The molecular basis of tumors has long been studied; nevertheless, its importance has been restricted to the pathogenesis of these disorders. More recently, aberrant signal transduction has been increasingly studied from a therapeutic viewpoint.33,34
However, such studies have shown several new issues, such as the differences in involved molecular pathways in patients with a similar histological tumor type. Another closely associated issue is the differences in tissue specificity of transcriptional targets of a pathway. These are among a few challenges that daunt custom chemotherapy. In our effort to address the tissue specificity of a pathway that has been shown to be aberrantly activated in multiple tumors and for which no specific therapy is available, we generated a liver-specific transgenic mouse that overexpresses β-catenin, a component of the Wnt pathway.
Activation of the Wnt/β-catenin pathway has been observed during early liver development and during liver regeneration.9,27–29
These events are characterized by nuclear translocation of β-catenin, as well as some stabilization and increased transcription of this protein. Although mutations in ctnnb1
or in other components of this pathway have been shown to occur in certain subsets of HCCs and hepatoblastomas, transgenic mice that have been generated by using such mutated β-catenin have not displayed any liver tumors.35
Our results from the transgenic mice that overexpress wild-type β-catenin (nonmutated) in their livers are also in agreement because of the absence of any spontaneous hepatic tumorigenicity in these animals. The lack of tumors in our transgenic mice could be multifactorial. One prominent reason could be the strain of mice used for generating these mice. C57BL/6 mice are known to be resistant to hepatocarcinogenesis, and we used this model in an effort to reproduce the patient scenario.36–38
This might be supplemented by the successful degradation of overexpressed wild-type β-catenin protein, which kept the total β-catenin levels regulated enough that they were unable to induce spontaneous tumors. This is supported by a recent study that shows spontaneous hepatomas in APC conditional knockout mice secondary to increased and activated β-catenin, although most HCC patients with β-catenin stabilization have not shown any mutations in the APC gene.39–41
As shown in another study, it is possible that β-catenin alone is insufficient to initiate HCC and that it acts in concert with other pathways, such as H-Ras.42
However, an overall similar (although milder) phenotype that consists of liver enlargement was observed in our transgenic mice. This could clearly be the influence of the nondegradable form of β-catenin or might be due to a dissimilar promoter/enhancer system used by other investigators. We observed an increase in total β-catenin protein, its cytoplasmic stabilization, and its nuclear translocation and activation in most transgenic mice. These events resulted in stimulating liver growth secondary to increased basal proliferation of hepatocytes in the nonchallenged transgenic livers. It is interesting to note that we did not observe any major difference in apoptosis as could have been expected according to our earlier results in ex vivo liver cultures, in which a decrease in β-catenin accelerated apoptosis in developmental hepatocytes and biliary epithelial cells.28
In light of these differences, β-catenin might be playing a role in regulating apoptosis during prenatal liver development, in which more apoptotic nuclei are normally evident as compared with normal adult livers, which show minimal apoptosis (unpublished data).
Although most of the transgenic mice displayed hepatomegaly secondary to increased proliferation, β-catenin, and EGFR, a subset of these mice were able to regulate their β-catenin levels. This was sufficient to normalize their liver weights, as was evident by an increase in the serine 45/threonine 41–phosphorylated fraction of β-catenin in these animals. We do not have an adequate explanation for the molecular basis of this observed difference in the ability of some of these transgenic mice to be able to successfully down-regulate β-catenin by enhancing its phosphorylation and, hence, degradation, and it might be due to the existence of some additional autocrine feedback loops. Several such loops are known to exist in this pathway at many different levels. One such example is that many of the positive and negative regulators of this pathway, such as sFRP-2, axin 2, and TCF-1, are in fact downstream targets of this pathway.43–45
Studies relating the qualitative and quantitative changes in these proteins and other crucial components of the pathway are ongoing.
Gene-array studies were aimed at giving a global molecular mechanism of the hepatomegaly observed in the β-catenin transgenic mice. Some of the genes that are showing increased expression in the transgenic livers are related to the synthetic and metabolic functions of the hepatocytes. These are genes such as cytochrome P450, 7b1, amino levulinate synthase, inositol polyphosphate-5-phosphatase, sterol 12-α hydroxylase cytochrome P450 8B1 (Cyp8b1
), glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. However, although these do define a metabolically heightened environment, the specific genes involved in glutamine metabolism that have been previously shown to be regulated by mutated β-catenin, including glutamate transporter 1, glutamine synthetase, and ornithine aminotransferase, remained unchanged.46
These differences might be due to differences in the transcriptional activation capability and specificity of mutated vs wild-type β-catenin. However, we believe that varied expression of these genes might be a function of multiple other factors, such as diet, time of the day, species, age, and other such factors.
The transgenic livers also displayed up-regulation of some factors, such as ZT2, Hey1, and ring finger protein 3, which have not yet been investigated for their importance in the liver.47,48
Hey1 might be of specific interest because of its involvement in proliferation and because it is a direct target of the Notch pathway.49
Others, such as FGF-4, are known targets of this pathway.50
We have also previously defined changes in β-catenin distribution secondary to exogenous FGF application, and, thus, interactions between the 2 pathways might be at multiple levels.51
However, some of the more prominent targets, such as c-myc and cyclin D1, remained unaffected in the transgenic livers, thus indicating tissue or stage specificity of the β-catenin targets.14,15
These results are also in accordance with the mutated β-catenin transgenic mice.35
Other relevant genes included frizzled 7 (Wnt receptor) and Nedd8-conjugating enzyme (ubiquitination), which might be part of the autoregulatory loop within the Wnt/β-catenin pathway. Some of the other genes showing an up-regulation had a more specific function, such as aquaporin 1 (expressed in bile ducts and intrahepatic cholangiocytes and required for fat absorption), annexin VII (tumor-suppressor gene in prostate carcinoma and HCC), and adrenergic receptor β1 (nor-epinephrine-mediated comitogen effect on liver).52–55
Some of the genes that display a decrease in their expression are the liver-specific transcription factors HNF-1 β and HNF-3 (forkhead homolog 1), which have been shown to correlate with decreased proliferation and increased differentiation of hepatocytes.56,57
Other specific genes that are more defined growth inhibitors, such as Cdk4 and Cdk6 inhibitor p19 protein and the growth arrest–specific 1 protein, also show a significant decrease in transgenic livers.58,59
Other genes that show a decreased expression possess developmental and/or tumor-suppressor properties, such as Dutt1 mRNA, SDF-1, and Notch gene.60–64
Other down-regulated genes with specific functions were Tie1 (angiogenesis) and metallothionein (cytoprotective against heavy metals and metabolism of trace elements).65,66
The collective premise that emerges from the microarray is an up-regulation of pro-mitogenic factors and a down-regulation of antimitogenic factors, thus giving a basis of the observed phenotype in the transgenic livers.
Although enhanced proliferation and liver size may be multifactorial, we attempted to examine the EGFR/β-catenin interaction in depth. EGFR up-regulation and its therapeutic inhibition have been shown to be effective in many cancers, such as those of the lung, breast, and prostate.67,68
The availability of EGFR inhibitors has greatly influenced the prognosis of these malignancies. More importantly, the availability of such drugs that affect individual signal transduction pathways at various levels provides an opportunity to customize therapies in many of the cancers.33
In our study, we show higher levels of EGFR mRNA and protein in β-catenin–overexpressing livers. The functionality of this interaction was further substantiated by the presence of activated EGFR in the transgenic livers that is probably secondary to the presence of normally higher levels of EGF that are synthesized by the Brunner’s glands and make their way into the portal circulation. Thus, EGFR up-regulation is sufficient to increase the sensitivity of hepatocytes to the available EGF to further prove to be mitogenic. This was further strengthened by the observed Stat3 activation in these livers. Stat3 activation is responsible for inducing proliferation in various tumors, such as those of the breast and skin.69,70
More than 80 target genes are known to be functioning in response to the Wnt/β-catenin pathway, and their tissue and stage specificity are becoming increasingly evident (http://www.stanford.edu/~rnusse/pathways/targets.html
). As mentioned, we did not find any changes in c-myc or cyclin D1 levels, similar to the mutated β-catenin transgenic mice.35
Also, because of the unavailability of molecular therapies against β-catenin, it is vital to define any newer downstream effectors of this pathway that might be targeted during the activation of this pathway in an organ-specific manner. The EGFR promoter showed a TCF-binding site that is a binding cofactor of β-catenin to support transcriptional activation effectively. The relevance of this site is clear from the EGFR/luciferase reporter activation in the presence of Wnt-3A–conditioned media that is abrogated by the presence of sFRP-1 in the same.29,32,71
Nuclear translocation of β-catenin in response to Wnt-3A–conditioned media has been described extensively.10,32
This observation was also extended to hepatoblastoma tumors. These pediatric tumors are known to harbor mutations in the Ctnnb1
gene, thus resulting in activation of the β-catenin pathway.20,24
Upon examination, we detected a concomitant increase and nuclear translocation of β-catenin and EGFR in a significant number of these tumors. Nuclear EGFR has been reported previously and indicates a positive correlation with cell proliferation.72–74
This observation suggests that EGFR up-regulation might be one of the downstream signaling events secondary to β-catenin activation and that it might be relevant to interfere with EGFR signaling in β-catenin–activated states. Indeed, when EGFR inhibitor was administered to the transgenic mice, there was an approximately 15% decrease in their liver weights to normal levels as compared with the control-treated animals. Again, we cannot rule out the possibility of interaction of the Wnt/β-catenin and EGF pathway at more than 1 level, as has been shown previously.75,76
Our study thus shows a direct regulation of EGFR expression by β-catenin, thus highlighting the relevance of therapeutic EGFR inhibition in β-catenin–increased states. Thus, although these distinct mitogenic pathways might have cooperative detrimental pathogenic implications, there might also be exploitable therapeutic benefits of such a relationship.