Laura Elisa Buitrago-Molina

The degree of liver injury determines the role of p21 in liver regeneration and hepatocarcinogenesis in mice

Hepatocellular carcinoma (HCC) frequently arises in the context of chronic injury that promotes DNA damage and chromosomal aberrations. The cyclin‐dependent kinase inhibitor p21 is an important transcriptional target of several tumor suppressors, which promotes cell cycle arrest in response to many stimuli. The aim of this study was to further delineate the role of p21 in the liver during moderate and severe injury and to specify its role in the initiation and progression of HCC. Deletion of p21 led to continuous hepatocyte proliferation in mice with severe injury allowing animal survival but also facilitated rapid tumor development, suggesting that control of compensatory proliferation by high levels of p21 is critical to the prevention of tumor development. Unexpectedly, however, liver regeneration and hepatocarcinogenesis was impaired in p21‐deficient mice with moderate injury. Mechanistically, loss of p21 was compensated by activation of Sestrin2, which impaired mitogenic mammalian target of rapamycin (mTOR) signaling and activated cytoprotective Nrf2 signaling. Conclusion: The degree of liver injury and the strength of p21 activation determine its effects on liver regeneration and tumor development in the liver. Moreover, our data uncover a molecular link in the complex mTOR, Nrf2, and p53/p21‐signaling network through activation of Sestrin2, which regulates hepatocyte proliferation and tumor development in mice with liver injury. (Hepatology 2013;53:1143–1152)

The strength of the Fas ligand signal determines whether hepatocytes act as type 1 or type 2 cells in murine livers.

The BH3‐interacting domain death agonist Bid has been shown to be critical for Fas‐induced hepatocellular apoptosis. Furthermore, some studies have suggested that phosphorylation of Bid may determine its apoptotic function and may act as a switch to nonapoptotic functions. The aim of this study was to evaluate the role of Bid and phosphorylated Bid for Fas ligand (FasL)‐induced apoptosis in murine livers. The monoclonal antibody Jo2 and a hexameric form of sFasL (MegaFasL) were used to induce apoptosis in wild‐type, Bid‐deficient (Bid−/−), Bid transgenic mice expressing a nonphosphorable form of Bid and Fas receptor‐deficient lpr mice. Apoptosis sensitivity was determined in healthy mice and in mice following bile duct ligation, partial hepatectomy, or suramin pretreatment. As previously reported, loss of Bid protects mice against Jo2‐induced liver failure. Remarkably however, Bid−/− mice are highly sensitive to MegaFasL‐induced apoptosis. MegaFasL‐treated Bid−/− mice showed a typical type I cell signaling behavior with activation of caspase‐3 without Bax translocation to the mitochondria and no cytochrome C/Smac release into the cytosol. In contrast to previous in vitro findings, phosphorylation of Bid does not affect the sensitivity of hepatocytes to Fas receptor‐mediated apoptosis in vivo. Conclusion: Our data suggest that Bid mainly amplifies a weak death receptor signal in quiescent and nonquiescent hepatocytes rendering the liver more sensitive to FasL‐induced apoptosis. Thus, depending on the efficacy of Fas receptor activation, hepatocytes and nonparenchymal cells can either behave as type I or type II cells. (HEPATOLOGY 2009.)

Rapamycin delays tumor development in murine livers by inhibiting proliferation of hepatocytes with DNA damage

In this study, everolimus (RAD001) was used to determine the role of mammalian target of rapamycin (mTOR) in hepatocarcinogenesis. We show that RAD001 effectively inhibits proliferation of hepatocytes during chronic liver injury. Remarkably, the ability of RAD001 to impair cell cycle progression requires activation of the DNA damage response; loss of p53 significantly attenuates the antiproliferative effects of mTOR inhibition. RAD001 modulates the expression of specific cell cycle–related proteins and the assembly of cyclin–cyclin‐dependent kinase complexes to prevent cell cycle progression. Furthermore, RAD001 sustains the apoptosis sensitivity of hepatocytes during chronic liver injury by inhibiting p53‐induced p21 expression. Long‐term treatment with RAD001 markedly delays DNA damage–induced liver tumor development. Conclusion: We provide evidence that mTOR inhibition has a substantial effect on sequential carcinogenesis and may offer an effective strategy to delay liver tumor development in patients at risk. (HEPATOLOGY 2009;50:500–509.)

Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3‐kinase inhibitor BEZ235 and the PI3‐kinase inhibitor BKM120 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide with only few therapeutic options for patients with advanced disease. There is growing evidence indicating that activation of the PI3K/Akt/mTOR pathway plays an important role in HCC and therefore represents a promising target for novel therapeutic approaches. The aim of this study was to evaluate and compare the antitumour activity of the mTOR inhibitor RAD001, the dual mTOR and PI3‐kinase inhibitor BEZ235 and the PI3‐kinase inhibitor BKM120 in vitro and in vivo.

mTor as a potential target for the prevention and treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a global health problem and responsible for up to 500.000 deaths annually. It usually occurs secondary to infections with hepatitis B or C viruses, alcohol consumption, non-alcoholic steatohepatitis or hereditary liver diseases. The prognosis for patients with advanced disease is dismal; therefore, new strategies to prevent or treat this malignancy are urgently needed. Over recent years, several molecular pathways have been identified contributing to the molecular pathogenesis of this devastating disease, among them the PI3K/AKT/mTOR pathway. mTOR is effectively inhibited by rapamycin and its derivatives such as temsirolimus and everolimus. The anti-tumor activity of rapamycin was identified more than 30 years ago in a screen performed at the National Cancer Institute, but was subsequently not developed for cancer treatment. In the 1990s, activation of the mTOR pathway was recognized in various malignancies spurring again the interest in mTOR inhibitors for anti-cancer treatment. In 2007, the US Food and Drug Administration approved the first mTOR inhibitor, temsirolimus, for the treatment of renal cell carcinoma. Currently, several clinical studies are underway to define the role of mTOR inhibitors for the treatment of hepatocellular carcinoma. The aim of this review is to outline the role of mTOR for hepatocarcinogenesis. We will also discuss the latest preclinical and clinical data of mTOR inhibitors for the prevention and treatment of HCC.

p21 promotes sustained liver regeneration and hepatocarcinogenesis in chronic cholestatic liver injury

Background and aims The cyclin-dependent kinase inhibitor p21 has been implicated as a tumour suppressor. Moreover, recent genetic studies suggest that p21 might be a potential therapeutic target to improve regeneration in chronic diseases. The aim of this study was to delineate the role of p21 in chronic liver injury and to specify its role in hepatocarcinogenesis in a mouse model of chronic cholestatic liver injury. Methods The degree of liver injury, regeneration and tumour formation was assessed in Mdr2−/− mice and compared with Mdr2/ p21−/− mice. Moreover, the role of p21 was evaluated in hepatoma cells in vitro and in human hepatocellular carcinoma (HCC). Results Mdr2−/− mice developed HCCs as a consequence of chronic inflammatory liver injury. In contrast, tumour development was profoundly delayed in Mdr2/ p21−/− mice. Delayed tumour development was accompanied by markedly impaired liver regeneration in Mdr2/ p21−/− mice. Moreover, the regenerative capacity of the Mdr2/ p21−/− livers in response to partial hepatectomy declined with age in these mice. Hepatocyte transplantation experiments revealed that impaired liver regeneration was due to intrinsic factors within the cells and changes in the Mdr2/ p21−/− microenvironment. In human HCCs, a subset of tumours expressed p21, which was associated with a significant shorter patient survival. Conclusions We provide experimental evidence that p21 is required for sustained liver regeneration and tumour development in chronic liver injury indicating that p21 needs to be tightly regulated in order to balance liver regeneration and cancer risk. Moreover, we identify p21 as a negative prognostic marker in human HCC.

Activation of Nuclear Factor E2-Related Factor 2 in Hereditary Tyrosinemia Type 1 and Its Role in Survival and Tumor Development

In tyrosinemia type 1 (HT1), accumulation of toxic metabolites results in oxidative stress and DNA damage, leading to a high incidence of hepatocellular carcinomas. Nuclear factor erythroid‐2 related factor 2 (Nrf2) is a key transcription factor important for cellular protection against oxidative stress and chemical induced liver damage. To specifically address the role of Nrf2 in HT1, fumarylacetoacetate hydrolase (Fah)/Nrf2−/− mice were generated. In acute HT1, loss of Nrf2 elicited a strong inflammatory response and dramatically increased the mortality of mice. Following low grade injury, Fah/Nrf2−/− mice develop a more severe hepatitis and liver fibrosis. The glutathione and cellular detoxification system was significantly impaired in Fah/Nrf2−/− mice, resulting in increased oxidative stress and DNA damage. Consequently, tumor development was significantly accelerated by loss of Nrf2. Potent pharmacological inducers of Nrf2 such as the triterpenoid analogs 1[2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oyl]imidazole have been developed as cancer chemoprevention agents. Pretreatment with 1[2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oyl]imidazole dramatically protected Fah−/− mice against fumarylacetoacetate (Faa)‐induced toxicity. Our data establish a central role for Nrf2 in the protection against Faa‐induced liver injury; the Nrf2 regulated cellular defense not only prevents acute Faa‐induced liver failure but also delays hepatocarcinogenesis in HT1. (HEPATOLOGY 2008;48:487–496.)

The BH3‐only protein BID impairs the p38‐mediated stress response and promotes hepatocarcinogenesis during chronic liver injury in mice

Apoptosis is critical for maintaining tissue homeostasis, and apoptosis evasion is considered as a hallmark of cancer. However, increasing evidence also suggests that proapoptotic molecules can contribute to the development of cancer, including liver cancer. The aim of this study was to further clarify the role of the proapoptotic B‐cell lymphoma 2 homology domain 3 (BH3)‐only protein BH3 interacting‐domain death agonist (BID) for chronic liver injury (CLI) and hepatocarcinogenesis (HCG). Loss of BID significantly delayed tumor development in two mouse models of Fah‐mediated and HBsTg‐driven HCG, suggesting a tumor‐promoting effect of BID. Liver injury as well as basal and mitogen‐stimulated hepatocyte proliferation were not modulated by BID. Moreover, there was no in vivo or in vitro evidence that BID was involved in DNA damage response in hepatocytes and hepatoma cells. Our data revealed that CLI was associated with strong activation of oxidative stress (OS) response and that BID impaired full activation of p38 after OS. Conclusion: We provide evidence that the tumor‐promoting function of BID in CLI is not related to enhanced proliferation or an impaired DNA damage response. In contrast, BID suppresses p38 activity and facilitates malignant transformation of hepatocytes. (Hepatology 2015;62:816–828)