Endometrial carcinoma (EC) is the most common malignant tumour of the female genital tract. EC falls into two different types, i.e. Types I and II. Type I tumours are characterized by microsatellite instability and mutations in PTEN, PIK3CA, K-RAS and CTNNB1 (beta-catenin) type II tumours exhibit TP53 mutations and chromosomal instability.
Endometrial carcinoma (EC) is the most common malignant tumour of the female genital tract. EC falls into two different types, i.e. Types I and II. Type I tumours are characterized by microsatellite instability and mutations in PTEN, PIK3CA, K-RAS and CTNNB1 (beta-catenin) type II tumours exhibit TP53 mutations and chromosomal instability.
Endometrial carcinoma (EC) is the most common malignant tumour of the female genital tract. EC falls into two different types, i.e. Types I and II. Type I tumours are characterized by microsatellite instability and mutations in PTEN, PIK3CA, K-RAS and CTNNB1 (beta-catenin) type II tumours exhibit TP53 mutations and chromosomal instability.
Xavier Matias-Guiu 1 & Jaime Prat 2 1 Department of Pathology and Molecular Genetics and Research Laboratory, Hospital Universitari Arnau de Vilanova, University of Lleida, IRBLLEIDA, Lleida, and 2 Department of Pathology, Hospital de la Santa Creu i Sant Pau, Institut dInvestigacio Biomedica (IIB) Sant Pau, Autonomous University of Barcelona, Barcelona, Spain Matias-Guiu X, & Prat J (2013) Histopathology 62, 111123 Molecular pathology of endometrial carcinoma This review paper discusses the main molecular alter- ations of endometrial carcinoma, the most common cancer of the female genital tract. Two clinicopatho- logical variants are recognized: the oestrogen-related (type I, endometrioid carcinoma) and the non-oestro- gen-related (type II, non-endometrioid carcinoma). Whereas type I shows microsatellite instability and mutations in PTEN, PIK3CA, K-RAS and CTNNB1 (beta-catenin), type II exhibits TP53 mutations and chromosomal instability. Recent investigations regarding the role of non-coding RNA have provided important information regarding tumour progression. Understanding pathogenesis at the molecular level is essential for identifying biomarkers of potential use in targeted therapies. Keywords: apoptosis, beta-catenin, chromosomal instability, E-cadherin, endometrial carcinoma, K-RAS, microsatellite instability, molecular genetics, PTEN, PIK3CA, TP53 Molecular features of endometrial carcinoma In western countries, endometrial carcinoma (EC) is the most common malignant tumour of the female genital tract, accounting for 1020 per 100 000 per- son-years. From a pathogenetic viewpoint, EC falls into two different types, i.e. types I and II. 1 Type I tumours are low-grade oestrogen-related endometri- oid carcinomas (EEC) that usually develop in peri- menopausal women and coexist with or are preceded by complex and atypical endometrial hyperplasia (Figure 1A). In contrast, type II tumours are aggres- sive non-endometrioid carcinomas (NEEC) (mainly serous and clear cell carcinomas) that occur mainly in older women and are unrelated to oestrogen stim- ulation (Figure 1B). Occasionally, type II carcinomas may arise in association with so-called serous endo- metrial intraepithelial carcinoma, either from atro- phic endometrium or endometrial polyps. It has been shown that the molecular alterations involved in the development of EEC (type I) carcinomas differ from those of NEEC (type II) carcinomas. 24 EEC shows mi- crosatellite instability (MI) and mutations in the PTEN, K-RAS, PIK3CA and CTNNB1 (beta-catenin) genes, whereas NEEC exhibit alterations of p53, loss of heterozygosity (LOH) on several chromosomes, as well as other molecular alterations (STK15, p16, E-cadherin and c-erb-B2). MI has been demonstrated in 75% of EC associated with hereditary non-polyposis colon cancer (HNPCC), but also in 2530% of sporadic EC 59 (Figure 2). In sporadic EC, MI occurs more frequently in EEC (30%) than in NEEC, and is secondary to MLH-1 promoter hypermethylation. Although data are controversial regarding the prognostic signicance of MI, it is usu- ally associated with a high histological grade. The MI-associated mismatch repair deciency leads to the accumulation of many mutations in coding and non- coding DNA sequences, including short-tandem repeats, named microsatellites. Some small short-tan- Address for correspondence: X Matias-Guiu, Department of Pathology and Molecular Genetics, Hospital Universitari Arnau de Vilanova, Av. Alcalde Rovira Roure 80, 25198 Lleida, Spain. e-mail: xmatias@arnau.scs.es 2012 Blackwell Publishing Limited. Histopathology 2013, 62, 111123. DOI: 10.1111/his.12053 dem repeats, such as mononucleotide repeats, are located within the coding sequence of some impor- tant genes (BAX, IGFIIR, hMSH3, hMSH6, MBD4, CHK-1, Caspase-5, ATR, ATM, BML, RAD-50, BCL-10 and Apaf-1) (Figure 3), and they may be potential targets in the process of tumour progression of MI + , EC. 10,11 PTEN, located on chromosome 10q23.3, is fre- quently abnormal in EC. 1216 LOH at the PTEN region occurs in 40% of EC. Somatic PTEN mutations are also common and found predominantly in EEC, occurring in 3761% of cases (Figure 4). PTEN muta- tions are found in 6086% of MI-positive EEC, but in only 2435% of the MI-negative tumours. Identical PTEN mutations have been detected in hyperplasias coexisting with MI-positive EEC, which suggests that PTEN mutations are early events in tumour development. Although data regarding the prognostic A B Figure 1. A, Endometrioid carcinoma. Upper left: Polypoid tumour with only supercial myometrial invasion. Upper right: Well-differentiated (grade 1) adenocarcinoma. B, Non-endometrioid carcinoma. Lower left: large haemorrhagic and necrotic tumour with deep myometrial invasion; lower right: serous carcinoma (grade 3) exhibiting stratication of anaplastic tumour cells and abnormal mitoses. Microsatellite instability (MSI) Hereditary non-polyposis colorectal carcinoma (HNPCC) Sporadic cancers: colon, stomach, pancreas, ovary and endometrium MSH2 MSH2 MSH6 MSH6 PMS2 PMS2 TUMOR TUMOR (Mutations) n Inactivation by promoter hypermethylation MLH1 MLH1 MLH1 Figure 2. Microsatellite instability in hereditary and sporadic endometrial carcinoma. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. 112 X Matias-Guiu & J Prat signicance of PTEN mutations in EC are controver- sial, some results suggest an association with favour- able prognostic factors. Several studies have shown that EECs with PTEN mutations have genomic instability. For this reason, some authors suggest treating patients with poly (ADP- ribose) polymerase (PARP) inhibitors. Mutations in PIK3CA contribute to alteration of the PI3KAKT signalling pathway in EC. 1724 PI3K (phos- phatidylinositol 3-kinase) is a heterodimeric enzyme consisting of a catalytic subunit (p110) and a regula- tory subunit (p85). The PIK3CA gene, located on chro- mosome 3q26.32, codes for the p110a catalytic subunit of PI3K. A high frequency of mutations in the PIK3CA gene has been reported recently in EC. Muta- tions are located predominantly in the helical (exon 9) and kinase (exon 20) domains, but they can also occur in exons 17. PIK3CA mutations occur in 2439% of the cases, and coexist frequently with PTEN mutations (Figure 5). PIK3CA mutations, particularly in exon 20, have been associated with adverse prognostic factors such as high histological grade and myometrial inva- sion. Although described initially in EEC, PI3KCA mutations also occur in NEEC and mixed EECNEEC. Furthermore, different gene expression proles in the PI3KAKT signalling pathway serve to separate two subgroups of high-grade EC with distinct molecular alterations (PI3KAKT pathway versus p53 alterations) that may play different roles in endometrial MSP Bat-25 Bat-26 MLH1 promoter hypermethylation Loss of MLH1 expression MI BAX BAX T1 T1 T2 T2 IGFIIR MSH3 PTEN Casp-5 Bcl-10 APAF-1 U U M M N T N+T Figure 3. MLH1 inactivation by promoter hypermethylation is the most common cause of the microsatellite instability (MI) phenotype in endometrial carcinoma. Progressive accumulation of alterations secondary to MI affects important regulatory genes and promotes carcinogenesis. BAX somatic frameshift mutations are distributed heterogeneously throughout the tumour and provide selective growth advantage. T1: BAX-non-mutated component of endometrioid endometrial carcinoma; T2: BAX-mutated component of endometrioid endometrial carcinoma. MSP: methylation specic PCR, U: unmethylated DNA, M: methylated DNA. N: normal tissue, T: tumour tissue. Tumour in the histological section on the left failed to react with anti-MLH-1 antibody, demonstrating negative immunoreaction in tumour cells with MLH-1 promoter hypermethylation. The section on the right was incubated with an antibody against the carboxy-terminus of BAX. The negative area (T2) corresponds to tumour cells having mutations in the mononucleotide tract of BAX, which is typical of endometrial carcinomas with MI. PTEN MUTATION PROMOTER HYPERMETHYLATION LOH T N 76 79 82 85 88 Figure 4. Inactivation of tumor suppressor gene PTEN may occur by several mechanisms such as point mutation, promoter hypermethylation, or deletion (loss of heterozygosity [LOH]) at 10q23. T: tumour DNA, N: normal tissue DNA. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. Endometrial carcinoma 113 carcinogenesis 24 (Figure 6). Moreover, mutations in PIK3RI, the gene encoding p85a, the inhibitory sub- unit of PI3K, have been detected in 43% of EEC and 12% of NEEC. The RAS-RAF-MEK-ERK signalling pathway plays an important role in tumorigenesis. The frequency of K-RAS mutation in EC ranges between 10 and 30%. In some series, K-RAS mutations have been reported to be more frequent in EEC showing MI. 25 BRAF, another member of the RASRAFMEKERK pathways, is mutated very infrequently in EC. 26 RAS effectors such as RASSF1A are thought to provide an inhibitory growth signal, which needs to be inactivated during tumorigenesis. RASSF1A inactivation by promoter hypermethylation may contribute signicantly to increased activity of the RASRAFMEKERK signal- ling pathway. 27 PIP 2 Dephosphorylation Phosphorylation PIP 3 P PTEN P13K ATP ADP AKT-P Cell proliferation and survival Figure 5. Phosphatidylinositol 3-kinase (PI3K)PTEN function. Phosphorylation by PI3K converts phosphatidylinositol biphosphate (PIP2) into phosphatidylinositol triphosphate (PIP3), promoting cell proliferation and survival. PTEN regulates PI3K signalling negatively by dephosphorylation of PIP3. P21 AKT1 FAK PAK1 CCND1 FAS FOXO1A P27 P53 XIAP NFKB GSK3B mTOR E-CADH CASP3 PIK3CA AKT2 MDM2 PTEN p53 alterations PIK3CA exon 20 mutations PIK3CA exon 9 mutations PTEN mutations MI High-grade High-stage >50% myometrial infiltration Vascular invasion Yes No Not assessed Cluster 3 U p r e g u l a t i o n D o w n r e g u l a t i o n Cluster 2 Cluster 1 Non-endometrioid p16 overexpression Figure 6. Hierarchical clustering analysis of mRNA expression of 19 genes in 38 endometrial carcinomas. Up-regulated and down-regulated expression are indicated as red and green cubes, respectively. Genes that did not vary in their expression level are shown in black, and genes with unsatisfactory results labelled in grey. Enclosed in the clustering image, results of p53, p16, PIK3CA, PTEN and microsatellite instability (MI) analysis, as well as clinicopathological parameters such as high-grade (grade 3), non-endometrioid, high-stage (stage 2 or higher), myometrial invasion (>50%) and vascular invasion, are represented graphically for each case. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. 114 X Matias-Guiu & J Prat Several studies suggest that the broblast growth factor (FGF) signalling pathway is important in EC. It has been shown that EC presents frequent inactiva- tion of SPRY-2, a protein involved in the negative regulation of the FGF receptor (FGFR) pathway by promoter methylation. 28 Reduced SPRY2 immunoex- pression is seen in almost 20% of EC and is associated strongly with increased cell proliferation. Moreover, somatic mutations in the receptor tyrosine kinase FGFR2 have been detected recently in 612% of EC, particularly in EEC. 2931 Interestingly, FGFR2 muta- tions and K-RAS mutations are mutually exclusive events, while mutations in FGFR2 and PTEN fre- quently coexist. FGFR2 is of special interest as a potential target therapy, and FGFR-2 inhibitors are currently under consideration. The beta-catenin gene (CTNNB1) maps to 3p21. Beta-catenin is a component of the E-cadherincatenin unit, which plays an important role in cell differentiation and maintenance of normal tissue architecture. Muta- tions in exon 3 of CTNNB1 result in stabilization of the beta-catenin protein, cytoplasmic and nuclear accu- mulation, and participation in signal transduction and transcriptional activation through the formation of complexes with DNA binding proteins (Figure 7). Mutations in exon 3 of CTNNB1 with nuclear accumu- lation of beta-catenin occur in 1444% of EC. 3136 Alterations in CTNNB1 have been described in endome- trial hyperplasias that contain squamous metaplasia (morules). Although data are controversial regarding the prognostic signicance of CTNNB1 mutations in EC, they probably occur in tumours associated with a good prognosis. In contrast to EEC, NEEC show TP53 mutations (90%), markedly reduced expression of E-cadherin (8090%), c-erb-B2 (HER-2) amplication (30%), alterations in genes involved in regulation of the mitotic spindle checkpoint (STK15) and loss of hetero- zygosity at multiple loci reecting chromosomal insta- bility. While TP53 mutations occur in 90% of NEEC, they are present in only 1020% of EEC (Figure 8), mainly grade 3 tumours. 3739 Reduced expression of CTNNB1 Codon 37 TCT CCT Ser Pro Exon 3 Figure 7. Upper left, CTNNB1 (beta-catenin gene) mutations shown by single-strand conformation polymorphism (SSCP) analysis with abnormal extra band; and upper right, corresponding partial representative nucleotide sequence demonstrating a missense mutation in exon 3. Different patterns of beta-catenin immunostaining in endometrioid carcinoma: lower left, membranous immunoreaction; lower middle, membranous immunostaining with occasional positive nuclei; and lower right, membranous and nuclear immunostaining in squamous morules. The latter two indicate aberrant protein expression and are associated with CTNNB1 mutation. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. Endometrial carcinoma 115 E-cadherin is frequent in EC, and may be caused by LOH or promoter hypermethylation. In fact, LOH at 16q22.1 is seen in almost 60% of NEEC, but in only 22% of EEC. C-erb-B2 overexpression and amplica- tion are also seen more frequently in NEEC (43% and 29%, respectively) than in EEC. 40 However, the most typical molecular feature of NEEC is the presence of widespread chromosomal gains and losses which reect the presence of aneuploidy. 41 cDNA arrays have demonstrated that NEEC usually show up-regu- lation of genes (STK15, BUB1, CCNB2) involved in the regulation of the mitotic spindle checkpoint. One of these genes, STK15, essential for chromosome seg- regation and centrosome functions, is amplied fre- quently in NEEC. Recently documented potential biomarkers of serous carcinoma are epithelial cell adhesion molecule (EpCAM), claudin-3 and claudin-4 receptors, serum amyloid A, folate binding protein, mesothelin and insulin-like growth factor-II mRNA binding protein 2 (IMP2). Among NEEC, clear cell carcinomas show specic features. Endometrial clear cell carcinomas appear to represent a heterogeneous group of tumours that may arise through different pathogenetic pathways. Based on morphological similarities between ovarian and endometrial clear cell carcinomas, it has been sug- gested that both tumour types may exhibit similar alterations, including mutations in PIK3CA and PTEN. Mutation of the ARID1A gene and loss of the corre- sponding protein BAF250a have been described as fre- quent events in clear cell and endometrioid carcinomas of the ovary. In a recent study, however, these changes have been found in 29% of grades 1 or 2 and 39% of grade 3 EECs, 18% of serous NEEC and 26% of clear cell NEEC. Uterine low-grade endometrioid carcinomas have also shown loss of ARID1A expression (26%) and ARID1A mutations (40%). 42,43 cDNA array studies have shown that the expres- sion proling of EEC differs from that of NEEC. 4447 Genes up-regulated in serous carcinomas were IGF2, PTGS1, FOLR and p16, whereas genes up-reg- ulated in EEC included TFF3, FOXA2 and MSX2. Moreover, two members of the secreted frizzled related protein family (SFRP1 and SFRP4) were down-regulated more frequently in EC with MI. 48 Interestingly, by comparing the expression proles of similar histological subtypes of ovarian and endo- metrial carcinomas it was found that clear cell carcinomas had a similar prole, regardless of the organ of origin. In contrast, differences were strik- ing between endometrioid and serous carcinomas of ovarian and endometrial origin. Classication of EC into two groups (types I and II) is articial and too rigid, and the dualistic model has been challenged recently. In daily practice, pathologists are faced with tumours showing mixed, combined or hybrid morphological and molecular characteristics (often endometrioid and serous carcinomas). Further- more, though serous and clear cell carcinomas have been classied within the same category of NEEC, based mainly on their common high nuclear grade and aggressive behaviour, recent studies have shown that these carcinomas are, in fact, distinct tumour types exhibiting different clinical, immunohistochemical and molecular features. Based on molecular analyses, it has been suggested that in mixed EECNEEC the NEEC component originates from a pre-existing EEC as a result of tumour progression and, not infrequently, these tumours retain their typical EEC molecular alter- ations (Figure 9). This hypothesis would explain the existence of mixed EECNEEC, the presence of MI and the alterations in PTEN, K-RAS or beta-catenin in some cases of NEEC. Another controversial scenario is the grey zone between high-grade (predominantly solid) PTEN MI B-Catenin K-Ras PIK3CA Exon 9 Exon 20 Exon 20 EEC Low-grade EEC High-grade Mixed EEC - NEEC NEEC mRNA overexpression P53 Figure 8. Microsatellite instability (MI) and PTEN, PIK3CA, K-RAS, CTNNB1 (beta-catenin) and TP53 mutations are the most common molecular genetic alterations in endometrial carcinomas. EEC: endometrioid endometrial carcinoma; NEEC: non-endometrioid endometrial carcinoma. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. 116 X Matias-Guiu & J Prat EEC and NECC. Distinction between these two types of tumours is difcult; conversely, high-grade EEC occa- sionally exhibit molecular alterations typical of NEEC such as TP53 mutations. Undifferentiated carcinomas of the endometrium are epithelial tumours that fail to show evidence of either glandular or squamous differentiation. They represent 110% of EC, and show a proliferation of medium-sized, monotonous, epithelial cells growing in solid sheets. Occasionally, undifferentiated carcino- mas arise from pre-existing well- or moderately-differ- entiated EEC. The term de-differentiated carcinoma has been used to designate such special tumour types. Several groups of investigators have conrmed that MI is the predominant molecular feature encountered in this type of carcinoma. 49 However, MI is not seen in all cases, and some tumours may have TP53 mutations suggesting transformation into a high- grade carcinoma. Malignant mixed m ullerian tumours (MMMT), also referred to as uterine carcinosarcomas or sarcomatoid carcinomas, are rare uterine tumours accounting for less than 5% of EC. Molecular studies have suggested recently that MMMT should be regarded as metaplastic carcinomas. Like sarcomatoid carcinomas of other locations, carcinosarcomas probably result from endo- metrial carcinomas through epithelial-to-mesenchymal transition (EMT). EMT is a process of cellular transdif- ferentiation in which epithelial cells lose polarity and cellcell contacts, reorganize their cytoskeleton, acquire expression of mesenchymal markers and mani- fest a migratory phenotype. EMT can be induced by different signals and pathways, such as those medi- ated by transforming growth factor (TGF)-b, tyrosine kinase receptors and/or Wnt, depending on the spe- cic cellular context. Activation of one or more of these pathways converges frequently in a group of transcription factors, Snail1, Slug, ZEB1, ZEB2, E47, E2-2 and Twist, most of them with the ability to repress E-cadherin, a master regulator of cell adhe- sion and polarity. While the transient presence of EMT features is important for myometrial invasion in conventional endometrial carcinomas, MMMT show permanent expression of these features leading to repression of epithelial markers (E-cadherin) and increased expression of mesenchymal markers, including proteins involved in skeletal muscle devel- opment. All these molecular changes are responsible for the appearance of the sarcomatous areas as well as the presence of heterologous elements. It has been shown recently that MMMT show a microRNA signature typical of EMT. 50 Non-coding RNA The classic perception that cancer resulted mainly from alterations in coding genes has been challenged recently by demonstration of the important roles of non-coding RNAs, such as microRNA and long non- coding RNA (lncRNA). M I C R O R N A MicroRNAs (miRNAs) are small, approximately 20- nucleotide-long, non-coding single-stranded RNA molecules regulating the expression of target genes by imperfect (in animals) binding to the 3-untrans- lated region (UTR) and possibly 5-UTR of mRNA. To become the mature form, miRNAs are processed by enzymatic complexes Dorsha and Dicer, and they repress translation or lead to the degradation of the mRNA of their target genes. Currently approximately 2000 human miRNA sequences have been identied, and this number continues to grow. Regardless of the relatively small number of miRNAs, as each single miRNA targets several hundred genes, and a single target gene can bind to multiple miRNAs, thus mak- ing the whole network very complex, it is believed that approximately 30% of all human genes are a target for miRNA regulation. There is also evidence that these small molecules are expressed in a tissue-/ cell-specic manner, being reserved restrictively to specic cell type or associated ubiquitously with dif- ferent human body compartments. It was shown pre- viously that dysfunctional expression of miRNAs is a MI PTEN KRAS beta-catenin PIK3CA (9,20) Tumor progression Gene alterations PIK3CA (20) P53 NE Low-grade Endometrioid Ca Non-Endometrioid Ca High-grade Endometrioid Ca PIK3CA (20) Cadherin E Cyclic D1 Cyclic E Her-2/neu STK15 Figure 9. Pathogenesis of endometrial carcinoma: an alternative to the dualistic model. Exons 9 and 20 of PIK3CA are those frequently mutated in endometrial carcinoma. Ca: carcinoma; NE: normal endometrium. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. Endometrial carcinoma 117 frequent attribute of malignant behaviour. Nowadays, aberrant expression of specic miRNAs is associated with all cancer types. Germline and somatic muta- tions as well as polymorphisms in the mRNAs tar- geted by miRNAs can also lead to cancer predisposition and progression. Growing evidence points to miRNA being implicated in oncogenic pro- cesses, suggesting that miRNA expression proling can distinguish tumours (according to diagnosis and cancer stages) more accurately than traditional gene expression analyses. Small non-coding RNAs can play a dual role in tumorigenesis, acting as oncogenes (e.g. miR-155 of miR-17-92 cluster family members) or tumour suppressors (e.g. miR-15a and miR-16). To date, there are three proposed mechanisms impli- cating miRNA deregulation in cancer. These pro- cesses involve chromosomal lesions at regions encoding miRNAs, failure in their biosynthetic path- way machinery, and epigenetic regulation. Among the miRNAs that have been demonstrated to be up-regu- lated in EC are miR-185, miR-106a, miR-181a, miR- 210, miR-423, miR-103, miR-107, miR-Let7c, miR-205, miR-200c, miR-449, miR-429, miR-650, miR-183, miR-572, miR-200a, miR-182, miR-622, miR-34a and miR-205. In contrast, other miRNAs have been found to be down-regulated, including miR-Let7e, miR-221, miR-30c, miR-152, miR-193, miR-204, miR-99b, miR- 193b, miR-204, miR-99b, miR-193b, miR-411, miR- 133, miR-203, miR-10a, miR-31, miR-141, miR-155, miR-200b and miR-487b. 5155 These miRNAs target important genes in tumour development and progres- sion, such as KCNMB1, IGFBP-6, ENPP2, TBL1X, CNN1, MYH11, KLF2, TGFB1/1, MYL9, SNCAIP, RAMP1, FOXO1, FOXC1 E2F3, MET and Rictor. In one series, high levels of miR-205 expression were associated with poor patient overall survival; 53 the miRNA signature was different between EEC and ser- ous carcinoma, and included miR-19a, miR-19b, miR-30e-5p, miR-101, miR-452, miR-15a, miR-29c and miR-382. 51 We have shown recently that the miRNAs Lin28B and let-7b were involved in the reg- ulation of the HMGA-2 gene, a factor that regulates epithelial to mesenchymal transition, and which is expressed frequently in MMMT and NEEC. 56 L O N G N O N - C O D I N G R N A S Long non-coding RNAs (lncRNAs) are long (>200 nucleotide) transcripts from intergenic and intronic regions of the human genome that lack protein-coding capacity. They probably have important roles in recruitment of histone-modifying complexes to chro- matin and regulation of transcription and splicing. Only a few lncRNA have been well characterized, but expectations are that they will outnumber protein- coding genes. Aberrant expression of these transcripts has been documented in different types of cancer. Some of them may play a role in EC. NC25 is a can- didate tumour suppressor lncRNA, mapped to 6q13, that has been shown to be expressed in EC, and to exhibit mutations in almost half of EC samples that have been tested. 57 Another lncRNA that may have a role in EC is the PTEN pseudogene, PTENP1, which can regulate PTENs tumour suppressor role by com- petitive binding to common miRNAs. 58 Resistance to apoptosis, hypoxia and radiation therapy in endometrial carcinoma There is a great deal of evidence suggesting that alteration of apoptosis is important in the develop- ment and progression of EC (Figure 10). Several of the molecular abnormalities that have been detected in EC may be associated with apoptosis deregulation. EEC show a high frequency of mutations in PTEN, leading to constitutively active AKT which, in turn, suppresses apoptosis triggered by various stimuli. Moreover, the recent evidence that NF-jB activation is frequent in endometrial carcinoma 59 may explain the presence of apoptosis resistance by activation of target genes such as FLIP and Bcl-xL. p53 altera- tions, which are characteristic of NEEC, may also occur in endometrioid tumours, particularly in those neoplasms showing overlapping features between types I and II tumours, and they may have an impact on apoptosis at several different levels. Also, members of the Bcl-2 family of genes are abnormal in EC. In EC, divergent observations have been reported with respect to Bcl-2 and Bcl-xL (Fig- Death factors Death receptors Growth factors PI3K BCLxL AP AF-1 CYT-C Caspase 9 Caspase 3 Caspase 8 Adapter protein APOPTOSIS FLIP BAX PTEN AKT NFKB Survivin Figure 10. Apoptosis: intrinsic (mitochondrial) and extrinsic (death receptor-initiated) pathways. 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. 118 X Matias-Guiu & J Prat ure 10). Some authors have found up-regulated Bcl- xL and Bcl-2 in EC compared to normal tissue, and they have also been reported to be involved in devel- opment of metastases. Many pathways can control Bcl-2 expression, and typical EC molecular alterations such as those involved in exacerbated PI3KAKT sig- nalling could trigger Bcl-2 family members overex- pression. However, other non-canonical molecular events in EEC, such as those involving the NF-jB pathway which plays an important role in tumori- genesis, have been correlated in immunohistochemi- cal studies with strong immunoreactivity for Bcl-xL. The Bax family contains three former members: Bax, Bak and Bok. All Bax family members contain Bcl-2 homology domains 13 and are therefore capable of binding to anti-apoptotic Bcl-2 proteins. This is thought to be the mechanism by which Bcl-2 anti- apoptotic proteins inhibit cell death. Once activated, Bax and Bak trigger permeabilization of the outer mitochondrial membrane that, in turn, will promote release of cytochrome C and other mitochondrial pro- apoptotic factors to the cytosol, such as second mito- chondria-derived activator of caspase (Smac)/DIA- BLO, Omi/HtrA2 protease, endonuclease G and apoptosis-inducing factor (AIF). Interestingly, Bax is a target gene for mutations in EEC with MI, and may have a role in resistance to apoptosis in these tumours. Cancer cells can evade the extrinsic apoptotic path- way by several mechanisms. FLICE-like inhibitory protein (FLIP), one of the most important regulators of death receptor signalling (Figure 10), is a protein that shares high homology with caspase-8 but lacks proteolytic activity. Like caspase-8, it contains two death-effector domains (DED) that allow it to interact either with DED within Fas-associated death domain protein (FADD) or caspase-8, thereby inhibiting death-inducing signalling complex (DISC) assembly and caspase-8 activation. Hence, FLIP represents a potent inhibitor of cell death initiated by death recep- tors. Moreover, FLIP overexpression seems to play a key role in tumour evasion of the immune system. Thus, FLIP-induced cell survival through inhibition of the extrinsic apoptotic pathway appears to be critical for the survival of many tumour cells. Direct evidence for the role of FLIP in the resistance of endometrial carcinoma cells to apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) is provided by treatment with specic small interfering RNAs (siR- NA) targeting FLIP. 60 Transfection of endometrial carcinoma cell lines with FLIP siRNA results in a marked decrease in cell viability after TRAIL exposi- tion. This is accompanied by activation of both cas- pase-8 and caspase-3, suggesting activation of the extrinsic pathway. Moreover, in EEC, FLIP can be reg- ulated transcriptionally by casein kinase-2 (CK2), a Ser/Thr kinase implicated in the development and progression of many neoplasias. These data point fur- ther to CK2 as an important modulator of TRAIL sen- sitivity. In fact, the CK2 beta regulatory subunit has been found overexpressed in endometrial cancer com- pared to normal tissue and is thought to regulate cell proliferation and anchorage-independent cell growth. Recent studies have shown that FLIP may be regulated by a cellular complex, including CK2BRAFKSR1. 61,62 Interestingly, this regulation allows connection of apop- tosis resistance with the RASRAFMEKERK signalling pathway. The KSR1 (kinase suppressor of RAS 1) is considered a scaffold protein that regulates the MAP kinase pathway. KSR1 interacts with different kinases of the RAFMEKERK signalling pathway to enhance its activation and also modulates the apoptotic response to death receptors. Recently, it has been shown that the expression of KSR1 is increased in EC, suggesting its possible role in endometrial carci- nogenesis. 63 Tumour hypoxia is known to render tumours more resistant to radiotherapy. By reacting with the radia- tion-created broken ends of DNA, oxygen xes the damage and thus enhances radiation-induced cell death. This phenomenon, known as the oxygen enhancement effect, could render oxygenated cells three times more radiosensitive than hypoxic cells. Under hypoxic conditions, the oxygen enhancement effect is lost and cells become more radioresistant. Although the absence of oxygen is the major factor inducing radioresistance under hypoxic conditions, there is increasing evidence that signalling pathways activated under hypoxia may modulate cancer cell radioresistance. Some investigators have addressed the molecular mechanisms involved in resistance to radiotherapy in EC, including progesterone receptor (PR) expression and a single specic polymorphism in the gene coding for PR (so-called PROGINS allele). De-novo MLH-1 promoter methylation is found occasionally during EC progression in patients receiving radiotherapy. By comparing immunohistochemically tissue microarrays from post-radiation recurrences of EC and a group of primary EC, interesting information has been obtained regarding the mechanisms of radioresistance. It has been shown that post-radiation recurrences exhibited increased expression of beta-catenin. 64 Recent work has revealed hypoxia-induced beta-catenin nuclear translocation in EC Ishikawa and HEC-1A cell lines. Moreover, hypoxia induces an increase in TCF-4 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. Endometrial carcinoma 119 reporter (Wnt reporter) activity in both endometrial cell lines. Hypoxia-inducible factor 1-alpha (HIF-1a) is another candidate that could confer radioresistance to EC cells. HIF-1 is the most important mediator of hypoxia, as it controls the expression of more than 100 genes. It has been shown recently that HIF-1a expression increases in post-radiation recurrences compared to primary EC. HIF-1a controls the classical NF-jB activation pathway and survival under hypoxia through RelA (p65) nuclear accumulation. Possibly, HIF-1a expression results in increased radio- resistance. 65 Targeted therapies in endometrial carcinoma The PI3KAKT pathway is one of the most frequently abnormal signalling pathways in EEC, often resulting from mutations in the tumour suppressor gene PTEN and activating mutations in PIK3CA. The importance of the PI3KPTENAKT survival pathway in EC raises the possibility that PI3K inhibitors may be used as potential anticancer agents. In fact, a decrease of AKT phosphorylation and increased apoptosis are seen in mutated PTEN human endometrial cancer cells in the presence of PI3K inhibitor. It has been shown recently that EC tumours with PTEN muta- tions have a high level of genetic instability and defects in repair of DNA double-strand breaks by homologous recombination, similar in some ways to that seen for breast and ovarian cancers with altera- tions in BRCA-1 and BRCA-2. For this reason, some investigators have suggested using PARP inhibitors in the treatment of patients with PTEN mutated EC. 66 Of particular interest among AKT targets is the downstream effector mammalian target of rapamycin (mTOR). AKT activates mTOR via direct phosphoryla- tion of tuberous sclerosis 2 (TCS2) protein and by the inhibition of 5-AMP-activated protein kinase (AMP- PK), thereby activating Rheb (Ras homologue enriched in brain) and mTORRaptor activity. Upon activation, mTORRaptor phosphorylates S6K and 4EBP1, resulting in initiation of translation. The acti- vation of mTORC1 downstream targets leads to pro- tein synthesis, such as cell cycle regulating proteins, vascular endothelial growth factor (VEGF) or c-Myc. mTOR inhibitors (rapamycin and rapamycin deriva- tives also called rapalogues) have been developed recently as potential anticancer agents. Tumours associated with PTEN inactivation, like EC, are partic- ularly susceptible to the therapeutic effects of mTOR inhibitors. Several mTOR inhibitors are available for clinical trials: the prototype rapamycin and three ra- pamycin derivatives, CCI-779 (temsirolimus), RAD001 (everolimus) and AP23573 (ridaforolimus). PTEN
mice are a good model for testing the sensitiv-
ity of EC to anticancer drugs, because they develop complex atypical hyperplasia and endometrial carci- noma. The use of dual PI3KmTOR has been proposed as a targeted therapy in EC. 67,68 The p110 subunits of PI3K and mTOR share similar structures. Pharmaco- logical inhibitors of p110 may also inhibit mTOR. It has been shown that mTOR inhibitors often lead to feedback activation of PI3K. Dual PI3K and mTOR inhibitors may inactivate PI3K and mTOR, but also reduce the feedback activation of PI3K caused by mTOR inhibition, thus obtaining increased thera- peutic effects. Considering the high frequency of mutations in both PIK3CA and PTEN in EC, a dual PI3KmTOR inhibitor, such as BEZ235, would be a promising molecular-targeted therapy in endometrial cancer. Tyrosine kinase receptors are also good targets for anticancer therapies. The epidermal growth factor (EGF) receptor family and its growth factors are known to play critical roles in cell growth and differ- entiation. Increased expression of EGF-related protein and epidermal growth factor receptor (EGFR) may contribute to a drug-resistant phenotype. EGFR has intrinsic tyrosine kinase activity which is activated upon ligand binding. Inhibition of EGFR with mono- clonal antibodies leads to growth arrest, and a similar and potentially synergistic effect is anticipated with inhibition of EGFR tyrosine kinase activity. Potential agents are getinib, trastuzumab and lapatinib. Erloti- nib, an EGF inhibitor, was associated with a response rate of only 13% of EC patients. Sunitinib is an inhib- itor of multiple tyrosine kinase receptors, including c-kit, VEGFR, platelet-derived growth factor receptor (PDGFR) and EGFR. It has also been shown recently that sunitinib targets NF-jB. Several multi-target kinase inhibitors targeting FGFRs have been also proposed. These receptors mediate signalling from their high-afnity ligands, FGFs. FGF binding leads to FGFR dimerization, fol- lowed by receptor autophosphorylation and activa- tion of downstream signalling pathways. These signalling pathways have been shown to contribute to FGFR-mediated cell proliferation and migration. Mutations in FGFR2 have been described in EC, pro- viding a rationale for targeting FGFR2 to treat patients with refractory tumours. Inhibition of FGFR2 kinase activity in EC cell lines bearing such FGFR2 mutations inhibits transformation and survival. Sev- 2012 Blackwell Publishing Ltd., Histopathology, 62, 111123. 120 X Matias-Guiu & J Prat eral agents with activity against FGFRs are currently in clinical trials. Sorafenib is a potent receptor tyrosine kinase inhib- itor with antiproliferative and anti-angiogenic activi- ties that may result in clinical benet for a minority of EC patients. There is recent evidence showing that sorafenib sensitizes EC cells to TRAIL-induced apopto- sis by down-regulating FLIP and Mcl-1. 69 Bevacizumab, a recombinant humanized immuno- globulin monoclonal antibody to VEGF, has been used in a small series of patients with recurrent EC, with response in 20% and disease stabilization in 30%. Oestrogen and progesterone are the most important steroid hormones that modulate endometrial cell pro- liferation and differentiation. Medroxiprogesterone acetate has been studied in EC patients. Interestingly, there is preclinical and clinical evidence that mTOR inhibition may have a synergistic effect on hormonal therapy. Apoptosis may be also subjected to targeted ther- apy. The recent evidence that NF-jB activation is fre- quent in EC may explain the presence of apoptosis resistance by activation of target genes such as FLIP and Bcl-xL. Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to trigger cell growth arrest or apoptosis on several tumours. 70 In many different types of tumour cells, bortezomib and other proteasome inhibitors cause cell death by blocking NF-jB activity. Interestingly, block- ade of NF-jB activity by tyrosine kinase inhibitor sunitinib increases cell death in bortezomib-treated EC cell lines. 71 There is increasing evidence that epigenetic altera- tions contribute to cancer initiation and progression. In contrast to genetic mutations, epigenetic changes are reversible and are therefore an attractive target for cancer therapy. Histone deacetylase inhibitors (HDACi), such as vorinostat, are a relatively new class of drugs that play important roles in epigenetic or non-epigenetic regulation, inducing death, apopto- sis and cell cycle arrest in cancer cells. Current inves- tigations have suggested that HDACi have antitumour activity against solid tumours. 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