Overexpression of KIAA0101 Predicts High Stage, Early Tumor Recurrence, and Poor Prognosis of Hepatocellular Carcinoma

Purpose: KIAA0101 is a proliferating cell nuclear antigen–associated factor and involved in cell proliferation. This study is to elucidate its role in the progression, early tumor recurrence (ETR), and prognosis of hepatocellular carcinoma (HCC).

Experimental Design: KIAA0101 mRNA was measured by reverse transcription-PCR in 216 resected, unifocal, primary HCCs and its protein in 164 cases by immunohistochemistry.

Results: KIAA0101 mRNA was overexpressed in 131 (61%) HCCs, and protein was detected in 105 (64%). KIAA0101 mRNA overexpression correlated with higher tumor grade (P = 0.0001), higher tumor stage with vascular invasion and various extents of intrahepatic spread (P = 1 × 10⁻⁸), ETR (P = 1.8 × 10⁻⁶), and lower 5-year survival (P = 0.0026). Multivariate analysis confirmed that KIAA0101 overexpression was an independent risk factor associated with high-grade tumor (P = 0.0001), high-stage tumor (P < 0.0001), and ETR (P = 0.0052) and thus contributed to poor prognosis. KIAA0101 protein–positive tumor cells accumulated at the borders of tumor macrotrabeculae and were more abundant in tumor thrombi than in the main tumors. Hence, KIAA0101 may contribute to growth advantage and resistance to hypoxic insult. In this series, p53 mutation was detected in 93 of 184 (51%) HCCs. In both p53-mutated and non–p53-mutated HCCs, KIAA0101 overexpression correlated with higher vascular invasion (stages IIIA to IV; all Ps < 0.0001) and, accordingly, led to lower 5-year survival rates (P = 0.011 and 0.029, respectively).

Conclusion: KIAA0101 correlates with enhanced metastatic potential and is a significant prognostic factor of HCC.

Hepatocellular carcinoma (HCC), one of the most common fatal malignancies in Taiwan and many other countries in Asia and Africa (1), is the leading cause of cancer mortality in Taiwan (2), and its incidence is increasing in Western countries (1, 3). Surgical resection provides an opportunity for cure, but the outcome of the surgical HCC patients remains grave mainly due to the frequent tumor recurrence (4–6). Although recent studies have unraveled many aberrantly expressed genes in HCC (7–9), the molecular factors that can help to identify early tumor recurrence (ETR) and serve as potential therapeutic target remain limited.

KIAA0101 is a 15-kDa protein containing a conserved proliferating cell nuclear antigen (PCNA)-binding motif (10–12) and known as p15^PAF (PCNA-associated factor; ref. 13), OEATC-1 (overexpressed in anaplastic thyroid carcinoma-1; ref. 14), and L5 (15). The PCNA-binding motif is also present in many PCNA-binding proteins, such as p21^WAF, p57^Kip2, and p33ING1b (16–18). PCNA is a supplementary factor for DNA polymerase and required for DNA replication or repair (19, 20). High PCNA labeling index correlated with tumor size and prognosis of HCC (21). The p21^WAF, p57^Kip2, and p33ING1b can form complexes with PCNA and compete with one another (13, 18) as well as other PCNA-binding proteins to prevent PCNA from binding to DNA polymerase, which in turn leads to inhibition of DNA synthesis and cell cycle progression (17, 22, 23) and eventually G₁ cell cycle arrest (24). Hence, KIAA0101 is involved in the regulation of DNA repair (25), cell cycle progression (25), and cell proliferation (13). After UV exposure, the increased interaction of the elevated nuclear p21^WAF and p33 (ING1b) with PCNA leads to DNA repair (26) and programmed cell death (18). In contrast, KIAA0101 can interact with p33ING1b, leading to a decrease in UV-induced cell death. Thus, KIAA0101 overexpression can protect cells from UV-induced cell death (25).

KIAA0101, which was expressed in a highly restricted pattern in a spatiotemporal manner in mouse embryo (25), was highly expressed in thymus and colon but low or absent in most of the other tissues, including adult liver (27). KIAA0101 is overexpressed in tumors of the breast, uterine cervix, brain, kidney, esophagus (13), lungs (15), and colon (28) and also in several anaplastic thyroid carcinoma cell lines (14). Despite these observations, the KIAA0101 expression and its clinicopathologic significance in human HCC remain unclear. This study was aimed to elucidate the clinicopathologic significance of KIAA0101 expression in HCC, particularly intrahepatic metastasis, ETR, and prognosis, and the interaction with p53 mutation.

Tissue samples. From 1984 to 1997, 216 surgically resected, unifocal, primary HCCs, which received detailed pathologic assessment and regular follow-up at the National Taiwan University Hospital, were recruited into this study as described (29–31). This study was conducted according to the regulations of the Ethical Committee, and the specimens were anonymous and analyzed in a blinded manner. These patients were 170 males and 46 females, with a mean age of 56.4 years (range, 14-88 years). Serum hepatitis B surface antigen (HBsAg) was detected in 149 cases and anti-hepatitis C virus antibody in 70, including 20 positive for both. Liver cirrhosis was found in 88 cases (41%). All patients had adequate liver function reserve at the time of surgery, and none had received transhepatic arterial embolization or chemotherapy before surgery.

Histologic study and tumor staging. Tumor grade was divided into three groups: well differentiated (grade 1, 49 cases), moderately differentiated (grade 2, 88 cases), and poorly differentiated (grade 3, 59 cases, and grade 4, 20 cases). The tumor stage of unifocal HCC closely correlated with prognosis as described (31–33). Stage IIIA to IV HCC had vascular invasion and various extents of intrahepatic metastasis, whereas stage I and II HCC did not. The tumor staging was classified into I (well-encapsulated minute HCC ≤ 2 cm, 5 cases), II (encapsulated or unencapsulated HCC without vascular invasion, 87 cases), IIIA (with invasion of vessels in tumor capsule, 39 cases), IIIB (with invasion of portal vein branch, 30 cases), and IV (with distant portal vein branch, 55 cases).

Measurements of KIAA0101 mRNA by reverse transcription-PCR. According to the data files in National Center for Biotechnology Information,⁷

there are two variant transcripts of KIAA0101. The variant 1 consists of 1,508 bp and four exons and encodes a protein of 111 amino acids, which has the PCNA-binding motif. The variant 2 consists of 1,345 bp and three exons, and its protein lacks PCNA-binding motif. We designed three sets of primers: primers spanning the PCNA-binding motif to detect the differential expression of both variants, primers specific for the variant 1 that contains PCNA-binding motif, and primers at the COOH-terminal for both variants. Reverse transcription-PCR (RT-PCR) was used to determine the mRNA levels of KIAA0101 using ribosomal protein S26 (RPS26) mRNA as the internal control (34, 35). PCR was stopped at the exponential phase, 28 cycles for KIAA0101 and 22 for RPS26. PCR was done in an automatic DNA thermal cycler 480 (Perkin-Elmer Co.), with initial heating at 94°C for 2 min followed by 28 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 1 min, and final 72°C for 10 min for KIAA0101. RPS26 primers were added at cycle 7. Primers spanning the PCNA-binding motif to detect the differential expression of both variants were KIAA0101-B-F (5′-CGGGAAAGAGACCAATATAAAC-3′) and KIAA0101-B-R (5′-AAAGCTAATTGGAACAAACACAT-3′); primers specific for the variant 1 that contains PCNA-binding motif were KIAA0101-P-F (5′-AAATAAATATGCAGGAGGGAACC-3′) and KIAA0101-P-R (5′-AGCAAGAACTAGACACTTAATTTGG-3′). The primers at the COOH-terminal common for both variants and used for RT-PCR measurements of KIAA0101 mRNA levels were KIAA0101-C-F (5′-AAAAGCATGTCCTTTGCAACC-3′) and KIAA0101-C-R (5′-TGGCACCATTCCAATAATCA-3′). The primers for RPS26 have been described (4). After PCR, 5 μL of reaction products were electrophoresed on a 2% agarose gel. The KIAA0101 mRNA expression level was determined by the ratio of signal intensity of KIAA0101 to that of RPS26 measured by 1D Image Analysis Software (Kodak Digital Science) and scored as high (ratio ≥ 1.0; 102 tumors), intermediate (0.5 < ratio < 1.0; 29 tumors), low (0.2 ≤ ratio < 0.5; 8 tumors), and trace or negative (ratio < 0.2; 77 tumors). With the exception of four cases, none of the remaining 192 nontumorous livers examined had a KIAA0101 to RPS26 ratio of >0.3. Hence, a ratio of >0.5 was defined as KIAA0101 overexpression.

Immunohistochemical stain of KIAA0101 protein. The KIAA0101 protein was examined on formalin-fixed, paraffin-embedded sections in 164 tumors by the Super Sensitive Polymer-HRP IHC Detection System after antigen retrieval using polymer-enhancing reagents (BioGenex Laboratories, Inc.). The commercially obtained monoclonal antibody against KIAA0101 (H0009768-M01, 1:500 dilution; Abnova) was used. The proportion of tumor cells positive for KIAA0101 immunostain varied considerably, ranging from diffuse positive (>50%), heterogeneous, nodular, and trace to negative. In the nontumorous liver, KIAA0101 protein was detected only in very few isolated liver cells. Hence, HCC with 1% of tumor cells showing nuclear immunostaining for KIAA0101 was regarded as positive and scored as 1+ (1-5%; 60 cases), 2+ (6-10%; 32 cases), and 3+ (>10%; 14 cases), respectively.

Cell lines and cell culture. The cell lines used include (a) cancer cells [HCC cell lines (HuH-7, HA22T, Hep3B, and HCC36), hepatoblastoma cell HepG2, HeLa, and MCF-7 cells] and (b) nontumor transformed cells (HEK293 and HEK 293T). The cells were grown in DMEM.

Western blot. Protein samples (60 μg each) were separated in 4% to 20% SDS-PAGE gel and then electrotransferred onto nitrocellulose membrane (Amersham Pharmacia Biotech). The membranes were allowed to react with primary and secondary antibodies at optimum dilution, and the immunoreactive signals were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Analysis of p53 mutation. Mutations of the p53 tumor suppressor gene were analyzed in 184 tumors by direct sequencing spanning exon 2 to exon 11 as described previously (36, 37).

Follow-up observation, ETR, and treatment. The intrahepatic tumor recurrence or distant metastasis detected by imaging diagnosis within 12 months after tumor resection was designated as ETR as described (4, 32). Among the 216 study patients, 215 cases (99.5%) had been followed for more than 5 years or until death, 72 patients (33%) survived more than 5 years, and 194 (90%) were eligible for the evaluation of ETR. Among the 95 cases with ETR, 86 (44%) received tumor resection and/or transhepatic arterial embolization.

Statistical analysis. The data analyses were carried out using the Epi Info (version 3.3.2; Centers for Disease Control and Prevention) and Statistical Analysis System (version 9.1; SAS Institute, Inc.) software. The χ², Fisher's exact test, and log-rank test were used for univariate analysis. Multivariate analyses of grade and stage were conducted by fitting multiple logistic regression models (38), and then times to ETR and death were analyzed by fitting multiple Cox's proportional hazards models (39). Basic model-fitting techniques for (a) variable selection, (b) goodness-of-fit assessment, and (c) regression diagnostics (including residual analysis, influence analysis, and check of multicollinearity) were used in our regression analyses to assure the quality of analysis results (38, 39). Two-tailed P < 0.05 was considered statistically significant.

Expression of KIAA0101 mRNA and protein in liver and HCC. To determine the expression patterns of the variant forms of KIAA0101 in tissues and cell lines, we used RT-PCR for semiquantitative measurements of the mRNA levels and one set each of specific primers for the differential expression of variants 1 and 2, variant 1, and for common region of both variants, respectively. We found that both variants were overexpressed in HCC but variant 1 predominated, with a variant 1 to variant 2 ratio ranging from 2.5 to 7.8. All three different reactions gave similar results (Fig. 1). We then used primers common for the common regions of both variants at the COOH termini to measure the mRNA levels of KIAA0101. The KIAA0101 mRNA levels were increased (KIAA0101 to RPS26 ratio > 0.5) in 131 of 216 (61%) HCCs and in only 4 of 196 (2%) nontumorous livers (Fig. 2A).

The KIAA0101 protein level was also increased in HCC and expressed abundantly in almost all the proliferated cell lines examined, including HCC and breast cancer cells (Fig. 2B). Immunohistochemical stain revealed the predominant nuclear expression of KIAA0101 protein in 105 of 164 (64%) HCCs examined (Fig. 3): being positive in 85 of 102 HCCs with KIAA0101 mRNA overexpression and in 20 of 62 HCCs without the overexpression, giving a concordance rate of 76%. In the nontumorous liver, KIAA0101 protein was detected only occasionally in isolated hepatocytes and rarely exceeding 0.1% of the liver cells (Fig. 3A and B). The KIAA0101 protein expression in HCC varied considerably and showed heterogeneous distribution (Fig. 3C-E), sometimes with “tumor in tumor” pattern (Fig. 3F). KIAA0101 protein–positive tumor cells tended to accumulate at the borders of the tumor trabeculae facing the capillaries and dilated vessels (Fig. 4A and B) and at the tumor peripheries. KIAA0101 protein was often absent in the trabecular centers where tumor cells frequently showed degenerative changes with pyknotic nuclei or ischemic necrosis. Notably, tumor cells of the tumor thrombi in portal vein branches exhibited more diffuse KIAA0101 protein expression than those of the main tumor (Fig. 4C and D).

KIAA0101 mRNA expression in relation to clinicopathologic features and prognosis. KIAA0101 mRNA overexpression occurred more commonly in the patients of younger age (≤56; P = 0.002) and was associated with serum HBsAg positivity (P = 0.0014) and high serum α-fetoprotein (AFP; >200 ng/mL; P = 0.017; Table 1). Histopathologically, KIAA0101 overexpression was closely correlated with higher-grade (grades 2-4) tumors (P = 0.0001). HCC with KIAA0101 overexpression had a >2-fold higher chance of having high-stage (stages IIA to IV) tumors [odds ratio (OR), 11.8; P = 1 × 10⁻⁸; Table 1] and a lower 5-year survival (P = 0.00026; Fig. 5A) than HCC without the overexpression. KIAA0101 expression did not correlate with gender, cirrhosis, and tumor size.

Verification of clinicopathologic correlations of KIAA0101 overexpression in learning and test sets of HCC patients. To test the reliability of KIAA0101 overexpression in relation to HCC tumor progression and prognosis, we divided the cases into two groups evenly, learning set (108 cases) and test set (108 cases), according to the chronological order of pathologic examination. The two sets of cases did not differ in any of the major clinical and pathologic variables, such as age, gender, chronic hepatitis B virus infection, liver cirrhosis, tumor size, grade and stage, frequencies of ETR and p53 mutation, and treatment modalities for ETR (data not shown). The correlations of KIAA0101 overexpression with age, HBsAg seropositivity, tumor grade and stage, and 5-year survival rate were confirmed in both sets of patients (Table 2; Fig. 5B and C).

KIAA0101 overexpression predicts ETR and poor prognosis. ETR is the most crucial clinical event associated with poor prognosis of surgical HCC before death (4, 6) and might be regarded as an important event earlier than mortality. As listed in Table 3, multivariate analysis showed that KIAA0101 overexpression (P = 0.0052), cirrhosis (P = 0.0090), stage III tumor (P = 0.0066), stage IV tumor (P < 0.0001), and larger tumor with p53 mutation (P = 0.0011) were significant independent risk factors in the Cox's proportional hazards model for the time to ETR. Furthermore, older age (P = 0.0114), cirrhosis (P = 0.0414), grade 4 tumor (P = 0.0001), stage III tumor (P = 0.0206), stage IV tumor (P < 0.0001), ETR (P < 0.0001), and larger tumor with p53 mutation (P = 0.0198) were significant independent risk factors associated with poor patient's survival. Most importantly, we found that overexpression of KIAA0101 was an independent risk factor associated with high-grade tumor (P = 0.0003), high-stage tumor (P < 0.0001), and ETR (P = 0.0052) and hence contributed to poor survival of the patients (Table 3). However, KIAA0101 overexpression did not exert prognostic effect independent of tumor stage.

KIAA0101 overexpression versus p53 mutation in HCC progression and poor prognosis. The mutation of tumor suppressor p53 is the most common genetic alteration in HCCs and associated with tumor progression and poor prognosis (36). In this study, p53 mutation occurred in 93 of 184 (51%) HCCs examined and was closely correlated with KIAA0101 overexpression (P = 0.0006; Table 1). Further, KIAA0101 overexpression was associated with more frequent vascular invasion (stage IIIA to IV HCC) in both non–p53-mutated and p53-mutated HCCs (P = 0.000018 and 0.0000027, respectively; Table 4). Accordingly, both non–p53-mutated and p53-mutated HCCs with concomitant KIAA0101 overexpression had lower 5-year survival compared with those without the overexpression (P = 0.029 and 0.011, respectively; Fig. 5D and E). Moreover, 86% of HCC with p53 mutation and concomitant KIAA0101 overexpression had vascular invasion (stage IIIA to IV HCC; Table 4).

The frequent KIAA0101 overexpression (61%) in HCC accorded with the observations in other types of human cancer (13–15, 28). The KIAA0101 overexpression in HCC was correlated with serum HBsAg positivity, high AFP (>200 ng/mL), and high-grade (grades 2 to 4) tumor. By immunohistochemistry, KIAA0101 protein tended to express at the tumor borders and the peripheries of macrotrabeculae but was absent in the trabecular centers where tumor cells often had degenerative changes with pyknotic nuclei or ischemic necrosis. These findings suggest that KIAA0101 protein–positive tumor cells have growth advantage and are more resistant to hypoxia insult, leading to poor differentiation and high AFP (29, 32). Consistent with this suggestion are the observations that KIAA0101 colocalizes with PCNA in the highly proliferating cell compartments in the hair follicle and colonic crypts (25). KIAA0101 can interact with PCNA competitively with p21^WAF (13) and overcome the p21^WAF-mediated inhibitory function in cell cycle progression (40). Furthermore, down-regulation of KIAA0101 by small interfering RNA treatment results in growth suppression of KTA2 cells (13). Taken together, these findings indicate that KIAA0101 expression facilitates cell proliferation in both physiologic conditions and human cancers (13–15, 25, 28), including HCC. This suggestion, however, was in disagreement with the observations of Guo et al. (41) who showed decreased KIAA0101 protein expression in HCC. The reasons for this discrepancy are unclear but not related to the differential expression of the KIAA0101 variants.

Intrahepatic tumor spread through the portal vein system is the most crucial histologic feature for high-stage HCC associated with poor prognosis (4), but the related molecular factors remain limited. We showed that high-stage (stages IIIA, IIIB, and IV) HCC, which had vascular invasion and various extents of intrahepatic spread, had more frequent KIAA0101 overexpression by >2-fold compared with low-stage (stages I and II) HCC. Importantly, HCC with KIAA0101 overexpression had lower 5-year survival. Besides, the KIAA0101 protein–positive tumor cells were more numerous in the portal vein tumor thrombi than in the main tumor. These findings led us reasonably to believe that KIAA0101 overexpression is an important factor related to the enhanced metastatic potential of HCC and hence poor prognosis.

To further substantiate the role of KIAA0101 in vascular invasion and prognosis, we compared its expression with the well-known important molecular prognostic factors, p53 mutation (36, 37), which is the most commonly mutated gene in HCC and associated with vascular invasion and tumor recurrence (36, 37, 42). By pair-wise analyses, KIAA0101 overexpression was associated with more frequent vascular invasion, regardless of the presence or absence of p53 mutation (all Ps < 0.00002). Notably, both non–p53-mutated and p53-mutated HCCs with concomitant KIAA0101 overexpression had lower 5-year survival rates compared with those without the overexpression (P = 0.029 and 0.011, respectively). However, it was also noted that HCC with absence of p53 mutation and KIAA0101 overexpression had the highest survival followed by HCC with p53 mutation alone or KIAA0101 overexpression alone, whereas HCC with both events had the lowest survival (Fig. 5D and E). Moreover, the great majority (87%) of HCC that had KIAA0101overexpression and concomitant p53 mutation had vascular invasion. These observations indicate that KIAA0101 overexpression is an imperative molecular prognostic factor of HCC, which contributes toward poor prognosis in cooperation with p53 mutation.

Although surgical resection provides an opportunity of cure for HCC, the prognosis for surgical HCC patients remains grave because of the high ETR (4–6). Hence, the identification of more molecular markers for the prediction of ETR will help develop better strategy for patient management. In addition to the well-known histopathologic factors (large tumor, high-grade tumor, and high-stage tumor) and several previously reported molecular factors, such as high AFP, p53 mutation, and overexpression of osteopontin, stathmin, and L2DTL (4, 6, 31, 32, 43–45), we showed that HCC with KIAA0101 overexpression had a >4-fold higher chance of ETR than HCC without the overexpression. Hence, KIAA0101 overexpression is an important molecular factor associated with vascular invasion, leading to high-stage HCC and poor prognosis, and may serve as a useful new predictive molecular marker for ETR.

We thank Mei-Ling Chen for her technical assistance and Chia-Chi Cheng for her assistance in statistical computing.