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Received: October 22^nd 2014

Accepted: May 15^th 2015

Epigenetic alterations in cervical cancer progression

Magdalena Ríos-Romero,^a Ana Guadalupe Soto-Valladares,^a Patricia Piña-Sánchez^a

^aUnidad de Investigación Médica en Enfermedades Oncológicas, Laboratorio de Oncología Molecular, Hospital de Oncología, Centro Médico Nacional Siglo XXI, Distrito Federal, México

Communication with: Patricia Piña-Sánchez

Telephone: (55) 2454 6476

Email: 1307patricia@gmail.com

Despite the use of the screening test, such as Papanicolaou, and the detection of human papillomavirus (HPV), cervical cancer remains as a public health problem in México and it is the second leading cause of death for malignant neoplasias among women. High-risk HPV infection is the main risk factor for the development of premalignant lesions and cervical cancer; however, HPV infection is not the only factor; there are various genetic and epigenetic alterations required for the development of neoplasias; some of them have been described and even in some cases they have been suggested as biomarkers for prognosis. However, in contrast with other cancer types, such as breast cancer, in cervical cancer the use of biomarkers has not been established for clinical applications. Unlike genetic alterations, epigenetic alterations are potentially reversible; in this sense, their characterization is important, since they have not only a potential use as biomarkers, but they also could represent new therapeutic targets for treatment of cervical cancer. This review describes some of the more common epigenetic alterations in cervical cancer and its potential use in routine clinical practice.

Keywords: Cervical cancer, Epigenetics, Biomarkers.

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Cervical cancer

Cervical cancer (CC) is the second leading cause of death from malignancy in women in Mexico, so it remains a public health problem. In 2011 CC represented 10.4% of all deaths from malignancies in our country.¹ CC is preceded by premalignant lesions known as low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL), according to the Bethesda classification, or intraepithelial neoplasia grade I, II, and III according to Richard’s classification.² Its main etiological factor is persistent infection with human papillomavirus (HPV) of high oncogenic potential.³ However, infection alone is not enough for the development of CC, since many women may be infected with HPV and not develop cancer, so there are other cofactors involved in development of this neoplasia.⁴ Cancer is considered as a set of diseases associated with genetic alterations. In CC a large number of genetic alterations have been characterized such as chromosomal aberrations, which include gains in 3q21-q22, 5p15.2, and 5p13 regions, and losses in chromosomal regions 3p12, 3p14.2, 13q14, and others.^5,6 Regarding overall expression profiles, genes have been identified via Wnt.⁷

However, cancer is not just a product of genetic alterations. Since the early eighties epigenetic alterations in cancer have been reported. Some events, such as hypermethylation of promoter regions, are involved in the initiation and progression of various cancers, and are considered a classic mechanism of inactivation of tumor suppressor genes.⁸ It is currently considered that alterations in expression and mutations can derive from epigenetic alterations, whose functional consequences can be equivalent to those generated by genetic changes.⁹

Epigenetic alterations in CC

Epigenetics is the study of heritable changes in gene expression that do not involve alterations in DNA sequence. Four epigenetic modifications that are closely related to each other are known: DNA methylation, histone modifications, gene imprinting, and regulation mediated by non-coding RNAs (Figure 1).¹⁰ Much of the epigenetics research focuses on changes in chromatin structure, such as DNA methylation and histone modifications. 

DNA methylation

DNA methylation is the main modification in eukaryotic genomes. It is the covalent addition of a methyl group (CH3) to the carbon 5 of cytosines in the context of CpG dinucleotides by DNA methyl transferase (DNMT). CpG islands (CGI) (GpC-rich regions associated with gene promoters, at least 200 bp in length) are involved in the regulation of expression. It is estimated that approximately 60-70% of human genes have CGI in their regulatory regions. Under normal conditions, CGI are protected from methylation, which is associated with open chromatin configuration, characterized by the presence of acetylated histones.¹¹

Local hypermethylation is generally related with silencing of the associated gene. The increase in DNA methylation causes an impediment in the binding of transcription factors and thus downregulation of gene expression. Hypermethylation is a classic phenomenon of inactivation of tumor suppressor genes (such as Rb, VHL, CDKN2, BRCA, APC, and GSTP1) which are associated with the development of cancer.¹² In some cancers, such as breast, colon, and rectum, it is suggested that there is a "CGI methylator phenotype" in which multiple genes in malignant cells are frequently methylated; this, associated with particular clinical features, could serve as a basis to classify different tumor subtypes.¹³ It has been observed that some of the genes that are part of this phenotype are classic tumor suppressors, like p16, MLH1, APC, and HIC1, among others.¹⁴

A large number of genes have been described whose methylation profile is differential, when comparing epithelium without neoplastic alterations with precursor lesions or cervical cancer. However, only some are consistently methylated in various studies: DAPK1, RASSF1, CDH1, CDKN2A, MGMT, RARB, APC, FHIT, MLH1, TIMP3, GSTP1, CADM1, CDH13, HIC1, and TERT; some of these data are derived from global methylation studies that aimed to identify new markers associated with CaCU.¹⁵ Hypermethylation of SOX and WT1 in epithelia without neoplastic changes is associated with the development of precursor lesions,¹⁶ while hypermethylation of hTERT in precursor lesions is associated with progression to CC.¹⁷ It has been suggested that hypermethylation of genes such as BRCA1 and RARbeta can be used as a predictive marker of poor prognosis in CC due to the failure to respond to treatment based on radiotherapy.¹⁸ It is also suggested that DAPK and FAS are predictors of response to chemotherapy and radiotherapy, as patients with a poor response to therapy had higher rates of methylation than patients with good response.¹⁹

In cancer, the genome is globally hypomethylated; it has been shown that Dnmt3 inactivation generates chromosomal instability and immortalization.²⁰ Thus, global hypomethylation has been associated with the progression of CC precursor lesions.²¹ However, despite hypomethylation being the first epigenetic alteration described in cancer, this is not as extensively studied as the phenomenon of hypermethylation.

Histone code

Histones are organized into octamers consisting of three types of histones: H2A, H2B, and H3 and H4. Histones have an amino-terminal region rich in lysine residue (K), which are susceptible to post-translational modifications, of which acetylation and methylation are the most studied. These changes affect the DNA-histone interaction, which leads to changes in gene transcription, DNA repair, DNA replication, and chromosome organization.⁸ The set of histone modifications is a code that indicates whether that region of chromatin must be activated or inactivated. There are well-characterized examples of histone marks associated with the opening or closing of chromatin, such as the acetylation of lysine 9 on histone 4 (H4K9 ac), which is associated with active gene transcription, and trimethylation of lysine 27 on histone 3 (H3K27me3) which is associated with transcriptional repression. The histone code pattern suggests that the combination of these marks promotes an activation or silencing phenotype and not the individual modifications.²²

In precursor lesions, alterations are described in the phosphorylated and acetylated forms of histone H3 associated with the progression of CIN I to CIN II and CIN II to CIN III.²³ The gene RARbeta2 has been found silenced due to promoter methylation; however, its silencing has been reported associated with H3 and H4 deacetylation and reduced H3me2K4 levels and high H3me2K9 levels, regardless of promotor hypermethylation.²⁴ Histone variants such as H2AX are phosphorylated (Ser139) in response to breakthroughs of double-stranded DNA; in vivo studies have suggested the presence of g-H2AX as a predictor of response in the treatment of tumors with cisplatin and radiotherapy; however, these studies are still in experimentation.²⁵

Noncoding RNA

Non-coding RNA (ncRNA) constitute a large group of RNA that are not translated into proteins. They are divided into two groups according to their size: small ncRNA are 20-200 nt, which are formed by miRNA, siRNA, piRNA and are involved in regulating the stability and efficiency of mRNA translation; and long ncRNA (lncRNA) are more than 200 nt, and function as regulators of transcription by recruiting chromatin remodeling complexes.²⁶

MiRNAs are sequences of 22 nt RNA whose function is to regulate gene expression at the posttranscriptional level, inhibiting protein translation, by binding to the 3'UTR end of mRNA. A number of reports have been published on their biogenesis, processing, annotation, function, and in relation to their role in diseases such as cancer. The database MiRBase (www.mirbase.org) reports 1872 human miRNA. The expression profiles of miRNAs in cancer are proposed as traces associated with the diagnosis, stage, progression, prognosis, and response to treatment.²⁷ Particularly in CC there is abundant information about alterations in miRNA expression. For example, Pereira et al.²⁸ identified that in the sequences miR-143, miR-145, miR-99a, miR-26a, miR-203, miR-513, miR-29a, and miR-199a, expression is significantly decreased in precursor lesions and CC relative to normal samples, in contrast to miR-148a, miR-302b, miR-10a, miR-196a, and miR-132, which are overexpressed. Moreover, it has been determined that miR-375 and miR-224 overexpression is associated with acquired resistance to paclitaxel²⁹ and poor prognosis,³⁰ respectively, so evaluation is important for their use as potential markers of progression and response to therapy. In particular, the expression of miRNAs has been evaluated in connection with HPV infection, which suggests deregulation of expression of the miR-15/16 cluster, the miR-17-92 family, miR-21, miR 23b, miR-34th E6-p53 pathway, and miR-106b/93/25 cluster E7-pRb pathway.³¹ 

Like miRNA, lncRNA have been associated with various cancers, including colon, bladder, breast, hepatocellular, neuroblastoma, prostate, leukemia, thyroid, and recently cervical.³² One of the first studies that reported changes in the expression of lncRNA involved the imprinted gene H19; later, aberrations in imprinting or deletions were identified in 58% of cases of cervical cancer, so their participation is suggested in tumor progression.³³ In cellular models (CasKi), the lncRNA MALAT1 participates in events such as apoptosis, and its expression is associated with the regulation of caspase 3, caspase 8, Bax, and BclXL.³⁴ Landerer et al.³⁵ reported low expression of mitochondrial lncRNA ASncmtRNAs-1 and ASncmtRNAs-2 and suggested their involvement in neoplastic transformation and progression. Later, lncRNA were identified differentially expressed in precursor lesions, but they are not entirely characterized.³⁶

Biomarkers and epigenetic therapies in cancer

Detection of epigenetic alterations can be used for diagnosis, prognosis, and response to therapy in various cancers. It is estimated that silencing by methylation is 10 times more represented than inactivation by genic mutation.³⁷ In low grade gliomas and glioblastoma, MGMT methylation is used as a marker of response to temozolomide.³⁸

Although the use of some epigenetic biomarkers in cancer is well supported (e.g. MGMT in glioblastomas), histone marks and lncRNA expression have been little studied in cancer in general and particularly in CC. DNA methylation, ncRNA, and histone codes are closely related, so that integration of these phenomena in the processes of transformation and progression is important not only in the context of their use as biomarkers, but in understanding processes that lead to cancer. Since there are no well-established biomarkers in CC (as in the case of breast or prostate cancer), it is necessary to study candidate molecules that are able to predict clinical factors such as progression, survival, and response to treatment. So far we have described evidence supporting a relevant role of epigenetic alterations in cervical cancer that are an early and widespread phenomenon in these tumors. These features make it an ideal target for the development of new biomarkers and new therapies. 

Treatment of cervical cancer and perspective from an epigenetic approach

Unlike genetic markers, epigenetic markers are potentially reversible. The Food and Drug Administration (FDA) of the United States approved two drugs that induce hypomethylation: 5-azacytidine and decitabine (5-aza-deoxycytidine). Both drugs inhibit DNMT1 and bond to it stoichiometrically, which prevents the enzymatic reaction of methylation from happening.39 5-azacytidine and decitabine are drugs often used as last-line treatment for patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). On the other hand, HDAC inhibitors are another class of drugs that have been tested in the clinic for the treatment of hematologic malignancies. An example of these is Vorinostat, which was approved by the FDA in 2007 to treat advanced cutaneous T-cell lymphoma (CTCL).⁴⁰

It has been suggested that the combination of demethylating agents and HDAC inhibitors such as valproic acid, may increase clinical response, as this leads to the re-expression of genes that were previously silenced, as well as sensitizing tumor cells to treatment with other chemotherapeutic agents or radiotherapy.⁴¹

Some clinical trials have shown benefit from epigenetic therapy based on valproic acid and hydralazine in patients with cervical cancer in advanced stages. These trials have shown improved survival, progression-free (median 10 months) compared to the control group (mean 6 months); however, the molecular characteristics associated with this response have not been characterized.⁴²

Moreover, there are some studies in clinical trials evaluating the use of antisense RNA in the treatment of solid tumors, such as trabedersen, which was evaluated in a phase II trial for the treatment of glioblastomas,⁴³ and oblimersen, which targets the BCL-2 family and has been used in patients with breast cancer.⁴⁴ Currently several in vitro trials are being developed to assess inhibition of some ncRNA, such as miR-214, as a measure to increase sensitivity to cisplatin, and the use of ribozymes directed against HPV;⁴⁵ but so far no published clinical trials have evaluated ncRNA as therapeutic targets in CC.

Conclusions and prospects

Epigenetic alterations involve multiple phenomena that together contribute to carcinogenesis in cervical cancer. Evidence in recent years suggests that these changes can be used as markers of clinical or predictive prognosis. The list of hypermethylated genes in cancer with potential clinical use continues to grow, suggesting that it is a prevalent phenomenon that could be used more extensively in various malignancies. One of the trends towards which research conducted in this field tends, relates to evaluating multiple epigenetic markers in parallel, which greatly increases their sensitivity and specificity and, therefore, their predictive or prognostic value. The integration of epigenetic information generated in basic and clinical research will be essential to improve diagnosis, prognosis, and treatment of cancers such as cervical cancer.

Acknowledgements

To Dr. Luis Felipe Jave Suarez and Dr. Guadalupe Martinez Silva for review and suggestions on this work.

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