Abstract
Transforming growth factor beta1 (TGFbeta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. We recently demonstrated that colon carcinoma cells transfected with oncogenic cellular K-rasV12, but not oncogenic cellular H-rasV12, switched from TGFbeta1-insensitive to TGFbeta1-growth-stimulated and also became more invasive (Yan, Z., Deng, X., and Friedman, E. (2001) J. Biol. Chem. 276, 1555-1563). We now demonstrate that TGFbeta1 growth stimulation of colon carcinoma cells is Ras-dependent and smad-independent. In U9 colon carcinoma cells, which are responsive to TGFbeta1 by growth stimulation, a truncating mutation at Gln-311 was found in the smad4 gene. Very little smad4 protein was detected in these cells. Loss of smad4 protein was confirmed by functional studies. In U9 cells co-transfected wild-type smad4, but not mutant smad4, mediated response of the 3TP-lux and pSBE promoter reporter constructs to TGFbeta1. Proliferation initiated by TGFbeta1 in U9 cells required Ras-mediated down-regulation of p21cip1 protein. Less p21cip1 was associated with cdk2 small middle dotcyclin complexes in TGFbeta1-treated U9 cells, and the cdk2 complexes had increased kinase activity. Elevation of p21cip1 levels diminished proliferative response to TGFbeta1. U9 cells expressing DN-N17ras neither proliferated in response to TGFbeta1 nor down-regulated the cdk inhibitor p21cip1, and TGFbeta1 activation of 3TP-lux in U9 cells was inhibited by DN-N17ras in a dose-dependent manner. TGFbeta1 also decreased p21cip1 levels and stimulated proliferation in SW480 cells, which express mutant K-Ras but no smad4 protein. TGFbeta1 did not activate or inhibit the p21cip1 promoter construct in U9 cells even in the presence of co-transfected smad4, or alter p21cip1 mRNA levels. Thus the decrease in p21cip1 levels was mediated by a TGFbeta-initiated Ras-dependent, but smad-independent post-transcriptional mechanism.
Highlights
Implicated in diverse phenomena, including growth control, cell adhesion and motility, production of extracellular matrix components, and alteration of cell phenotype [1, 2]
In our earlier studies about 40% of resected colon cancers placed into primary culture, including most metastatic tumors, responded to Transforming growth factor 1 (TGF1) by growth stimulation; the oncogenic K-Ras-expressing cell lines serve as models for this biological response [19, 20]
transforming growth factor  (TGF)1 down-regulated the abundance of the cdk inhibitor p21cip1 when it stimulated cell proliferation in colon carcinoma cells expressing transfected oncogenic
Summary
TGF, transforming growth factor ; TRI, -II, and -III, type I–III transmembrane serine/threonine kinases; SBE, smad binding element; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CMV, cytomegalovirus; DME, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescence protein; Ab, antibody; DN, dominant-negative. TGF1 activation of its receptors leads, by as yet unknown pathways, to activation of a series of signaling pathways, in addition to the smads These pathways include Ras ERK [8], Rho JNK [9], RhoA3p160ROCK [10], Tak13p38 MAPK [11, 12], protein phosphatase 2A3 S6 kinase [13], and possibly others. Elevated TGF1 levels could mediate increased tumor aggressiveness in several ways, and autocrine TGF1 pathways have been shown to mediate increased invasiveness and metastasis in carcinomas [18] Another autocrine response to TGF1 is increased proliferation, which has been observed in resected carcinomas in primary culture [19, 20] as well as established cell lines such as U9 colon carcinoma cell line. The colon carcinoma cells, which exhibited increased proliferation in response to TGF1, have become more invasive in vitro and in vivo, suggesting. In the current study we demonstrate that the TGF1-initiated signaling pathways, which mediate increased proliferation are Rasdependent and smad-independent
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