Epigenetic deregulations in chordoma

Abstract

Chordoma is a rare type of malignant bone tumour arising from remnant notochord and prognosis of patients with it remains poor as its molecular and genetic mechanisms are not well understood. Increasing evidence has demonstrated that epigenetic mechanisms (DNA methylation, histone modification and nucleosome remodelling), play a crucial role in the pathogenesis of many diseases. Aberrant epigenetic patterns are present in patients with chordoma, indicating a potential role for epigenetic mechanisms inthis malignancy. Furthermore, epigenetic alterations may provide novel biomarkers for diagnosis and prognosis as well as therapeutic targets for treatment. In this review, we discuss relevant epigenetic findings associated with chordoma, and their potential application for diagnosis, prognosis and treatment.

Introduction

Chordoma is a rare malignant neoplasm of bone arising from remnant notochordal tissues. It usually arises in the axial skeleton, 50% of these tumours being located in the sacrum 1. Surgery remains the best standard treatment, but adequate excision is difficult due to close relationships between elements of the central nervous system and other vital structures 2, 3, 4. Thus, prognosis of patients with chordoma remains poor, with local recurrence levels of over 40%; overall median survival is approximately only 6 years 5. Although morphology and immunoprofile of chordoma cells are well characterized, its molecular and genetic mechanisms are not well understood. Identification of novel molecular pathways will contribute to finding new targets for chordoma therapy and epigenetic deregulation has recently emerged as new pathogenic mechanism.

Genetics is the study of variation in the genetic code that results in changes in phenotype 6. Epigenetics is the study of various mechanisms that result in mitotically heritable changes in gene expression that do not involve any alterations in underlying DNA sequences 7, 8, 9. Epigenetic modifications mainly include DNA methylation and histone modifications, by non‐coding RNAs.

Traditionally, aetiology and pathogenesis of human diseases mainly focused on determining genetic and environmental factors. However, accumulating evidence has shown that deregulation in epigenetic networks plays significant roles in many pathological processes, particularly in cancers. The DNA sequence remains stable in different tissues but epigenetic status of the genome varies considerably. In addition, genetic alterations are not reversible, whereas the epigenetic changes are reversible with use of pharmacological agents. Several epigenetic treatment strategies have been achieved in various cancers. Thus, identifying epigenetic changes in disease may provide insights into their pathogenesis, as well as development of new therapeutic targets. This review provides an introduction into epigenetics, focusing on epigenetic changes associated with chordoma.

Overview of epigenetic mechanisms

The definition of epigenetics has undergone decades of debate and research since the term “epigenetics” was originally coined by Conrad Waddington in 1942 10. Its most commonly used definition is ‘the study of mitotically heritable changes in gene expression without any alteration in the underlying DNA sequence’. The three main epigenetic mechanisms include DNA methylation, histone modifications and post‐transcriptional gene regulation by non‐coding RNA, commonly referred to as miRNA.

DNA methylation

DNA methylation is the transfer of a methyl group (‐CH3) to a cytosine nucleotide within in a given DNA sequence, which is catalysed by DNA methyltransferases (DNMTs) 11, 12. DNA methylation correlates with transcriptional silencing and takes place almost exclusively at DNA sequences that contain cytosines adjacent to a guanine nucleotide (CpG site) 13, 14. CpG islands are unmethylated under normal conditions except in some biological processes such as silencing of imprinted genes and X chromosome inactivation 15. There are about 30 million CpG sites in the human genome, with certain regions of DNA containing a high proportion of CpG sites, known as CpG islands 16. DNA methylation regulates various cellular processes including transcription, genomic imprinting and X chromosome inactivation. Abnormal DNA methylation is often associated with inappropriate transcriptional silencing and diseases. For example, aberrant methylated CpG islands in cancer may silence tumour suppressor genes and subsequently uncontrolled cell growth and cancer progression result 17, 18.

Histone modification

Once, histones were only considered to be packaging proteins for DNA without any role in regulation of gene expression. However, since the 1990s histone modification has been identified as a branch of epigenetic regulation and the field has received much attention. The nucleosome is the basic subunit of chromatin; each is comprised of a 146‐bp (base ‐pair) length stretch of DNA wrapped around an octamer, which consists of two copies of each of the four core histones, H2A, H2B, H3 and H4 19, 20. Histones themselves are proteins (with inherent positive charge) that bind to DNA (with its inherent negative charge). Histone modifications include addition of acetylation, methylation, phosphorylation, ubiquitination, ADP ribosylation, deimination⁄citrullination and proline isomerization tags 21, 22. Acetylation of lysine residues is the most common type of histone modification, often resulting in transcriptional activation 23. Histone modifications are associated with regulation of many biological and pathological processes such as of the cell cycle, differentiation and apoptosis.

MicroRNAs

MicroRNAs (miRNAs or miRs) are a class of non–protein‐coding RNAs of approximately 22‐nucleotides, that post‐transcriptionally modulate gene expression by binding to messenger RNA, leading to transcript degradation or translational inhibition 24, 25. Thus, miRNAs exert their roles by regulating target genes. It has been proven that miRNAs regulate expression of more than a third of protein‐encoding mRNAs. miRNAs play significant roles in various cell processes, such as in the cell cycle, cell differentiation, apoptosis, and immune functions. Moreover, deregulated miRNA expression is involved in many diseases, specially cancers 26, 27. They are frequently deregulated in malignancies and function as oncogenes (when up‐regulated) or tumour suppressors (when down‐regulated) 28.

Epigenetic alterations in chordoma

The earliest evidence for epigenetic influence in cancers comes from gene expression and DNA methylation studies. Major epigenetic mechanisms implicated in the pathogenesis of cancers (DNA methylation, histone modification and post‐transcriptional gene regulation by non‐coding RNA, miRNA) have also been involved in chordoma pathogenesis.

More importantly, as recent pre‐clinical successes of inhibitors against bromodomain‐containing protein 4 (BRD4, acetyl‐lysine chromatin‐binding protein), have entered pre‐clinical trials, epigenetic cancer therapies have accrued more attention 29, 30, 31.

Aberrant DNA methylation in chordoma

Since aberrant DNA methylation was first identified as cancer‐related epigenetic alteration, increasing cancer‐related epigenetic changes have been observed, including hypermethylation of tumour suppressor genes and genome‐wide DNA hypomethylation 18, 32, 33. Aberrant DNA hypermethylation takes place mainly in unmethylated CpG islands, located in promoters of many tumour suppressor genes (TSGs). The hypermethylation leads to silencing of TSG expression and loss of normal TSG function in the tumourigenic process.

Although aberrant DNA methylation was the first cancer‐related epigenetic alteration reported, there are numbers of reports concerning its deregulation in chordoma. Rinner et al. showed 20 hyper/hypomethylated genes using AITCpG360 methylation assay; these included C3, XIST, TACSTD2, FMR1, HIC1, RARB, DLEC1, KL and RASSF1. Chordoma showed a characteristic DNA methylation pattern, resulting in significant genomic instability 34. These findings provide novel targets in chordoma pathogenesis, diagnosis and therapeutic options. MGMT (O6‐methylguanine‐DNA methyltransferase) gene promoter methylation was previously proven to be present in gliomas, acting as a prognostic and predictive marker 35. Presence of methylated MGMT is positively linked to efficacity of temozolomide (TMZ), an anti‐cancer drug. Marucci et al. showed that a high portion of methylated MGMT promoter was present in recurring clival chordoma, whereas its promoter was always unmethylated in clival chordoma without recurrence 35; that study helped predict clinical post‐operative outcomes of chordoma. In a recent investigation, DNA methylation profiling revealed that 8819 loci (2.9%) were differentially methylated in chordoma, among which 5868 probes (66.5%) were hypomethylated and 2,951 (33.5%) were hypermethylated 36. In addition, 104 probes (104/2951) demonstrated cancer‐specific hypermethylation in chordoma. These findings indicated that cancer‐specific hypermethylation loci might also play a crucial role in development of chordoma, which still needs further investigation. Longoni et al. 37 found seven (7/13) chordoma specimens with methylated DNA correlating with transcriptional silencing of the gene. CDKN2A gene, located on 9p21, has been shown to be homo‐ or heterozygously lost in 70% of chordomas. Using methylation specific PCR, tumour suppressor proteins CDKN2A and PTEN promoter hypermethylation were evaluated using methylation specific PCR (MSP). Only one case, CDKN2A, showed definitive promoter methylation, indicating that promoter methylation may explain CDKN2A gene silencing in only a small subset of chordomas. In addition, transcriptional silencing of PTEN was not found to be associated with methylation status in the promoter region of the gene 37, 38, 40. Le et al. observed methylated DNA sequences in two and four of fifteen cases for CDNK2A and PTEN respectively 38.

Histone modification in chordoma

Histone deacetylases (HDACs) are a class of enzymes that can remove acetyl groups from histones, altering accessibility of transcription factors to gene promoter regions 41. Besides influencing histone, HDACs can affect malignant transformation by acting on non‐histone proteins too. HDAC inhibitors are a novel class of anti‐cancer drugs being tested in various clinical trials for various cancers 42. Vorinostat (SAHA) and Panobinostat (LBH‐589) are Pan‐HDAC inhibitors. Abnormal expression of HDACs,which regulate balance of histone acetylation and deacetylation, has been observed in chordoma. Using immunochemistry, Scheipl et al. observed expression of HDACs 2–6, with the strongest expression of HDAC6 in chordoma 43. Furthermore, Pan‐HDAC inhibitors, SAHA and LBH‐589, significantly promote apoptosis and change cell cycle distribution in chordoma cells in vitro. This study has suggested potential use in future, for HDAC inhibitors as therapeutic options for chordoma.

MicroRNAs in chordoma

Over recent years, accumulating data have suggested that miRNAs play significant roles in pathogenesis of various cancers, including chordoma, and to date, several studies have investigated roles of miRNAs in it 44, 45. As well as regulating genes involved in the cell cycle, cell proliferation, apoptosis, angiogenesis, invasion and metastasis, miRNAs play a crucial role in pathogenesis and development of chordoma 45, 46.

Expression of miR‐1 and miR‐206 are reduced in both chordoma tissues and cell lines. In addition, Met and HDAC4 have been proven to be miR‐1 targets overexpressed in chordomas 44. Met is a proto‐oncogene over‐expressed in many human cancers, including chordoma; restoration of miR‐1 expression suppresses migratory and invasive activities of chordoma cells. These findings implicate a potential effect of miR‐1 on chordoma pathogenesis, and by downregulating Met, miR‐1 might act as a tumour suppressor here. Furthermore, Duan et al. showed that miR‐1 expression levels were also associated with clinical prognosis 45. They also found that miR‐1 reduced cell population growth and proliferation in chordoma cells in vitro. A further study has also shown that miR‐1 reduced cell proliferation, as well as migratory and invasive activities of chordoma cells. In addition, Slug, over‐expressed in chordoma cell lines and advanced chordoma lesions, has been identified to be the direct target of miR‐1. Thus, the miR‐1–Slug pathway has crucial roles in chordoma cell population growth and migration and may itself serve as a potential therapeutic target 46. Zou et al. have identified 29 differentially expressed miRNAs in chordomas compared to normal nucleus pulposus tissues. In addition, patients with low miR‐1237‐3p expression had the worse recurrence‐free survival of patients, with high chordoma invasion 47.

To determine miRNA expression profiles in chordoma, Bayrak et al. performed miRNA arrays of chordoma samples compared to those of normal nucleus pulposus 48. Thirty up‐regulated and 23 down‐regulated miRNAs were identified in chordoma. Among all deregulated miRNAs, the most up‐regulated included miR‐140‐3p and miR‐148a, and most down‐regulated were miR‐31 and miR‐222, confirmed by polymerase chain reaction. In addition, miR‐31 promoted apoptosis in chordoma cells by down‐regulating c‐MET and radixin. Kuang et al. showed that miR‐10a and miR‐125a were reduced in chordoma compared to controls 49; miR‐140‐3p was up‐regulated in chordomas, positively correlating with muscle invasion. In addition, over‐expression of miR‐140‐3p predicted poorer recurrence‐free survival and served as an independent prognostic factor for chordoma patients. miR‐608 and miR‐34a expressions were down‐regulated in human chordoma cell lines UCH1 and UCH2. Their restoration reduced cell proliferation and invasion and induced apoptosis 50. In addition, EGFR and Bcl‐xL, oncogenes, were proven to be direct targets of miR‐608, while MET was the direct target of miR‐34a. Expression of miR‐34a and miR‐608 was inversely correlated to MET and EGFR expression, respectively, in chordoma cells. Furthermore, EGFR and MET activations increased cell proliferation and invasion, while pharmacological inhibition of EGFR and MET reduced proliferation and survival. These findings indicate that miR‐608 and miR‐34a act as tumour suppressors in chordoma by targeting these oncogenes (Table 1). Fragile histidine triad (FHIT) is a tumour suppressor, implicated in cancers of tissues arising from all three germ cell layers. Diaz et al. found high rates of low‐FHIT protein expression in sacral (98%) and skull base chordoma (67%) while there was only 21% classic clival chordoma with chromosomal loss or gain encompassing the FHIT locus. These results imply that epigenetic mechanisms in addition to genomic instability regulate FHIT expression in chordoma 51.

Table 1.

Functional characterization of the deregulated miRNAs in chordoma

Name	Up‐ or down‐regulation	Target gene	Role	Reference
miR‐1	Down	Met, HDAC4, Slug	Tumour suppressor	44, 45, 46
miR‐206	Down		Tumour suppressor	44
miR‐1237‐3p	Down		Tumour suppressor	47
miR‐140‐3p	Up		Oncogene	7
miR‐148a	Up		Oncogene	48
miR‐31	Down	c‐MET, radixin	Tumour suppressor	48
miR‐222	Down		Tumour suppressor	48
miR‐10a	Down		Tumour suppressor	49
miR‐125a	Down		Tumour suppressor	49
MiR‐140‐3p	Up		Oncogene	50
MiR‐608	Down	EGFR, Bcl‐xL	Tumour suppressor	50
miR‐34a	Down	MET	Tumour suppressor	50

Potential therapeutic methods based on epigenetic mechanisms

Management of chordoma is a clinical challenge, requiring multimodal therapy – yet there is poor overall survival. Significant improvements have made by discovery of epigenetic involvement in this disease. However, targeting or modulating epigenetic remodelling enzymes and transcription factor can reprogram epigenetic deregulation and attenuate the malignant phenotype. For example, by use of inhibitors of DNA methyltransferases DNMTs and HDACs, epigenetically silenced genes have been re‐expressed successfully in vitro. Traditional DNMT inhibitors along with second‐generation DNMT inhibitors greatly improve efficacy of epigenetic therapies with fewer side effects. Furthermore, the US Food and Drug Administration have approved some of these compounds for elective treatment of myelodysplastic syndromes 5‐azacytidine and 5‐aza‐20‐deoxycytidine [DNMT inhibitors] and cutaneous T‐cell lymphomas, suberoyl anilide hydroxamic acid [SAHA] and romidepsin, which are HDAC inhibitors (HDACi) 52, 53, 54. For example, pan‐HDAC‐ inhibitors Vorinostat and Panobinostat (LBH‐589) promote apoptosis and change cell cycle distribution in chordoma cells in vitro 43. Patients with chordoma will benefit from additional treatment options with epigenetic alterations. In addition, miRNAs are also expected to provide relevant targets for development of specific anti‐cancer molecules that may simultaneously target several key pathways. By down‐regulating Met, miRNA‐1 seems to be able to act as a tumour suppressor in chordoma 46.