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Cell Communication Network Factor 4 (CCN4/WISP1) Shifts Melanoma Cells from a Fragile Proliferative State to a Resilient Metastatic State

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Abstract

Introduction

Cellular communication network factor 4 (CCN4/WISP1) is a secreted matricellular protein that stimulates metastasis in multiple malignancies but has an unclear impact on phenotypic changes in melanoma. Recent data using cells edited via a double-nickase CRISPR/Cas9 approach suggest that CCN4/WISP1 stimulates invasion and metastasis of melanoma cells. While these data also suggest that loss of CCN4/WISP1 increases cell proliferative, the CRISPR approach used may be an alternative explanation rather than the loss of gene function.

Methods

To test whether CCN4/WISP1 also influences the proliferative phenotype of melanoma cells, we used mouse melanoma models and knocked out Ccn4 using a homology-directed repair CRISPR/Cas9 system to generate pools of Ccn4-knockout cells. The resulting edited cell pools were compared to parental cell lines using an ensemble of in vitro and in vivo assays.

Results

In vitro assays using knockout pools supported previous findings that CCN4/WISP1 promoted an epithelial–mesenchymal-like transition in melanoma cells and stimulated invasion and metastasis. While Ccn4 knockout also enhanced cell growth in optimal 2D culture conditions, the knockout suppressed certain cell survival signaling pathways and rendered cells less resistant to stress conditions. Tumor cell growth assays at sub-optimal conditions in vitro, quantitative analysis of tumor growth assays in vivo, and transcriptomics analysis of human melanoma cell lines were also used to quantify changes in phenotype and generalize the findings.

Conclusions

In addition to stimulating invasion and metastasis of melanoma cells, the results suggested that CCN4/WISP1 repressed cell growth and simultaneously enhanced cell survival.

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Abbreviations

CCLE:

Cancer Cell Line Encyclopedia

CCN4:

Cell Communication Network factor 4

CMV:

Cytomegalovirus

CRISPR:

Clustered regularly interspaced short palindromic repeats

ELISA:

Enzyme-linked immunosorbent assay

EMT:

Epithelial-mesenchymal transition

GFP:

Green fluorescent protein

HDR:

Homology-directed repair

KO:

Knock-out

NSG:

NOD-scid IL2Rgammanull

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Acknowledgments

This work was supported by National Science Foundation (NSF CBET-1644932 to DJK) and National Cancer Institute (NCI 1R01CA193473 to DJK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSF or NCI. Small animal imaging and image analysis were performed in the West Virginia University Animal Models & Imaging Facility, which has been supported by the West Virginia University Cancer Institute and NIH Grants P20 RR016440 and P30RR032138/P30GM103488. No human studies were carried out by the authors for this article. All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the appropriate institutional committees.

Conflict of interest

Wentao Deng, Audry Fernandez, Sarah L. McLaughlin, and David J. Klinke II declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV, 26505, USA

    Wentao Deng, Audry Fernandez & David J. Klinke II

  2. WVU Cancer Institute, West Virginia University, Morgantown, WV, 26505, USA

    Wentao Deng, Audry Fernandez, Sarah L. McLaughlin & David J. Klinke II

  3. Animal Models and Imaging Facility, West Virginia University, Morgantown, WV, 26505, USA

    Sarah L. McLaughlin

  4. Department of Chemical and Biomedical Engineering, West Virginia University, Morgantown, WV, 26505, USA

    David J. Klinke II

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  1. Wentao Deng
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Correspondence to David J. Klinke II.

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Deng, W., Fernandez, A., McLaughlin, S.L. et al. Cell Communication Network Factor 4 (CCN4/WISP1) Shifts Melanoma Cells from a Fragile Proliferative State to a Resilient Metastatic State. Cel. Mol. Bioeng. 13, 45–60 (2020). https://doi.org/10.1007/s12195-019-00602-2

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  • DOI: https://doi.org/10.1007/s12195-019-00602-2

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