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• W1982920006 abstract "Transforming growth factor-β1 (TGF-β1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras G12V transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivothan controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928–30936). We now show that stable expression of oncogenic Ki-ras G12V converts the HD6-4 colon cancer cell line from insensitive to TGF-β1 to growth-promoted by TGF-β1. Each of three Ki-ras G12V transfectants responded to TGF-β1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-β1. The wild-type Ki-rastransfectants lack functional TGF-β receptors, whereas all three Ki-ras G12V transfectants expressed functional TGF-β receptors that bound 125I-TGF-β1. The previous studies showed that in cells with wild-type Ki-ras, TGF-β receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-β receptor III (TβRIII) was incomplete. We now show that the betaglycan form of TβRIII is highly modified following translation when transiently expressed in Ki-ras G12V cells, whereas no such post-translational modification of TβRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TβRIII post-translational modification in Ki-ras G12V cells and TGF-β1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TβRIII post-translational modification. Transforming growth factor-β1 (TGF-β1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras G12V transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivothan controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928–30936). We now show that stable expression of oncogenic Ki-ras G12V converts the HD6-4 colon cancer cell line from insensitive to TGF-β1 to growth-promoted by TGF-β1. Each of three Ki-ras G12V transfectants responded to TGF-β1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-β1. The wild-type Ki-rastransfectants lack functional TGF-β receptors, whereas all three Ki-ras G12V transfectants expressed functional TGF-β receptors that bound 125I-TGF-β1. The previous studies showed that in cells with wild-type Ki-ras, TGF-β receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-β receptor III (TβRIII) was incomplete. We now show that the betaglycan form of TβRIII is highly modified following translation when transiently expressed in Ki-ras G12V cells, whereas no such post-translational modification of TβRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TβRIII post-translational modification in Ki-ras G12V cells and TGF-β1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TβRIII post-translational modification. transforming growth factor-β transforming growth factor-β receptor mitogen-activated protein kinase extracellular signal-regulated kinase thymidine base pair(s) Dulbecco's modified Eagle's medium reverse transcription-polymerase chain reaction polyacrylamide gel electrophoresis The TGF-β1 family of proteins number >25 and regulate cell growth and differentiation as well as morphogenesis and angiogenesis (1Kingsley D. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1732) Google Scholar, 2Gold L. Crit. Rev. Oncog. 1999; 10: 303-360PubMed Google Scholar). There are three mammalian isoforms, TGF-β1, TGF-β2, and TGF-β3, which are structurally very similar with nine conserved cysteines. The TGF-βs belong to a superfamily of structurally related proteins including the activins, inhibins, and bone morphogenic proteins (3Cui W. Akhurst R. Bondy C. LeRoith D. Growth Factors and Cytokines in Health and Disease. JAI Press, Greenwich, CT1996Google Scholar). The TGF-βs induce diverse biological responses by binding to the high affinity receptors TβRI (53 kDa) and TβRII (75 kDa), which function as a heterodimer. Both receptors have a cysteine-rich extracellular domain, one transmembrane segment, and a cytoplasmic tail that includes a serine/threonine kinase domain (4Lin H. Wang X.-F. Ng-Eaton E. Weinberg R. Lodish H. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (969) Google Scholar, 5Franzen P. ten Dijke P. Ichijo H. Yamashita H. Schultz P. Heldin D. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar). Constitutively phosphorylated TβRII binds TGF-β1, which then recruits TβRI into the complex. TβRI is transphosphorylated by TβRII and propagates the signal by its kinase activity to downstream substrates (6Wrana J. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2120) Google Scholar). Two other cell-surface TGF-β-binding proteins are the type III receptors betaglycan and endoglin, which modulate cellular responses to TGF-β, but have no signaling sequences. Betaglycan and endoglin may function by regulating TGF-β access to TβRII (7Lopez-Casillas F. Cheifetz S. Doody J. Andres J. Lane W. Massague J. Cell. 1991; 67: 785-795Abstract Full Text PDF PubMed Scopus (553) Google Scholar, 8Lastres P. Letamendia A. Zhang H. Rius C. Almendro N. Raab U. Lopez L. Langa C. Fabra A. Letarte M. Bernabeu C. J. Cell Biol. 1996; 133: 1109-1121Crossref PubMed Scopus (283) Google Scholar, 9Wang X.-F. Lin H. Ng-Eaton E. Downward J. Lodish H. Cell. 1991; 67: 797-805Abstract Full Text PDF PubMed Scopus (543) Google Scholar). TβRIII receptors are not found in every TGF-β-responsive cell and are down-regulated during myoblast differentiation into myotubes (10Ewton D. Spizz G. Olson E. Florini J. J. Biol. Chem. 1988; 263: 4029-4032Abstract Full Text PDF PubMed Google Scholar). The chief mediators of TGF-β signaling are the SMAD family of structurally related proteins. Receptor-activated SMAD proteins form heterotrimeric complexes and translocate to the nucleus, where they interact with other transcription factors to drive TGF-β1-induced transcription (11Derynck R. Zhang Y. Feng X.-H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar). Transcription factors interacting with SMAD proteins include FAST-1 and FAST-2 (12Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Nature. 1997; 389: 85-89Crossref PubMed Scopus (494) Google Scholar, 13Liu F. Pouponnot C. Massague J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (398) Google Scholar) and the c-Jun/c-Fos heterodimer (14Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (688) Google Scholar). The cooperation of SMAD proteins with other transcription factors suggests that TGF-β1 induces multiple parallel signaling pathways. Additional reported members of the TGF-β signaling pathways include the SMAD-interacting protein SARA and related proteins (15Tsukazaki T. Chiang T. Davison A. Attisano L. Wrana J. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar), Ras (16Mulder K. Morris S. J. Biol. Chem. 1992; 267: 5029-5031Abstract Full Text PDF PubMed Google Scholar, 17Yan Z. Winawer S. Friedman E. J. Biol. Chem. 1994; 269: 13231-13237Abstract Full Text PDF PubMed Google Scholar, 18Hartsough M. Frey R. Zipfel P. Buard A. Cook S. McCormick F. Mulder K. J. Biol. Chem. 1996; 271: 22368-22375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), the Raf homolog TAK-1 (19Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguichi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar), TAK-1-associated proteins TAB-1 and TAB-2 (20Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar), and the FK506- and rapamycin-binding protein FKBP-12 and the α-subunit of farnesyltransferase (21Ventura F. Liu F. Doody J. Massague J. J. Biol. Chem. 1996; 271: 13931-13934Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 22Wang T. Danielson P. Li B.-Y. Shah P. Kim S. Donahoe P. Science. 1996; 271: 1120-1122Crossref PubMed Scopus (107) Google Scholar, 23Wang T. Donahoe P. Zervos A. Science. 1994; 265: 674-676Crossref PubMed Scopus (312) Google Scholar). Several investigators have reported that expression of oncogenic Ras confers resistance to the growth inhibitory properties of TGF-β (24Sipes N. Lyons R. Moses H. Mol. Carcinog. 1990; 3: 12-19Crossref PubMed Scopus (10) Google Scholar, 25Houck K. Michalopoulos G. Strom S. Oncogene. 1989; 4: 19-25PubMed Google Scholar, 26Filmus J. Zhao J. Buick R. Oncogene. 1992; 7: 521-526PubMed Google Scholar, 27Longstreet M. Miller B. Howe P. Oncogene. 1992; 7: 1549-1556PubMed Google Scholar). In mouse mammary epithelial cells, oncogenic Ras was shown to constitutively activate the MAPKs ERK1 and ERK2. Activated ERK1/ERK2 phosphorylate multiple sites within the linker regions of SMAD2 and SMAD3, decreasing, but not totally eliminating, SMAD translocation into the nucleus following TGF-β stimulation (28Kretzschmar M. Doody J. Timokhina I. Massague J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (856) Google Scholar). Oncogenic Ras, by this mechanism, eliminates the antimitogenic activity of TGF-β. However, treatment of the same oncogenic Ras-transformed cells with TGF-β1 causes an epithelial-to-fibroblastoid conversion to a more invasive phenotype (29Oft M. Peli J. Rudaz C. Schwarz H. Berg H. Reichman E. Genes Dev. 1996; 10: 2462-2477Crossref PubMed Scopus (565) Google Scholar), showing that these cells maintained some TGF-β signaling pathways, which mediated tumor aggressiveness. In the mouse skin model of chemical carcinogenesis, one Ha-ras gene is activated by mutation and the normal is allele lost, causing the cells to undergo a morphological transformation to a highly invasive spindle phenotype that can be induced in synergy with TGF-β1 (30Akhurst R. Balmain A. J. Pathol. 1999; 187: 82-90Crossref PubMed Scopus (160) Google Scholar). We now report another system in which oncogenic Ras and TGF-β1 work together to enhance tumorigenicity and also report initial studies on the mechanism of this response. Goblet cells compose ∼20% of the colon epithelial cell population and are resistant to growth inhibition by TGF-β1 in vivo, becoming enriched relative to other colon epithelial cells in mice injected with recombinant TGF-β1 (31Deng X. Bellis S. Yan Z. Friedman E. Cell Growth Differ. 1999; 10: 11-18PubMed Google Scholar). Colon goblet cell lines are also resistant to the growth inhibitory effects of TGF-β1 (32Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar, 33Yan Z. Hsu S. Winawer S. Friedman E. Oncogene. 1992; 7: 801-805PubMed Google Scholar). We now show that oncogenic Ras up-regulates post-translational modification of the type III TGF-β receptor in the HD6-4 colon goblet cell line. Unexpectedly, TGF-β1 induces cell proliferation in the presence of oncogenic Ki-ras and, in parallel, down-regulates the Cdk inhibitor p21Cip1/Waf1 and the tumor suppressor phosphatase PTEN. 125I-TGF-β1 and [3H[TdR were obtained from PerkinElmer Life Sciences. Human recombinant TGF-β1 was from R&D Systems. Protein A-Sepharose was obtained from Amersham Pharmacia Biotech. Polyvinylidene difluoride transfer paper (Immobilon-P) was from Millipore Corp. Antibody TED1 to PTEN was the kind gift of Dr. Hong Sun (Department of Genetics, Yale University School of Medicine). Antibodies to TβRI/Alk5, TGF-β1, and TβRII were purchased from Santa Cruz Biotechnology; antibodies to phospho-SMAD2 and total SMAD2 were from Upstate Biotechnology, Inc.; antibody to betaglycan/TβRIII was from R&D Systems; and mouse monoclonal IgG2a clone 70 to p21Cip1 and anti-phosphotyrosine monoclonal antibody PY69 were purchased from Transduction Laboratories. Phosphorothiolated oligodeoxynucleotides were a gift of Dr. Brett Monia (Isis Pharmaceuticals, Carlsbad, CA). Isis-13177 is a 20-mer of random sequence, and Isis-6957 is a 20-mer targeted to the 5′-untranslated region of Ki-Ras (CAG-TGC-CTG-CGC-CGC-GCT-CG). This sequence is found within the promoter region of the c-Ki-ras2 gene cloned into pMiKVal12 (see below) ∼60 bp upstream of the translational start site. Endoglycosidase F (N-glycosidase F-free), also known as endo-β-N-acetylglucosidase F, was purchased from Roche Molecular Biochemicals. All other reagents, including chondroitinase ABC and heparitinase III, were from Sigma. Treatment of Ki-ras transfectants was essentially as described (34Chen G. Oh S. Monia B. Stacey D. J. Biol. Chem. 1996; 271: 28259-28265Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Cells were seeded at 2 × 105/well in six-well plates. 48 h later, the cells were washed with prewarmed serum-free insulin transferrin/selenious acid-supplemented DMEM and then incubated in this medium with a fixed ratio of oligonucleotide to Lipofectin (2.4 μl of Lipofectin/100 μmoligonucleotide) for 4 h. The oligonucleotide-containing medium was then replaced with normal growth medium, and growth was continued for 48 h to allow Ras turnover plus reduced Ras mRNA levels to result in reduced Ras protein levels (34Chen G. Oh S. Monia B. Stacey D. J. Biol. Chem. 1996; 271: 28259-28265Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Total RNA was isolated from monolayer cultures with TRIzol (Life Technologies, Inc.) according to the supplier's manual. RNA was treated with DNase I (amplification-grade; Life Technologies, Inc.) and RT-PCR was performed on a PerkinElmer Life Sciences DNA Thermal Cycler 480 using a Promega Access RT-PCR kit as follows and using the primers and conditions described previously (31Deng X. Bellis S. Yan Z. Friedman E. Cell Growth Differ. 1999; 10: 11-18PubMed Google Scholar). The amplification products were analyzed by ethidium bromide-agarose gel electrophoresis. The primer set used for RT-PCR of betaglycan mRNA yielded an expected amplification product of 592 bp, whereas primers for glyceraldehyde-3-phosphate dehydrogenase fromCLONTECH gave a product of 452 bp. Derivation of the oncogenic Ki-ras and wild-type Ki-ras transfectants was as described (35Yan Z. Chen M. Perucho M. Friedman E. J. Biol. Chem. 1997; 272: 30928-30937Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). All cell lines were maintained in low glucose DMEM buffered with 25 mm Hepes (Atlanta Biologicals, Inc.) and containing 7% fetal bovine serum as described (32Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar). Cell density was carefully controlled in these experiments. Within 1 day of growth to confluency, cells were plated at one-third confluent density and then used 2 days later. Serum-free transferrin/selenious acid-supplemented DMEM was used for the TGF-β1 growth experiments (33Yan Z. Hsu S. Winawer S. Friedman E. Oncogene. 1992; 7: 801-805PubMed Google Scholar). TGF-β1-treated cells were less adherent, so both adherent and floating cells were analyzed for [3H[TdR incorporation after 3 h of labeling as described (33Yan Z. Hsu S. Winawer S. Friedman E. Oncogene. 1992; 7: 801-805PubMed Google Scholar). Parallel wells that were not seeded with cells were washed to remove nonspecifically bound label, which was subtracted from all experimental values. The c-Myc-tagged rat betaglycan expression plasmid pCMV-mycBGL was received from J. Massague (7Lopez-Casillas F. Cheifetz S. Doody J. Andres J. Lane W. Massague J. Cell. 1991; 67: 785-795Abstract Full Text PDF PubMed Scopus (553) Google Scholar) and subcloned into the retroviral vector pLXSN (CLONTECH). The recombinant retrovirus was used to infect mutant Ki-ras transfectant cells and control cells. Cells were incubated with 100 μCi/ml [35S]methionine in methionine-free DMEM for 6 h before lysates were prepared. 500 μg of cell lysates were incubated overnight at 4 °C with 2 μg of anti-TβRIII antibody and 40 μl of protein G Plus-agarose (Santa Cruz Biotechnology), pelleted at 2500 rpm, and washed three times with lysis buffer. After the final wash, the pellet was mixed with 50 μl of SDS sample buffer and boiled in a water bath for 5 min. Aliquots were analyzed by SDS-PAGE, and TβRIII was detected by Western blotting using anti-TβRIII antibody and horseradish peroxidase-conjugated rabbit anti-goat IgG (Zymed Laboratories Inc.) and ECL. Cell lysates were diluted 1:5 with detergent-free lysis buffer and then incubated for 36 h with chondroitinase ABC (2 units/ml), heparitinase (2 units/ml), and N-glycosidase F (5 units/ml) before SDS-PAGE and Western blot analysis. Cells grown in complete medium were lysed in buffer containing 25 mm Tris (pH 7.4), 1% Triton X-100, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 0.5 mmphenylmethylsulfonyl fluoride, 200 μm sodium orthovanadate, and 20 mm sodium fluoride. Depending on the experiment, 30–70 μg of cell lysate proteins were blotted onto polyvinylidene difluoride membranes after separation by SDS-PAGE. The blots were blocked in blocking buffer (Tris-buffered saline containing 0.05% Tween 20 and 4% nonfat dry milk) for 1 h at room temperature. Primary antibodies were used in Tris-buffered saline containing 0.05% Tween 20 and 1% dry milk at the indicated concentrations (anti-p21Cip1/Waf1, 1 μg/ml; anti-TβRI, 1 μg/ml; anti-TβRII, 1 μg/ml; and anti-TβRIII (betaglycan), 1 μg/ml) and dilutions (1:1000 TGF-β1, 1:1000 PTEN, 1:1000 phospho-SMAD2 and total SMAD2, and 1:1000 phosphotyrosine) and then incubated for 2–3 h with primary antibody, and proteins were subsequently detected by ECL (Amersham Pharmacia Biotech). For p21Cip1 blots, cells were lysed in radioimmune precipitation assay buffer, and blocking was performed in 1% dry milk plus 4% bovine serum albumin. For SMAD2 blots, the first antibody was diluted in 5% bovine serum albumin, and the second in 2.5% dry milk. Affinity labeling of TGF-β receptors with125I-TGF-β1 was performed essentially as described (33Yan Z. Hsu S. Winawer S. Friedman E. Oncogene. 1992; 7: 801-805PubMed Google Scholar). Cell-surface proteins were biotinylated essentially as described by the vendor (Amersham Pharmacia Biotech). Briefly, exponentially growing cells were washed once with cold phosphate-buffered saline and once with 40 mm sodium bicarbonate (pH 8.6). Cells were then incubated for 30 min at 4 °C in 40 mm bicarbonate buffer containing 50 μl of biotin reagent/ml of buffer, 4 ng/ml TGF-β1, and 1 mg/ml bovine serum albumin (fatty acid-free). Following two washes with cold phosphate-buffered saline, cells were lysed in 25 mm Tris buffer containing 1% Triton X-100, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 200 μm sodium orthovanadate, and 20 mm sodium fluoride. 300 μg of each cell lysate were incubated overnight at 4 °C with 200 μl of streptavidin coupled to agarose beads (Sigma). The beads were then washed extensively and resuspended in SDS-PAGE sample buffer. Cell-surface TβRI and TβRII were immunoprecipitated with anti-TβRII antibody C-16 with or without TβRII peptide C-16 (Santa Cruz Biotechnology) or with anti-TβRI antibody R-20 (Santa Cruz Biotechnology)Biotechnology; antibodies were bound with horseradish peroxidase-streptavidin after washing and then detected with ECL. Calculations measuring the difference between two means were performed by the t test. The Ki-ras G12V transfectant cloned sublines V, V1, and V2 were isolated following stable transfection of HD6-4 colon carcinoma cells with a mini-gene construct of the cellular Ki-ras4B gene mutated at codon 12 to valine. Three stable sublines (G, G2, and G3) were isolated following transfection with the wild-type mini-gene construct of cellular Ki-ras4B (35Yan Z. Chen M. Perucho M. Friedman E. J. Biol. Chem. 1997; 272: 30928-30937Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 36Yan Z. Deng X. Chen M. Xu Y. Abram M. Sloane B. Friedman E. J. Biol. Chem. 1997; 272: 27902-27907Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The Ki-ras G12V transfectant lines expressed oncogenic Ki-Ras protein, whereas the wild-type Ki-ras transfectant G lines expressed more wild-type Ki-Ras protein as shown by Western blotting with Ki-Ras-specific and pan-RasVal12-specific antibodies (35Yan Z. Chen M. Perucho M. Friedman E. J. Biol. Chem. 1997; 272: 30928-30937Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The parental line is not responsive to TGF-β1 (31Deng X. Bellis S. Yan Z. Friedman E. Cell Growth Differ. 1999; 10: 11-18PubMed Google Scholar, 32Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar, 33Yan Z. Hsu S. Winawer S. Friedman E. Oncogene. 1992; 7: 801-805PubMed Google Scholar), but unexpectedly became TGF-β1-responsive following expression of oncogenic Ki-Ras protein. Ki-ras G12Vtransfectant V cells exhibited a rapid increase in tyrosine phosphorylation of an unknown cellular protein of ∼60 kDa after addition of TGF-β1, whereas no increase in tyrosine phosphorylation of this or any other protein was found in control cells treated with TGF-β1 (Fig. 1 A). The 60-kDa protein showed increased tyrosine phosphorylation following 5 and 15 min of TGF-β1 treatment (Fig. 1 A, arrow), whereas the abundance of phosphotyrosine in a 200-kDa protein remained constant, demonstrating equal loading. The chief mediators of TGF-β signaling are the SMAD family of structurally related proteins. Upon TGF-β receptor activation, TβRI activates phosphorylation of SMAD2 and/or SMAD3, each of which can form an association with the common mediator SMAD4 and then translocate to the nucleus and mediate transcription (reviewed in Refs. 11Derynck R. Zhang Y. Feng X.-H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar and 37Heldin C. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar). SMAD2 is activated by phosphorylation of serines 465 and 467 (38Abdollah S. Macias-Silva M. Tsukasaki T. Hayashi H. Attisano L. Wrana J. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). 30 min of TGF-β1 treatment caused a striking increase in the activation of phosphorylation of SMAD2 in the oncogenic Ki-ras V transfectant, but no activation in control cells (Fig. 1 B). A slight increase in the total amount of SMAD2 and SMAD3 in the Ki-ras V transfectant was also detected by Western blotting. Thus, expression of mutant Ki-Ras protein unexpectedly restored an initial step in a TGF-β signaling pathway in these cells. The Ki-ras G12Vtransfectant lines were more tumorigenic in vivo and more invasive in vitro than the control cells (35Yan Z. Chen M. Perucho M. Friedman E. J. Biol. Chem. 1997; 272: 30928-30937Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 36Yan Z. Deng X. Chen M. Xu Y. Abram M. Sloane B. Friedman E. J. Biol. Chem. 1997; 272: 27902-27907Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Mutant Ki-Ras caused aberrant post-translational glycosylation of β1 integrin, loss of cellular adhesion to the extracellular matrix, blocked cell polarization, and decreased cell-cell adhesion properties through up-regulation of carcinoembryonic antigen and down-regulation of N-cadherin (35Yan Z. Chen M. Perucho M. Friedman E. J. Biol. Chem. 1997; 272: 30928-30937Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 36Yan Z. Deng X. Chen M. Xu Y. Abram M. Sloane B. Friedman E. J. Biol. Chem. 1997; 272: 27902-27907Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These decreases in attachment to the extracellular matrix and intercellular adhesion provide one basis for selection for Ki-rasmutations in colon cancers. We had noted in past experiments that certain highly aggressive or metastatic colon cancer cells responded to TGF-β1 by increased cell growth (39Schroy P. Rifkin J. Coffey R. Winawer S. Friedman E. Cancer Res. 1990; 50: 261-265PubMed Google Scholar, 40Hsu S. Huang F. Hafez M. Winawer S. Friedman E. Cell Growth Differ. 1994; 5: 267-275PubMed Google Scholar, 41Huang F. Newman E. Kerbel R. Friedman E. Cell Growth Differ. 1995; 6: 1635-1642PubMed Google Scholar, 42Kansra S. Ewton D. Wang J. Friedman E. Int. J. Cancer. 2000; 87: 373-378Crossref PubMed Scopus (45) Google Scholar) and wondered if the increased aggressiveness of the Ki-ras G12V transfectant cells in vivoreflected increased growth in response to autocrine or exogenous TGF-β1. TGF-β1 induced a statistically significant, dose-dependent increase in growth up to 2-fold in Ki-ras G12V transfectant V cells, whereas TGF-β1 induced no change in proliferation of parental cells grown in parallel (Fig. 2 A, upper panel). The p value for mean growth ± 1 ng/ml TGF-β1 was <0.01, whereas the p values for growth ± 2 and 4 ng/ml TGF-β1 were both <0.001, all statistically significant differences. This growth response to TGF-β1 was generalized by assaying two additional Ki-ras G12V transfectant cell lines, V1 and V2, and three other control lines at the optimal TGF-β1 concentration (Fig. 2 B, upper panel). TGF-β1 at 4 ng/ml increased [3H[TdR incorporation 1.5- to >2-fold in V1 and V2 cells, with statistically significant p values of <0.0005. In contrast, TGF-β1 had no effect on cycling of wild-type Ki-ras transfectant G2 and G3 cells or the parental line with endogenous wild-type Ki-ras (Fig.2 B). The mechanism of this unexpected proliferative response to TGF-β1 was explored by assaying two proteins associated with tumorigenicity: the Cdk inhibitor p21Cip1/Waf1 (43El-Deiry W. Tokino T. Velculescu V. Levy D. Parsons R. Trent J. Lin D. Mercer W. Kinzler K. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar, 44Harper J. Adami G. Wei N. Keyomarsi K. Elledge S. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar) and the tumor suppressor phosphatase PTEN (45Li D. Sun H. Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar, 46Maehama T. Dixon J. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). p21Cip1/Waf1 inhibits cell cycling by targeting most Cdk-cyclin complexes, binding to them and inhibiting their kinase activity. PTEN is a phospholipid phosphatase, deleted or mutated in a wide variety of tumors (46Maehama T. Dixon J. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Loss of PTEN activity would up-regulate the activity of the serine/threonine kinase Akt, which is known to play an important role in generating cell survival signals (47Khwaja A. Nature. 1999; 401: 33-34Crossref PubMed Scopus (276) Google Scholar, 48King W. Mattaliano M. Chan T. Tsichlis P. Brugge J. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar, 49Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1190) Google Scholar). Ki-ras G12Vtransfectant V cells and control cells were treated for 2 days with 0, 1, 2, 4, or 8 ng/ml TGF-β1, and then cell lysates were blotted for abundance of p21 and PTEN (Fig. 2 A, lower panel). In control cells with wild-type Ki-ras, TGF-β1 caused no alteration in the levels of either protein. However, in duplicate experiments, TGF-β1 induced a dose-dependent decrease in the abundance of the Cdk inhibitor p21Cip1/Waf1, with the optimal growth-stimulating concentration of 4 ng/ml TGF-β1 reducing p21 levels to 16% of the control. PTEN levels were decreased 6-fold by 2, 4, or 8 ng/ml TGF-β1 only in the mutant Ki-Ras transfectant V cells. Coomassie Blue staining of the blot demonstrated equal loading (Fig. 2 A, lower panel). The generality of these responses was determined by assaying several more cell lines. Levels of p21Cip1/Waf1 and PTEN were examined in two other oncogenic Ki-ras transfectant lines (V1 and V2), two wild-type Ki-ras transfectant lines (G2 and G3), and the parental line with endogenous wild-type ras. Treatment with the optimal concentration of 4 ng/ml TGF-β1 decreased p21 levels in V1 and V2 cells expressing mutant Ki-Ras by 52 and 90%, respectively, whereas no decreases in p21 levels were observed in G1 and G3 lines with wild-type ras (Fig. 2 B,lower panel). Treatment with 4 ng/ml TGF-β1 decreased PTEN levels in V1 and V2 cells expressing mutant Ki-Ras by 50 and 30%, respectively, in duplicate experiments. In contrast, no decreases were observed in PTEN levels in G2 and G3 cells expressing only wild-type Ki-ras. Thus, TGF-β1 acts like a mitogen in mutant Ki-ras-transformed colon carcinoma cells, possibly by decreasing the abundance of the Cdk inhibitor p21Cip1/Waf1. The loss of PTEN expression with TGF-β1 treatment may contribute to the aggressive growth of each Ki-ras G12V transfectant cell line in vivo, as cells can respond to TGF-β1 from autocrine or paracrine sources (35Yan Z. Chen M. Perucho M. Friedman E. J. Biol. Chem. 1997; 272: 30928-309" @default.
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• W1982920006 title "Oncogenic Ki-ras Confers a More Aggressive Colon Cancer Phenotype through Modification of Transforming Growth Factor-β Receptor III" @default.
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