Brief ReportTumor suppression by collagen XV is independent of the restin domain
Highlights
► Collagen XV increases adhesion of cells to collagen I. ► N-terminal/collagenous domains of collagen XV mediate collagen I adhesion. ► The C-terminal restin domain does not increase adhesion to collagen I. ► Mutation of a cysteine residue in the collagenous domain disrupts adhesion. ► Restin does not suppress tumorigenicity of cervical cancer cells in vivo.
Introduction
Our interest in collagen XV as a potential tumor suppressor arose from previous data (reviewed in Harris, 2003) that mapped a locus for suppression of malignancy to a region between bands A4 and C3 on mouse chromosome 4 (Jonasson et al., 1977). The activity of this locus was dependent on gene dosage (Evans et al., 1982) and correlated with the expression of a dense collagenous matrix (Harris, 1985). A search for potential tumor suppressor and/or collagen genes within the A4–C3 region identified the gene encoding procollagen XVa1 as the most likely candidate. The α1 chain of human type XV collagen (COL15A1) encodes a 1388 amino acid (aa) protein, with a 25 aa putative signal peptide, a 530 aa N-terminal non-collagenous domain, a 577 residue discontinuous collagenous sequence and a 256 aa C-terminal non-collagenous domain (Hagg et al., 1998). Within the collagenous part of the molecule nine collagenous domains (containing the characteristic Gly-X-Y motif where X and Y may be any other amino acid but an excess of proline and hydroxyproline residues is found) are interrupted by 8 non-collagenous sequences (Fig. 1A). The N-terminal non-collagenous domain of type XV and type XVIII collagen, to which it is highly similar, shows sequence homology to thrombospondin. Collagen XV was originally described in a sequence derived from a placental cDNA clone (Myers et al., 1992) though it is widely distributed in many human tissues. It is strongly associated with vascular, neuronal, mesenchymal and some epithelial basement membranes implicating a function in adhering the basement membrane to adjacent connective tissue stroma (Myers et al., 1996). More recently a very close association between collagen XV and the fibrillar collagen network was shown subjacent to the basement membrane, a location from which it is ideally placed to contribute to signal transduction pathways (Amenta et al., 2005). Type XV collagen exists in two core protein forms of 250 and 225 kDa, which differ in their carboxyl termini; however, the core proteins are modified by O-linked chondroitin/dermatan sulfate side chains and so may exhibit a much larger apparent molecular weight of about 400 kDa or greater (Li et al., 2000, Amenta et al., 2005). Collagen XV contains a trimerization domain located between the collagenous region and the C-terminal restin domain (Wirz et al., 2011). The trimers are linked by interchain disulfide bonds involving only the two cysteine residues (at amino acids 733 and 965) in the collagenous domain and not the other 8 cysteines located elsewhere in the molecule (Li et al., 2000) (Fig. 1A).
Not only does the location of collagen XV subjacent to the basement membrane support a role in the suppression of growth of malignant tumors, invasion of the basement membrane by human ductal breast carcinoma cells and colon carcinomas was preceded by the disappearance of this molecule (Amenta et al., 2000, Amenta et al., 2003). This phenomenon is also true for skin carcinomas and melanomas (Fukushige et al., 2005) and we have preliminary evidence for loss of collagen XV from pancreatic cancers (data not shown). We demonstrated that expression of recombinant collagen XV in a human cervical carcinoma cell line that does not normally express the protein, suppresses tumorigenesis in a dose-dependent manner (Harris et al., 2007). More recently, recombinant collagen XV was also shown to inhibit the adhesion and migration of fibrosarcoma cells in vitro (Hurskainen et al., 2010).
One potential mechanism for the tumor suppressor functions of collagen XV is the reported anti-angiogenic properties of the endostatin domains of collagen XVIII, XV (restin) (Ramchandran et al., 1999, John et al., 2005) and C-terminal fragments of collagen IV α3 (tumstatin, arresten and canstatin) (reviewed in Cooke and Kalluri, 2008). The active peptides are derived from the non-triple helical carboxyl-terminal NC1 domains of these collagens, which are cleaved by proteases and released as trimers, though they are not active until converted to monomeric forms. Though endostatin derived from collagen XVIII has been shown to inhibit angiogenesis (the sprouting of new blood vessels) and endothelial cell migration and to reduce tumor growth in animal models (O'Reilly et al., 1997) these data are controversial (Harris, 2005, Brideau et al., 2007). Also the sequence, structure and function of collagen XV- and collagen XVIII-derived endostatins are divergent (Sasaki et al., 2000, Gaetzner et al., 2005). We previously proposed (Harris et al., 2007) that the tumor suppressor properties of collagen XV reported here involve mechanisms that are different from those mediated by endostatin-like activities for a number of reasons: 1. Collagen XV alters the growth properties of cervical carcinoma cells in three-dimensional culture in vitro where angiogenesis is not relevant. 2. We observe the effects in vivo when an incipient tumor is well below the dimensions at which angiogenesis would be relevant. 3. At high levels of collagen XV expression suppression of malignancy is complete, in contrast to the effects of high doses of endostatin on solid tumors. In the experiments described here we first demonstrate that expression of collagen XV increases adhesion of cervical cancer cells to collagen I substrates, which may recapitulate early events in tumor growth, and that this is conferred by the N-terminal and/or collagenous domains of the protein but not by the restin domain. Moreover, the increased adhesion is dependent on the ability of collagen XV to form wild-type intermolecular interactions. Next, we show that the expression of the restin domain of collagen XV alone, does not inhibit tumor growth in vivo. Thus collagen XV-mediated tumor suppression is not caused by potential anti-angiogenic properties of the restin domain.
Section snippets
Generation of D98 AP2 clones expressing the restin domain of collagen XV; collagen XV without the restin domain; and collagen XV with mutations in a critical cysteine residue
The restin construct was transiently expressed in COS-7 cells to demonstrate that it generated a protein of the expected size (~ 32 kDa) (Fig. 1B). Three additional constructs were evaluated in transient transfection: a FLAG epitope-tagged version of human collagen XV (hcolXV), (F-hcolXV); a construct that encompassed the N terminal and collagenous domains of collagen XV without the restin domain (F-hcolXV(−)restin) (Fig. 1C); and an hcolXV construct with the cysteine residue at 733 mutated to
Discussion
We previously showed that human collagen XV altered the growth properties of cervical cancer cells in vitro and suppressed growth of these cells as tumors in vivo, in a dose-dependent manner. It was possible that these observations were due to functions associated with sequence similarity between the carboxyl terminal restin domain of collagen XV and the endostatin domain of collagen XVIII. Endostatin is a potent inhibitor of angiogenesis and so can partially inhibit tumor growth in mice (
Collagen XV expression constructs
The full-length human collagen XV cDNA (hcolXV, NM_001855) was kindly donated by Dr. Taina Pihlajaniemi and transferred to pcDNA3.1(−)Neo as described previously (Harris et al., 2007). A schematic of the collagen XV protein is shown in Fig. 1A. All PCR primers and oligonucleotides used for mutagenesis or cloning are shown in Suppl. Table 1. Two restin domain constructs were generated: 1) includes the collagen XV signal sequence (amino acids 1–27), followed by a large part of NC10 (1130–1388);
Acknowledgments
This work was supported in part by NIH grants CA129258 to AH and P50CA072712, and U01CA111294 to MAH. We thank Dr. Taina Pihlajaniemi, for the human colXV cDNA.
References (25)
- et al.
Type XV collagen in human colonic adenocarcinomas has a different distribution than other basement membrane zone proteins
Hum. Pathol.
(2000) - et al.
Chapter 1. Molecular mechanism of type IV collagen-derived endogenous inhibitors of angiogenesis
Methods Enzymol.
(2008) - et al.
Endostatin's heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants
Matrix Biol.
(2005) - et al.
Complete exon–intron organization of the human gene for the alpha1 chain of type XV collagen (COL15A1) and comparison with the homologous COL18A1 gene
J. Biol. Chem.
(1998) - et al.
Recombinant human collagen XV regulates cell adhesion and migration
J. Biol. Chem.
(2010) - et al.
Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin
Biochim. Biophys. Acta
(2005) - et al.
Basement membrane zone type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan in human tissues and cultured cells
J. Biol. Chem.
(2000) - et al.
Endostatin: an endogenous inhibitor of angiogenesis and tumor growth
Cell
(1997) - et al.
Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin
Biochem. Biophys. Res. Commun.
(1999) - et al.
Endostatins derived from collagens XV and XVIII differ in structural and binding properties, tissue distribution and anti-angiogenic activity
J. Mol. Biol.
(2000)
Crystal structure of the human collagen XV trimerization domain: a potent trimerizing unit common to multiplexin collagens
Matrix Biol.
Loss of types XV and XIX collagen precedes basement membrane invasion in ductal carcinoma of the female breast
J. Pathol.
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