Superfund Research Program

Effects of Superfund Toxicants on Liver Cancer Progenitor Cells

Project Leader: Michael Karin
Grant Number: P42ES010337
Funding Period: 2000-2017

Project-Specific Links

Final Progress Reports

Year:   2016  2009  2004 

Dr. Karin’s component:

Researchers have discovered that the Sestrins are stress inducible proteins that inhibit TOR signaling through their ability to interact with and activate AMPK. As mammalian genomes contain 3 separate Sestrin loci, researchers have resorted to study Sestrin function in Drosophila, as this model organism contain only a single Sestrin locus which encodes a protein that is highly similar to the mammalian Sestrins. Researchers have confirmed that Drosophila Sestrin is identical in its biochemical function to the mammalian Sestrins, being able to stimulate AMPK activity and in turn inhibit TOR activation. Consistent with its ability to inhibit TOR activation, Drosophila Sestrin is an inhibitor of cell growth. An important insight to Sestrin function was provided by the analysis of Sestrin-null flies. Although these flies exhibit normal morphology at hatching, biochemical analysis of larval fat bodies revealed a 50% increase in TOR activity and a 50% decrease in AMPK activity. This was accompanied by a 35% increase in triglyceride accumulation affecting the entire fly. Researchers also found that TOR activation results in increased Sestrin gene transcription in a manner dependent on activation of the transcription factor FoxO. These results indicate that Drosophila Sestrin is a critical part of a feedback loop in which prolonged TOR activation leads to Sestrin induction, resulting in activation of AMPK and inhibition of TOR.

Importantly, 5 days old Sestrin-null flies exhibit accumulation of defective mitochondria in their flight muscles and when 20 days old, they exhibit massive muscle degeneration. Sestrin-null flies also exhibit cardiac arrhythmias, that are usually seen only in very old (>90 days) flies. Cardiac arrhythmia in Sestrin-null flies is accompanied by structural degeneration of the heart muscle. Degeneration of the flight and cardiac muscles in Sestrin-null flies is associated with accumulation of aggregates of ubiquitinated proteins, reactive oxygen species (ROS) and apoptotic cells. These signs suggested that loss of Sestrin expression may result in inhibition of autophagy due to persistent TOR activation. To test this hypothesis, researchers knocked down expression of ATG1, a component of the autophagy machinery whose function is inhibited by TOR. Indeed, ATG1 knockdown resulted in the same degenerative, premature aging, phenotype caused by loss of Sestrin. Overall, these results identify Sestrin as an important physiological regulator that is responsible for maintenance of homeostasis and prevention of premature tissue aging and degeneration. Through its ability to keep TOR activity under control, Sestrin provides an important link between stress and metabolic control.

In mammals, TOR activity is stimulated by obesity, which was recently shown to increase cancer risk in addition to its known ability to increase the risk of cardiovascular disease and metabolic disorders. Of all cancers, the effect of obesity is most pronounced on hepatocellular carcinoma (HCC), the most common form of liver cancer. Researchers have established several mouse models in which obesity greatly enhances the induction of HCC upon administration of the procarcinogen DEN, a close relative of the Superfund toxic substance dimethylnitrosamine (DMN) which is known to cause HCC in humans. In addition to developing more HCC in response to DEN administration, obese mice were found to be much more susceptible to DEN-induced liver damage. The major mechanism by which obesity increases HCC risk entails the elevated production of the inflammatory cytokines IL-6 and TNF, which are needed for establishment of hepatosteatosis (liver fat accumulation) and steatohepatitis (liver inflammation due to fat accumulation). Mice deficient in either IL-6 or type 1 TNF receptor (TNFR1) were resistant to the HCC promoting effect of obesity.

These findings are likely to be of human clinical relevance as elevated HCC risk is closely linked not only to obesity but also to hepatosteatosis and steatohepatitis, both of which are the cardinal signs of non-alcoholic fatty liver disease (NAFLD). In addition, NAFLD and obesity are known to be associated with elevated IL-6 and TNF production.

Dr. Leffert’s component:

Researchers have shown that IkkßΔhep hepatocytes display growth advantages over IkkßF/F hepatocytes in vivo and in vitro. The significance of the in vitro findings is that they suggest that such growth advantage are cell-autonomous, occurring independently of liver non-parenchymal or extrahepatic cells. These observations are an important advance because they suggest new ways of thinking about mechanisms that augment DEN-induced HCC in IkkßΔhep mice. For example, increased HCC development in IkkßΔhep mice may also be caused by growth advantages of Ikkß-deleted hepatocyte survivors that are independent of ROS formation, cell death and paracrine growth factor regulation of surviving IkkßΔhep hepatocytes exerted by non-parenchymal cells.

The specific findings consist of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G0 →G1 transitions); and, enhanced recovery efficiency, cyclin D1 expression and cell proliferation in vitro. Ex vivo deletion of Ikkß (by infection of cultured IkkßF/F hepatocytes with Ad-Cre adenovirus) also accelerates hepatocyte growth, and eliminates problems of interpretation associated with non-equivalent recovery efficiency. IkkßΔhep hepatocyte proliferative responses show heightened sensitivity to TGF_a and TNF_a (well-known hepatocyte mitogens), and heightened expression of fibronectin, collagens I/III, nidogen, ß-actin and integrin ß1 mRNAs (which encode cell matrix proteins with specialized functions). The combined findings suggest that altered mitogen signaling and potentially upregulated expression of extracellular matrix and its associated protein components underlie IkkßΔhep hepatocyte growth-autonomous advantages. In vitro investigations of the effects of DEN on growth regulatory processes in cultured IkkßF/F and IkkßΔhep hepatocytes are ongoing.