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

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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.

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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.

Author information

Author notes

  1. Emily R. McMullen and Maria E. Gonzalez have contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA

    Emily R. McMullen, Maria E. Gonzalez, Stephanie L. Skala, Mai Tran, Dafydd Thomas, Sabra I. Djomehri, Boris Burman & Celina G. Kleer

  2. Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, 48109, USA

    Maria E. Gonzalez, Mai Tran, Sabra I. Djomehri, Boris Burman, Kelley M. Kidwell & Celina G. Kleer

  3. Department of Biostatistics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA

    Kelley M. Kidwell

  4. Department of Pathology, University of Michigan Medical School, 4217 Rogel Cancer Center, 1500 E. Medical Center Dr., Ann Arbor, MI, 48109, USA

    Celina G. Kleer

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  1. Emily R. McMullen
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Correspondence to Celina G. Kleer.

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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).

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

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10549_2018_4960_MOESM1_ESM.pptx

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)

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McMullen, E.R., Gonzalez, M.E., Skala, S.L. et al. CCN6 regulates IGF2BP2 and HMGA2 signaling in metaplastic carcinomas of the breast. Breast Cancer Res Treat 172, 577–586 (2018). https://doi.org/10.1007/s10549-018-4960-2

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