Integrin a2 31 in Tumorigenic Human Osteosarcoma Cell Lines Regulates Cell Adhesion, Migration, and Invasion by Interaction with Type I Collagen1

Human osteosarcomas are aggressive bone tumors. Here we propose that their progression requires altered cell interaction with extracellular matrix. Since type I collagen is the main matrix molecule found in bone and thus obligated to interact with tumor cells, we analyzed the expression and function of different integrin-type collagen receptors in tumor cell-collagen interaction by using eight human osteogenic sarcoma (HOS) cell lines. Virally (Kirsten sarcoma virus) transformed derivatives of HOS cells (KHOS-NP) and chemically [N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)] transformed tumorigenic subclones of human osteogenic sarcoma cells (HOS-MNNG) expressed a2131 integrin in remarkably larger amounts than the six other nontumorigenic cell lines (HOS, MG-63, Saos2, KHOS-240, KHOS-312, and G292). Concomitantly, Mg2 -dependent adhesion of tumorigenic cells to type I collagen was increased. We also show that the migration of tumorigenic cells on and invasion through type I collagen is faster than that of HOS cells. HOS cells forced to express a2 integrin by cDNA transfections showed increased Mg2 -dependent cell adhesion to type I collagen and also accelerated migration and invasion rate, indicating that the overexpression of a2 31 integrin in tumorigenic cells alone explains the altered cell-collagen interaction. Finally, HOS cells forced to express a2 integrin subunit did not grow s.c. in athymic mice, suggesting that overexpression of a2 integrin is not efficient to make these cells tumongenic. Received 9/10/95; revised 1/22/96; accepted 2/1/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 This work was supported by grants from the Finnish Cancer Association, the Sigrid Jus#{233}lius Foundation, the Academy of Finland, and the Technology Development Centre (TEKES). 2 To whom requests for reprints should be addressed. Phone: 011-35821-633-7005; Fax: 011-358-21-633-7000. Introduction In malignant transformation, the expression pattern of cell surface adhesion receptors, including the integrin family, may change. The integrins needed for maintenance of normal cell phenotype can be down-regulated, and those needed in invasion, matrix degradation, and cell movement are often activated (1). An integnn consists of an a and a p subunit, which bind together to form a cell adhesion receptor (2). The 3l integrin subfamily comprises 10 different a subunits, i.e., al -a9, and aV. These heterodimers include al /31 and a2 l integrins, collagen receptors which can also bind to laminin-l . lntegrin a3/31 is a receptor for Iaminin-5, and this integrin may also bind fibronectin. a4/31 heterodimer is a fibronectin receptor, which is also known as a lymphocyte-homing receptor. Integrin a3f31 binds the same RGD sequence in the fibronectin molecule as the a5 3l integrin, but the binding area of the a4f31 heterodimer in this same ligand differs from the RGD sequence. Integrins a6j31 and a7/3l are specialized in laminin-l binding, whereas the ligands for the a8/3l integrins are fibronectin, vitronectin, and tenascin. Integrin a9f31 is a tenascin receptor (3), and avj3l integrin is a receptor for fibronectin and vitronectin. The interaction of cells with matrix molecules, e.g., type I collagen, might have a special role in the movement of tumor cells through connective tissue. Recently, the altered expression of a2 l integrin seen in many different transformed cell lines (4-6) has suggested its importance as a mediator of the metastatic potential of cells and cell-collagen interaction as a fundamental phenomenon in the process of invasion. The high expression levels of a2/3l integrin have been shown to be associated with tumor progression of human melanoma (4), and it is up-regulated in non-small cell lung cancer (5), as well as in cancer of pancreas (6). In some other carcinomas, a2 integrin is down-regulated, suggesting that in some organs, its main function is to control normal structure (7, 8). Integrin a2f31 collagen receptor has been shown to mediate the attachment of cells to type I collagen (9) and the contraction of three-dimensional collagen gels (1 0, 1 1). Furthermore, up-regulation of a2 integrin by transforming growth factor /3 leads to enhancement of collagen gel contraction (12). Integrin a23l may also be involved in the cell migration on type I collagen (13). Many cell types also recognize different collagens by another integnn-type receptor, the al l integrin. Integrin al f31, as well as a2J31 integrin, contains an interactive domain (I domain), which has been shown to function in ligand binding (1 4, 15). It has been suggested that type I collagen contains separate binding sites for al /31 and a2f31 integrins (16). Furthermore, the cellular signals generated by the interaction 440 Cell Migration and Invasion by n2 l lntegrin of these two receptors with collagen might be different and eventually lead to distinct alterations in cell behavior (1 7). The binding specificity of a2j3l integrin is known to be modulated with cell type-specific factors (18-20). In some but not all cell lines, it is also a receptor for laminin-l and tenascin (21). In this study, we have examined the role of the a2 l collagen receptor integrin in the first steps of cell invasion. We show that tumorigenic human osteosarcoma cells, overexpressing a2j31 integrin, show increased adhesion to, migration on, and invasion through type I collagen. We propose that a2 integrin expression is not sufficient to make the cells tumorigenic in vivo, but it might be essential for the penetration of malignant cells through collagenous stroma. Results The Expression Pattern of Putative Collagen Receptor Integrins Is Not Similar in Different Human Osteosarcoma Cell Lines. We analyzed the expression levels of putative collagen receptor integrins in eight human osteosarcoma cell lines. HOS3 cells are osteogenic cells, which are originally cultured from human osteosarcoma but behave as nontumorigenic cells. KHOS-240, KHOS-NP, and KHOS-3l 2 are Kirsten sarcoma virus-transformed derivatives of HOS cells. The HOS-MNNG cell line is a chemically transformed subclone of HOS cells, which is, like the KHOS-NP cell line, tumorigenic in athymic mice (22). MG-63, G292, and SAOS-2 are all nontumorigenic cell lines, primarily cultured from human osteogenic sarcomas. The analysis of integrin expression was done by immunoprecipitating the octylglycoside-extracted proteins from metabolically steady state-labeled cells by different anti-integrin antisera. Immunoprecipitations of all cell lines with anti-/3l integrin antiserum created three different bands (data not shown). These bands represented multiple comigrating a subunits (Mr 140,000), /31 integrin subunit (Mr 130,000), and an intracellular precursor for pi integrin subunit (Mr 110,000), as described earlier (23). In addition, a fourth band (Mr 190,000), shown to represent the al integrin subunit (23), was constantly seen in all cell lines except in MG-63 (Fig. 1). It has been shown earlier that the al integrin subunit cannot be detected in MG-63 cells, but its expression can be induced by treatment with cytokines, i.e., interleukin 1/3 and tumor necrosis factor-a (24). Immunoprecipitations with a subunit-specific antisera showed that in combination with the l integrin subunit, transformed HOS variants, MG-63 and G292 cells, expressed the a2 integrin subunit (Fig. 1). However, in original HOS cells or SAOS-2 cells, there was no detectable a2 integrin subunit expression (Fig. 1). In HOS wild-type cells, some expression of the a2 integrin was detected when the cells had been subcultured several times (data not shown). These cells were not, however, used in the experiments. In KHOS-NP and HOS-MNNG cells, a2 integrin expression was remarkably stronger than in other cell lines. 3 The abbreviations used are: HOS, human osteogenic sarcoma; MNNG, N-methyl-N’ -nitro-N-nitrosoguanidine; wt, wild type; MMP, matrix metalloproteinase. AL #{176} <‘, I /‘ # S. / ,%‘ L_ai_ ::u’4 MM!, .. ...