Background
Cervical cancer remains a serious global health problem, with an estimated 528,000 new cases and 266,000 deaths in 2012 [
1]. Approximately 80% of cervical cancers occur in developing countries [
2]. Certain types of the
human papilloma virus (HPV) infection, particularly HPV 16 and HPV 18, are the greatest risk factors for cervical cancer. Screenings, such as the Papanicolaou and HPV tests, have been largely successful in preventing cervical cancer, but it is still the second most common cause of cancer death among women worldwide, resulting in 275,000 deaths annually [
3].
Not all individuals who are infected with high-risk HPVs develop genital cancer, which indicates that HPV infection is necessary but not sufficient for malignant development [
4‐
6]. Additional genetic alterations, either independent or in conjunction with HPV infection, are required for tumor development. When cells are persistently infected with HPV, the primary viral oncoproteins E6 and E7 are reportedly involved in the disruption of many normal functions [
7‐
10]. Consequently, these lead to an accumulation of somatic mutations. Early reports have frequently observed somatic mutations in cervical cancer [
11,
12]. These genetic alterations can be equally important for cell transformation. An in-depth characterization of the underlying genetic events is important for understanding tumor progression, which can guide the development of effective targeted therapies.
The large volume of data currently generated by the TCGA provides a rich resource and a new opportunity for exploring the genetic alterations in cervical cancer. In this study, we set out to explore the pathogenic genes by investigating the somatic mutations in 194 cases of cervical cancer exome-seq data from the TCGA. The analysis showed that the chromatin modification pathway was significantly altered. Half of the epigenetic regulators involved in this pathway harbored mutations capable of disrupting the phosphorylation sites. Of all of the altered epigenetic regulators, only the histone methyltransferase MLL2 mutations were associated with poor survival. Around half of the mutations’ peptides were predicted to be immunoreactive, which indicates that patients are likely to benefit from immunotherapy. This study highlights the emerging role of epigenetic regulators, particularly MLL2, and suggests potential epigenetic dysregulation, in cervical cancer tumorigenesis.
Discussion
Papanicolaou smear and colposcopy programs can prevent cervical cancer development; however, 80% of diagnosed cases have already progressed to the later stages [
34]. Women with cervical cancer remain a high-risk population for whom effective treatment options and reliable therapy targets are limited. In this study, we demonstrated that the chromatin modification pathways of cervical cancer patients were significantly altered. Mutations in
MLL2, a histone methyltransferase, were associated with poor survival. This study indicates that genetic mutations in epigenetic regulators and potential epigenetic dysregulation play a role in the development of cervical cancer. As a result, our understanding of the pathogenesis of cervical cancer is greatly improved, and new therapeutic strategies are suggested.
Aberrant epigenetic changes in cervical cancer have been widely studied, and the main focus has been on DNA methylation [
35‐
37], such as the hypermethylation of oncogenic genes [
35]. In contrast, histone modification changes in cervical cancer have not been studied as extensively. Alterations of epigenetically modified genes were not examined in our study, but it was shown that epigenetic regulator genes were actually recurrently mutated in cervical cancer. It was also shown that they mainly had a histone methyl-transferation function. Thus, the mutation of these factors may consequently lead to abnormal histone modification in the genome. Interestingly, mutations in the histone methyltransferase
MLL2 that methylates the Lys-4 position of histone H3 exhibited worse overall survival. It is highly probable that the cervical cancer genome may harbor abnormal H3K4 methylation, which may shape a new epigenetic landscape that contributes to cancer deterioration. Approaches such as Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) for the H3K4me3, and other histone modifications in the patient tissues, deserve further study.
Half of the epigenetic regulators harbored mutations in or around the phosphorylation sites in their enzymes. Interestingly, the blocking of
EP300’s phosphorylation was reported to decrease the proliferation and metastasis activity of lung cancer cells. The molecular mechanism showed that phosphorylation blocking in this protein disrupted chromatin condensation and increased the acetylation of histone H3 [
38]. The phosphorylation of
MLL2, controlled by CDK2, facilitated its recruitment to developmental genes in G1 in human pluripotent cells, consequently leading to changes in the developmental genes’ chromosome architecture [
39]. Mutations occurring on the phosphorylation site in
CREBBP were shown to result in inappropriate activation of gluconeogenesis [
40]. The inhibition of
SETD2’s phosphorylation by long non-coding RNA HOTAIR has been reported to trigger a reduction of trimethylation on histone H3 thirty-sixth lysine, consequently promoting human liver cancer stem cell malignant growth [
41].
SMARCA4, also known as
BRG1, was shown to modulate DNA double-strand break repair by its phosphorylation [
42]. It has been suggested that the enzymatic activity of
DNMT1 is possibly modulated by phosphorylation [
43], and it has been demonstrated that its phosphorylation by AKT1 kinase increases its stability and abundance [
15]. The phosphorylation of
NCOR1 was shown to play a role in transcriptional regulation in prostate cancer [
44] and in the liver in mice [
45].
EZH2, despite of its ability to trimethylate lysine 27 in histone H3, when phosphorylated, suppressed its methyltransferase activity [
46,
47], and switched to a coactivator for its oncogenic function in prostate cancer [
48]. The phosphorylation of
DNMT3A was found to be required for its localization to
heterochromatin and capable of shaping the CpG methylome [
49]. Phosphorylated
DAXX was reported to facilitate DNA damage-induced
p53 activation [
50]. Thus, it seems evident that phosphorylation is very important to these proteins’ normal functions. In some epigenetic regulators, phosphorylation is associated with cancer cell malignant growth. It will be interesting to explore the functional links between those specific phosphorylation events and the epigenetic regulators’ activities in cervical cancer.
Although the chromatin modification pathway was predominantly mutated across all clinical stages, others, such as the RAS, PI3K, NOTCH and apoptosis pathways, were also recurrently mutated at certain stages. These pathways and the recurrently mutated genes involved therein, such as
EP300,
ARID1A,
FBXW7,
NFE2L2,
PIK3CA, and
ERBB2, were all found to be pathogenic in previous cervical cancer studies [
11,
12]. However, only mutations in
ERBB2 were associated with worse survival.
ERBB2 is involved in both the RAS and PI3K pathways.
MAP2K1 and
MAP3K1 were also mutated in the RAS pathway. Thus, the signal cascade, which should be activated when normally phosphorylated, may be disrupted. We observed that the mutations in some epigenetic regulators occurred around the phosphorylation sites. Currently, the temporal mutational order relationship or the association between these genetic events is unknown. The genetic mutations appear to have disrupted phosphorylation, which could together lead to a series of disorders in the cervical cancer cells.
One previous study of 115 cervical cancer samples from Norway and Mexico identified previously unknown somatic mutations that recurrently occurred in
EP300,
FBXW7,
NFE2L2,
TP53, and
ERBB2 [
11]. Another study in 15 cervical cancer patients from Hong Kong revealed frequently altered genes, including
FAT1,
ARID1A,
ERBB2, and
PIK3CA [
51]. One recent study of 228 cervical cancers using TCGA data identified
SHKBP1,
ERBB3,
CASP8,
HLA-A, and
TGFBR2 as novel significantly mutated genes, and previously identified pathogenic genes including
PIK3CA,
EP300,
ARID1A, and
NFE2L2 were also confirmed [
52]. Similarly, all of these genes were identified in each study using the same approach of analyzing the significantly mutated genes, whereas we only focused on the driver genes and core pathways that play significant roles in tumorigenesis. Some of the gene mutations reported in our study, such as
MLL3 and
MLL2, were not previously identified in those studies, which may be because they did not satisfy the significance criteria. In contrast, other altered genes in this study, including
EP300,
ARID1A,
FBXW7,
NFE2L2,
PIK3CA, and
ERBB2, were reported as pathogenic genes in the aforementioned previous studies and were consistent with those cervical cancer genome studies. Interestingly,
CASP8, which was newly identified as a significantly mutated gene in the recent study [
52], was also included in the 138 driver genes in our study, and was frequently mutated in the apoptosis pathway which was significantly altered at Stage III (Fig.
1; Additional file
6: Figure S3). Additionally, unlike this study, other cervical cancer genome studies did not report the chromatin modification pathway as being predominately mutated as compared to other core cancer pathways. Among the genes involved in this pathway, we found that the mutations in
MLL2 were associated with poor survival in cervical cancer. The role of epigenetic regulator mutations has been identified as increasingly important in other cancers’ tumorigenesis [
19,
53]; however, it has not been extensively explored in cervical cancer. This study is the first to highlight mutations in the epigenetic regulators, particularly the emerging role of
MLL2 in cervical carcinogenesis. Our results shed more light on the epigenetic mechanism underlying cervical cancer pathogenesis.
Most epigenetic therapy agents used in treatment are analogue inhibitors [
35]. Clinical studies, however, have demonstrated their limitations, such as poor activity against solid tumors and toxicity [
35,
54]. Thus, the merits of this targeted therapy have not yet been established. Accordingly, it is imperative that possible therapy strategies be identified, and the evidence increasingly suggests that T cells can provide clinical responses by recognizing unique neo-antigens expressed by somatic mutations in tumors [
55‐
57]. By screening tumor-specific neo-antigens and identifying mutation-specific T cells, the immune targeting of cancer mutations has demonstrated therapeutic potential [
30,
58]. The numerous neo-epitopes in our data derived from the mutations indicate that an anti-mutation T cell response might be feasible. Further investigation into potential immunotherapies for cervical cancer is warranted.