Maria Rosaria Muroni

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.)

Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC

Background and aim: Previous studies indicate unrestrained cell cycle progression in liver lesions from hepatocarcinogenesis-susceptible Fisher 344 (F344) rats and a block of G1–S transition in corresponding lesions from resistant Brown Norway (BN) rats. Here, the role of the Forkhead box M1B (FOXM1) gene during hepatocarcinogenesis in both rat models and human hepatocellular carcinoma (HCC) was assessed. Methods and results: Levels of FOXM1 and its targets were determined by immunoprecipitation and real-time PCR analyses in rat and human samples. FOXM1 function was investigated by either FOXM1 silencing or overexpression in human HCC cell lines. Activation of FOXM1 and its targets (Aurora Kinose A, Cdc2, cyclin B1, Nek2) occurred earlier and was most pronounced in liver lesions from F344 than BN rats, leading to the highest number of Cdc2–cyclin B1 complexes (implying the highest G2–M transition) in F344 rats. In human HCC, the level of FOXM1 progressively increased from surrounding non-tumorous livers to HCC, reaching the highest levels in tumours with poorer prognosis (as defined by patients’ length of survival). Furthermore, expression levels of FOXM1 directly correlated with the proliferation index, genomic instability rate and microvessel density, and inversely with apoptosis. FOXM1 upregulation was due to extracellular signal-regulated kinase (ERK) and glioblastoma-associated oncogene 1 (GLI1) combined activity, and its overexpression resulted in increased proliferation and angiogenesis and reduced apoptosis in human HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of human HCC cell lines. Conclusions: FOXM1 upregulation is associated with the acquisition of a susceptible phenotype in rats and influences human HCC development and prognosis.

Altered methionine metabolism and global DNA methylation in liver cancer: relationship with genomic instability and prognosis

Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c‐Myc and c‐Myc/Tgf‐α transgenic mice and human HCCs. S‐adenosyl‐L‐methionine/S‐adenosylhomocysteine ratio and liver‐specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c‐Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patients survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c‐Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c‐Myc/Tgf‐α lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c‐Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c‐Myc/Tgf‐α HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c‐Myc/Tgf‐α tumors, may develop in the absence of alterations in DNA methylation.

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.

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.

Polygenic control of hepatocarcinogenesis in Copenhagen × F344 rats

Cop and CFF1 rats exhibit resistance to hepatocarcinogenesis, associated with high rates of remodeling of neoplastic lesions. We have mapped hepatocarcinogenesis susceptibility, resistance and remodeling loci affecting the number, volume and volume fraction of neoplastic nodules induced by the “resistant hepatocyte” model in male CFF2 rats. Three loci in significant linkage with the number or volume of nonremodeling lesions were identified on chromosomes 1, 4 and 18. Suggestive linkage with number or volume fraction of total, nonremodeling or remodeling lesions was found for 7 loci on chromosomes 1, 2, 13, 14 and 15. All of these loci showed significant allele‐specific effects on the phenotypic traits. We also detected by analysis of variance 19 2‐way interactions inducing phenotypic effects not predictable on the basis of the sum of separate effects. These novel epistatic loci were in significant linkage with the number and/or volume of total, nonremodeling or remodeling nodules. These data indicate that susceptibility to hepatocarcinogenesis in Cop rats is controlled by a complex array of genes with several gene–gene interactions and that different genetic mechanisms control remodeling and nonremodeling liver nodules. Frequent deregulation in human liver cancer of genes positioned in chromosomal segments syntenic to rat susceptibility/resistance loci suggests some similarities between the genetic mechanisms involved in hepatocarcinogenesis in rats and humans.

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.

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Background Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.