Tumor p16INK4 gene expression and prognosis in colorectal cancer
- Authors:
- Published online on: November 26, 2018 https://doi.org/10.3892/or.2018.6884
- Pages: 1367-1376
Abstract
Introduction
Colorectal cancer (CRC) is the third most frequent malignancy worldwide and the fourth most common cause of cancer-associated mortalities (1). Globally, ~1.36 million people are diagnosed with CRC each year and approximately one-half will succumb to this disease (1,2). A number of genes that are mutated in the multistep process of colorectal carcinogenesis and progression have been demonstrated to influence the prognosis of patients with CRC and their response to treatment (3–6). In addition to these somatic genetic mutations, epigenetic alterations and particularly the aberrant hypermethylation of gene promoter regions leading to transcriptional silencing are suggested to be important in CRC tumorigenesis (7). This mechanism is responsible for the functional inactivation of numerous tumor suppressor genes in CRC (7,8), including human MutL homolog 1, tissue inhibitor of metalloproteinase-3, p14, death-associated protein kinase, adenomatous polyposis coli, O-6-methylguanine-DNA methyltransferase and p16INK4 [(p16) or cyclin-dependent kinase (CDK) inhibitor 2a] (9–11).
In normal cells, p16 and retinoblastoma (Rb) proteins serve an important role in regulating the cell cycle pathway (12,13). Rb is phosphorylated by the cyclin D1-CDK4/6 complex, resulting in its dissociation from transcription E2 factor (E2F) (13). The subsequent transcriptional activation of E2F leads to progression of the cell cycle from G1 to S phase (14). As p16 interferes with cell cycle progression by inactivating CDK4/6, decreased expression or inactivation of p16 attenuates the ability of Rb to inhibit cell proliferation (15). The p16-Rb pathway is suppressed in a number of cancer types via genetic or epigenetic alterations in Rb and/or p16, through overexpression of the cyclin D1/CDK4 complex, in addition to a number of other mechanisms (12,13,15,16). In particular, deletion or mutation of the p16 gene is frequently observed in cancer of the biliary tract, lung, pancreas and esophagus and in brain tumors (17–21). Deletion of p16 has been associated with a late clinical stage in esophageal cancer, and with lymphatic invasion and distant metastasis in pancreatic cancer (22,23). In gall bladder and lung cancer, p16 deletions and mutations are associated with poor prognosis (24,25).
Decreased p16 expression due to hypermethylation of the p16 promoter was detected in 32–55% CRC cases (26–30). Although p16 mRNA expression is inversely correlated with tumor size and lymph node metastasis (31), its influence on the prognosis of patients with CRC remains unclear (32). A previous Japanese study identified that p16 promoter hypermethylation in the primary tumors of patients with CRC was associated with a shorter survival (33). This result was supported by a subsequent meta-analysis (34). In the present study, it was investigated whether p16 mRNA expression was associated with methylation of the p16 gene promoter and with patient prognosis in CRC.
Materials and methods
Patients with CRC and tissues
The present study included 101 patients with primary CRC who underwent surgery at the Kanazawa University Hospital (Kanazawa, Japan) between April 1999 and December 2002. Eligible patients were aged 20 years or older and had histologically proven adenocarcinoma of the colon and rectum. Exclusion criteria included absolute contraindications to general anesthesia and/or surgery. Their clinicopathological characteristics are presented in Table I. The survival status was determined for all patients and the median follow-up period was 54.5 months. In total, 54 patients (53.5%) received postoperative 5-fluorouracil (5-FU)-based adjuvant chemotherapy.
 | Table I.Association between p16 methylation status or p16 mRNA expression and clinicopathological features in colorectal cancer. |
Tumor tissue samples collected from the fresh surgical specimen were cryopreserved in liquid nitrogen and stored at −80°C for extraction of DNA and RNA. The remaining surgical specimen was fixed with 10% neutral-buffered formalin for 1–2 days at room temperature and embedded in paraffin for histopathological examination. The tumor stage was determined according to the Union for International Cancer Control tumor, node and metastasis (TNM) classification (35). Genomic DNA and total RNA were extracted from the same tumor tissues using the QIAmp DNA Mini kit and the RNeasy Mini kit (both from Qiagen GmbH, Hilden, Germany), respectively, according to the manufacturer's protocols.
The present study was performed in accordance with the Declaration of Helsinki. The design and protocol for the present study were approved by the Kanazawa University Human Genome and Gene Analysis Research Ethics Committee, and written informed consent was obtained from the majority of the patients.
Quantification of p16 methylation by the MethyLight assay
Bisulfite conversion of genomic DNA was performed as previously described (36). DNA was denatured using 0.2 M NaOH and subsequently incubated with bisulfite for 16 h at 50°C. The bisulfite-converted DNA was purified using the Wizard DNA purification kit (Promega Corporation, Madison, WI, USA) and precipitated with ethanol. The DNA sample was resuspended in water and stored at −30°C.
MethyLight, a fluorescence-based real-time PCR assay was used to measure the level of p16 promoter methylation as previously described (37). The sense and antisense primers used for amplifying the bisulfite-converted p16 promoter were: 5′-TGGAATTTTCGGTTGATTGGTT-3′ and 5′-AACAACGTCCGCACCTCCT-3′, respectively (37). These primers were used with the probe 5′-6FAM-ACCCGACCCCGAACGCG-TAMRA-3′ to measure CpG methylation of the p16 promoter region by real-time PCR (38). The specificity for amplification of methylated DNA was confirmed separately using human sperm DNA (unmethylated) and SssI (New England BioLabs, Inc., Ipswich, MA, USA)-treated sperm DNA (fully methylated) in the assay. Actin was amplified as a control for the total amount of DNA using the sense primer, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and antisense primer, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′; and probe, 5′-6FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA-3′ (38). The percentage of fully methylated fraction [percentage of methylated reference (PMR)] at a specific gene locus was calculated by dividing the gene:actin ratio of the sample DNA by the gene:actin ratio of the SssI-treated sperm DNA and multiplying by 100 (38). PMR values obtained using MethyLight were classified into high (PMR ≥4) and low (PMR <4) methylation categories, according to previous studies (9,39,40).
Analysis of p16 mRNA expression
Reverse transcription (RT)-PCR was used to measure p16 mRNA expression, as previously described (41). The relative expression of mRNA was quantified using the 2−ΔΔCq method (42). The expression of actin was measured as an internal standard and the level of p16 mRNA expression in each tumor sample was normalized to the expression of actin. The cut-off value for high and low levels of p16 mRNA expression was defined as the median expression level for all tumor samples. Cut-off values for p16 PMR and mRNA expression were used to compare p16 promoter methylation and mRNA expression in the same tumors.
Proliferating cell nuclear antigen (PCNA) mRNA expression levels were additionally measured, using actin expression level as the internal standard. p16 mRNA expression relative to PCNA (p16/PCNA) was calculated as the p16 mRNA:actin ratio divided by the PCNA mRNA:actin ratio in the same cDNA sample. The cut-off value for p16/PCNA expression was defined as the median p16/PCNA level for all tumor samples. This was used to investigate the influence of p16 mRNA expression on the survival of patients with CRC.
Statistical analysis
For statistical comparison of tumor p16 methylation and mRNA expression with clinicopathological factors and tumor stage, the Fisher's exact test was used to study non-continuous variables, and the Mann-Whitney U test and Kruskal-Wallis test were used to study continuous variables. The Mann-Whitney U test was used to compare p16 methylation with p16 mRNA expression. The survival of patients was evaluated using Kaplan-Meier analysis. Univariate and multivariate analyses of survival were conducted using Cox proportional hazards regression. All statistical analyses were performed with EZR (version 1.29, Jichi Medical University, Shimotsuke, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics (43).
Results
p16 promoter methylation and p16 mRNA expression in CRC
The levels of p16 gene promoter methylation and p16 mRNA expression in the tumor samples of all patients with CRC are presented in Table I in association with clinicopathological features. p16 methylation (PMR >0) was detected in 67 cases (66.3%) and the median PMR value was 0.34 (range, 0.00–468.6; Fig. 1A). A PMR cut-off value of ≥4 was used to define high methylation and PMR <4 for low methylation, according to previous studies (9,38–40). Using this definition, the high methylation group comprised 18 (17.8%) of the 101 patients (Table II), in agreement with previous studies (9,33). The range of relative p16 mRNA expression levels was between 0 and 154.0, with a median expression level of 1.98 (Fig. 1B). As p16 mRNA expression is likely to be associated with cell cycle (44), the p16 mRNA expression level was normalized to that of PCNA expression in the same tumor and designated as p16/PCNA expression. The relative values of p16/PCNA ranged between 0 and 1,957.6, with a median value of 11.46 (Fig. 1C). To evaluate the influence of p16 mRNA expression on survival outcome, patients were divided into two groups (high and low) according to the median value for p16 mRNA or p16/PCNA expression.
| Table II.Association between p16 methylation status or p16/PCNA expression and clinicopathological features in colorectal cancer. |
Comparison of p16 mRNA expression between the p16 high and low methylation groups identified a significant inverse association (P=0.034; Fig. 2A). When a higher PMR cut-off value of 10 was used rather than 4, a strong inverse association with p16 mRNA expression was observed between the high (n=12; 11.9%) and low (n=89; 87.1%) p16 methylation groups (P<0.001; Fig. 2B). Subsequently, p16/PCNA expression was compared between the p16 high and low methylation groups. Using a PMR value of 4 to classify p16 methylation, no significant association was observed between p16 methylation and p16/PCNA expression (P=0.168; Fig. 2C). However, when classified according to the higher PMR cut-off value of 10, a significant inverse association was observed between p16 methylation and p16/PCNA expression (P=0.006; Fig. 2D).
Tumor p16 methylation and prognosis of patients with CRC
Comparison of tumor p16 methylation levels with clinical and histopathologic characteristics of patients with CRC is presented in Table II. High p16 methylation (PMR >4) level was more frequently detected in mucinous (P=0.017) compared with the other histological types and was significantly associated with later clinical stage (P=0.008); however, not with the sex or age of the patient, tumor site, T stage or adjuvant chemotherapy. There was no significant difference in prognosis between the high and low p16 methylation groups (P=0.94; Fig. 3).
Tumor p16 mRNA expression and prognosis of patients with CRC
No significant differences in p16 mRNA expression levels (high or low) were observed according to sex or age of the patient, or with tumor site, histology, T stage, or adjuvant chemotherapy (Table III). The survival of patients with CRC with high p16 mRNA expression was worse compared with patients with low expression; however, this did not reach statistical significance (P=0.109; Fig. 4A). The majority (83/101; 82%) of patients with CRC demonstrated low levels (PMR <4) of p16 methylation (Table II; Fig. 2A). When these patients were divided into high and low p16 mRNA expression groups defined by a median level of value (2.15), the high expression group (n=42) demonstrated a worse outcome compared with the low expression group (n=41; P=0.076; Fig. 4B).
| Table III.Association between p16 mRNA expression and clinicopathological features in colorectal cancer. |
Patients with CRC were additionally divided into p16/PCNA high (n=50) and low (n=51) groups according to the median value (11.46). No differences in any of the clinical and histopathologic parameters were observed between these two groups (Table II), nor was there a significant difference in patient survival between the high and low p16/PCNA groups (P=0.122; Fig. 4C). The 83 patients with low tumor p16 methylation were further examined by dividing them into groups with high or low p16/PCNA expression according to the median value (12.49). In this analysis, patients with high p16/PCNA expression demonstrated a significantly worse survival (P=0.026; Fig. 4D).
The patient group with a low p16 methylation (PMR <4) was additionally evaluated using Cox regression analysis for the prognostic significance of various clinical and histopathologic features and for p16 mRNA and p16/PCNA expression levels. p16/PCNA mRNA expression was the only significant prognostic factor in univariate analysis (Table IV; P=0.026). The influence of T stage, tumor stage and adjuvant chemotherapy on the overall survival of patients with CRC was analyzed using the Kaplan-Meier method. There was no association between any of these factors and the prognosis of the patients (Fig. 5). Multivariate analysis identified that low p16/PCNA expression was an independent factor for better survival in patients with low p16 methylation (hazard ratio 0.287; 95% confidence interval 0.110–0.747; P=0.011; Table IV).
| Table IV.Univariate and multivariate analysis for the prognostic significance of clinicopathological factors and p16/PCNA mRNA. |
Discussion
Tumor p16 methylation has previously been identified as a prognostic factor for a worse outcome in CRC (33,34,41). However, other previous studies demonstrated that p16 methylation has no impact on prognosis (32) or predicts worse outcome only in patients with poorly differentiated CRC (45). A possible mechanism for the putative association between tumor p16 methylation and survival of patients with CRC is that expression of p16 protein is diminished, thereby promoting tumor cell proliferation and invasion (46,47). In the present study, however, no significant association was observed between tumor p16 methylation and the outcome of patients with CRC, thus supporting a previous study (32). A number of technical reasons, in addition to the number of patients examined may account for the inconsistent results demonstrated by different previous studies and the present study. The technical reasons include differences in the tissue samples analyzed (fresh compared with fixed tissues), the methods used to quantify p16 methylation levels and the cut-off values used in the analyses (32–34,41,45).
p16 promoter methylation is associated with decreased expression of p16 mRNA in clinical samples of CRC (26). In the present study, an inverse association between tumor p16 methylation and the expression of its transcript was additionally identified. This was most pronounced in tumors with high methylation levels (PMR >10). However, within the high methylation group (PMR ≥4) a number of cases with relatively increased expression of p16 mRNA were identified, in agreement with previous studies, which demonstrated that tumor cells with p16 promoter methylation may express p16 mRNA (31,41). Therefore, p16 mRNA expression may not be controlled exclusively through promoter methylation; however, may additionally be influenced by other factors, including the Ras signaling pathway (48), which is frequently activated in CRC due to KRAS proto-oncogene GTPase and NRAS proto-oncogene GTPase mutations.
Previous studies demonstrated that low p16 protein expression in CRC was associated with larger tumor size, lymph node metastasis and faster tumor proliferation (31,49,50), and is thus likely to account for an association with worse prognosis (51). However, little is known regarding the prognostic impact of p16 mRNA expression in patients with CRC. Although an inverse association between tumor p16 methylation and mRNA expression was observed in the present study, relatively few patients (18/101) demonstrated high levels of p16 methylation, defined as PMR ≥4. Therefore, patients with low tumor p16 methylation levels were investigated, as it was hypothesized that p16 mRNA expression may affect patient survival, as those patients may exhibit a wide range of p16 mRNA expression for analysis. An unexpected result of the present study was that patients with high p16/PCNA mRNA expression demonstrated significantly worse survival. Furthermore, this was demonstrated by multivariate analysis to be independent of other factors that potentially influence patient survival, including tumor stage and histological types. Despite its well-recognized tumor suppressor role (52), p16 is overexpressed at the mRNA and protein expression levels in tumor tissues compared with adjacent normal mucosa (31,51). Similar to the present results, patients with breast (53,54) and prostate cancer (55) with high p16 expression were additionally identified to have a worse prognosis. Notably, although p16 is a critical cell cycle regulator and its mRNA expression is likely to be associated with cell cycle (44), none of the previous studies investigating the association of p16 promoter methylation with prognosis of patients with CRC (32,34) scored a copy number of p16 mRNA. Therefore, the clinical implications of p16 mRNA and protein expression in different cancer types and association with p16 methylation require further investigation.
Established tumor cell lines with Rb deletion demonstrated activated p16 transcription and increased p16 protein expression (56). It has additionally been demonstrated that cell cycle regulation by p16 is lost in tumor cells with inactivated Rb (57), and that the efficacy of exogenously expressed p16 in cancer cells depends on Rb function (58). Furthermore, the overexpression of transcription factor E2F1 promotes p16 transcription (59), whereas CDK4 overexpression in sarcoma cells is thought to increase p16 expression through a feedback loop (60). This putative feedback regulation in the expression and function of Rb pathway mediators may explain the paradoxical association observed in the present study and in others between high p16 expression and worse patient survival.
In summary, p16 is a CDK4 inhibitor that counteracts the cell cycle process by sustaining the Rb-mediated pathway. Accordingly, p16 has been recognized as a tumor suppressor that is lost or inactivated through gene mutation, deletion or promoter methylation in various cancer types, including CRC. Previous studies demonstrated an association between p16 gene promoter hypermethylation and worse prognosis in patients with CRC, in addition to an inverse association between p16 expression and tumor progression. However, the effect of p16 mRNA expression on the prognosis of patients with cancer is controversial. In the present study, it was demonstrated that p16 mRNA expression in the tumors was inversely associated with the levels of p16 promoter methylation. In addition, multivariate analysis determined that high p16 mRNA expression normalized to PCNA mRNA expression (p16/PCNA) was an independent prognostic factor for poor survival of patients with CRC. These results identified a previously unrecognized and paradoxical association between high expression of p16 mRNA and worse prognosis of patients with CRC, although a similar association has been demonstrated in other cancer types.
Acknowledgements
The authors would like to thank Dr Barry Iacopetta (University of Western Australia, Crawley, Australia) for critically reading and editing the manuscript.
Funding
The present study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (grant nos. 20390353 and 23390321).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
HK made substantial contributions to the design and conception of the study, and the acquisition, analysis and interpretation of the data, and drafted the manuscript. HT and TM collected the clinical samples and contributed to the interpretation of the data. TM helped to draft and finalize the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was performed in accordance with the Declaration of Helsinki. As the tissues used in the present study were from the patients diagnosed between 1999 and 2002, written informed consent was obtained from the patients prior to the tissue sample collection. In accordance with Japanese ethical guidelines and law, the study protocol was reviewed and approved by the Kanazawa University Human Genome/Gene Analysis Research Ethics Committee (approval no. 181; Kanazawa, Japan). At the start of the study, it was not possible to directly contact the specific patients to explain the present study. According to the Human Genome/Gene Analysis Research Ethics Committee, the present study was announced on our website, providing these patients an opportunity to opt out of the present study. By the date indicated, none of them refused to be included in the present study. All samples were anonymized before analysis was performed to guarantee the protection of privacy.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
CRC |
colorectal cancer |
|
5-FU |
5-fluorouracil |
|
PCNA |
proliferating cell nuclear antigen |
|
PCR |
polymerase chain reaction |
|
PMR |
percentage of methylated reference |
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