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REVIEW

Molecular pathology of endometrial carcinoma

Xavier Matias-Guiu 1 & Jaime Prat 21 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, InstitutdInvestigaci o 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 commoncancer 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 andmutations in PTEN , PIK3CA , K-RAS and CTNNB1

(beta-catenin), type II exhibits TP53 mutations andchromosomal instability. Recent investigationsregarding the role of non-coding RNA have providedimportant information regarding tumour progression.Understanding pathogenesis at the molecular level isessential for identifying biomarkers of potential use intargeted therapies.

Keywords: apoptosis, beta-catenin, chromosomal instability, E-cadherin, endometrial carcinoma, K-RAS ,microsatellite instability, molecular genetics, PTEN , PIK3CA , TP53

Molecular features of endometrialcarcinomaIn western countries, endometrial carcinoma (EC) isthe most common malignant tumour of the femalegenital tract, accounting for 10 20 per 100 000 per-son-years. From a pathogenetic viewpoint, EC fallsinto two different types, i.e. types I and II. 1 Type Itumours 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) (mainlyserous and clear cell carcinomas) that occur mainlyin older women and are unrelated to oestrogen stim-ulation (Figure 1B). Occasionally, type II carcinomasmay arise in association with so-called serous endo-

metrial intraepithelial carcinoma, either from atro-phic endometrium or endometrial polyps. It has beenshown that the molecular alterations involved in thedevelopment of EEC (type I) carcinomas differ fromthose of NEEC (type II) carcinomas. 2

4 EEC shows mi-crosatellite instability (MI) and mutations in thePTEN, K-RAS, PIK3CA and CTNNB1 (beta-catenin)genes, whereas NEEC exhibit alterations of p53, lossof heterozygosity (LOH) on several chromosomes, aswell as other molecular alterations (STK15, p16,E-cadherin and c-erb-B2).

MI has been demonstrated in 75% of EC associatedwith hereditary non-polyposis colon cancer (HNPCC),but also in 25 30% of sporadic EC 5

9 (Figure 2). Insporadic EC, MI occurs more frequently in EEC (30%)than in NEEC, and is secondary to MLH-1 promoterhypermethylation. Although data are controversialregarding the prognostic signicance of MI, it is usu-ally associated with a high histological grade. TheMI-associated mismatch repair deciency leads to theaccumulation of many mutations in coding and non-coding DNA sequences, including short-tandemrepeats, named microsatellites. Some small short-tan-

Address for correspondence: X Matias-Guiu, Department of Pathology and Molecular Genetics, Hospital Universitari Arnau deVilanova, Av. Alcalde Rovira Roure 80, 25198 Lleida, Spain.e-mail: [email protected]

2012 Blackwell Publishing Limited.

Histopathology 2013, 62, 111123. DOI: 10.1111/his.12053

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dem repeats, such as mononucleotide repeats, arelocated within the coding sequence of some impor-tant genes ( BAX , IGFIIR , hMSH3, hMSH6, MBD4 ,CHK-1 , Caspase-5 , ATR, ATM , BML, RAD-50 , BCL-10and Apaf-1 ) (Figure 3), and they may be potentialtargets in the process of tumour progression of MI + ,

EC.10,11

PTEN , located on chromosome 10q23.3, is fre-quently abnormal in EC. 12

16 LOH at the PTEN region occurs in 40% of EC. Somatic PTEN mutationsare also common and found predominantly in EEC,occurring in 37 61% of cases (Figure 4). PTEN muta-tions are found in 60 86% of MI-positive EEC, but inonly 24 35% of the MI-negative tumours. IdenticalPTEN mutations have been detected in hyperplasiascoexisting with MI-positive EEC, which suggests thatPTEN mutations are early events in tumourdevelopment. 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 tumourwith deep myometrial invasion; lower right: serous carcinoma (grade 3) exhibiting stratication of anaplastic tumour cells and abnormalmitoses.

Microsatellite instability (MSI)

Hereditary non-polyposis colorectal carcinoma (HNPCC)

Sporadic cancers: colon, stomach, pancreas, ovary andendometrium

MSH2

MSH2

MSH6

MSH6

PMS2

PMS2 TUMOR

TUMOR

(Mutations) n

Inactivation by promoterhypermethylation

MLH1

MLH1

MLH1

Figure 2. Microsatellite instability in hereditary and sporadicendometrial carcinoma.

2012 Blackwell Publishing Ltd., Histopathology , 62, 111123.

112 X Matias-Guiu & J Prat

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signicance of PTEN mutations in EC are controver-sial, some results suggest an association with favour-able prognostic factors. Several studies have shownthat EECs with PTEN mutations have genomicinstability. For this reason, some authors suggest

treating patients with poly (ADP- ribose) polymerase(PARP) inhibitors.

Mutations in PIK3CA contribute to alteration of thePI3K AKT signalling pathway in EC. 17

24 PI3K (phos-phatidylinositol 3-kinase) is a heterodimeric enzymeconsisting of a catalytic subunit (p110) and a regula-tory subunit (p85). The PIK3CA gene, located on chro-mosome 3q26.32, codes for the p110 a catalyticsubunit of PI3K. A high frequency of mutations in thePIK3CA 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 1

7. PIK3CA mutations occur in 24

39% of the cases, and coexist frequently with PTEN mutations(Figure 5). PIK3CA mutations, particularly in exon 20,have been associated with adverse prognostic factorssuch as high histological grade and myometrial inva-sion. Although described initially in EEC, PI3KCAmutations also occur in NEEC and mixed EEC NEEC.Furthermore, different gene expression proles in thePI3K AKT signalling pathway serve to separate twosubgroups of high-grade EC with distinct molecularalterations (PI3K AKT pathway versus p53alterations) that may play different roles in endometrial

MSP

Bat-25 Bat-26

MLH1 promoterhypermethylation

Loss ofMLH1expression

MI

BAX

BAX

T1

T1

T2

T2

IGFIIRMSH3PTENCasp-5Bcl-10APAF-1

U UM M

N

T

N+T

Figure 3. MLH1 inactivation by promoter hypermethylation is the most common cause of the microsatellite instability (MI) phenotype inendometrial carcinoma. Progressive accumulation of alterations secondary to MI affects important regulatory genes and promotescarcinogenesis. BAX somatic frameshift mutations are distributed heterogeneously throughout the tumour and provide selective growthadvantage. T1: BAX-non-mutated component of endometrioid endometrial carcinoma; T2: BAX-mutated component of endometrioidendometrial 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 tumourcells 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.

PTENMUTATION

PROMOTERHYPERMETHYLATION

LOH

T

N

76 79 82 85 88

Figure 4. Inactivation of tumor suppressor gene PTEN may occurby several mechanisms such as point mutation, promoterhypermethylation, or deletion (loss of heterozygosity [LOH]) at10q23. T: tumour DNA, N: normal tissue DNA.


Endometrial carcinoma 113

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carcinogenesis 24 (Figure 6). Moreover, mutations inPIK3RI , the gene encoding p85 a , the inhibitory sub-unit of PI3K, have been detected in 43% of EEC and12% of NEEC.

The RAS-RAF-MEK-ERK signalling pathway playsan important role in tumorigenesis. The frequency of K-RAS mutation in EC ranges between 10 and 30%. Insome series, K-RAS mutations have been reported to bemore frequent in EEC showing MI. 25 BRAF , anothermember of the RAS RAF MEK ERK pathways, ismutated very infrequently in EC. 26 RAS effectors suchas RASSF1A are thought to provide an inhibitorygrowth signal, which needs to be inactivated duringtumorigenesis. RASSF1A inactivation by promoterhypermethylation may contribute signicantly toincreased activity of the RAS RAF MEK ERK signal-

ling pathway.27

PIP 2Dephosphorylation

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 phosphatidylinositolbiphosphate (PIP2) into phosphatidylinositol triphosphate (PIP3),promoting cell proliferation and survival. PTEN regulates PI3K

signalling negatively by dephosphorylation of PIP3.

P21AKT1FAKPAK1CCND1FASFOXO1AP27P53XIAPNFKBGSK3BmTORE-CADHCASP3PIK3CAAKT2MDM2PTEN

p53 alterations

PIK3CA exon 20 mutationsPIK3CA exon 9 mutationsPTEN mutations

MIHigh-grade

High-stage>50% myometrial infiltrationVascular 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-regulatedexpression are indicated as red and green cubes, respectively. Genes that did not vary in their expression level are shown in black, andgenes with unsatisfactory results labelled in grey. Enclosed in the clustering image, results of p53, p16, PIK3CA, PTEN and microsatelliteinstability (MI) analysis, as well as clinicopathological parameters such as high-grade (grade 3), non-endometrioid, high-stage (stage 2 orhigher), myometrial invasion ( > 50%) and vascular invasion, are represented graphically for each case.



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Several studies suggest that the broblast growthfactor (FGF) signalling pathway is important in EC. Ithas been shown that EC presents frequent inactiva-tion of SPRY-2, a protein involved in the negativeregulation of the FGF receptor (FGFR) pathway bypromoter methylation. 28 Reduced SPRY2 immunoex-pression is seen in almost 20% of EC and is associatedstrongly with increased cell proliferation. Moreover,somatic mutations in the receptor tyrosine kinase

FGFR2 have been detected recently in 6

12% of EC,particularly in EEC. 29 31 Interestingly, FGFR2 muta-

tions and K-RAS mutations are mutually exclusiveevents, while mutations in FGFR2 and PTEN fre-quently coexist. FGFR2 is of special interest as apotential target therapy, and FGFR-2 inhibitors arecurrently under consideration.

The beta-catenin gene ( CTNNB1 ) maps to 3p21.Beta-catenin is a component of the E-cadherin cateninunit, which plays an important role in cell differentiationand 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 andtranscriptional 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 14 44% of EC.31

36

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 goodprognosis.

In contrast to EEC, NEEC show TP53 mutations(90%), markedly reduced expression of E-cadherin(80 90%), c-erb-B2 (HER-2 ) amplication (30%),alterations in genes involved in regulation of themitotic 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 10 20% of EEC (Figure 8),mainly grade 3 tumours. 37

39 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 withabnormal 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.



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E-cadherin is frequent in EC, and may be caused byLOH or promoter hypermethylation. In fact, LOH at16q22.1 is seen in almost 60% of NEEC, but in only22% of EEC. C-erb-B2 overexpression and amplica-tion are also seen more frequently in NEEC (43% and29%, respectively) than in EEC. 40 However, the mosttypical molecular feature of NEEC is the presence of widespread chromosomal gains and losses whichreect the presence of aneuploidy. 41 cDNA arrayshave demonstrated that NEEC usually show up-regu-lation of genes ( STK15 , BUB1 , CCNB2 ) involved inthe regulation of the mitotic spindle checkpoint. Oneof these genes, STK15 , essential for chromosome seg-regation and centrosome functions, is amplied fre-quently in NEEC. Recently documented potentialbiomarkers of serous carcinoma are epithelial celladhesion molecule (EpCAM), claudin-3 and claudin-4receptors, serum amyloid A, folate binding protein,mesothelin and insulin-like growth factor-II mRNAbinding protein 2 (IMP2).

Among NEEC, clear cell carcinomas show specicfeatures. Endometrial clear cell carcinomas appear torepresent a heterogeneous group of tumours that mayarise through different pathogenetic pathways. Basedon morphological similarities between ovarian and

endometrial clear cell carcinomas, it has been sug-gested that both tumour types may exhibit similaralterations, 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 carcinomasof the ovary. In a recent study, however, these changeshave been found in 29% of grades 1 or 2 and 39% of grade 3 EECs, 18% of serous NEEC and 26% of clearcell NEEC. Uterine low-grade endometrioid carcinomashave also shown loss of ARID1A expression (26%) andARID1A mutations (40%). 42,43

cDNA array studies have shown that the expres-sion proling of EEC differs from that of NEEC. 44 47

Genes up-regulated in serous carcinomas wereIGF2 , PTGS1 , FOLR and p16 , whereas genes up-reg-ulated in EEC included TFF3 , FOXA2 and MSX2 .Moreover, two members of the secreted frizzledrelated protein family (SFRP1 and SFRP4) weredown-regulated more frequently in EC with MI. 48

Interestingly, by comparing the expression prolesof similar histological subtypes of ovarian and endo-metrial carcinomas it was found that clear cellcarcinomas had a similar prole, regardless of theorgan 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) isarticial and too rigid, and the dualistic model hasbeen challenged recently. In daily practice, pathologistsare faced with tumours showing mixed, combined orhybrid morphological and molecular characteristics(often endometrioid and serous carcinomas). Further-more, though serous and clear cell carcinomas havebeen classied within the same category of NEEC, basedmainly on their common high nuclear grade andaggressive behaviour, recent studies have shown that

these carcinomas are, in fact, distinct tumour typesexhibiting different clinical, immunohistochemical andmolecular features. Based on molecular analyses, it hasbeen suggested that in mixed EEC NEEC the NEECcomponent originates from a pre-existing EEC as aresult of tumour progression and, not infrequently,these tumours retain their typical EEC molecular alter-ations (Figure 9). This hypothesis would explain theexistence of mixed EEC NEEC, the presence of MI andthe alterations in PTEN , K-RAS or beta-catenin in somecases of NEEC. Another controversial scenario is thegrey zone between high-grade (predominantly solid)

PTEN

MIB-Catenin

K-Ras

PIK3CA

Exon 9Exon 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 commonmolecular genetic alterations in endometrial carcinomas. EEC: endometrioid endometrial carcinoma; NEEC: non-endometrioid endometrialcarcinoma.



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EEC and NECC. Distinction between these two types of tumours is difcult; conversely, high-grade EEC occa-sionally exhibit molecular alterations typical of NEECsuch as TP53 mutations.

Undifferentiated carcinomas of the endometriumare epithelial tumours that fail to show evidence of either glandular or squamous differentiation. Theyrepresent 1 10% of EC, and show a proliferation of medium-sized, monotonous, epithelial cells growingin solid sheets. Occasionally, undifferentiated carcino-mas arise from pre-existing well- or moderately-differ-entiated EEC. The term de-differentiated carcinomahas been used to designate such special tumour types.Several groups of investigators have conrmed thatMI is the predominant molecular feature encounteredin this type of carcinoma. 49 However, MI is not seenin all cases, and some tumours may have TP53mutations suggesting transformation into a high-grade carcinoma.

Malignant mixed m

ullerian tumours (MMMT), also

referred to as uterine carcinosarcomas or sarcomatoidcarcinomas, are rare uterine tumours accounting forless than 5% of EC. Molecular studies have suggestedrecently that MMMT should be regarded as metaplasticcarcinomas. Like sarcomatoid carcinomas of otherlocations, carcinosarcomas probably result from endo-metrial carcinomas through epithelial-to-mesenchymaltransition (EMT). EMT is a process of cellular transdif-ferentiation in which epithelial cells lose polarity andcell cell 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 , tyrosinekinase 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 torepress E-cadherin, a master regulator of cell adhe-sion and polarity. While the transient presence of EMT features is important for myometrial invasionin conventional endometrial carcinomas, MMMTshow permanent expression of these features leadingto repression of epithelial markers (E-cadherin) andincreased 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 wellas the presence of heterologous elements. It hasbeen shown recently that MMMT show a microRNAsignature typical of EMT. 50

Non-coding RNAThe classic perception that cancer resulted mainlyfrom alterations in coding genes has been challengedrecently 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 RNAmolecules regulating the expression of target genesby imperfect (in animals) binding to the 3 -untrans-lated region (UTR) and possibly 5 -UTR of mRNA. Tobecome the mature form, miRNAs are processed byenzymatic complexes Dorsha and Dicer, and theyrepress translation or lead to the degradation of themRNA of their target genes. Currently approximately2000 human miRNA sequences have been identied,

and this number continues to grow. Regardless of therelatively small number of miRNAs, as each singlemiRNA targets several hundred genes, and a singletarget gene can bind to multiple miRNAs, thus mak-ing the whole network very complex, it is believedthat approximately 30% of all human genes are atarget for miRNA regulation. There is also evidencethat these small molecules are expressed in a tissue-/cell-specic manner, being reserved restrictively tospecic cell type or associated ubiquitously with dif-ferent human body compartments. It was shown pre-viously that dysfunctional expression of miRNAs is a

MIPTENKRASbeta-catenin

PIK3CA (9,20)

TumorprogressionGene alterationsPIK3CA (20)

P53

NE

Low-gradeEndometrioid Ca

Non-Endometrioid Ca

High-gradeEndometrioid Ca

PIK3CA (20)Cadherin ECyclic D1

Cyclic E

Her-2/neuSTK15

Figure 9. Pathogenesis of endometrial carcinoma: an alternative tothe dualistic model. Exons 9 and 20 of PIK3CA are thosefrequently mutated in endometrial carcinoma. Ca: carcinoma; NE:normal endometrium.



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frequent attribute of malignant behaviour. Nowadays,aberrant expression of specic miRNAs is associatedwith all cancer types. Germline and somatic muta-tions as well as polymorphisms in the mRNAs tar-geted by miRNAs can also lead to cancerpredisposition and progression. Growing evidencepoints to miRNA being implicated in oncogenic pro-cesses, suggesting that miRNA expression prolingcan distinguish tumours (according to diagnosis andcancer stages) more accurately than traditional geneexpression analyses. Small non-coding RNAs can playa 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 regionsencoding miRNAs, failure in their biosynthetic path-way machinery, and epigenetic regulation. Amongthe 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 andmiR-205. In contrast, other miRNAs have been foundto 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. 51 55 These miRNAs targetimportant 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 . Inone series, high levels of miR-205 expression wereassociated with poor patient overall survival; 53 themiRNA 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-29cand miR-382. 51 We have shown recently that themiRNAs Lin28B and let-7b were involved in the reg-

ulation of the HMGA-2 gene, a factor that regulatesepithelial to mesenchymal transition, and which isexpressed 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 ( > 200nucleotide) transcripts from intergenic and intronicregions of the human genome that lack protein-codingcapacity. They probably have important roles inrecruitment of histone-modifying complexes to chro-matin and regulation of transcription and splicing.

Only a few lncRNA have been well characterized, butexpectations are that they will outnumber protein-coding genes. Aberrant expression of these transcriptshas 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 toexhibit mutations in almost half of EC samples thathave been tested. 57 Another lncRNA that may havea role in EC is the PTEN pseudogene, PTENP1 , whichcan regulate PTEN s tumour suppressor role by com-petitive binding to common miRNAs. 58

Resistance to apoptosis, hypoxia andradiation therapy in endometrial carcinoma

There is a great deal of evidence suggesting thatalteration of apoptosis is important in the develop-ment and progression of EC (Figure 10). Several of the molecular abnormalities that have been detectedin 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- j B activationis frequent in endometrial carcinoma 59 may explainthe presence of apoptosis resistance by activation of target genes such as FLIP and Bcl-xL. p53 altera-tions, which are characteristic of NEEC, may alsooccur in endometrioid tumours, particularly in thoseneoplasms showing overlapping features betweentypes I and II tumours, and they may have an impacton apoptosis at several different levels.

Also, members of the Bcl-2 family of genes areabnormal in EC. In EC, divergent observations havebeen reported with respect to Bcl-2 and Bcl-xL (Fig-

Death factors

Death receptors

Growth factors

PI3K

BCLxLAP AF-1CYT-CCaspase 9Caspase 3

Caspase 8Adapter protein

APOPTOSIS

FLIP

BAXPTEN

AKT

NFKB

Survivin

Figure 10. Apoptosis: intrinsic (mitochondrial) and extrinsic (deathreceptor-initiated) pathways.



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ure 10). Some authors have found up-regulated Bcl-xL and Bcl-2 in EC compared to normal tissue, andthey have also been reported to be involved in devel-opment of metastases. Many pathways can controlBcl-2 expression, and typical EC molecular alterationssuch as those involved in exacerbated PI3K AKT sig-nalling could trigger Bcl-2 family members overex-pression. However, other non-canonical molecularevents in EEC, such as those involving the NF- j Bpathway 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-2homology domains 1 3 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 outermitochondrial membrane that, in turn, will promoterelease 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 andapoptosis-inducing factor (AIF). Interestingly, Bax isa target gene for mutations in EEC with MI, and mayhave a role in resistance to apoptosis in thesetumours.

Cancer cells can evade the extrinsic apoptotic path-way by several mechanisms. FLICE-like inhibitoryprotein (FLIP), one of the most important regulatorsof death receptor signalling (Figure 10), is a proteinthat shares high homology with caspase-8 but lacksproteolytic activity. Like caspase-8, it contains twodeath-effector domains (DED) that allow it to interacteither with DED within Fas-associated death domainprotein (FADD) or caspase-8, thereby inhibitingdeath-inducing signalling complex (DISC) assemblyand caspase-8 activation. Hence, FLIP represents apotent 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 criticalfor the survival of many tumour cells. Direct evidencefor the role of FLIP in the resistance of endometrialcarcinoma cells to apoptosis induced by TNF-relatedapoptosis-inducing ligand (TRAIL) is provided bytreatment with specic small interfering RNAs (siR-NA) targeting FLIP. 60 Transfection of endometrialcarcinoma cell lines with FLIP siRNA results in amarked decrease in cell viability after TRAIL exposi-tion. This is accompanied by activation of both cas-

pase-8 and caspase-3, suggesting activation of theextrinsic pathway. Moreover, in EEC, FLIP can be reg-ulated transcriptionally by casein kinase-2 (CK2), aSer/Thr kinase implicated in the development andprogression 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 hasbeen found overexpressed in endometrial cancer com-pared to normal tissue and is thought to regulate cellproliferation and anchorage-independent cell growth.Recent studies have shown that FLIP may be regulatedby a cellular complex, including CK2 BRAF KSR1.61,62

Interestingly, this regulation allows connection of apop-tosis resistance with the RAS RAF MEK ERK signallingpathway. The KSR1 (kinase suppressor of RAS 1) isconsidered a scaffold protein that regulates the MAP

kinase pathway. KSR1 interacts with different kinasesof the RAF MEK ERK signalling pathway to enhanceits activation and also modulates the apoptoticresponse to death receptors. Recently, it has beenshown that the expression of KSR1 is increased inEC, suggesting its possible role in endometrial carci-nogenesis. 63

Tumour hypoxia is known to render tumours moreresistant to radiotherapy. By reacting with the radia-tion-created broken ends of DNA, oxygen xes thedamage and thus enhances radiation-induced celldeath. This phenomenon, known as the oxygenenhancement effect, could render oxygenated cellsthree times more radiosensitive than hypoxic cells.Under hypoxic conditions, the oxygen enhancementeffect is lost and cells become more radioresistant.Although the absence of oxygen is the major factorinducing radioresistance under hypoxic conditions,there is increasing evidence that signalling pathwaysactivated under hypoxia may modulate cancer cellradioresistance.

Some investigators have addressed the molecularmechanisms involved in resistance to radiotherapy inEC, including progesterone receptor (PR) expressionand a single specic polymorphism in the gene coding

for PR (so-called PROGINS allele). De-novo MLH-1promoter methylation is found occasionally during ECprogression in patients receiving radiotherapy. Bycomparing immunohistochemically tissue microarraysfrom post-radiation recurrences of EC and a group of primary EC, interesting information has been obtainedregarding the mechanisms of radioresistance. It hasbeen shown that post-radiation recurrences exhibitedincreased expression of beta-catenin. 64 Recent workhas revealed hypoxia-induced beta-catenin nucleartranslocation in EC Ishikawa and HEC-1A cell lines.Moreover, hypoxia induces an increase in TCF-4



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reporter (Wnt reporter) activity in both endometrialcell lines.

Hypoxia-inducible factor 1-alpha (HIF-1 a ) isanother candidate that could confer radioresistanceto EC cells. HIF-1 is the most important mediator of hypoxia, as it controls the expression of more than100 genes. It has been shown recently that HIF-1 aexpression increases in post-radiation recurrencescompared to primary EC. HIF-1 a controls the classicalNF-j B activation pathway and survival underhypoxia through RelA (p65) nuclear accumulation.Possibly, HIF-1 a expression results in increased radio-resistance. 65

Targeted therapies in endometrialcarcinomaThe PI3K AKT pathway is one of the most frequentlyabnormal signalling pathways in EEC, often resultingfrom mutations in the tumour suppressor gene PTEN and activating mutations in PIK3CA . The importanceof the PI3K PTEN AKT survival pathway in EC raisesthe possibility that PI3K inhibitors may be used aspotential anticancer agents. In fact, a decrease of AKT phosphorylation and increased apoptosis areseen in mutated PTEN human endometrial cancercells in the presence of PI3K inhibitor. It has beenshown recently that EC tumours with PTEN muta-tions have a high level of genetic instability anddefects in repair of DNA double-strand breaks byhomologous recombination, similar in some ways tothat seen for breast and ovarian cancers with altera-tions in BRCA-1 and BRCA-2 . For this reason, someinvestigators have suggested using PARP inhibitorsin the treatment of patients with PTEN mutated EC. 66

Of particular interest among AKT targets is thedownstream effector mammalian target of rapamycin(mTOR). AKT activates mTOR via direct phosphoryla-tion of tuberous sclerosis 2 (TCS2) protein and by theinhibition of 5 -AMP-activated protein kinase (AMP-PK), thereby activating Rheb (Ras homologue

enriched in brain) and mTOR Raptor activity. Uponactivation, mTOR Raptor phosphorylates S6K and4EBP1, 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 developedrecently as potential anticancer agents. Tumoursassociated with PTEN inactivation, like EC, are partic-ularly susceptible to the therapeutic effects of mTORinhibitors. 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 developcomplex atypical hyperplasia and endometrial carci-noma.

The use of dual PI3K mTOR has been proposed asa 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. Ithas been shown that mTOR inhibitors often lead tofeedback activation of PI3K. Dual PI3K and mTORinhibitors may inactivate PI3K and mTOR, but alsoreduce the feedback activation of PI3K causedby mTOR inhibition, thus obtaining increased thera-

peutic effects. Considering the high frequency of mutations in both PIK3CA and PTEN in EC, a dualPI3K mTOR inhibitor, such as BEZ235, would be apromising molecular-targeted therapy in endometrialcancer.

Tyrosine kinase receptors are also good targets foranticancer therapies. The epidermal growth factor(EGF) receptor family and its growth factors areknown to play critical roles in cell growth and differ-entiation. Increased expression of EGF-related proteinand epidermal growth factor receptor (EGFR) maycontribute to a drug-resistant phenotype. EGFR hasintrinsic tyrosine kinase activity which is activatedupon ligand binding. Inhibition of EGFR with mono-clonal antibodies leads to growth arrest, and a similarand potentially synergistic effect is anticipated withinhibition of EGFR tyrosine kinase activity. Potentialagents are getinib, trastuzumab and lapatinib. Erloti-nib, an EGF inhibitor, was associated with a responserate of only 13% of EC patients. Sunitinib is an inhib-itor of multiple tyrosine kinase receptors, includingc-kit, VEGFR, platelet-derived growth factor receptor(PDGFR) and EGFR. It has also been shown recentlythat sunitinib targets NF- j B.

Several multi-target kinase inhibitors targeting

FGFRs have been also proposed. These receptorsmediate 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. Thesesignalling pathways have been shown to contributeto FGFR-mediated cell proliferation and migration.Mutations in FGFR2 have been described in EC, pro-viding a rationale for targeting FGFR2 to treatpatients with refractory tumours. Inhibition of FGFR2kinase activity in EC cell lines bearing such FGFR2mutations inhibits transformation and survival. Sev-



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eral agents with activity against FGFRs are currentlyin 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 minorityof EC patients. There is recent evidence showing thatsorafenib 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 usedin a small series of patients with recurrent EC, withresponse in 20% and disease stabilization in 30%.

Oestrogen and progesterone are the most importantsteroid hormones that modulate endometrial cell pro-liferation and differentiation. Medroxiprogesteroneacetate has been studied in EC patients. Interestingly,

there is preclinical and clinical evidence that mTORinhibition may have a synergistic effect on hormonaltherapy.

Apoptosis may be also subjected to targeted ther-apy. The recent evidence that NF- j B activation is fre-quent in EC may explain the presence of apoptosisresistance by activation of target genes such as FLIPand Bcl-xL. Proteasome inhibitors are currently usedas chemotherapeutic drugs because of their ability totrigger cell growth arrest or apoptosis on severaltumours. 70 In many different types of tumour cells,bortezomib and other proteasome inhibitors cause celldeath by blocking NF- j B activity. Interestingly, block-ade of NF-j B activity by tyrosine kinase inhibitorsunitinib increases cell death in bortezomib-treated ECcell lines. 71

There is increasing evidence that epigenetic altera-tions contribute to cancer initiation and progression.In contrast to genetic mutations, epigenetic changesare reversible and are therefore an attractive targetfor cancer therapy. Histone deacetylase inhibitors(HDACi), such as vorinostat, are a relatively newclass of drugs that play important roles in epigeneticor non-epigenetic regulation, inducing death, apopto-sis and cell cycle arrest in cancer cells. Current inves-

tigations have suggested that HDACi have antitumouractivity against solid tumours.

AcknowledgementsThis review was supported by grants FIS PI100922,FIS PI11-01561, Fundaci on Mutua Madrile

~

naAP75732010, 2009SGR794, RTICC RD06/0020/1034 and RD06/0020/0015; Fundaci on Asociaci onEspa

~

nola contra el Cancer, and programa de intensi-caci on de la investigaci on, Instituto Carlos III, Depart-ment of Health, Spain.

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