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The tumor suppressor RASSF1A is inactivated in many human cancers and is implicated in regulation of microtubule stability, cell cycle progression and apoptosis. However, the precise mechanisms of RASSF1A action and their regulation remain unclear. Here we show that Skp2, an oncogenic subunit of the Skp1–Cul1–F–box ubiquitin ligase complex, interacts with, ubiquitinates, and promotes the degradation of RASSF1A at the G1–S transition of the cell cycle. This Skp2-dependent destruction of RASSF1A requires phosphorylation of the latter on serine-203 by cyclin D–cyclin-dependent kinase 4. Interestingly, mutation of RASSF1A-phosphorylation site Ser203 to alanine results in a delay in cell cycle progression from G1 to S phase. Moreover, enforced expression of Skp2 abolishes the inhibitory effect of RASSF1A on cell proliferation. Finally, the delay in G1–S progression after Skp2 removal is normalized by depletion of RASSF1A. These findings suggest that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival.Oncogene (2008) 27, 3176–3185; doi:10.1038/sj.onc.1210971; published online 10 December 2007 [ABSTRACT FROM AUTHOR]

Although TGF-beta1, a growth inhibitor, is known to also induce apoptosis, the molecular mechanism of this apoptosis is largely undefined. Here, we identify the mechanism of TGF-beta1-induced apoptosis in SNU-16 human gastric cancer cells. Cell cycle and TUNEL analysis showed that, upon TGF-beta1 treatment, cells were initially arrested at the G1 phase and then driven into apoptosis. Of note, caspase-3 was activated in accordance with TGF-beta1-induced G1 arrest. Activated caspase-3 is targeted to cleave p21(cip1), p27(kip1), and Rb, which play important roles in TGF-beta-induced G1 arrest, into inactive fragments. Subsequently, Cdk2 was aberrantly activated due to the cleavage of p21 and p27. We found that the inhibition of Cdk2 activity efficiently blocks TGF-beta1-induced apoptosis, whereas it did not prevent caspase-3 activation or the subsequent cleavage of target proteins. In contrast, the suppression of caspase-3 activity inhibited the cleavage of target proteins, the activation of Cdk2, and the induction of apoptosis. Taken together, our results suggest that activation of caspase-3 by TGF-beta1 may initiate the conversion from G1 cell cycle arrest to apoptosis via the cleavage of p21, p27 and Rb, which in turn causes Cdk2 activation and, most significantly, Cdk2 activation as a downstream effector of caspase is a critical step for the execution of TGF-beta1-induced apoptosis.

Transcriptional repression of the TGF-beta type II receptor (RII) is one of the mechanisms leading to TGF-beta resistance. The newly identified epithelium-specific ets transcription factor ERT/ESX/ELF-3/ESE-1/jen binds to the TGF-beta RII promoter and induces promoter activity. The human gastric cancer cell lines, which show undetectable level of TGF-beta RII mRNA, do not express ERT mRNA. To study the molecular mechanisms of loss of ERT expression, we have cloned and characterized the human ERT promoter. DNA transfection experiments and electrophoretic mobility shift assays have revealed the existence of a distinct enhancer element (-186 to -177) which we named ESE (ERT promoter specific element). Deletion of the ESE markedly decreased expression of the target gene. ESE interacts with two distinct nuclear protein complexes, at least one of which appears to be inactivated in a cell line which does not express the ERT mRNA, compared to a cell line expressing the ERT mRNA. These results suggest the possibility that inactivation of the sequence-specific DNA binding protein to the region from -186 to -177 contributes to the loss of ERT expression, leading to the loss of TGF-beta type II receptor mRNA in human gastric cancer cell lines.

The tumor suppressor SMAD4, also known as DPC4, deleted in pancreatic cancer, is a central mediator of TGF-beta signaling. It was previously shown that mice homozygous for a null mutation of Smad4 (Smad4-/-) died prior to gastrulation displaying impaired extraembryonic membrane formation and endoderm differentiation. Here we show that Smad4+/- mice began to develop polyposis in the fundus and antrum when they were over 6 - 12 months old, and in the duodenum and cecum in older animals at a lower frequency. With increasing age, polyps in the antrum show sequential changes from hyperplasia, to dysplasia, in-situ carcinoma, and finally invasion. These alterations are initiated by a dramatic expansion of the gastric epithelium where Smad4 is expressed. However, loss of the remaining Smad4 wild-type allele was detected only in later stages of tumor progression, suggesting that haploinsufficiency of Smad4 is sufficient for tumor initiation. Our data also showed that overexpression of TGF-beta1 and Cyclin D1 was associated with increased proliferation of gastric polyps and tumors. These studies demonstrate that Smad4 functions as a tumor suppressor in the gastrointestinal tract and also provide a valuable model for screening factors that promote or prevent gastric tumorigenesis.

The epithelium-specific transcription factor, ERT/ESX/ESE-1/ELF3, binds to the TGF-beta RII promoter in a sequence specific manner and regulates its expression. In this study, we investigated whether ERT could regulate endogenous TGF-beta RII expression in Hs578t breast cancer cells. Analyses of the Hs578t parental cell line revealed low RII mRNA expression and resistance to the growth inhibitory effects of TGF-beta. Infection of this cell line with a retroviral construct expressing ERT induced higher levels of endogenous RII mRNA expression and protein expression relative to cells infected with chloramphenicol acetyltransferase (CATneo) as a control. Relative to control cells, the ERTneo-expressing Hs578t cells show approximately a 50% reduction in cell growth in the presence of exogenous TGF-beta1, as well as a fourfold higher induction of activation in transient transfection assays using the 3TP-luciferase reporter construct. When transplanted into athymic mice, ERT-expressing Hs578t cells showed decreased and delayed tumorigenicity compared with control cells. This data strongly suggests that ERT plays an important role as a transcriptional activator of TGF-beta RII expression, and that deregulated ERT expression may play a critical role in rendering Hs578t human breast cancer cells insensitive to TGF-beta's growth inhibitory effects.

The transforming growth factor-beta (TGF-beta) signaling pathway subserves an essential tumor suppressor function in various cell types. A heteromeric complex composed of TGF-beta type I (RI) and type II (RII) receptors is required for TGF-beta signaling. We have identified a subset of human gastric cancer cell lines which are insensitive to TGF-beta and which express a low level of TGF-beta type I receptor mRNA relative to a gastric cancer cell line which is highly responsive to TGF-beta. Using these cells, we show that hypermethylation of a CpG island in the 5' region of the TGF-beta RI gene provides another potentially important mechanism of escape from negative growth control by TGF-beta. This hypermethylation was found in four of five human gastric cancer cell lines and five out of 40 (12.5%) primary tumors examined. In human gastric cancer cell lines, treatment with the demethylating agent, 5-aza-2'-deoxycytidine, resulted in increased expression of the TGF-beta RI gene, but not the RII gene. Transient transfection of an RI expression vector into the TGF-beta resistant SNU-601 cell line restores TGF-beta responsiveness. These findings suggest that one of the mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve aberrant methylation of CpG islands in the 5' region of the TGF-beta RI gene.

The transforming growth factor-beta (TGF-beta receptor system has been implicated in the development of resistance to the growth-inhibitory effects of TGF-beta. It has been reported that resistance to TGF-beta correlates with inactivation of the TGF-beta type II receptor (RII). In the present report, we examine the genetic changes in the TGF-beta RII gene of human gastric cancer cell lines, SNU-5 and SNU-668, which we had previously reported to express truncated TGF-beta RII transcripts. By independent PCR and Southern hybridization analysis of genomic DNA, we found that the genomic sequence of TGF-beta RII is truncated after exon 2 in SNU-5 and after exon 3 in SNU-668. This was confirmed by sequencing the TGF-beta RII cDNA cloned from a SNU-5 cDNA library. Predicted TGF-beta RII protein of SNU-5 cells based on sequencing data contains only a part of extracellular domain of TGF-beta RII. We demonstrate that cotransfection of 3TP-Lux and wild type TGF-beta RII restores the TGF-beta responsiveness in SNU-5 cells, suggesting that genetic changes in the TGF-beta RII gene of SNU-5 cells are responsible for the loss of sensitivity to TGF-beta. This is the first report demonstrating that truncation of the TGF-beta RII gene is an alternative mechanism to inactivate the TGF-beta signal transduction pathways.

Human hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma (HCC). The virus encodes a 17 kDa protein, X, which is known to be a causative agent in the formation of HCC. An insulin-like growth factor-II (IGF-II) is expressed during the formation of HCC. Among the four promoters of the IGF-II gene, promoters 2, 3 and 4 become activated during the formation of HCC. The high frequency of detection of hepatitis B virus X (HBV-X) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that the expressions of HBV-X and IGF-II are associated. Studies were carried out to test the relationship between the HBV-X gene product and the activation of IGF-II promoter 4. We demonstrated that the HBV-X protein increases the endogenous IGF-II expression from promoter 3 and 4 of IGF-II gene. Analysis of the fourth promoter of IGF-II gene showed that the HBV-X gene product positively regulates transcription. Two copies of a motif are responsible for conferring HBV-X regulation on the fourth promoter of IGF-II. These motifs have been identified as Sp1 binding sites. Sp1 binding to IGF-II P4 promoter was identified by gel mobility shift assay using purified Sp1. By using a GAL4-Sp1 fusion protein it was demonstrated that HBV-X positively regulates the Spl mediated transcriptional activity of IGF-II in vivo. A protein-affinity chromatography experiment showed that HBV-X protein does not bind directly to Sp1, but HBV-X does augment the DNA binding activity of the phosphorylated form of Sp1 in HepG2 cells. Sp1 was phosphorylated by HBV-X and its DNA-binding activity was up-regulated upon HBV-X transfections. Various HBV-X mutant expression vectors were used for the demonstration of specific interactions between Sp1 and HBV-X. These results indicate that HBV-X functions as a positive regulator of transcription, and that Sp1 is a direct target for the transcriptional regulation of IGF-II. Increasing the DNA binding ability of the phosphorylated form of Sp1 by HBV-X might be an important mechanism for regulating the IGF-II gene expression and possibly promoting cell division during hepatic carcinogenesis. Our experimental results suggest that expression of HBV-X might induce the expression of IGF-II and the IGF-II might play a role in hepatitis B virus pathogenesis during the formation of HCC.

Transformation of interleukin-3 dependent (IL-3) 32D-123 myeloid cells by p120-v-Abl produced the factor-independent 32D-abl cell line. In 32D-abl cells, myc expression was found to be significantly higher than in the parental cells and was correlated with increased E2F-1 protein expression and DNA binding ability. Surprisingly, in 32D-abl cells, TGF-beta1, a potent G1/S inhibitor of 32D-123 and 32D-abl cell growth, increased E2F transactivation as shown by increased c-myc promoter-CAT and GAL4-E2F-1 activity. In addition, TGF-beta1 was also found to increase E2F-1 protein levels but had no effect on steady-state retinoblastoma (RB) protein levels or phosphorylation state. In the absence of TGF-beta1, transient expression of RB in v-Abl expressing cells resulted in decreased c-myc transcription, inhibition of GAL4-E2F-1 driven transactivation and inhibition of cellular proliferation. RB and v-Abl were found to physically associate in vivo and in vitro via v-Abl's ATP binding region. In summary, these studies established that in myeloid cells: (1) v-Abl binds RB resulting in increased E2F-1-driven c-myc transcription, and (2) an alternative pathway exists for TGF-beta1-mediated growth inhibition of v-Abl-transformed cells, in which increased rather than decreased E2F-mediated c-myc transcription is observed.