PCNA-associated factor, the protein encoded by the KIAA0101/PCLAF gene, is a cell-cycle regulated oncoprotein that functions through the interaction with PCNA [3, 6-14]. KIAA0101 is overexpressed in various cancers [3-6, 15, 16]. However, few studies have directly investigated KIAA0101 expression in HCC, with contradictory results [17-19]. Furthermore, it remains unknown whether KIAA0101 overexpression is coupled to gene amplification in HCC. The present study aimed to investigate potential correlations between KIAA0101 mRNA overexpression, detected by quantitative real-time PCR, and KIAA0101 gene copy number alterations detected by ddPCR in our collected liver tissues. The study also aimed to investigate potential correlations between KIAA0101 protein expression, detected by IHC, and other clinicopathological parameters.
In hepatocellular carcinoma (HCC), only a few studies have directly investigated the protein expression of KIAA0101, and interpretation of these studies are hampered by the use of different antibodies. First, as the antibody for KIAA0101 was not commercially available, Guo et al. prepared their own polyclonal rabbit antibody against a full-length KIAA0101-His tag and they reported down-regulated KIAA0101 protein expression in HCC as compared with non-cancerous liver tissues . Yuan et al. used semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) for measurements of KIAA0101 mRNA, and a mouse monoclonal antibody against a full-length KIAA0101-GST tag (clone 3C11-1F11, Abnova) for immunohistochemical (IHC) analysis. They reported overexpression of KIAA0101 at both mRNA and protein levels in approximately 60% of HCCs, and found the association of KIAA0101 overexpression with higher tumor grade, higher tumor stage, and early tumor recurrence concordant with poor prognosis . Yuan et al. also found a correlation of KIAA0101 overexpression with p53 mutations; p53 mutation, which occurred in approximately 50% of HCCs in their study . Lastly, Liu et al. reported that the KIAA0101 gene can be alternatively spliced to produce 2 transcriptional variants, which are translated to 2 protein variants: (1) KIAA0101 tv1 protein, the canonical sequence of 111 amino acid residues containing the PIP-box, and (2) KIAA0101 tv2 protein, the alternative sequence of 65 amino acid residues not containing the PIP-box [19, 28]. Liu et al. showed overexpression of KIAA0101 tv1 at both mRNA and protein levels in ~70% of HCC tissues (~ 40% in stage I-II, and ~80% in stage III-IV HCCs) as assessed by semiquantitative RT-PCR, virtual northern blot, western blot, and IHC analysis . For IHC analysis, Liu et al. used a goat polyclonal antibody against a peptide mapping at the C-terminus of KIAA0101 (sc‐65163 antibody, Santa Cruz Biotechnology). In addition, Liu et al. found that doxorubicin (Adriamycin, ADR) treatment down-regulated the expression of KIAA0101 tv1 whereas the treatment increased the acetylation of p53 . Recently, Liu et al. showed that KIAA0101 tv2 was highly expressed in adjacent non-tumorous liver tissues (NTs) as compared to HCC tissues, and KIAA0101 tv2 could induce cell cycle arrest and apoptosis . Using transfection, Liu et al. showed that KIAA0101 tv2 could inhibit the function of KIAA0101 tv1 by partially down-regulating KIAA0101 tv1, by acting similar as a short hairpin RNA (shRNA) .
In our present study, as expected, KIAA0101 mRNA and protein expression levels were significantly higher in HCC than in the matched non-cancerous liver tissues, consistently with previous studies by Yuan et al., and Liu et al. [18, 19], but in contrast to Guo et al.. We found no amplification of KIAA0101 gene in HCC, and no significant difference of KIAA0101 gene copy numbers in HCC and matched non-cancerous tissues, as well as no correlation between KIAA0101 gene copy numbers and KIAA0101 RNA expression. Our results indicate that KIAA0101 overexpression in HCC is not secondary to KIAA0101 gene amplification. Previous studies have suggested that KIAA0101 expression is regulated by the p53-p21 pathway , and the Rb/E2F pathway , as well as the NF-kB (p50) pathway , and the cAMP-dependent transcription factor ATF-3 . Recently, Zhang, et al. demonstrated that KIAA0101 is a direct transcriptional target of Forkhead box protein M1 (FoxM1), which may account for KIAA0101 overexpression in HCC .
This study also found significant correlations of KIAA0101 protein overexpression with p53 tumor suppressor protein overexpression, and with Ki-67 proliferation marker protein overexpression. The TP53 tumor suppressor gene, the most frequently mutated gene in cancer, encodes the p53 tumor suppressor protein [32, 33] The wild-type p53 protein is a sequence-specific DNA-binding transcription factor, which upregulates many important genes regulating various cellular processes, including cell cycle arrest, DNA repair, and cell death (apoptosis). The p53 protein has been known as the most important tumor suppressor and “the guardian of the genome”.
The p53 tumor suppressor protein is active as a tetramer, with four identical chains of 393 residues. Domain structure of the full-length p53 protein consists of an N-terminal transactivation domain, followed by a proline-rich region, the central DNA-binding domain responsible for sequence-specific DNA binding, the tetramerization domain regulating the oligomerization state of p53, and the extreme C-terminus . According to Cancer Genome Atlas Research Network, the TP53 gene is inactivated by mutation in about 30% of HCC patients; the mutations are truncating mutations (frame-shift, nonsense, and splice-site mutations) in about one-third, and non-truncating mutations (missense) in about two-third. Most missense mutations of TP53 occur in the central DNA-binding domain and result in the defective p53 function .
The significant risk factors for HCC include chronic infections with the hepatitis B (HBV) or C (HCV) virus and exposure to dietary aflatoxin B1 (AFB1) or alcohol consumption. TP53 gene mutations occur in more than 50% of AFB1-induced HCC, in up to 45% of HBV-related HCC, and in 13% of HCV-related HCC . AFB1 frequently generates the hotspot TP53-R249S mutation and cooperates with HBV in causing TP53 mutations in HCC. Chronic HBV and HCV infection also induces reactive nitrogen/oxygen species that can damage DNA and mutate cancer-related genes such as TP53.  HBx, the X gene of HBV, is the most common open reading frame integrated into the hepatic genome, and the integrated HBx is frequently mutated in HCC . Mutant HBx proteins still retain their capability to bind to p53 protein and attenuate DNA repair and p53-mediated apoptosis. Thus, both viruses and chemicals are the causative agents of TP53 mutations during the molecular pathogenesis of HCC . The p53 tumor suppressor protein is a short-lived transcription factor promoting cell-cycle arrest or apoptosis when cells encounter stress stimuli such as oncogene activation or DNA damage. The p53 protein is usually kept at low levels in unstressed cells by continuous ubiquitylation, primarily via the p53 protein interaction with the E3 ubiquitin-protein ligase Mdm2, then degradation by the 26S Proteasome . However, in stressed cells, p53 ubiquitylation is suppressed, and p53 protein is stabilized and accumulates in the nucleus, where it forms a tetramer. Only tetrameric p53 protein appears to be fully active as a transcriptional activator or repressor of distinct target genes containing p53 sequence-specific DNA binding sites. Furthermore, in stressed cells, p53 protein is subject to various post-translational modifications, including phosphorylation and acetylation, that influence p53 target genes' expression. Phosphorylation and acetylation of p53 protein generally result in stabilization and accumulation of p53 protein in the nucleus, promoting transcriptional activation/repression of target genes. Missense-mutant p53 proteins generally show intense phosphorylation and acetylation at sites known to stabilize wild-type p53 protein. Thus, these post-translational modifications possibly facilitate the accumulation of dysfunctional mutant p53 in the nucleus .
An analysis of the TP53 gene and pathway alterations in 32 cancer subtypes, including HCC, from The Cancer Genome Atlas (TCGA), has been recently published . More than 90% of TCGA Cancers with TP53 mutations exhibit second allele loss by mutation, chromosomal deletion, or copy-neutral loss of heterozygosity (LOH). TP53 mutations are associated with enhanced chromosomal instability, including increased amplification of oncogenes (CCND1, CCNE1, ERBB2, and MYC) and deep deletion of tumor suppressor genes (RB1, PTEN, and WWOX) . As compared with their non-mutated tumors, tumors with TP53 mutations display enhanced expression of cell cycle progression genes and proteins (cyclin B1, cyclin E1, FOXM1, and CDK1). Mutant TP53 cancers contained enhanced p53 protein expression, presumably the mutant p53 proteins derived from non-truncating (missense) mutations. Besides, proteins associated with the DNA damage response were also upregulated in mutant TP53 cancers . . In HCC, TP53 gene mutation is significantly correlated to p53 protein overexpression . Consequently, p53 protein overexpression determined by IHC can predict P53 gene mutations in HCC patients . Therefore, the correlation between KIAA001 protein overexpression and p53 protein overexpression, found in this study, is consistent with the correlation between KIAA0101 overexpression and P53 gene mutation in HCC as reported by Yuan et al. .
The Ki-67 proliferation marker protein is the nuclear antigen recognized Ki-67 antibody; this antigen is present in proliferating cells but absent in resting cells . The Ki-67 protein is a protein phosphatase 1 (PP1)-binding protein that organizes the mitotic chromosome periphery . The Ki-67 protein acts as a biological surfactant and is required to maintain individual mitotic chromosomes dispersed in the cytoplasm following nuclear envelope disassembly . The Ki-67 protein serves as the proliferation marker and correlated with tumor growth rate and poor prognosis in HCC [46, 47]. Furthermore, KIAA0101 also serves as a proliferation marker and a poor prognosis marker in HCC . Correlation between KIAA0101 overexpression and the Ki-67 protein has been reported in breast cancer by Kais Z et al.. However, to the best of our knowledge, the present study is the first report that shows the correlation of KIAA0101 protein overexpression and the Ki-67 protein in HCC.
Our study shows the significant correlations of KIAA0101 protein overexpression with p53 protein and Ki-67 protein overexpression. Thus, it indicates that p53 protein accumulation and overexpression in HCC, most likely caused by non-truncating (missense) TP53 mutations , links with the enhanced cell cycle progression that requires the overexpression of KIAA0101 protein to cooperate with the PCNA for DNA synthesis and repair [10, 11]. The enhanced cell cycle progression also involves the overexpression of Ki-67 proliferation marker protein for maintaining individual mitotic chromosomes dispersed in the cytoplasm during the enhanced cell division .
Currently, the development of targeted cancer therapies are being actively investigated against KIAA0101/p15PAF by agents with inhibitory functions similar to KIAA0101 tv2 , against p53 by anti-mutant p53 agents or MDM2/4 antagonists [49, 50], against Ki-67 by exploitation of MKI67 promoter to drive the expression of siRNAs or therapeutic genes , and against PCNA by inhibitors that are targeted to modified PCNA involved in DNA repair . These attempts might translate into the novel therapeutics for HCC in the near future. Our study suggests that the candidates for these novel targeted therapies would be patients with KIAA0101 overexpression in their HCC tissues, which also correlated with p53 overexpression/mutation and Ki-67 overexpression.