1. Kanker Endometrium Di Indonesia
2. Kanker Endometrium Pdf

Most sporadic endometrial cancers (ECs) can be histologically classified as endometrioid, serous, or clear cell. Each histotype has a distinct natural history, clinical behavior, and genetic etiology.

Endometrioid ECs have an overall favorable prognosis. They are typified by high frequency genomic alterations affecting PIK3CA, PIK3R1, PTEN, KRAS, FGFR2, ARID1A (BAF250a), and CTNNB1 (β-catenin), as well as epigenetic silencing of MLH1 resulting in microsatellite instability. Serous and clear cell ECs are clinically aggressive tumors that are rare at presentation but account for a disproportionate fraction of all endometrial cancer deaths. Serous ECs tend to be aneuploid and are typified by frequent genomic alterations affecting TP53 (p53), PPP2R1A, HER-2/ERBB2, PIK3CA, and PTEN; additionally, they display dysregulation of E-cadherin, p16, cyclin E, and BAF250a. The genetic etiology of clear cell ECs resembles that of serous ECs, but it remains relatively poorly defined.

A detailed discussion of the characteristic patterns of genomic alterations that distinguish the three major histotypes of endometrial cancer is reviewed herein. Microsatellite instability (MSI) A MSI phenotype is marked by a high frequency of mutations at sites of short nucleotide repeats (microsatellites) within the genome. MSI is the result of unrepaired errors that arise during DNA replication. It is detectable in 20% of unselected endometrial tumors, – and is more frequent among EECs than non-EECs (NEECs)., In sporadic endometrial tumors, MSI-positivity reflects an increased mutation rate resulting from somatic alterations in DNA mismatch repair genes. Most presumed sporadic, MSI-positive EECs are associated with epigenetic silencing of MLH1, via promoter hypermethylation. – This occurs early in EEC progression; MLH1 promoter hypermethylation has been documented in 3% of complex endometrial hyperplasias, and 33% of atypical hyperplasias.

A smaller fraction of MSI-positive EECs have somatic mutations in MSH6, or loss of MSH2 protein expression., Somatic mutations in MSH3 have also been described in sporadic EC but, because they occur within a mononucleotide repeat tract, it has been suggested that they may be a consequence, rather than a cause, of defective mismatch repair. Likewise, certain MSH6 mutations have recently been proposed to occur secondarily to MSI.

MLH1 and other mismatch repair genes are among the so-called “caretaker genes” that normally function to preserve genomic stability; loss of their function leads to the accumulation of mutations in other target genes that drive tumorigenesis. A number of target genes have been described in EC, although it is worth noting that most studies do not state whether the tumors occurred sporadically or in the context of Lynch syndrome. Within MSI-high EECs, the presence of somatic mutations involving simple nucleotide repeats in BHD (13%), BAX (29%–53%),– IGFIIR (14%–21%), TGFβ-RII (10%–37%), E2F4 (21%), MLH3 (21%), MSH3 (14%–33%), MHS6 (7%–36%), CDC25C (7%), DNAPKcs (34%), RAD50 (17%), MRE11 (15%–50%), ATR (14%–15%), BRCA1 (15%), CtIP (12%), CHK1 (7%–28%), and MCPH1 (12%), implicates these genes as targets of MSI and potential drivers of MSI-positive endometrial tumorigenesis. Many of these genes, including ATR, are involved in the DNA damage response.

MSI-associated truncating mutations in ATR are loss-of-function mutations that are significantly associated with both disease-free survival and overall survival in multivariate analyses., Early-stage EECs with and without MSI exhibit distinct gene expression profiles. It has been suggested that this might be either a direct effect of their differing MSI status, or alternatively, it might result from differences in the global methylation status of MSI+ and MSI− tumor subgroups, and therefore be indirectly associated with MSI caused by MLH1 hypermethylation. The PI3K pathway The most frequently altered biochemical pathway in EECs is the PI3K-PTEN-AKT signal transduction pathway, which regulates numerous cellular processes including proliferation, growth, and survival. In the most comprehensive evaluation of PI3K pathway alterations in EECs to date, more than 80% of tumors had one or more somatic alterations affecting the pathway. These alterations consist of high frequency mutations in PIK3R1 (p85α), PIK3CA (p110α), and PTEN; PIK3CA amplification (7%–33% of EECs); PTEN promoter methylation or loss of PTEN expression; as well as rare mutations in AKT1 (2%) and PIK3R2 (p85β) (5%). – In EEC, an additional level of dysregulation of mTOR is achieved by loss of expression of TSC2 and LKB1, which have been documented in 13% and 21% of EECs, respectively.

The interplay between the various PI3K pathway alterations in EECs is complex. PIK3R1 and PIK3CA mutations are generally mutually exclusive, suggesting functional redundancy., In contrast, PTEN mutations frequently coexist, and can functionally cooperate, with PIK3R1 and PIK3CA mutations., Although PTEN is an important regulator of the PI3K-AKT pathway, it also has PI3K-independent functions.

For example, PTEN plays an important role in the maintenance of genomic integrity. Recent work revealed that PTEN-deficient EC cell lines are sensitive to PARP inhibitors, pointing to a potential Achille’s heel for targeted therapy in EC. Endometrial tumors have a tissue specific pattern of PIK3CA mutations, with a significantly higher frequency of mutations in the ABD and C2 domains of p110α than any other tumor type that has been comprehensively evaluated., The reason for this tissue specificity is unclear but it is intriguing that p85α, which binds the ABD and C2 domains of p110α, is somatically mutated at high frequency in EC but only rarely in other tumors. Together, these observations suggest that disrupting the p85α-p110α interaction may confer a tissue specific selective advantage in endometrial tumorigenesis. PTEN mutation is one of the earliest known events in the genesis of EEC, occurring in 20%–27% of endometrial hyperplasias, and in 55% of endometrial intraepithelial neoplasias. PTEN mutations are believed to precede mismatch repair defects in the progression of sporadic EECs. In contrast to PTEN, PIK3CA mutations are rare in complex atypical hyperplasia, and appear to be later events in the progression of EEC.

The RAS-RAF-MEK-ERK pathway The RAS family of oncogenes are frequently activated in a variety of human cancers. RAS proteins mediate signal transduction via both the RAF-MEK-ERK and PI3K-PTEN-AKT pathways, and thus regulate numerous processes including cell proliferation and cell survival. Somatic mutations in KRAS were first described in EC over two decades ago, and were subsequently found to be significantly more frequent in EEC than in serous EC. – On average, KRAS is mutated in 18% of EECs compared with 3% of serous ECs. KRAS mutations occur early in the genesis of EECs, having been documented in atypical endometrial hyperplasia.

– However, MSI appears to precede KRAS mutation in the progression of EEC. In EC, KRAS mutations can coexist with mutations in PTEN, PIK3CA, and PIK3R1, suggesting that KRAS mutations are not functionally redundant with PI3K pathway mutations., This is supported by the results of a recent comprehensive genomics and proteomics analysis of the RAS-RAF-MEK-ERK and PI3K-PTEN-AKT pathways in EC in which Cheung et al showed that KRAS mutations were associated with increased phosphorylation of MEK1/2, ERK1/2, and p38MAPK. Oda et al have also shown functional synergy between mutant KRAS and mutant PIK3CA in the transformation of HMLE cells. Finally, a conditional mouse model of EC in which PTEN was ablated and KRAS was activated in the reproductive tract, showed an acceleration in the development of EC as compared to mice with only a single lesion. In contrast to KRAS mutations, somatic mutations in codons 11 and 15 of BRAF, the sites of hotspot mutations in other cancers, are infrequent in EECs,– and are mutually exclusive with KRAS mutations and hypermethylation of RASSF1A. The overall BRAF mutation frequency in ECs is 1%.

Kanker Endometrium Di Indonesia

Only one study noted a high frequency (21%) of BRAF mutations in EC. It has been suggested that this high frequency of mutations might reflect ethnic differences between study populations, although this has not yet been verified. RASSF1A is a multifunctional tumor suppressor that has been implicated in the regulation of numerous cellular processes and pathways, including the RAS signal transduction pathway.

Hypermethylation of the RASSF1A promoter is frequent in EECs (62%–74%) and correlates with reduced expression of RASSF1A. – RASSF1A promoter methylation has been documented in histologically normal tissue adjacent to EEC, and in complex hyperplasia with and without atypia., In EECs, methylation of the RASSF1A promoter is significantly associated with advanced stage disease. RASSF1A promoter hypermethylation is significantly more frequent in microsatellite unstable tumors than in microsatellite stable tumors, leading to the proposal that this reflects an underlying methylator phenotype that targets the MLH1 mismatch repair gene and other genes, including RASSF1A. RASSF1A methylation is also more frequent in tumors lacking a KRAS mutation than in tumors with mutant KRAS (38% vs 14%), although the difference did not achieve statistical significance. Several other genes that modulate the activity of the RAS-RAF-MAPK pathway are also subjected to aberrant methylation in EECs.

These include RASSF2A, HDAB2IP, BLU, SPROUTY-2, and RSP6KA6 ( RSK4). FGFR2 Somatic mutations in the FGFR2 receptor tyrosine kinase have been described in 12% of EECs., FGFR2 mutations are mutually exclusive with KRAS mutations, indicating functional redundancy, whereas most (77%) FGFR2-mutant ECs are PTEN-mutant. In EC, the vast majority of FGFR2 mutations are missense mutations within the extracellular, transmembrane, and kinase domains of the protein. Codon 252 (S252) forms a prominent mutation hotspot within a region of the extracellular domain that mediates ligand binding. The S252W mutant is oncogenic and accounts for 41% of all mutations reported in EC to date., EC cell lines that harbor the FGFR2-S252W mutant appear to be dependent upon expression of the mutant protein for their survival., Importantly, EC cell lines with an activating mutation in FGFR2 are more sensitive to killing by PD173074, a pan-FGFR inhibitor, than FGFR2-wildtype EC cell lines, thus pointing to mutant FGFR2 as a potential therapeutic target. CTNNB1(β-Catenin) CTNNB1 encodes β-catenin, an integral member of the canonical WNT signaling pathway. Somatic mutations in CTNNB1 and stabilization of β-catenin are common features of EEC., CTNNB1 mutations occur in up to 45% of EECs; they have not been found in NEECs, but only a small number of tumors have been evaluated.

Similarly, nuclear expression of β-catenin has been observed in 31%–47% of EECs, compared with 0%–3% of NEECs. A significant correlation between β-catenin accumulation and CTNNB1 mutations has been noted ( P.

ARID1A (BAF250a) ARID1A is a recently described tumor suppressor gene that encodes BAF250a, a component of the SWI/SNF chromatin-remodeling complex. Dysregulation of ARID1A and BAF250a has been implicated in a large fraction of EECs. Loss of BAF250a expression has been observed by immunohistochemistry (IHC) in 26%–29% of low-grade (G1 or G2) EECs, and in 39% of high-grade (G3) EECs., Consistent with this observation, somatic mutations in ARID1A were detected among 40% of low-grade EECs; 50% of mutated tumors showed loss of BAF250a expression.

TP53 (p53) The most frequently altered cancer gene in serous EC is the TP53 tumor suppressor gene. Game genie save editor serial key. In early landmark studies, 80%–86% of serous tumors showed positive immunostaining for p53, and 53%–90% of tumors had somatic TP53 mutations.,– TP53/p53 aberrations occur very early in the genesis of serous EC.

They are present in morphologically benign endometrial glands or epithelium adjacent to serous EC, the so-called “p53 signature”, as well as in EmGD, and EIC., In 50% of uteri with coexisting “p53 signatures,” EmGD, EIC, and serous EC, identical p53 mutations were observed in all four entities. An increasing frequency of TP53 mutations has also been noted between the normal endometrium (0%), EmGD (43%), EIC (72%), and serous-EC (96%). These observations, coupled with detailed pathologic descriptions of “p53 signatures,” EmGD, and EIC, present a new model for the evolution of serous EC.

This model posits a transition from the normal resting epithelium, to latent precancerous “p53 signatures,” to precancerous EmGD, to EIC, and finally to serous EC., In contrast to serous ECs, EECs have a significantly lower overall incidence of p53 positivity (3%–52%) and TP53 mutation (12%–23%).,– The incidence of TP53 mutations is greater in high-grade (G3) EECs than in low-grade EECs (43% of G3, 8% of G2, 0% of G1). However the incidence of p53 positivity and TP53 mutation in high-grade EECs is still subject to interpretation, as some of the reported high-grade EEC cases may actually be serous EC, due to the occasional histological ambiguity between these two subtypes. The frequency of TP53 mutations in clear cell EC has not been well defined although one study noted mutations in 9% of tumors. PPP2R1A The PP2A serine-threonine phosphatase is a trimeric holoenzyme composed of a catalytic subunit (PP2Ac; subunit C), a scaffolding subunit (PR65; subunit A) and one of a number of variable regulatory (B) subunits (reviewed by Eichhorn et al). The scaffolding subunits are encoded by PPP2R1A (PR65α) or PPP2R1B (PR65β).

They contain 15 HEAT (Huntington/elongation/A-subunit/TOR) motifs; HEAT motifs 2–7 mediate binding to the regulatory subunits, whereas HEAT motifs 11–15 mediate binding to the catalytic subunit of the holoenzyme. Somatic mutations in PPP2R1A (PR65α) occur at very high frequency (17%–41%) in serous EC.

– In contrast, PPP2R1A is infrequently (5%–7%) mutated in EECs. – It remains to be determined whether PPP2R1A is mutated in pure clear cell ECs; only five primary tumors of this subtype have been sequenced and no mutations were detected., Resequencing of PPP2R1A in ECs has thus far been confined to exons 5 and 6, based on earlier observations that PPP2R1A mutations in ovarian cancer localized exclusively within these two exons.

Interestingly, the distribution of PPP2R1A mutations within exons 5 and 6 differs between endometrial and ovarian cancers. The majority (72%, 18 of 25 mutations) of mutations in ovarian cancer involve codons 182 and 183 whereas the majority of mutations in EC (77%, 30 of 39) involve codons 179, 256, and 257. The significance of this tissue-specific difference is currently unclear but has been suggested to possibly reflect different underlying mechanisms of mutagenesis, or perhaps tissue-specific functional effects. Only a small number of mutations in PPP2R1A have been described in EECs, but it is noteworthy that they were more frequent in codons 182/183 than in codons 256/257. – The mechanism whereby PPP2R1A mutations contribute to tumorigenesis is currently unclear.

HER-2/ERBB2 Protein overexpression and genomic amplification of the HER-2/ERBB2 receptor tyrosine kinase are significantly more frequent among serous ECs than among EECs. – In serous EC, overexpression of HER-2/ERBB2 by IHC has been noted in 17%–80% of cases.,– HER-2/ ERBB2 amplification, determined by FISH, has been noted in 17%–68% of serous tumors that overexpress the protein, and in 17%–42% of serous tumors overall., A number of factors have been suggested to account for the inter-study variability in the frequency of HER-2/ERBB2 overexpression, including the small number of samples in some studies, differences in study populations, and variability in IHC, including inconsistencies in scoring HER-2/ERBB2 positivity. Several studies have observed correlations between HER-2/ERBB2 status and clinicopathological characteristics of serous ECs. The PI3K pathway Somatic alterations in the PI3K pathway are significantly less frequent in serous EC than EEC. Nonetheless, the combined frequency of PI3K pathway alterations in serous EC is appreciable (39%), resulting from mutations in PTEN (13%), PIK3CA (35%), and PIK3R1 (8%)., Compared to serous ECs, clear cell ECs do not show a statistically significant difference in the mutation frequency of PTEN (5%), PIK3CA (30%), and PIK3R1 (20%), although only a small number of clear cell tumors have been analyzed., Overall, 35% of clear cell ECs had a PI3K pathway mutation in one series. The spectrum of PIK3R1 mutations in NEECs differs somewhat from that of EECs.

Most PIK3R1 mutants found in NEECs are truncation mutations, which preferentially co-occur with PIK3CA mutations and are currently of unknown functional significance. This is in contrast to PIK3R1 mutations in EECs, which tend to be small in-frame deletions that are mutually exclusive with PIK3CA mutations and, in some cases, have an impaired ability to inhibit AKT activation. ARID1A (BAF250a) Loss of BAF250a expression has recently been reported in 18% of serous ECs and in 26% of clear cell ECs. The frequency of BAF250a loss is significantly lower in serous ECs than in high-grade endometrioid carcinomas ( P. CCNE (cyclin E) High levels of cyclin E, measured by IHC staining, have been reported in 51%–80% of poorly differentiated ECs, compared with 31%–45% of well- to moderately-differentiated ECs; in some studies, this difference attained statistical significance. – Cyclin E overexpression is also statistically significantly more frequent among NEECs than EECs (54.5% vs 27.5%; P = 0.035).

There are at least two underlying molecular mechanisms that account for high levels of cyclin E in EC. The first mechanism is amplification of CCNE, which is present in 16% of ECs overall, and in 30% of ECs that over express cyclin E. The second mechanism is loss-of-function mutations within the FBXW7/CDC4/hAGO tumor suppressor gene., FBXW7 encodes the substrate recognition component of an SCF-ubiquitin ligase complex that targets cyclin E for ubiquitin-mediated proteosomal degradation., Somatic mutations within FBXW7 have been reported at variable frequency among endometrial carcinomas. Two studies found FBXW7 mutations only rarely (3%) in endometrial carcinomas, though neither specified the histology of tumors analyzed for mutations., In contrast, Suehiro et al identified a high frequency of FBXW7 mutations in EECs (46.8%). Spruck et al reported a moderate frequency (16%) of FBXW7 mutations in endometrial tumors that had elevated levels of cyclin E or phosphorylated cyclin E, although the tumor histotype was not specified. Thus, the frequency of FBXW7 mutations in NEECs remains to be elucidated. CDKN2A (p16) The CDKN2A/p16 tumor suppressor is a negative regulator of G1/S cell cycle progression.

Recent studies on large tumor panels have revealed that serous ECs nearly uniformly show strong diffuse staining of p16, indicative of high expression. – This is in stark contrast to the weak focal staining of endometrioid tumors of all grades.

Though the prognostic significance and molecular basis for p16 overexpression has yet to be determined, it has been suggested that p16 expression may serve as a potent biomarker that might be useful in the molecular classification of ECs, particularly for high-grade tumors. – In addition, CDKN2A is mutated in 10%–28% of EECs compared with 44% of NEECs, although the latter observation is based on a small sample size. Claudins and other cellular adhesion proteins In 2005, Santin et al noted differential expression of numerous genes by microarray analysis between primary short-term cultures of serous ECs and normal endometrial cells, including several genes that regulate cell adhesion. Among these genes, claudins-3 and -4, which encode cell adhesion proteins present at tight junctions, were upregulated in serous EC. RT-PCR confirmed the upregulation of claudin-3 (8-fold) and claudin-4 (12-fold) in serous cultures compared with normal endometrial cell cultures. Immunohistochemistry for claudin-4 on corresponding primary tumor specimens, as well as a small number of additional serous tumors, revealed stronger staining for claudin-4 in serous EC compared with normal endometrial cells. In a subsequent study of a large number of endometrial tumors, Konecny et al showed that positive immunohistochemical staining for claudins-3 and -4 is significantly more frequent among serous (78% and 56%) and clear cell (61% and 44%) ECs than among EECs (38% and 9%).

Kanker Endometrium Pdf

In multivariate analyses, claudin expression was not a significant independent prognostic indicator. In contrast, Sobel et al failed to find an association between claudin-3 and -4 levels and histotype of EC in a small series of tumors. One possible reason for the variability between studies might be the differences in sample sizes. The observation that claudins-3 and -4 are upregulated in serous EC holds promise for the development of targeted therapy, since Clostridium perfringens enterotoxin (CPE) targets claudins-3 and -4 and causes cytolysis upon binding.

The original study that uncovered upregulation of claudins-3 and -4 in serous EC, compared with normal endometrium, also noted upregulation of several other genes that encode cell adhesion proteins, including L1CAM ( L1 cellular adhesion molecule), and EpCAM ( Epithelial Cell Adhesion Molecule). The observation of L1CAM upregulation was consistent with an earlier report that immunohistochemical expression of L1CAM was more frequent among serous ECs than EECs (75% vs 16%), although the number of serous tumors evaluated was small. The upregulation of EpCAM expression in serous EC has also been verified immunohistochemically; in one study, EpCAM staining was shown to be significantly higher in serous ECs than in normal endometrium. Serous EC cell lines that were positive for EpCAM were sensitive to MT201 (adecatumumab), a human monoclonal antibody against EpCAM, suggesting that high EpCAM levels may represent a druggable target for serous ECs. As discussed below, E-cadherin, another cell adhesion molecule, has a well-established role in serous EC. E-cadherin The CDH1 tumor suppressor gene encodes E-cadherin, a calcium-dependent cell adhesion molecule.

Loss of E-cadherin expression is a characteristic feature of the epithelial to mesenchymal transition. Negative or reduced expression of E-cadherin has been described in ECs, and is significantly more frequent among serous and clear cell endo-metrial tumors than among EECs 83% vs 53%; P = 0.002, 62% vs 5%; P.

Conclusion and future prospects In conclusion, our understanding of the genetic etiology of EECs and serous ECs has advanced considerably over the past 20 years, and reflects a large body of research on individual genes, gene families, and pathways, as reviewed herein. As for most cancers, the rate-limiting step in dissecting the genetic alterations that underlie EC has been the availability of sufficiently high-resolution genomic technologies. However, within the past 5 years, the development and implementation of so-called next generation sequencing has resulted in a massive paradigm shift in cancer genomics because it provides the tools to systematically interrogate cancer genomes, exomes, and transcriptomes, nucleotide by nucleotide, for somatic alterations in gene sequence, structure, and copy number.

The Cancer Genome Atlas is currently conducting large-scale, integrated genomic and epigenomic analyses of low-grade EECs, high-grade EECs, and serous ECs using massively parallel sequencing and other high resolution genomic and epigenomic approaches. The resulting catalogs of somatic alterations are eagerly awaited because they will reveal, for the first time, the most comprehensive view of the genomic, transcriptomic, and epigenomic landscape of ECs. This will provide a solid foundation for future studies to determine whether the altered genes are relevant to the biology and clinical management of women with EC.

1, 1, Maja Anko 1, Neli Hevir 1, Katja Vouk 1, Aleš Jerin 2, 3 and 1. 1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. 2Institute of Clinical Chemistry and Biochemistry, University Medical Centre, Ljubljana, Slovenia. 3Division of Obstetrics and Gynecology, Department of Pathology, University Medical Centre, Ljubljana, Slovenia Endometrial cancer (EC) is the most common estrogen-dependent gynecological malignancy in the developed World. To investigate the local formation of estradiol (E2), we first measured the concentrations of the steroid precursor androstenedione (A-dione) and the most potent estrogen, E2, and we evaluated the metabolism of A-dione, estrone-sulfate (E1-S), and estrone (E1) in cancerous and adjacent control endometrium. Furthermore, we studied expression of the key genes for estradiol formation via the aromatase and sulfatase pathways.

A-dione and E2 were detected in cancerous and adjacent control endometrium. In cancerous endometrium, A-dione was metabolized to testosterone, and no E2 was formed. Both, E1-S and E1 were metabolized to E2, with increased levels of E2 seen in cancerous tissue. There was no significant difference in expression of the key genes of the aromatase ( CYP19A1) and the sulfatase ( STS, HSD17B1, HSD17B2) pathways in cancerous endometrium compared to adjacent control tissue.

The mRNA levels of CYP19A1 and HSD17B1 were low, and HSD17B14, which promotes inactivation of E2, was significantly down-regulated in cancerous endometrium, especially in patients with lymphovascular invasion. At the protein level, there were no differences in the levels of STS and HSD17B2 between cancerous and adjacent control tissue by Western blotting, and immunohistochemistry revealed intense staining for STS and HSD17B2, and weak staining for SULT1E1 and HSD17B1 in cancerous tissue. Our data demonstrate that in cancerous endometrium, E2 is formed from E1-S via the sulfatase pathway, and not from A-dione via the aromatase pathway. Introduction Endometrial cancer (EC) is the fifth-most-common cancer in women in Western Europe and the USA, with the majority of cases arising after menopause (; ). EC can be classified into estrogen-dependent type I, which comprises 80% of all cases, and the poorly differentiated, more aggressive, type II.

Although, type II EC was considered to be estrogen independent (; ), experimental data suggest involvement of estrogens (; ). Local estrogen formation has an important role in the development of EC and increased estradiol (E2) concentrations have been detected in cancerous, as compared to normal endometrium.

Locally, E2 can be formed either via the so-called aromatase pathway from androstenedione (A-dione), which originates from dehydroepiandrosterone-sulfate (DHEA-S) and DHEA, or from testosterone (T), by the actions of aromatase and the reductive 17β-hydroxysteroid dehydrogenases (enzymes 17β-HSD, HSD17B; Figure ). These are NADPH dependent enzymes, which due to high intracellular concentration ratio NADPH/NADP + act preferentially as reductases in a cellular context. The most potent estrogen, E2 can also be formed from estrone-sulfate (E1-S) via the sulfatase pathway by the actions of sulfatase (STS) and the reductive enzymes HSD17B (Figure ). Estrogen biosynthesis. Formation of estrogens via the aromatase pathway from androstenedione and testosterone, by the actions of aromatase (CYP19A1), and the reductive 17β-hydroxysteroid dehydrogenases types 1, 7, and 12 (HSD17B1, HSD17B7, HSD17B12) and type 5 (AKR1C3). Formation of estrogens via the sulfatase pathway from estrone-sulfate, by the action of sulfatase (STS), and the reductive HSD17B1, HSD17B7, and HSD17B12.

The oxidative 17β-HSD types 2, 4, 8, and 14 (HSD17B2, HSD17B4, HSD17B8, HSD17B14) catalyze the inactivation of estradiol to estrone and sulfotransferase (SULT1E1) catalyzes conjugation of estrone. The aromatase pathway depends on availability of A-dione or T. A-dione, with 1–8 nM concentrations in blood, originates mainly from adrenal gland (zona reticularis), from ovaries in premenopausal women and also from conversions of DHEA-S and DHEA in peripheral tissues. Aromatase (CYP19A1) converts A-dione and T into estrone (E1) and E2, respectively. As the plasma concentrations of A-dione in postmenopausal women are 4-fold higher than those of T (; ), aromatase mainly converts A-dione to E1.

Currently, the data on aromatase expression in EC are controversial, with everything from high levels, to no significant differences between diseased and normal tissues, to no expression being reported (;;;;;;;;;; ). E1 formed from A-dione should be further activated by the reductive estrogenic 17β-HSD type 1 (HSD17B1), to form E2. Although, several groups have failed to detect expression of HSD17B1 in normal and cancerous endometrium, others have seen low mRNA levels in both tissues (;;;; ) with decreased mRNA levels in EC compared to adjacent control endometrial tissue (; ). In contrast showed increased mRNA levels of HSD17B1 in ERα positive grade 1 EC compared to control tissue. In addition to HSD17B1, three other reductive estrogenic 17β-HSDs, types 7 and 12 (HSD17B7 and HSD17B12), and type 5 (aldo-keto reductase 1C3, AKR1C3), that can form E2 from E1, albeit with lower catalytic efficiencies, can contribute to E2 formation.

Also the expression of these genes in EC is rather controversial as has been reviewed. There were no significant differences in mRNA levels of AKR1C3 (;; ), the expression of HSD17B7 was reported as decreased or unchanged (; ) and expression of HSD17B12 was unchanged (; ) or increased in EC compared to control tissue. The expression of the oxidative NAD + dependent estrogenic 17β-HSDs, types 2, 4, 8, and 14 (HSD17B2, HSD17B4, HSD17B8, and HSD17B14), can also affect local E2 concentrations. These enzymes catalyze inactivation of E2 to E1. Previous studies by Lepine et al., and our group revealed increased mRNA levels of HSD17B2 (; ) in EC, while Cornel et al. Found no significant difference in ERα positive grade 1 EC. For HSD17B4 and HSD17B8 we saw no changes in gene expression in EC compared to adjacent control tissue , while expression of HSD17B14 has not yet been studied in EC.

E2 can also be formed via the sulfatase pathway from E1-S by the actions of STS and the reductive enzymes HSD17B1, HSD17B7, HSD17B12, and also AKR1C3. Unchanged , and increased expression of STS have previously been reported in EC. Sulfotransferase SULT1E1 catalyzes conjugation of estrogens and our previous studies show that gene encoding this enzyme is not differentially expressed in EC as compared to adjacent control tissue , while Lepine et al., reported borderline increased mRNA levels in cancer tissue.

There is a great need for a better understanding of the local formation of E2 in cancerous endometrium, which may reveal novel targets for treatment of this most common gynecological malignancy. The aims of the present study were thus to investigate E2 formation in paired samples of EC and adjacent control endometrium at different levels. Our goals were: (i) to determine concentrations of steroid precursor A-dione and the most potent estrogen E2, (ii) to examine capacity for formation of A-dione, E1-S and E1; (iii) to re-examine the mRNA levels of individual genes involved in the aromatase pathway and the sulfatase pathway of E2 formation, (iv) to evaluate protein levels of the key players in the sulfatase pathway, STS, SULT1E1, HSD17B2, and HSD17B1 and their prognostic potential. Materials and Methods Endometrial Tissue The specimens of EC and paired adjacent control endometrium were obtained from 55 patients undergoing hysterectomies for histologically proven EC (Table, Supplementary Table 1). The study was approved by the National Medical Ethics Committee of the Republic of Slovenia with written informed consent required from all subjects involved. The patients were all treated in the Department of Gynecology and Obstetrics at the University Medical Centre Ljubljana, from 2003 to 2010.

The samples used for steroid concentration measurements, metabolism studies, qPCR, Western blotting and immunohistochemical staining have been selected chronologically. Steroid Concentration Measurements Ten paired samples of EC and adjacent control tissue were frozen in liquid nitrogen and ground to a fine powder. Prior to extraction, 100–200 mg of homogenate was suspended in 0.1 M sodium phosphate buffer (pH 7.4).

The extraction was performed three times with 4 mL of diethyl ether; the extracts were pooled and evaporated under a stream of nitrogen. Before analysis, the samples were resuspended in phosphate buffer. The levels of A-dione were measured using a double antibody radioimmunoassay with interassay CV. A-dione Is Metabolized to T in Cancerous and Adjacent Control Endometrium A-dione formed locally in EC or A-dione from circulation might serve as precursor for E2 formation. We thus examined the ability of EC tissue for aromatization. We studied the metabolism of 80 nM 3H labeled A-dione in seven paired samples of EC and adjacent control endometrium. In all of these samples, only conversion to T and much lower levels of 5α-androstanedione were detected by autoradiography after TLC in the first mobile phase (Figure ), while in the second mobile phase, 5α-dihydrotestosterone (5α-DHT) was also seen in two EC samples (Figure ).

Aromatase activity was observed only in the control tissue, human placenta, where A-dione was metabolized to T, E1, and E2 (Figures ). As the first experiment included relatively high concentration of A-dione, we also examined the metabolism of 8 nM 3H labeled A-dione in the presence of the NADPH regeneration system in three paired samples of EC and adjacent control endometrium. In this experiment the products were separated by HPLC, where T was the major metabolite in all paired samples (Figure ) with increased formation of T seen in EC (Figure ). This is in agreement with our previous study in nine EC samples where 10 nM 3H labeled A-dione was metabolized mainly to T with no E2 seen (Vouk and Rizner, Unpublished data; Supplementary Figure 1). A-dione metabolism in the cancer and adjacent control endometrium.

(A) Identification of the reaction products by autoradiography (TLC developed in cyclohexane/ ethyl acetate 1:1, v/v) showing conversion of 3H labeled A-dione (80 nM) to testosterone (T) and 5α-androstanedione (5α-A) in cancer, T, and corresponding control, C, tissues. In placenta, P, A-dione was metabolized to T, E1, and E2.

(B) Autoradiography of the same samples that were extracted from silica gel and developed in cyclohexane/ ethanol (95:5, v/v), confirming the presence of 5α-dihydrotestosteone (5α-DHT) in two EC samples. (C) HPLC profiles demonstrates metabolism of 3H labeled A-dione (8 nM) to T in control tissue and matched tumor tissue. (D) Formation of T (fmol/mg powdered tissue). E1-S Is Metabolized to E2 in Cancerous and Adjacent Control Endometrium with Increased Formation of E2 Seen in Cancer Tissue Since no E2 were formed by metabolism of A-dione in EC specimens, we next studied the ability of this tissue for metabolism of the major circulating estrogen, E1-S. This study was performed in five paired samples of EC and adjacent control tissue. Sixteen nM E1-S was metabolized to E1 and E2 in both, EC and adjacent control tissue, with significantly higher levels of E2 formed in cancerous tissue ( p = 0.0085; Figures ).

In addition to E2 and E1 also several unidentified polar metabolites were formed (Figure ). The metabolism of 10 nM E1 was further examined in 12 paired samples of EC and adjacent control endometrium. The formation of E2 was detected in all of the samples (Figures ); with a 2-fold, but non-significantly increased median levels seen in cancerous tissue ( p = 0.151; Figure ). In seven samples of EC there was an increased E2 formation, in one sample there was no difference and in four samples of EC originating from one patient with serous EC and three patients with well-differentiated EC (G1) there was a decreased E2 formation as compared to adjacent control endometrial tissue (Figure ).

E1-S metabolism in the EC and adjacent control endometrium. (A) Table for the conversion of E1-S to E1 and E2 (fmol/mg powdered tissue) in control and corresponding EC. (B) Histogram with logarithmic scale demonstrates increased tumor/ control tissue ratio for E2 formation in all paired samples. (C) HPLC elution profiles shows the formation of E2 and E1 from 3H labeled E1-S (16 nM) for the representative samples of control (C64) and corresponding cancer (T64) tissues.

Rf of E1-S and formation of several unidentified polar metabolites is also shown. (D) Before-and-after graph demonstrates levels of E2 formed in control endometrium (Control) and the corresponding EC (Tumor) for samples 61, 63, 64, 65, and 66. Genes for Local Formation of E2 Via the Aromatase and Sulfatase Pathways are Expressed in Cancerous Endometrium Genes that are involved in local E2 formation from A-dione or E1S (Figure ) are expressed in cancerous and adjacent control tissue. In the same cohort of EC patients, we previously reported no statistically significant differences in the expression of the majority of the genes involved in E2 formation: CYP19A1, STS, HSD17B4, HSD17B8, HSD17B12, and SULT1E1; however, HSD17B1, and HSD17B7, which promote E1 activation to E2, were down-regulated, and HSD17B2, which has the opposite role, was upregulated in the EC samples (; ). In the present study, we re-examined the mRNA levels of CYP19A1, STS, and HSD17B1 on a larger cohort of samples and found very low and unaltered mRNA levels of CYP19A1 and HSD17B1. The mRNA levels of STS were about 1,000-fold higher than the levels of HSD17B1, but still unchanged in the EC and control tissues (Figure ). With an arbitrary threshold of 1.2 for the ratio of mRNA levels in pairs of EC and adjacent control tissue we saw increased ratios in 8 out of 22 pairs for CYP19A1, 10 out of 27 pairs for STS and 9 out of 27 pairs for HSD17B1.

We also investigated the mRNA levels of the oxidative HSD17B14, which has not yet been studied in EC and found high, but statistically significantly decreased levels ( p. Further, stratification according to clinical data (menopausal status and vital status of the patients' and FIGO stage) and histopathological data (histological type and grade of the tumor, depth of myometrial invasion, and presence of lymphovascular invasion) revealed differences in STS, SULT1E1, and HSD17B14 expression (Table ). STS was significantly downregulated ( p = 0.0439) only in high grade tumors (G3) while in lower grade tumors (G1 and G2) STS levels did not differ between cancer and adjacent control tissue. The expression of SULT1E1 was significantly downregulated ( p = 0.0392) in cancer tissue from premenopausal women, with significantly lower levels seen in cancer and adjacent control tissue from postmenopausal women as compared to premenopausal women. The expression of HSD17B14 changed in more invasive cancers, there was an extensive downregulation in cancer compared to adjacent control tissue form patients with lymphovascular invasion ( p = 0.0298). High Protein Levels of STS and HSD17B2 Are Seen in Cancerous and Adjacent Control Endometrium As mRNA levels do not necessary correlate with protein levels and enzymatic activity, we also examined protein levels of STS, SULT1E1, HSD17B1, and HSD17B2. With the specific antibodies we performed Western blot analysis to evaluate protein levels of these enzymes in paired samples (Figure ).

We found high protein levels of STS, with increased levels in 12 EC samples out of 24, where this difference was not statistically significant (Figures ). SULT1E1 protein levels were very low in all but one tumor sample (T53, Figure ) thus it was not possible to accurately estimate differences in protein levels between control compared to EC tissue. We were not able to detect HSD17B1 protein in EC tissues using two different antibodies (rabbit monoclonal antibody, EP1682Y, Abcam UK; and rabbit polyclonal antibodies from Solvay Pharmaceuticals; data not shown) although these antibodies recognized HSD17B1 in placenta tissue and in homogenates of E. Coli overexpressing HSD17B1. Protein levels of HSD17B2 were seen in the majority of samples, with increased levels in 7 out of 17 pairs, but with no statistically significant difference between EC and adjacent control tissue (Figures ). The stratification according to clinical and histopathological data confirmed the effects of tumor differentiation on STS expression (Table ). The significantly lower STS protein ( p = 0.0039) levels in EC as compared to adjacent control tissue were seen only in high grade tumors (G3), while in well-differentiated tumors (G1, G2) there were no differences in STS levels between EC and control tissue (Table ).

However, this trend was not supported by further immunohistochemical staining. Protein levels of STS, SULT1E1, and HSD17B2 in the EC and adjacent control tissue. (A) Before-and-after graph shows STS protein levels in 24 paired samples of control endometrium (Control) and corresponding cancer tissue (Tumor).

The data was quantified and normalized to GAPDH levels. (B) Histogram with logarithmic scale demonstrates higher STS levels in cancer endometrium in 12 paired samples out of 24. (C) Representative membrane with STS and GAPDH staining. (D) Representative membrane with SULT1E1and GAPDH staining. (E) Before-and-after graph shows HSD17B2 protein levels in 17 paired samples of control endometrium (Control) and corresponding cancer tissue (Tumor). The data was quantified and normalized to GAPDH levels. (F) Histogram with logarithmic scale demonstrates protein ratio between paired samples of tumor and control tissue.

(G) Representative membrane with HSD17B2 and GAPDH staining. C, control endometrium; T, EC tissue; HIEEC, control epithelial cell line of normal endometrium; HEC1A, endometrial cancer cell line; HepG2, liver cancer cell line. With the same set of antibodies as previously used for Western blotting we performed immunohistochemical staining of tissue microarrays, which included 44 pairs of cancer and adjacent control tissue.

We observed staining for STS, HSD17B2, HSD17B1, and SULT1E1 in EC and adjacent control tissue (Figures ). STS staining indicated clear cytoplasmic reaction with several samples showing distinct luminal accumulation of this protein. Scoring and further statistical analysis revealed overall significantly lower levels of STS ( p = 0.0219) in cancer compared to adjacent control tissue but with unchanged levels in 12 pairs, increased levels in 10 pairs and decreased levels in 22 out of 44 pairs (Figures, Supplementary Table 4). HSD17B2 showed granulated cytoplasmic reaction in all samples.

Protein levels of HSD17B2 were in general significantly increased ( p = 0.0236) in cancer as compared to adjacent control tissue with unchanged levels in 5 pairs, decreased levels in 11 pairs, and increased levels in 24 out of 40 pairs (Figures, Supplementary Table 4). Staining for HSD17B1 with Abcam EP1682Y antibodies was weak but indicated distinct and clear cytoplasmic reaction with clearly negative control staining and intense staining in placenta tissue, which served as a positive control (Figure ). Weak staining for HSD17B1 was seen in 38 control and 36 cancer samples out of 42 pairs investigated. With Solvay antibodies against HSD17B1 moderate staining was seen in control and cancer tissue in epithelial and stromal cells, with cytoplasmic but also some positive nuclear staining, and intense staining in placenta tissue (data not shown). Staining for SULT1E1 in EC and adjacent control endometrial tissue samples was cytoplasmic with no significant difference between cancer and the adjacent control tissue, but with decreased levels in 16 pairs, increased levels in six pairs and no staining in 8 out of 31 pairs (Figures; Supplementary Table 4). The same antibody intensively stained small intestine and duodenum tissue, which served as positive controls and weakly lung tissue, which was a negative control (data not shown). Stratification of the experimental data according to clinical and histopathological characteristics of patients revealed no effects on STS, HSD17B2, and SULT1E1 levels (Table ).

Immunohistochemical staining for STS, HSD17B2, HSD17B1, and SULT1E1 in endometrial cancer. (A) Before-and-after graphs show scoring for STS, HSD17B2, and SULT1E1 in control endometrium (Control) and the corresponding cancer endometrium (Tumor). (B) Table with number of samples with increased, decreased or unchanged levels of protein in tumor tissue compared to adjacent control endometrium. (C) representative staining of adjacent control endometrium (C) and cancerous tissue (T) for STS, HSD17B2, HSD17B1, and SULT1E1 (samples 54 and 62), and placenta (P) as a positive control. Samples were stained with hematoxylin and eosin (HE staining) and with specific antibodies against STS, HSD17B2, HSD17B1, and SULT1E1. 400 × magnifications are shown.