Here, we show that two non-genotoxic hepatotoxins, DDC and CCl4
, can cooperate with MYC to induce robust mitotic cellular division and the near immediate onset of liver tumorigenesis. Our results have implications for the mechanisms by which oncogenes such as MYC induce and are restrained from causing HCC. Previously, we have shown that MYC overexpression induces robust cellular proliferation in embryonic and neonatal hepatocytes, but, in marked contrast, induced cellular growth and DNA replication without mitotic cellular division in the adult liver 
. We speculated that for MYC to induce liver cancer in adult hosts, the liver context must be changed, presumably through factors that stimulate hepatocyte proliferation. Indeed, both DDC and CCl4
, toxins that target distinct regions of the liver 
were found to markedly increase the ability of MYC to induce mitotic cellular division and permit the almost immediate onset of HCC. Our results suggest that oncogenes such as MYC can become latently active in the liver but only upon subsequent exposure to a hepatotoxin is their malignant potential manifested.
Our data are consistent with the general notion that the liver is most permissive to malignant transformation under circumstances when hepatocytes are undergoing cellular proliferation 
. Moreover, our observations further validate previous reports that MYC can cooperate with agents that modulate the liver and thereby stimulate hepatocyte proliferation. Most notably, MYC has been shown to cooperate with TGF-alpha and phenobarbital to induce accelerated tumorigenesis in the adult murine liver 
. Our model recapitulates the latency of tumor formation associated with adult onset HCC. Hence, we should be able to define factors that either enhance or repress tumorigenesis. To our knowledge, our report is the first to describe that hepatotoxins cooperate with MYC overexpression to induce accelerated HCC specifically in the context of an adult murine liver.
A possible explanation for our findings is that MYC appears to be only capable of inducing the activation of Cyclin B1-Cyclin-dependent kinase 1 (Cdk1) in embryonic/neonatal hepatocytes or in adult livers treated with hepatotoxins. Elevated Cyclin B1 levels often precede the onset of tumor cell immortalization and aneuploidy 
. When we compared Cyclin B1 expression levels in the different cohorts of mice, we found that treatment with DDC or CCl4
was associated with elevated Cyclin B1 expression relative to a normal or MYC ON liver. Furthermore, when MYC was overexpressed in conjunction with either DDC or CCl4
we unveiled evidence for a further increase in Cyclin B1 expression levels. Notably, Cyclin B1 has been shown to be a direct transcriptional target of MYC 
. Hence, one likely explanation for these differences is that the Cyclin B1 promoter region may be more accessible to MYC in hepatotoxin-treated livers, possibly by regulation of chromatin structure 
. Alternatively, the increased expression of Cyclin B1 could be attributed to mitotic signals from the regenerative response initiated in the liver by CCl4
or DDC damage. Future experiments will be directed at determining if MYC is directly activating the transcription of Cyclin B1 in hepatotoxin-treated livers.
Previously, we were able to show that a loss of p53 is associated with MYC-induced tumorigenesis in the normal adult liver 
. In contrast, there was no evidence for p53 protein loss in MYC ON/DDC and MYC ON/CCl4 liver tumors, consistent with our observations in embryonic or neonatal MYC-induced tumors. Interestingly, Yin and colleagues recently showed that loss of p53 function cooperates with MYC to induce increased Cyclin B1 expression 
. Our earlier report supports this mechanism in that loss of p53 in MYC-induced adult tumors was associated with elevated Cyclin B1 mRNA. Interestingly, we did not observe a correlation between p53 protein status and Cyclin B1 expression in hepatotoxin-treated livers. One possible explanation for this discordance is that, after exposure of the liver to DDC or CCl4
, the already elevated levels of Cyclin B1 enable MYC to bypass the p53 checkpoint and further drive hepatocyte proliferation.
We hypothesized that one possible mechanism by which DDC facilitates MYC-induced HCC is by inducing oval cell expansion. Since these cells more closely resemble embryonic hepatoblasts, we posited that they may be more susceptible to MYC-induced tumorigenesis 
. However, when assayed by A6 immunohistochemistry, there was no evidence for a difference in oval cell number between MYC OFF/DDC and MYC ON/DDC livers. One possible explanation for a lack of change in oval cell number between MYC ON/DDC and MYC OFF/DDC livers is that some oval cells were undergoing neoplastic transformation, thereby losing their oval cell properties and were no longer expressing the A6 antigen. This idea is further supported by the fact that the first appearance of neoplastic foci and microcarcinomas in the MYC ON/DDC livers occurs as early as 10 days after MYC activation, and is predominantly located in the periportal area of the liver. Alternatively, these oval cells could have also lost their oval cell properties by quickly differentiating to immature hepatocytes. Our model should be useful in further investigating the role of oval cells in MYC-induced tumorigenesis.
In contrast to DDC, continuous exposure to CCl4
damages the liver by causing massive centrilobular necrosis and stimulating an inflammatory response through the activation of kupffer cells. Activated kupffer cells produce pro-inflammatory cytokines, such as Tumor necrosis factor-alpha (TNF-alpha), that can exacerbate chemical-induced liver injury as well as functioning as hepatic mitogens 
. TNF-alpha is commonly induced almost immediately after CCl4
exposure and is responsible for the activation of nuclear transcription factors that promote the expression of genes involved in cell cycle progression and mitotic division 
. It will be interesting to subsequently investigate the role of CCl4
-induced TNF-alpha signaling in MYC-induced liver tumorigenesis.
In this report, we demonstrate that toxin-mediated liver damage is sufficient to accelerate MYC-induced HCC formation in adults. Interestingly, this effect is irrespective of the target of the hepatotoxin or whether it functions independently as a carcinogen. Our results are consistent with a model whereby oncogenic events, such as MYC overexpression, may be generally silent in adult somatic hepatocytes unless the host is exposed to a liver toxin that promotes a permissive environment for tumorigenesis.