Spontaneous Liver Tumorigenesis Research Articles

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy

Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

SQSTM1/p62 Inhibits whereas Nrf2 Promotes Tumorigenesis by Inducing Cell Population Remodeling and Metabolic Reprograming in Mouse Livers with mTORC1 Activation and Defective Autophagy

Tsc1 (Tuberous Sclerosis 1) and Tsc2 are tumor suppressor genes and negative regulators of mTORC1, and the mutation of Tsc1 or Tsc2 is associated with various cancers including hepatocellular carcinoma (HCC). mTORC1 activation promotes anabolic protein synthesis and inhibits catabolic autophagy process. Either hepatic persistent activation of mTORC1 due to the loss of Tsc1 or impaired hepatic autophagy due to the loss of Atg5 leads to spontaneous liver tumorigenesis in mice. However, how autophagy impacts mTORC1-mediated liver metabolic changes and tumorigenesis is less clear. Here we show that deletion of Atg5 in liver-specific Tsc1 knockout (L-Tsc1 KO) mice inhibited liver tumorigenesis but increased mortality of L-Tsc1 KO mice. Histological and unbiased RNA-seq as well as metabolomic analysis revealed that lack of hepatic autophagy dramatically induced the remodeling of liver cell population and hepatic metabolic reprogramming in L-Tsc1 KO mice. RNA-seq analysis revealed increased cholangiocyte, hepatic stellate cell and macrophage but decreased hepatocyte gene signatures in liver-specific Tsc1 and Atg5 double knockout (DKO) mice. Histologically, Atg5 and Tsc1 DKO mice had increased hepatomegaly, ductular reactions, increased number of macrophage/Kupffer cells and infiltrated neutrophils as well as massive fibrosis, and the DKO mice died at the age of approximately 8-months-old with no detectable liver tumors. Metabolomic analysis revealed marked increased hepatic glutathione and glutathione metabolites but decreased hepatic levels of glucose and lipid with accumulation of TCA cycle intermediate metabolites and fatty acid oxidation intermediates in Atg5 and Tsc1 DKO mice. Deletion of hepatic p62 partially but deletion of Nrf2 almost completely rescued the liver cell type remodeling and metabolic reprogramming and the mortality of Atg5 and Tsc1 DKO mice. Deletion of p62 promoted whereas deletion of Nrf2 suppressed the liver tumorigenesis of Atg5 and Tsc1 DKO mice. These results reveal a critical role of Nrf2 in regulating liver cell type remodeling and metabolic reprogramming in mice lack of hepatic autophagy with mTORC1 activation. Our data may provide a mechanistic basis for a precision medicine to target Nrf2 pathway for treating as subset of HCC, especially in HCV- or alcohol-associated liver cancers that may have defective autophagy and mTOR activation.

Spontaneous Development of Hepatocellular Carcinoma and B-Cell Lymphoma in Mosaic and Heterozygous Brca2 and Cdkn1a Interacting Protein Knockout Mice

Hepatocellular carcinoma (HCC) is the most common form of liver tumors. Although HCC is associated with chronic viral infections, alcoholic cirrhosis, and nonalcoholic fatty liver disease, genetic factors that contribute to the HCC risk remain unknown. The BRCA2 DNA repair associated (BRCA2) and cyclin-dependent kinase inhibitor 1A (CDKN1A) interacting protein, known as BCCIP, are essential for cell viability and maintenance of genomic stability. In this study, we established a new genetically engineered mouse model with Bccip deficiency. Mosaic or heterozygous Bccip deletion conferred an increased risk of spontaneous liver tumorigenesis and B-cell lymphoma development at old age. These abnormalities are accompanied with chronic inflammation, histologic features of nonalcoholic steatohepatitis, keratin and ubiquitin aggregates within cytoplasmic Mallory-Denk bodies, and changes of the intracellular distribution of high-mobility group box 1 protein. Our study suggests BCCIP dysregulation as a risk factor for HCC and offers a novel mouse model for future investigations of nonviral or nonalcoholic causes of HCC development.

Mice with hepatocyte-specific FXR deficiency are resistant to spontaneous but susceptible to cholic acid-induced hepatocarcinogenesis.

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily with its endogenous ligands bile acids. Mice with whole body FXR deficiency develop liver tumors spontaneously, but the underlying mechanism is unclear. Moreover, it is unknown whether FXR deficiency in liver alone serves as a tumor initiator or promoter during liver carcinogenesis. This study aims to evaluate the effects of hepatocyte-specific FXR deficiency (FXR(hep-/-)) in liver tumor formation. The results showed that FXR(hep-/-) mice did not show spontaneous liver tumorigenesis with aging (up to 24 mo of age). Therefore FXR(hep-/-) mice were fed a bile acid (cholic acid)-containing diet alone or along with a liver tumor initiator, diethylnitrosamine (DEN). Thirty weeks later, no tumors were found in wild-type or FXR(hep-/-) mice without any treatment or with DEN only. However, with cholic acid, while only some wild-type mice developed tumors, all FXR(hep-/-) mice presented with severe liver injury and tumors. Interestingly, FXR(hep-/-) mouse livers increased basal expression of tumor suppressor p53 protein, apoptosis, and decreased basal cyclin D1 expression, which may prevent tumor development in FXR(hep-/-) mice. However, cholic acid feeding reversed these effects in FXR(hep-/-) mice, which is associated with an increased cyclin D1 and decreased cell cycle inhibitors. More in-depth analysis indicates that the increased in cell growth might result from disturbance of the MAPK and JAK/Stat3 signaling pathways. In conclusion, this study shows that hepatic FXR deficiency may only serve as a tumor initiator, and increased bile acids is required for tumor formation likely by promoting cell proliferation.

Targeted Deletion of ER Chaperone GRP94 in the Liver Results in Injury, Repopulation of GRP94-Positive Hepatocytes, and Spontaneous Hepatocellular Carcinoma Development in Aged Mice

Hepatocellular carcinoma (HCC) often results from chronic liver injury and severe fibrosis or cirrhosis, but the underlying molecular pathogenesis is unclear. We previously reported that deletion of glucose regulated protein 94 (GRP94), a major endoplasmic reticulum chaperone, in the bone marrow and liver leads to progenitor/stem cell expansion. Since liver progenitor cell (LPC) proliferation can contribute to liver tumor formation, here we examined the effect of GRP94 deficiency on spontaneous liver tumorigenesis. Utilizing liver-specific Grp94 knockout mice driven by Albumin-Cre (cGrp94f/f), we discovered that while wild-type livers are tumor free up to 24 months, cGrp94f/f livers showed abnormal small nodules at 15 months and developed HCC and ductular reactions (DRs) by 21 months of age, associating with increased liver injury, apoptosis and fibrosis. cGrp94f/f livers were progressively repopulated by GRP94-positive hepatocytes. At 15 months, we observed expansion of LPCs and mild DRs, as well as increase in cell proliferation. In examining the underlying mechanisms for HCC development in cGrp94f/f livers, we detected increase in TGF-β1, activation of SMAD2/3, ERK, and JNK, and cyclin D1 upregulation at the premalignant stage. While epithelial-mesenchymal transition (EMT) was not evident, E-cadherin expression was elevated. Correlating with the recurrence of GRP94 positive-hepatocytes, the HCC was found to be GRP94-positive, whereas the expanded LPCs and DRs remained GRP94-negative. Collectively, this study uncovers that GRP94 deficiency in the liver led to injury, LPC expansion, increased proliferation, activation of oncogenic signaling, progressive repopulation of GRP94-positive hepatocytes and HCC development in aged mice.

Bile Acids Activate YAP to Promote Liver Carcinogenesis

Elevated bile acid levels increase hepatocellular carcinoma by unknown mechanisms. Here, we show that mice with a severe defect in bile acid homeostasis due to the loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, and Yes-associated protein (YAP) activation and develop spontaneous liver tumorigenesis. This phenotype mirrors mice with loss of hippo kinases or overexpression of their downstream target, YAP. Bile acids act as upstream regulators of YAP via a pathway dependent on the induction of the scaffold protein IQGAP1. Patients with diverse biliary dysfunctions exhibit enhanced IQGAP1 and nuclear YAP expression. Our findings reveal an unexpected mechanism for bile acid regulation of liver growth and tumorigenesis via the Hippo pathway.

NO INHIBITORY EFFECTS OF (-)-EPIGALLOCATECHIN GALLATE AND LYCOPENE ON SPONTANEOUS HEPATOTUMORIGENESIS IN C3H/HeN MICE

Although several studies have indicated that (-)-epigallocatechin gallate (EGCG) and lycopene, representative dietary antioxidants, inhibit chemically induced animal tumorigenesis, only a few studies have examined the inhibitory effects of these compounds on spontaneous liver tumorigenesis in rodents. In this study, we investigated the inhibitory effects of these compounds on the formation of spontaneous liver tumors in C3H/HeN mice. We used xeroderma pigmentosum group A (XPA) gene-deficient mice to simultaneously examine whether the knockout mice could be used as a sensitive animal model. Inaddition, we examined the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG)--a marker of reactive oxygen species-induced DNA injury--in liver tissue. Male XPA +/+, XPA +/-, and XPA -/- mice with a C3H/HeN genetic background were divided into 3 groups: control, EGCG, and lycopene. Autopsy at 18 months of age revealed that EGCG and lycopene did not exhibit obvious suppressive effects on the development of liver tumors in any XPA genotype; further, the XPA genotype did not influence any susceptibility to liver tumors. With regard to 8-OHdG levels in non-tumorous liver tissue at 8 months of age, EGCG showed no significant inhibitory effects and lycopene showed significant inhibitory effects only in XPA +/- mice. The present study demonstrates that contrary to previous reports of the inhibitory effects of EGCG and lycopene on the development of various carcinogen-induced animal tumors, these compounds exert no chemopreventive effects on spontaneous liver tumorigenesis in C3H/HeN mice. EGCG and lycopene may inhibit carcinogen-induced tumors through properties other than their antioxidant abilities.

Suppression of tumor suppressor Tsc2 and DNA repair glycosylase Nth1 during spontaneous liver tumorigenesis in Long-Evans Cinnamon rats

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson's disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16-17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16-24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.

PIK3IP1, a Negative Regulator of PI3K, Suppresses the Development of Hepatocellular Carcinoma

Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, and survival in most cell types. Recently, we characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1), which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about the role of PIK3IP1 in normal and neoplastic growth in vivo. Proper liver function and development depend on intact PI3K signal transduction; when dysregulated, the PI3K pathway is linked to the development of liver cancer. To begin to dissect the contribution of PIK3IP1 to hepatic PI3K signaling in vivo and to liver tumorigenesis in particular, we formulated the following hypothesis: because PIK3IP1 down-regulates PI3K signaling and uncontrolled PI3K signaling is associated with liver cancer, then PIK3IP1-mediated down-regulation of the PI3K pathway should inhibit hepatocellular carcinoma (HCC) development. To test this idea, we generated transgenic mice overexpressing PIK3IP1 in hepatocytes in a mouse strain prone to develop HCC. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility, and survival compared with controls. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics compared with controls. We also observed that human HCC expressed less PIK3IP1 protein than adjacent matched liver tissue. Our data show that PIK3IP1 is an important regulator of PI3K in vivo, and its dysregulation can contribute to liver carcinogenesis.

Low frequency of H-ras activation in naturally occurring hepatocellular tumors of C3H/HeNCr mice.

Previous reports from several laboratories have consistently shown that approximately 30% of spontaneous hepatocellular adenomas and 70-80% of spontaneous hepatocellular carcinomas found in aged B6C3F1 [C57BL/6 (liver tumor resistant) x C3H (liver tumor susceptible)] male mice contain one of three missense point mutations in codon 61 of the H-ras oncogene, CAA-->AAA, CGA or CTA. Irrespective of subline, the C3H mouse, the paternal parent strain of the B6C3F1 hybrid, is more susceptible to spontaneous liver tumorigenesis than the B6C3F1 mouse. However, the role of H-ras in the pathogenesis of hepatocellular tumors in C3H mice is less clear, as widely different frequencies of activation of this gene, but by the same point mutations in codon 61, have been reported by various laboratories. The present study was undertaken to characterize H-ras involvement in hepatocellular tumors of aged C3H/He mice from the NCI-Frederick Cancer Research and Development Center Colony (C3H/HeNCr). Oncogene activation was evaluated in 45 C3H/HeNCr hepatocellular tumors by the NIH 3T3 transfection assays, and point mutations in the H-ras oncogene were detected and characterized in DNA fragments amplified by PCR, using dot blot hybridization analysis with mutation-specific oligonucleotide probes and direct dideoxy sequencing of PCR products. The only transforming gene detected in these tumors by NIH 3T3 transfection was H-ras. Only 17% (1/6) of spontaneous carcinomas and 8% (3/39) of spontaneous adenomas contained transforming H-ras sequences, each with a point mutation in codon 61. In all four cases with H-ras mutations, mutated sequences comprised a minor fraction of total H-ras gene copies in DNA extracted from primary tumors. H-ras mutations thus appear to have arisen relatively late in the pathogenesis of the neoplasms. For comparison, sections of formalin-fixed, paraffin-embedded hepatocellular tumors that occurred in untreated B6C3F1 hybrid mice sired by C3H/HeNCr males were assayed for the same H-ras mutations by PCR and dot blot hybridization. Nine of 13 such tumors (4/6 carcinomas, 5/7 adenomas) were positive. The overall difference in frequency of H-ras codon 61 mutations in hepatocellular tumors in C3H/HeNCr (4/45) versus B6C3F1 (9/13) was highly significant (P = 0.000035, Fisher's exact test). These data indicate that point mutations in H-ras do not generally play a major or an initiating role in spontaneous hepatocarcinogenesis of inbred C3H/HeNCr mice and contrast with the high rate of ras mutations in liver tumors of the B6C3F1 hybrid.

Effect of Konjac mannan on spontaneous liver tumorigenesis and fecal flora in [formula omitted] male mice

The effect of Konjac mannan (KM) on spontaneous liver tumorigenesis and fecal flora was studied in C3H He male mice maintained on a diet containing 10% KM. The weight gain in the 10% KM diet group was lower than that in a control diet group throughout the experimental period. At 12 months of age, the number of liver tumor nodules per mouse in the KM diet group was significantly lower than that in the control diet group ( P < 0.05). Frequency of occurrence of bifidobacteria and bacterial counts of enterobacteriaceae in the KM diet group were significantly higher than those in the control diet group ( P < 0.05). Thus, the present study demonstrates that dietary KM inhibited spontaneous liver tumorigenesis and altered fecal flora in C3H He male mice.