Carcinogenic mechanisms of endometrial cancer: involvement of genetics and epigenetics.

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doi:10.1111/jog.12442

J. Obstet. Gynaecol. Res. Vol. 40, No. 8: 1957–1967, August 2014

Carcinogenic mechanisms of endometrial cancer: Involvement of genetics and epigenetics Kouji Banno, Megumi Yanokura, Miho Iida, Kenta Masuda and Daisuke Aoki Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan

Abstract Endometrial cancer is increasing worldwide and the number of patients with this disease is likely to continue to grow, including younger patients. Many endometrial cancers show estrogen-dependent proliferation, but the carcinogenic mechanisms are unknown or not completely explained beyond mutations of single oncogenes and tumor suppressor genes. Possible carcinogenic mechanisms include imbalance between endometrial proliferation by unopposed estrogen and the mismatch repair (MMR) system; hypermethylation of the MMR gene hMLH1; mutation of PTEN, β-catenin and K-ras genes in type I endometrial cancer and of HER-2/neu and p53 genes in type II endometrial cancer; hypermethylation of SPRY2, RASSF1A, RSK4, CHFR and CDH1; and methylation of tumor suppressor microRNAs, including miR-124, miR-126, miR-137, miR-491, miR-129-2 and miR-152. Thus, it is likely that the carcinogenic mechanisms of endometrial cancer involve both genetic and epigenetic changes. Mutations and methylation of MMR genes induce various oncogenic changes that cause carcinogenesis, and both MMR mutation in germ cells and methylation patterns may be inherited over generations and cause familial tumorigenesis. Determination of the detailed carcinogenic mechanisms will be useful for prevention and diagnosis of endometrial cancer, risk assessment, and development of new treatment strategies targeting MMR genes. Key words: DNA hypermethylation, DNA mismatch repair, endometrial cancer, epigenetics, genetics.

Introduction A total of 288 000 patients were newly diagnosed with endometrial cancer worldwide in 2008.1 More than 4000 women died from endometrial cancer in the USA in 2011.2 In Japan, the annual morbidity increased from 48 in the 20–30 years in 1975 to 478 in 2005.3 The annual mortality per 100 000 population in Japan increased from 0.4 in 1975 to 3.2 in 2012 and the total morbidity increased from 229 to 2092.4 The incidence of endometrial cancer is likely to continue to increase based on these recent trends. Discovering the causes of the increase and establishment of prophylactic measures and new therapeutic strategies requires an improved understanding of the carcinogenic mechanisms of endometrial cancer.

Environmental factors, including estrogen, an abnormal mismatch repair (MMR) system, genetic abnormalities, and aberrant methylation of DNA and microRNA, are currently proposed as major mechanisms of carcinogenesis in endometrial cancer. Endometrial cancer is defined as type I or II based on clinicopathological properties. Type I endometrial cancer more commonly develops in premenopausal or perimenopausal women and occurs in an estrogendependent manner via atypical endometrial hyperplasia. The tumor is positive for the estrogen receptor and progesterone receptor, shows well-differentiated endometrioid adenocarcinoma, has a lower frequency of lymph node metastasis, shows little muscular invasion, and often has a relatively favorable prognosis. In contrast, type II endometrial cancer tends to develop in

Received: February 18 2014. Accepted: March 3 2014. Reprint request to: Dr Kouji Banno, Department of Obstetrics and Gynecology, School of Medicine, Keio University, Shinanomachi 35 Shinjuku-ku, Tokyo 160-8582, Japan. Email: [email protected]

© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology

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postmenopausal women in an estrogen-independent manner, and is thought to be due to de novo carcinogenesis that develops directly from the normal endometrium, rather than via endometrial hyperplasia or undiagnosed precancerous lesions. The tissue type is specific, including extremely poorly differentiated endometrioid adenocarcinoma and serous adenocarcinoma, and the prognosis is often poor. This review focuses on the mechanisms of carcinogenesis in endometrial cancer that have recently emerged.

Carcinogenic Mechanisms Involving Estrogen Estrogen is a steroid hormone that promotes the development of female genitalia, including the endometrium, vagina, vulva and mammary gland. Estrogen passes through the cell membrane and binds to estrogen receptor (ER) in the cytoplasm. ER forms dimers and regulates gene expression via estrogen response elements in promoter regions of target genes. ER has ligand- and DNA-binding domains, and ligandindependent activation function (AF)-1 and liganddependent AF-2 transcriptional activation domains.5 The balance of transcriptional activation domains varies among tissues, with dominance of AF-2 in mammary gland cells and AF-1 in endometrial cells.6,7 Miyamoto et al.8 suggested that mismatch repair (MMR) deficiency was the most important abnormality in early-stage endometrial cancer, and examined the correlation between MMR and estrogen. Expression of hMLH1 and hMSH2, which are important MMR proteins, was examined by immunostaining and showed a strong positive correlation with blood estrogen. MMR activity in endometrial epithelial cells in vitro also showed a dose-dependent increase with higher estrogen levels. This suggests that cancer is unlikely to occur in a high estrogen environment because increased cell growth is paralleled by increased MMR activity. In contrast, hMLH1 and hMSH2 were absent or had extremely low expression at estrogen levels ranging from 20 to 60 pg/mL, but some cell growth still occurred. Therefore, cells dividing in a low-estrogen environment are more likely to accumulate genetic errors due to low repair activity and may be at high risk for carcinogenesis. Based on these results, Miyamoto et al.8 suggested that the incidence of growth-induced genetic errors should be low in young women with high estrogen levels and sufficient repair activity of MMR proteins, making carcinogenesis unlikely. In

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Low estrogen

Relatively low

High estrogen

(E2 < 15 pg/mL)

estrogen

(E2 > 80 pg/mL)

(E2 20–60pg/mL) CP = MMR activity

CP > MMR activity

CP < MMR activity

Non-carcinogenic

Carcinogenic

Non-carcinogenic

Cancer window

Figure 1 Hypothesis of endometrial carcinogenesis based on the estrogen environment and mismatch repair (MMR) activity. CP, cell proliferation.

older women with lower estrogen but an atrophic endometrium, carcinogenesis would also be unlikely because of the absence of cell growth. However, under perimenopausal conditions, the carcinogenic risk would be increased because sufficient estrogen is present to promote cell division, but MMR activity is low. This intermediate status was defined as the cancer window (Fig. 1).

Carcinogenic Mechanisms of Abnormal Mismatch Repair System The mismatch repair (MMR) system is responsible for repairing base mismatches that arise during DNA replication. Typical MMR proteins include hMLH1, hMSH2, hPMS2, hMSH3 and hMSH6. Genes encoding these proteins are called MMR genes and aberrations in these genes prevent correct repair of mismatched bases, resulting in DNA strands with different lengths. This phenomenon occurs in microsatellite regions of the human genome and is referred to as microsatellite instability (MSI). Microsatellites or short tandem repeats (STR) are repeating sequences of one to five base pairs of DNA, such as CA and CAG. Some STR occur in regions encoding phosphatase and tensin homolog deleted on chromosome ten (PTEN), a lipid phosphatase that is a tumor suppressor gene; TGF-βR2 and IGF2R, which are associated with inhibition of cell proliferation; K-ras, which is involved in cell proliferation; and BAX, which is related to apoptosis induction. Therefore, MSI is implicated in carcinogenesis.9 Aberrations in MMR genes are involved in carcinogenesis of type I endometrial cancer. These aberrations are caused by epigenetic changes independent of the DNA sequence, that is, gene inactivation by aberrant


Carcinogenesis of endometrial cancer

hypermethylation of promoter regions. Such inactivation of MMR genes permits accumulation of gene mutations and leads to carcinogenesis. In endometrial cancer, carcinogenesis most frequently involves aberrant methylation of hMLH1 and mutation of hMLH1 is detected in 30% of cases. Mutations of hMLH1 are also found in atypical endometrial hyperplasia, which suggests that hMLH1 is implicated in the early stage of carcinogenesis.10,11 Muraki et al.12 reported aberrant hMLH1 hypermethylation in 40.4% of patients with endometrial cancer and found significantly reduced hMLH1 protein levels in these patients (P < 0.01). However, none of the four cancer-related genes were aberrantly methylated in the normal endometrium. MMR genes are also causative genes in Lynch syndrome (hereditary nonpolyposis colorectal cancer). Lynch syndrome is a typical familial tumor with autosomal dominant inheritance. Female patients with Lynch syndrome complicate with endometrial cancer at a high incidence. In the revised 1999 Amsterdam II Criteria (AC II), endometrial cancer was included as a cancer with similar features to colon, small intestine, ureteral and kidney cancers.13 The prevalence of Lynch syndrome is 0.9–2.7%14 and approximately 2.3% of cases of endometrial cancer occur due to Lynch syndrome.15 The lifetime risk of endometrial cancer is 28–60% in women with aberrant genes associated with Lynch syndrome.16 hMLH1, hMSH2 and hMSH6 mutations are particularly important in families of patients with Lynch syndrome. Most mutations occur in hMLH1 and hMSH2, whereas hMHS6 mutations are important in tumorigenesis in patients with endometrial cancer.17,18 Kawaguchi et al.19 proposed a possible new cascade in which hMSH6 mutation is induced by

silencing of hMLH1 due to aberrant DNA hypermethylation in endometrial cancer. Westin et al.20 showed that the incidence of Lynch syndrome was 1.8% in endometrial cancer in total and 9% in endometrial cancer in women aged less than 50 years old, but 29% in cases with lower uterine segment cancer (LUS) and germ cell mutation of hMSH2. These results suggest a correlation between endometrial cancer of the uterine isthmus and Lynch syndrome. Masuda et al.21 also found germ cell mutations of hMLH1 in 1.4% of patients with LUS. Based on these findings, Lynch syndrome may be clinically predictive of the onset site of endometrial cancer.

Carcinogenic Mechanisms of Gene Mutation Several gene mutations have emerged as candidates for roles in carcinogenesis of type I and II endometrial cancer (Fig. 2), based on observation of the mutation in endometrial hyperplasia and at least a similar incidence of mutation in endometrial cancer. Different genes are involved in carcinogenesis of the two types of endometrial cancer. Gene mutations found in type I endometrial cancer include those in PTEN, β-catenin and K-ras. PTEN is a tumor suppressor gene on chromosome 10 and has been identified as a disease gene in three autosomal dominant disorders (Cowden disease, LhermitteDuclos disease and Bannayan-Zonana syndrome). PTEN inactivation is also found in malignant melanoma, brain tumors, and endometrial, ovarian, thyroid, breast and prostate cancers. PTEN protein induces

PTEN mutation hMLH1 methylation hMSH6 mutation

PTEN mutation K-ras mutation b-catenin mutation

p53 mutation

Type I Normal endometrium

Type II

Figure 2 Gene mutations in the carcinogenesis of endometrial cancer.

Normal endometrium Atrophic endometrium

Atypical endometrial hyperplasia

Endometrioid cancer (low-grade)

Endometrioid cancer (high-grade)

p53 mutation HER-2/neu mutation Non-endometrioid cancer


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apoptosis and carcinogenesis occurs in cells with PTEN mutation due to avoidance of apoptosis. PTEN mutations have been detected in 20–33% of cases of atypical endometrial hyperplasia and 33–50% of cases of endometrial cancer;22–24 thus, PTEN appears to be involved in the early stage of carcinogenesis, which is a pattern that differs from that in late-onset cancer, including rectal cancer. Furthermore, in endometrial cancer, PTEN mutations are frequently detected in cases with aberrant methylation in promoter regions of hMLH1 that cause inactivation of the MMR gene; therefore, PTEN is thought to be a target of MMR genes.25 β-catenin (CTNNB1) mutations are found in 20–40% of cases of type I endometrial cancer.26–28 β-catenin is a component of E-cadherin, which has an important role in cell adhesion and is involved in the Wnt signaling pathway that regulates cell proliferation and differentiation. β-catenin degradation is prevented by mutations and the transcription levels of target genes of β-catenin increase. These mutations are also detected in atypical endometrial hyperplasia; therefore, β-catenin mutations are implicated in the early stage of carcinogenesis.29 The K-ras oncogene encodes a protein of 21 kDa that has a signaling function from activated membrane receptors in the MAPK pathway. If mutations occur, K-ras continuously functions as activated Ras and excessive signaling causes cell proliferation and induces carcinogenesis.30 K-ras mutations have been detected in 6–16% of cases of endometrial hyperplasia31 and 10–31% cases of endometrial cancer.32,33 Tsuda et al.34 showed that the incidence of K-ras mutation was significantly higher in tumors with invasive proliferation (P < 0.002) and that the incidence of mutation in well-differentiated (Grade 1) tumors was significantly higher than that in moderately (Grade 2) and poorly differentiated (Grade 3) tumors (P < 0.025). These results suggest that K-ras is involved in two stages of carcinogenesis: a shift from endometrial hyperplasia to endometrial cancer and invasive proliferation of well-differentiated tumor cells. Lagarda et al.35 found that the incidence of K-ras mutation was significantly higher in MSI-positive endometrial cancer and was related to aberrant methylation of MMR genes. Mutations in type II endometrial cancer are thought to be linked to the oncogene HER-2/neu and tumor suppressor gene p53. HER-2/neu is a tyrosine kinase membrane receptor in the epidermal growth factor (EGF) receptor family. Mutations of this gene are also found in breast and ovarian cancers. HER-2/neu expression in endometrial cancer has a strong inverse corre-

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lation with differentiation.36 However, the incidence of gene amplification differs from 14% to 63% in all cancers37–40 and overexpression of the protein ranges from 9% to 74%.41,42 A p53 gene mutation is the most frequent mutation in human cancer. Normal p53 regulates cell proliferation, apoptosis induction and DNA repair. Point mutations in p53 are found in 90% of cases of type II endometrial cancer, but in only 10–20% of cases of Grade 3 type I endometrial cancer. The incidence is low in endometrial hyperplasia and type I endometrial cancer of other grades.43,44 Feng et al.45 showed that p53 gene mutations occurred only at sites with positive p53 protein expression in endometrioid adenocarcinoma, which were poorly differentiated regions of cancer tissues. p53 is also implicated in the early stage of carcinogenesis of serous adenocarcinoma. Zheng et al.46,47 found strong positive p53 immunostaining in normal endometria and suggested that this ‘p53 signature’ reflected potential serous adenocarcinoma lesions. The p53 signature is found frequently in normal endometria adjacent to serous adenocarcinoma, but rarely detected in other normal endometria or tissues adjacent to endometrioid adenocarcinoma. Based on these findings, Zheng et al. proposed a carcinogenic model in which genetic changes occur prior to morphological changes. Atypical epithelium develops features similar to serous adenocarcinoma and covers endometrial cortical layers, but is not invasive. This state is referred to as serous endometrial intraepithelial carcinoma (EIC) and finally progresses to serous adenocarcinoma. RB and cyclin may also be involved in carcinogenesis of endometrial cancer. RB was the first gene to be identified as a disease gene in retinoblastoma in children. Non-phosphorylated RB protein inhibits cell proliferation in the G0 and early G1 phases. After phosphorylation by the complex of cyclin and cyclin-dependent kinase (CDK), pRB releases the transcription factor E2F, which then increases DNA polymerase activity and promotes cell proliferation. RB gene mutations have been found in small cell lung, bladder and esophageal cancers. In endometrial cancer, loss of heterozygosity (LOH) was found in 18% of RB genes and pRB downregulation was consistent with LOH.48 The incidence of mutations increased with advancement of the clinical stage.49 Cyclin is a protein that controls the cell cycle in cooperation with CDK and is overexpressed in endometrial cancer. Shih et al.50 showed that expression of cyclin A was an independent poor prognostic factor. Overexpression of cyclin D1 is induced by mutations in sites with ubiquitin degradation in the same



gene.51 Cables, an inhibitor of CDK2 that negatively regulates cell proliferation, was recently indicated to be involved in onset of endometrial cancer through a relation with cyclin. Endometrial hyperplasia and well-differentiated endometrial cancer occur in Cablesknockout mice and Cables is downregulated in human endometrial cancer, regardless of the tissue type, which implicates Cables mutation in the onset of endometrial cancer.52,53

Carcinogenic Mechanisms of Epigenetic Changes Epigenetic regulation of gene expression includes effects of DNA methylation, histone modification and Polycomb group proteins.54 DNA methylation is imprinted at the time of cell division and has been widely studied in mammals. Genomic DNA methylation in vertebrates is based on addition of a methyl group to a cytosine base at a CpG sequence by DNA methyltransferase. This includes methylation of a CpG in a new DNA strand to maintain the methylation pattern found in the template DNA strand, and de novo methylation of a CpG that was not previously methylated. Maintenance methylation permits inheritance of DNA methylation patterns, while de novo methylation creates new methylation patterns in cell development and differentiation, aging and tumorigenesis processes. DNA methylation in CpG islands, which are regions dense with CpG sites, in promoters upstream of gene transcription start sites is critical in gene expression.55 If the DNA in this region is not methylated, a nucleosome does not form and transcription occurs, while methylation of the same DNA allows nucleosome formation and blocks transcription.56,57 Many tumor suppressor genes in cancer cells are inactivated by aberrant DNA methylation in promoter CpG islands, which suggests that aberrant DNA methylation may cause carcinogenesis similarly to gene mutations.58 MMR gene methylation is particularly important and, as described above, Muraki et al.12 detected aberrant methylation of hMLH1 in 40.4% of patients with endometrial cancer. Inactivation of MMR genes that repair mismatches induces MSI in many tumor suppressor genes, including PTEN, TGF-βR2, IGF2R and BAX, and contributes to carcinogenesis. For example, TGF-βR2 encodes receptors of TGF-β, a cytokine that inhibits epithelial cell proliferation. Sakaguchi et al.59 showed downregulation of TGF-βR2 in endometrial cancer and suggested that the major

cause was hMLH1 methylation and that TGF-βR2 was a target gene of MMR genes. PTEN and K-ras mutations are found in cases with aberrant methylation of the hMLH1 promoter region and MSI-positive cases, suggesting that PTEN and K-ras are also MMR target genes.25,35 In addition to hMLH1, genes inactivated by DNA methylation in endometrial cancer include SPRY2 (Sprouty2), Ras association domain family 1 isoform A (RASSF1A), ribosomal 56 kinase4 (RSK4), adenomatous polyposis coil (APC), checkpoint with FHA and RING (CHFR), p73, caspase-8 (CASP8), G-protein coupled receptor 54 (GPR54), cadherin 1 (CDH1), homeobox A11 (HOXA11) and catechol-O-methyltransferase (COMT).12,60–67 SPRY2 is an antagonist of the fibroblast growth factor (FGF) receptor, and inhibits cell proliferation and differentiation and angiogenesis by inhibiting the RAS-MAPK pathway downstream of the FGF receptor. Velasco et al.60 found that SPRY2 expression depended on the menstrual cycle in normal endometria and proposed involvement of SPRY2 in development of glandular structures. SPRY2 expression is extremely low in highly invasive cancer other than endometrioid adenocarcinoma.60 RASSF1A is also a tumor suppressor gene that negatively regulates the RAS-MAPK pathway. Pallarés et al.61 found aberrant hypermethylation of RASSF1A promoters and downregulation of RASSF1A in advanced endometrial cancer associated with MSI. RSK4 is a tumor suppressor gene in the FGFR2/RAS/ERK pathways that inhibits cell proliferation. Dewdney et al.62 showed that RSK4 expression was downregulated by methylation in atypical endometrial cancer (and particularly in high-grade endometrial cancer), as well as in rectal, breast and kidney cancers. APC is also a tumor suppressor gene and APC protein induces degradation of β-catenin, a Wnt-signaling factor. Aberrant APC methylation is found in endometrial hyperplasia and early endometrial cancer. Ignatov et al.68 showed that the incidence of APC methylation decreased with progression of endometrial cancer, which suggests that aberrant APC methylation may be an important marker of early carcinogenesis of endometrial cancer. CHFR is an M phase checkpoint gene that regulates progression of the cell cycle. Satoh et al.69 and Wang et al.70 showed that CHFR downregulation by aberrant hypermethylation increases the paclitaxel sensitivity of gastric and endometrial cancers. These findings suggest that examination of CHFR expression could form the basis of personalized cancer treatment. p73 is a homolog of the tumor suppressor gene p53 that


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regulates DNA repair, cell growth arrest and apoptosis, similar to p53. CASP8 is an apoptosis-related gene involved in cell death via Fas ligands.71 Both p73 and CASP8 have been found to be methylated in endometrial cancer.63 GPR54 is a gene encoding endogenous receptors of kisspeptin (KISS1), a cancer metastasis suppressor. Kang et al.64 found significantly higher survival in patients with endometrial cancer with high GPR54 expression (P < 0.05) and showed that the expression was epigenetically regulated by methylation. Yi et al.65 showed that CDH1, a promoter of E-cadherin involved in cell adhesion, was methylated in endometrial cancer, and the consequent downregulation of E-cadherin had effects on both cancer progression in clinical pathology and 5-year survival rates. These findings suggest that aberrant methylation of GPR54 and CDH1 promotes invasion and metastasis of cancer cells and worsens the prognosis of endometrial cancer. HOXA11 is involved in proliferation and differentiation of the endometrium. Whitcomb et al.66 showed that methylation of the HOXA11 promoter was more frequent in recurrent endometrial cancer than in primary cases. COMT is an enzyme that degrades catechol estrogen. Sasaki et al.67 found that methylation of the COMT promoter selectively inactivated membrane-bound COMT and was implicated in carcinogenic mechanisms of endometrial cancer via estrogen. Overall organization of gene methylation can be described using the concept of the CpG island methylator phenotype (CIMP). CpG island methylation in colon cancer is found genome-wide or in specific regions. Toyota et al.72 proposed classification of the cancer type based on CIMP. Thus, cancer with genomewide methylation is classified as CIMP-positive because of breakdown of regulation of methylation. Weisenberger et al.73 suggested that CIMP could be a new tumor marker. In endometrial cancer, Zhang et al.74 examined the methylation status of five genes (p14, p16, ER, COX-2 and RASSF1A) and found CIMPpositive cancer tissues and adjacent normal endometrial tissues. These findings suggest that CIMP could be a marker for early carcinogenesis in endometrial cancer.

Epimutation and Inheritance Epimutation refers to epigenetic changes in germ cells that inhibit transcription of genes for which expression

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is not usually inhibited or activate genes that are usually inhibited.75,76 Typical epigenetic changes include DNA methylation and histone acetylation. Epimutation may be the first stage or a direct cause of carcinogenesis. Development of endometrial cancer may involve epimutation of MMR genes, including hMLH1 and hMSH2. Kondo et al.77 showed that epigenetic silencing of hMLH1 was more frequent than that of hMSH2. hMLH1 mutation is particularly found in multiple primary neoplasms, including Lynch syndrome; however, epimutation may exist in germ cells without mutation of hMLH1 itself.78 Hitchins et al.79 suggested that epigenetic errors can be resolved by demethylation during oogenesis, but that small amounts of methylation remain; consequently, epimutation is maternally inherited. However, the frequency of inheritance is lower than that of variants in germ cell lines. Goel et al.80 described a case of hMLH1 epimutation in the paternal allele, which suggests that new epimutation can also occur after fertilization and is inherited. In 2006, Chan et al.81 showed hMSH2 epimutation in germ cell lines of a family including three parents who developed colon or endometrial cancer in young adulthood. hMSH2 mutation did not exist, but protein deficiency was found and MSI was shown to be associated with epimutation of maternally inherited hMSH2. Inheritance of the same epimutation from three parents to three children suggested that not only DNA sequence mutation but also epimutation may be inherited through multiple generations. Also, epimutation did not exist in all cells and methylation levels differed among tissues. This mosaic status of methylation may be the first hit of the two-hit theory of onset and suggests new genetic mechanisms that do not comply with Mendel’s laws. Methylation levels were the highest in the rectal mucosa and colon cancer tissues, and the lowest in leukocytes, leading to the suggestion that epimutation may be overlooked in common gene mutation assays using leukocytes.81 The EPCAM gene encodes epithelial cell adhesion molecules and is overexpressed in most cancers. There are various opinions on the role of EPCAM in carcinogenesis. EPCAM is a homophilic intracellular adhesion molecule that may promote metastasis of cancer cells by inhibiting intracellular adhesion due to E-cadherin.82 Ligtenberg et al.83 showed that epigenetic mutation in the 3′-upstream region of EPCAM inactivated hMSH2 and was involved in carcinogenesis of endometrial cancer.



Aberrant DNA hypermethylation

Estrogen

hMLH1 hMSH6 hMSH2

Reduction of TS-miRNA expression miR-152, miR-129-2

EPCAM

Mismatch repair deficiency

APC RASSF1A RSK4 p53 p73 GPR54 CHFR CDH1 CASP8 Inactivation of tumor suppressor genes

Microsatellite instability

DNMT1 E2F3 MET Rictor

SOX4

Activation of oncogenes

PTEN, TGF-βR2, IGF2R, K-ras, BAX ?

Mutation of tumor-associated genes

Normal endometrium

Atypical endometrial hyperplasia

Endometrial cancer (well-differentiated)

?

?

Endometrial cancer (poorly differentiated)

Figure 3 Carcinogenic mechanisms caused by aberrant DNA hypermethylation in type I endometrial cancer.

Carcinogenic Mechanisms Involving micro-RNA microRNAs (miRNAs) are short noncoding RNAs of 18–25 base pairs that regulate gene expression. miRNAs that inhibit DNA methylation in cancers are referred to as tumor suppressor miRNAs (TSmiRNA), and include miR-124, miR-126, miR-137 and miR-491.84–88 Huang et al.89 showed that miR-129-2 functions as a TS-miRNA through negative regulation of SRY-related high-mobility group box 4 (SOX4), an oncogene that is overexpressed in endometrial cancer. Methylation of miR-129-2 was found in 68% of 117 patients with endometrial cancer with elevated SOX4 expression. Methylation of miR-129-2 is also related to MSI and hypermethylated hMLH1. Therefore, oncogene activation may be caused by methylation of a miRNA that has an inhibitory action on oncogene

expression, in addition to direct promoter demethylation. Tsuruta et al.90 similarly showed that expression of miR-152 is reduced by aberrant DNA methylation and can be recovered by the demethylating action of 5-aza-dC. Screening of methylation and expression showed that miR-152 is also a TS-miRNA in endometrial cancer. miR-152 methylation levels are also changed in acute lymphoblastic leukemia, gastrointestinal cancer and cholangiocarcinoma.91–93 DNA methyltransferase 1 (DNMT1) is a well-known target of miR-152; and E2F3, MET and Rictor have been identified as new targets. miR-152 inhibits expression of all of these genes. E2F3 is an E2F family transcriptional inhibitor and may be an oncogene;94 MET is a cell surface receptor for hepatocyte growth factor and a known oncogene;95 and Rictor is part of the mTOR complex 2 (mTORC2) and is important for cancer cell proliferation.96,97


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Conclusion In this review, we summarized new findings on the carcinogenic mechanisms of endometrial cancer. Carcinogenesis cannot be completely explained by endometrial proliferation due to estrogen and a single gene mutation. However, the core carcinogenic mechanisms of type I endometrial cancer are DNA methylation (an epigenetic change) and subsequent breakdown of the MMR system (Fig. 3). These actions cause oncogene mutation, inactivation of tumor suppressor genes, and oncogene activation via TS-miRNA silencing, and contribute to chaotic cell proliferation, that is, carcinogenesis. Methylation patterns of MMR genes may be inherited over generations and may cause familial tumorigenesis, including Lynch syndrome, while estrogen may control both cell proliferation and MMR activity. However, the carcinogenic mechanisms remain largely unknown, particularly with regard to de novo carcinogenesis of type II endometrial cancer. Improved diagnosis, risk assessment, and new treatment strategies targeting MMR genes will require establishment of the details of these mechanisms in endometrial cancer.

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Acknowledgments The authors gratefully acknowledge grant support from the Japan Society for the Promotion of Science (JSPS) through a Grant-in-Aid for Scientific Research (KAKENHI), a Grant-in-Aid for Scientific Research (C) (22591866), and a Grant-in-Aid for Young Scientists (B) (24791718); the Medical Research Encouragement Prize of The Japan Medical Association; and the Keio Gijyuku Academic Development Fund.

Disclosure

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None disclosed. 15.

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Genetics of endometrial cancer.

Interplay between Epigenetics and Genetics in Cancer.

Genetics. Genetics driving epigenetics.

Genetics and epigenetics of gliomas.

The genetics of cognitive epigenetics.

Genetics and Epigenetics of Eating Disorders.

Genetics and epigenetics of adrenocortical tumors.

Genetics and epigenetics of primary biliary cirrhosis.

Epigenetics and Genetics of Viral Latency.

Genetics and epigenetics of aging and longevity.

Genetics and Epigenetics of Diabetic Nephropathy.

Genetics and epigenetics of endocrine neoplasia.

Cancer Epigenetics: Mechanisms and Crosstalk of a HDAC Inhibitor, Vorinostat.

Inflammatory Bowel Disease: Genetics, Epigenetics, and Pathogenesis.

Epigenetics: where environment, society and genetics meet.

Genetics and epigenetics of arrhythmia and heart failure.

Congenital heart disease: the crossroads of genetics, epigenetics and environment.

Gene amplification: mechanisms and involvement in cancer.

Survival analysis of endometrial cancer patients with cervical stromal involvement.

Genetics, epigenetics, and regulation of drug-metabolizing cytochrome p450 enzymes.

Molecular mechanisms of tamoxifen-associated endometrial cancer (Review).

Integrating genetics and epigenetics in breast cancer: biological insights, experimental, computational methods and therapeutic potential.

The genetics of loneliness: linking evolutionary theory to genome-wide genetics, epigenetics, and social science.

Osteosarcoma Genetics and Epigenetics: Emerging Biology and Candidate Therapies.