Maria Antonietta Seddaiu

SKP2 and CKS1 Promote Degradation of Cell Cycle Regulators and Are Associated With Hepatocellular Carcinoma Prognosis

BACKGROUND & AIMS The cell cycle regulators P21(WAF1), P27(KIP1), P57(KIP2), P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. METHODS Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, <3 years survival) and HCC cell lines were analyzed. RESULTS The promoters of P21(WAF1), P27(KIP1), and P57(KIP2) were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. CONCLUSIONS Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.

Role of HSP90, CDC37, and CRM1 as modulators of P16INK4A activity in rat liver carcinogenesis and human liver cancer

Current evidence indicates that neoplastic nodules induced in liver of Brown Norway (BN) rats genetically resistant to hepatocarcinogenesis are not prone to evolve into hepatocellular carcinoma. We show that BN rats subjected to diethylnitrosamine/2‐acetylaminofluorene/partial hepatectomy treatment with a “resistant hepatocyte” protocol displayed higher number of glutathione‐S‐transferase 7‐7(+) hepatocytes when compared with susceptible Fisher 344 (F344) rats, both during and at the end of 2‐acetylaminofluorene treatment. However, DNA synthesis declined in BN but not F344 rats after completion of reparative growth. Upregulation of p16INK4A, Hsp90, and Cdc37 genes; an increase in Cdc37‐Cdk4 complexes; and a decrease in p16INK4A‐Cdk4 complexes occurred in preneoplastic liver, nodules, and hepatocellular carcinoma of F344 rats. These parameters did not change significantly in BN rats. E2f4 was equally expressed in the lesions of both strains, but Crm1 expression and levels of E2f4‐Crm1 complex were higher in F344 rats. Marked upregulation of P16INK4A was associated with moderate overexpression of HSP90, CDC37, E2F4, and CRM1 in human hepatocellular carcinomas with a better prognosis. In contrast, strong induction of HSP90, CDC37, and E2F4 was paralleled by P16INK4A downregulation and high levels of HSP90‐CDK4 and CDC37‐CDK4 complexes in hepatocellular carcinomas with poorer prognosis. CDC37 downregulation by small interfering RNA inhibited in vitro growth of HepG2 cells. In conclusion, our findings underline the role of Hsp90/Cdc37 and E2f4/Crm1 systems in the acquisition of a susceptible or resistant carcinogenic phenotype. The results also suggest that protection by CDC37 and CRM1 against growth restraint by P16INK4A influences the prognosis of human hepatocellular carcinoma.(HEPATOLOGY 2005;42:1310–1319.)

Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis

gamma-Glutamyltranspeptidase (GGT)-positive and glutathione S-transferase (placental-GST-P) positive foci were induced in male Wistar rats by initiation with diethylnitrosamine (DENA), followed by selection and phenobarbital (PB). GGT- and GST-P-positive foci occupied 20-46% and 27-68% of liver parenchyma, respectively, 5-9 weeks after initiation. A high DNA synthesis was found in GGT-positive foci. Decrease in S-adenosyl-L-methionine (SAM) level and SAM/S-adenosylhomocysteine (SAH) ratio, and overall DNA hypomethylation occurred in the liver during the development of enzyme altered foci (EAF). These parameters underwent very small and transient changes in the liver of uninitiated rats at the 5th week, when EAF occupied 0.7-1.4% of the liver. At the 9th week, high RNA transcripts of c-myc, c-Ha-ras, and c-Ki-ras were found in the liver of initiated rats, but not in that of uninitiated rats. Immunohistochemical evaluation of c-myc gene product showed overexpression in GST-P-positive cells. SAM treatment of initiated rats caused inhibition of EAF growth, recovery of SAM/SAH ratio and DNA methylation, and decrease in protooncogene expression proportional to the dose and length of treatment. Liver SAM/SAH ratio was positively correlated with DNA methylation, and negatively correlated with transcript levels of the three protooncogenes. Thus, decrease in SAM/SAH ratio and DNA hypomethylation are early features of hepatocarcinogenesis promotion in rats fed a diet containing adequate lipotrope amounts, paralleled by overexpression of growth-related genes and rapid growth. Re-establishment of a physiologic SAM level makes it possible to inhibit protooncogene expression and EAF growth and to prevent late liver lesion development.

Correlation of c‐myc overexpression and amplification with progression of preneoplastic liver lesions to malignancy in the poorly susceptible wistar rat strain

Persistent liver nodules (Pns) and hepatocellular carcinomas (HCCs) induced in F344 rats by the resistant hepatocyte (RH) model exhibit c‐myc overexpression and amplification. The role of these changes in progression of PN was investigated in nodules with different propensities to evolve to HCC in resistant Wistar rats and, for comparison, in susceptible F344 rats. Initiation of rats with diethylnitrosamine was followed by selection with 2‐acetylaminofluorene (AAF) plus partial hepatectomy (RH groups). Two additional Wistar rat groups received a second AAF treatment without (RH+AAF) and with a necrogenic dose of Ccl4 (RH+AAF/CCl4) 15 d after selection. The number to liver ratio and volume of glutathione‐S‐transferase placental form–positive lesions were lower in the Wistar than the F344 RH groups 9 and 32 wk after initiation and increased after a second AAF cycle treatment with and without Ccl4. DNA synthesis in glutathione‐S‐transferase placental form–positive lesions was low in Wistar RH group at 9 wk and was stimulated by additional AAF treatments. HCCs developed at 57–60 wk in F344 RH, Wistar RH+AAF, and RH+AAF/CCl4 rats. Tumor incidence and multiplicity were lower in RH+AAF rats than in RH+AAF/CCl4 and F344 rats. At 32 wk, PN exhibited c‐myc overexpression that increased from RH to RH+AAF rats and to RH+AAF/CCl4 Wistar rats. This was associated with c‐myc amplification in Wistar RH+AAF/CCl4 rats. These results showed correlation of c‐myc overexpression and amplification with nodule propensity to progress to HCC in poorly susceptible Wistar rats and suggested a possible genetic mechanism for susceptibility to hepatocarcinogenesis. The experimental system used in this work may be a valuable tool for studies on molecular mechanisms underlying liver growth and tumorigenesis supported by c‐myc overexpression. Mol. Carcinog. 25:21–29, 1999.

c-myc amplification in pre-malignant and malignant lesions induced in rat liver by the resistant hepatocyte model

We have investigated by restriction fragment analysis genomic abnormalities involving the c‐myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the “resistant hepatocyte” protocol in diethylnitrosamine‐initiated male F344 rats. Southern‐blot analysis of EcoRI‐restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c‐myc, in all tissues. c‐myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c‐myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII‐restricted DNA, and in HCCs by differential PCR. c‐myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13‐kb DNA extraband, hybridizing with c‐myc, was found in the HindIII‐restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1‐kb extra band was present in EcoRI‐digested DNA from one LA. EcoRI‐restricted DNA from some adenomas exhibited a decrease in intensity of the 18‐kb fragment, and an increase in intensity of the 3.2‐kb fragment. No alteration in banding pattern occurred in the β‐actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c‐myc gene, in pre‐malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c‐myc rearrangement in the progression of adenomas to malignancy.

Activation of v‐Myb avian myeloblastosis viral oncogene homolog‐like2 (MYBL2)‐LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53

Up‐regulation of the v‐Myb avian myeloblastosis viral oncogene homolog‐like2 B‐Myb (MYBL2) gene occurs in human hepatocellular carcinoma (HCC) and is associated with faster progression of rodent hepatocarcinogenesis. We evaluated, in distinct human HCC prognostic subtypes (as defined by patient survival length), activation of MYBL2 and MYBL2‐related genes, and relationships of p53 status with MYBL2 activity. Highest total and phosphorylated protein levels of MYBL2, E2F1‐DP1, inactivated retinoblastoma protein (pRB), and cyclin B1 occurred in HCC with poorer outcome (HCCP), compared to HCC with better outcome (HCCB). In HCCP, highest LIN9‐MYBL2 complex (LINC) and lowest inactive LIN9‐p130 complex levels occurred. MYBL2 positively correlated with HCC genomic instability, proliferation, and microvessel density, and negatively with apoptosis. Higher MYBL2/LINC activation in HCC with mutated p53 was in contrast with LINC inactivation in HCC harboring wildtype p53. Small interfering RNA (siRNA)‐mediated MYBL2/LINC silencing reduced proliferation, induced apoptosis, and DNA damage at similar levels in HCC cell lines, irrespective of p53 status. However, association of MYBL2/LINC silencing with doxorubicin‐induced DNA damage caused stronger growth restraint in p53−/− Huh7 and Hep3B cells than in p53+/+ Huh6 and HepG2 cells. Doxorubicin triggered LIN9 dissociation from MYBL2 in p53+/+ cell lines and increased MYBL2‐LIN9 complexes in p53−/− cells. Doxorubicin‐induced MYBL2 dissociation from LIN9 led to p21WAF1 up‐regulation in p53+/+ but not in p53−/− cell lines. Suppression of p53 or p21WAF1 genes abolished DNA damage response, enhanced apoptosis, and inhibited growth in doxorubicin‐treated cells harboring p53+/+. Conclusion: We show that MYBL2 activation is crucial for human HCC progression. In particular, our data indicate that MYBL2‐LIN9 complex integrity contributes to survival of DNA damaged p53−/− cells. Thus, MYBL2 inhibition could represent a valuable adjuvant for treatments against human HCC with mutated p53. (HEPATOLOGY 2011;)

Ras‐driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1‐S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf‐1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1‐driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1‐dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro‐apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.

The Degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the disease

Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21WAF1, p27KIP1, p57KIP2 and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21WAF1, p27KIP1, p57KIP, p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1‐Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21WAF1, P27KIP1, P57KIP2 and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators.

Mybl2 expression is under genetic control and contributes to determine a hepatocellular carcinoma susceptible phenotype.

BACKGROUND & AIMS MYBL2 is implicated in human malignancies and over expressed in hepatocellular carcinoma (HCC). We investigated Mybl2 role in the acquisition of susceptibility to HCC and tumor progression. METHODS MYBL2 mRNA and protein levels were evaluated by quantitative RT-PCR and immunoblotting, respectively. MYBL2 expression in HCC cell lines was controlled through MYBL2 cDNA or anti-MYBL2 siRNA transfection. Gene expression profile of cells transfected with MYBL2 was analyzed by microarray. RESULTS Low induction of Mybl2 and its target Clusterin mRNAs, in low-grade dysplastic nodules (DN), progressively increased in fast growing high-grade DN and HCC of F344 rats, susceptible to hepatocarcinogenesis, whereas no/lower increases occurred in slow growing lesions of resistant BN rats. Highest Mybl2 protein activation, prevalently nuclear, occurred in F344 than BN lesions. Highest Mybl2, Clusterin, Cdc2, and Cyclin B1 expression occurred in fast progressing DN and HCC of E2f1 transgenics, compared to c-Myc transgenics, and anti-Mybl2 siRNA had highest anti-proliferative and apoptogenic effects in cell lines from HCC of E2f1 transgenics. MYBL2 transfected HepG2 and Huh7 cells exhibited increased cell proliferation and G1-S and G2-M cell cycle phases. The opposite occurred when MYBL2 was silenced by specific siRNA. MYBL2 transfection in Huh7 cells led to upregulation of genes involved in signal transduction, cell proliferation, cell motility, and downregulation of oncosuppressor and apoptogenic genes. CONCLUSIONS mybl2 expression and activation are under genetic control. Mybl2 upregulation induces fast growth and progression of premalignant and malignant liver, through cell cycle deregulation and activation of genes and pathways related to tumor progression.

Phenotypic reversion of rat neoplastic liver nodules is under genetic control

Low DNA synthesis and high redifferentiation (remodeling) characterize neoplastic nodules induced by chemical carcinogens in hybrid BFF1 rats, generated by crossing the susceptible F344 and resistant BN strains. We performed whole‐genome scanning of BFF2 rats to identify loci controlling remodeling of nodules induced, 32 weeks after initiation with diethylnitrosamine, by the RH protocol. Remodeling nodules were identified as areas lacking uniformity of GST‐P immunostaining and with irregular margins. Two loci in suggestive linkage with the percentage of remodeling nodules were identified on chromosomes 7 and 1 (LOD scores 3.85 and 2.9 at D7Rat25 and D1Mgh14). Significant dosage‐negative effect of the B allele on remodeling and additive interaction between these loci were found. Significant epistatic interactions, showing a recessive, remodeling‐enhancing effect of B alleles, occurred between D1Mit3 and D11Rat11 (corrected p = 0.0013) and between D6Rat14 and D8Rat46 (corrected p = 0.028). These data show that remodeling of neoplastic nodules during rat hepatocarcinogenesis is under genetic control. Loci affecting remodeling map to chromosomal regions syntenic to chromosomal segments of human HCC showing structural abnormalities.