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Implications of Genetic and Epigenetic Alterations of CDKN2A (p16
INK4a
)
in Cancer
Ran Zhao, Young Choi Bu, Mee-Hyun Lee, Ann M. Bode, Zigang Dong
PII: S2352-3964(16)30152-9
DOI: doi: 10.1016/j.ebiom.2016.04.017
Reference: EBIOM 570
To appear in: EBioMedicine
Received date: 10 February 2016
Revised date: 1 April 2016
Accepted date: 14 April 2016
Please cite this article as: Zhao, Ran, Bu, Young Choi, Lee, Mee-Hyun, Bode, Ann M.,
Dong, Zigang, Implications of Genetic and Epigenetic Alterations of CDKN2A (p16
INK4a
)
in Cancer, EBioMedicine (2016), doi: 10.1016/j.ebiom.2016.04.017
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1
Implications of Genetic and Epigenetic Alterations of CDKN2A (p16
INK4a
) in Cancer
Ran Zhao
1
, Bu Young Choi
2
, Mee-Hyun Lee
1*
, Ann M. Bode
3
, and Zigang Dong
1,3*
1
China-US (Henan) Hormel Cancer Institute, No.127, Dongming Road, Jinshui District,
Zhengzhou, Henan, 450008, China,
2
Department of Pharmaceutical Science and Engineering,
Seowon University, Cheongju, 361-742, Korea,
3
The Hormel Institute, University of Minnesota,
Austin, MN55912, USA
*
Address correspondence to:
Professor Mee-Hyun Lee
China-US (Henan) Hormel Cancer Institute, No.127, Dongming Road, Jinshui District,
Zhengzhou, Henan, 450008, China.
Fax) +86-371-65587670; Phone) +86-371-65587008; E-mail) meehyun-lee@outlook.com
Professor Zigang Dong
The Hormel Institute, University of Minnesota, 801 16
th
Ave NE, Austin, MN55912, USA
Fax) +1-507-437-9606; Phone) +1-507-437-9600; E-mail) zgdong@hi.umn.edu
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ABSTRACT
Aberrant gene silencing is highly associated with altered cell cycle regulation during
carcinogenesis. In particular, silencing of the CDKN2A tumor suppressor gene, which encodes
the p16
INK4a
protein, has a causal link with several different types of cancers. The p16
INK4a
protein plays an executional role in cell cycle and senescence through the regulation of the
cyclin-dependent kinase (CDK) 4/6 and cyclin D complexes. Several genetic and epigenetic
aberrations of CDKN2A lead to enhanced tumorigenesis and metastasis with recurrence of
cancer and poor prognosis. In these cases, the restoration of genetic and epigenetic
reactivation of CDKN2A is a practical approach for the prevention and therapy of cancer. This
review highlights the genetic status of CDKN2A as a prognostic and predictive biomarker in
various cancers.
Keywords: CDKN2A, p16
INK4a
, genetic alterations, epigenetic alterations, cancer
Highlights
- The status of CDKN2A provides epigenetic/genetic information for the cancer patient.
- The correlation of p16
INK4a
and related biomarkers should be considered for prognosis of
cancers.
- Epigenetic/genetic modulation of changes in CDKN2A might be a promising cancer
preventive/therapeutic strategy.
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Introduction
Cancer comprises a collection of complex genetic and epigenetic diseases that arise through
multistep processes (Tallen and Riabowol, 2014). Tumor cells acquire common properties,
including unlimited proliferation potential, self-sufficiency in growth signaling, neovascularization
for nutrient and oxygen supply, and resistance to anti-proliferative and apoptotic stimuli
(Hanahan and Weinberg, 2011). In resting cells, the cell cycle is strictly managed by a set of
regulatory proteins that control the various cell cycle checkpoints (Collado et al., 2007, Collins et
al., 1997). This cell cycle machinery is often deregulated in cancer as a consequence of the
silencing of various tumor suppressor genes (Collins et al., 1997, Tallen and Riabowol, 2014). In
fact, loss of tumor suppressor genes and their encoded proteins through deletion, inactivating
mutations, epigenetic silencing or post-translational modification results in tumorigenesis. The
progression of the mammalian cell cycle from G1 to mitosis is regulated by various cyclin
proteins and their catalytic subunits referred to as cyclin-dependent kinases (CDKs) (Nabel,
2002). A family of cyclin–CDK inhibitor proteins (CDIs), which bind and inactivate the CDKs, has
been isolated. This family includes the p16
INK4a
, p21
CIP1
, p27
KIP1
, and associated proteins
p15
INK4b
, p18
INK4c
, p19
INK4d
and p57
KIP2
(Nabel, 2002). These proteins potentially act as tumor
suppressors and their inactivation corresponds with human carcinogenesis.
One of the tumor suppressor proteins that is inactivated in cancer is the p16
INK4a
protein,
which is encoded by the cyclin-dependent kinase inhibitor 2A (CDKN2A) or multiple tumor
suppressor 1 (MTS1) gene (Figure 1) (Witcher and Emerson, 2009). The CDKN2A gene is
located within the frequently deleted chromosomal region 9 of p21 (Gil and Peters, 2006). This
gene (8.5 kb full length) contains two introns and three exons and encodes the p16
INK4a
protein.
The p16
INK4a
protein is a nuclear phosphor-protein consisting of 156 amino acids with a
molecular weight of 16 kDa and is a negative regulator of the cell cycle (Serrano et al., 1993). In
addition to p16
INK4a
, CDKN2A encodes a completely unrelated tumor suppressor protein,
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alternate open reading frame (ARF or p19
Arf
in mice), which interacts with the p53 regulatory
protein, mouse double minute 2 homolog (MDM2) (Pomerantz et al., 1998). The simple tandem
arrangement is complicated by the presence of an additional exon 1β, which is transcribed from
its own promoter. The resulting RNA incorporates exons 2 and 3, but specifies a distinct protein
because the exons are translated by an alternative reading frame. Thus, while exons 2 and 3
are shared by the two mRNAs, they encode different protein products, p16
INK4a
and ARF (Quelle
et al., 1995). The specific binding of the p16
INK4a
protein to CDK4 or CDK6 induces an allosteric
conformational change in these proteins and inhibits the formation of the complex between
CDK4 or 6 and cyclin D (Serrano et al., 1993). The lack of this complex formation maintains the
retinoblastoma protein (Rb) in its hypo-phosphorylated and growth-suppressive states. This
leads to the induction of G1 phase cell cycle arrest through the formation of the Rb/E2Fs-
repressive complex (Figure 1) (Weinberg, 1995). The loss of p16
INK4a
is increasingly common
with advancing stages of various neoplasms, suggesting that p16
INK4a
inactivation may
contribute to cancer progression. The frequent inactivation of p16
INK4a
induced by homozygous
deletion or promoter hyper-methylation and point mutation has been observed in various
cancers (Table 1).
Epigenetic alterations are suggested to regulate gene expression without affecting the
base sequence. These modifications include genomic DNA-methylation, histone modifications,
chromatin remodeling and miRNA/non-coding RNA-induced regulation of gene expression
(Hauptman and Glavac, 2013, Portela and Esteller, 2010, Sarkar et al., 2015). The polycomb
group (PcG) genes, first identified in Drosophila melanogaster, encode the highly conserved
PcG proteins, which form two large macromolecular complexes classified as polycomb
repressive complex-1 (PRC1) and -2 (PRC2). The PRC1 complex consists of B lymphoma Mo-
MLV insertion region 1 homolog (BMI1), mPh1/2, Pc/chromobox (CBX) and RING1A/B, and
sustains silencing of chromatin. The mammalian PRC1 might harbor specificity against selecting
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one of the multiple Pc/CBX homologs. The PRC2 complex is composed of embryonic ectoderm
development (EED), suppressor of zeste (SUZ)12 and enhancer of zeste homolog (EZH)1/2,
and initiates repression of target genes by di- and trimethylation of lysine 27 of histone H3
(H3K27me2 and H3K27me3) (Bracken et al., 2007, Cao et al., 2002). Thus, activation of PRC1
and PRC2 induces gene silencing through histone modification ultimately affecting the
chromatin structures. Long non-coding RNAs are transcripts longer than 200 nucleotides and
lack protein-coding capacity. These RNAs might play a major role in programming the
epigenome by cooperating and coordinating with the PcG complexes to impose chromatin
states in a dynamic manner (Aguilo et al., 2011). The purpose of this mini-review is to shed light
on the molecular mechanisms of genetic and epigenetic changes in p16
INK4a
and the
implications in carcinogenesis.
Genetic alterations of CDKN2A in various cancers
The changes in CDKN2A/p16
INK4a
status are highly variable depending on the type of cancer.
The following section describes the mechanisms of p16
INK4a
inactivation in various cancers
(Table 1; Figure 2).
Lymphoma
Several studies have demonstrated that promoter hyper-methylation leads to the loss of p16
INK4a
expression in lymphoma. Gastric lymphoma is an extra-nodal non-Hodgkin’s lymphoma and is
associated with 2-8% of all gastric cancers (Alevizos et al., 2012). Huang et al. reported that
26.5% (13 of 49) of primary gastric lymphomas exhibited hyper-methylation of the CDKN2A
promoter, suggesting that expression of p16
INK4a
is a clinical risk factor for gastric lymphoma
(Huang et al., 2007). Burkitt’s lymphoma is a common subtype of B-cell non-Hodgkin’s
lymphoma in children and adolescents (Molyneux et al., 2012). A recent analysis of 51 Burkitt’s
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lymphoma tumor samples revealed that methylation of theCDKN2A promoter occurred in 72.5%
of the samples and nuclear expression of the p16
INK4a
protein remained undetectable in about
41% of the samples (Robaina et al., 2015). In this study, CDKN2A promoter methylation was
detected in 32 patient samples (80%) at stage III/IV of the cancer (Robaina et al., 2015).
Skin cancer and melanoma
Solar ultraviolet (UV) radiation is the most common risk factor for the initiation and promotion of
melanoma and non-melanoma skin carcinogenesis (de Gruijl, 1999). The CDKN2A gene is a
melanoma susceptibility gene and its mutations are present in 20 to 40% of familial and 2 to 3%
of sporadic melanomas (Kostaki et al., 2014). The inactivation of CDKN2A in skin cancer
involves histone modifications as well as DNA methylation. Chronic exposure of HaCaT skin
keratinocytes to UVA radiation has been reported to cause 80 to 90% histone methylation
(H3K4m3) and 50 to 70% DNA methylation in the CDKN2A promoter region (Chen et al.,
2012a). Deletion of p16
INK4a
has also been detected in 50% of melanomas and its inactivation by
point mutations occurs in about 9% of cases, which correlates with an increased risk of
metastases and disease progression. Methylation of the CDKN2A gene promoter occurs in
about 5 to 19% of sporadic melanomas, whereas promoter methylation of the gene was found in
27 to 33% of melanoma metastases (Kostaki et al., 2014). In cutaneous melanoma metastases,
the CDKN2A promoter is hyper-methylated in about 25% (15/59) of cases and non-synonymous
mutation of the gene was observed in about 16% (9/56) of cases examined (Jonsson et al.,
2010).
ANRIL (antisense noncoding RNA in the INK4 locus) mediates a cis-acting silencing
mechanism. ANRIL binds with PRC1 at the RNA-binding domains of CBX7 and methylates the
CDKN2A promoter region at H3K27, which inhibits CDKN2A gene expression (Sarkar et al.,
2015, Yap et al., 2010, Sato et al., 2010).
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Head and neck cancer
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease that arises in
the areas of the nasal and oral cavities, pharynx and larynx. Although HNSCC can be caused
by infection with human papilloma virus (HPV) (Bishop et al., 2013), approximately 90% of HPV-
negative HNSCC tumors exhibit low expression of CDKN2A. This is largely due to mutations,
loss of heterozygosity, and DNA hyper-methylation of the gene (Cancer Genome Atlas, 2015).
In one study, 57% of HPV-negative HNSCC from the TCGA (The Cancer Genome Atlas)
dataset was due to mutation or loss of the CDKN2A gene (Chung et al., 2015). In primary
HNSCC with deficient expression of CDKN2A, 66.7% (20/30) of the tumors exhibited DNA
methylation of the CDKN2A promoter. The number of methylations was found to be 4
methylations at exon 1, 7 at exon 2 and 9 each at exons 1 and 2 (El-Naggar et al., 1997).
Oral cancer includes a group of neoplasms affecting any region of the oral cavity, pharyngeal
regions and salivary glands. More than 90% of all oral neoplasms are oral squamous cell
carcinomas (OSCCs) (Markopoulos, 2012). Genetic alterations in OSCC might activate
mutations or methylation of cell cycle regulatory genes, thereby promoting cell survival and
proliferation (Al-Kaabi et al., 2014). In OSCC patients, hyper-methylation of the CDKN2A
promoter was observed 60% of the time and oral leukoplakia patients also exhibited 60% hyper-
methylation in the CDKN2A promoter (Asokan et al., 2014).
Pancreatic cancer
Because of their severe metastatic potential, pancreatic tumors are the most aggressive types
of cancer. Expression of CDKN2A has been reported to be aberrant in ~95% of pancreatic
adenocarcinomas, and approximately 15% of those (El-Naggar et al., 1997) and 24.6% in
another study (Jiao et al., 2007) were attributed to promoter hyper-methylation. The CDKN2A
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gene is abnormally methylated in 27% of pancreatic cancer cell lines (Moore et al., 2001). In
addition, the mutation of CDKN2A occurred 11.8% of the time in hereditary pancreatic cancer
patients (Salo-Mullen et al., 2015).
Lung cancer
Lung cancer is the leading cause of cancer death and most patients with lung cancer suffer from
non-small cell lung cancer (NSCLC) (Siegel et al., 2015). Alterations in CDKN2A were
examined in 63 NSCLCs samples of which 30 samples were primary resected NSCLCs with
metastatic involvement of thoracic lymph nodes and 33 NSCLCs were without lymph node
metastases (Marchetti et al., 1997). The analysis revealed that 6 NSCLC tumor samples had
somatic aberrations of the CDKN2A gene, including 4 mutations, 1 frame-shift and 1
homozygous deletion (Marchetti et al., 1997). These alterations of CDKN2A were significantly
associated with lymph node metastasis of NSCLC. Further analysis of 40 NSCLC
adenocarcinoma cell lines revealed that CDKN2A was inactivated in 75% (30/40) of cases
including 16 homozygous deletions, 10 methylations and 4 mutations (Tam et al., 2013). In
another study, alteration of CDKN2A in NSCLC adenocarcinoma tissue samples was noted as
38% (17/45) and included 10 homozygous deletions, 4 methylations and 3 mutations (Tam et al.,
2013).
Esophageal cancer
Esophageal cancer is the 6
th
leading cause of cancer death worldwide (Zhang, 2013).
Esophageal squamous cell carcinoma (ESCC) is more common in the developing world and
arises from the epithelial cells. The promoter region of CDKN2A was highly methylated in tissue
samples from Chinese subjects (81.7% or 210/257) (Chen et al., 2012b). Among 38 Japanese
ESCC samples examined, 13% showed a loss of heterozygosity of the CDKN2A gene and 67
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tumors showed 6% with CDKN2A mutations (Kuwabara et al., 2011). A novel 7 base pair
(TCCAGCC) deletion in 22 of 106 (~20%) esophageal tumor samples was observed in exon 2
of CDKN2A (Qureshi et al., 2012). An in silico study revealed that the deletion of these 7 base
pairs resulted in premature termination and caused instability of the p16
INK4a
/CDK6 complex
(Qureshi et al., 2012).
Gastric cancer
Gastric (stomach) cancer develops in the lining of the stomach. In a meta-analysis of Chinese
gastric cancer patients, hyper-methylation of the CDKN2A promoter was recorded as 43.3% of
the median average (28.3-64.4%), and was significantly correlated with CDKN2A promoter
methylation and carcinogenesis (Peng et al., 2014, Wang et al., 2014). Infection with the
Epstein-Barr virus (EBV) is one risk factor for gastric cancer. EBV-associated gastric carcinoma
(EBVaGC) accounts for approximately 8-10% of all gastric carcinomas (Liang et al., 2014).
Hyper-methylation of the CDKN2A promoter was detected in 81.6% and 33.3% of the EBVaGC
and EBVnGC (EBV-negative gastric carcinomas), respectively (He et al., 2015). These findings
suggest that a high level of CDKN2A promoter methylation is a risk factor for developing EBV-
associated gastric cancers. Another important etiologic factor for gastric carcinogenesis is
Helicobacter pylori (H. pylori) infection. Matsusaka et al. demonstrated that H. Pylori infection
promotes CDKN2A DNA methylation from 21.3% to 45.0%, which correlates with poor tumor
differentiation, increased lymph node metastasis, and lower survival rates of gastric cancer
patients (Matsusaka et al., 2014, Qu et al., 2013).
Colorectal cancer
The development of colorectal cancer involves genetic and epigenetic changes of several tumor
suppressor genes, including CDKN2A (Chan et al., 2002). Similar to other cancers, the
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CDKN2A gene is silenced through promoter hyper-methylation during the course of colorectal
cancer pathogenesis (Kim et al., 2010). Kim et al. reported that promoter methylation of
CDKN2A occurred in 11.9% of 285 sporadic colorectal cancers. Among these CDKN2A hyper-
methylated cancer patients, 10.6% of 264 patients exhibited microsatellites with low frequency,
whereas 28.6% of 21 patients showed microsatellites with high frequency (Kim et al., 2010). In
another study, CDKN2A promoter methylation occurred in 17.1% of 497 patients and its
regulation was highly associated with poor survival in stage II and III colorectal cancer patients
treated with adjuvant fluoropyrimidine (Lee et al., 2015). The prognostic value of concurrent
methylation of the CDKN2A promoter was associated with gender variation and was significant
only in male patients (Lee et al., 2015).
Ovarian cancer
Ovarian cancer is the most common form of gynecological cancer and a leading cause of
cancer-related mortality among women (Jayson et al., 2014). This cancer is diagnosed usually
at an advanced stage and has a high rate of recurrence (Hennessy et al., 2009). In a recent
study, Bhagat et al. demonstrated that the frequency of CDKN2A promoter methylation was
43% in 134 invasive cancer cases including 22% of 23 low malignancy patient tumors and 42%
of 26 benign cases (Bhagat et al., 2014). Down-regulation of CDKN2A mRNA expression and
hyper-methylation of the CDKN2A promoter are significantly associated.
Prostate cancer
Prostate cancer presents mostly as adenocarcinomas and is the second leading cause of
cancer death in men (Siegel et al., 2015). Silencing of the CDKN2A gene plays a critical role in
prostate cancer progression. Methylation of the CDKN2A promoter has been reported to occur
in 47.6% of 21 patients with poor prognosis (Ameri et al., 2011). Epigenetically, ANRIL and
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H3K2me are involved and alternatively bind with CBX7 of PRC1, thereby repressing CDKN2A
gene transcription (Yap et al., 2010).
Renal cell carcinoma
Accumulation of various genetic aberrations, including the CDKN2A gene, underlies the
development of renal cell carcinomas (Dulaimi et al., 2004). Evaluation of the methylation
status of the promoter CpG island of CDKN2A and immunohistochemical detection of the
p16
INK4a
-encoded protein in 57 patients with renal carcinomas revealed that the CDKN2A gene
was hyper-methylated in 22.9% of patients and none of them expressed the p16
INK4a
protein
product (Vidaurreta et al., 2008). The lack of p16
INK4a
protein expression was detected in 52.9%
of the tumors, suggesting the involvement of another genetic alteration or post-transcriptional
modification of the protein (Vidaurreta et al., 2008).
Molecular switches associated with p16
INK4a
aberrations
The inactivation of CDKN2A in cancer is neither an isolated event nor does it appear to have a
direct link with carcinogen exposure (Table 2). Analysis of the current literature reveals that
promoter methylation of CDKN2A is not associated with HPV infection, which is a cause of
head and neck squamous cell carcinomas (Chung et al., 2015). However, the epigenetic
changes in CDKN2A are associated with simultaneous genetic or epigenetic alterations in
other cancer-related oncogenes or tumor suppressor genes (Veganzones et al., 2015, Chung et
al., 2015, Kostaki et al., 2014, Tam et al., 2013, Wilson et al., 2010, Jonsson et al., 2010). Thus,
the study of the concordance between p16
INK4a
inactivation with other tumor-related
genetic/epigenetic lesions could provide new avenues for developing anticancer therapies. Tam
et al. have demonstrated that mutations of the epidermal growth factor receptor (EGFR) and
KRAS are major oncogenic drivers in the pathogenesis of lung adenocarcinomas. The mutation
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of EGFR is reportedly associated with CDKN2A homozygous deletion or mutation, whereas
KRAS mutation is related to hyper-methylation of the CDKN2A promoter (Tam et al., 2013).
However, changes in STK11 (also known as LKB1), a gene frequently mutated in lung
adenocarcinomas, have no impact on CDKN2A inactivation mechanisms (Tam et al., 2013).
Veganzones and colleagues investigated the frequency of simultaneous methylation of
CDKN2A and hMLH1, a gene that encodes a DNA repair enzyme, in colorectal cancer patients
with microsatellite instability (MSI) (Veganzones et al., 2015). The outcome of the analysis of 51
samples of MSI-positive sporadic colorectal cancer was expressed by a new variable referred to
as combined methylation of CDKN2A and hMLH1 (CMETH2). Results indicated that 17 of 51
patients (33.3%) tested positive for CMETH2 expression, which was associated with poorly
differentiated tumors in proximal locations. The clinic-pathological implication of CMETH2 was
reflected in the overall survival of patients with distal tumors (Veganzones et al., 2015).
Histone modification, especially histone 3-lysine-9 methylation (H3K9) is a common
mechanism of transcriptional repression of gene expression (Yoruker et al., 2012). SETDB1,
which belongs to the family of SET domain histone methyltransferases including Suv39H, EZH2
and G9a, contains a highly conserved motif of 150-amino acids and is involved in the
modulations of chromatin structure (Yang et al., 2002). The SETDB1 protein structure contains
a CpG DNA methyl-binding domain, which regulates H3K9 methylation activity (Ceol et al.,
2011). The CpG island methylation of the CDKN2A gene promoter and expression of SETDB1
in sporadic cutaneous melanomas are highly correlated with histologic prognostic parameters
(Kostaki et al., 2014). Methylation of the CDKN2A promoter is associated with 52% (12/23) of
NRAS-mutated cutaneous melanomas compared with BRAF-mutated (7%, 2/27) tumors
(Jonsson et al., 2010). EZH2, a histone methyltransferase and a catalytic subunit of the PRC2
polycomb repressor, mediates gene silencing by tri-methylating H3K27 of various target gene
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promoters (Bracken et al., 2007, Cao et al., 2002). SNF5, one of the core subunits of Brahma-
associated factor (BAF) chromatin remodeler complexes, down-regulates EZH2 transcription
(Wilson et al., 2010). Thus, the loss of SNF5 function leads to increased EZH2 expression,
thereby repressing the expression of CDKN2A through the tri-methylation of H3K27 in the
CDKN2A promoter region (Wilson et al., 2010).
Epigenetic induction of CDKN2A
Reactivation of silenced CDKN2A or the inhibition of epigenetic repression of the gene could be
a rational strategy for the prevention or treatment of various cancers. While epigenetic silencing
of CDKN2A is caused by DNA hyper-methylation and histone modification (Bracken et al., 2007,
Cao et al., 2002, Collado et al., 2007), restoration of p16
INK4a
transcriptional activation can be
achieved by a set of intracellular regulatory genes as well as a series of small molecule
epigenetic modifiers (Table 3, Figures 3, 4) (Crea et al., 2009, Feng et al., 2009, Hassler et al.,
2012, Kollmann et al., 2011, Li et al., 2013a, Majid et al., 2008, Nandakumar et al., 2011,
Valdez et al., 2015, Zhang and Tong, 2014).
Induction of CDKN2A by regulatory genes
The FOXA1 (Forkhead box A1) protein is a transcription factor that regulates the expression of
p16
INK4a
(Li et al., 2013a, Zhang and Tong, 2014). In hormone-dependent cancers, such as
breast and prostate cancers, the expression of FOXA1 is positively correlated with the
expression of p16
INK4a
, but inhibits the expression of EZH2, which is a component of PRC2 and
an epigenetic repressor of CDKN2A (Nanni et al., 2006). FOXA1 directly binds to the C-terminal
histone-binding motif and inhibits EZH2 methyltransferase activity, thereby inducing the
expression of p16
INK4a
and suppressing cancer cell growth (Zhang and Tong, 2014).
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ZBP-89, a four-zinc finger transcription factor, plays a role in the regulation of target
genes by binding to the GC-rich DNA elements (Wu et al., 2007). ZBP-89 directly recruits
histone deacetylase-3 (HDAC3) to the CDKN2A promoter and represses the expression
CDKN2A by histone hypo-acetylation (Feng et al., 2009). Therefore knockdown of ZBP-89
(ZBP-89i) by a specific small interfering RNA vector induced the expression of p16
INK4a
and cell
senescence in NCI-H460 human lung cancer cells by promoting histone acetylation (Feng et al.,
2009).
Jmjd3 (jumonji domin containing 3) is a histone lysine demethylase, which especially
works to remove the tri-methylation of histone H3 at lysine 27. Jmjd3 induces expression of
p16
INK4a
and senescence of Schwann cells under the conditions of nerve regeneration and
tumorigenic stimulation (Gomez-Sanchez et al., 2013). The UHRF1 (ubiquitin-like plant
homeobox domain (PHD) and RING finger containing 1) protein interacts with DNA
methyltransferase 1 (DNMT1) and recognizes hemi-methylated CpG dinucleotides through its
SRA (SET- and RING-associated domain) region (Bostick et al., 2007). The aromatic cage
mutant of tandem tudor domain (TTD, 124-285 aa) within UHRF1 cannot bind to H2K9me3 and
therefore induces the expression of p16
INK4a
(Nady et al., 2011).
The c-Jun protein is a component of activator protein (AP)-1 and a common regulator of
cell cycle components and was reported to have a direct role in regulating gene transcription of
e.g., p53 and cyclin D1. Although JunB, as a tumor enhancer, reportedly inhibits promoter
methylation of Cdk6 in BCR-ABL-induced leukemia (Ott et al., 2007), binding of c-Jun to the
promoter region shows a protective function and prevents methylation and silencing of these
genes. Because the promoter region of the CDKN2A gene includes binding sites for AP-1, c-Jun
can inhibit methylation of the CDKN2A promoter (Kollmann et al., 2011)
Induction of p16
INK4a
by reagents
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A number of natural and synthetic small molecule modifiers of epigenetic regulation of gene
expression have been identified (Figure 4). Many of these small molecules are capable of
restoring the expression of p16
INK4a
in various cancers. Genistein, an isoflavone present in
soybean, is a cancer chemopreventive agent that inhibits cell proliferation and induces
apoptosis (Gullett et al., 2010). Li et al. reported that genistein represses early breast
tumorigenesis through epigenetic regulation of CDKN2A by impacting histone modifications as
well as by recruiting the BMI1-c-MYC complex to the regulatory region in the CDKN2A
promoter (Li et al., 2013b). In prostate cancer, treatment with genistein increased acetylated
histones 3 and 4 of the CDKN2A transcription start sites (Majid et al., 2008). Thus, soybean
products containing genistein might be useful in preventing breast and prostate cancer through
transcriptional activation of CDKN2A by modulating its epigenetic silencing (Li et al., 2013b,
Majid et al., 2008). Sulforaphane, a major component of broccoli and other cruciferous
vegetables, is an inducer of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), which
regulates the transcriptional activation of a battery of genes encoding various phase 2
detoxification enzymes (Fahey et al., 1997, Kensler et al., 2013). Rajendran et al. reported that
Nrf2 wildtype mice showed a higher incidence of colon tumors than Nrf2 heterozygous (Nrf2
+/-
)
mice when treated with 1,2-dimethylhydrazine. Tumors from wildtype mice exhibited higher
HDAC3 levels globally and deregulated p16
INK4a
levels locally. Treatment with sulforaphane
markedly reduced the tumor burden in wildtype mice but not in Nrf2
+/-
mice. Sulforaphane, an
inhibitor of HDAC3, a repressor of p16
INK4a
, induced p16
INK4a
expression and reduced the tumor
burden in this model (Rajendran et al., 2015).
Epigallocatechin-3-gallate (EGCG), a main polyphenol component of green tea,
stimulated expression of p16
INK4a
by inhibiting DNA methylation and increasing histone
acetylation in the CDKN2A promoter of human skin cancer cells (Nandakumar et al., 2011).
Trichostatin A (TSA) is an HDAC4 inhibitor that elevated the acetylation of HBP1 (K419), a
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member homologous to the sequence-specific high mobility group (HMG) family of transcription
factors, and increased the expression of p16
INK4a
, thereby inducing apoptosis and differentiation
(Wang et al., 2012). The combination treatment with inhibitors of DNMTs and HDACs might be
a representative therapeutic strategy in cancer. Treatment of CA46 lymphoma cells with EGCG
(24 μg/ml) or the combination treatment of EGCG (6 μg/ml) and TSA (15 ng/ml) resulted in
lower proliferative indices when compared to the other groups. Co-treatment with EGCG and
TSA decreased the DNA methylation of CDKN2A, which coincided with increased CDKN2A
mRNA and protein expression. Thus, EGCG and TSA synergistically induced CDKN2A
expression by reducing promoter methylation, which might decrease CA46 lymphoma cell
proliferation (Wu et al., 2013).
5-Aza-2’-dexoycytidine (5-aza-CdR, decitabine, Dacogen) is a DNMT inhibitor, which
has been approved by the Food and Drug Administration (FDA) for the treatment of
myelodysplastic syndrome, a hematological malignancy (Scandura et al., 2011). Treatment of
anaplastic large cell lymphoma cells with 5-aza-CdR resulted in the inhibition of proliferation
through G1 phase cell cycle arrest in vitro and in vivo through re-expression of p16
INK4a
(Hassler
et al., 2012). Furthermore, combination treatment with TSA and 5-aza-CdR exerted synergic cell
growth inhibition and apoptosis in EBV-associated gastric carcinoma through the induction of
CDKN2A by inhibiting methylation (He et al., 2015). On the other hand, co-treatment with 5-aza-
CdR and irinotecan, a topoisomerase-1 inhibitor, caused a more sensitive cytotoxic effect in
p53-mutated colon cancer cells, such as HT-29, SW620, and WiDr cells (Crea et al., 2009). The
synergistic effect of 5-aza-CdR and irinotecan was significantly associated with topoisomerase I
up-regulation by 5-aza-CdR, and combined to induce CDKN2A expression through promoter
demethylation as well as Sp1 up-regulation. Expression of p16
INK4a
enhanced cell cycle arrest
after irinotecan treatment (Crea et al., 2009).
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Cladribine and clofarabine are second-generation analogues of 2’-deoxyadenosine.
Fludarabine (Fludara) and Busulfan (Myeran) and are cell cycle non-specific alkylating
antineoplastic agents. Although both cladribine and clofarabine induced p16
INK4a
expression,
cladribine was effective at a lower concentration compared to clofarabine (Valdez et al., 2015).
The combination of cladribine or clofarabine with fludarabin and busulfan caused synergic
cytotoxicity in acute myeloid leukemia. Moreover, the addition of panobinostat, an HDAC
inhibitor, and 5-aza-CdR with these combinations also enhanced cytotoxicity. Inclusion of
panobiostat and 5-aza-CdR increased histone modifications and DNA demethylation, and
increased the levels of the p16
INK4a
, p15
INK4b
and p21
Waf1/Cip1
proteins (Valdez et al., 2015).
Treatment with doxorubicin and FUMI (5-fluorouracil and mitomycin C) in breast cancer
resulted in demethylation of the CDKN2A promoter by 19.3% and 9.1%, respectively (Klajic et
al., 2014). The CDKN2A gene was differentially methylated before/after treatment with
doxorubicin and before/after treatment between responders. Lower levels of methylation were
observed after treatment in the responder groups. Also, CDKN2A methylation levels among the
non-responders were significantly changed (Klajic et al., 2014).
Conclusion
A high frequency of genetic and epigenetic alterations (e.g., promoter hyper-methylation,
homozygous deletion or mutation) in the CDKN2A gene has been observed in human cancer
cell lines derived from various tumor types. However, a significantly lower frequency of CDKN2A
abnormalities has been reported in fresh, non-cultured primary tumors and cell lines. Therefore,
regulation of CDKN2A abnormalities will have benefits for the cancer prevention and/or therapy.
Outstanding Questions
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Besides genetic and epigenetic aberrations, the regulation of gene expression is also
associated with the status of many other proteins, such as p53, BRAF, ataxia talengectia
mutated (ATM), phosphatase and tensin homolog deleted at chromosome 10 (PTEN), KRAS
and phosphatidylinositol 3-kinase (PI3-K) as well as the components of the CDKN2A promoter,
such as the PRC1 and PRC2 complexes and long noncoding RNAs. HPV, a small DNA virus
(major types HPV-16 and HPV-18) expresses E6 and E7 oncoproteins and is linked causally to
most cervical cancers that show a high expression of p16
INK4a
(Kobalka et al., 2015, Munger et
al., 2013, Pauck et al., 2014, Nehls et al., 2008). The E6 and E7 oncoproteins directly block
p53 and pRb tumor suppressors resulting in the accumulation of p16
INK4a
, which is upstream of
of p53 and pRb (Dyson et al., 1989, Scheffner et al., 1993). HPV-infection-associated cervical
cancer also mediates the high expression of p16
INK4a
by epigenetic modulation of the CDKN2A
upstream promoter (Nehls et al., 2008, McLaughlin-Drubin et al., 2013). Thus in HPV-infection-
associated cancer, we should consider the p16
INK4a
as a biomarker in an opposite manner than
what is generally expected. Although smoking and/or drinking is a risk factor for lung, head and
neck, and pancreatic cancers (Gillison et al., 2012, Jiao et al., 2007, Tam et al., 2013),
meaningful correlations of p16
INK4a
expression with smoking or drinking are not yet supported by
strong evidence (Jiao et al., 2007, Gillison et al., 2012, Tam et al., 2013). Because p16
INK4a
is a
critical regulator of cell proliferation and is inactivated during the course of tumorigenesis,
methods for restoration of p16
INK4a
function could provide good therapeutic strategies. To
achieve this goal, the focus has been directed toward understanding the molecular mechanisms,
especially genetic and epigenetic changes that lead to p16
INK4a
inactivation in cancer, and
developing small molecule modifiers of these mechanistic switches. For example, ZBP-89 can
induce the expression of p16
INK4a
(Zhang et al., 2010). Likewise, FOXA1 and jmjd3 enhance
CDKN2A expression by direct transcriptional activation and promoter di-methylation,
respectively. Thus, discovery of small molecule activators of ZBP-89, jmjd3 or FOXA1 might be
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a future research goal for developing novel anticancer therapies. On the other hand, factors that
repress CDKN2A gene expression, such as SETDB1, EZH2, or PcB proteins, could be potential
therapeutic targets to induce p16
INK4a
functionality and tumor suppression. Although several
natural and synthetic compounds, such as genistein, sulforaphane, EGCG and 5-aza-CdR have
been shown to intervene against the epigenetic silencing of CDKN2A, mechanism-based
development of p16
INK4a
activators warrants further studies.
Search Strategy and Selection Criteria
This review was prepared by searchin in PubMed with querying key words: p16
INK4a
, epigenetics,
alteration, gene alteration, gene silencing, cancer, promoter alteration and induction of p16
INK4a
.
The search was conducted for research articles including clinical reports up to 28 December
2015 and we especially summarized research articles in each cancer type.
Acknowlegements
We wish to thank Dr. Joydeb Kumar Kundu for helpful comments on the manuscript
Financial Support: This work was supported by grant funding from the National Institutes of
Health CA166011, CA187027 and CA196639, The Hormel Foundation and Henan Provincial
Government, China. Funders had no role in producing this manuscript.
Disclosure of Potential Conflicts of Interests.
No potential conflicts of interest are disclosed.
Authors’ contribution. RZ contributed to the literature search and collection of articles,
assisted with designing the figures and writing; BYC contributed to the design and organization
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20
of the manuscript; MHL did most of the original writing; AMB did all the editing and confirmation
of references and accurate information; ZD supervised the studies and allocated the funding.
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Figure legends
Figure 1. Schematic structure of the INK4a/ARF locus and the role of p16
INK4a
in cells. CDKN2A
is produced by alternative splicing of E1, E2 and E3. The p16
INK4a
protein binds to the cyclin D
and CDK4/6 complexes and inhibits the activation of the transcription factor, E2F1, which
induces proteins to move from the G1 phase to S phase in the cell cycle.
Figure 2. Frequency of CDKN2A alterations in various cancer types. Based on the types of
aberrant CDKN2A, the bars represent the percentage of changes reported.
Figure 3. The epigenetic induction of p16
INK4a
by regulatory genes. FOXA1, Si-ZBP-89, Jmjd3,
Mutant UHRF1 and c-JUN induce p16
INK4a
protein expression by re-activation of the CDKN2A
promoter.
Figure 4. The epigenetic induction of p16
INK4a
by reagents. Phytochemicals such as genistein,
SFN and EGCG and synthetic chemicals, TSA, 5-aza-2’-deoxycytidine, irinotecan, cladribine,
clofarabine, doxorubicin, FUMI (5-fluorouracil+mitomycin C) and combinations reactivate
p16
INK4a
protein expression by prohibiting alterations in the CDKN2A promoter.
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Table 1. Changes in CDKN2A in various cancers
Cancer types
Status of alteration
Frequency (%)
Reference
Gastric
lymphoma
Promoter hypermethylation
29.7% (11/37)
(Huang et al., 2007)
Burkitt’s
lymphoma
Promoter hypermethylation
72.5% (37/51)
(Robaina et al., 2015)
Skin cancer
Promoter hypermethylation,
histone modification
50-70%, 80-90%
(Chen et al., 2012a)
Melanoma
Promoter hypermethylation
25.9% (15/58)
(Kostaki et al., 2014)
Histone modification
(Sarkar et al., 2015)
Promoter hypermethylation,
non-synonymous mutation
25% (15/59), 16%
(9/56)
(Jonsson et al., 2010)
Head and neck
squamous cell
carcinoma
Mutation/ promoter
hypermethylation
57% (138/243)
(Chung et al., 2015)
Promoter hypermethylation
54.5% (6/11 cell
line) and 66.7%
(20/30)
(El-Naggar et al.,
1997)
Oral cancer
Promoter hypermethylation
60% (6/10)
(Asokan et al., 2014)
Pancreatic
adenocarcinoma
Mutation
11.8% (2/17)
(Salo-Mullen et al.,
2015)
Promoter hypermethylation
24.6% (14/57)
(Jiao et al., 2007)
Non-small cell
Lung cancer
(NSCLC)
Homozygous deletion (HD)
/mutation/ Promoter
hypermethylation
53%/13%/33% in
cell lines
(Tam et al., 2013)
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Mutation/frameshift/HD
19% (12/63)
(Marchetti et al., 1997)
Esophageal
squamous cell
carcinoma
Loss of heterozygocity (LOH)
/mutation
13% (5/38)/6%
(4/67)
(Kuwabara et al.,
2011)
Promoter hypermethylation
81.7% (210/257)
(Chen et al., 2012b)
Deletion
20% (22/106)
(Qureshi et al., 2012)
Gastric cancer
Hypermethylation
81.6% (40/49,
EBVaGC), 33.3%
(15/45, EBVnGC)
(He et al., 2015)
Colorectal
cancer
Promoter hypermethylation
11.9% (34/285)
(Kim et al., 2010)
Promoter hypermethylation
17.1% (87/497)
(Rajendran et al.,
2015)
Epithelial ovarian
caricnoma
Promoter hypermethylation
43% (58/134)
(Bhagat et al., 2014)
Prostate cancer
Expression of ANRIL, CBX7,
and EZH2
(Yap et al., 2010)
Promoter hypermethylation
47.6% (10/21)
(Ameri et al., 2011)
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Table 2. Genes and factors correlated with changes in CDKN2A
Genes/Factors
Cancer types
Note
Reference
HPV
Head and neck
squamous cell
carcinoma
CDKN2A alteration in
non-HPV-cancer
(Chung et al., 2015)
EGFR
Lung adenocarcinoma
CDKN2A
homozygous deletion
or mutation is
correlated with EGFR
mutation
(Tam et al., 2013)
KRAS
Hypermethylation of
CDKN2A promoter is
correlated with KRAS
mutation
hMLH1 (human mutL
homologue 1)
Colorectal cancer
(microsatellite
instability)
Hypermethylation of
both CDKN2A and
hMLH1 is 33.3%
(17/51)
(Veganzones et al.,
2015)
SETDB1
(sporadic cutaneous)
Melanoma
Cytoplasmic SETDB1
expression correlates
with higher frequency
of CDKN2A
methylation and
p16
INK4A
expression
(Kostaki et al., 2014)
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NRAS
Cutaneous melanoma
NRAS-mutated
tumors have CDKN2A
promoter methylation
(52%, 12/23)
(Jonsson et al., 2010)
SNF5
-
Inactivation of H3K27
tri-methylation of
CDKN2A by EZH2 in
deficient SNF5
(Wilson et al., 2010)
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Table 3. Epigenetic induction of p16
INKa
Inducer
Mechanisms
Cancer types
References
Genes
FOXA1
EZH2 inhibition
Breast cancer
and prostate
cancer
(Zhang and Tong,
2014, Li et al.,
2013a)
Si-ZBP-89
HDAC3 inhibition
NCI-460 human
lung cancer cells
(Feng et al., 2009)
Jmjd3
Histone demethylation
Neurofibroma
Schwann cells
(Gomez-Sanchez
et al., 2013)
Mutant UHRF1
H3K9me3 inhibition
(Nady et al., 2011)
c-JUN
Protect DNA
methylation
(Kollmann et al.,
2011)
Compounds
Genistein
Induction of histone
acetyl transferase
(HAT)
Prostate cancer
(Majid et al.,
2008)
Modification of histone
and Inhibition of binding
BMI1 and c-MYC to
CDKN2A promoter
Breast cancer
(Li et al., 2013b)
Sulforaphane (SFN)
HDAC3 inhibition by
induction of Nrf2
Colon cancer
(Rajendran et al.,
2015)
EGCG
DNA demethylation and
histone acetylation
Skin cancer cell
(Nandakumar et
al., 2011)
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TSA
HBP1 transcription
factor acetylation
(Wang et al.,
2012)
EGCG and TSA
DNA demethylation
Lymphoma
(Wu et al., 2013)
TSA and 5-aza-2’-
deoxycytidine
DNA demethylation
Epstein–Barr
virus-associated
gastric cancer
cells
(He et al., 2015)
5-aza-2’-
deoxycytidine
DNA demethylation
Large cell
lymphoma
(Hassler et al.,
2012)
5-aza-2’-
deoxycytidine and
irinotecan
DNA demethylation
Colorectal cancer
cells
(Crea et al., 2009)
Cladribine, clofarabine
DNA demethylation
Acute myeloid
leukemia
(Valdez et al.,
2015)
Doxorubicin, FUMI (5-
fluorouracil+mitomycin C)
DNA demethylation
Breast cancer
(Klajic et al.,
2014)
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