SKP2 and CKS1 promote degradation of cell cycle regulators and are associated with hepatocellular carcinoma prognosis.
OBJECTIVE
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.
Calvisi
Diego F
DF
Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy.
Ladu
Sara
S
Pinna
Federico
F
Frau
Maddalena
M
Tomasi
Maria L
ML
Sini
Marcella
M
Simile
Maria M
MM
Bonelli
Piero
P
Muroni
Maria R
MR
Seddaiu
Maria A
MA
Lim
Dae-Sik
DS
Feo
Francesco
F
Pascale
Rosa M
RM
D042846:CDC2-CDC28 Kinases; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology*; D002352:Carrier Proteins / Q000502:physiology*; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology*; D044786:S-Phase Kinase-Associated Proteins / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D054875:Ubiquitination
C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; C093090:CKS1B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D047908:Intracellular Signaling Peptides and Proteins; C411762:RASSF1 protein, human; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; D018844:Cyclin-Dependent Kinases