Decorin-induced Growth Suppression Is Associated with Up-regulation of p21, an Inhibitor of Cyclin-dependent Kinases

The secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-β. Moreover, decorin expression is markedly up-regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-β- and p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin-expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo. The secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-β. Moreover, decorin expression is markedly up-regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-β- and p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin-expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo. INTRODUCTIONThe genes that regulate the transition from a proliferative to a quiescent state are beginning to be understood at the molecular level. It has been recognized, however, that several key molecules in cancer development are proteins that operate outside the nucleus and often interact at the cell-matrix boundaries either by detecting changes in the extracellular environment or by relaying messages from receptor-transducer proteins to their control machineries. Under the appropriate circumstances, the latter genes may act as tumor suppressor genes. Several lines of evidence indicate that decorin (1Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (412) Google Scholar), a member of the small leucine-rich proteoglycan gene family (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar), is intimately associated with growth suppression activity (3Yamaguchi Y. Ruoslahti E. Nature. 1988; 336: 244-246Crossref PubMed Scopus (208) Google Scholar, 4Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar). For example, this proteoglycan is markedly up-regulated during quiescence in human diploid fibroblasts (5Coppock D.L. Kopman C. Scandalis S. Gilleran S. Cell Growth 4: 483-493PubMed Google Scholar, 6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and vascular smooth muscle cells (7Asundi V.K. Dreher K.L. Eur. J. Cell Biol. 1992; 59: 314-321PubMed Google Scholar), and this effect is transcriptionally regulated and long lasting (6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Transcriptional activation of decorin gene expression also occurs when HeLa cervical carcinoma cells are rendered quiescent by serum deprivation (6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Moreover, decorin is rarely expressed by actively dividing normal cells as well as in SV40 transformed cells (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar, 5Coppock D.L. Kopman C. Scandalis S. Gilleran S. Cell Growth 4: 483-493PubMed Google Scholar), and its expression is totally abrogated in several tumor cell lines via methylation of the genomic control regions (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar, 8Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar). In contrast, both mRNA and protein levels are markedly augmented in the peritumorous stroma of human colon cancer (8Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar, 9Adany R. Iozzo R.V. Biochem. J. 1991; 276: 301-306Crossref PubMed Scopus (39) Google Scholar), a process that may reflect a regional response of the host connective tissue cells to the invading neoplastic cells (10Iozzo R.V. J. Biol. Chem. 1985; 260: 7464-7473Abstract Full Text PDF PubMed Google Scholar, 11Iozzo R.V. Cohen I. Experientia (Basel). 1993; 49: 447-455Crossref PubMed Scopus (104) Google Scholar). Using a gene transfer approach in human colon WiDr/HT29 carcinoma cells, which do not constitutively express this gene (12Iozzo R.V. J. Cell Biol. 1984; 99: 403-417Crossref PubMed Scopus (103) Google Scholar), we discovered that the de novo expression of decorin reverted the cells to a “normal” phenotype: the cells lost anchorage-independent growth, failed to generate tumors in scid/scid mice, and were arrested in the G1 phase of the cell cycle (13Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar). The cells could reenter the cell cycle when decorin expression was abrogated by decorin-specific antisense oligodeoxynucleotide treatment (13Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar). Of note, these effects were due to neither clonal selection nor integration site, indicating that decorin might be a component of a negative loop that controls cell growth.In this paper, we show that the decorin-induced growth suppression is associated with an up-regulation of p21WAF1/CIP1 (p21) 1The abbreviations used are: p21the inhibitor of cyclin-dependent kinase activity p21WAF1/CIP1CDKcyclin-dependent kinaseTGF-βtransforming growth factor-βPAGEpolyacrylamide gel electrophoresis. and that this leads to a suppression of cyclin and cyclin-dependent kinase (CDK) activity in a TGF-β- and p53-independent pathway. Abrogation of decorin expression by decorin-specific antisense oligodeoxynucleotide treatment leads to suppression of expression of p21 and restoration of cellular growth. These results demonstrate for the first time that an extracellular matrix proteoglycan can regulate the cell cycle and that p21 is a downstream effector of this biological process.DISCUSSIONAn emerging concept in cancer biology is that the cyclin·CDK complexes are relatively well regulated in relation to the coordination of DNA replication and mitosis (23Pines J. Semin. Cancer Biol. 1995; 6: 63-72Crossref PubMed Scopus (170) Google Scholar, 24Morgan D.D. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2920) Google Scholar, 32Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3205) Google Scholar). However, transformed cells are unable to regulate these complexes vis-á-vis DNA damage or extracellular signals (23Pines J. Semin. Cancer Biol. 1995; 6: 63-72Crossref PubMed Scopus (170) Google Scholar, 24Morgan D.D. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2920) Google Scholar, 25MacLachlan T.K. Sang N. Giordano A. Crit. Rev. Eukaryotic Gene Expr. 1995; 5: 127-156Crossref PubMed Scopus (300) Google Scholar). Two independent lines of evidence point to decorin as a potential extracellular growth regulator. The first derives from the observation that decorin is up-regulated during quiescence in human diploid fibroblasts (5Coppock D.L. Kopman C. Scandalis S. Gilleran S. Cell Growth 4: 483-493PubMed Google Scholar, 6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and in growth-arrested vascular smooth muscle cells (7Asundi V.K. Dreher K.L. Eur. J. Cell Biol. 1992; 59: 314-321PubMed Google Scholar). Decorin is one of several genes, named quiescins, whose expression is up-regulated 10-40-fold vis-á-vis rapidly proliferating cells (5Coppock D.L. Kopman C. Scandalis S. Gilleran S. Cell Growth 4: 483-493PubMed Google Scholar). Of note, various types of collagen, such as types I, III, and VI, are members of the quiescins. Because, all these collagenous proteins interact specifically with the small leucine-rich proteoglycans (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar), including decorin (33Vogel K.G. Paulsson M. Heinegård D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (701) Google Scholar, 34Bidanset D.J. Guidry C. Rosenberg L.C. Choi H.U. Timpl R. Höök M. J. Biol. Chem. 1992; 267: 5250-5256Abstract Full Text PDF PubMed Google Scholar), it is possible that part of the complex regulation of cell cycle arrest, e.g. during contact inhibition of normal diploid cells, may also include coordinately regulated extracellular matrix genes. The up-regulation of decorin is long lasting, up to 16 days after reaching confluence, and is transcriptionally suppressed by tumor necrosis factor-α (6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The second line of evidence is based on experiments employing gene transfer and forced expression of decorin in cells that do not otherwise express this proteoglycan. For example, ectopic expression of decorin in Chinese hamster ovary cells leads to growth suppression via a block of TGF-β (3Yamaguchi Y. Ruoslahti E. Nature. 1988; 336: 244-246Crossref PubMed Scopus (208) Google Scholar, 4Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar), whereas de novo expression of decorin in human colon carcinoma cells abrogates the malignant phenotype by arresting the cells in G1 (13Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar). In the present study, we extended our original observation and discovered a link between a secreted proteoglycan and the cell cycle machinery. Specifically, we discovered that decorin-induced cell cycle arrest of colon carcinoma cells was accompanied by a marked inhibition of cyclin and CDK activities without any significant change in their protein levels. The generalized reduction in kinase activity suggested the action of a potent inhibitory molecule acting at multiple cell cycle checkpoints. Our results demonstrate that this growth arrest was due to an induction of p21 message and protein levels via a mechanism that is independent of either TGF-β or p53 and that p21 exists in a multimeric complex with cyclins and CDKs in the nuclei of growth-arrested cells. Indeed, p21 is a potent inhibitor of CDK complexes (27Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar), is induced in senescent cells (35Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1309) Google Scholar), and is transcriptionally regulated by p53 (16El-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. Wiman K.G. Mercer W.E. Kastan M.B. Kohn K.W. Elledge S.J. Kinzler K.W. Vogelstein B. Cancer Res. 1994; 54: 1169-1174PubMed Google Scholar). Recent evidence indicates that p21 acts as an inhibitory buffer whose levels dictate the threshold kinase activity required for cell cycle progression (36Harper J.W. Elledge S.J. Keyomarsi K. Dynlacht B. Tsai L.-H. Zhang P. Dobrowolski S. Bai C. Connell-Crowley L. Swindell E. Fox M.P. Wei N. Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (855) Google Scholar). Thus, the marked increase in p21 complexed with various cyclins and CDKs could provide a likely explanation for the action of decorin in the expressing cells: the induction of p21 would prevent phosphorylation of critical CDK substrates and block cell cycle progression. In the wild-type colon carcinoma cells with inactive p53 (22Rodrigues N.R. Rowan A. Smith M.E.F. Kerr I.B. Bodmer W.F. Gannon J.V. Lane D.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7555-7559Crossref PubMed Scopus (967) Google Scholar), this pathway would be defective, thereby permitting unregulated growth.Through the use of antisense oligodeoxynucleotides to lower the expression of decorin, we provide evidence that the levels of decorin transcript are directly linked to those of p21 and that reduction of both genes leads to growth stimulation. Of note, a recent report (37Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (354) Google Scholar) has demonstrated that transformed fibroblasts maintain constantly activated cyclin E·CDK2 complexes regardless of attachment, whereas in normal fibroblasts these complexes are activated only upon cell attachment. The lack of cyclin E·CDK2 activity in suspended cells is the result of an increased expression of CDK2 inhibitors, including p21 (37Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (354) Google Scholar). These results are in support of our finding in colon carcinoma cells (13Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar) and those reported here insofar as augmentation of nuclear p21 parallels a loss of anchorage-independent growth and a decline in cyclin·CDK activity without significantly affecting their steady-state protein levels.How general is this decorin-induced tumor suppression? Our recent unpublished results have shown that ectopic expression of decorin suppresses growth not only in colon carcinoma cells but also in several mammalian tumor cell lines of diverse histogenetic origin, including fibrosarcoma, osteosarcoma, melanoma, and leukemic tumor cells. In all instances the attenuated growth kinetics were associated with an up-regulation of p21 mRNA and a concomitant increase in the proportion of cells in the G1 phase of the cell cycle. Thus, decorin appears to be a general growth suppressor protein acting from the “outside” of the cell. Appearance of this gene around certain epithelial neoplasms (8Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar) could represent a protective mechanism by which the normal stroma counteracts the invasive malignant cells. INTRODUCTIONThe genes that regulate the transition from a proliferative to a quiescent state are beginning to be understood at the molecular level. It has been recognized, however, that several key molecules in cancer development are proteins that operate outside the nucleus and often interact at the cell-matrix boundaries either by detecting changes in the extracellular environment or by relaying messages from receptor-transducer proteins to their control machineries. Under the appropriate circumstances, the latter genes may act as tumor suppressor genes. Several lines of evidence indicate that decorin (1Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (412) Google Scholar), a member of the small leucine-rich proteoglycan gene family (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar), is intimately associated with growth suppression activity (3Yamaguchi Y. Ruoslahti E. Nature. 1988; 336: 244-246Crossref PubMed Scopus (208) Google Scholar, 4Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar). For example, this proteoglycan is markedly up-regulated during quiescence in human diploid fibroblasts (5Coppock D.L. Kopman C. Scandalis S. Gilleran S. Cell Growth 4: 483-493PubMed Google Scholar, 6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and vascular smooth muscle cells (7Asundi V.K. Dreher K.L. Eur. J. Cell Biol. 1992; 59: 314-321PubMed Google Scholar), and this effect is transcriptionally regulated and long lasting (6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Transcriptional activation of decorin gene expression also occurs when HeLa cervical carcinoma cells are rendered quiescent by serum deprivation (6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Moreover, decorin is rarely expressed by actively dividing normal cells as well as in SV40 transformed cells (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar, 5Coppock D.L. Kopman C. Scandalis S. Gilleran S. Cell Growth 4: 483-493PubMed Google Scholar), and its expression is totally abrogated in several tumor cell lines via methylation of the genomic control regions (2Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (545) Google Scholar, 8Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar). In contrast, both mRNA and protein levels are markedly augmented in the peritumorous stroma of human colon cancer (8Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar, 9Adany R. Iozzo R.V. Biochem. J. 1991; 276: 301-306Crossref PubMed Scopus (39) Google Scholar), a process that may reflect a regional response of the host connective tissue cells to the invading neoplastic cells (10Iozzo R.V. J. Biol. Chem. 1985; 260: 7464-7473Abstract Full Text PDF PubMed Google Scholar, 11Iozzo R.V. Cohen I. Experientia (Basel). 1993; 49: 447-455Crossref PubMed Scopus (104) Google Scholar). Using a gene transfer approach in human colon WiDr/HT29 carcinoma cells, which do not constitutively express this gene (12Iozzo R.V. J. Cell Biol. 1984; 99: 403-417Crossref PubMed Scopus (103) Google Scholar), we discovered that the de novo expression of decorin reverted the cells to a “normal” phenotype: the cells lost anchorage-independent growth, failed to generate tumors in scid/scid mice, and were arrested in the G1 phase of the cell cycle (13Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar). The cells could reenter the cell cycle when decorin expression was abrogated by decorin-specific antisense oligodeoxynucleotide treatment (13Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar). Of note, these effects were due to neither clonal selection nor integration site, indicating that decorin might be a component of a negative loop that controls cell growth.In this paper, we show that the decorin-induced growth suppression is associated with an up-regulation of p21WAF1/CIP1 (p21) 1The abbreviations used are: p21the inhibitor of cyclin-dependent kinase activity p21WAF1/CIP1CDKcyclin-dependent kinaseTGF-βtransforming growth factor-βPAGEpolyacrylamide gel electrophoresis. and that this leads to a suppression of cyclin and cyclin-dependent kinase (CDK) activity in a TGF-β- and p53-independent pathway. Abrogation of decorin expression by decorin-specific antisense oligodeoxynucleotide treatment leads to suppression of expression of p21 and restoration of cellular growth. These results demonstrate for the first time that an extracellular matrix proteoglycan can regulate the cell cycle and that p21 is a downstream effector of this biological process.