In this manuscript, we showed that CCND1 expression is up-regulated in liver tumors induced by hMet/β-catenin. Consistent with the studies by Cadoret A. and colleagues (35
), we found that CCND1 is not a direct target of activated β-catenin in normal mouse hepatocytes. On the other hand, over-expression of CCND1 has been found to be correlated with β-catenin activation in multiple mouse liver tumor models (31
). Recently, Zeng G. et. al.
reported that RNA-mediated β-catenin knockdown in human HCC cell lines with activated β-catenin mutations leads to decreased expression of CCND1 (37
). Together, all the data suggest that CCND1 is likely to be induced by activated β-catenin during hepatic carcinogenesis. Our experiments demonstrate that CCND1 can partially substitute activated β-catenin and cooperates with hMet to induce liver cancer formation in vivo,
thus providing additional evidence of CCND1 as a target of β-catenin during malignant transformation.
While co-expression of CCND1 and hMet can induce liver cancer formation in mice, we found that the tumors induced by CCND1/hMet require longer latency, form at a lower frequency, and appear to be more benign compared with tumors induced by β-catenin/hMet. These observations indicate that CCND1 is only part of the signaling triggered by β-catenin activation, and other molecules may be required to fully transduce the aberrant β-catenin signaling. Other targets of β-catenin include Tbx3 (38
) and Gpr49 (39
), both found to be up-regulated in liver tumors induced by hMet/β-catenin (Patil M.A. et al
., unpublished results). Tbx3, a member of the T-box transcriptional repressor family, has been found be over-expressed in melanoma, breast cancer, and ovarian cancer (40
). In addition, Tbx3 has been shown to be a potent inhibitor of p19Arf and hence, a regulator of the p19Arf-MDM2-p53 pathway (43
). Thus, Tbx3 may provide a novel link between activated β-catenin and p19Arf tumor suppressor pathways. Gpr49, also known as Lgr5, is an orphan G protein-coupled receptor. Gpr49 has been found to be over-expressed in human colon and ovarian tumors and is proven to be a marker for intestinal stem cells (44
). Altogether, it would be of great importance to elucidate how Tbx3, Grp49, and CCND1 function together and mimic the activity of β-catenin in cooperation with c-Met to induce liver cancer.
In our study, we have shown that expression of CCND1 is not required in murine HCC pathogenesis induced by activated β-catenin and hMet. Therefore, while CCND1 expression is upregulated in liver cancer cells, cyclin D1 expression appears to be dispensable for c-Met and β-catenin’s activity in liver tumor development. Unexpectedly, we observed that hMet and ΔN90-β-catenin induced tumor development was accelerated by the loss of cyclin D1. Tumor cells appeared to be more aggressive, with frequent E-cadherin negative tumors present in the HCCs of CCND1−/−
mice. Interestingly, increased breast tumorigenesis has also been observed in CCND1−/−
mice when they were crossed with mice expressing activated β-catenin targeted to the mammary gland (26
). Our study provides additional evidence that cyclin D1 plays divergent roles under different oncogenic signals and in diverse cell types. For each specific oncogenic signal and each cell type, one has to assay for the tumorigenic activity of the oncogene in a CCND1 null background in order to elucidate the requirements of cyclin D1 in the specific circumstance.
What are the molecular mechanisms for the accelerated and more aggressive phenotype observed in CCND1 knockout mice? One possible clue comes from our investigation of the expression of other D-type cyclins and Cdks in the CCND1−/−
tumor samples. Increased expression of CCND2 in CCND1+/−
tumors strongly suggests that this member of D-type cyclin family can replace cyclin D1. In addition, lack of cyclin D1 seems to decrease the Cdk4 protein level, likely through reduced stability of free Cdk4. Whether CCND2/Cdk6 is more efficient in the phosphorylation of Rb than CCND1/Cdk4 during liver tumor development warrants further investigation, and may provide functional roles for CCND2 in hepatic carcinogenesis. A recent study supports a positive role for cyclin D2 in tumorigenesis, where cyclin D2 transgenic mice are more susceptible to developing skin tumors, a characteristic that is not shared by cyclin D1 and D3 transgenic mice generated under the same promoter (46
). In addition, D-type cyclins play Cdk-independent roles in certain cell types. For example, D-type cyclins bind to nuclear receptors such as androgen, estrogen, and vitamin D receptors to regulate the expression of several genes in prostate, mammary gland and skin keratinocytes (48
). While the interaction of D-type cyclins with nuclear receptors has not been described in liver, whether cyclin D1 acts through this pathway during liver tumorigenesis clearly needs to be evaluated. A third possibility is that D-type cyclins may modulate tumorigenesis via regulating cell types other than hepatocytes. For example, D type cyclins may regulate tumor immunity or angiogenesis, and the more rapid tumor growth in CCND1 null mice may be due to the reduced immune response or more robust angiogenesis since CCND1 is deleted in all cell types in mice. This hypothesis can be tested by generating hepatocyte specific deleted CCND1 mice using the albumin Cre system. If we fail to observe this accelerated tumor growth phenotype in these mice, the result will support an additional non-hepatocyte role of D type cyclins during HCC pathogenesis.
It has been speculated that small molecules targeted against CCND1/Cdk4 may be useful as therapeutic reagents against human tumors. However, our study and the study by Rowlands et al suggest that we need to be cautious about such treatments, since it could lead to unfavorable consequences under certain conditions. For example, CCND1/Cdk4 inhibitors may not be suitable for patients who have chronic HBV or HCV infection, as these patients are at a greater risk of developing HCC, and loss of CCND1/CDK4 activity may accelerate the progression of this malignancy.