SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

KIAA0101 overexpression was detected in numerous malignant solid tumors and involved in tumor progression; however, the correlation between KIAA0101 expression level and human hepatocellular carcinoma (HCC) was controversial. Our data revealed abnormal expression of the KIAA0101 transcript variant 1 (KIAA0101 tv1) at both messenger RNA and protein levels in HCC tissues and cell lines assessed by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), virtual northern blot, western blot, and immunohistochemical analysis, especially in stage 3-4 HCCs. NIH3T3 cells transfected with KIAA0101 tv1 induced colony formation in vitro and tumor xenorafts in vivo, implying the oncogenic potential of KIAA0101 tv1. Semiquantitative RT-PCR, real-time quantitative RT-PCR, and western blot analysis demonstrated that doxorubicin (Adriamycin, ADR) treatment down-regulated expression of the KIAA0101 tv1, whereas it increased the acetylation of the p53 protein. Additionally, KIAA0101 tv1 prevented cells from apoptosis caused by ADR through suppressing the acetylation of p53 at Lys382. Immunoprecipitation analysis and mammalian two-hybrid assay indicated that KIAA0101 tv1 bound to the transactivation region (1-42 amino acids) of p53 and strongly inhibits its transcriptional activity. Taken together, our data suggest that KIAA0101 tv1 played an important role in the late stage of metastatic HCC and prevented apoptosis after chemotherapeutic drug treatment through inhibiting the transcriptional activity of the p53 gene. Conclusion: KIAA0101 tv1 may function as a regulator, promoting cell survival in HCC through regulating the function of p53. Suppression of the KIAA0101 tv1 function is likely to be a promising strategy to develop novel cancer therapeutic drugs. (HEPATOLOGY 2012;56:1760–1769)

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world.1, 2 Approximately 564,000 individuals are expected to be diagnosed with HCC per year.3 Because of a paucity of efficient therapy, HCC has one of the worst mortality rates among the frequent common malignancies.1, 2 As a result, it is necessary to identify the molecular targets in HCC.

KIAA0101, a proliferating cell nuclear antigen (PCNA)-associated factor, is also called p15 (PAF),4 OEATC-1 (overexpressed in anaplastic thyroid carcinoma-1),5 or NS5ATP9 (HCV NS5A-transactivated protein 9).6 The KIAA0101 gene has two transcriptional variants: the KIAA0101 transcript variant 1 (KIAA0101 tv1) and KIAA0101 tv2.2 The KIAA0101 tv1 protein contains a conserved PCNA-binding motif (also named PIP-box domain).4 PCNA plays an important role in DNA replication. KIAA0101 tv1 was observed to colocalize with PCNA in replication foci in S phase of the pancreatic cancer cell cycle and was speculated to be a member of the DNA replication complex.7 However, tv2 does not have the PIP-box domain.

KIAA0101 is also involved in the regulation of DNA repair,8, 9 cell cycle progression, and cell proliferation.7-9 In numerous malignant solid tumors, KIAA0101 is overexpressed and promotes tumor progression, including pancreatic cancer and colorectal adenocarcinoma, among others.4, 5, 7, 10-12 However, there are arguments regarding the correlation between KIAA0101 expression level and HCC.4, 12, 13 Guo et al.13 reported that the KIAA0101 protein was decreased in HCC, whereas KIAA0101 overexpression was observed in HCC by Yuan et al.12 Our data showed different expressions of KIAA0101 tv1 in HCC and adjacent nontumorous tissues (NTs) by differential hybridization of arrayed complementary DNA membranes and differential display reverse-transcription polymerase chain reaction (RT-PCR) (F. Zhu, unpublished data). It will be interesting to know the role of KIAA0101 in HCC progression.

KIAA0101 overexpression in HCC was closely correlated with p53 mutation.12 Loss of p53 function, either directly through mutations or indirectly through other mechanisms, plays an important role in the development of cancer,14-16 including HCC.17 However, the regulatory mechanism of p53 and KIAA0101 in the development of HCC remains to be elucidated.

In this study, overexpression of KIAA0101 tv1 in HCCs was observed compared with NTs, especially in stage 3-4 HCCs. NIH3T3 cells expressing KIAA0101 tv1 increased colony formation in vitro and induced tumor formation in vivo compared with parental NIH3T3 cells, indicating the oncogenic potential of KIAA0101 tv1. Doxorubicin (Adriamycin, ADR) treatment down-regulated the expression of KIAA0101 tv1 and resulted in the acetylation of p53. Interestingly, KIAA0101 tv1 abrogated the ADR-mediated apoptosis by suppressing the acetylation of p53 at Lys382. Further experiments showed that KIAA0101 tv1 bound to the transactivation region (1-42 amino acids) of p53 and inhibited the promoter activity of p53.

As a result, KIAA0101 tv1 may function as a regulator, promoting cell survival at the late stage of HCC through inhibiting the transcriptional activity of the p53 gene. Therefore, our results provided valuable information for further understanding the mechanism of HCC, and also identified a potential marker for prognostic detecting and a promising target molecule for the development of novel anticancer therapies.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Tissue Samples.

One hundred twenty-one paired samples of HCC and the corresponding NTs were obtained at Ren-Min Hospital of Wuhan University (Wuhan, P.R. China) from 2002 to 2008. All tissues were classified according to the tumor-node-metastasis classification system of the Union for International Cancer Control,18 and were stored in liquid nitrogen until use. Sample collections were under consensus agreements, and were approved by the Institutional Review Board of Wuhan University, School of Medicine in Human Production.

Immunohistochemistry.

Immunohistochemistry stainings for KIAA0101 tv1 were performed using the standard streptavidin-biotin-peroxidase immunostaining procedure. The sc-65163 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used for KIAA0101 tv1. The second antibody was peroxidase-conjugated rabbit anti-goat immunoglobulin G (sc-2768, Santa Cruz Biotechnology). The staining intensity of KIAA0101 tv1 was scored 0, 1+, 2+, 3+, or 4+ as described by Chung et al.,19 and the samples with scores up to 2+ were considered as positive.

Cell Culture.

HepG2, HepG2.2.15, HuH-7, HCCLM3, BEL-7402, HEK293T, and NIH3T3 cells were purchased from American Type Cluture Collection (Manassas, VA). All cell lines were maintianed in Dulbecco's modified Eagle's medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate at 37°C with 5% CO2.

Flow Cytometry.

After the floating and detached cells were collected and combined, cells were treated by an Annexin V-FITC/propidium iodide apoptosis detection kit (Promega) according to the manufacturer's protocol. Cells were analyzed via flow cytometry (Epics Altra II; Beckman Coulter, Brea, CA). The apoptosis rate of cells was measured by a Beckman Coulter Epics Altra with Expo32 software (Beckman Coulter).

Luciferase Reporter Assay.

The Dual-Luciferase reproter assay system (Promega) was used according to the manufacturer's instructions. The activity of firefly luciferase was detected using a luminometer. The transfection efficiency was normalized via Renilla reniformis activity.

Statistical Analyses.

Statistical analyses were performed using a chi-square test and a paired Student t test when comparing multiple means. The data were obtained from at least three independent trials. P < 0.05 was considered statistically significant. All experiments were performed blindly.

The description of other methods used in this study is provided in the Supporting Information.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

KIAA0101 tv1 Messenger RNA and Protein Were Overexpressed in HCC Tissues.

The messenger RNA (mRNA) of KIAA0101 tv1 was remarkably expressed in HCC samples, whereas it was undetectable in paired NTs (Supporting Information Fig. 1A). Semiquantitative RT-PCR data (Supporting Table 1 and Supporting Fig. 1B) showed that the mRNA of KIAA0101 tv1 was detected in 83 (68.59%) HCC specimens, compared with only 19 (15.70%) in NTs (P < 0.005). Virtual northern blot analysis (Supporting Fig. 1C) revealed that the mRNA of KIAA0101 tv1 was up-regulated in 84 (69.42%) HCC samples. In contrast, only 19 (15.70%) NTs were positive for KIAA0101 tv1 (Table 1, Supporting Fig. 1D; P < 0.005).

Table 1. KIAA0101 tv1 mRNA Levels Analyzed via Virtual Northern Blot Analysis
Tissue GroupTotal No. of TissuesNo. with KIAA0101 TV1 OverexpressionRatio (%)P
HCC1218469.42<0.005
NT1211915.70

Western blot analysis revealed that KIAA0101 tv1 protein was also overexpressed in HCC samples compared with paired NTs (Fig. 1A). As shown in Supporting Table 2 and Fig. 1B, increased expression of KIAA0101 tv1 was observed in 85 (70.25%) HCC samples compared with only 20 (16.53%) NTs (P < 0.005). Furthermore, the expression of KIAA0101 tv1 was assessed via immunohistochemical analysis (Fig. 1C), and the result showed that 88 (72.73%) HCC specimens were positively stained, whereas only 20 (16.53%) NTs were positive for KIAA0101 tv1 (Table 2, Fig. 1D; P < 0.005).

thumbnail image

Figure 1. The level of KIAA0101 transcript variant 1 (tv1) protein was up-regulated in human HCC tissues compared with adjacent NTs, especially in HCCs at stages 3-4. (A,B) KIAA0101 tv1 protein level was analyzed via western blotting in 121 pairs of HCC and NTs. KIAA0101 tv1 overexpression was detected in 85 HCCs but only 20 NTs. Two pairs of human HCC samples were exhibited (A). (C,D) Immunohistochemical analysis. Intensive staining was observed in the nuclei of HCC cells, whereas less staining was showed in NTs (original magnification ×200) (C). The expression of KIAA0101 tv1 in 121 pairs of HCCs and NTs was assessed via immunohistochemical staining using anti-KIAA0101 tv1 polyclonal antibody (Santa Cruz Biotechnology) (D). (E) The expression of KIAA0101 tv1 protein by immunohistochemistry was analyzed in 32 HCCs at stage 1-2 and 89 HCCs at stage 3-4. The graph shows the mean ± SE of KIAA0101 tv1 protein levels. **P < 0.005 (B, D, E).

Download figure to PowerPoint

Table 2. KIAA0101 tv1 Protein Levels Measured with Immunohistochemical Analysis in Tissue Array
Tissues GroupTotal No. of TissuesNo. with KIAA0101 TV1 OverexpressionRatio (%)P
HCC1218872.73<0.005
NT1212016.53

Taken together, both the mRNA and protein expression levels of KIAA0101 tv1 were significantly higher in HCC tissues than in NTs, suggesting a strong correlation between human HCC and the expression level of KIAA0101 tv1.

KIAA0101 tv1 Protein Was Highly Expressed in Stage 3-4 HCCs.

To further examine the expression of KIAA0101 tv1 in the progress of HCC, 121 HCC tissues were classified into four stages according to the tumor-node-metastasis classification system of the Union for International Cancer Control.18 As shown in Table 3 and Fig. 1E, among the 32 HCCs at stages 1-2 examined via immunohistochemical analysis, 14 (43.75%) showed positive staining in the nuclei, whereas 74 of 89 (83.15%) HCCs at stages 3-4 were detected with predominant nuclear expression of KIAA0101 tv1 (P < 0.005). Therefore, the expression level of KIAA0101 tv1 was positively correlated with the stage of HCC. Given that HCCs at stage 3-4 have stronger invasive and metastatic potential compared with those at stage 1-2,18 these results suggest that KIAA0101 tv1 overexpression might be involved in the progression of human HCC.

Table 3. KIAA0101 tv1 Protein Levels Detected in 32 HCCs at Stages 1-2 and 89 HCCs at Stages 3-4 via Immunohistochemical Analysis
Tissues GroupNo. of TissuesNo. with KIAA0101 TV1 OverexpressionRatio (%)P
Stage 1-2 HCC321443.75<0.005
Stage 3-4 HCC897483.15

Expressions of KIAA0101 tv1 mRNA and Protein Were Up-regulated in HCC Cell Lines.

To determine the expression of KIAA0101 tv1 in HCC cell lines, both semiquantitative RT-PCR and real-time RT-PCR were used to examine the mRNA level using KIAA0101 tv1 primers. As shown in Figs. 2A,B, the mRNA of KIAA0101 tv1 was detectable in all of the HCC cell lines. The KIAA0101 tv1 protein in HCC cell lines was also detected using antibody specific to the C-terminus of KIAA0101 isoform 1; however, the mRNA and protein levels of KIAA0101 tv1 were undetectable in NT, suggesting a high expression of KIAA0101 protein in HCC cell lines (Fig. 2C,D).

thumbnail image

Figure 2. Both mRNA and protein levels of KIAA0101 tv1 were overexpressed in HCC cell lines. (A, B) The mRNA level of KIAA0101 tv1 in HCC cell lines (HepG2, HepG2.2.15, Huh-7, HCCLM3, BEL-7402) was analyzed via semiquantitative RT-PCR and real-time quantitative RT-PCR. NT was used as negative control. β-Actin mRNA was used as an internal control. (C, D) The protein level of KIAA0101 tv1 in HCC cell lines was assessed via western blotting using an antibody specific to the C-terminus of KIAA0101 tv1. NT was the negative control. Expression of β-actin was used as the internal control. At least four reproducible experiments have been performed.

Download figure to PowerPoint

Exogenous Overexpression of KIAA0101 tv1 Induced NIH3T3 Cells Transformation In Vitro and In Vivo.

To further investigate the oncogenic potential of KIAA0101 tv1, mouse fibroblast NIH3T3 cells, which did not express a homologue of human KIAA0101,7 were transfected with pcDNA3.1(−)-KIAA0101 tv1 plasmids, followed by G418 selection. The expression of exogenous KIAA0101 tv1 in G418-resistant cells (NIH3T3-K) was analyzed via semiquantitative RT-PCR and western blot analysis (Supporting Fig. 2A). NIH3T3 cells transfected with pcDNA3.1(−) [NIH3T3-(−)] were used as a negative control, while HepG2 cells with high level of endogenous KIAA0101 tv1 expression were used as a positive control.

Colony formation assays (Supporting Fig. 2B,C, Supporting Table 3) showed that a large number of colonies were formed by NIH3T3-K cells and HepG2 cells, whereas few foci were observed in control cells [NIH3T3 and NIH3T3-(−)]. Furthermore, NIH3T3-K cells and HepG2 cells induced tumor formation at the flank of nude mice, whereas NIH3T3 and NIH3T3-(−) cells did not form tumors (Supporting Fig. 2D,E, Supporting Table 4). These results suggest that KIAA0101 tv1 has oncogenic potential.

ADR Treatment Down-regulated the Expression of KIAA0101 tv1.

To explore the role of KIAA0101 tv1 in DNA damage response, HepG2 cells, which highly expressed endogenous KIAA0101 tv1, were treated with the genotoxic agent ADR. As shown in Fig. 3A, the viability of HepG2 cells underwent reduction in a dose-dependent manner as examined by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide (MTT) assay. The expression level of KIAA0101 tv1 was examined. Of interest, semiquantitative RT-PCR, real-time RT-PCR, and immunoblotting showed that the mRNA and protein levels of KIAA0101 tv1 decreased by 70.6% (Fig. 3B,C; P = 0.00028<0.001) and 76.9% (Fig. 3D,E; P = 0.0023<0.005), respectively. The down-regulation of KIAA0101 tv1 by ADR treatment further demonstrated that KIAA0101 tv1 might be associated with HCC.

thumbnail image

Figure 3. ADR treatment down-regulated KIAA0101 tv1 and promoted the acelytation of p53. HepG2 cells were treated with the indicated concentrations of ADR for 48 hours. (A) MTT assays. Cell viability was determined via MTT assay. The data are presented as the mean ± SD of five independent experiments. (B) Semiquantitative RT-PCR. Total mRNAs were prepared. The mRNA level of KIAA0101 tv1 was down-regulated after ADR treatment. (C) Real-time quantitative RT-PCR. The relative KIAA0101 tv1 mRNA level is shown as the mean ± SD based on three independent experiments. ***P < 0.001. (D) Western blot analysis. Total proteins were prepared. The down-regulation of KIAA0101 tv1 protein caused by ADR was confirmed by western blot analysis. It also showed that the level of acetylated p53 was up-regulated after ADR treatment. All experiments were repeated three times. (E) The bands are shown as the mean ± SD. **P < 0.005, ***P < 0.001.

Download figure to PowerPoint

KIAA0101 tv1 Overexpression Prevented ADR-Induced Apoptosis.

To investigate the function of KIAA0101 tv1 in ADR-induced apoptosis, HepG2 cells transfected with KIAA0101 tv1 (HepG2-K) were treated with different doses of ADR for 48 hours; HepG2 cells transfected with empty vector were used as a control. MTT assay (Supporting Fig. 3A,B) revealed that ADR treatment resulted in decreased viability of both HepG2-K and HepG2-(−) cells in a concentration-dependent manner, with decreases between 6.4% and 12.0% (P = 0.00098<0.001), 18.0% and 36.9% (P = 0.00006<0.001), 39.9% and 58.2% (P = 0.00025<0.001), 58.0% and 77.8% (P = 0.00104<0.005), respectively, by mean over a 0-2.4 μM concentration range of ADR. Experiments using NIH3T3 cells yielded similar results (Supporting Fig. 3C,D). After ADR treatment, the viabilities of NIH3T3-K and NIH3T3-(−) cells were down-regulated by 7.5% and 12.7% (P = 0.0044<0.005), 18.1% and 37.8% (P = 0.00033<0.001), 37.9% and 54.8% (P = 0.006<0.05), and 57.5% and 79.7% (P = 0.0025<0.005), respectively, by mean over a 0-2.4 μM ADR concentration range.

The role of KIAA0101 tv1 in apoptosis was further confirmed by flow cytometry analysis: ADR-induced apoptosis of both HepG2-(−) and HepG2-K cells were in a dose-dependent manner. However, the percentage of apoptotic HepG2-K cells significantly decreased compared with HepG2-(−) cells at the same ADR concentration (Fig. 4A-C). The average percentages of apoptotic HepG2-K and HepG2-(−) cells were 34.0% and 53.6%, respectively, at the concentration of 0.3 μM ADR (P = 0.0001<0.001); 52.2% and 76.6%, respectively, at the concentration of 1.2 μM ADR (P = 0.009<0.05); and 65.7% and 91.3%, respectively, at the concentration of 4.8 μM ADR (P = 0.006<0.05), suggesting that KIAA0101 tv1 overexpression promoted cell survival after ADR treatment.

thumbnail image

Figure 4. KIAA0101 tv1 prevented HepG2 cells from ADR-induced apoptosis by inhibiting the acetylation of p53. HepG2 cells were transfected with either the plasmid encoding the KIAA0101 tv1 (HepG2-K) or an empty vector [HepG2-(−)], and then were treated with serial dilutions of ADR (0, 0.3, 1.2, and 4.8 +M). (A-C) The apoptosis rates of cells were measured with Annexin V-FITC/propidium iodide by flow cytometry (A). The apoptotic cells are shown as the mean ± SD based on three independent experiments. ***P < 0.001, *P < 0.05 (B, C). (D) Cell survival assays. HepG2-pShp53 and HepG2-pSh(−) cells were incubated with serial dilutions of ADR, then the cell survivals were examined by MTT assay. The cell viabilities are shown as the mean ± SD based on five independent experiments. (E, F) KIAA0101 tv1 inhibited the activity of p53 by down-regulating the level of acetylation of p53 in response to ADR. The graph in (F) shows the mean ± SD of protein levels by western blot analysis based on three independent experiments. ***P < 0.001.

Download figure to PowerPoint

The endogenous KIAA0101 tv1 mRNA was knocked down by a plasmid encoding a short hairpin RNA (shRNA) targeted to KIAA0101 tv1 in HepG2 cells, whereas the KIAA0101 tv1 mRNA level has no changes in cells transfected with empty vector and the scrambled shRNA (Supporting Fig. 4A). MTT assays showed that silencing of KIAA0101 tv1 in HepG2 cells (HepG2-pShK) resulted in an increased sensitivity to ADR treatment compared with control cells [HepG2-pSh(−)] (Supporting Fig. 4B).

Flow cytometry analysis was used to confirm the antiapoptotic effect of KIAA0101 tv1. Twenty-four hours after transfection with pSh-KIAA0101 tv1 plasmids, cells were treated with different concentrations of ADR (0, 0.3, or 1.2μM) for another 48 hours, and HepG2 cells transfected with vector were used as a control. As expected, shRNA for KIAA0101 tv1 resulted in a significant increase of apoptosis in response to ADR (Supporting Fig. 4C-E). The average percentages of apoptosis of HepG2-pShK and HepG2-pSh(−) cells were 65.0% and 52.9% at 0.3 μM ADR (P = 0.040<0.05) and 92.6% and 76.3% at 1.2 μM ADR (P = 0.022<0.05), respectively.

Based on the above observations, we proposed that KIAA0101 tv1 overexpression may protect HCC cells from apoptosis in response to ADR-induced DNA damage.

p53 Was Involved in ADR-Mediated Apoptosis in HepG2 Cells.

As shown in Supporting Fig. 4F, p53 mRNA was significantly suppressed in HepG2 cells transiently transfected with shRNA for p53 compared with cells transfected with empty vector and the scrambled shRNA. MTT assay showed that after p53 was silenced, there was a significant increase (1.1- to 2.0-fold; P < 0.05) in cell viability compared with the control HepG2 cells (Fig. 4D), indicating that ADR-induced apoptosis in HepG2 cells was regulated by p53.

KIAA0101 tv1 Prevented ADR-Induced Apoptosis in HepG2 Cells by Inhibiting the Acetylation of p53 at Lys382.

After ADR treatment, immunoblotting revealed that the level of p53 acetylation in HepG2 was increased by 14.1-fold (Fig. 3D,E; P = 0.00064<0.001). To further identify the relationship between p53 and KIAA0101 tv1, p53 acetylation in HepG2 cells transfected with KIAA0101 tv1 (HepG2-K) was examined. As shown in Fig. 4E,F, the total level of p53 was constant, however, the acetylation of p53 was significantly reduced (74.7%; P = 0.00064<0.001) in HepG2-K cells 48 hours after ADR treatment. At the same time, some targeting genes of p53 (such as p21WAF1, Bax, PUMA, and Noxa) were analyzed via semiquantitative RT-PCR and real-time RT-PCR. As shown in Supporting Fig. 5A,B, the levels of p53 targeting genes were all decreased in HepG2-K cells 48 hours after ADR treatment. These data suggest that KIAA0101 tv1 may protect cells from apoptosis by inhibiting acetylation of p53 at Lys382.

KIAA0101 tv1 Inhibited p53 Promoter Activity.

To examine the effect of KIAA0101 tv1 on the p53 promoter activity, HEK293T cells were transiently transfected with the p53 promoter/luciferase reporter with or without the different amounts of the KIAA0101 tv1 expression plasmid for the promoter reporter assay. As shown in Fig. 5A, p53 promoter activity was reduced after KIAA0101 tv1 plasmid transfection. Furthermore, the decrease was dependent on the concentration of transfected KIAA0101 tv1 plasmid. Approximately 11.0% (P = 0.009<0.05) and 31.2% (P = 0.00044<0.001) reductions of p53 promoter activity were observed with the transfection ratio of 0.25:1 and 0.5:1 (the weight ratio of KIAA0101 tv1 plasmid/luciferase vector) (Fig. 5A). When the HEK293T cells were cotransfected with equal concentration of KIAA0101 tv1 and luciferase vector, the activity of p53 promoter was decreased by approximately 40.2% (P = 0.0015<0.005). These results suggest that KIAA0101 tv1 inhibited the transcriptional activity of p53 in HEK293T cells.

thumbnail image

Figure 5. KIAA0101 tv1 inhibited the transcriptional activity of p53 promoter and physically interacted with p53. (A) Dual-luciferase activity assay. HEK293T cells were transiently cotransfected with a different ratio of KIAA0101 tv1 expression plasmid : pGL3-p53 promoter or pGL3-basic. Luciferase activity was measured following 48 hours of incubation. (B) Co-immunoprecipitation. Twenty-four hours after transfection, whole cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-FLAG M5 antibody, followed by immunoblotting (IB) with the anti-p53 antibody. SV40 T-antigen was used as a positive control. (C, D) Mammalian two-hybrid assay. Plasmids encoding KIAA0101 tv1 (pBIND-K) and p53 (pACT-p53) and the pG5luc vector were cotransfected into HEK293T cells (C). pACT plasmids carrying p53(full), p53(1-42), p53(1-101), or p53(102-393) were further used to examine which part of p53 is responsible for the physical interaction (D). Protein-protein association was measured by quantitating luciferase expression using luciferase activity assay. All of the graphs are presented as the mean ± SD based on three independent experiments and are normalized with the Renilla reniformis luciferase activity. *P < 0.05, **P < 0.005, ***P < 0.001.

Download figure to PowerPoint

KIAA0101 tv1 Physically Interacted with p53 Protein.

Interaction between KIAA0101 tv1 protein and p53 protein was examined by immunoprecipitation analysis. Antibodies against Flag and p53 were used for precipitation and immunoblotting, respectively. As shown in Fig. 5B, the band in lane 4 corresponding to p53-KIAA0101 tv1 complex was detected. The interaction between SV40 large T antigen and p53 was shown in lane 3. The data suggest that KIAA0101 tv1 forms a complex with p53.

The mammalian two-hybrid system was used to further confirm the interaction between KIAA0101 tv1 and p53. Cell lysates of HEK293T cells following pBIND-K and pACT-p53 cotransfection (48 hours) were subjected to luciferase activity assay. As shown in Fig. 5C, cotransfection of KIAA0101 tv1 with p53 resulted in a significant increase (approximately 4.4- to 7.5-fold; P < 0.005) in the activity of firefly luciferase compared with negative controls, suggesting that KIAA0101 tv1 protein interacted directly with p53.

KIAA0101 tv1 Could Bind to the Transactivation Region (1-42 Amino Acids) of p53.

To further determine which region of p53 is required for the interaction with KIAA0101 tv1, HEK293T cells were transiently cotransfected with pG5luc, the pBIND vector or pBIND-K, together with the pACT plasmids carrying p53(full), p53(1-42), p53(1-101), or p53(102-393). As shown in Fig. 5D, all of the p53 deletion mutants except p53(102-393) led to an increase of luciferase activity. Furthermore, the first 42 amino acids of p53 were critical for increased luciferase activity compared with control (approximately 2.58-fold; P = 0.0014<0.005). These results suggest that the transactivation region (1-42 amino acids) of p53 is required for the interaction with KIAA0101.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

KIAA0101 is reported to be involved in the regulation of DNA replication,7 DNA repair,8, 9, 20 cell cycle progression,20, 21 and cell proliferation.4, 7 It has been revealed that the expression of KIAA0101 was up-regulated in many types of human cancers, including pancreatic cancer,4, 7 colorectal adenoma and adenocarcinoma,10 non–small cell lung cancer,11 and anaplastic thyroid carcinoma.5 However, only few papers reported a yet ambiguous correlation between KIAA0101 and liver cancer.4, 12, 13 Guo et al.13 reported decreased KIAA0101 protein expression in HCCs compared with paired NTs. Our study showed that both the mRNA and protein levels of KIAA0101 tv1 were up-regulated in HCC tissues compared with those in NT tissues, which were completely contrary to the data reported by Guo et al.13 The reason for this might be that the polyclonal antibody used by Guo's group was a nonspecific antibody. It could not only detect KIAA0101 tv1, it could also detect KIAA0101 tv2. In fact, KIAA0101 tv2 was highly expressed in NTs. In addition, the antibody we used was specific to the C-terminus of KIAA0101 tv1, which is merely presented in KIAA0101 tv1. Actually, KIAA0101 tv2 was detected in some normal tissues; it might play an important role in the regulation of KIAA0101 tv1 expression (F. Zhu, unpublished data). Furthermore, the mRNA and protein levels of KIAA0101 tv1 were also up-regulated in five HCC cell lines, suggesting that KIAA0101 tv1 was associated with HCC.

Expression of KIAA0101 tv1 in HCC tissues at different stages further showed that 83.15% of HCCs at stages 3-4 were positively stained in the nuclei, which were significantly higher than those at stages 1-2 (43.75%). These results are consistent with those of Yuan et al.12 Because HCCs at stages 3-4 have stronger vascular invasive and metastatic potential than those at stages I-II,18 it is possible that KIAA0101 tv1 is involved in the invasion and metastasis of HCC. KIAA0101 also found to be correlated with advanced tumor progression and lymphnode metastasis of lung adenocarcinomas,22 and the metastasis of adrenocortical carcinoma.23 In additon, the KIAA0101 protein-positive tumor cells were observed more numerous in the portal vein tumor thrombi than in the main HCC tumor,12 further suggesting the metastasis potential of KIAA0101 in HCC. To further study metastasis potential of KIAA0101 tv1, scrape motility assays were used. It revealed that KIAA0101 tv1 overexpression, but not tv2, promoted the invasion of NIH3T3 cells compared with the parental NIH3T3 cells (Zhu F, unpublished data), combined with the observation that suppressing KIAA0101 expression in adrenocortical carcinoma cells resulted in suppression of growth and invasion,23 suggesting that KIAA0101 tv1 played growth, invasion and metastasis promoting roles in various malignant tumors.

ADR has been demonstrated to induce apoptosis in a variety of tumor cell lines through DNA damage,24, 25 down-regulation of oncogenes (survivin) or up-regulation of apoptotic genes (bcl-2, bcl-XL).26, 27 In this study, both mRNA and protein levels of KIAA0101 tv1 decreased in response to ADR, suggesting oncogenic potential of KIAA0101 tv1. Furthermore, similar to Hosokawa's study,7 exogenous overexpression of KIAA0101 tv1 induced tumor formation in nude mice (Supporting Fig. 2), which further confirmed the oncogenic potential of KIAA0101 tv1. In this study, KIAA0101 tv1 overexpression also transformed NIH3T3 cells in vitro (Supporting Fig. 2), while knockdown of KIAA0101 by shRNA introduction suppressed colony formation,7 indicating the carcinogenesis function of KIAA0101 tv1. Interestingly, the level of p53 acetylation was up-regulated after ADR treatment (Fig. 3D,E), our further study indicated that several p53 downstream target genes (p21WAF1, Bax, PUMA, and Noxa) were also significantly up-regulated in response to ADR treatment (Zhu F, unpublished data), which strongly suggested p53 was activated. Additionally, the knockdown of p53 in HepG2 cells led to a remarkable decrease in the proportion of apoptosis caused by ADR (Fig. 4D), suggesting that ADR-induced HepG2 cells apoptosis might be regulated by p53.

There are evidences to show that KIAA0101 was anti-apoptotic in response to DNA damage.8, 21 In our study, after transfected with KIAA0101 tv1, survival rates of HepG2 increased and apoptotic rates reduced in response to ADR treatment (Supporting Fig. 3, Fig. 4A-C). Consistent with these results, the shRNA-mediated knockdown of the endogenous KIAA0101 tv1 in HepG2 cells led to a significant decrease in the survival rate and a dramatic increase in the apoptotic rate after ADR treatment (Supporting Fig. 4B-E). These findings indicated that KIAA0101 tv1 inhibited ADR induced-apoptosis, and further suggested oncogenic potential of KIAA0101 tv1.

In one study, KIAA0101 expression was down-regulated, whereas p53 and p21 were up-regulated after ADR treatment, it was considered that KIAA0101 expression was regulated by p53-p21 pathway.7 However, in our study, it was interesting to observe that KIAA0101 tv1 overexpression could down-regulate the level of p53 acetylation at Lys382 (Fig. 4E,F). Inhibition of p53 acetylation could counteract p53-mediated G1-phase cell-cycle arrest and apoptosis,28 which was consistent with our observations. Since Lys382 of p53 is acetylated by p300,29 the mRNA of p300 was examined. It showed that p300 was also down-regulated by KIAA0101 tv1 overexpression in HepG2 cells after ADR treatment (Zhu F, unpublished data). After that, the activity of p53 was decreased, which was reflected in the down-regulation of a series of p53 downstream genes (p21WAF1, Bax, PUMA, and Noxa) (Supporting Fig. 5). From above on, it was supposed that ADR-induced apoptosis was inhibited by KIAA0101 tv1 through suppressing the acetylation of p53 at Lys382.

Our further study showed that overexpression of KIAA0101 tv1 reduced the activity of the p53 promoter (Fig. 5A), suggesting that overexpression of KIAA0101 tv1 inhibited the transcriptional activity of p53. Further study showed that KIAA0101 tv1 physically interacted with p53 (Fig. 5B,C) by binding to the transactivation domain of p53 (Fig. 5D). While the transactivation domain of p53 was important for CBP/p300 binding,30 which played a positive role in the accumulation of p53 protein in stress response, and contribute to p53 stability.31 It was speculated that KIAA0101 tv1 might be a competitor with CBP/p300 to the binding of the p53 transactivation domain, and might also be an inhibitor to CBP/p300 expression. Additional studies should be necessary to clarify these issues.

Taken together, our study suggested KIAA0101 tv1 might play an important role in the progression of HCC, and its overexpression might predict high stage and high transfer of human HCC. KIAA0101 tv1 possessed the oncogenic potential to transform NIH3T3 cells to tumor cells. Additionally, KIAA0101 tv1 inhibited the transcriptional activity of p53 by directly suppressing the p53 promoter. Furthermore, KIAA0101 tv1 physically interacted with p53 by binding to the transactivation region (1-42 amino acids) and inhibited the acetylation of p53 at Lys382, thereby suppressing the transcriptional activity of p53.

In conclusion, our study indicated that KIAA0101 tv1, as a regulator, may regulate the function of the p53 gene and promote cell survival in HCC. Therefore, KIAA0101 tv1 overexpression in HCC specimens could be used for the diagnosis and prognosis of HCC. In addition, it might also be a promising candidate target for the development of novel liver cancer therapeutic drugs.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
  • 1
    Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003; 362: 1907-1917.
  • 2
    Moradpour D, Wands JR. The molecular pathogenesis of hepatocellular carcinoma. J Viral Hepat 1994; 1: 17-31.
  • 3
    Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004; 127( 5 Suppl. 1): S5-S16.
  • 4
    Yu P, Huang B, Shen M, Lau C, Chan E, Michel J, et al. p15(PAF), a novel PCNA associated factor with increased expression in tumor tissues. Oncogene 2001; 20: 484-489.
  • 5
    Mizutani K, Onda M, Asaka S, Akaishi J, Miyamoto S, Yoshida A, et al. Overexpressed in anaplastic thyroid carcinoma-1 (OEATC-1) as a novel gene responsible for anaplastic thyroid carcinoma. Cancer 2005; 103: 1785-1790.
  • 6
    Li K, Ma Q, Shi L, Dang C, Hong Y, Wang Q, et al. NS5ATP9 gene regulated by NF-kappaB signal pathway. Arch Biochem Biophys 2008; 479: 15-19.
  • 7
    Hosokawa M, Takehara A, Matsuda K, Eguchi H, Ohigashi H, Ishikawa O, et al. Oncogenic role of KIAA0101 interacting with proliferating cell nuclear antigen in pancreatic cancer. Cancer Res 2007; 67: 2568-2576.
  • 8
    Turchi L, Fareh M, Aberdam E, Kitajima S, Simpson F, Wicking C, et al. ATF3 and p15PAF are novel gatekeepers of genomic integrity upon UV stress. Cell Death Differ 2009; 16: 728-737.
  • 9
    Cheung KJ Jr, Mitchell D, Lin P, Li G. The tumor suppressor candidate p33(ING1) mediates repair of UV-damaged DNA. Cancer Res 2001; 61: 4974-4977.
  • 10
    Notterman DA, Alon U, Sierk AJ, Levine AJ. Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays. Cancer Res 2001; 61: 3124-3130.
  • 11
    Petroziello J, Yamane A, Westendorf L, Thompson M, McDonagh C, Cerveny C, et al. Suppression subtractive hybridization and expression profiling identifies a unique set of genes overexpressed in non-small-cell lung cancer. Oncogene 2004; 23: 7734-7745.
  • 12
    Yuan RH, Jeng YM, Pan HW, Hu FC, Lai PL, Lee PH, et al. Overexpression of KIAA0101 predicts high stage, early tumor recurrence, and poor prognosis of hepatocellular carcinoma. Clin Cancer Res 2007; 13: 5368-5376.
  • 13
    Guo M, Li J, Wan D, Gu J. KIAA0101 (OEACT-1), an expressionally down-regulated and growth-inhibitory gene in human hepatocellular carcinoma. BMC Cancer 2006; 6: 109.
  • 14
    Balint EE, Vousden KH. Activation and activities of the p53 tumour suppressor protein. Br J Cancer 2001; 85: 1813-1823.
  • 15
    Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991; 253: 49-53.
  • 16
    Olivier M, Petitjean A, Marcel V, Petre A, Mounawar M, Plymoth A, et al. Recent advances in p53 research: an interdisciplinary perspective. Cancer Gene Ther 2009; 16: 1-12.
  • 17
    Hsu HC, Tseng HJ, Lai PL, Lee PH, Peng SY. Expression of p53 gene in 184 unifocal hepatocellular carcinomas: association with tumor growth and invasiveness. Cancer Res 1993; 53: 4691-4694.
  • 18
    Hermanek P, Scheibe O, Spiessl B, Wagner G. TNM classification of malignant tumors: new edition 1987 [in German]. Rofo 1987; 146: 732-733.
  • 19
    Chung KY, Shia J, Kemeny NE, Shah M, Schwartz GK, Tse A, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol 2005; 23: 1803-1810.
  • 20
    Emanuele MJ, Ciccia A, Elia AE, Elledge SJ. Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate. Proc Natl Acad Sci U S A 2011; 108: 9845-9850.
  • 21
    Simpson F, Lammerts van BK, Butterfield N, Bennetts JS, Bowles J, Adolphe C, et al. The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b. Exp Cell Res 2006; 312: 73-85.
  • 22
    Kato T, Daigo Y, Aragaki M, Ishikawa K, Sato M, Kaji M. Overexpression of KIAA0101 predicts poor prognosis in primary lung cancer patients. Lung Cancer 2012; 75: 110-118.
  • 23
    Jain M, Zhang L, Patterson EE, Kebebew E. KIAA0101 is overexpressed, and promotes growth and invasion in adrenal cancer. PLoS One 2011; 6: e26866.
  • 24
    Muller I, Jenner A, Bruchelt G, Niethammer D, Halliwell B. Effect of concentration on the cytotoxic mechanism of doxorubicin—apoptosis and oxidative DNA damage. Biochem Biophys Res Commun 1997; 230: 254-257.
  • 25
    Kaina B. DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling. Biochem Pharmacol 2003; 66: 1547-1554.
  • 26
    Lotem J, Sachs L. Regulation of bcl-2, bcl-XL and bax in the control of apoptosis by hematopoietic cytokines and dexamethasone. Cell Growth Differ 1995; 6: 647-653.
  • 27
    Wang L, Zhang GM, Feng ZH. Down-regulation of survivin expression reversed multidrug resistance in adriamycin-resistant HL-60/ADR cell line. Acta Pharmacol Sin 2003; 24: 1235-1240.
  • 28
    Avantaggiati ML, Ogryzko V, Gardner K, Giordano A, Levine AS, Kelly K. Recruitment of p300/CBP in p53-dependent signal pathways. Cell 1997; 89: 1175-1184.
  • 29
    Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, et al. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev 1998; 12: 2831-2841.
  • 30
    Grossman SR, Perez M, Kung AL, Joseph M, Mansur C, Xiao ZX, et al. p300/MDM2 complexes participate in MDM2-mediated p53 degradation. Mol Cell 1998; 2: 405-415.
  • 31
    Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997; 90: 595-606.
  • 32
    Zhu F, Li WX, Jiang DH, Gou DM. Differential expression of the Quox-1 gene in normal human cells, early human embryo, and tumor cells. Cell Tissue Res 2002; 308: 333-337.
  • 33
    Huang W, Li S, Hu Y, Yu H, Luo F, Zhang Q, et al. Implication of the env gene of the human endogenous retrovirus W family in the expression of BDNF and DRD3 and development of recent-onset schizophrenia. Schizophr Bull 2011; 37: 988-1000.
  • 34
    Wei W, Huang W, Pan Y, Zhu F, Wu J. Functional switch of viral protein HBx on cell apoptosis, transformation, and tumorigenesis in association with oncoprotein Ras. Cancer Lett 2006; 244: 119-128.
  • 35
    Nakanishi M, Ozaki T, Yamamoto H, Hanamoto T, Kikuchi H, Furuya K, et al. NFBD1/MDC1 associates with p53 and regulates its function at the crossroad between cell survival and death in response to DNA damage. J Biol Chem 2007; 282: 22993-23004.
  • 36
    Iyer NG, Chin SF, Ozdag H, Daigo Y, Hu DE, Cariati M, et al. p300 regulates p53-dependent apoptosis after DNA damage in colorectal cancer cells by modulation of PUMA/p21 levels. Proc Natl Acad Sci U S A 2004; 101: 7386-7391.
  • 37
    Paola Secchiero. Tumor necrosis factor-related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway. In: Arianna Gonelli, ed. 100 ed. 2011. 2421-2429.
  • 38
    Zwart W, Griekspoor A, Berno V, Lakeman K, Jalink K, Mancini M, et al. PKA-induced resistance to tamoxifen is associated with an altered orientation of ERalpha towards co-activator SRC-1. EMBO J 2007; 26: 3534-3544.
  • 39
    Dutta D, Chattopadhyay S, Bagchi P, Halder UC, Nandi S, Mukherjee A, et al. Active participation of cellular chaperone Hsp90 in regulating the function of rotavirus nonstructural protein 3 (NSP3). J Biol Chem 2011; 286: 20065-20077.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25834_sm_SuppFig1.tif3738KSupporting Information Figure. S1. Levels of KIAA0101 tv1 mRNA were up-regulated in human HCC tissues. (A) Semi-quantitative RT-PCR showed the high level of KIAA0101 tv1 mRNA in HCCs and its expression was weak in NT. Four pairs of HCC samples were exhibited here. (B) The graph shows the mean±standard error of KIAA0101 tv1 mRNA levels by semi-quantitative RT-PCR. **P<0.005. (C) Increased expression of KIAA0101 tv1 in HCC (lane 1) was detected by virtual northern blot analysis. And its expression was hardly detectable in NT (lane 2). GAPDH was used as control. Lane 3 is a marker. (D) The graph shows the mean±standard error of KIAA0101 tv1 mRNA levels by virtual northern blot. **P<0.005.
HEP_25834_sm_SuppFig2.tif9946KSupporting Information Figure. S2. Exogenous overexpression of KIAA0101 tv1 induced transformation of NIH3T3. NIH3T3 cells transfected with KIAA0101 tv1 (NIH3T3-K) or vector (NIH3T3-(-)) were selected with G418. (A) RT-PCR and western blot analysis. The resistant clones were analyzed by KIAA0101 tv1-specific primers or antibody. HepG2 was used as positive control. β-actin was used as a loading control. (B and C) Colony formation assay. NIH3T3-K cells resulted in significant increase of the numbers of colonies, comparing with the NIH3T3-(-) cells (B). The graph shows the mean±standard deviation of the colony number based on three independent experiments. **P<0.005 (C). (D and E) Tumor formation in nude mice. NIH3T3, NIH3T3-(-) and NIH3T3-K cells were inoculated into the flank of 6-week-old nude mice. After 4 weeks, NIH3T3-K clones formed a large mass at the right flank of nude mice. HepG2 was used as positive control (D). The growth curve was measured by the tumor volume of each five days after injection. It shows the mean±standard deviation of tumor volume based on five independent experiments. **P<0.005 (E).
HEP_25834_sm_SuppFig3.tif760KSupporting Information Figure. S3. KIAA0101 tv1 prevented HepG2 and NIH3T3 cells from ADR-induced apoptosis. Cell survival assays. HepG2 cells (A and B) or NIH3T3 cells (C and D) were transfected with either the plasmid encoding the KIAA0101 tv1 (NIH3T3-K) or an empty vector (NIH3T3-(-)), and then treated with serial dilutions of ADR. 48 hours post ADR treatment, cell viability was determined by MTT assays. The cell viability is shown as the mean±standard deviation based on five independent experiments. *P<0.05, **P<0.005, ***P<0.001.
HEP_25834_sm_SuppFig4.tif15209KSupporting Information Figure. S4. shRNA mediated down-regulation of the endogenous KIAA0101 tv1 promoted ADR-induced apoptosis in HepG2 cells. HepG2 cells were transiently transfected with shRNA against KIAA0101 tv1 (HepG2-pShK), vector control (HepG2-pSh(-)) or scrambled shRNA (HepG2-pShCntrl). 48 hours after transfection, cells were treated as indicated. (A) Check the level of KIAA0101 tv1. mRNA were prepared and processed for semi-quantitative RT-PCR with primers for KIAA0101 tv1 (upper panel). β-actin was also analyzed as a control (lower panel). (B) Cell survival assays. The cell survivals were examined by MTT assay after treatment with different concentration of ADR for 48 hours. The cell viabilities are shown as the mean±standard deviation based on four independent experiments. (C, D and E) Cell apoptosis assay by flow cytometry. Cell apoptosis was detected with Annexin V-FITC/propidium iodide after ADR treatment (C). The apoptotic cells are shown as the mean±standard deviation based on three independent experiments. *P<0.05 (D and E). (F) Knockdown of p53. HepG2 cells were transiently transfected with shRNA against p53 (HepG2-pShp53), vector control (HepG2-pSh(-)) or scrambled shRNA (HepG2-pShCntrl). Knockdown effect was tested with semi-quantitative RT-PCR.
HEP_25834_sm_SuppFig5.tif2151KSupporting Information Figure. S5. Exogenous expression of KIAA0101 tv1 inhibited the activity of p53 in response to ADR treatment. (A) HepG2 cells were transiently transfected with 2 μg of the empty plasmid (HepG2-(-)) or with the expression plasmid for KIAA0101 tv1 (HepG2-K). 48 hours after transfection, cells were treated with 0.6 μM ADR. After another 48 hours treatment, total mRNA were also prepared and subjected to quantitative RT-PCR using specific primers. β-actin was used as control. The experiments were performed at least three times. (B) The graph shows the mean±standard deviation of mRNA levels by real-time RT-PCR based on three independent experiments. *P<0.05, **P<0.05.
HEP_25834_sm_SuppInfo.rtf79KSupporting Information
HEP_25834_sm_SuppTables1_4.doc38KSupporting Information Tables.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.