Mmp10 expression correlates with cancer stem cell genotypes and metastasis in human lung tumors.

<p><b>A</b>) Gene expression data from primary human lung adenocarcinomas were divided into two groups of 30 samples consisting of lowest (Low) and highest (High) Mmp10 expressing lung tumors. n = 30; * p = 2.8×10⁻³³. <b>B</b>) Mmp10 in normal lung versus lung tumors with (<b><i>Met.</i></b>) and without (<b><i>No Met.</i></b>) bone metastases. n = 3, normals, n = 9, No Mets, n = 7, Met.; NS =  not significant, *p = 0.008; **p = 0.04. <b>C</b>) Mmp10 mRNA expression in human tumors. Data are expressed as fold-change from matched normal.</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 Overexpressed in many Human Cancer Types.

<p>MMP10 is Overexpressed in many Human Cancer Types.</p

Mmp10 is required for <i>Kras</i>-induced expansion and transformation of BASCs <i>in vitro.</i>

<p>BASCs isolated from Ntg, <i>Mmp10^−/−</i>, <i>LSL-Kras</i>, and <i>LSL-Kras/Mmp10^−/−</i> mice were treated with AdCre and plated in three-dimensional Matrigel culture as described in <i>Experimental Procedures</i>. <b>A</b>) Flow cytometry of isolated BASCs for SPC and CCSP <b>B</b>) QPCR for Mmp10 in BASCs from Ntg, <i>LSL-Kras,</i> and <i>LSL-Kras/Mmp10^−/−</i> mice. Fold of Ntg +/<i>−</i>SEM. n = 3, *p<0.000008. <b>C</b>) Morphology of BASC colonies from Ntg, <i>Mmp10^−/−</i>,<i>LSL-Kras</i>, and <i>LSL-Kras/Mmp10^−/−</i> mice. <b>D</b>) Analysis of BASC colony size. %Ntg +/−SEM. n = 85 Ntg, 56 <i>Mmp10^−/−</i>,30 (<i>LSL-Kras</i>) and 80 (<i>LSL-Kras/Mmp10^−/−</i>). *p<0.00001 Ntg vs, <i>LSL-Kras;</i> **p<0.00001 <i>LSL-Kras</i> vs. <i>LSL-Kras/Mmp10^−/−</i>.</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

The Effects of Knockdown of Rho-Associated Kinase 1 and Zipper-Interacting Protein Kinase on Gene Expression and Function in Cultured Human Arterial Smooth Muscle Cells

<div><p>Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and ZIPK have diverse, but predominantly distinct regulatory functions in vascular SMC and that ROCK1-mediated activation of ZIPK is not involved in most of these functions.</p></div

Mmp10 plays a promotive role in urethane-induced lung tumorigenesis.

<p><i>Mmp10^−/−</i> mice and Ntg littermates were injected with urethane and analyzed as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#s4" target="_blank">Materials and Methods</a></i>. <b>A</b>) H & E and immunohistochemical staining for Mmp10 in urethane-induced lung tumors. Higher magnification image of Mmp10 immunostaining is shown in the inset. Quantitative analysis of tumor number <b>B</b>), tumor size <b>C</b>) and tumor burden <b>D</b>) in urethane-treated Ntg (n = 7) and <i>Mmp10^−/−</i> (n = 12) mice. Mean +/−SEM; p<. 0.012 tumor number; p<0.019 tumor burden; p = 0.034 tumor size). <b>E</b>) Urethane-induced tumors from Ntg and Mmp10<i>⁻</i>^/<i>−</i> mice were graded as hyperplasia, adenoma or adenocarcinoma using published criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone.0026439-KellySpratt1" target="_blank">[26]</a>. Results are presented as the percentage of total tumors of each grade. Statistical analysis using Mann-Whitney U test revealed no statistically significant difference in tumor grade between urethane-induced tumors in Ntg and Mmp10<i>⁻</i>^/<i>−</i> mice (p = 0.39).</p

The 10 most highly correlated signatures associated with high Mmp10 in lung cancer.

<p>Gene sets marked in bold text contain Mmp10 as part of the gene signature.</p

Validation of the association between high Mmp10 in lung tumors and cancer stem cell signatures.

<p>Gene sets marked in bold text were also significantly enriched in the high Mmp10 lung tumor gene set analysis outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone-0026439-g005" target="_blank">Figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone-0026439-t001" target="_blank">Table 1</a>.</p

Association of metastatic lung cancer genes with cancer stem cell signatures.

<p>Gene sets marked in bold text are cancer stem cell signatures also identified as highly correlated with lung tumors expressing high Mmp10.</p