Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

The transforming growth factor ␤ (TGF␤) 1 family of secreted factors is involved in the control of different biological processes including cell proliferation, differentiation, and apoptosis (1). TGF␤ signals through the activation of heteromeric complexes of TGF␤ type I (T␤RI) and type II (T␤RII) receptors (1, 2). Activated T␤RI phosphorylates receptor-associated Smads (Smad2 and Smad3), which then bind Smad4 and translocate to the nucleus where they regulate transcription of target genes (3, 4). TGF␤ exhibits a tumor suppressor activity, and components of its signaling pathway are frequently mutated or silenced in colon and pancreatic cancers (1, 5). However, accumulating data indicate that TGF␤ can positively affect tumorigenesis and contribute to the progression and invasiveness of tumors (5-8). Moreover, it was recently reported that inhibition of autocrine TGF␤ signaling in carcinoma cells reduces cell invasiveness and tumor metastases (9, 10). These effects of TGF␤ are associated with its ability to induce an epithelial to mesenchymal transition (EMT) and stimulate cell migration. The EMT induced by TGF␤ results in the disruption of the polarized morphology of epithelial cells, formation of actin stress fibers, and enhancement of cell migration (8, 9). Two species of T␤RI, Alk2 and Alk5, have been implicated in the induction of EMT by TGF␤ in mammary epithelial cells (11,12). It has also been reported that high levels of ectopic Smad2 and Smad3 can induce some features of EMT in mammary epithelial cells in the context of expression of an activated type I receptor (12). However, considering the complexity of TGF␤ signaling (3,(13)(14)(15)(16), it is conceivable that other molecules can also contribute to EMT. For example, members of the AP-1 family of transcription factors have been shown to induce EMT and promote tumor invasiveness (17, 18). AP-1 complexes can be activated in response to , physically interact with Smads (13,14), and cooperate with Smads in the control of gene expression (19 -21). In addition, several other downstream signaling pathways can also be activated by TGF␤, including p38Mapk (21), c-jun N-terminal kinase (22, 23), and phosphatidylinositol 3-OH kinase (PI3K) (24, 25). These signaling pathways can potentially contribute to TGF␤1-mediated EMT, but their significance for EMT and cell migration mediated by TGF␤ remains unclear.In this study, we used the NMuMG mammary epithelial cell line as a model for TGF␤1-induced EMT (11). Two metastatic breast tumor cell lines, 4T1 and EMT6, that express high levels of TGF␤ ligands and TGF␤ receptors were used in transcription and migration studies. We report that TGF␤-induced EMT

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.

A new technique has been developed for the facile location of pseudouridylate (psi) residues in any RNA molecule. The method uses two known modification procedures which in combination uniquely identify U residues which have been converted into psi. The first procedure involves reaction of all U-like and G-like residues with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), followed by alkaline removal of all CMC groups except those linked to the N3 of psi. This stops reverse transcription, resulting in a gel band which identifies the U residue. The second procedure is uridine-specific hydrazinolysis which cleaves the RNA chain at all U residues and produces a gel band upon reverse transcription. psi residues, being resistant to hydrazinolysis, are not cleaved and do not stop reverse transcription. This leads to the absence of a band at psi residues. The combined method can also distinguish psi from 5-methyluridine, 4-thiouridine, uridine-5-oxyacetic acid, and 2-thio-5-methylaminomethyluridine as shown by treating rRNA and tRNA species known to contain these modified bases at defined sites. By this procedure, four new sites for psi in Escherichia coli 23S RNA were discovered, and one was disproven. The four new sites are at positions 2457, 2504, 2580, and 2605. The erroneous site is at position 2555. These four new psi residues, which are all in or within 2-3 residues of the peptidyltransferase ring, are thus in a position to play a functional and/or structural role at the peptidyltransferase center.(ABSTRACT TRUNCATED AT 250 WORDS)

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