A predicted protein, KIAA0247, is a cell cycle modulator in colorectal cancer cells under 5-FU treatment

Background Colorectal cancer (CRC) is the predominant gastrointestinal malignancy and the leading cause of cancer death. The identification of genes related to CRC is important for the development of successful therapies and earlier diagnosis. Methods Molecular analysis of feces was evaluated as a potential method for CRC detection. Expression of a predicted protein with unknown function, KIAA0247, was found in feces evaluated using specific quantitative real-time polymerase chain reaction. Its cellular function was then analyzed using immunofluorescent staining and the changes in the cell cycle in response to 5-fluorouracil (5-FU) were assessed. Results Gastrointestinal tissues and peripheral blood lymphocytes ubiquitously expressed KIAA0247. 56 CRC patients fell into two group categories according to fecal KIAA0247 mRNA expression levels. The group with higher fecal KIAA0247 (n = 22; ≥ 0.4897) had a significantly greater five-year overall survival rate than the group with lower fecal KIAA0247 (n = 30; < 0.4897) (66.0 ± 11.6%; p = 0.035, log-rank test). Fecal expression of KIAA0247 inversely related to CRC tumor size (Kendall's tau-b = -0.202; p = 0.047). Immunofluorescent staining revealed that the cytoplasm of CRC cells evenly expresses KIAA0247 without 5-FU treatment, and KIAA0247 accumulates in the nucleus after 40 μM 5-FU treatment. In HCT116 p53-/- cells, which lack p53 cell cycle control, the proportion of cells in the G2/M phase was larger (13%) in KIAA0247-silent cells than in the respective shLuc control (10%) and KIAA0247-overexpressing cells (7%) after the addition of low dose (40 μM) 5-FU. Expression of three cyclin genes (cyclin A2, cyclin B1, and cyclin B2) also downregulated in the cells overexpressing KIAA0247. Conclusions This is the first description of a linkage between KIAA0247 and CRC. The study's data demonstrate overexpression of KIAA0247 associates with 5-FU therapeutic benefits, and also identify the clinical significance of fecal KIAA0247 in CRC.


Background
Colorectal cancer (CRC) is the predominant gastrointestinal malignancy and the leading cause of cancer death [1]. CRC usually arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation [2]. The identification of CRC-related genes is important for the development of successful therapies and earlier diagnosis [3][4][5].
Genes involved in cell growth, cell cycle, apoptosis, angiogenesis, or invasion could have a crucial role in CRC tumorigenesis [6,7]. In particular, some promising targets responsible for the control of cell cycle progression have attracted a great deal of attention for drug discovery [8,9]. In recent decades, researchers developed several agents with the function of regulating the degree of cell cycle arrest for cancer treatment [10,11]. Enhancement of the effects of defects in the G2/M arrest checkpoint that make a damaged cell enter * Correspondence: chunchao@tmu.edu.tw 8 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital and Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan Full list of author information is available at the end of the article mitosis and undergo apoptosis might increase the effective cytotoxicity of chemotherapy [8].
The novel gene, KIAA0247, previously identified as one of the CRC-related candidates, is a speculated target of the tumor suppressor gene, p53, because of a p53-responsive element in the promoter region [12,13]. This implies that KIAA0247 might participate in the p53 pathway of CRC tumorigenesis. Previous studies have identified that many molecules have altered expression in the feces of CRC patients [14,15]; some of these novel candidate genes with unknown function. The detailed characteristics of KIAA0247 are still unknown. Further understanding of the cellular functions in CRC of this predicted protein may provide an alternative target for CRC treatment.
The present study, therefore, aimed to investigate the molecular function of KIAA0247 in CRC tumorigenesis. Firstly, the clinical significance of KIAA0247 was evaluated from fecal samples of CRC patients using specific quantitative real-time polymerase chain reaction (qRT-PCR). Its cellular function was then evaluated using immunofluorescent staining and the changes in the cell cycle in response to 5-fluorouracil (5-FU) were assessed. Results demonstrated that, in CRC patients, the expression of KIAA0247 influences the effects of treatment with 5-FU at a relatively low concentration.

Patients
Solid fecal samples (approximately 0.5 g) from 56 CRC patients from the Cathay General Hospital (CGH) or Taipei Veterans General Hospital were taken before surgery or any application of chemotherapy with Institutional Review Board (IRB)-approved informed consent at the CGH IRB. Follow-up data were obtained prospectively, and the mean follow-up time was 34.9 months (SD, 26.8; median, 23). The patients' initial tumor stage and other clinical information are listed in Table 1. Presence of distant metastasis was routinely confirmed by abdominal computed tomography.

Colonic cell lines and human multiple tissue cDNA
The p53-null HCT116 cell line (HCT116 p53 -/-, a gift from Prof. Bert Vogelstein) was cultured in Dulbecco's modified Eagles medium with 5 mM glutamine according to routine culture procedures. The cDNAs of multiple gastrointestinal tissues and PBL for qRT-PCR were selected from the human multiple tissue cDNA panels (BD Biosciences Clontech, Mountain View, CA).

Total RNA extraction and reverse transcription reaction
Total RNA from these cultured cells was extracted using the Easy Pure Total RNA Mini Kit (Bioman, Taipei, Taiwan) according to the manufacturer's instructions and fecal RNA was prepared as reportedly previously [16].
One microgram of cellular total RNA or fecal RNA was reverse transcribed to single-stranded cDNA using an oligo(dT) 12 primer with the ABI Reverse Transcriptase Kit (Applied Biosystems, Carlsbad, CA) according to the manufacturer's protocol. Synthesized cDNA could be used directly in the following qRT-PCR analyses.

qRT-PCR
The qRT-PCR for quantifying targets in multiple tissue cDNA, cellular cDNA, and fecal cDNA was performed using a TaqMan probe, from the Human Universal Probe Library (Roche Diagnostics, Mannheim, Germany), as described previously [17,18] except for fecal KIAA0247 (NM014734). To quantify fecal KIAA0247, the amount of each primer was elevated to 4 pmol in a 10 μL reaction volume. Each fecal sample run also included human reference cDNA (Clontech, Mountain View, CA) as a standard to estimate the relative expression levels in feces. The relative levels of expression of genes in various samples were determined by normalizing their expression to that of 18S ribosomal (r)RNA (X03205) [19]. The primers and universal probes used to quantify KIAA0247, cyclin A2 (NM001237), cyclin B1 (NM031966), and cyclin B2 (NM004701) are listed in Table 2.

Lentivirus-mediated RNA interference (RNAi) and overexpression of KIAA0247
The lentiviral constructs encoding the siKIAA0247 hairpin (pLKO.1-KIAA0247: TRCN0000134410) for gene silencing (shKIAA0247) or the KIAA0247 cDNA for gene overexpression (overKIAA0247) were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan. pLKO.1-Luc (TRCN0000072246) acted as a control (shLuc) for the previously mentioned two lentiviruses. Infection of each lentivirus into colonic cells was performed as described previously. Changes in the expression of KIAA0247 were determined using qRT-PCR.

Cell cycle analysis by flow cytometry
To determine the cellular effects of KIAA0247 in colonic cells, cell cycle analysis was performed using flow cytometry by analyzing the DNA content [20] of propidium iodide (PI)-stained nuclei as described previously [21]. Colonic cells transfected with shKIAA0247, shLuc, or overKIAA0247 were plated, at a density of 5 × 10 6 cells/well in 6-well dishes, and cultured for 24 h. These subconfluent cells were incubated with DNA analogue 5-FU (40 μM) (Sigma-Aldrich, St. Louis, MO) for another 24 h. The control cells were treated with medium alone. Thereafter, cells were trypsinized, washed twice with PBS, and fixed in 70% ethanol for 5 h at 4°C. These fixed cells were washed twice more with PBS, incubated with 1 μg/ml RNase A for 1 h at 37°C, and stained with 5 μg/ml PI for 1 h at room temperature. The percentage of cells in the G0/G1 phase, S phase, and G2/M phase were determined according to relative DNA content analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) [22].

Statistical analysis
Survival probabilities were estimated using the Kaplan-Meier method and compared using the log-rank test. Chi-squared or Fisher's exact tests were used for group comparisons. Kendall's tau-b correlation and linear regression analysis were applied to analyze correlations between the relative levels of fecal KIAA0247 and sizes of colonic tumor [23]. The Student's t test was used to compare the mRNA levels of cyclins in different groups. These statistical analyses were performed using SPSS 13.0 software (SPSS, Chicago, IL). The Medcalc software statistical package was employed to generate receiveroperating characteristic (ROC) curves. A p value < 0.05 was considered statistically significant.

Results
Expression of KIAA0247 in multiple gastrointestinal tissues and colonic cell lines qRT-PCR determined the expression of the uncharacterized gene, KIAA0247, in human gastrointestinal tissues and colonic cell lines. Results indicated that KIAA0247 ubiquitously expresses in gastrointestinal tissues and in peripheral blood leukocytes (PBL), with highest expression in PBL and lowest expression in the small intestine ( Figure 1). To obtain a more comprehensive understanding of the ability of KIAA0247 to reduce the G2/M population, qRT-PCR quantified the mRNA levels of genes belonging to the highly conserved cyclin family. As shown in Figure 5, CRC cells that overexpressed KIAA0247 simultaneously downregulated the expression of three cyclin genes (cyclin A2, cyclin B1, and cyclin B2) after 40 μM 5-FU treatment. For example, the mRNA level of cyclin A2 in 5-FU-treated KIAA0247-overexpressing cells was 69% of that in these cells without 5-FU treatment. However, this cyclin A2 downregulation was not detected in the shLuc cells. Cyclin B1 and cyclin B2 mRNA levels demonstrated similar trends after the same treatment.

Intracellular localization of KIAA0247 in colonic cells
Immunofluorescent staining of overexpressed KIAA0247 in HCT116 p53 -/cells identified that, under 5-FU-free conditions, the cytoplasm of CRC cells weakly expressed endogenous KIAA0247 (red fluorescence). This endogenous KIAA0247 demonstrated a tendency to move into the nucleus after treatment of cells with 40 μM 5-FU ( Figure 6A, indicated as white arrowhead). In the KIAA0247-overexpressing cells KIAA0247 clearly accumulated in the nucleus ( Figure 6B, indicated as white arrowhead). KIAA0247 overexpressed in the cytoplasm of most CRC cells without 5-FU treatment and accumulated in the nucleus after cellular DNA damage by 40 μM 5-FU.

Discussion
Cell cycle checkpoints are important control mechanisms which ensure the proper passage of genetic codes and genome stability [24,25]. One of the checkpoints, the G2/M checkpoint, blocks the entry into mitosis after DNA damage [26]. Many previous reports indicated that p53 can regulate the G2/M transition via induction of p21 and 14-3-3σ [27,28] or associated apoptosis [29]. The findings of two investigations indicated that a p53independent control also coordinates activation of the G2/M checkpoint [30,31].
This study demonstrated that KIAA0247 is under p53independent control in CRC cells despite speculation that it is a p53-responsive target [12]. The predicted p53-responsive elements in the KIAA0247 promoter region demonstrated no electrophoretic mobility shift with p53 protein in a gel shift assay (data not shown). Higher expression of KIAA0247 occurred in fecal samples from early-stage CRC patients with a greater fiveyear overall survival rate. Use of a p53-null CRC cell line at Dukes' stage B, HCT116 p53 -/-, as a target cell, excluded the influence of p53 on the cell cycle to correspond with the clinical findings.
Molecular markers are needed to assess CRC patients at Dukes' stage B who could benefit from adjuvant therapy [32]. Clinicians widely and routinely use 5-FU as one of the components in the therapeutic regimen [33,34] and a cytotoxic effect occurs during the intracellular metabolism of 5-FU. Such adjuvant chemotherapy is also beneficial to patients at Dukes' stage C [35]. In the present study's findings with a CRC cell line at Dukes' stage B, 40 μM 5-FU decreased the number of cells in G2/M in the presence of KIAA0247 expression. The presence of KIAA0247 expression and 5-FU also negatively modulated three common cell cycle activators. These data emphasize that early-stage CRC cells that are able to overexpress KIAA0247 could impede the progression of the cell cycle at the G2/M phase if an appropriate amount of 5-FU damages the cellular DNA.
The DNA damage response activates in precancerous lesions to permit CRC progression [36]. As reviewed by Wei et al., the prevention of DNA instability and uracil misincorporation might reduce the risk of the early    [24]. When testing the cDNA from multiple tissues, KIAA0247 expression was highest in PBL and at various levels in gastrointestinal tissues. These results suggest that fecal KIAA0247 provides a more useful therapeutic reference for early-stage CRC patients than blood KIAA0247. This translocation of KIAA0247 from the cytoplasm to the nucleus might be involved in the control of the G2/M checkpoint. The cellular effect of KIAA0247 is very similar to that of 14-3-3σ, whose overexpression could also cause G2/M cell cycle arrest, although 14-3-3σ is a p53-dependent inhibitor of G2/M progression [26].
In the group's previous studies of fecal gene expression, advanced microarray technology defined global changes in gene expression detectable in feces [18,38]. Results identified a novel gene for a homologue of the Drosophila headcase protein (HECA) as a classifier of early-stage CRC [38]. Comprehensive results for HECA and KIAA0247 indicate both fecal molecules could be markers of early-stage CRC. In this study, levels of fecal KIAA0247 inversely related to CRC tumor size with patients with high levels of fecal KIAA0247 having a longer five-year overall survival. Cell line results identifying that overexpressed KIAA0247 could move into the  nucleus and repress the progression of the cell cycle at the G2/M phase supported the clinical findings. The downregulation of three cyclins may partly cause this repression. However, the exact mechanism by which KIAA0247 operates remains unclear. A high priority is to study other factors that lead to growth arrest, senescence, and apoptosis.

Conclusions
This study describes and characterizes, for the first time, KIAA0247 from CRC patients using flow cytometry and qRT-PCR analysis. Results indicate that fecal KIAA0247 expression is a useful indicator of the need for 5-FU treatment in CRC, especially in cases diagnosed at early stages.