Methylation status of homeobox genes in common human cancers

YGENO-08844; No. of pages: 9; 4C: Genomics xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Genomics journal homepage: www.elsevier.com/locate/ygeno

Review

Methylation status of homeobox genes in common human cancers Maria Fernanda Setúbal Destro Rodrigues a, Carina Magalhães Esteves, DDS, PhD a, Flávia Caló Aquino Xavier, DDS, PhD b, Fabio Daumas Nunes Professor a,⁎ a b

Department of Oral Pathology, School of Dentistry, University of São Paulo, São Paulo, Brazil Department of Stomatology, School of Dentistry, Federal University of Bahia, Salvador, Brazil

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 18 May 2016 Received in revised form 27 September 2016 Accepted 1 November 2016 Available online xxxx

Approximately 300 homeobox loci were identified in the euchromatic regions of the human genome, of which 235 are probable functional genes and 65 are likely pseudogenes. Many of these genes play important roles in embryonic development and cell differentiation. Dysregulation of homeobox gene expression is a frequent occurrence in cancer. Accumulating evidence suggests that as genetics disorders, epigenetic modifications alter the expression of oncogenes and tumor suppressor genes driving tumorigenesis and perhaps play a more central role in the evolution and progression of this disease. Here, we described the current knowledge regarding homeobox gene DNA methylation in human cancer and describe its relevance in the diagnosis, therapeutic response and prognosis of different types of human cancers. © 2016 Elsevier Inc. All rights reserved.

Keywords: DNA methylation Homeobox genes Epigenetic Solid tumors

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . DNA methylation in normal cells . . . . Cancer epigenetics and homeobox genes 3.1. Bladder cancer . . . . . . . . . 3.2. Endometrial cancer . . . . . . 3.3. Ovarian cancer . . . . . . . . 3.4. Lung cancer . . . . . . . . . . 3.5. Esophageal cancer . . . . . . . 3.6. Gastric cancer . . . . . . . . . 3.7. Prostate cancer . . . . . . . . 3.8. Pancreatic cancer . . . . . . . 3.9. Glioma . . . . . . . . . . . . 3.10. Colorectal cancer . . . . . . . 3.11. Breast cancer . . . . . . . . . 3.12. Head and neck . . . . . . . . 3.13. Others tumors . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author at: Universidade de São Paulo, Department of Oral Pathology, Av. Prof. Lineu Prestes, 2227, Cidade Universitária, Brazil. E-mail address: [email protected] (F.D. Nunes).

An epigenetic process is defined as a change in gene expression by altering the chromatin structure without modifying the DNA sequence [1]. Since the 1940s, when Conrad Waddington first described epigenetics, discoveries about its implication in normal and disease biology

http://dx.doi.org/10.1016/j.ygeno.2016.11.001 0888-7543/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: M.F.S.D. Rodrigues, et al., Genomics (2016), http://dx.doi.org/10.1016/j.ygeno.2016.11.001

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have increased significantly, compiling a huge amount of knowledge in the past decade. Epigenetic change is a regular, natural and reversible phenomenon that can also be influenced by several factors, including age, environment/lifestyle, and disease state. The molecular basis of epigenetic processes is complex, involving DNA methylation, histone modifications, gene regulation by non-coding RNAs and altered expression and function of factors implicated in regulating assembly and remodeling of nucleosomes [2]. Epigenetic mechanisms provide essential mechanisms for normal development, cellular diversity and maintenance of tissue-specific gene expression patterns in mammals. Disruption of molecular events that control epigenetic processes can lead to deregulated gene function, resulting in gene silencing or oncogene activation [3]. An epigenetic mechanism that has been prominently described in cancer research is DNA methylation, which is also one of the most active fields of epigenetic research. Homeobox genes act as master regulators of morphogenesis and cell differentiation and participate in the maintenance of adult cellular identity [4]. These genes contain a highly conserved DNA sequence that encodes the homeodomain proteins (HD). HD proteins act as transcription factors that specifically bind to DNA motifs controlling target genes involved in cell adhesion, proliferation and differentiation. In the human genome, at least 200 homeobox genes have been identified, and the vast majority are dispersed throughout the entire genome. Only 39 homeobox genes, namely the HOX gene family, are organized on chromosome clusters [5]. Recently, altered expression of homeobox genes has been associated with different solid tumors, including breast, bladder, lung, prostate and oral cancer [6–10]. Aberrant expression of homeobox genes in cancer includes three main categories. First, these genes can be re-expressed in malignant cells derived from embryonic cells that normally express homeobox genes during development. This is the main category in which deregulated homeobox genes contribute to cancer. There are many examples in this category, including overexpression of HOXB7 and HOXB9 in breast cancer [11,12]; up-regulation of HOXC11 in renal cell carcinoma [13]; high expression of HOXA13, B6, C13, D1 and D13 in ovarian cancer [14]; and HOXA1 overexpression in oral squamous cell carcinoma [15]. Second, homeobox genes can be expressed only in tumor cells, but they are not normally expressed during development. There are few examples in this category, including the expression of PAX in medulloblastoma and lack of expression in cerebellum cells [16]. Third, homeobox genes can be downregulated in malignant cells derived from a tissue in which these genes are normally expressed in adult differentiated cells [5]. For example, HOXA5 and HOXA9 downregulation is a frequent event in breast cancer and is also associated with aggressive tumors, metastasis and poor prognosis [17,18]; additionally, low HOXA11 expression contributes to cell proliferation in gastric cancer. Most importantly, homeobox genes exhibit tissue-specific features and can promote tumorigenesis as a consequence of their gain or loss of function, leading to inappropriate effects on growth and differentiation [5]. Moreover, there are different mechanisms that can lead to deregulated expression of homeobox genes in cancer, including translocations, loss of heterozygosity, gene amplification and non-coding RNA. Detailed deregulation of homeobox genes as causal evidence of solid tumors was recently reviewed by Haria and Naoria [19] and is beyond the scope of this review. Epigenetics, mainly DNA methylation, is one of the mechanisms by which homeobox genes are aberrantly expressed in cancer [20]. 2. DNA methylation in normal cells This cellular process is defined by the addition of a methyl group to the 5-carbon position of cytosine in a CpG dinucleotide, which results in the regulation of gene expression [3]. This process is performed by DNA methyltransferases (DNMTs) that catalyze the substitution of the

methyl group from S-adenosyl l-methionine (SAM) to the cytosine in CpG dinucleotide [21]. Three main DNA DNMTs are responsible for methylation: DNMT1, which is responsible for the maintenance of the existing methylation patterns, and DNMT3A and DNMT3B (and “de novo” enzymes) that target previously unmethylated CpGs [2]. Potentially ‘methylable’ CpG dinucleotides are not randomly distributed in the human genome; instead, CpG-rich regions also identified as CpG islands, which span the 5′ end region (promoter, untranslated region and exon 1) of many genes, are typically unmethylated in normal cells [22]. On the other hand, DNA demethylation is also an important cellular process that regulates patterns of gene expression during embryonic development and in adult tissues. Active DNA demethylation mechanisms lead to methyl group release by carbon-carbon bond breakage. However, passive DNA demethylation is the result of DNMT1 downregulation or inhibition during successive cycles of DNA duplication. Although passive DNA demethylation is generally understood and accepted, the subject of active DNA demethylation has been controversial [23]. Accumulating data have increased the precise understanding of aberrant DNA methylation and may ultimately form the basis for novel therapeutic strategies and targets for the treatment of cancer. DNA methylation is one of the mechanisms that regulates embryogenesis, gene expression, transposons silencing and host defense against viral sequences [24]. DNA methylation also plays an important role in X chromosome inactivation and genomic imprinting [2]. Many imprinted genes regulate different cellular processes, such as cell growth, cell signaling pathways, cell cycle and embryogenesis [25]. In cancer, hypermethylated CpG islands of tumor suppressor genes are noted as a frequent epigenetic marker, leading to gene expression inhibition by changing its open euchromatic structure to a compact heterochromatic structure [26]. Thus, the control of DNA methylation can provide a selective advantage to cancer cells. However, promoter hypermethylation of these genes is important depending on its activity in a particular cellular context. In head and neck cancer, DNMT3B exhibited overexpression in invasive cell lines and is associated with E-cadherin methylation and promotion of the epithelial-mesenchymal transition [27]. Tumor suppressor genes, including BRCA1 and CDKN2A, and metastasis-related genes, such as E-cadherin and CEACAM6, are frequently methylated in breast cancer [28]. Additionally, breast cancer cell lines also have elevated DNMT3B protein expression, which results in increased DNMT activity and methylation of target genes, such as Ecadherin and ESR1 [28]. In colorectal cancer, CpG island methylation is a frequent event and is also associated with BRAF mutation, contributing to key events in the development of this type of cancer [29]. On the other hand, there is a positive relationship between low DNA promoter methylation and KRAS mutation in colorectal tumors [30]. 3. Cancer epigenetics and homeobox genes The cancer epigenome is characterized by a global change in the pattern of DNA methylation. Commonly, DNA hypomethylation or hypermethylation of specific promoters is observed in early and invasive tumors and benign tumors. For example, DNA methylation is altered in normal tissues derived from patients with cancer, and these changes increase during malignant transformation [31]. In Barrett's esophagus, epigenetic modifications can be observed before the development of malignancy [31]. Thus, it becomes clear that epigenetic deregulation may precede the classic genetic events that drive cancer development, such as mutation of tumor suppressor genes, proto-oncogenes and genomic instability [2]. CpG island hypermethylation as a direct driver of carcinogenesis is associated with tumor suppressor gene silencing. Hypermethylation of BRCA1 is commonly found in breast and ovarian cancer [32], and inactivation of CDKN2A by DNA methylation was associated with development and progression of prostate cancer [33]. APC hypermethylation was strongly related to non-small cell lung cancer [34], hepatocellular carcinoma [35] and sporadic colon cancers [36].

Please cite this article as: M.F.S.D. Rodrigues, et al., Genomics (2016), http://dx.doi.org/10.1016/j.ygeno.2016.11.001

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Homeobox genes are transcriptional regulators that integrate multiple signals, inducing phenotypic changes, through the control of downstream target genes where they can act as activators or repressors of gene transcription [37]. In many types of cancer, inhibition of homeobox genes by hypermethylation of CpG islands in their promoters contributes to the inactivation of regulatory or DNA repair genes, contributing to tumorigenesis (Fig. 1). Here, we provide a review of the literature highlighting the current status of homeobox gene DNA methylation in human cancers and its relevance for the diagnosis, therapeutic response and prognosis of different types of human cancers. All data regarding types of cancer and DNA methylation reviewed here are summarized in Table 1.

Table 1 Homeobox genes methylation in human cancer, according with type of cancer, locus, alteration and reference. Type of cancer

Gene

Locus

Alteration

Ref

Bladder

TLX3 HOXA9 HOXA1 ISL1 HOXA9 HOXA10 HOXA9 LMX1A HOXA10 HOXB5 SOX1 PAX1 MSX1 HOXA1 HOXA11 PITX2 SHOX2 HOXA9 DLX4 HOXA5 CDX2 SIX3 CDX2 MEIS1 PDX1 HOXD10 CDX2 IRX1 HOXP-β EVX1 PITX2 HOXD3 GSH2 HOPX HOXA3 HOXA7 HOXA9 HOXA10 CDX1 CDX2 HOPX HOXD1 ALX4 MEIS1 PITX2 HOXA10 HOXB13 HOXD13 PROX1 HOXA9 IRX1 PROX1

5q35.1 7p15.2 7p15.3 5q11.1 7p15.2 7p15.2 7p15.2 1q24.1 7p15.2 17q21.3 13q34 20p11.2 4p16.2 7p15.3 7p15.2 4q25 3q25.32 7p15.2 17q21.33 7p15.2 13q12.3 2p21 13q12.3 2p21 13q12.1 2q31.1 13q12.3 5p15.3 4q12 7p15.2 4q25 2q31.1 4q12 4q12 7p15.2 7p15.2 7p15.2 7p15.2 5q32 13q12.3 4q12 2q31.1 11p11.2 2p21 4q25 7p15.2 17q21.2 2q31.1 1q41 7p15.2 5p15.3 1q41

Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypomethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypomethylated Hypermethylated Hypomethylated Hypomethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermthylated Hypermethylated Hypermethylated Hypermethylate Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated Hypermethylated No methylated

[20,22] [20] [21] [20] [25] [26] [28–31] [32,33] [32] [28] [33] [33] [34] [36,38] [38] [39] [39,40–45] [37] [46–47] [8] [36] [48] [50] [51] [55,56] [54] [55,56,59,60] [57] [58] [9] [62–64] [65] [67–68] [69] [70] [70] [70] [70] [72–75] [76,77] [78] [79] [80] [81] [82–85] [6] [6,86] [87] [88] [90,91] [92] [64]

IRX1 HOXA9

5p15.3 7p15.2

Hypomethylated Hypermethylated

[94] [95]

Endometrial Ovarian

3.1. Bladder cancer Urinary bladder cancer is the fifth most common neoplasm in industrialized countries [38]. The high recurrence rate, progression risk, cisplatin resistance and longer follow-up make bladder cancer the most expensive cancer to treat [7]. Microarray-based studies have been recently used to detect novel epigenetic markers of nonmuscle invasive bladder cancer that enable the prediction of prognosis and the establishment of appropriate treatment strategies [39]. DNA methylation profiling of 181 bladder cancer samples revealed significant associations among Homeobox A9 (HOXA9), ISL LIM homeobox 1 (ISL1) and ALDH1A3 methylation (either single or combined) with decreased mRNA expression levels, advanced stage, and higher tumor grade as well as an independent factor to predict prognosis [39]. In cancer cell lines, elevated expression of the histone demethylase KDM3A, which is an enzyme that mediates chromatin remodeling and induces gene expression, is associated with the up-regulation of the HOXA1 gene by binding to its promoter region. As a result, the demethylation of HOXA1 contributes to bladder cancer development, leading to a significant increase in cell proliferation [40]. The homeobox gene Tcell leukemia homeobox 3 (TLX3) encodes a DNA-binding nuclear transcription factor that is involved in cell differentiation and cell proliferation. TLX3 DNA methylation was observed in bladder cancer samples and confers cisplatin resistance to malignant cells. On the other hand, TLX3 expression promotes cell proliferation in bladder cancer cells and probably increases cisplatin intercalation in the cell genome, resulting in cisplatin toxicity [41].

Lung

Esophagus Gastric

Prostate

Pancreatic Glioma

Colorectal

Breast

3.2. Endometrial cancer Endometrial cancer (EC) is the most common gynecological malignancy in developed countries, and its prevalence is increasing. This tumor is commonly classified as type I, associated with estrogen production and mainly develops from endometrial hyperplasia, and

Fig. 1. Homeobox genes downregulation can be implicated in numerous cellular processes involved in cancer progression, including angiogenesis, proliferation, invasion, morphological differentiation, cell adhesion and migration.

3

Head and neck

Others tumors Osteosarcoma Hepatocarcinoma

type II, which result from “de novo” mutations acquired by normal endometrium cells [42]. Actually, it is becoming clear that EC comprises a range of heterogeneous diseases with distinct genetic, epigenetic and molecular features [43]. Chen and co-workers [44] described five genes that are methylated in EC, including HOXA9 homeobox gene. The HOXA9 methylation rate was increased (20.8%) in endometrial hyperplasia compared with normal endometrium. Moreover, there was also a significant difference between the methylation profiles of HOXA9 in EC (80.8%) in relation to endometrial hyperplasia, indicating that HOXA9 methylation status may be an important event in EC progression. EC samples also exhibited a significant HOXA10 hypermethylation profile compared with normal endometrial tissues, suggesting a possible role for HOXA10 epigenetic deregulation in EC development [45].

Please cite this article as: M.F.S.D. Rodrigues, et al., Genomics (2016), http://dx.doi.org/10.1016/j.ygeno.2016.11.001

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3.3. Ovarian cancer Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and accounts for 90% of ovarian cancer. Most patients are diagnosed at an advanced stage (FIGO III and IV), when the disease has spread throughout the abdomen. Patients with advanced-stage disease have a 5-year survival rate of only 30% in contrast to early-stage disease confined to the ovaries, for which the 5-year survival rate exceeds 80% [46]. DNA hypermethylation of tumor suppressor genes seems to play an important role in ovarian carcinogenesis, and knowledge of the global genomic/epigenomic changes in this tumor is important to develop a more effective (including epigenetic) treatment strategy [47]. The overall risk of ovarian cancer is increased 12.3-fold by high HOXA9 methylation for all cancer stages and 14.8-fold for early-stage cancers, independent of age, phase of the menstrual cycle and tumor microscopic features [48]. HOXA9 is an important transcription factor that regulates serous differentiation, and its methylation in ovarian cancer may reflect a shift toward epithelial cell dedifferentiation [48]. Montavon et al. (2012) reported a 95% frequency of HOXA9 DNA methylation in high-grade serous ovarian cancer, and the combination of HOXA9 and EN1 (engrailed homeobox 1) methylation was reported to discriminate malignant from benign tumors with a sensitivity of 98.8% and a specificity of 91.7% when pre-operative CA125 levels are also incorporated [49]. Xing and co-workers also suggested that a methylation panel with HOXA9 and others non-homeobox genes, such as Ras association (RalGDS/AF-6) domain family member 1 (RASSF1A) and opioid binding protein/cell adhesion molecule-like (OPCML), is associated with CA125 levels with high sensitivity (85.7%) and specificity (100%) for discriminating ovarian cancer from normal ovarian tissues [50]. Additionally, the homeobox genes LIM homeobox transcription factor 1, alpha (LMX1A) and HOXA10 are also epigenetically silenced in ovarian cancer and are implicated in tumor progression [51,52]. Promoter hypermethylation of HOXA9, HOXB5, SCGB3A1 and CRABP1 genes was identified as a common mechanism involved in ovarian carcinogenesis. Additionally, HOXA9 hypermethylation was more frequently noted in tumors from patients older than 60 years, and there was a significant difference in HOXA9 methylation frequency among the histological types. [47]. Su and co-workers investigated the DNA methylation status of SFRP1, SFRP2, SFRP4, SFRP5, SOX1, PAX1 and LMX1A genes in samples obtained from patients with a benign ovarian tumor, borderline malignancy or invasive ovarian cancers using methylation-specific PCR (MS-PCR) [52]. All genes exhibited increased methylation rates in ovarian cancer samples compared with benign or borderline malignancy, suggesting that epigenetic silencing may be involved in the tumorigenesis and progression of ovarian cancers. Moreover, the presence of homeobox gene LMX1A methylation was associated with worse survival and increased risk for cancer-related death. Bonito and co-workers have recently investigated the methylation profile of Msh homeobox 1 gene (MSX1) in high-grade serous ovarian cancer. Lower levels of DNA methylation were observed in patients with recurrent disease after 6 months compared with patients with recurrence after 12 months. Moreover, MSX1 methylation was also associated with poor response to treatment and progression-free survival [7].

3.4. Lung cancer The majority of patients with non-small cell lung cancer (NSCLC) are diagnosed with advanced disease, and survival remains poor [53]. To date, the pathogenesis of NSCLC is difficult to determine, and it is critical to identify NSCLC cancer-specific events involved in tumor development. In this context, DNA methylation represents a common event in

lung cancer and exhibits great promise as a cancer-specific marker that can improve lung cancer screening tools and early diagnosis [54]. Wrangle and co-workers (2014) have recently validated a threemethylated gene test in a large database and two independent cohorts to demonstrate a highly sensitive and specific diagnostic test for NSCLC. The DNA methylation of HOXA9, CDO1 (cysteine dioxygenase type 1) and/or TAC1 (tachykinin, precursor 1) was a common event in the United States and may be used to improve early diagnosis and patient prognosis [55]. Additionally, the data presented by Selamat et al. (2011) [56] indicated that distinct epigenetic events occur with the transition to hyperplasia, carcinoma in situ and invasive lung cancer. CpG islands at HOXA1 and HOXA11 genes are significantly hypermethylated in adenocarcinoma in situ, contributing to cancer progression. Tsou et al. (2007) reported a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma, including the HOXA1 gene and other genes such as cyclin-dependent kinase inhibitor 2A (CDKN2A EX2), caudal type homeobox 2 (CDX2), and opioid binding protein/cell adhesion molecule-like (OPCML) [57]. In non–small-cell lung cancer, DNA methylation of the homeobox genes paired-like homeodomain 2 (PITX2) and short stature homeobox 2 (SHOX2) is noted as an independent prognostic biomarker. High methylation of SHOX2 and PITX2 was a significant predictor of progression-free survival, and patients with low methylation of either PITX2 and/or SHOX2 exhibited a significantly higher risk of disease progression compared with patients with higher methylation of both genes [57]. SHOX2 DNA hypermethylation has recently emerged as a candidate biomarker for lung cancer and a powerful tool for the detection of patients with this type of cancer [58–61]. Studies have revealed that the methylation level of the SHOX2 gene CpG islands was significantly increased in tissues and cells of lung cancer compared with normal tissues and cells [62]. Additionally, Schmidt and co-workers demonstrated in a pilot study that the measurement of the extracellular methylated SHOX2 DNA (mSHOX2) levels in plasma during therapy appears to be useful for the monitoring of a treatment response for advanced-stage lung cancer patients [63]. The distal-less homeobox 4 (DLX4) gene is involved in cell motility and in vivo lung cancer metastasis inhibition [64]. Harada and coworkers identified DLX4 methylation as a promising biomarker for stage I NSCLC patients that is associated with poor prognosis and high risk of recurrence even after curative resection [65]. HOXA5 gene expression was identified by Zhang et al. (2015) [8] to be regulated by DNA methylation in NSCLC cells. Additionally, HOXA5 overexpression reduced cell proliferation and invasion, whereas HOXA5 knockdown promoted cell growth [8]. Thus, decreased HOXA5 expression may represent a negative prognostic factor and higher risk for NSCLC patients. Mo and co-workers (2013) investigated the potential of SIX3 expression as a prognostic biomarker for patients with lung adenocarcinoma [66]. The majority of tumors exhibited significantly reduced SIX3 expression compared with normal tissues as well as hypermethylation of the gene promoter. Moreover, SIX3 mRNA expression was related to improve overall survival and progression-free survival, suggesting that this gene may be a novel prognostic marker for patients with lung cancer. 3.5. Esophageal cancer This type of cancer is associated with high mortality and low quality of life of patients receiving curative treatment. There are two major histological subtypes: squamous cell carcinoma and adenocarcinoma. Genetic and epigenetic changes are associated with the development of this malignant tumor that arises from an altered mucosa [67]. Increased methylation of homeobox gene CDX2 was observed in primary squamous esophageal cancers compared with adenocarcinomas

Please cite this article as: M.F.S.D. Rodrigues, et al., Genomics (2016), http://dx.doi.org/10.1016/j.ygeno.2016.11.001

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and normal esophageal tissues in which the CDX2 methylation rate is low and absent, respectively. In addition, loss of CDX2 expression was associated with reduced MUC2 gene expression, which is associated with reduced protection of esophageal mucosa due to decreased mucin production. Thus, MUC2 inactivation or downregulation may be important for esophageal tumor development [68]. Decreased expression of MEIS1 homeobox gene mRNA and protein was observed in ESCC tumor samples compared with normal adjacent tissues and was also correlated with lymph node involvement, poor tumor differentiation and worse prognosis [69]. Interestingly, MEIS1 downregulation was not associated with promoter hypermethylation but with the expression of the epigenetic factor Enhancer of zeste homolog 2 (EZH2). This gene belongs to the polycomb repressive complex 2 that mediates gene silencing by histone modifications. These modifications lead to epigenetic regulation of signaling pathways involved in oncogene activation and tumor suppressor gene silencing as well as metastasis, contributing to cancer development and progression [70]. 3.6. Gastric cancer Gastric cancer is the third most common cause of death associated with cancer worldwide [71]. This type of cancer arises from precancerous gastric lesions as gastritis and intestinal metaplasia. Different genetic alterations including molecular alterations in gastric cancer have been extensively studied, including TP53, BRCA2 and chromatin remodeler gene mutations; deregulated expression of cell adhesion, cytoskeleton and cell motility-related genes; and chromosomal instability and epigenetic changes. [72,73]. HOXD10 plays a critical role in cell differentiation and morphogenesis during development, and it is commonly down-regulated in gastric cancer tissues and cell lines compared with normal stomach tissues. Functionally, re-expression of HOXD10 results in significant inhibition of cell survival, apoptosis activation and cell migration and invasion. HOXD10 is inactivated through promoter hypermethylation and can act as a tumor suppressor gene in gastric cancer [74]. Moreover, other homeobox genes, including CDX2 and pancreatic and duodenal homeobox 1 (PDX1), also showed promoter hypermethylation in gastric cancer, contributing to tumor development. High CDX2 expression in tumor-derived cell lines is associated with decreased proliferation and increased apoptosis [75,76]. IRX1 expression is significantly decreased in gastric cancer, and transcriptional silencing via methylation on CpG sites of its promoter is the main mechanism responsible for IRX1 downregulation [77]. The mean methylated levels of IRX1 in primary tumor tissues and in plasma showed a decrease in methylation only in plasma of tumor samples vs. control samples. Additionally, higher IRX1 methylation levels were closely associated with increased age and TNM staging and serve as a molecular marker for gastric cancer. The frequency of HOPX-β (HOP homeobox gene spliced variant β) promoter methylation was also increased in gastric cancer compared with normal tissues, leading to HOPX-β inactivation. Functionally, loss of HOPX-β expression is associated with an increase in cell proliferation and invasion and a decrease in apoptosis. Thus, HOPX-β promoter methylation may indicate an aggressive behavior of this cancer and can be considered in the future as a possible therapeutic target for advanced gastric cancer [78]. The CDX2 homeobox gene is an essential transcription factor for cellular proliferation and differentiation of intestinal epithelial cells, and its downregulation is associated with high tumor grade, advanced stage, and poor prognosis [79]. Kameoka and co-workers [80] have investigated the methylation status of the CDX2 promoter in non-cancerous and cancerous areas of intestinal-type gastric cancer, revealing increased CDX2 hypermethylation in deep invasive areas compared with superficial areas, which is also associated with decreased CDX2 gene expression.

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3.7. Prostate cancer The predominant tools for predicting prostate cancer outcomes consist of clinical and pathologic nomograms incorporating PSA, clinical stage, Gleason grade and biopsy results [81]. Despite the tumor grade, a wide range of aggressive behavior is observed. Even-skipped homeobox 1 (EVX1) gene is hypermethylated in 83% of patients with prostate carcinomas, and these patients had only a 51% recurrence-free survival compared with 91% in patients without hypermethylation. Thus, EVX1 is a very specific methylation marker that can be used to predict tumor recurrence and tumor grade [9]. Paired-like homeodomain 2 (PITX2) and other non-homeobox genes, such as glutathione S-transferase pi 1 (GSTP1), adenomatous polyposis coli (APC), and retinoic acid receptor beta (RARβ2), are frequently methylated in prostate cancer, demonstrating that methylation is not only a common event in prostate cancer but can also be used as a sensitive tool for the detection of neoplastic lesions in the prostate [82]. Weiss and co-workers investigated the prognostic value of PITX2 methylation in prostate cancer. The data obtained from a large cohort of patients revealed that patients with high PITX2 methylation had advanced tumors, worse prognosis and a 4-fold increased chance of developing recurrence within 8 years compared with patients with low PITX2 methylation. [83,84]. Moreover, the methylation profile was able to distinguish prostate tumors as high and low risk for recurrence [84]. Interestingly, the two-gene PITX2 + HOXD3 hypermethylation profile in prostate cancer proved to be a better predictor of disease-free survival compared with the single markers PITX2 and HOXD3 [85]. 3.8. Pancreatic cancer Pancreatic cancer is a highly aggressive tumor associated with the highest mortality rate of patients with cancer, with an overall fiveyear survival rate of b 5% [86]. Effective treatment remains elusive, and new treatment options are necessary for pancreatic cancer patients. Homeobox gene GSH2 is a downstream target of the Shh signaling pathway, which participates during embryogenesis in pancreatic development [87]. This gene was hypermethylated in pancreatic tumor samples and tumor tissues compared with the surrounding normal tissues and normal pancreatic tissue derived from healthy patients. Moreover, GSH2 hypermethylation is associated with advanced disease and may be an important tool to predict prognosis in patients with pancreatic cancer [88]. Additionally, downregulation of HOPX, which is associated with promoter hypermethylation, may be associated with an aggressive phenotype of pancreatic cancer [89]. 3.9. Glioma The majority of tumors that arise from central nervous system tumors exhibit glial origin (40%) and are categorized into four grades, varying from curable tumors and pilocytic astrocytoma to aggressive glioblastoma. HOXA3, HOXA7, HOXA9, and HOXA10 genes are methylation target genes in high-grade glioma, and the hypermethylation of HOXA9 and HOXA10 is important for glioblastoma patient prognosis. The results indicate that altered methylation of these homeobox genes may be useful to clinically discriminate subgroups of glioma patients who could respond to target therapy [90]. 3.10. Colorectal cancer Colorectal cancer is associated with deregulation of epigenetic events, including DNA methylation [91]. CDX1 is a homeobox gene that participates in the control of epithelial differentiation and proliferation [91]. Some studies have demonstrated that this gene is down-regulated in colorectal cancer samples and cancer-derived cell lines due to promoter hypermethylation [92–95]. Zhang and co-workers [92]

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suggest that oxidative stress can lead to inhibition of the CDX1 tumor suppressor gene through epigenetic mechanisms and may participate in the progression of colorectal cancer. CDX1 and CDX2 (caudal type homeobox 2) play roles as tumor suppressor genes in colorectal cancer, as their expression is lost in colorectal carcinomas due to hypermethylation but preserved in adenomas [96]. Another study suggests that CDX2 inactivation in colon cancer results from defects in CDX2 transcription regulation instead of DNA methylation [97]. Methylation of other homeobox genes, including HOPX, plays an important role in the development of this cancer. Increased HOPX methylation is associated with poorly differentiated carcinomas [98]. Hypermethylation of HOXD1 is an event in premalignant lesions and increases during tumorigenesis. [99] ALX homeobox 4 (ALX4) is also methylated in the majority of both colorectal cancer and premalignant adenomas and is rarely observed in normal epithelia [100]. The BRAF gene mutation BRAFp.V600E is the most common mutation of this gene in colorectal cancer and leads to an increase in protein kinase activity [101]. The methylation of homeobox gene MEIS1 in colorectal cancer is associated with a decrease in MEIS1 gene expression and BRAF mutation [100]. 3.11. Breast cancer Aberrant epigenetic changes could lead to breast cancer development through neoplastic initiation and progression. Knowledge of epigenetic regulation in cancer is useful to understand carcinogenesis and for the development of epigenetic drugs [6], as well as to further elucidate the resistance to treatment of estrogen receptor-positive breast cancer. PITX2 methylation status was identified by Maier and co-workers (2007) as an important biomarker to predict the risk of distant recurrence in patients with primary breast tumors treated with tamoxifen. The methylation status of PITX2 was useful to discriminate patients with node-negative and -responsive tumors to tamoxifen [102]. Additionally, PITX2 DNA methylation is also considered as a prognostic marker for patients with hormone receptor-positive disease and is associated with early distant metastasis and poor overall survival [102]. On the other hand, in invasive ductal carcinomas [103], PITX2 methylation status together with progesterone receptor expression may be indicative of very good prognosis [104]. Hypermethylation of HOXA10 and HOXB13 is associated with high expression of estrogen and progesterone receptors [6]. Another study suggests that hypermethylation of HOXB13 is also implicated in late events of breast tumorigenesis and can be considered as a poor prognostic indicator of node-positive cancer patients [105]. DNA methylation of HOXD13 was detected in 113 of 196 sporadic breast cancer patients and was significantly associated with tumor size and poor clinical outcome compared with patients with negative HOXD13 DNA methylation [106]. However, no association between HOXD13 methylation and other clinico-pathological factors was detected. Finally, PROX1 was identified as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer [107]. 3.12. Head and neck One of the most significant clinical factors responsible for death from oral squamous cell carcinoma (OSCC) is metastasis to the cervical (neck) lymph nodes. Methylation of HOXA9 was identified as a frequent event in oral squamous cell carcinoma (OSCC), and its methylation status was increased in tumors with positive nodes compared with tumors with negative nodes [108]. In OSCC cell lines, HOXA9 methylation is associated with reduced HOXA9 gene expression, conferring a growth advantage to tumor cells [108]. Furthermore, hypermethylation of HOXA9 and Nidogen 2 (NID2), a non-homeobox gene, may be useful as a panel for early detection and cancer prevention studies. In the prevalence

screen, these genes exhibited almost perfect agreement with histologic diagnosis according to Guerrero-Preston et al. (2011) [109]. In patients with HNSCC, methylation of HOXA9 combined with EDNRB (endothelin receptor type B) methylation was identified as a powerful predictor of locoregional recurrence and poor recurrence-free survival and overall survival [110]. IRX1 methylation was observed in 45% (21 of 47) of OSCC samples and associated with a significant decreased in mRNA expression compared with normal oral mucosa [111]. Although there was no difference in the methylation status of the PROX1 gene between non-tumoral margins and OSCC samples, Rodrigues and co-workers reported that PROX1 downregulation is associated with increased cell proliferation [10]. 3.13. Others tumors As the most common primary malignant bone tumor, osteosarcoma typically affects children and adolescents and frequently metastasizes, which is the main cause of treatment failure and patient death [112]. IRX1 promoter hypomethylation was identified by Lu et al. [113] as a metastasis-driving gene in osteosarcoma. Decreased methylation levels in the promoter region of IRX1 are significantly associated with increased mRNA expression. The IRX1 promoter sequence was also hypomethylated in 34.2% of serum patients with osteosarcoma without metastasis, whereas 62.1% of serum patients with metastasis exhibited IRX1 hypomethylation. Additionally, IRX1 hypomethylation in the serum is associated with worse lung-metastasis-free survival, indicating that IRX1 hypomethylation could be an important screening tool for early lung metastasis detection and for the development of new therapeutic drugs. In hepatocellular carcinoma (HCC), the HOXA9 methylation profile is a potential biomarker for HCC detection [114]. This gene was hypermethylated in 67.7% of HCC compared with controls, and this methylation was also detected in plasma but not in normal plasma. 4. Conclusion This review has focused on the involvement of homeobox gene methylation in solid cancers. It is evident that epigenetic regulation features are a major mechanism in carcinogenesis, and several pathways that control cancer progression appear to be specifically epigenetically deregulated. Targeting epigenetic alteration of homeobox genes as novel agents is a rational approach for treating specific types of cancer. Additionally, the use of epigenetic biomarker panels including homeobox genes may be clinically applicable in screening, diagnosis, prediction of response and prognosis of different solid tumors. Conflict of interest The authors have no conflict of interest. Acknowledgements This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP, São Paulo, Brazil (grants 2008/06223-3 and 2010/08720-4). References [1] R. Shaw, The epigenetics of oral cancer, Int. J. Oral Maxillofac. Surg. 35 (2006) 101–108. [2] M. Rodriguez-Paredes, M. Esteller, Cancer epigenetics reaches mainstream oncology, Nat. Med. 17 (2011) 330–339. [3] S. Sharma, T.K. Kelly, P.A. Jones, Epigenetics in cancer, Carcinogenesis 31 (2010) 27–36. [4] R. Lopez, E. Garrido, G. Vazquez, P. Pina, C. Perez, I. Alvarado, M. Salcedo, A subgroup of HOX Abd-B gene is differentially expressed in cervical cancer, Int. J. Gynecol. Cancer 16 (2006) 1289–1296.

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