Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters

  • Authors:
    • Anna E. Kowalczyk
    • Bartlomiej E. Krazinski
    • Janusz Godlewski
    • Jolanta Kiewisz
    • Przemyslaw Kwiatkowski
    • Agnieszka Sliwinska-Jewsiewicka
    • Jacek Kiezun
    • Piotr M. Wierzbicki
    • Gabriel Bodek
    • Marian Sulik
    • Zbigniew Kmiec
  • View Affiliations

  • Published online on: June 29, 2015     https://doi.org/10.3892/ijo.2015.3067
  • Pages: 951-962
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Abstract

Pleomorphic adenoma gene-like 1 gene (PLAGL1) encodes a zinc-finger nuclear transcription factor which promotes apoptosis and cell cycle arrest. Loss or downregulation of its expression has been observed in various human neoplasms. This study compared PLAGL1 expression in colorectal cancer (CRC) tissue and colon mucosa of healthy subjects at the mRNA and protein levels, and estimated its prognostic value. The PLAGL1 mRNA levels were also determined in CRC cell lines. We collected paired tumor tissue and unchanged mucosa of the large intestine from 121 CRC patients as well as 72 colon biopsies of healthy subjects obtained during screening colonoscopy. PLAGL1 mRNA levels were determined by quantitative PCR, while PLAGL1 protein expression was estimated by western blotting and immunohistochemistry. PLAGL1 mRNA level in tumor tissue was ~2-fold lower than in samples of corresponding unchanged tissues and biopsies of healthy colon mucosa. Downregulated expression of PLAGL1 mRNA was also observed in all tested CRC cell lines. Although the average content of PLAGL1 protein did not differ significantly between tumor and unchanged tissues of CRC patients or colon mucosa of healthy individuals, the decreased PLAGL1 protein levels in tumor specimens correlated with lymph node involvement, the presence of metastases and higher TNM disease stage. The PLAGL1 expression level did not correlate significantly with patient overall survival; however, the hazard ratio for patients whose tumor tissues showed reduced PLAGL1 immunohistochemical staining was twice higher than in patients with increased PLAGL1 immunoreactivity. In conclusion, these results suggest that dysregulation of PLAGL1 expression may be involved to some extent in the progression of CRC, but the so far collected patient survival data do not confirm applicability of the PLAGL1 expression level as a prognostic factor in CRC.

Introduction

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide (1). The main cause of death in CRC patients is distant metastasis. Identification of factors and mechanisms underlying CRC development and progression, as well as detection of early-stage disease markers could effectively prevent death from CRC.

Alterations in genes are one of the mainsprings of CRC. This includes inactivation of tumor suppressor genes by genetic and epigenetic mechanisms. Tumor suppressor genes are typically inactivated by mutation, deletion, or promoter methylation which silences gene expression (2). Pleomorphic adenoma gene-like 1 gene (PLAGL1, also known as ZAC1 or LOT1) is the candidate tumor suppressor gene that encodes a protein with antiproliferative activity which is due to the induction of apoptosis and cell cycle arrest (3).

PLAGL1 is widely expressed in normal tissues (4), and localized on chromosome 6q24-25, a region maternally imprinted (5), and frequently deleted in many tumors (6). It was shown that PLAGL1 may be transcriptionally silenced by DNA methylation of CpG islands and histone deacetylation (5). Altered expression of PLAGL1 gene was observed in several types of human cancers and tumor-derived cell lines such as breast (7), ovary (8,9), and prostate tumors (10), as well as in nonfunctioning pituitary adenomas (11), head and neck squamous cell carcinoma (12), and basal cell carcinoma (6). Expression profiling of colorectal samples of CRC patients with cDNA microarrays also demonstrated altered expression of PLAGL1 mRNA in CRC as compared to proximal non-cancerous colorectal mucosa (13), but there is no previous comparison of PLAGL1 expression in CRC tissue and colon mucosa of healthy subjects at the mRNA and protein levels. Moreover, the prognostic value of the PLAGL1 expression level in CRC progression and patient survival remains unknown. Therefore, the main objective of our study was to analyze and compare PLAGL1 gene expression in samples of tumor and unchanged colorectal tissues of CRC patients as well as in mucosal colon biopsies in a group of healthy subjects by quantitative real-time PCR (qPCR), western blotting and immunohistochemical (IHC) techniques. Furthermore, we have examined levels of PLAGL1 mRNA in CRC cell lines. To estimate the prognostic significance of the PLAGL1 expression level, we analyzed correlations between the expression level of PLAGL1 and clinicopathological features of CRC patients, as well as the overall survival of the patients.

Materials and methods

Ethics

This study was performed in accordance with the ethical standards, and approved by the Bioethics Committee of the University of Warmia and Mazury in Olsztyn (decision no. 3/2010 and 34/2010), and informed written consent regarding the use of tissue was obtained from each patient included in the study.

Patients and collection of colorectal samples

The specimens were collected at the Hospital of the Ministry of Internal Affairs and Administration in Olsztyn from 2010 to 2013. The study included 121 patients with CRC (demographic and clinicopathological data are presented in Table I). None of the CRC patients had a second neoplastic disease or had previously undergone chemo- or radiotherapy. The control group consisted of 72 healthy individuals (24 males and 48 females, average age 57.2±6.65 years, range 36–82 years; mean ± SD) who underwent colonoscopy as a part of a routine screening for CRC (within the National screening program for early detection of colorectal cancer). Control subjects had no family history of CRC. None of the CRC patients or control subjects suffered from inflammatory bowel disease. Clinical and demographic data were obtained at the time of enrollment. Data on the overall survival were collected for all patients. Median follow-up time was 36.1 months.

Table I

Demographic and clinicopathological characteristics of studied CRC patients.

Table I

Demographic and clinicopathological characteristics of studied CRC patients.

ParameterNo. of casesPercentage (%)
Total121100.0
Gender
 Male6755.4
 Female5444.6
Age (years)
 ≤676049.6
 >676150.4
Localization
 Cecum, ascending, and transverse colon4436.4
 Descending and sigmoid colon2924.0
 Rectum4839.7
Depth of invasion (pT status)
 T143.3
 T21613.2
 T38066.1
 T42117.4
Lymph nodes (pN status)
 N06352.1
 N13831.4
 N22016.5
Metastasis (pM status)
 M010687.6
 M11512.4
TNM stage
 I1814.9
 II4133.9
 III4738.8
 IV1512.4

CRC samples were obtained during the partial surgical resection of the large intestine, and control group specimens were collected during colonoscopy. In CRC patient group, two types of matched samples were taken within 20 min after tumor resection: i) tumor tissue and ii) macroscopically unchanged mucosa from a distant part of resected large intestine. Specimens were immediately cut in two pieces for qPCR and western blot analyses, frozen in liquid nitrogen, and stored at −80°C, whereas for routine histological evaluation and immunohistochemistry, the samples were fixed in 10% neutral buffered formalin and further processed into paraffin blocks. In the control group of healthy patients, one biopsy was fixed in 10% neutral buffered formalin for routine histological examination, and two specimens from the adjacent location to the biopsy site were collected for qPCR or western blot assays.

Cell lines

Human CRC cell lines with different characteristics (HT-29, SW-480, LoVo) (14) and a control line, the CCD 841 CoN cell line (epithelial-like, established from normal colonic tissue) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured under conditions recommended by the manufacturer, and were harvested at ~80% confluence.

Total RNA extraction and reverse transcription

Total RNA was extracted from cell lines as well as paired samples of cancer tissue and unchanged mucosa derived from 121 CRC patients and 40 colonoscopic biopsies of healthy subjects using a Total RNA Prep Plus kit (A&A Biotechnology, Gdynia, Poland), following the manufacturer's protocol. Isolated RNA was quantified with spectrophotometry (NanoDrop 1000, NanoDrop products, Wilmington, DE, USA). Reverse transcription was carried out in a vial containing 20 μl reaction mixture of 2 μg of total RNA, 0.5 μg of oligo dT primers (Sigma-Aldrich, St. Louis, MO, USA), 200 U of RevertAid™ Reverse Transcriptase, 20 U of RiboLock™ RNase inhibitor, and 1 mM of each dNTP (all Thermo Scientific, Waltham, MA, USA). Reactions were performed according to the manufacturer's instructions, and resulting complementary DNAs (cDNAs) were stored at −20°C after 10-fold dilution with nuclease-free water to be used as the template in qPCR analysis.

Real-time quantitative PCR

Quantification of PLAGL1 gene expression was carried out using ABI 7500/7500 Fast Real-Time PCR system (Life Technologies, Applied Biosystems, Foster City, CA, USA). Hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene was used as an internal control to normalize the transcript levels of PLAGL1. The levels of PLAGL1 and HPRT1 cDNAs in collected isolates were determined using TaqMan®Fast Advanced Master Mix and a respective TaqMan® Gene Expression assay (all from Life Technologies, Applied Biosystems) according to the manufacturer's instructions. The primers and TaqMan probes used were PLAGL1: Hs00414677_m1, and HPRT1: Hs02800695_m1. All samples were amplified in duplicates using the following conditions: polymerase activation for 20 sec at 95°C, followed by 40 cycles of denaturation at 95°C for 3 sec and annealing/extension at 60°C for 30 sec. No template control reactions were performed for each qPCR run. Standard curves consisting of serial dilutions of the appropriate cDNA were used to control the efficiency of qPCR reactions. Relative quantification of PLAGL1 expression was evaluated using the ΔΔCt method (15). The fold change in the relative PLAGL1 gene expression was determined by calculating the 2−ΔΔCt value. Fold increase above 1 (2−ΔΔCt >1) indicated PLAGL1 overexpression in CRC tissue, and fold decrease under 1 (2−ΔΔCt <1) indicated PLAGL1 downregulation.

Protein extraction and western blot analysis

Paired samples of tumor tissue and unchanged mucosa derived from 95 CRC patients and 32 colonoscopic biopsies were homogenized in RIPA lysis buffer supplied with 1:100 protease inhibitor cocktail, 1:100 phosphatase inhibitor cocktail 2 and 5 mM EDTA (all from Sigma-Aldrich). Homogenates were briefly centrifuged to remove tissue debris. Then, samples were centrifuged twice at 9,000 × g for 10 min at 4°C. After centrifugation, supernatants were collected and the total protein content was determined by the Bradford method (16). Samples were aliquoted and stored at −80°C until further analyses.

To determine the level of PLAGL1 protein in tissue lysates, the SDS-PAGE followed by western blotting assays were performed. Isolated protein samples were denatured for 5 min at 95°C and loaded on polyacrylamide gel (40 μg/lane). Gels were run at the 10 mA/gel during migration in the stacking gel and 15 mA/gel in the separating gel (10%). Proteins were transferred onto PVDF membrane (western blotting membrane, Roche, Mannheim, Germany). Blots were blocked in 5% non-fat dry milk dissolved in Tris-buffered saline pH 7.5 with 0.1% Tween-20 (TBS-T) followed by overnight incubation at 4°C with respective primary antibody. Rabbit anti-human monoclonal antibodies against PLAGL1/ZAC (diluted 1:1,000 in TBS-T; #ab129063, Abcam, Cambridge, UK), and polyclonal antibodies anti-actin (ACTB; 1:100; #A2066, Sigma-Aldrich) were used. ACTB level was used as the internal protein load control. After the incubation, primary antibodies were washed out with TBS-T. Then, the membranes were treated with the specific HPR-conjugated goat anti-rabbit IgG secondary antibodies (1:40,000; #A0545, Sigma-Aldrich) for 90 min at room temperature (RT), developed with an enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific), and visualized with G:BOX iChemi XR imaging system (Syngene, Cambridge, UK). For the negative control, a primary antibody was omitted and substituted with phosphate-buffered saline (PBS). Protein extracts from 293T cells or HeLa cells (both from Abcam) were used as the positive controls for PLAGL1 immunoblotting. Molecular weight standard (Spectra Multicolor Broad Range Protein Ladder, Thermo Scientific) was included into each blotting experiment to confirm the molecular weight of detected bands. Band intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA) (17).

PLAGL1 protein optical density (OD) was normalized on the basis of ACTB protein OD. OD ratios between tumor and the corresponding unchanged tissue of CRC patients were calculated. The ratios >1 indicated that the expression of PLAGL1 protein was upregulated in CRC tissue while those <1 were regarded as downregulated.

Immunohistochemistry

PLAGL1 immunoreactivity was analyzed in matched tumor and unchanged colorectal tissues of 60 CRC patients. Immunohistochemistry was performed on 4-μm-thick paraffin sections. Sections were subjected to antigen retrieval procedure by microwaving for 20 min in retrieval solution buffer, pH 6.0 (Leica, Wetzlar, Germany), followed by the incubation in 3% H2O2 in methanol for 10 min, and next in 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 30 min. The sections were incubated overnight at 4°C with rabbit anti-human monoclonal antibody against PLAGL1/ZAC (diluted 1:2,000 in PBS; #ab129063, Abcam). After washing with PBS, the sections were treated with HRP-conjugated secondary antibody (ready-to-use dilution; ImmPRESS Universal reagent Anti-Mouse/Rabbit Ig, Vector Laboratories) for 30 min at RT. Then, the sections were immersed in 3′,3′-diaminobenzidine (DAB; Dako, Glostrup, Denmark), counterstained with Harris' haematoxylin (Sigma-Aldrich), dehydrated in ethanol, cleared in xylene, and mounted with DPX (Sigma-Aldrich). For each set of staining, the negative controls were performed by omitting the primary antibody.

The PLAGL1 immunostained sections were evaluated using Olympus BX41 light microscope (Olympus, Tokyo, Japan) by an independent pathologist in a blinded manner regarding the clinical data of the patients. Immunoreactivity of PLAGL1 was assessed in cancer cells of CRC sections and enterocytes of the unchanged colon mucosa using the scale based on the reaction intensity (0, no reaction; 10, ≤10%; 30, 11–30%; 60, 31–60%; 80, 61–80% and 100, >80%). Ratios in PLAGL1 score between tumor cells and cells of the matched unchanged tissue of CRC patients were calculated. The ratios >1 indicated that the immunoreactivity of PLAGL1 in CRC tissue was upregulated while those <1 were regarded as down-regulated.

Statistical analyses

Statistical analyses were performed using Prism 6 (GraphPad, La Jolla, CA, USA) and Statistica v.10 (StatSoft, Tulsa, OK, USA) software. The differences in mRNA and protein levels between matched tumor and unchanged samples of CRC patients were examined by the Wilcoxon matched-pairs test, whereas differences between colon mucosa biopsies of healthy subjects and tissues of CRC patients as well as between cell lines were assessed by the Mann-Whitney U test. The correlations between the demographic, clinicopathological, and molecular parameters were analyzed by the Fisher's exact and Chi-square tests and confirmed using the Mann-Whitney U test and Kruskal-Wallis test. Survival curves were plotted using Kaplan-Meier method. The statistical significance of differences in survival between groups of patients was evaluated using the log-rank test and confirmed by Cox regression method. In all the analyses, results were considered statistically significant when p<0.05.

Results

PLAGL1 mRNA expression in CRC tissues and cell lines is downregulated

To determine the expression of PLAGL1 at the mRNA level, matched tumor and unchanged tissues derived from CRC patients and colonic biopsies of healthy group were subjected to qPCR analysis. PLAGL1 mRNA was found in all studied tissue samples of CRC patients and colonic biopsies of healthy individuals. Among the 121 tumor specimens tested, the relative PLAGL1 mRNA level (tumor tissue vs. matching unchanged mucosa of CRC patients) was decreased in 87 (71.9%) tumors while it was increased in 34 (28.1%) cases (Table II). The expression of PLAGL1 mRNA was significantly decreased in the tumor tissues when compared to unchanged tissue of CRC patients and the colon mucosa of healthy individuals (0.44±0.02 vs. 0.83±0.02 and 1.00±0.04, respectively; p<0.0001; Fig. 1). The levels of PLAGL1 mRNA in unchanged tissues of CRC patients did not differ significantly from those in colonic biopsies of the healthy group (0.83±0.02 vs. 1.00±0.04; p>0.05; Fig. 1).

Table II

Associations between demographic and clinicopathological features of studied CRC patients and the relative mRNA expression of PLAGL1 in colorectal tumor tissues.

Table II

Associations between demographic and clinicopathological features of studied CRC patients and the relative mRNA expression of PLAGL1 in colorectal tumor tissues.

PLAGL1 mRNA levels in tumor vs. unchanged tissues of CRC patients

ParameterNo. of casesPercentage (%)Down (ratio <1)Percentage (%)Up (ratio >1)Percentage (%)p-values
Total121100.08771.93428.1
Gender
 Male6755.45277.61522.40.1548
 Female5444.63564.81935.2
Age (years)
 ≤676049.64066.72033.30.2295
 >676150.44777.01423.0
Localization
 Cecum, ascending, and transverse colon4436.43272.71227.30.0800
 Descending and sigmoid colon2924.02586.2413.8
 Rectum4839.73062.51837.5
Depth of invasion (pT status)
 T1+T22016.51365.0735.00.5864
 T3+T410183.57473.32726.7
Lymph nodes (pN status)
 N06352.14266.72133.30.2259
 N1+N25847.94577.61322.4
Metastasis (pM status)
 M010687.67570.83129.20.5533
 M11512.41280.0320.0
TNM stage
 I+II5948.83966.12033.90.3484
 III+IV6251.24877.41422.6

QPCR analysis was also used to determine the expression of PLAGL1 gene in CRC cell lines and the control line established from normal colonic tissue. PLAGL1 mRNA levels in HT-29, LoVo and SW-480 cells were significantly lower than those in the CCD 841 CoN cell line (0.0004±0.0003, 0.024±0.0006 and 0.047±0.0087, respectively, vs. 1.00±0.0087; p<0.05; Fig. 2). Among the CRC cell lines the lowest level of PLAGL1 expression was observed in HT-29 cells (p<0.05; Fig. 2). The level of PLAGL1 mRNA in the LoVo cell line was significantly lower than that in SW480 cells (p<0.05; Fig. 2).

The lack of correlation between PLAGL1 mRNA expression in CRC and demographic or clinicopathological features

To assess the impact of PLAGL1 expression at the mRNA level on CRC pathogenesis, the relationships between PLAGL1 mRNA content and selected demographic and clinicopathological parameters were tested (Table II). The relative PLAGL1 mRNA level did not correlate with the parameters gender, age, tumor localization, TNM disease stage, depth of invasion, lymph node involvement, or the presence of metastases (p>0.05; Table II).

Heterogeneous expression of PLAGL1 protein in CRC tissues

To determine the expression of the PLAGL1 gene at the protein level, matched tumor and unchanged tissues derived from CRC patients and colonic biopsies of healthy subjects were analyzed by western blotting. PLAGL1 protein was found in all studied tissues of CRC patients and in 30/32 (93.75%) colonic biopsies of healthy subjects. The average content of PLAGL1 protein did not differ significantly between tumor and unchanged tissues of CRC patients or the colon mucosa of healthy individuals (3.47±0.27 vs. 3.12±0.24 and 2.73±0.26, respectively; p>0.05; Fig. 3). Among 95 tumor tissue specimens tested, the relative content of PLAGL1 protein (tumor tissue vs. matching unchanged mucosa of CRC patients) was downregulated in 47 (49.5%) tumors while it was upregulated in 48 (50.5%) cases (Table III).

Table III

Associations between demographic and clinicopathological features of studied CRC patients and the relative PLAGL1 protein levels (assessed by western blotting) in colorectal tumor tissues.

Table III

Associations between demographic and clinicopathological features of studied CRC patients and the relative PLAGL1 protein levels (assessed by western blotting) in colorectal tumor tissues.

PLAGL1 protein levels in tumor vs. unchanged tissues of CRC patients

ParameterNo. of casesPercentage (%)Down (ratio <1)Percentage (%)Up (ratio >1)Percentage (%)p-values
Total95100.04749.54850.5
Gender
 Male5355.82649.12750.91.0000
 Female4244.22150.02150.0
Age (years)
 ≤674749.52042.62757.40.2204
 >674850.52756.32143.8
Localization
 Cecum, ascending, and transverse colon3233.71650.01650.00.9931
 Descending and sigmoid colon2223.21150.01150.0
 Rectum4143.22048.82151.2
Depth of invasion (pT status)
 T1+T21717.9741.21058.80.5939
 T3+T47882.14051.33848.7
Lymph nodes (pN status)
 N05254.71936.53363.50.0074a
 N1+N24345.32865.11534.9
Metastasis (pM status)
 M08185.33543.24656.80.0037a
 M11414.71285.7214.3
TNM stage
 I + II4951.61632.73367.30.0010a
 III+IV4648.43167.41532.6

a Statistically significant p-values.

Heterogeneous PLAGL1 immunohistochemical staining in CRC patient tissues

PLAGL1 immunoreactivity was observed in enterocytes (Fig. 4A) as well as cancer cells of the analyzed tissues (Fig. 4B, D, E, G, H and J–M). Immunoreactivity of the PLAGL1 protein was noted in cancer cells in 49/60 (81.7%) of the analyzed CRC cases, whereas in enterocytes of unchanged large intestine tissue its expression was observed in 57/60 (95%) cases. The average intensity of PLAGL1 immunostaining did not differ significantly between tumor and unchanged tissues of CRC patients (18.5±2.07 vs. 24.83±2.85, respectively; p>0.05). Among 60 tumor tissue specimens tested, the relative intensity of PLAGL1 staining was decreased in 27 (45%) tumors and elevated in 15 (25%) cases, while 18 (30%) tumor samples demonstrated similar level of PLAGL1 immunoreactivity as cells of the corresponding unchanged tissues (Table IV).

Table IV

Associations between demographic and clinicopathological features of studied CRC patients and the relative PLAGL1 immunoreactivity in tumor cells.

Table IV

Associations between demographic and clinicopathological features of studied CRC patients and the relative PLAGL1 immunoreactivity in tumor cells.

PLAGL1 immunoreactivity in tumor vs. unchanged tissues of CRC patients

ParameterNo. of casesPercentage (%)Down (ratio <1)Percentage (%)Up (ratio >1)Percentage (%)p-values
Totalb42100.02764.31535.7
Gender
 Male2047.61470.0630.00.5311
 Female2252.41359.1940.9
Age (years)
 ≤671842.91055.6844.40.3465
 >672457.11770.8729.2
Localization
 Cecum, ascending, and transverse colon1228.6975.0325.00.5802
 Descending and sigmoid colon921.4666.7333.3
 Rectum2150.01257.1942.9
Depth of invasion (pT status)
 T1+T2716.7342.9457.10.2252
 T3+T43583.32468.61131.4
Lymph nodes (pN status)
 N02457.11145.81354.20.0081a
 N1+N21842.91688.9211.1
Metastasis (pM status)
 M03890.52463.21436.81.0000
 M149.5375.0125.0
TNM stage
 I + II2354.81043.51356.50.0031a
 III+IV1945.21789.5210.5

a Statistically significant p-values.

b Tumor samples (n=18) showing the same intensity of immunostaining as cells of the corresponding unchanged tissue were excluded from this analysis.

Correlations between PLAGL1 protein content in CRC and demographic or clinicopathological features

Possible correlations of PLAGL1 expression at the protein level with selected demographic and clinicopathological parameters were analyzed based on the results obtained by western blot and immunohistochemical analyses. The relative PLAGL1 protein levels in tumor specimens did not correlate with the patient gender, age, tumor localization, and depth of invasion. However, PLAGL1 protein content was lower in tumor specimens derived from patients diagnosed with: i) lymph node involvement [p=0.0074, the Fisher's exact test, Table III; confirmed by the Mann-Whitney U test: 1.66±0.18 vs. 1.21±0.17 (N0 vs. N1+N2), p=0.0122, Fig. 5A], ii) the presence of metastases [p=0.0037, the Fisher's exact test, Table III; confirmed by the Mann-Whitney U test: 1.57±0.14 vs. 0.80±0.16 (M0 vs. M1), p=0.0309, Fig. 5B], and iii) a higher TNM disease stage [p=0.001, the Fisher's exact test, Table III; confirmed by the Mann-Whitney U test: 1.73±0.19 vs. 1.17±0.16 (I+II vs. III+IV), p=0.0014, Fig. 5C].

Similarly, decreased intensity of PLAGL1 immunohistochemical staining in tumor specimens was associated with lymph node involvement [p=0.0081, the Fisher's exact test, Table IV; confirmed by the Mann-Whitney U test: 1.10±0.14 vs. 0.45±0.11 (N0 vs. N1+N2), p=0.0020, Fig. 4D–F] and a higher TNM disease stage [p=0.0031, the Fisher's exact test, Table IV; confirmed by the Mann-Whitney U test: 1.12±0.14 vs. 0.46±0.10 (I+II vs. III+IV), p=0.0014, Fig. 4J–N].

Expression of the PLAGL1 gene and overall survival of the patients

To estimate the significance of the PLAGL1 expression level as a prognostic factor, all patients were followed up for 36.1 months. During this observation period, 43 (35.5%) patients died.

The levels of PLAGL1 mRNA and protein expression, or the intensity of PLAGL1 immunostaining did not correlate significantly with the patient overall survival (Table V; Fig. 6A–C); however, the hazard ratio (HR) for patients whose tumor tissues showed reduced immunostaining of the PLAGL1 protein was twice higher than in patients whose tumor tissues revealed increased PLAGL1 immunoreactivity (Table V). Of the analyzed demographic and clinicopathological parameters, advanced age at diagnosis (>67 years; p=0.0014; Table V and Fig. 6D), depth of invasion (p=0.0477; Table V and Fig. 6E), lymph node involvement (p<0.0001; Table V and Fig. 6F), the presence of metastases (p<0.0001; Table V and Fig. 6G), and a high TNM disease stage (p<0.0001; Table V and Fig. 6H) were associated with poor patient outcome.

Table V

Analysis of overall survival of CRC patients in relation to their demographic, clinicopathological and molecular characteristics.

Table V

Analysis of overall survival of CRC patients in relation to their demographic, clinicopathological and molecular characteristics.

ParameterDeaths/casesPercentage (%)Hazard ratio95% CI of HRp-values
Gender
 Male23/6734.31.0070.5530–1.8330.9823
 Female20/5437.0(1.00)
Age (years)
 ≤6713/6021.70.3610.2055–0.68280.0014a
 >6730/6149.2(1.00)
Localization
 Cecum, ascending, and transverse colon12/4427.30.6910.3437–1.4070.3125
 Descending and sigmoid colon12/2941.41.3160.6286–2.8290.4541
 Rectum19/4839.6(1.00)
Depth of invasion (pT status)
 T1+T23/2015.00.3250.2144–0.99160.0477a
 T3+T440/10139.6(1.00)
Lymph nodes (pN status)
 N012/6319.00.2810.1579–0.5299<0.0001a
 N1+N231/5853.4(1.00)
Metastasis (pM status)
 M030/10628.30.148 0.003700–0.04558<0.0001a
 M113/1586.7(1.00)
TNM stage
 I2/1811.10.0630.01695–0.1578<0.0001a
 II6/4114.60.084 0.004435–0.05342<0.0001a
 III22/4746.80.2660.04809–0.3322<0.0001a
 IV13/1586.7(1.00)
PLAGL1 mRNA
 Downregulated33/8737.91.4480.7378–2.7030.2997
 Upregulated10/3429.4(1.00)
PLAGL1 protein
 Downregulated18/4738.31.5850.7928–3.1940.1916
 Upregulated14/4829.2(1.00)
PLAGL1 relative immunoreactivity
 Downregulated11/2740.72.0860.6393–5.6510.2482
 Upregulated3/1520.0(1.00)

a Statistically significant p-values; the log-rank test.

Discussion

Allelic loss at long arm of chromosome 6 occurs in many types of human cancers, suggesting the presence of at least one tumor suppressor gene within this region (8,9,18). The inactivation of tumor suppressor genes that control a variety of cellular processes including the cell cycle and apoptosis, can promote tumor formation. Genes which are differentially expressed in tumors as compared to healthy tissues may be considered as potential biomarkers for cancer detection, prognostic factors, or therapeutic targets. PLAGL1, localized at 6q24-q25, encodes a transcription factor with antiproliferative potential (19) and is ubiquitously expressed in normal tissues (5), with especially abundant expression shown in the pituitary and adrenal glands, as well as in the kidney (20). Expression of this gene was also confirmed in the colon tissue (20), therefore, we undertook the present study to compare the level of PLAGL1 expression in tissues of CRC patients and the healthy colon mucosa, and also to estimate its prognostic value. This is the first comprehensive report describing PLAGL1 expression in CRC and healthy colon tissues at both mRNA and protein levels, examined by the combination of three different techniques (qPCR, western blotting, and IHC), as well as in relation to demographic and clinicopathological features of CRC patients and their overall survival.

Previous studies revealed that PLAGL1 expression is frequently lost or decreased in several human cancer types (612); however, in the case of salivary gland tumors, Enlund et al (21) found no significant changes in the expression of this gene assessed at the mRNA level. We found PLAGL1 gene expression in all studied tissues of CRC patients and healthy subjects; however, 72% of CRC tumors showed decreased PLAGL1 mRNA levels. The results of our study are corroborated by the findings of Dai et al (13), who observed downregulated PLAGL1 expression in CRC using cDNA microarray. Our study is the first to report downregulated PLAGL1 gene expression in commonly used CRC cell lines as opposed to non-cancerous cells. Previous studies revealed undetectable or reduced levels of PLAGL1 mRNA in breast tumor cell lines (7), and human ovarian carcinoma cell lines (9). We observed the lowest level of PLAGL1 expression in the HT-29 cell line, which had been shown to have a high metastatic capacity (14). Xenografts, which were established by injection of HT-29 cells into mice, produced lymph node metastases in 83% of tumor-bearing animals (14). Moreover, the level of PLAGL1 mRNA was lower in the LoVo cell line in comparison to the SW-480 cells. The LoVo cell line corresponds to cells of Dukes' type C, grade IV, colorectal adenocarcinoma that was derived from metastatic site, whereas SW-480 cells are derived from Dukes' type B, colorectal adenocarcinoma (according to ATCC). These results suggest that downregulated PLAGL1 expression may be associated with the progression of CRC. In prostate cancer it was found that loss of PLAGL1 expression is associated with progression from benign to metastatic prostate tumors (10).

So far, little is known about the clinical significance of the altered PLAGL1 expression in cancers. Our study failed to reveal any relationship between the level of PLAGL1 mRNA and clinicopathological and demographic features of the CRC patients. Interestingly, we found significant correlations between the reduced PLAGL1 protein content in CRC tissues and unfavorable clinical parameters. Although we detected no significant differences between the average content of PLAGL1 protein in samples of healthy colon mucosa and CRC, we observed significant downregulation of PLAGL1 protein expression in tumor specimens derived from patients diagnosed with lymph node involvement, the presence of metastases, and advanced TNM stage. These findings support the suppressor role of PLAGL1 and suggest that altered expression of PLAGL1 gene may promote progression of CRC. Moreover, our assumptions are in agreement with observations of Jarmalaite et al (22), who suggested an involvement of PLAGL1 loss in a more aggressive course of pheochromocytoma. Although we observed an increased hazard ratio for CRC patients with reduced intensity of PLAGL1 immunohistochemical staining of tumor tissue, the PLAGL1 expression level was not significantly correlated with overall survival of the patients. This may be partly due to the fact that the analysis of survival was limited by a relatively short period of follow-up.

We did not investigate mechanisms underlying the observed downregulation of PLAGL1 expression in CRC. Previous studies established that the transcriptional silencing of this gene may be regulated by epigenetic processes, such as methylation of CpG islands and histone deacetylation (5). Moreover, it has been reported that downregulation of PLAGL1 expression may be mediated via epidermal growth factor receptor (EGFR) (4,23), which upon binding to its ligands activates signaling pathways that promote tumor growth, including cell invasion and metastasis (24,25). EGFR has been reported to be overexpressed in CRC (26); furthermore, Cheirsilpa et al (27), using immunohistochemistry, demonstrated associations between overexpression of EGFR and lymph node status, as well as the advanced TNM stages. Our findings indicate analogous correlations between mentioned clinicopathological parameters and decreased PLAGL1 immunoreactivity in CRC tissues. These observations raise the hypothesis that cellular responses activated via the EGFR signaling cascade, such as proliferation, migration, and apoptosis, may be partially mediated by altered PLAGL1 expression. It has been also reported that the protein encoded by the PLAGL1 gene may act as a regulator of nuclear receptor activity, including the glucocorticoid receptor (28), whose expression in colorectal cancer correlated with the expression of cell cycle-related molecules, such as Rb protein and p16 (29). Further studies are necessary to unveil mechanisms underlying the regulation of PLAGL1 expression in CRC and their implications for the role of the PLAGL1 protein in the pathogenesis of CRC.

In conclusion, the results of our study suggest that altered PLAGL1 expression, associated with unfavorable clinicopathological parameters, may be involved in the progression of colorectal cancer. However, the expression of PLAGL1 at the mRNA and protein levels failed to correlate with the patient survival. The latter calls into question the applicability of the PLAGL1 expression level as a prognostic factor in colorectal cancer until a longer period of follow-up will be available for survival analysis.

Acknowledgements

This study was supported by the National Science Centre grant no. NN402 452339.

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September 2015
Volume 47 Issue 3

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APA
Kowalczyk, A.E., Krazinski, B.E., Godlewski, J., Kiewisz, J., Kwiatkowski, P., Sliwinska-Jewsiewicka, A. ... Kmiec, Z. (2015). Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters. International Journal of Oncology, 47, 951-962. https://doi.org/10.3892/ijo.2015.3067
MLA
Kowalczyk, A. E., Krazinski, B. E., Godlewski, J., Kiewisz, J., Kwiatkowski, P., Sliwinska-Jewsiewicka, A., Kiezun, J., Wierzbicki, P. M., Bodek, G., Sulik, M., Kmiec, Z."Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters". International Journal of Oncology 47.3 (2015): 951-962.
Chicago
Kowalczyk, A. E., Krazinski, B. E., Godlewski, J., Kiewisz, J., Kwiatkowski, P., Sliwinska-Jewsiewicka, A., Kiezun, J., Wierzbicki, P. M., Bodek, G., Sulik, M., Kmiec, Z."Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters". International Journal of Oncology 47, no. 3 (2015): 951-962. https://doi.org/10.3892/ijo.2015.3067