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A spontaneous metastasis model reveals the significance of claudin-9 overexpression in lung cancer metastasis

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Abstract

Metastasis causes most cancer related mortality but the mechanisms governing metastatic dissemination are poorly defined. Metastasis involves egression of cancer cells from the primary tumors, their survival in circulation and colonization at the secondary sites. Cancer cell egression from the primary tumor is the least defined process of metastasis as experimental metastasis models directly seed cancer cells in circulation, thus bypassing this crucial step. Here, we developed a spontaneous metastasis model that retains the egression step of metastasis. By repeated in vivo passaging of the poorly metastatic Lewis lung carcinoma (3LL) cells, we generated a cell line (p-3LL) that readily metastasizes to lungs and liver from subcutaneous (s.c.) tumors. Interestingly, when injected intravenously, 3LL and p-3LL cells showed a similar frequency of metastasis. This suggests enhanced egression of p-3LL cells may underlie the enhanced metastatic spread from primary tumors. Microarray analysis of 3LL and p-3LL cells as well as the primary tumors derived from these cells revealed altered expression of several genes including significant upregulation of a tight junction protein, claudin-9. Increased expression of claudin-9 was confirmed in both p-3LL cells and tumors derived from these cells. Knockdown of claudin-9 expression in p-3LL cells by si-RNA significantly reduced their motility, invasiveness in vitro and metastasis in vivo. Conversely, transient overexpression of claudin-9 in 3LL cells enhanced their motility. These results suggest an essential role for claudin-9 in promoting lung cancer metastasis.

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Abbreviations

LLC/3LL cells:

Lewis lung carcinoma

s.c.:

Subcutaneous

i.v.:

Intravenous

p-3LL:

Passaged 3LL cells

EMT:

Epithelial to mesenchymal trans-differentiation

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Acknowledgments

This study was funded in part from NIH R01 (CA138623), Kentucky Lung Cancer Research Program (RKS), The James Graham Brown Cancer Center and a grant from The University of Louisville School of Medicine (RKS). We thank John W. Eaton for critical reading of the manuscript. No writing assistance was utilized in the production of this manuscript.

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Authors and Affiliations

  1. James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA

    Rajesh K. Sharma, Biswa Pratim Das Purkayastha, Venkatakrishna R. Jala & Bodduluri Haribabu

  2. Department of Medicine, University of Louisville, Louisville, KY, 40202, USA

    Rajesh K. Sharma

  3. Department of Microbiology and Immunology, University of Louisville, Louisville, KY, 40202, USA

    Rajesh K. Sharma, Zinal S. Chheda, Venkatakrishna R. Jala & Bodduluri Haribabu

  4. The Hiram C. Polk Jr, MD, Department of Surgery, University of Louisville, Louisville, KY, 40202, USA

    Jorge G. Gomez-Gutierrez

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  1. Rajesh K. Sharma
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  2. Zinal S. Chheda
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Sharma, R.K., Chheda, Z.S., Das Purkayastha, B.P. et al. A spontaneous metastasis model reveals the significance of claudin-9 overexpression in lung cancer metastasis. Clin Exp Metastasis 33, 263–275 (2016). https://doi.org/10.1007/s10585-015-9776-4

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  • DOI: https://doi.org/10.1007/s10585-015-9776-4

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