Conceived and designed the experiments: MJ LZ EK. Performed the experiments: MJ LZ. Analyzed the data: MJ LZ EEP EK. Contributed reagents/materials/analysis tools: MJ LZ EK. Wrote the paper: MJ LZ EEP EK.
The authors have declared that no competing interests exist.
KIAA0101 is a proliferating cell nuclear antigen-associated factor that is overexpressed in some human malignancies. Adrenocortical neoplasm is one of the most common human neoplasms for which the molecular causes are poorly understood. Moreover, it is difficult to distinguish between localized benign and malignant adrenocortical tumors. For these reasons, we studied the expression, function and possible mechanism of dysregulation of KIAA0101 in human adrenocortical neoplasm.
KIAA0101 mRNA and protein expression levels were determined in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 adrenocortical carcinoma (ACC). SiRNA knockdown was used to determine the functional role of KIAA0101 on cell proliferation, cell cycle, apoptosis, soft agar anchorage independent growth and invasion in the ACC cell line, NCI-H295R. In addition, we explored the mechanism of KIAA0101 dysregulation by examining the mutational status. KIAA0101 mRNA (9.7 fold) and protein expression were significantly higher in ACC (p<0.0001). KIAA0101 had sparse protein expression in only a few normal adrenal cortex samples, which was confined to adrenocortical progenitor cells. KIAA0101 expression levels were 84% accurate for distinguishing between ACC and normal and benign adrenocortical tumor samples. Knockdown of KIAA0101 gene expression significantly decreased anchorage independent growth by 80% and invasion by 60% (p = 0.001; p = 0.006). We found no mutations in KIAA0101 in ACC.
KIAA0101 is overexpressed in ACC. Our data supports that KIAA0101 is a marker of cellular proliferation, promotes growth and invasion, and is a good diagnostic marker for distinguishing benign from malignant adrenocortical neoplasm.
Adrenal neoplasms are one of the most common human neoplasms, often detected incidentally
ACC may be associated with hereditary cancer syndromes such as Beckwith-Wiedemann syndrome (associated with germline 11p15 chromosomal alterations leading to
Genome–wide gene expression profiling provides important insight into the molecular pathways that are dysregulated in cancer and may be involved in tumor initiation and progression. Since the molecular mechanism of adrenocortical carcinogenesis is poorly understood, cDNA microarray analysis of adrenocortical tumors has been used to reveal genes whose misexpression is associated with ACC
In this study, we addressed these issues and showed that KIAA0101 is overexpressed in ACC and a marker of cellular proliferation. Furthermore, reducing KIAA0101 expression in ACC resulted in growth suppression and invasion suggesting that KIAA0101 plays an oncogenic role in ACC.
The National Cancer Institute review board approved this research protocol after informed written consent was obtained from all participants. Adrenal tissues were snap frozen at the time of surgery and stored at −80°C. In this study, 112 human adrenocortical tissue specimens were analyzed including 21 normal adrenocortical tissues, 80 benign adrenocortical tumors, and 11 primary adrenocortical carcinomas(78 of the benign adrenocortical tumors and 11 of the primary adrenocortical carcinomas were previously analyzed)
The NCI-H295R ACC cell line (ATCC, Rockville, MD) was grown and maintained in DMEM supplemented with 1% ITS+ Premix (BD Biosciences, San Jose, CA), 2.5% Nu-Serum I (BD Biosciences), and 10,000 U/mL penicillin/streptomycin in a standard humidified incubator at 37°C in a 5% CO2 atmosphere. Twenty-four hours after cells were seeded in 24 and 6 well plates (4×10^4 cells in 0.5 ml and 1.6×10^5 cells in 2 ml), cells were transfected with a nonspecific negative control siRNA and with a KIAA0101 specific siRNA at a final concentration of 40 and 80 nM (AM4636 and AM46235, respectively, Applied Biosystems, Foster City, CA). The TransIT siQuest reagent (Mirus Bio, LLC) was used to deliver siRNA to the cells according to manufacturer instructions. The knockdown efficiency was similar for 40 and 80 nM. All in vitro assays were done with both 40 and 80 nM concentration of KIAA0101 specific siRNAs and nonspecific negative control.
RNA was isolated using the TRIzol reagent following the manufacturer's instructions (Invitrogen Inc., Carlsbad, CA). RNA quantity and quality was assessed by using NanoDrop (NanoDrop Technologies, Inc., Thermo Fischer) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively.
RIPA buffer was used to prepare tissue lysates and whole cell lysate was prepared with 1% SDS plus 10 mM Tris [pH 7.5] buffer. The protein concentration was determined using the BioRad RC DC protein assay (Hercules, CA).
Total RNA (125 ng) was reverse transcribed using the RT script cDNA synthesis kit (USB Corporation, Cleveland, OH). Real time quantitative PCR was used to measure mRNA expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. Normalized gene expression level = 2 – (Ct of gene of interest – Ct of GAPDH)×100%, where Ct is the PCR cycle threshold. The PCR primers and probes for KIAA0101 (Hs00207134_m1) and GAPDH (Hs99999905_m1) were obtained from Applied Biosystems (Assay-on-Demand kit®, Foster City, CA). All PCR reactions were performed in a final volume of 20 µl with 1 µl of cDNA template on an ABI PRISM®7900 Sequence Detection System (Applied Biosystems). The PCR thermal cycler condition was 95°C for 12 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All experiments were performed in triplicate and repeated three times.
Protein samples (40 µg) were separated in 4% to 20% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Western blotting was performed following standard procedures using primary mouse monoclonal antibodies, anti-KIAA0101 (Abcam; ab56773) at 1∶100 dilution and anti-β-actin (sc-81178, Santa Cruz Biotechnology Inc, Santa Cruz, CA) at 1∶2,000 dilution. Signal detection was performed using HRP conjugated secondary antibody and an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).
Tumor tissues were formalin fixed, embedded in paraffin, and 5 micron thick sections were cut for immunostaining. Sections were incubated with the primary anti-KIAA0101 mouse monoclonal antibody at 1∶300 dilution overnight at 4°C (Abcam; ab56773) followed by biotinylated secondary antibody for 1 hr at room temperature (1∶150; Vector Laboratories, Burlingame, CA, USA). Sections were developed using 3,3′-diaminobenzidine DAB as the chromogen (ABC elite kit, Vector Laboratories, Burlingame, CA, USA) and hematoxylin as counterstain. The sections were rehydrated and mounted with vectamount mounting medium (Vector Laboratories, Burlingame, CA, USA). The slides were scanned under Olympus light microscope (Nikon, Tokyo, Japan) and pictures were taken at 20X and 40X magnifications. A semiquantitative scoring system was used to analyze KIAA0101 expression; expression level was classified as no staining (0), <30% of cells staining (1), 30–50% cells (2) and >50% of cells. Two observers, who were blinded to the tumor type, independently scored each sample. The scores were averaged to obtain the final KIAA0101 expression score.
Immuunofluoroscence staining was done using primary anti-KIAA0101mouse monoclonal antibody at 1∶300 dilution overnight (Abcam; ab 56773) and rabbit anti-SF-1 (Millipore; 07-618, 2 µg/ml) at 4°C. The primary antibodies were detected with fluorophore conjugated with RedTX anti-rabbit IgG (Invitrogen, Carlsbad, CA) and FITC anti-mouse IgG secondary antibodies (Vector Laboratories). The slides were then rinsed and mounted with DAPI (4′,6-diamidino-2-phenylindole) mounting solution. Images were analyzed with a Zeiss Axioskop-2 microscope at 20X and 40X magnifications.
Genomic DNA was isolated from 1 mg of tumor and normal samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), eluted in a total volume of 200 µL and stored at −20°C. DNA concentrations were measured by UV absorbance using NanoDrop (NanoDrop Technologies, Inc., Thermo Fischer).
The KIAA0101 exons 1, 2, 3 and 4 were sequenced. PCR primers were designed for coding region using UCSC genome browser, Primer 3 integrated software. The primer sequences are listed in
Genomic Primer Sequences | PCR product size in bp |
|
|
F |
617 |
R |
|
|
|
F |
358 |
R |
|
|
|
F |
412 |
R |
Cells were seeded in a 96-well plate at a concentration of 5×10^3 cells per 100 µL culture medium in six replicates. At each timepoint, the media was aspirated from the well and the cells were immediately frozen at −80°C for 24 hours. The plates were thawed at room temperature, and prepared for cell number quantification using the CyQUANT™ assay kit (Invitrogen, Carlsbad, CA). The CyQuant assay was performed according to the manufacturer's instructions, and analyzed on a fluorometric microplate reader (Molecular Devices, Sunnyvale, CA) at 480 nm/520 nm.
KIAA0101 and negative control siRNA treated cells were harvested, ethanol-fixed overnight at 4°C, and resuspended in 1x PBS to a concentration of 1×10^6 cells/mL. Cells were treated with DNase-free RNase (100 µg/ml) for 20 min at 37°C. The cells were stained with propidium iodide at concentration of 50 µg/ml and samples were stored at 4°C. Flow cytometric analysis was performed on a Becton Dickinson FACScan (BD Biosciences, Franklin Lakes, NJ). Data files were generated for 20,000 events (cells) using the CellQuest software. The fraction of the total cell population present in each of the G1, S and G2/M cell cycle phases was obtained from ModFit LT software (Verity Software House, Inc.). Apoptosis analysis was performed using Annexin V staining (ApoAlert® Annexin V Apoptosis Kit) according to the manufacturer's instruction (Clontech, Mountain View, CA).
Two-layered soft agar assays were performed in six-well plates. The bottom layer of agar (2 ml/well) contained 0.5% agar (Difco Noble Agar, Becton, Dickinson and Company, Sparks, MD) in maintenance medium. Five days after siRNA transfection, cells were trypsinized, counted, and 50,000 cells were mixed with 1.3 ml of top agar solution supplemented with 10% Nu-serum (0.3% agar in culture media). Solidified agar was overlayed with 1 ml of culture media containing 10% Nu-serum. The plates were cultured at 37°C in 5% CO2, and the media was changed twice a week. After 16 days of culture, cell colonies were stained with 0.2% crystal violet solution and examined by microscopy. Colonies were counted in 5 different fields per well and confirmed by TotalLab Quant v11 software (
The extent of cell invasion was assessed using the BD BioCoat™ Matrigel™ Invasion Chamber (BD Biosciences, Bedford, MA), according to the manufacturer's protocol. A total of 1×10^5 cells were seeded onto the inserts (8-µM pore sized polycarbonate membrane) coated with a thin layer of Matrigel Basement Membrane Matrix (BD Biosciences). The inserts were placed into a 24-well plate with 10% serum-containing culture medium or media without serum. The plates were incubated for 48 hrs at 37°C. Cells that invaded the Matrigel matrix to the lower surface of the membrane were fixed and stained with Diff Quik Stain (Dade Behring, Newark, DE, USA) and counted under a light microscope. Four fields in four separate quadrants of each membrane were counted and averaged.
The continuous data was represented as mean ± standard deviation (S.D.). Two-tailed ANOVA multi-comparison
The expression of KIAA0101 mRNA was significantly higher in ACC as compared to normal adrenocortical tissue and benign adrenocortical tumors (12-fold higher than normal and 9-fold higher than in benign tumors, p<0.0001) (
Given the significant expression difference in mRNA between benign and malignant tumors, we were interested in assessing if KIAA0101 was an accurate diagnostic predictor of tumor type. KIAA0101 expression was 84% accurate for distinguishing between ACC and normal and benign adrenocortical tumor samples (number of true positive and negative results divided by the total sample number using a cutoff level of 1.5). The area under the receiver operator characteristic (ROC) curve (AUC) was 0.78 for KIAA0101 mRNA expression (
In addition to high mRNA expression, KIAA0101 protein expression was also elevated in ACC as compared to normal adrenal cortex and benign adrenocortical tumor samples. On Western blot, we found higher expression in ACC (n = 3) than benign adrenocortical tumors (n = 13), and in normal adrenal cortex samples (n = 5) (
We observed only sparse KIAA0101 staining in a few normal adrenal cortex samples, which was confined to the subcapsular cortex region (
To determine whether the KIAA0101 overexpression was a consequence of gene sequence variations, we performed mutation analysis in the KIAA0101. We found one de
Variations | Position | Region | Distribution | ||
Normal (n = 8) |
Benign (n = 75) |
Malignant (n = 10) | |||
88T>C | EXON 1–2 | 5′ UTR | 1/8 (12.5%) | 5/75 (6.6%) | ND |
410T>C | EXON 1–2 | Intronic | 1/8 (12.5%) | ND |
ND |
15360T>C | EXON 4 | Intronic | ND |
1/75 (1.3%) | ND |
*Percentage of mutation is for the samples which were analyzed by category and available for sequencing.
**ND (not detected).
Given the significant overexpression of KIAA0101 in ACC (
Given the paradoxical effects, a modest increase in cellular proliferation and G1 arrest, we used additional functional assays to better understand the phenotypic changes associated with KIAA0101 knockdown. Specifically tumorogenicity and metastatic potential were evaluated. KIAA0101 knockdown in NCI-H295R cells reduced the number of colonies that were able to grow in soft agar by almost 80% (p = 0.001) at 16 days of culture (
In this study, we examined KIAA0101 expression and function in ACC. Our results indicate that KIAA0101 is overexpressed in ACC, is a good diagnostic marker for ACC, and is a marker of cellular proliferation. Suppressing KIAA0101 expression in adrenocortical carcinoma cells resulted in suppression of growth and invasion, suggesting that it plays a growth promoting role in ACC.
Previous studies have demonstrated altered expression of KIAA0101 in human malignancies and absent or low expression in normal tissue
Although we found that KIAA0101 protein expression was higher in malignant and benign tumors as compared to normal samples, we did detect KIAA0101 expression in a subset of normal samples. Its expression was confined to the subcapular region of the adrenal cortex and overlapped the SF-1 positive staining cells. In this region, SF-1 positive cells are hypothesized to be adrenal progenitor cells
Little is known about the function of the KIAA0101 gene in tumor cell biology. Previously, studies have suggested that KIAA0101 is involved in the regulation of DNA replication, cell cycle, apoptosis and cellular proliferation
While most genes upregulated in malignancy have a growth promoting function, one of the most common tumor suppressor genes in human malignancy, p53, is overexpressed and has a tumor suppressor function because of inactivating dominant negative mutations. In our study, KIAA0101 was also overexpressed, therefore, we hypothesized that sequence alterations may reveal the reason for dysregulation. However, we did not detect the presence of such mutations in coding region of KIAA0101 consistent with one other study
In summary, the results of our study provide the first evidence that KIAA0101 is a marker of cell proliferation and is overexpressed in ACC, therefore it suggests that its expression could be used as a molecular marker for the diagnosis of ACC. In addition, our functional studies suggests, it has modest effect on the cell proliferation and cell cycle progression. However, the majority of the results support that it is a potential tumor-promoting gene and contributes to metastatic potential in vitro.