Advertisement

Breast Cancer Research and Treatment

, Volume 172, Issue 3, pp 577–586 | Cite as

CCN6 regulates IGF2BP2 and HMGA2 signaling in metaplastic carcinomas of the breast

  • Emily R. McMullen
  • Maria E. Gonzalez
  • Stephanie L. Skala
  • Mai Tran
  • Dafydd Thomas
  • Sabra I. Djomehri
  • Boris Burman
  • Kelley M. Kidwell
  • Celina G. Kleer
Preclinical study
  • 103 Downloads

Abstract

Purpose

Metaplastic breast carcinomas are an aggressive subtype of triple-negative breast cancer (TNBC) in which part or all of the adenocarcinoma transforms into a non-glandular component (e.g., spindled, squamous, or heterologous). We discovered that mammary-specific Ccn6/Wisp3 knockout mice develop mammary carcinomas with spindle and squamous differentiation that share upregulation of the oncofetal proteins IGF2BP2 (IMP2) and HMGA2 with human metaplastic carcinomas. Here, we investigated the functional relationship between CCN6, IGF2BP2, and HMGA2 proteins in vitro and in vivo, and their expression in human tissue samples.

Methods

MMTV-cre;Ccn6fl/fl tumors and spindle TNBC cell lines were treated with recombinant CCN6 protein or vehicle. IGF2BP2 was downregulated using shRNAs in HME cells with stable CCN6 shRNA knockdown, and subjected to invasion and adhesion assays. Thirty-one human metaplastic carcinomas were arrayed in a tissue microarray (TMA) and immunostained for CCN6, IGF2BP2, and HMGA2.

Results

CCN6 regulates IGF2BP2 and HMGA2 protein expression in MMTV-cre;Ccn6fl/fl tumors, in MDA-MB-231 and − 468, and in HME cells. CCN6 recombinant protein reduced IGF2BP2 and HMGA2 protein expression, and decreased growth of MMTV-cre;Ccn6fl/fl tumors in vivo. IGF2BP2 shRNA knockdown was sufficient to reverse the invasive abilities conferred by CCN6 knockdown in HME cells. Analyses of the TCGA Breast Cancer Cohort (n = 1238) showed that IGF2BP2 and HMGA2 are significantly upregulated in metaplastic carcinoma compared to other breast cancer subtypes. In clinical samples, low CCN6 is frequent in tumors with high IGF2BP2/HMGA2 with spindle and squamous differentiation.

Conclusions

These data shed light into the pathogenesis of metaplastic carcinoma and demonstrate a novel CCN6/IGF2BP2/HMGA2 oncogenic pathway with biomarker and therapeutic implications.

Keywords

Breast cancer CCN6 WISP3 IGF2BP2 HMGA2 Metaplastic 

Notes

Acknowledgements

We thank all members of the Kleer laboratory for helpful discussion. We thank Tina Fields from the Histology laboratory for assistance with immunostaining.

Funding

This work was supported by National institutes of Health (NIH) Grants R01CA125577 and R01CA107469 (C.G.K.), and the University of Michigan Rogel Cancer Center support Grant P30CA046592.

Compliance with ethical standards

Conflict of interest

Dr. Dafydd Thomas is consultant of Resonant Therapeutics. The other authors have no conflicts of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed University of Michigan UCUCA protocol #: PRO00006984 (approval date 6/23/2016, expiration date 6/23/2019).

Human and animal rights

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

10549_2018_4960_MOESM1_ESM.pptx (1.1 mb)
Supplementary Figure 1. CCN6 overexpression in MDA-MB-231 cells reduces EMT transcription factor expression. Immunoblot of MDA-MB-231 cells stably transduced with Flag-Vector or Flag-CCN6 with the indicated antibodies. Supplementary material 1 (PPTX 1117 KB)

References

  1. 1.
    Oberman HA (1987) Metaplastic carcinoma of the breast. A clinicopathologic study of 29 patients. Am J Surg Pathol 11(12):918–929CrossRefGoogle Scholar
  2. 2.
    Rakha EA, Tan PH, Varga Z, Tse GM, Shaaban AM, Climent F, van Deurzen CH, Purnell D, Dodwell D, Chan T, Ellis IO (2015) Prognostic factors in metaplastic carcinoma of the breast: a multi-institutional study. Br J Cancer 112(2):283–289.  https://doi.org/10.1038/bjc.2014.592 CrossRefPubMedGoogle Scholar
  3. 3.
    Abouharb S, Moulder S (2015) Metaplastic breast cancer: clinical overview and molecular aberrations for potential targeted therapy. Curr Oncol Rep 17(3):431.  https://doi.org/10.1007/s11912-014-0431-z CrossRefPubMedGoogle Scholar
  4. 4.
    Liu T, Zhang X, Shang M, Zhang Y, Xia B, Niu M, Liu Y, Pang D (2013) Dysregulated expression of Slug, vimentin, and E-cadherin correlates with poor clinical outcome in patients with basal-like breast cancer. J Surg Oncol 107(2):188–194.  https://doi.org/10.1002/jso.23240 CrossRefPubMedGoogle Scholar
  5. 5.
    Song Y, Liu X, Zhang G, Song H, Ren Y, He X, Wang Y, Zhang J, Zhang Y, Sun S, Liang X, Sun Q, Pang D (2013) Unique clinicopathological features of metaplastic breast carcinoma compared with invasive ductal carcinoma and poor prognostic indicators. World J Surg Oncol 11:129.  https://doi.org/10.1186/1477-7819-11-129 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kleer CG, Zhang Y, Merajver SD (2007) CCN6 (WISP3) as a new regulator of the epithelial phenotype in breast cancer. Cells Tissues Organs 185(1–3):95–99.  https://doi.org/10.1159/000101308 CrossRefPubMedGoogle Scholar
  7. 7.
    Kleer CG, Zhang Y, Pan Q, Merajver SD (2004) WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer. Neoplasia 6(2):179–185.  https://doi.org/10.1593/neo.03316 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Pal A, Huang W, Li X, Toy KA, Nikolovska-Coleska Z, Kleer CG (2012) CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer. Cancer Res 72 (18):4818–4828.  https://doi.org/10.1158/0008-5472.CAN-12-0154 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Zhang Y, Pan Q, Zhong H, Merajver SD, Kleer CG (2005) Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells. Breast Cancer Res 7(6):R1080–R1089.  https://doi.org/10.1186/bcr1351 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Huang W, Zhang Y, Varambally S, Chinnaiyan AM, Banerjee M, Merajver SD, Kleer CG (2008) Inhibition of CCN6 (Wnt-1-induced signaling protein 3) down-regulates E-cadherin in the breast epithelium through induction of snail and ZEB1. Am J Pathol 172(4):893–904CrossRefGoogle Scholar
  11. 11.
    Kleer CG, Zhang Y, Pan Q, van Golen KL, Wu ZF, Livant D, Merajver SD (2002) WISP3 is a novel tumor suppressor gene of inflammatory breast cancer. Oncogene 21(20):3172–3180CrossRefGoogle Scholar
  12. 12.
    Martin EE, Huang W, Anwar T, Arellano-Garcia C, Burman B, Guan JL, Gonzalez ME, Kleer CG (2017) MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas. Oncogene 36(16):2275–2285.  https://doi.org/10.1038/onc.2016.381 CrossRefPubMedGoogle Scholar
  13. 13.
    Dai N, Ji F, Wright J, Minichiello L, Sadreyev R, Avruch J (2017) IGF2 mRNA binding protein-2 is a tumor promoter that drives cancer proliferation through its client mRNAs IGF2 and HMGA1. Elife.  https://doi.org/10.7554/eLife.27155 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Li Y, Francia G, Zhang JY (2015) p62/IMP2 stimulates cell migration and reduces cell adhesion in breast cancer. Oncotarget 6(32):32656–32668.  https://doi.org/10.18632/oncotarget.5328 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Morishita A, Zaidi MR, Mitoro A, Sankarasharma D, Szabolcs M, Okada Y, D’Armiento J, Chada K (2013) HMGA2 is a driver of tumor metastasis. Cancer Res 73 (14):4289–4299.  https://doi.org/10.1158/0008-5472.CAN-12-3848 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Degrauwe N, Suva ML, Janiszewska M, Riggi N, Stamenkovic I (2016) IMPs: an RNA-binding protein family that provides a link between stem cell maintenance in normal development and cancer. Genes Dev 30(22):2459–2474.  https://doi.org/10.1101/gad.287540.116 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cleynen I, Brants JR, Peeters K, Deckers R, Debiec-Rychter M, Sciot R, Van de Ven WJ, Petit MM (2007) HMGA2 regulates transcription of the Imp2 gene via an intronic regulatory element in cooperation with nuclear factor-kappaB. Mol Cancer Res 5(4):363–372.  https://doi.org/10.1158/1541-7786.MCR-06-0331 CrossRefPubMedGoogle Scholar
  18. 18.
    Huang W, Gonzalez ME, Toy KA, Banerjee M, Kleer CG (2010) Blockade of CCN6 (WISP3) activates growth factor-independent survival and resistance to anoikis in human mammary epithelial cells. Cancer Res 70 (8):3340–3350.  https://doi.org/10.1158/0008-5472.CAN-09-4225 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Gonzalez ME, DuPrie ML, Krueger H, Merajver SD, Ventura AC, Toy KA, Kleer CG (2011) Histone methyltransferase EZH2 induces Akt-dependent genomic instability and BRCA1 inhibition in breast cancer. Cancer Res 71 (6):2360–2370.  https://doi.org/10.1158/0008-5472.CAN-10-1933 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Leung BM, Lesher-Perez SC, Matsuoka T, Moraes C, Takayama S (2015) Media additives to promote spheroid circularity and compactness in hanging drop platform. Biomater Sci 3(2):336–344.  https://doi.org/10.1039/c4bm00319e CrossRefPubMedGoogle Scholar
  21. 21.
    Zhang Y, Toy KA, Kleer CG (2012) Metaplastic breast carcinomas are enriched in markers of tumor-initiating cells and epithelial to mesenchymal transition. Mod Pathol 25(2):178–184.  https://doi.org/10.1038/modpathol.2011.167 CrossRefPubMedGoogle Scholar
  22. 22.
    Lorenzatti G, Huang W, Pal A, Cabanillas AM, Kleer CG (2011) CCN6 (WISP3) decreases ZEB1-mediated EMT and invasion by attenuation of IGF-1 receptor signaling in breast cancer. J Cell Sci 124 (Pt 10):1752–1758.  https://doi.org/10.1242/jcs.084194jcs.084194 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Investig 121(7):2750–2767.  https://doi.org/10.1172/JCI45014 CrossRefPubMedGoogle Scholar
  24. 24.
    Hayes MJ, Thomas D, Emmons A, Giordano TJ, Kleer CG (2008) Genetic changes of Wnt pathway genes are common events in metaplastic carcinomas of the breast. Clin Cancer Res 14 (13):4038–4044.  https://doi.org/10.1158/1078-0432.CCR-07-4379 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Weigelt B, Ng CK, Shen R, Popova T, Schizas M, Natrajan R, Mariani O, Stern MH, Norton L, Vincent-Salomon A, Reis-Filho JS (2015) Metaplastic breast carcinomas display genomic and transcriptomic heterogeneity [corrected]. Mod Pathol 28(3):340–351.  https://doi.org/10.1038/modpathol.2014.142 CrossRefPubMedGoogle Scholar
  26. 26.
    Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K, Gilcrease MZ, Krishnamurthy S, Lee JS, Fridlyand J, Sahin A, Agarwal R, Joy C, Liu W, Stivers D, Baggerly K, Carey M, Lluch A, Monteagudo C, He X, Weigman V, Fan C, Palazzo J, Hortobagyi GN, Nolden LK, Wang NJ, Valero V, Gray JW, Perou CM, Mills GB (2009) Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 69 (10):4116–4124.  https://doi.org/10.1158/0008-5472.CAN-08-3441 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Ng CKY, Piscuoglio S, Geyer FC, Burke KA, Pareja F, Eberle CA, Lim RS, Natrajan R, Riaz N, Mariani O, Norton L, Vincent-Salomon A, Wen YH, Weigelt B, Reis-Filho JS (2017) The landscape of somatic genetic alterations in metaplastic breast carcinomas. Clin Cancer Res 23(14):3859–3870.  https://doi.org/10.1158/1078-0432.CCR-16-2857 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Badve S, Dabbs DJ, Schnitt SJ, Baehner FL, Decker T, Eusebi V, Fox SB, Ichihara S, Jacquemier J, Lakhani SR, Palacios J, Rakha EA, Richardson AL, Schmitt FC, Tan PH, Tse GM, Weigelt B, Ellis IO, Reis-Filho JS (2011) Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Mod Pathol 24(2):157–167.  https://doi.org/10.1038/modpathol.2010.200 CrossRefPubMedGoogle Scholar
  29. 29.
    Turner NC, Reis-Filho JS (2006) Basal-like breast cancer and the BRCA1 phenotype. Oncogene 25(43):5846–5853CrossRefGoogle Scholar
  30. 30.
    Turner NC, Reis-Filho JS, Russell AM, Springall RJ, Ryder K, Steele D, Savage K, Gillett CE, Schmitt FC, Ashworth A, Tutt AN (2007) BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene 26(14):2126–2132CrossRefGoogle Scholar
  31. 31.
    Nakamura Y, Weidinger G, Liang JO, Aquilina-Beck A, Tamai K, Moon RT, Warman ML (2007) The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling. J Clin Investig 117(10):3075–3086.  https://doi.org/10.1172/JCI32001 CrossRefPubMedGoogle Scholar
  32. 32.
    Barghash A, Helms V, Kessler SM (2015) Overexpression of IGF2 mRNA-binding protein 2 (IMP2/p62) as a feature of basal-like breast cancer correlates with short survival. Scand J Immunol 82(2):142–143.  https://doi.org/10.1111/sji.12307 CrossRefPubMedGoogle Scholar
  33. 33.
    Liu W, Li Y, Wang B, Dai L, Qian W, Zhang JY (2015) Autoimmune response to IGF2 mRNA-binding protein 2 (IMP2/p62) in breast cancer. Scand J Immunol 81(6):502–507.  https://doi.org/10.1111/sji.12285 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Wend P, Runke S, Wend K, Anchondo B, Yesayan M, Jardon M, Hardie N, Loddenkemper C, Ulasov I, Lesniak MS, Wolsky R, Bentolila LA, Grant SG, Elashoff D, Lehr S, Latimer JJ, Bose S, Sattar H, Krum SA, Miranda-Carboni GA (2013) WNT10B/beta-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer. EMBO Mol Med 5(2):264–279.  https://doi.org/10.1002/emmm.201201320 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wu J, Zhang S, Shan J, Hu Z, Liu X, Chen L, Ren X, Yao L, Sheng H, Li L, Ann D, Yen Y, Wang J, Wang X (2016) Elevated HMGA2 expression is associated with cancer aggressiveness and predicts poor outcome in breast cancer. Cancer Lett 376 (2):284–292.  https://doi.org/10.1016/j.canlet.2016.04.005 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Emily R. McMullen
    • 1
  • Maria E. Gonzalez
    • 1
    • 2
  • Stephanie L. Skala
    • 1
  • Mai Tran
    • 1
    • 2
  • Dafydd Thomas
    • 1
  • Sabra I. Djomehri
    • 1
    • 2
  • Boris Burman
    • 1
    • 2
  • Kelley M. Kidwell
    • 2
    • 3
  • Celina G. Kleer
    • 1
    • 2
    • 4
  1. 1.Department of PathologyUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Rogel Cancer CenterUniversity of Michigan Medical SchoolAnn ArborUSA
  3. 3.Department of BiostatisticsUniversity of Michigan Medical SchoolAnn ArborUSA
  4. 4.Department of PathologyUniversity of Michigan Medical School, 4217 Rogel Cancer CenterAnn ArborUSA

Personalised recommendations