In this study, we describe the consequences of genetically inactivating the Rb gene family in the liver of adult mice. We found that Rb family TKO mice develop HCC, providing a novel tractable mouse model for this deadly human cancer. Using this genetically defined model, we identified adult progenitor cells as initiating populations in HCC development upon inactivation of the RB pathway and we uncovered a potential tumor suppressor role for Notch signaling in human HCC.
RB family members display extensive functional overlap in many cell types, including in liver cells (Reed et al., 2009
). In fact, we found that the presence of one WT allele of p107
was sufficient to prevent HCC development for at least 6–8 mo after Cre activation, similar to our observation in the hematopoietic compartment, indicating that reintroduction of one Rb
family allele significantly impairs tumor development (Viatour et al., 2008
). We cannot exclude the possibility that these mice will eventually develop HCC at later time points; indeed two of the cell lines that we have isolated thus far from TKO mice have retained a WT p130
allele (unpublished data). Although it is difficult to directly compare the genetic deletion of Rb
family genes in the mouse system to the protein inactivation by degradation or phosphorylation observed in human HCC, these results support the observation that most events targeting the RB pathway in HCC simultaneously inactivate the three RB family proteins (see introduction).
An important question in the liver cancer field is whether HCC initiates from hepatocytes and/or earlier progenitors in the adult liver, a question which is challenging to address in patients. Cre activation in cTKO mice results in the proliferation and rapid expansion of cells with characteristics of liver stem/progenitor cells, including their morphology, their localization close to the portal triad, and their expression of specific markers. At the same time point, we observed only limited DNA replication in mature hepatocytes and no evidence of clonal expansion. These observations, together with the fact that a fraction enriched for nonparenchymal progenitor cells can recapitulate tumor development upon transplantation, strongly suggest that TKO HCC tumors initiate from the stem/progenitor compartment in the liver of mice. The stem/progenitor cell hypothesis is supported by evidence that Cullin 3, a regulator of Cyclin E (an inhibitor of the RB family), prevents liver cancer development from liver progenitors (Kossatz et al., 2010
Although EpCAM and CK19 have been proposed to mark some liver progenitors (Schmelzer et al., 2006
; Zhang et al., 2008
), immunostaining and FACS analysis (unpublished data) for these markers did not identify EpCAM+
cells in TKO early lesions. One interpretation of this observation is that TKO lesions initiate from progenitors that are distinct from EpCAM+
progenitors, which would underscore the heterogeneity of liver stem/progenitor cell populations. This idea is supported by the observation that progenitor cells expanding upon inactivation of the NF2 tumor suppressor display different characteristics than progenitor cells expanding in TKO mice (Benhamouche et al., 2010
). In addition, we found that some TKO liver progenitors are positive for markers that were originally developed in a model of DDC-induced liver progenitor expansion. However, the paucity of C3+
, or E10+
cells in TKO mice compared with DDC treatment (compare with Fig. S2, L–N), as well as the lack of organized structure in TKO livers versus the formation of atypical bile ducts in the DDC model (Fig. S2, O and P) further suggest that the populations of progenitor cells involved in the expansion in the two models are not equivalent. Nevertheless, the observation that some cells in early TKO lesions express Sox9 indicates that there are some similarities between the two systems. Future experiments will continue to compare markers expressed by adult liver progenitor cells identified in different systems to better define distinct subpopulations of these cells.
In the TKO model, we found that the colony-forming potential was restricted to two distinct mutant stem/progenitor cell populations in TKO mice, C3/C7/E10+ cells and Sca1+ cells. The decreased expression of Bmi1 and the increased expression of lineage markers, as well as the increased cell cycle and colony-forming activities observed in TKO C3/C7/E10+ cells suggest that these cells may represent a more mature population of progenitors when compared with Sca1+ cells. C3/C7/E10+ or Sca1+ cells were not able to recapitulate tumors upon transplantation, suggesting that either they do not represent the cell of origin or that transplantation protocols need to be improved to allow a small number of purified stem/progenitor cells to colonize recipient liver.
Our data strongly suggest that TKO liver tumors arise from subpopulations of stem/progenitor cells. After exiting quiescence and an initial phase of expansion, these TKO cells undergo some differentiation, and these fairly differentiated proliferating cells represent the bulk of the HCC tumors. This model fits with increasing evidence suggesting that loss of RB function initiates cancer from specific populations of stem/progenitor cells (Macpherson, 2008
; Viatour et al., 2008
; Calo et al., 2010
; Choi et al., 2010
; Jiang et al., 2010
), providing a general model for tumor suppression by the RB pathway. However, we cannot formally exclude at this point that an intermediate cell population (i.e., very late progenitor or early hepatocyte) could also be the cell of origin in TKO HCC. The recent identification of Cre-expressing mouse strains in liver progenitor populations (Dorrell et al., 2011
; Shin et al., 2011
) may provide novel means to perform lineage-tracing experiments and to formerly identify the cell of origin of the TKO tumors, including its differentiation potential and the role of the RB pathway in its cell fate.
Previous studies in neural progenitors have reported repression of Notch signaling by p107 (Vanderluit et al., 2004
). In this study, we found that RB family members directly control the transcription of multiple components of the Notch pathway in human and mouse cells, identifying a strong link between these two central cellular pathways in liver cells. However, the lack of induction of Notch pathway genes in TKO liver progenitor cells suggests that this link is context dependent. More work is needed to understand the regulation of Notch pathway genes by E2F family members in different cell types. Functionally, our data showing that increased Notch activity leads to cell cycle arrest in G2 and/or cell death might be related to observations that inactivation of Rb
family genes induces a G2 arrest in TKO fibroblasts (van Harn et al., 2010
In vivo modulation of Notch pathway activity with DAPT, a γ-secretase inhibitor and potent inhibitor of Notch signaling, resulted in accelerated cancer development in TKO mice. Interestingly, liver-specific inactivation of Notch1
expression, although not sufficient to promote HCC, leads to the proliferation of hepatocytes in mice (Croquelois et al., 2005
). In contrast, in vivo DAPT treatment failed to increase proliferation in TKO progenitor cells, either because these cells have already reached a very high level of cell cycle activity or because DAPT treatment is not sufficient to block Notch signaling in progenitor cells. In vitro, we found that activation of Notch signaling in mouse and human HCC cells was sufficient to block their expansion, as was reported in one human cell line before (Qi et al., 2003
). We further show that HCC cells with enforced expression of NICD undergo cell cycle arrest in G2 and display increased apoptotic activity. The Notch pathway interacts with multiple molecular effectors, and there are probably multiple mechanisms involved in inhibiting tumor cell progression. In the future, thorough investigation of Notch effectors should bring more mechanistic insights into HCC development.
Our findings suggest a model in which Notch signaling acts as a tumor suppressor feedback mechanism in response to activation of E2F transcription factors in TKO liver cells. Although this negative feedback is clearly not sufficient to prevent cancer initiation, activation of Notch may significantly slow the expansion of tumor cells in vivo, and we found that expression of a Notch pathway signature of 17 genes strongly predicts survival in HCC patients. Because signaling downstream of Notch1 may play a tumor-suppressor role in different cancer types (Nicolas et al., 2003
; Hanlon et al., 2010
; Ranganathan et al., 2011
), our observations indicate that local activation of the Notch pathway may provide novel treatment approaches in a large group of human cancers of epithelial origin.