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Hepatocarcinogenesis results from the accumulation of genetic and epigenetic changes in liver cells. A common mechanism through which these alterations induce liver cancer is by deregulating signaling pathways. A number of signaling pathways, including the PI3K/PTEN/AKT and transforming growth factor β (TGF-β) pathways have been implicated in normal liver development as well as in cancer formation. In this study, we assessed the effect of the TGF-β signaling pathway on liver tumors induced by phosphatase and tensin homolog (Pten) loss. Inactivation of only the TGF-β receptor type II, Tgfbr2, in the mouse liver (Tgfbr2(LKO)) had no overt phenotype, while inactivation of Pten alone (Pten(LKO)), resulted in the formation of both hepatocellular carcinomas and cholangiocarcinomas (CC). Interestingly, deletion of both Pten and Tgfbr2 (Pten(LKO);Tgfbr2(LKO)) in the mouse liver resulted in a dramatic shift in tumor type to predominantly CC. Assessment of the PI3K/PTEN/AKT pathway revealed increased phosphorylation of AKT and glycogen synthase kinase 3 beta (GSK-3β) in both the Pten(LKO) and Pten(LKO);Tgfbr2(LKO) mice, suggesting that this pathway is constitutively active regardless of the status of the TGF-β signaling pathway. However, phosphorylation of p70 S6 kinase was observed in the liver of all three phenotypes (Tgfbr2(LKO), Pten(LKO), Pten(LKO);Tgfbr2(LKO)) indicating that the loss of Tgfbr2 and/or Pten leads to an increase in this signaling pathway. Analysis of markers of liver progenitor/stem cells revealed that the loss of TGF-β signaling resulted in increased expression of c-Kit and CD133. Furthermore, in addition to increased c-Kit and CD133, Scf and EpCam expression were also increased in the double knock-out mice. These results suggest that the alteration in tumor types between the Pten(LKO) mice and Pten(LKO);Tgfbr2(LKO) mice is secondary to the altered regulation of stem-cell features induced by the loss of TGF-β signaling.

The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-β) signaling pathway, which is often inactivated by mutations of TGF-β type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in ∼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2(IEKO)) alone is not sufficient for intestinal tumor formation and lack of Pten (Pten(IEKO)) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (Pten(IEKO);Tgfbr2(IEKO)) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a β-catenin-independent mechanism. Inactivation of TGF-β signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-β signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.

MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a 'field defect' involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

The transforming growth factor beta (TGF-beta) signaling pathway, which is activated by the TGF-beta receptor complex consisting of type I and type II TGF-beta receptors (TGFBR1 and TGFBR2), regulates cell growth and death. TGF-beta and components of its signaling pathway, particularly TGFBR2, have been implicated as tumor suppressor genes and important antimitogenic factors in the gastrointestinal tract and liver. An in vivo approach to study these effects has been hindered by the embryonic lethality of Tgfbr2(-/-) mice and poor viability of the Tgfb1(-/-) mice. Consequently, we have developed a hepatocyte-specific Tgfbr2 knockout mouse, the Alb-cre Tgfbr2(flx/flx) mouse, to study the physiologically relevant effects of TGF-beta signaling on epithelial cell proliferation in vivo. After 70% hepatectomy, we observed increased proliferation and an increased liver mass : body weight ratio in the Alb-cre Tgfbr2(flx/flx) mice compared to Tgfbr2(flx/flx) mice. We also observed decreased expression and increased phosphorylation of p130 in the livers from the Alb-cre Tgfbr2(flx/flx) mice as well as increased expression of cyclin E, which is transcriptionally regulated, in part, by p130:E2F4. Consistent with these results, in a hepatocyte cell line derived from the Tgfbr2(flx/flx) mice, we found that TGF-beta increases the nuclear localization of E2F4, and presumably the transcriptional repression of the p130:E2F4 complex. Thus, we have demonstrated that TGF-beta signaling in vivo regulates the mitogenic response in the regenerating liver, affecting the liver mass : body weight ratio after partial hepatectomy, and that these mitogenic responses are accompanied by alterations in p130 expression and phosphorylation, implicating p130 as one of the proteins regulated in vivo by TGF-beta during liver regeneration.