Matrix Metalloproteinase-10 Is Required for Lung Cancer Stem Cell Maintenance, Tumor Initiation and Metastatic Potential

Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10−/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells

Abstract LB-204: Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential

Abstract Matrix metalloproteinase 10 (Mmp10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many members of which have been implicated in tumor progression, invasion and metastasis. However, emerging evidence suggests a possible role for Mmps in tumor initiation. Here we reveal an unexpected role for Mmp10 in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mouse lung cancer cell cultures enriched in stem-like cells grow as undifferentiated tumor “oncospheres” that express elevated levels of the cancer stem cell markers Aldh1, CD133, Nanog, Notch3, Notch 4, Hey 1 and Hey 2. These cells also express elevated levels of Mmp10 mRNA and secrete elevated levels of Mmp10 protein. Functionally, these lung CSCs exhibit self-renewal, the ability to clonally expand, enhanced transforming potential in vitro, and enhanced tumorigenic properties when injected orthotopically into the lungs of syngeneic mice. RNAi-mediated knockdown of Mmp10 in these cells leads to a loss of stem cell marker gene expression and a dramatic inhibition of oncosphere growth, clonal expansion, transformed growth in vitro, and lung tumor formation and metastasis in vivo. In contrast, oncospheres implanted into syngeneic non-transgenic or Mmp10−/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human tumors demonstrates that Mmp10 is elevated in many human tumor types including lung, head and neck, esophageal, colon, breast, melanoma, bladder, cervical, ovarian, prostate and brain. Furthermore, gene set enhancement analysis (GSEA) demonstrates that elevated Mmp10 expression correlates with tumor metastasis and with cancer stem-like genomic signatures in human lung tumors. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-204. doi:1538-7445.AM2012-LB-204</jats:p

MMP10 is required for urethane- and <i>Kras-</i>induced BASC expansion <i>in vivo</i>.

<p><b>A</b>) Immunofluorescent analysis of CCSP (green) and SPC (red) dual positive BASCs (white arrows) in terminal bronchioles (TB) of control Ntg, and urethane-treated Ntg and <i>Mmp10^−/−</i>mice (<b><i>upper panels</i></b>), and from Ntg, <i>Kras^LA2</i> and <i>Kras^LA2/Mmp10^−/−</i> mice (<b><i>lower panels</i></b>). <b>B</b>) Quantitative analysis of BASCs in control Ntg, and urethane treated Ntg and <i>Mmp10^−/−</i> mice. %TBs; bars +/−SEM, n = ≥30 TBs/genotype. p<0.0005 urethane treated NTg vs. <i>Mmp10^−/−</i>; p<0.0001 untreated Ntg vs. urethane-treated Ntg; No significant difference between untreated non-Ntg vs. urethane treated <i>Mmp10^−/−</i> mice. <b>C</b>) Quantitative analysis of BASCs in lung TBs of Ntg, <i>Kras^LA2</i> and <i>Kras^LA2/Mmp10^−/−</i> mice. Columns, percentage of TBs; bars  = /−SEM, n≥50 TBs/genotype; p<0.001 Ntg vs. <i>Kras^LA</i> mice; p<0.003 <i>Kras^LA</i> mice vs. <i>Kras^LA2/Mmp10^−/−</i> mice; no significant difference in BASC number or distribution was observed between Ntg and <i>Kras^LA2/Mmp10^−/−</i> (p = 0.76) or Mmp10<i>⁻</i>^/<i>−</i> (p = 0.76) mice.</p

Cancer Stem Cell Signatures correlate with high Mmp10 expression in lung cancer.

<p>Cancer Stem Cell Signatures correlate with high Mmp10 expression in lung cancer.</p

Mmp10 is necessary for <i>Kras^LA2</i>-induced lung tumorigenesis <i>in vivo.</i>

<p><b>A</b>) Immunohistochemical staining of <i>Kras^LA2</i> lung tumor for mouse MMP10. Higher magnification image of Mmp10 immunostaining is shown in the inset. Quantitative analysis of <b>B</b>) tumor number, <b>C</b>) tumor size and <b>D)</b> tumor burden in <i>Kras^LA2</i> and <i>Kras^LA2/Mmp10^−/−</i> mice. Columns, mean; bars, SEM, n = 13, (*) denotes p = 0.04. E) Tumors from <i>Kras^LA2</i> and <i>Kras^LA2/Mmp10^−/−</i> mice were categorized as advanced adenomatous hyperplasia (AAH), or grade 1,2 or 3 adenomas using the published scoring criteria described by Jackson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone.0026439-Jackson1" target="_blank">[28]</a>. Results are presented as the percentage of total tumors of each grade. Statistical analysis using Mann-Whitney U test revealed a significant decrease in higher grade tumors in <i>Kras^LA2/Mmp10^−/−</i> mice; *p<0.002.</p