpressed in the liver and plasma of bile duct ligated rats and its levels and glycosylation are altered in plasma of humans with cirrhosis. Int J Biochem Cell Biol. 2008;40(4):766–75.

    Article  PubMed  Google Scholar 

  10. Tseng WL, Chen TH, Huang CC, Huang YH, Yeh CF, Tsai HJ, et al. Impaired thrombin generation in reelin-deficient mice: a potential role of plasma reelin in hemostasis. J Thromb Haemost. 2014;12(12):1–11.

    Article  Google Scholar 

  11. Vázquez-Carretero MD, García-Miranda P, Calonge ML, Peral MJ, Ilundain AA. Dab1 and reelin participate in a common signal pathway that controls intestinal crypt/villus unit dynamics. Biol Cell. 2014;106(3):83–96.

    Article  PubMed  Google Scholar 

  12. Lutter S, Xie S, Tatin F, Makinen T. Smooth muscle-endothelial cell communication activates reelin signaling and regulates lymphatic vessel formation. J Cell Biol. 2012;197(6):837–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Khialeeva E, Lane TF, Carpenter EM. Disruption of reelin signaling alters mammary gland morphogenesis. Development. 2011;138(4):767–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hong SM, Kelly D, Griffith M, Omura N, Li A, Li CP, et al. Multiple genes are hypermethylated in intraductal papillary mucinous neoplasms of the pancreas. Mod Pathol. 2008;21(12):1499–507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dohi O, Takada H, Wakabayashi N, Yasui K, Sakakura C, Mitsufuji S, et al. Epigenetic silencing of RELN in gastric cancer. Int J Oncol. 2010;36(1):85–92.

    CAS  PubMed  Google Scholar 

  16. Perrone G, Vincenzi B, Zagami M, Santini D, Panteri R, Flammia G, et al. Reelin expression in human prostate cancer: a marker of tumor aggressiveness based on correlation with grade. Mod Pathol. 2007;20(3):344–51.

    Article  CAS  PubMed  Google Scholar 

  17. Wang Q, Lu J, Yang C, Wang X, Cheng L, Hu G, et al. CASK and its target gene reelin were co-upregulated in human esophageal carcinoma. Cancer Lett. 2002;179(1):71–7.

    Article  CAS  PubMed  Google Scholar 

  18. Stein T, Cosimo E, Yu X, Smith PR, Simon R, Cottrell L, et al. Loss of reelin expression in breast cancer is epigenetically controlled and associated with poor prognosis. Am J Pathol. 2010;177(5):2323–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pulaski BA, Ostrand-Rosenberg S. Mouse 4 T1 breast tumor model. Curr Protoc Immunol. 2001; Chapter 20:Unit 20.2.

  20. Takahara T, Ohsumi T, Kuromitsu J, Shibata K, Sasaki N, Okazaki Y, et al. Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exon. Hum Mol Genet. 1996;5(7):989–93.

    Article  CAS  PubMed  Google Scholar 

  21. De Bergeyck V, Nakajima K, Lambert de Rouvrait C, Naerhuyzen B, Goffinet AM, Miyata T, et al. A truncated reelin protein is produced but not secreted in the “Orleans” reeler mutation (Reln(rl-Orl)). Brain Res Mol Brain Res. 1997;50(1–2):85–90.

    Article  CAS  PubMed  Google Scholar 

  22. D’Arcangelo G, Homayouni R, Keshvara L, Rice DS, Sheldon M, Curran T. Reelin is a ligand for lipoprotein receptors. Neuron. 1999;24(2):471–9.

    Article  PubMed  Google Scholar 

  23. Ewald AJ. Isolation of mouse mammary organoids for long-term time-lapse imaging. Cold Spring Harb Protoc. 2013;2013(2):130–3.

    PubMed  Google Scholar 

  24. Kim EJ, Choi MR, Park H, Kim M, Hong JE, Lee JY, et al. Dietary fat increases solid tumor growth and metastasis of 4 T1 murine mammary carcinoma cells and mortality in obesity-resistant BALB/c mice. Breast Cancer Res. 2011;13(4):R78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. York AG, Williams KJ, Argus JP, Zhou QD, Brar G, Vergnes L, et al. Limiting cholesterol biosynthetic flux spontaneously engages type I IFN signaling. Cell. 2015;163(7):1716–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Falconer D. Two new mutants, ‘trembler'and 'reeler', with neurological actions in the house mouse (Mus musculus L.). J Genet. 1951;50(2):192–205.

    Article  CAS  PubMed  Google Scholar 

  27. DuPré SA, Redelman D, Hunter KW. The mouse mammary carcinoma 4 T1: characterization of the cellular landscape of primary tumours and metastatic tumour foci. Int J Exp Pathol. 2007;88(5):351–60.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Cho HJ, Jung JI, Lim DY, Kwon GT, Her S, Park JH, et al. Bone marrow-derived, alternatively-activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model. Breast Cancer Res. 2012;14(3):R81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Green-Johnson JM, Zalcman S, Vriend CY, Nance DM, Greenberg AH. Suppressed T cell and macrophage function in the“ reeler”(rl/rl) mutant, a murine strain with elevated cerebellar norepinephrine concentration. Brain Behav Immun. 1995;9(1):47–60.

    Article  CAS  PubMed  Google Scholar 

  30. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer. 2008;8(8):618–31.

    Article  CAS  PubMed  Google Scholar 

  31. Mills CD. M1 and M2 macrophages: oracles of health and disease. Crit Rev Immunol. 2012;32(6):463–88.

    Article  CAS  PubMed  Google Scholar 

  32. Ma J, Liu L, Che G, Yu N, Dai F, You Z. The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time. BMC Cancer. 2010;10:112.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002;23(11):549–55.

    Article  CAS  PubMed  Google Scholar 

  34. Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10(3):170–81.

    Article  CAS  PubMed  Google Scholar 

  35. Trinchieri G. Interleukin-10 production by effector T cells: Th1 cells show self control. J Exp Med. 2007;204(2):239–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pineda-Torra I, Gage M, de Juan A, Pello OM. Isolation, culture and polarization of murine bone marrow-derived and peritoneal macrophages. Methods Mol Biol. 2015;1339:101–9.

    Article  PubMed  Google Scholar 

  37. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75(2):163–89.

    Article  CAS  PubMed  Google Scholar 

  38. Boonstra A, Rajsbaum R, Holman M, Marques R, Asselin-Paturel C, Pereira JP, et al. Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals. J Immunol. 2006;177(11):7551–8.

    Article  CAS  PubMed  Google Scholar 

  39. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;3:1–13.

    Google Scholar 

  40. Pulaski BA, Ostrand-Rosenberg S. Reduction of established spontaneous mammary carcinoma metastases following immunotherapy with major histocompatibility complex class II and B7.1 cell-based tumor vaccines. Cancer Res. 1998;58(7):1486–93.

    CAS  PubMed  Google Scholar 

  41. Thomas DL, Fraser NW. HSV-1 therapy of primary tumors reduces the number of metastases in an immune-competent model of metastatic breast cancer. Mol Ther. 2003;8(4):543–51.

    Article  CAS  PubMed  Google Scholar 

  42. Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992;52(6):1399–405.

    CAS  PubMed  Google Scholar 

  43. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010;141(1):52–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Stubbs D, DeProto J, Nie K, Englund C, Mahmud I, Hevner R, et al. Neurovascular congruence during cerebral cortical development. Cereb Cortex. 2009;19(suppl 1):i32–41.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Guy J, Wagener RJ, Möck M, Staiger JF. Persistence of functional sensory maps in the absence of cortical layers in the somsatosensory cortex of reeler mice. Cereb Cortex. 2015;25(9):2517–28.

    Article  PubMed  Google Scholar 

  46. Kopmels B, Wollman EE, Guastavino JM, Delhaye-Bouchaud N, Fradelizi D, Mariani J. Interleukin-1 hyperproduction by in vitro activated peripheral macrophages from cerebellar mutant mice. J Neurochem. 1990;55(6):1980–5.

    Article  CAS  PubMed  Google Scholar 

  47. Bakalian A, Kopmels B, Messer A, Fradelizi D, Delhaye-Bouchaud N, Wollman E, et al. Peripheral macrophage abnormalities in mutant mice with spinocerebellar degeneration. Res Immunol. 1992;143(1):129–39.

    Article  CAS  PubMed  Google Scholar 

  48. Rivera-Baltanas T, Romay-Tallon R, Dopeso-Reyes IG, Caruncho HJ. Serotonin transporter clustering in blood lymphocytes of reeler mice. Cardiovasc Psychiatry Neurol. 2010;2010:396282.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Resende C, Ristimäki A, Machado JC. Genetic and epigenetic alteration in gastric carcinogenesis. Helicobacter. 2010;15(suppl I):34–9.

    Article  CAS  PubMed  Google Scholar 

  50. Berthier-Vergnes O, El KM, de la Fouchardière A, Pointecouteau T, Verrando P, Wierinckx A, et al. Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion. Br J Cancer. 2011;104(1):155–65.

    Article  CAS  PubMed  Google Scholar 

  51. Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature. 2012;481(7380):157–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Patricia Phelps, Dr. Cristina Ghiani, Dr. Catalina Abad Rabat and Dr. Diana Moughon for kindly providing reagents and for thoughtful discussion. We are thankful to Donna Crandall for assistance with figure preparation and Joseph Argus for assistance with manuscript editing.

Author information

Authors and Affiliations

  1. Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA

    Elvira Khialeeva & Ellen M. Carpenter

  2. Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA, USA

    Joan W. Chou

  3. Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA

    Denise E. Allen

  4. Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA, USA

    Alec M. Chiu

  5. Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA, USA

    Steven J. Bensinger

  6. Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA

    Steven J. Bensinger

  7. Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Steven J. Bensinger

  8. Department of Psychiatry and Biobehavioral Sciences, University of California Los Angeles, Los Angeles, CA, USA

    Ellen M. Carpenter

Authors
  1. Elvira Khialeeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joan W. Chou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Denise E. Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alec M. Chiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Steven J. Bensinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ellen M. Carpenter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elvira Khialeeva.

Ethics declarations

Grants

These studies were supported by the National Institute of Child Health and Development R03 HD075840 - https://www.nichd.nih.gov/Pages/index.aspx and the California Breast Cancer Research Program 161B-0110 - http://www.cbcrp.org/to EMC. EK was supported by the Whitcome Fellowship of the UCLA Molecular Biology Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethics Approval

All animal studies were conducted in accordance with the UCLA Office of Animal Research Oversight and Institutional Animal Care and Use Committee protocols.

Electronic supplementary material

Fig. S1

Metastasis of 4T1 cells in rl Orl −/− and rl Orl +/+ mice bearing similar-sized tumors. (a) Wet weight of primary tumors collected 25 days (rl Orl +/+, n = 8) and 29 days (rl Orl −/−, n = 9) after 4T1 cell injection. (b) Quantification of metastatic burden in the lungs. ns – not significant. Statistical significance determined using two-tailed, unpaired Student’s t-test. (GIF 8 kb)

High Resolution Image (TIFF 1977 kb)

Fig. S2

Cytokine expression levels in rl Orl −/− and rl Orl +/+ BMDM treated with 4T1-conditioned medium for 24 h. Gene expression levels are relative to those in rl Orl +/+ BMDM controls. *P < 0.05, ns – not significant. Statistical significance determined using two-tailed, unpaired Student’s t-test. (GIF 6 kb)

High Resolution Image (TIFF 1375 kb)

Fig. S3

Cytokine expression levels in rl Orl +/+ BMDM treated with IFNγ and LPS. Gene expression levels are relative to those in unstimulated rl Orl +/+ BMDM controls. (GIF 10 kb)

High Resolution Image (TIFF 2109 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Khialeeva, E., Chou, J.W., Allen, D.E. et al. Reelin Deficiency Delays Mammary Tumor Growth and Metastatic Progression. J Mammary Gland Biol Neoplasia 22, 59–69 (2017). https://doi.org/10.1007/s10911-017-9373-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10911-017-9373-z

Keywords

search

Navigation