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Bone-related Genes Expressed in Advanced Malignancies Induce Invasion and Metastasis in a Genetically Defined Human Cancer Model

2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês

10.1074/jbc.m211498200

1083-351X

Jeremy N. Rich, Qing Shi, Mark D. Hjelmeland, Thomas J. Cummings, Chien‐Tsun Kuan, Darell D. Bigner, Christopher M. Counter, Xiao‐Fan Wang,

TGF-β signaling in diseases

We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitrophenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis. We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitrophenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis. matrix metalloproteinase epidermal growth factor receptor platelet-derived growth factor receptor insulin-like growth factor-1 receptor severe combined immune deficiency Gene expression analyses of human cancers have yielded tremendous quantities of data. Unfortunately, the phenotypic consequences of many changes in gene expression pattern between cancers and their corresponding normal tissues are largely unclear. To address this problem, we took a functional genomics approach by using a genetically defined glioma model system to investigate genes involved in the acquisition of malignant phenotype associated with late stages of tumorigenesis. Particularly, we were interested in genes whose expression is not associated with normal brain tissues or astrocyte cultures but which are nevertheless overexpressed in gliomas. Among those candidate genes, several bone-related genes were noticeably overexpressed in a high proportion of gliomas. Of note, two structurally unrelated genes, osteonectin and osteoactivin, have been found previously to be overexpressed in several other types of cancers, but their precise contribution to the development of specific cancer phenotype has yet to be elucidated. Osteonectin, also known as secreted protein, acidic and rich in cysteine (SPARC) or BM-40, is a 43-kDa extracellular matrix protein. Osteonectin was originally discovered as one of the most abundant non-collagenous components of bone, but it is also expressed in a number of other cell types that are involved in active remodeling of tissues (1Bradshaw A.D. Sage E.H. J. Clin. Invest. 2001; 107: 1049-1054Crossref PubMed Scopus (524) Google Scholar). Thus, the primary physiological role of osteonectin has been postulated to be an important modulator of cell-extracellular matrix interactions during the processes of tissue remodeling (2Brekken R.A Sage E.H. Matrix Biol. 2001; 19: 816-827Crossref PubMed Scopus (57) Google Scholar, 3Porter P.L. Sage E.H. Lane T.F. Funk S.E. Gown A.M. J. Histochem. Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (195) Google Scholar). Osteonectin is also abnormally expressed in many cancers, including gliomas (4Rempel S.A. Golembieski W.A. Ge S. Lemke N. Elisevich K. Mikkelsen T. Gutierrez J.A. J. Neuropathol. Exp. Neurol. 1998; 57: 1112-1121Crossref PubMed Scopus (120) Google Scholar, 5Lal A. Lash A.E. Altschul S.F. Velculescu V. Zhang L. McLendon R.E. Marra M.A. Prange C. Morin P.J. Polyak K. Papadopoulos N. Vogelstein B. Kinzler K.W. Strausberg R.L. Riggins G.J. Cancer Res. 1999; 59: 5403-5407PubMed Google Scholar), medulloblastomas (6Rempel S.A. Ge S. Gutierrez J.A. Clin. Cancer Res. 1999; 5: 237-241PubMed Google Scholar), meningiomas (7MacDonald T.J. Brown K.M. LaFleur B. Peterson K. Lawlor C. Chen Y. Packer R.J. Cogen P. Stephan D.A. Nat. Genet. 2001; 29: 143-152Crossref PubMed Scopus (391) Google Scholar), and cancers of the gastrointestinal tract, breast, lung, kidney, adrenal cortex, prostate, and bladder (3Porter P.L. Sage E.H. Lane T.F. Funk S.E. Gown A.M. J. Histochem. Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (195) Google Scholar, 8Ledda F. Bravo A.I. Adris S. Bover L. Mordoh J. Podhajcer O.L. J. Invest. Dermatol. 1997; 108: 210-214Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 9Thomas R. True L.D. Bassuk J.A. Lange P.H. Vessella R.L. Clin. Cancer Res. 2000; 6: 1140-1149PubMed Google Scholar, 10Le Bail B. Faouzi S. Boussarie L. Guirouilh J. Blanc J.F. Carles J. Bioulac-Sage P. Balabaud C. Rosenbaum J. J. Pathol. 1999; 189: 46-52Crossref PubMed Scopus (90) Google Scholar, 11Porte H. Chastre E. Prevot S. Nordlinger B. Empereur S. Basset P. Chambon P. Gespach C. Int. J. Cancer. 1995; 64: 70-75Crossref PubMed Scopus (164) Google Scholar). In gliomas, osteonectin is expressed in all tumor grades, usually at the tumor-brain margin and sites of neoangiogenesis (4Rempel S.A. Golembieski W.A. Ge S. Lemke N. Elisevich K. Mikkelsen T. Gutierrez J.A. J. Neuropathol. Exp. Neurol. 1998; 57: 1112-1121Crossref PubMed Scopus (120) Google Scholar), suggesting that osteonectin expression may be involved in tumor cell invasion (14Golembieski W.A. Ge S. Nelson K. Mikkelsen T. Rempel S.A. Int. J. Dev. Neurosci. 1999; 17: 463-472Crossref PubMed Scopus (80) Google Scholar). In contrast, increased osteonectin expression in other tumor types is associated with a conversion to invasive and metastatic tumors and a correlation in some instances with elevated expression of matrix metalloproteinases (MMPs)1that is linked to increased tumor malignancy (11Porte H. Chastre E. Prevot S. Nordlinger B. Empereur S. Basset P. Chambon P. Gespach C. Int. J. Cancer. 1995; 64: 70-75Crossref PubMed Scopus (164) Google Scholar, 12Gilles C. Bassuk J.A. Pulyaeva H. Sage E.H. Foidart J.M. Thompson E.W. Cancer Res. 1998; 58: 5529-5536PubMed Google Scholar, 13Sternlicht M. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3256) Google Scholar). Importantly, reduced expression of osteonectin by an antisense approach was found to correlate with a reduction in tumor formation by melanoma cells (15Ledda M.F. Adris S. Bravo A.I. Kairiyama C. Bover L. Chernajovsky Y. Mordoh J. Podhajcer O.L. Nat. Med. 1997; 3: 171-176Crossref PubMed Scopus (209) Google Scholar). Thus, although the precise role of osteonectin in the pathological process of carcinogenesis remains to be elucidated, osteonectin overexpression is intimately correlated with the progression of tumorigenesis of multiple types of human cancers. Osteoactivin, also known as GPNMB or dendritic cell-associated, heparan sulfate proteoglycan-integrin ligand (DC-HIL), is a type I transmembrane glycoprotein that is localized to the cell surface and lysosomal membranes (16Shikano S. Bonkobara M. Zukas P.K. Ariizumi K. J. Biol. Chem. 2001; 276: 8125-8134Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), as well as in a secreted form (17Safadi F.F. Xu J. Smock S.L. Rico M.C. Owen T.A. Popoff S.N. J. Cell. Biochem. 2001; 84: 12-26Crossref PubMed Scopus (136) Google Scholar). Highly expressed in bone, the physiological function of osteoactivin is postulated to be involved in the regulation of osteoblast maturation (16Shikano S. Bonkobara M. Zukas P.K. Ariizumi K. J. Biol. Chem. 2001; 276: 8125-8134Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Interestingly, osteoactivin is found to be overexpressed in melanomas (18Weterman M.A. Ajubi N. van Dinter I.M. Degen W.G. van Muijen G.N. Ruitter D.J. Bloemers H.P. Int. J. Cancer. 1995; 60: 73-81Crossref PubMed Scopus (213) Google Scholar), gliomas (19Loging W.T. Lal A. Siu I.M. Loney T.L. Wikstrand C.J. Marra M.A. Prange C. Bigner D.D. Strausberg R.L. Riggins G.J. Genome Res. 2000; 10: 1393-1402Crossref PubMed Scopus (88) Google Scholar), and cancers of the breast, stomach, and pancreas. 2NCBI SAGE Genie web site. Although the precise role of osteoactivin in cancer development remains unknown, it is tempting to suggest that the significant elevation of this bone-related protein in gliomas has a role in brain tumor progression. To determine the specific contributions of osteonectin and osteoactivin expression to human cancer development, we employed a genetically defined model system in which the genetic effects of increased gene expression could be directly linked to phenotypic changes of tumorigenesis. In this system, transformed human astrocytes by the sequential introduction of the simian virus-40 large T antigen, the human telomerase catalytic subunit (hTERT), and oncogenic Ha-Ras display a phenotype mimicking that of low grade gliomas in the formation of non-invasive tumor mass in immune-compromised animals (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). The characteristics of this human tumor model system allowed us to test specifically the phenotypic changes associated with tumor progression derived from the expression of genes under investigation. Consequently, we found that expression of osteonectin or osteoactivin was associated with angiocentric intracranial invasion and increased production of MMPs by the tumor cells. Furthermore, osteonectin and osteoactivin expression caused the development of spontaneous metastases systemically when the tumor cells were implanted subcutaneously. Our results suggest that the expression of bone-related genes in advanced human cancers may represent a novel mechanism by which tumor cells acquire capabilities that are associated with the phenotypic changes in late stages of tumorigenesis dependent on tumor microenvironment. Moreover, the data strongly support the notion that the genetically defined model of human cancers (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar, 21Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1991) Google Scholar, 22Elenbaas B. Spirio L. Koerner F. Fleming M.D. Zimonjic D.B. Donaher J.L. Popescu N.C. Hahn W.C. Weinberg R.A. Genes Dev. 2001; 15: 50-65Crossref PubMed Scopus (689) Google Scholar, 23Sonoda Y. Ozawa T. Hirose Y. Aldape K.D. McMahon M. Berger M.S. Pieper R.O. Cancer Res. 2001; 61: 4956-4960PubMed Google Scholar) offers a useful tool for functional genomics in defining the pathological contributions of specific genes to late stages of tumorigenesis. A genetically defined human glioma cell line was generated as described previously (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). A 1.5-kb cDNA fragment (a generous gift from Sandra Rempel, Henry Ford Hospital) and a full-length cDNA fragment of GPNMB (a generous gift from H. P. Bloemers, University of Nijmegen, The Netherlands) were each cloned into a retroviral backbone with a bleo selection marker. Cells underwent a positive selection with Zeocin (400 μg/ml, Invitrogen). U87MG, U251MG (American Type Culture Collection, Manassas, VA), and D54MG (Duke University Medical Center) were infected with a retrovirus expressing either a puromycin resistance gene or osteonectin and puromycin resistance. Selection was undertaken with puromycin (1 μg/ml). Early passage polyclonal cultures were used for all experiments. Cells were analyzed for the expression of osteonectin, growth factor receptors, and MMPs by Western analysis. A 10-cm plate was lysed, and 50 μg of total cellular protein was used for each sample. Samples were subjected to SDS-PAGE analysis and transferred to a PDVF membrane. The membrane was blocked in Tris-buffered saline with 0.05% Tween 20 and 5% albumin. Primary antibodies for anti-osteonectin (Hematologic Technologies, Essex Junction, VT), anti-GPNMB (gift of Carol Wikstrand, Duke University), anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-tubulin (Sigma), anti-MMP-9 (Calbiochem), anti-MMP-3 (Calbiochem), anti-MMP-2 (Calbiochem), anti-epidermal growth factor receptor (EGFR) (gift of Carol Wikstrand, Duke University), anti-platelet-derived growth factor receptor β (PDGFRβ) (Santa Cruz Biotechnology), anti-PDGFRα (Santa Cruz Biotechnology), or anti-insulin-like growth factor-1 receptor (IGF-1R) (Calbiochem) antibodies were used. Secondary antibodies were either goat anti-rabbit (Bio-Rad) or sheep anti-rabbit antibodies (Amersham Biosciences). Cells were plated into 12-well plates at a density of 2 × 104 cells per well and labeled for the last 6 h with 4 μCi of [3H]thymidine, fixed in 10% trichloroacetic acid, and lysed in 0.2 N NaOH. [3H]thymidine incorporation into the DNA was measured with a scintillation counter. Each measurement was performed in triplicate. Cells were plated into 10-cm plates at a density of 5 × 105cells per well, serum-starved overnight after attachment, and then fed with media containing serum for 24 h. Cells were then trypsinized, fixed in 70% ethanol, washed once in phosphate buffered saline, and resuspended in RNaseA (100 μg/ml) and propidium iodide (50 μg/ml). Samples were analyzed on a FACScan (BD Biosciences) flow cytometer. Each experiment was performed in triplicate. For these assays, 35-mm plates were prepared with a base layer of Dulbecco's minimal essential media with 10% calf serum (Invitrogen) and 0.6% bacto agar (BD Biosciences). Cells were plated at a density of 5 × 104 cells per plate in a mix of Dulbecco's minimal essential media with 10% calf serum and 0.4% bacto agar. Plated cells were fed once a week with 0.5 ml of Dulbecco's minimal essential media plus 10% calf serum and 0.4% bacto agar. After 14 days, the plates were stained with 0.5 ml of 0.005% crystal violet. On each plate, colonies with more than 30 cells were counted. Each measurement was performed in triplicate. Intracranial tumor formation was tested with SCID-beige mice injected with 1 × 106glioma cells in 10 μl of Methocel (Dow Chemical Co., Midland, IL). Mice were sacrificed when they developed any neurological abnormalities. Brains were serially sectioned and examined by histopathology. Immunohistochemistry was performed on all tumors with hematoxylin and eosin, factor VIII, collagen IV, and Ki-67. Vascular characteristics of tumors were compared through an examination of vessel number and size in selected high power fields. Fields were selected to represent maximal tumor diameters without significant areas of necrosis. Internal organs were completely removed and evaluated grossly for macroscopic metastases. Selected livers and lungs were examined for microscopic metastases. SCID-beige mice (Taconic, Germantown, NY) were subcutaneously injected in the flank with 10 × 106 glioma cells per mouse in 100 μl of Matrigel (BD Biosciences) (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). Mice were regularly checked for tumor formation. Tumor volume was calculated with the formula 0.5 × (length) × (width)2. Tumors were removed and examined by immunohistopathology. Mice that were noted to have significant weight loss were sacrificed regardless of primary tumor size. All internal organs including the brain were removed at the time of sacrifice and grossly examined for metastases. Selected lungs, livers, and brains were sectioned and examined for microscopic metastases. Kits were purchased from BD Biosciences and used according to instructions. Briefly, 2.5 × 104 tumor cells were incubated in selected conditions (serum-free media, 1% Me2SO control, 100 μm MMP-2 Inhibitor I (Calbiochem), 100 μmMMP-2/9 Inhibitor I (Calbiochem), or 100 μm MMP-3 Inhibitor II (Calbiochem)) and then allowed to attach and invade. Cells were fixed 22 h after plating. The invasion was calculated as a ratio of that shown by control (uncoated) inserts to that shown by Matrigel-coated inserts. Experiments were performed in triplicate. Wilcoxon rank sum test was used in all analysis Differential gene expression analyses of primary brain tumors and normal brain by the SAGE technology have permitted the identification of numerous genes that are increased in expression in malignant gliomas, including osteonectin and osteoactivin (4Rempel S.A. Golembieski W.A. Ge S. Lemke N. Elisevich K. Mikkelsen T. Gutierrez J.A. J. Neuropathol. Exp. Neurol. 1998; 57: 1112-1121Crossref PubMed Scopus (120) Google Scholar, 19Loging W.T. Lal A. Siu I.M. Loney T.L. Wikstrand C.J. Marra M.A. Prange C. Bigner D.D. Strausberg R.L. Riggins G.J. Genome Res. 2000; 10: 1393-1402Crossref PubMed Scopus (88) Google Scholar). To understand the role of these two genes in the pathogenesis of gliomas, we determined the effects of overexpressing osteonectin or osteoactivin by using a genetically defined human glioma cell line (hereafter referred to as the THR glioma line) developed in our laboratory (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). When implanted intracranially in immune-compromised mice, THR cells display a non-invasive phenotype. This phenotype is in contrast to that of gliomas in patients that are universally invasive, suggesting that additional genetic alterations beyond those already present in this cell line are required to develop an invasive phenotype in vivo. Thus, the THR cells represent an ideal model system in which to study the contributions of specific genetic changes to late stages of glioma development. Through the use of a retroviral system with an independent selectable marker, we generated polyclonal THR glioma cells that ectopically expressed either osteonectin (Fig. 1 A) or osteoactivin (Fig. 1 B). Early passage cells were used in all experiments to minimize genetic changes associated with simian virus 40 (SV40) T antigen expression. To determine the potential impact of osteonectin or osteoactivin expression on tumor development, we implanted these engineered THR cell lines intracranially in suspensions. In this nature environment, the genetically defined glioma cells formed large extra-axial masses with histologic features consistent with a malignant neural tumor including pseudopallisading necrosis (Fig. 1 C), regardless of whether the tumors expressed the vector, osteonectin, or osteoactivin. Tumors derived from vector control cells were located predominantly within the subarachnoid space, and rare foci superficially invaded the Virchow-Robin spaces but extended no deeper than the molecular layer of the superficial cerebral cortex. The Virchow-Robin spaces and vasculature remained delicate without expansion by tumor, increased numbers of vessels, or hypertrophy of the endothelium (not shown). However, in 23 of 33 mice implanted with the osteonectin-expressing glioma cells, tumors were located within the subarachnoid space and displayed a striking phenotype of invasion (Fig. 1 D), as well as expansion of the perivascular Virchow-Robin spaces by masses of tumor cells that extended into the deep layers of the cerebral cortex (Fig. 1 E) similar to previous reports of glioma invasion in mouse models (24Laws Jr., E.R. Goldberg W.J. Bernstein J.J. Int. J. Dev. Neurosci. 1993; 11: 691-697Crossref PubMed Scopus (64) Google Scholar). In sharp contrast to the tumors derived from vector control glioma cells, blood vessels associated with the tumors were increased in size and number (Fig. 1 F). These features readily contrasted with adjacent brain tissues not invaded by the tumor. Neoplastic cells in rare nests and single cells were seen adjacent to the Virchow-Robin spaces in the parenchyma, a finding that may represent either true invasion of brain parenchyma or formation of an angiocentric perivascular zone outside the limits of the pia-glial membrane. The prominent tumor associated vasculature was readily detected by immunohistochemical staining of the factor VIII antigen, which showed enhanced proliferation and endothelial hypertrophy of the osteonectin-expressing glioma tumors in comparison with the normal vasculature seen in adjacent brain, as well as the tumors derived from the vector control cells. Osteoactivin-expressing cells formed intracranial tumors in a fashion similar to those expressing osteonectin but with a lower rate of brain invasion (Fig. 1, D and G) and fewer associated vascular alterations (Fig. 1 H). Taken together, our data suggest that the expression of osteonectin and osteoactivin induce glioma invasion in the context of brain environment associated with penetrating blood vessels. Having documented the dramatic effects of osteonectin and osteoactivin expression on tumor invasion in xenograph experiments, we next investigated the mechanism by which these genes exert tumorigenic effects. Although in vitro systems are incomplete models of tumor invasion and metastasis as they lack normal stromal interactions, they allow selected analysis of potential changes at the cellular level, including cell proliferation, apoptosis, and invasion. Previously, it was reported that osteonectin inhibits cell proliferation (25Bradshaw A.D. Francki A. Motamed K. Howe C. Sage E.H. Mol. Biol. Cell. 1999; 10: 1569-1579Crossref PubMed Scopus (88) Google Scholar) and induces apoptosis in some ovarian carcinoma cell lines (26Yiu G.K. Chan W.Y. Ng S.W. Chan P.S. Cheung K.K. Berkowitz R.S. Mok S.C. Am. J. Pathol. 2001; 159: 609-622Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). However, we find that expression of osteonectin or osteoactivin in our THR glioma cells did not cause any significant changes in cell proliferation (Fig.2 A), apoptosis (Fig.2 A), or DNA synthesis (Fig. 2 B). Disruption of osteonectin expression has been linked to loss of IGF-1R expression (27Basu A. Rodeck U. Prendergast G.C. Howe C.C. Cell Growth & Differ. 1999; 10: 721-728PubMed Google Scholar), but we found no differences in the expression levels of IGF-1R, PDGFR-α or -β, or EGFR between control and osteonectin-expressing cells (Fig. 2 C). Osteoactivin expression was associated with an increase in EGFR expression but a decrease in PDGFR-α expression and no change in PDGFR-β or IGF-1R expression (Fig. 2 C). In addition, the ectopic expression of osteonectin or osteoactivin in human astrocytes expressing SV40 T antigen and human telomerase catalytic subunit failed to provide a mitogenic stimulus to transform these cells in the absence of oncogenic Ras as measured by soft agar colony formation assays (Fig. 2 D). These results suggest that osteonectin and osteoactivin expression is mainly associated with the acquired abilities by the tumor cells in invasion rather than tumor initiation. To test this hypothesis, we examined the impact of osteonectin and osteoactivin expression on tumor cell invasion in vitro. As shown in Fig.3, A and B, osteonectin or osteoactivin expression significantly increased cell invasion in all cell lines tested as measured by the Matrigel invasion assay. Constitutive expression of osteonectin or osteoactivin appeared to increase the ability of tumor cells to degrade components of the extracellular matrix and increased motility, a critical aspect of invasive cancers.Figure 3Osteonectin and osteoactivin expression is associated with increased invasion and expression of MMP-9 and MMP-3. As shown in A, Matrigel invasion assays were performed with the genetically defined THR glioma cell line, D54MG, U87MG, and U251MG, each engineered to express osteonectin (ON). The expression of osteonectin was clearly linked to increased invasion relative to vector control (VEC). *,p = 0.01. As shown in B, Matrigel analysis of glioma cells expressing osteoactivin with specific MMP-2, MMP-2/9, and MMP-3 inhibitors ablated the increase in invasion associated with osteoactivin expression (OA) relative to vector controls. CON, control. *, p = 0.01 relative to vector control; **, p = 0.01 relative to untreated osteoactivin-expressing tumor. As shown in C, Western analysis of a genetically defined glioma cell line with vector control (V), osteonectin, or osteoactivin expression revealed increased expression of gelatinase B (MMP-9) and stromelysin-1 (MMP-3) but minimal change of gelatinase A (MMP-2). As shown inD, Matrigel analysis of glioma cells expressing either vector control or osteonectin showed no change in invasion with an MMP-2/9 inhibitor, but an MMP-3 inhibitor ablated the increase in invasion associated with osteonectin expression (*, p = 0.01 relative to untreated). As shown in E, parental genetically defined THR glioma cells were treated with 50 μg/ml purified human osteonectin (Hematologic Technologies, Essex, VT). Conditioned media were collected simultaneously with either no osteonectin or after treatment with osteonectin for specific times. The media (50 μl) was resolved by SDS-PAGE and immunoblotted for stromelysin-1 (MMP-3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further explore the mechanisms that mediate the invasive effects of osteonectin and osteoactivin, we examined the expression profiles of proteins intimately associated with tumor invasion, specifically the MMPs. As shown in Fig. 3 C, we found that osteonectin and osteoactivin expression significantly increased the production of MMP-9 and MMP-3 by the tumor cells with minimal changes in MMP-2 expression. To test whether this up-regulation of MMPs is directly linked to the invasive behavior of tumors expressing osteonectin and osteoactivin, we examined the effect of specific MMP inhibitors in the Matrigel invasion assay. Treatment of cells expressing osteonectin with an MMP-2/9 inhibitor had no effect (Fig. 3 D), suggesting that the increase in MMP-9 expression was not a major contributing factor to the changes in invasive behavior by the osteonectin-expressing tumor cells. However, MMP-3 inhibitor treatment substantially reduced invasion through Matrigel by the same tumor cells (Fig. 3 D), suggesting an important role for MMP-3 in mediating the in vitro invasive behavior of these cells. Consistent with this observation, treatment of parental THR cells with exogenous osteonectin induced the production of MMP-3 within 4 h (Fig. 3 E), whereas the expression of MMP-2 and MMP-9 did not change (data not shown). Similarly, invasion of Matrigel by THR cells expressing osteoactivin was also most sensitive to a blockage of MMP-3 activity, although this activity was partially inhibited by inhibitors specific for MMP-2 and MMP-2/9 (Fig.3 B). Taken together, the data suggest that osteonectin and osteoactivin may promote tumor invasion, at least in part, by increasing the production of specific MMPs. Since osteonectin and osteoactivin are expressed in other types of advanced cancers, we investigated the potential effects of these two genes on tumor progression in a different growth microenvironment. To do this, osteonectin-expressing and osteoactivin-expressing THR glioma cells were subcutaneously injected into the flanks of SCID-beige mice. The primary subcutaneous tumors derived from the glioma cells expressing osteonectin have an identical latency period (∼29 days) to a vector control cell line but grow to a larger volume (Fig.4 A). Tumors from the osteoactivin-expressing cell line display a longer latency (∼39 days) but subsequently grow to, on average, a larger size than vector controls (Fig. 4 A). Tumors expressing osteonectin and osteoactivin did not exhibit a difference in angiogenic features from the control cells (data not shown). Strikingly, both types of tumor cells developed massive spontaneous intrathoracic and/or intraperitoneal metastases (Fig. 4, B and C). Metastases were found in 50% (16 of 32) of mice injected with osteonectin-expressing glioma cells and 14% (3 of 15) of osteoactivin-expressing glioma cells (Fig. 4 D). In contrast, the THR vector control cell line exhibited a single small, discrete metastasis in only 1 of 15 mice, and no metastases developed in 25 mice implanted with the THR parental cell line (not shown). Interestingly, the development of metastases was independent of primary tumor size. Three of 16 mice that developed metastases from osteonectin-expressing cells had no grossly evident subcutaneous tumors at the initial site of injection at the time of euthanasia. Therefore, the data suggest that the development of a metastatic phenotype from osteonectin-expressing glioma cells is associated with an early stage of tumor development inside the animals. Upon close pathological examination, we found that tumors derived from osteonectin-expressing glioma cells display a phenotype of solid and circumscribed neoplasms capable of invading the pancreas, peripancreatic soft tissues, subcutaneous tissues, and liver (Fig.4, E and F). Examination of hematoxylin- and eosin-stained sections of tumors derived from osteonectin-expressing cells revealed a malignant phenotype of poorly differentiated neoplasm. Within the tumor mass, there were numerous areas of atypical mitoses and necrosis pseudopalisaded by neoplastic cells. Taken together, histological analyses of tumors derived from osteonectin-expressing cells revealed typical pathological features that are consistent with metastatic disease. Interestingly, we found that the metastasized tumors did not possess the features of prominently increased vasculature and hypertrophic endothelium that are often associated with the invasive brain tumors. Consistent with this observation, lymphatic/vascular invasion and perineural extension were not detected in the metastatic sites. To test whether the effects of osteonectin described above were cell line-specific, we investigated the behavior of tumor development by the human glioma cell lines D54MG, U87MG, and U251MG engineered to express higher levels of osteonectin (Fig. 4 G). Importantly, we found that ectopic expression of osteonectin by these glioma cell lines also induced spontaneous metastases when they were injected subcutaneously in the flanks of SCID-beige mice (Fig. 4 D). These results suggest that osteonectin expression can induce a non-metastatic cancer cell line to adopt a metastatic phenotype with the alteration in the expression of only a single gene. Technological improvements now permit comprehensive analysis of gene expression patterns in cancer specimens. The resulting data have created a wealth of information to be mined, but utilization of the data remains difficult due to a lack of functional information for specific genes. Here we demonstrate the use of a genetically defined human cancer model system to investigate the function of specific genes differentially expressed in human cancers. Previously developed model systems of cancer have included cell lines created from human tumors and murine models. Although each model system has been of great benefit to our understanding of the contributions that specific genetic alterations play in the development of specific phenotypes of cancer, each system has significant drawbacks. Human cell lines are generally derived from advanced cancers that have widespread genetic changes and genomic instability as well as changes from long passages in cell culture. Murine models permit genetic control but are labor-intensive and suffer from potential species-specific differences in the process of transformation and gene function (28Hamad N.M. Elconin J.H. Karnoub A.E. Bai W. Rich J.N. Abraham R.T. Der C.J. Counter C.M. Genes Dev. 2002; 16: 2045-2057Crossref PubMed Scopus (349) Google Scholar). Thus, a genetically defined human cancer model system offers a useful tool to determine the roles of specific genes in carcinogenesis, particularly the late stages of this multistep process. Using this system, we investigated genes that normally appear to be highly expressed in bone without presence in brain tissues but have significantly increased expression in human gliomas. Although the expression of both osteonectin and osteoactivin has been linked to tumor progression in previous studies, their precise contributions to late stages of tumorigenesis remain unclear. Through an ectopic expression strategy in this model system, we revealed that osteonectin expression induced a highly malignant phenotype with significantly increased brain invasion associated with vascular proliferation and spontaneous systemic metastasis, whereas osteoactivin expression resulted in a more modest increase in malignant phenotype with less frequent metastases and lower degrees of vascular change. Although the highly metastatic characteristics displayed by the tumor cells in our study do not fully replicate the precise behavior of gliomas in humans, the data strongly support the notion that expression of osteonectin and osteoactivin could significantly alter or modify tumor behavior. Considering the fact that these genes are also overexpressed in other types of human cancers that do display a metastatic phenotype, this finding is clearly relevant and significant to our understanding of the mechanism underlying metastasis associated with those types of human cancers. Furthermore, our results indicate that the cellular microenvironment potently modulates the phenotypic behavior of cancer cells that express those two genes since glioma cells expressing osteonectin, and to a lesser extent, osteoactivin, formed large spontaneous metastases with subcutaneous implantation but remained localized to the brain with intracranial implantation. The vascular and extracellular environment in the brain is radically different from that of other organs, with the presence of the blood-brain barrier, absence of lymphatics, and brain-specific extracellular matrix and cell-cell interactions associated with neuronal and glial migration during development. These differences may partially account for the differential behavior of cells expressing osteonectin implanted in the brain versus other parts of the body. This finding strongly suggests that contributions to the development of specific tumorigenic phenotypes by the expression of specific genes are dependent on the specific characteristics of the microenvironment associated with tumor progression, adding another layer of complexity to the molecular mechanisms underlying late stages of tumorigenesis. Another potentially important finding is that osteonectin and osteoactivin appear to promote a specific invasive phenotype intracranially involving tumor cell invasion along pre-existent blood vessels in the Virchow-Robin spaces and development of altered vasculature at the brain-tumor interface. This phenotype mimics the behavior of a subset of human gliomas and many invasive medulloblastomas. Osteonectin has been reported to have an effect on angiogenesis through regulation of VEGF activity (29Vajkoczy P. Menger M.D. Goldbrunner R. Ge S. Fong T.A. Vollmar B. Schilling L. Ullrich A. Hirth K.P. Tonn J.C. Schmiedek P. Rempel S.A. Int. J. Cancer. 2000; 87: 261-268Crossref PubMed Scopus (69) Google Scholar). In our studies, neither osteonectin nor osteoactivin induced significant changes in the vasculature as measured by vessel number or diameter within the primary tumors, whether formed subcutaneously or intracranially. In contrast, intracranial tumor cells expressing osteonectin and osteoactivin grow along penetrating blood vessels, and the blood vessels at the invasive front were markedly abnormal with vessel hypertrophy and hyperplasia. These results suggest that the production of osteonectin and osteoactivin by glioma cells may create a complex relationship between invading tumor and normal vasculature that may be co-opted during tumor invasion, consequently allowing expansion of the tumor mass without the induction of angiogenesis at a significant level. The malignant phenotypes mediated by osteonectin and osteoactivin likely involve multiple mechanisms at the molecular level, with the induction of MMP expression as an important component. The rapid increase in MMP-3 protein levels in response to osteonectin treatment by the THR tumor cells strongly suggests that osteonectin may regulate the production of this specific MMP through a more direct mechanism. Taken together, our results validate the use of a genetically defined human cancer model system in investigating the contributions of specific genes to late stages of tumorigenesis. This in vivosystem permits the application of a functional genomics approach in defining the specific activities of genes that have been identified to be abnormally expressed in human cancers, particularly those associated with tumor invasion, metastasis, and angiogenesis. We thank G. J. Riggins, A. Hjelmeland, J. Herndon, and R. McLendon for helpful discussions, S. Rempel for osteonectin cDNA, C. Wikstrand for GPNMB antibody, and S. Keir, Y. Yu, and R. Nelson for technical assistance. J. Parsons provided editorial support.