Cell senescence and aging in carcinogenesis and liver repopulation

The studies presented in my thesis delve into the complex relationship between cancer and aging using analytical approaches shared with the field of regenerative medicine. We took advantage of a model of orthotopic cell transplantation to explore the basic question as to whether the contribution of aging to the risk of cancer is, at least in part, related to alterations in the tissue microenvironment. Previous investigations conducted by our research group had revealed that isolated normal hepatocytes form larger clones of daughter cells when transplanted into the liver of old animals as compared to those formed in young recipients. In a relatively simple experiment we then utilized a similar approach to transplant preneoplastic cells isolated from hepatic nodules into either young or old animals of a syngeneic strain. Results were striking: very limited expansion of transplanted nodular hepatocytes was observed in the liver of young animals, while the same cell preparations grew significantly upon injection into older hosts, with a few cases of progression to hepatocellular carcinoma (HCC). The relevance of these findings is twofold. Firstly, they clearly indicate that preneoplastic cell populations isolated from hepatic nodules are not endowed with any measurable degree of growth autonomy, in that they were unable to form nodules when transferred into the liver of young hosts. Thus, their focal growth is dependent on stimuli originating from the local microenvironment. In this respect they behave like normal hepatocytes. Secondly, it is evident from these results that the microenvironment of the aged liver is able to foster the growth transplanted syngeneic nodular hepatocytes, again reproducing analogous findings already obtained with normal cells. Having established this fundamental fact, it became important to explore possible biological and underlying molecular mechanisms explaining the growth-promoting nature of the aged liver microenvironment. To this end, intriguing insights came again from the field of regenerative medicine. Our research group is involved in the characterization of an experimental model of massive liver repopulation through hepatocyte transplantation. This approach was set up in our laboratory and is based on the administration of retrorsine (RS), a naturally occurring alkaloid, as a preconditioning treatment. Rat liver exposed to RS and then transplanted with isolated normal hepatocytes is entirely replaced by donor-derived cells within a few months. Importantly, transplanted nodular hepatocytes can also selectively expand in the RS-treated liver, rapidly evolving to HCC. Again, neither normal, nor nodular hepatocytes are able to proliferate when injected into the liver of untreated recipients, as already noted above. This pattern of findings is similar to that observed in the aged liver, although the magnitude of the observed phenomena is far greater following exposure to RS. Moreover, it is apparent from the foregoing discussion that the growth of normal and nodular hepatocytes is controlled by fundamentally similar biological mechanisms, such that carcinogenesis and liver repopulation represent in fact “two sides of the same coin”, as it has been proposed. Given the similarities of biological responses between the RS-treated and the aged liver, we then probed into possible cellular and molecular analogies shared by the two systems. Much to our surprise, it was found that exposure to RS is a powerful inducer of cell senescence in hepatocytes in vivo. As reported in the preceding section of this report, several markers of cell senescence were expressed by RS-exposed hepatocytes, including senescence-associated β-galactosidase (SA-β-gal), cell hypertrophy, a persistent block in the cell cycle coupled with activation of cell growth pathways. The picture emerging from these findings is that RS treatment, which sets the stage for the selective growth of both normal and nodular transplanted hepatocytes, leading to massive liver repopulation and emergence of HCC, respectively, is also associated with the induction of a senescent phenotype in resident hepatocytes. Such a senescence phenotype is commonly found in aged tissues including the liver, as also shown in our studies. This phenotype is likely to explain, at least in part, the similarities observed between the microenvironments of the aged liver and that of RS-exposed liver. The last section of this thesis describes studies aimed at reversing the RS-induced alterations in the liver microenvironment and, in doing so, at verifying whether this would modulate the growth of preneoplastic lesions promoted by RS. Animals given the genotoxic agent diethylnitrosamine (DENA) followed by RS developed large hepatocyte nodules within a few months. However, the growth of preneoplastic nodules was significantly delayed in animals receiving hepatocyte transplantation following the DENA+RS regimen. These results represent a proof of principle that an altered microenvironment is indeed a powerful driving force in neoplastic progression; most importantly, they clearly indicate that strategies aimed at “normalizing” such an altered microenvironment might impact on the rate of progression of the neoplastic process.