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

Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.

Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic activity by binding tightly to the MMP active site. While each of the four TIMPs can inhibit most MMPs, binding data reveal tremendous heterogeneity in affinities of different TIMP/MMP pairs, and the structural features that differentiate stronger from weaker complexes are poorly understood. Here we report the crystal structure of the comparatively weakly bound human MMP-10/TIMP-2 complex at 2.1 Å resolution. Comparison with previously reported structures of MMP-3/TIMP-1, MT1-MMP/TIMP-2, MMP-13/TIMP-2, and MMP-10/TIMP-1 complexes offers insights into the structural basis of binding selectivity. Our analyses identify a group of highly conserved contacts at the heart of MMP/TIMP complexes that define the conserved mechanism of inhibition, as well as a second category of diverse adventitious contacts at the periphery of the interfaces. The AB loop of the TIMP N-terminal domain and the contact loops of the TIMP C-terminal domain form highly variable peripheral contacts that can be considered as separate exosite interactions. In some complexes these exosite contacts are extensive, while in other complexes the AB loop or C-terminal domain contacts are greatly reduced and appear to contribute little to complex stability. Our data suggest that exosite interactions can enhance MMP/TIMP binding, although in the relatively weakly bound MMP-10/TIMP-2 complex they are not well optimized to do so. Formation of highly variable exosite interactions may provide a general mechanism by which TIMPs are fine-tuned for distinct regulatory roles in biology

Crystallographic data collection and refinement statistics for MMP-10cd/TIMP-2 complex.

a<p>Values in parentheses are for the highest resolution shell (2.14–2.1 Å).</p>b<p>Ramachandran distribution is reported as defined by Molprobity/PDB validation.</p

Contacts at the MMP-10cd/TIMP-2 interface.

<p>MMP-10cd is rendered as a cartoon covered by semitransparent surface (slate) in the standard frontal orientation, with horizontally aligned TIMP-2 segments in stick representation (salmon). (A) Overview shows contacts of the TIMP-2 C-connector and N-terminal segment with MMP-10 substrate binding cleft (center), TIMP-2 AB loop contacts with MMP-10 S-loop and βIV-βV loop (upper left), and TIMP-2 C-terminal domain contacts with MMP-10 specificity and βV-hB loops (lower right). (B) Closer view of MMP-10 substrate binding cleft shows TIMP-2 C-connector residues occupying nonprimed subsites to the left of the catalytic zinc, while TIMP-2 N-terminal residues occupy primed subsites to the right of the zinc. (C) Closer view of the AB loop interactions reveals two interfacial H-bonds (dotted yellow lines highlighted by yellow arrows), and burial of the Ile-40 side chain in a hydrophobic pocket formed by MMP-10 residues Phe-170, Tyr-171, and Leu190. (D) GH loop residues 132–135 and multiple turn loop residues 151–157 on the C-terminal domain of TIMP-2 form minimal interactions with the MMP-10cd, including ring-stacking and cation-π interactions with MMP-10 specificity loop residues Phe-242 and Tyr-239.</p

Diversity of MMP contact surfaces interacting with TIMP N- and C-terminal domains.

<p>MMPs are shown in the standard frontal orientation, with catalytic zinc shown as a blue sphere. (A) MMP-10cd/TIMP-2: MMP-10cd surface is shown in grey with footprint of the surface buried by TIMP-2 N-terminal domain in magenta and C-terminal domain in orange. (B) MT1-MMPcd/TIMP-2 (PDB ID: 1BQQ) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075836#pone.0075836-FernandezCatalan1" target="_blank">[27]</a>: MT1-MMPcd surface is shown in wheat with footprint of the surface buried by TIMP-2 N-terminal domain in magenta and C-terminal domain in orange. (C) MMP-13cd/TIMP-2 (PDB ID: 2E2D) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075836#pone.0075836-Maskos1" target="_blank">[28]</a>: MMP-13cd surface is shown in brown with footprint of the surface buried by TIMP-2 N-terminal domain in magenta and C-terminal domain in orange. (D) MMP-10cd/TIMP-1 (PDB ID: 3V96) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075836#pone.0075836-Batra1" target="_blank">[24]</a>: MMP-10cd surface is shown in grey with footprint of the surface buried by TIMP-1 N-terminal domain in green and C-terminal domain in yellow. (E) MMP-3cd/TIMP-1 (PDB ID: 1UEA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075836#pone.0075836-GomisRuth1" target="_blank">[26]</a>: MMP-3cd surface is shown in dark grey with footprint of the surface buried by TIMP-1 N-terminal domain in green and C-terminal domain in yellow. The MMP contact surfaces shown are for atoms within 4.5 Å of the TIMP; figures were generated using PYMOL.</p

Buried Surface Area (BSA).

†<p>ΔG refers to changes in solvation energy on complex formation.</p

Comparison of MMP-10cd/TIMP-1 and MMP-10cd/TIMP-2 complexes.

<p>MMP-10cd/TIMP-2 molecules are shown in blue and raspberry, respectively, with MMP-10cd/TIMP-1 complex (PDB ID: 3V96) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075836#pone.0075836-Batra1" target="_blank">[24]</a> shown in white; complexes are superposed based on C_α atoms of all MMP-10cd residues. (A) The long AB loop of TIMP-2 forms a much more extensive contact area with the MMP-10cd than is seen with TIMP-1, while the C-terminal loops of TIMP-2 form fewer contacts than in the complex with TIMP-1. (B) TIMP-2 is rotated by ∼21° around an axis centered on the catalytic zinc when compared with TIMP-1.</p

MMP-10cd/TIMP-2 interface electrostatic interactions.

<p>MMP-10cd/TIMP-2 interface electrostatic interactions.</p