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BMC Genomics logoLink to BMC Genomics
. 2009 Dec 16;10:613. doi: 10.1186/1471-2164-10-613

Increased epithelial stem cell traits in advanced endometrial endometrioid carcinoma

Shing-Jyh Chang 3,4, Tao-Yeuan Wang 5,6, Chan-Yen Tsai 1, Tzu-Fang Hu 1, Margaret Dah-Tsyr Chang 4, Hsei-Wei Wang 1,2,7,
PMCID: PMC2810306  PMID: 20015385

Abstract

Background

It has been recognized cancer cells acquire characters reminiscent of those of normal stem cells, and the degree of stem cell gene expression correlates with patient prognosis. Lgr5(+) or CD133(+) epithelial stem cells (EpiSCs) have recently been identified and these cells are susceptible to neoplastic transformation. It is unclear, however, whether genes enriched in EpiSCs also contribute in tumor malignancy. Endometrial endometrioid carcinoma (EEC) is a dominant type of the endometrial cancers and is still among the most common female cancers. Clinically endometrial carcinoma is classified into 4 FIGO stages by the degree of tumor invasion and metastasis, and the survival rate is low in patients with higher stages of tumors. Identifying genes shared between advanced tumors and stem cells will not only unmask the mechanisms of tumor malignancy but also provide novel therapeutic targets.

Results

To identify EpiSC genes in late (stages III-IV) EECs, a molecular signature distinguishing early (stages I-II) and late EECs was first identified to delineate late EECs at the genomics level. ERBB2 and CCR1 were genes activated in late EECs, while APBA2 (MINT2) and CDK inhibitor p16 tumor suppressors in early EECs. MAPK pathway was significantly up in late EECs, indicating drugs targeting this canonical pathway might be useful for treating advanced EECs. A six-gene mini-signature was further identified to differentiate early from advanced EECs in both the training and testing datasets. Advanced, invasive EECs possessed a clear EpiSC gene expression pattern, explaining partly why these tumors are more malignant.

Conclusions

Our work provides new insights into the pathogenesis of EECs and reveals a previously unknown link between adult stem cells and the histopathological traits of EECs. Shared EpiSC genes in late EECs may contribute to the stem cell-like phenotypes shown by advanced tumors and hold the potential of being candidate therapeutic targets and novel prognosis biomarkers.

Background

Tumor development, progression, and prognosis remain at the front position of medical research. Two hypotheses of the origin of cancer have existed for many decades. One hypothesis postulates that adult stem or precursor cell is the cell of origin for cancer, whereas the other declares a somatic cell can be mutated and then be dedifferentiated or be reprogrammed to regain properties associated with both cancer cells and stem cells [1-3]. The discovery of a subpopulation of tumor stem cells (TSCs) in leukemia and solid cancers has strengthened the stem cell hypothesis [4]. Glioblastomas also possess characters and gene expression patterns of local neural stem cells (NSCs) [5], and artificially introducing cancer-associated mutations into stem or lineage-restricted precursor cells can indeed turn them into cancer initiating cells and all mice received mutations developed medulloblastomas [6,7]. Another example that the adult stem cell represents the cell of origin of cancer has recently been made in chronic myeloid leukemia (CML): by restricting BCR-ABLp210 expression to mouse Sca1(+) hematopoietic stem cells, it is sufficient to induce CML formation that recapitulates the human disease [8]. These evidences support the idea that mutations of stem cells may initiate the carcinogenic process of certain, although not necessary all, tumors.

On the other hand, the importance of somatic or tumor cell mutation and dedifferentiation has not been excluded completely. It has been recognized that during malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Human tumor cells can be created from healthy somatic cells with defined genetic elements [9]. Even though cancers were originated from mutated stem cells, newly acquired mutations in tumors still contribute in cell malignancy and therapy resistance. It has been recognized that cancer cells acquire characters reminiscent of those of normal stem cells. Clinically cancer cells with poor differentiated pathological grading usually have worse therapy response than those with well differentiated morphology. The degree of embryonic gene re-expression correlates with pivotal tumor features and patient prognosis [10,11]. It is known that colon cancers adopt a broad program encompassing embryonic colon development [12]. In poorly differentiated breast cancer, gliomas and bladder carcinoma, an embryonic stem cell (ESC)-like gene expression signature is exhibited and the degree of ESC program recapitulation correlates with tumor stages and patient survival [13]. Recent studies demonstrated that Snail, a potent oncogene which can induce epithelial-mesenchymal transition (EMT), contributes to the acquisition of stem cell traits in breast cancer cells [14,15]. Pre-existing cancerous lesions may become more malignant by the accumulation of new oncogenic mutations (such as Snail) that can induce cell dedifferentiation. Identifying genes shared between transformed cells, especially the more malignant ones, and stem cells will help to unmask the pathogenesis of tumors, as well as provide us with novel therapeutic targets and prognosis biomarkers.

Endometrial carcinoma of the female genital tract can be divided into two forms: endometrial endometrioid carcinoma (EEC; Type I) which account for 70-80% of cases and are estrogen-related; whereas the Type II tumors (papillary serous or clear cell tumors) account for 20% of cases unrelated to estrogen stimulation [16]. Clinically endometrial carcinoma is classified into 4 FIGO stages by the degree of invasion and metastasis: stage I tumors are limit to the uterine body and stage II tumors extend to the uterine cervix. Both stages are considered as less invasive, although stage IIB cases are characterized by a less favorable prognosis. In contrast, tumors of stages III-IV are invasive: for stage III there is regional tumor spread and for stage IV there is bulky pelvic disease or distant spread [17]. Approximately 72% of endometrial carcinomas are stage I, 12% are stage II, 13% are stage III, and 3% are stage IV [17]. The survival rate is also low in patients with higher stages of tumors: 80-90% in stage I, 70-80% in stage II, 40-60% in stage III, and 20% in stage IV [17]. Identifying genes abundant in late EECs can not only unmask the mechanisms of tumor malignancy but also provide us with novel therapeutic targets. Recently Lgr5- or CD133-positive crypt stem cells of the intestinal track were identified and these cells were proven to be one of the original cells of intestinal cancer [18,19]. OLFM4 is also a new, robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells [20]. Disruption of beta-catenin in cells positive for CD133 resulted in a gross disruption of crypt architecture and a disproportionate expansion of CD133(+) cells at the crypt base [19]. It is unclear, however, whether genes high expressed in epithelial stem cells (EpiSCs) also contribute in tumor invasiveness, malignancy and therapy resistance. A broad description of stem cell traits reminiscent in EECs is therefore crucial.

In this study we dealt with the molecular bases of endometrial cancer and assessed the expression of epithelial precursor genes in advanced EEC. To examine the shared genes between EpiSC and late EECs, we first need to unmask the gene compositions in different stages of EECs. For this purpose we applied gene expression microarray and machine learning algorithms to filtrate genes differentially expressed in early (stages I-II) and late (stages III-IV) EECs. After obtaining genes unique in EECs of different stages, we then related transcriptional programs in EpiSCs and late EECs. This approach helped to discover a total of 217 probe sets differentiating EECs of different stages, and, moreover, showed late EECs possess a clear EpiSC gene expression pattern, partly explaining why these tumors are more malignant and fatal.

Results

Molecular signatures of early and late stage EECs

To identify epithelial stem cell genes in late EECs, we first delineated early (FIGO stages I and II) and late (FIGO stages III and IV) EECs at the genomics level. We explored genes differentially expressed between early and late EEC tissues using the Affymetrix U133 Plus 2.0 array. The demographics of patients in the training and testing cohorts are in Tables 1 and 2, respectively. Tumor samples were compared to each other to minimize stromal and myometrial contamination as well as female-specific genes. A multidimensional scaling (MDS) plot using the whole transcriptome showed that the mRNA profiles of normal and cancerous tissues are different (Figure 1A). We then searched for genes distinguishing early and late EECs according to a statistical pipeline we used [21,22]. A total of 678 probe sets could differentiate early and late stage samples, as well as discriminate 23 normal endometrium and 33 tumor tissues (Figure 1B; the positive false discovery rate (pFDR) cutoff q values are shown).

Table 1.

Characteristics of 34 EEC patients used in the training cohort.

GSE No. TNM FIGO stage Histology FIGO grade Patient Age Ethnic Background
(Isolation site: Endometrium)
GSM117600 T1aN0M0 1A Adenocarcinoma 1 60-70 Asian
GSM152644 T1bN0M0 1B Endometrioid 2 60-70 Caucasian
GSM152660 T1bN0M0 1B Endometrioid 2 40-50 Caucasian
GSM137960 T1bN0M0 1B Endometrioid 2 60-70 Caucasian
GSM137968 T1bN0M0 1B Endometrioid 2 60-70 Caucasian
GSM137980 T1bN0M0 1B Endometrioid 3 40-50 Caucasian
GSM117586 T1bN0M0 1B Endometrioid 2 50-60 African-American
GSM117643 T1bN0M0 1B Endometrioid 1 70-80 Caucasian
GSM117667 T1bN0M0 1B Endometrioid 2 60-70 Caucasian
GSM117703 T1bN0M0 1B Endometrioid 2 50-60 Caucasian
GSM117704 T1bN0M0 1B Endometrioid 2 50-60 Caucasian
GSM117722 T1bN0M0 1B Endometrioid 2 70-80 Caucasian
GSM117724 T1bN0M0 1B Endometrioid 2 60-70 Caucasian
GSM117739 T1bN0M0 1B Endometrioid 3 60-70 Caucasian
GSM89034 T1bN0 Endometrioid 2 40-50 Caucasian
GSM89089 T1bN0M0 1B Endometrioid 1 70-80 Caucasian
GSM76499 T1bN0M0 1B Endometrioid 2 70-80 Caucasian
GSM76638 T1bN0M0 1B Endometrioid 2 60-70 Caucasian
GSM117697 T1cN0M0 1C Endometrioid 3 60-70 Caucasian
GSM89076 T1cN0M0 1C Endometrioid 3 70-80 African Indian
GSM76507 T1cN0M0 1C Endometrioid 2 60-70 Caucasian
GSM137955 T2aN0M0 2A Endometrioid 2 60-70 African-American
GSM102425 T2bN0M0 2B Endometrioid 2 50-60 Caucasian
GSM102444 T2bN0M0 2B Endometrioid 1 60-70 Caucasian
GSM46912 T2bN0M0 2B Endometrioid 1 60-70 Caucasian
GSM117708 T3aN0M0 3A Endometrioid 3 70-80 Caucasian
GSM117712 T3aN0M0 3A Endometrioid 2 60-70 Caucasian
GSM38067 T3aN0M0 3A Endometrioid 3 60-70 Caucasian
GSM38084 T4NXM0 4A Endometrioid 3 60-70 Caucasian
GSM89087 T3aNXM1 (*) 4B Endometrioid 3 80-90 Caucasian
GSM46867 T3aN1M1 (**) 4B Endometrioid 3 60-70 Caucasian
(Isolation site: outside endometrium)
GSM89079 T3aNXM1($) 4B Endometrioid 3 40-50 Caucasian
GSM203686 T3aN0M0 ($) 3A Endometrioid 2 60-70 Caucasian
GSM46932 TXNXM1(@) 4B Endometrioid 2 50-60 Caucasian

Endometrioid: Endometrioid carcinoma

*: Hepatic metastasis

**: Lymph node metastasis

$: Isolated from ovary

@: Isolated from abdominal wall fascia

Table 2.

Characteristics of another 15 early EEC patients used in the testing set.

GSE No. TNM FIGO stage Histology FIGO grade Patient Age Ethnic Background
GSM88952 T1aN0M0 1A Endometrioid 2 50-60 Caucasian
GSM76487 T1bN0M0 1B Endometrioid 1 30-40 Caucasian
GSM102469 T1bN0M0 1B Endometrioid 1 60-70 Caucasian
GSM117579 T1bN0MX 1B Endometrioid 1 80-90 African-American
GSM117589 T1bN0MX 1B Endometrioid 1 80-90 Caucasian
GSM117767 T1bN0MX 1B Endometrioid 2 60-70 Caucasian
GSM137961 T1bN0MX 1B Endometrioid 2 60-70 Caucasian
GSM117590 T1cN0MX 1C Endometrioid 2 70-80 Caucasian
GSM117729 T2aN0M0 2A Endometrioid 1 50-60 Caucasian
GSM53176 T2aN0MX 2A Endometrioid 1 60-70 Caucasian
GSM117582 T2aN0MX 2A Endometrioid 2 40-50 Caucasian
GSM88966 T2aN1M0 2A Endometrioid 2 80-90 Caucasian
GSM53174 T2bN0MX 2B Adenosarcoma 2 60-70 Caucasian
GSM76525 T1aN0M0 1A Endometrioid (Mix) 3 80-90 Caucasian
GSM76632 T1bN0M0 1B Adenocarcinoma 2 50-60 Caucasian

Mix: Mixed endometrioid and serous adenocarcinoma

Figure 1.

Figure 1

Identification of genes in different EECs. (A) A multidimensional scaling (MDS) plot drawn by all probe sets (~54600 ones) on the chip. Normal endometrium (Normal) and EECs of all 4 stages are included. Each spot represents an array. (B) A Venn diagram summarizing genes differentially expressed between normal and tumor tissues or between early (Stages 1 & 2) and late (Stages 3 & 4) EEC samples in the training cohort. (C) Narrowing down the existing gene signature using a machine learning strategy. When probe sets were ranked by signal-to-noise ratios (weights), the top 217 features was the largest panel to give the lowest error rate (i.e., a best classification effect; upper panel). (D) The discrimination ability of the 217-probeset signature. A prediction strength plot [25] shows the prediction strengths of the identified 217 probe sets in discriminating early from late EECs in the training cohort. Samples 1B and 2B denote 2 early EECs (Stages 1B and 2B, respectively) which express late EEC gene signatures. (E) A MDS plots using the above 217 probe sets. 2 misgrouped early EECs are indicated. (F) Signature evaluation by an independent testing data set. One Stage 1B case, which expresses late EEC gene signatures, is grouped into the late EEC area (separated by a red line).

The discrimination ability of these 678 probe sets were evaluated by a supervised machine learning strategy, which combines the weighted voting algorithm and leave-one-out cross validation (LOOCV) [23-25]. An error rate of 12.1% (2 out of 24 early cancers and 2 out of 9 late samples; P < 0.001 by permutation test) was found (Figure 1C and Additional file 1). However, we found the top 217 features (ranked by the weighted value of each probe set [25]) is the largest panel to have better discrimination ability than that of the 678-probeset signature (error rate 6.1% vs. 12.1%; Figure 1C, upper panel): 2 out of 24 early EEC tissues are classified into the late group while all 9 late ones are correct (Figure 1D). MDS analysis supports the superior classification power of these 217 probe sets: only 2 early samples express late EECs gene signatures and are grouped together with the late cases (Figure 1E). When applying these 217 probe sets on another independent testing data set containing 15 early EEC cases, 1 out of 15 early tissues (error rate 6.7%; P < 0.001 by permutation test) is misgrouped (Figure 1F).

In-depth exploration of EEC-related genes

To have a better idea how the filtrated genes distribute in early and late EECs, a gene expression heat map for those 217 probe sets was drawn (Figure 2). This heat map showed the unique gene expression patterns between early or late EEC tumor tissues. Consistent with the classification data obtained by prediction strength (PS) analysis in Figure 1D, hierarchical clustering showed that only 2 early cases in the training data set are misclassified (indicated by arrows; Figure 2).

Figure 2.

Figure 2

Molecular fingerprint of EEC subtypes. A heat map shows the 217 probes sets differentiating early and late EECs in the training data set, as well as discriminating normal endometrium and tumor tissues. Columns represent tumor samples; rows represent probe sets. In red, increased; in blue, decreased. Arrows indicates two early EECs which express a late EEC gene signature (black, Stage 1B; red, Stage 2B).

Those 217 probe sets correspond to 177 known genes (with gene symbols) and 29 cDNAs, which have no gene symbols been assigned yet (all in Additional file 2). Among them 58 genes/cDNAs are predominantly up in early ECCs while 25 being down (Figure 2). In contrast, 48 genes/cDNAs are particularly high in late EECs while another 75 being low (Figure 2). The details of known genes (especially those with known function) are in Tables 3, 4, 5, 6 and 7 respectively. Many of these genes, such as CD163 [26], MSR1 (CD204) [27], ERBB2 oncogene (also known as HER-2/neu) [28,29], CSTA (stefin A) [30] and CCR1 [31], have been associated with tumor malignancy and poor patient outcomes in EEC or other cancers (Table 3, bold). CD163 and MSR1 (macrophage scavenger receptor 1; CD204) are markers for M2 macrophages, whose infiltration in tumor lesions is correlated with the histological grade of the gliomas [27] (Table 3, bold). These consistent findings support the reliability of our gene lists. We also validated our array data by performing immunohistochemical staining on Taiwanese EEC cases. ERBB2 was indeed more abundant in stages III and IV EEC tissues (Figure 3).

Table 3.

Up-regulated known genes in late stage EECs.

Probe Set ID UniGene ID Gene Title Gene Symbol Chromosomal Location
213532_at Hs.404914 ADAM metallopeptidase domain 17 ADAM17 chr2p25
223660_at Hs.281342 adenosine A3 receptor ADORA3 chr1p13.2
200966_x_at Hs.513490 aldolase A, fructose-bisphosphate ALDOA chr16q22-q24
205568_at Hs.104624 aquaporin 9 AQP9 chr15q22.1-22.2
224376_s_at Hs.584985 chromosome 20 open reading frame 24 C20orf24 chr20q11.23
224972_at Hs.472564 chromosome 20 open reading frame 52 C20orf52 chr20q11.22
200625_s_at Hs.370581 CAP, adenylate cyclase-associated protein 1 CAP1 chr1p34.2
201850_at Hs.516155 capping protein (actin filament), gelsolin-like CAPG chr2p11.2
205098_at Hs.301921 chemokine (C-C motif) receptor 1 CCR1 chr3p21
203645_s_at Hs.504641 CD163 molecule CD163 chr12p13.3
209396_s_at Hs.382202 chitinase 3-like 1 (cartilage glycoprotein-39) CHI3L1 chr1q32.1
204971_at Hs.518198 cystatin A (stefin A) CSTA chr3q21
202190_at Hs.172865 cleavage stimulation factor, subunit 1, 50kDa CSTF1 chr20q13.31
1554863_s_at Hs.473133 docking protein 5 DOK5 chr20q13.2
224336_s_at Hs.536535 dual specificity phosphatase 16 DUSP16 chr12p13
218282_at Hs.632276 ER degradation enhancer mannosidase a-like 2 EDEM2 chr20q11.22
216836_s_at Hs.446352 v-erb-b2 erythroblastic leukemia viral oncogene ERBB2 (HER2) chr17q11.2-q12
203561_at Hs.78864 IgG Fc fragment, IIa, receptor (CD32) FCGR2A chr1q23
210889_s_at Hs.352642 IgG Fc fragment, IIb, receptor (CD32) FCGR2B chr1q23
210992_x_at Hs.78864 IgG Fc fragment, IIc, receptor (CD32) FCGR2C chr1q23.3
204007_at Hs.176663 IgG Fc fragment, IIIb, receptor (CD16b) FCGR3B chr1q23
217782_s_at Hs.268530 G protein pathway suppressor 1 GPS1 chr17q25.3
212355_at Hs.558466 KIAA0323 KIAA0323 chr14q12
203364_s_at Hs.410092 KIAA0652 KIAA0652 chr11p11.2
230252_at Hs.155538 lysophosphatidic acid receptor 5 LPAR5 chr12p13.31
228360_at Hs.357567 hypothetical protein LOC130576 LOC130576 chr2q23.2
226710_at Hs.105685 similar to RIKEN cDNA C030006K11 gene MGC70857 chr8q24
224324_at Hs.131072 maestro MRO chr18q21
226241_s_at Hs.355935 mitochondrial ribosomal protein L52 MRPL52 chr14q11.2
214770_at Hs.632045 macrophage scavenger receptor 1 MSR1 (CD204) chr8p22
205460_at Hs.156832 neuronal PAS domain protein 2 NPAS2 chr2q11.2
209222_s_at Hs.473254 oxysterol binding protein-like 2 OSBPL2 chr20q13.3
210907_s_at Hs.478150 programmed cell death 10 PDCD10 chr3q26.1
238693_at Hs.529592 Polyhomeotic like 3 (Drosophila) PHC3 chr3q26.2
203691_at Hs.112341 peptidase inhibitor 3, skin-derived (SKALP) PI3 chr20q12-q13
226577_at Hs.593811 Presenilin 1 (Alzheimer diseas 3) PSEN1 chr14q24.3
217811_at Hs.369052 selenoprotein T SELT chr3q25.1
222523_at Hs.401388 SUMO1/sentrin/SMT3 specific peptidase 2 SENP2 chr3q27.2
227518_at Hs.585896 solute carrier family 35, member E1 SLC35E1 chr19p13.11
1552671_a_at Hs.496057 solute carrier family 9 (Na/H exchanger), 7 SLC9A7 chrXp11.3-11.23
222410_s_at Hs.583855 sorting nexin 6 SNX6 chr14q13.2
203114_at Hs.25723 Sjogren's syndrome/scleroderma autoantigen 1 SSSCA1 chr11q13.1
223478_at Hs.530373 translocase of inner mitochondrial 8 homolog B TIMM8B chr11q23.1-q23.2
212769_at Hs.287362 transducin-like enhancer of split 3 TLE3 chr15q22
204787_at Hs.8904 V-set and immunoglobulin domain containing 4 VSIG4 chrXq12-q13.3
221247_s_at Hs.900069 Williams-Beuren syndrome region 16 WBSCR16 chr7q11.23
202939_at Hs.591501 zinc metallopeptidase (STE24 homolog, yeast) ZMPSTE24 chr1p34
219050_s_at Hs.121025 zinc finger, HIT type 2 ZNHIT2 chr11q13

Table 4.

Down-regulated known genes in late stage EECs.

Probe Set ID UniGene ID Gene Title Gene Symbol Chromosomal Location
211224_s_at Hs.158316 ATP-binding cassette, sub-family B, 11 ABCB11 chr2q24
232948_at Hs.444414 AF4/FMR2 family, member 3 AFF3 chr2q11.2-q12
207133_x_at Hs.99691 alpha-kinase 1 ALPK1 chr4q25
1562271_x_at Hs.508738 Rho guanine nucleotide exchange factor 7 ARHGEF7 chr13q34
243899_at Hs.579108 ADP-ribosylation factor-like 17 pseudogene 1 ARL17P1 chr17q21.32
211076_x_at Hs.143766 Atrophin 1 ATN1 chr12p13.31
214256_at Hs.128041 ATPase, Class V, type 10A ATP10A chr15q11.2
237716_at Hs.434253 Chromosome 9 open reading frame 3 C9orf3 chr9q22.32
233844_at Hs.522805 CD99 molecule-like 2 CD99L2 chrXq28
243640_x_at Hs.127411 CDC14 cell division cycle 14 homolog A CDC14A chr1p21
233630_at Hs.472027 CDP-diacylglycerol synthase 2 CDS2 chr20p13
210701_at Hs.461361 craniofacial development protein 1 CFDP1 chr16q22.2-q22.3
238863_x_at Hs.130849 Component of oligomeric golgi complex 8 COG8 chr16q22.1
215377_at Hs.501345 C-terminal binding protein 2 CTBP2 chr10q26.13
1561616_a_at Hs.591570 dynein, axonemal, heavy polypeptide 6 DNAH6 chr2p11.2
1560042_at Hs.591566 family with sequence similarity 82, A FAM82A chr2p22.2
243588_at Hs.403917 FERM, RhoGEF & pleckstrin domain protein 1 FARP1 chr13q32.2
243876_at Hs.189409 Formin binding protein 1 FNBP1 chr9q34
1560094_at Hs.155090 Guanine nucleotide binding protein, β 5 GNB5 chr15q21.2
210855_at Hs.467733 GREB1 protein GREB1 chr2p25.1
1557289_s_at Hs.334930 GTF2I repeat domain containing 2 GTF2IRD2 chr7q11.23
232889_at Hs.620129 glucuronidase, beta pseudogene 1 GUSBP1 chr5q13.2
1555685_at Hs.463511 Hexose-6-phosphate dehydrogenase H6PD chr1p36
240482_at Hs.519632 Histone deacetylase 3 HDAC3 chr5q31
1559600_at Hs.632767 Hypermethylated in cancer 2 HIC2 chr22q11.21
1557329_at Hs.371350 Holocarboxylase synthetase HLCS chr21q22.1
1553111_a_at Hs.534040 kelch repeat and BTB domain containing 6 KBTBD6 chr13q14.11
231875_at Hs.374201 kinesin family member 21A KIF21A chr12q12
232814_x_at Hs.20107 Kinesin 2 KNS2 chr14q32.3
242112_at Hs.631954 LSM11, U7 small nuclear RNA associated LSM11 chr5q33.3
232418_at Hs.30824 leucine zipper transcription factor-like 1 LZTFL1 chr3p21.3
1560033_at Hs.167531 Methylcrotonoyl-Coenzyme A carboxylase 2 MCCC2 chr5q12-q13
216783_at Hs.187866 Neuroplastin NPTN chr15q22
217802_s_at Hs.632458 nuclear casein kinase and CDK substrate 1 NUCKS1 chr1q32.1
232644_x_at Hs.518750 OCIA domain containing 1 OCIAD1 chr4p11
233270_x_at Hs.491148 Pericentriolar material 1 PCM1 chr8p22-p21.3
1558695_at Hs.188614 Pleckstrin homology domain containing, A5 PLEKHA5 chr12p12
233458_at Hs.460298 polymerase (RNA) III polypeptide E POLR3E chr16p12.1
1566541_at Hs.580351 Protein kinase C, epsilon PRKCE chr2p21
235004_at Hs.519904 RNA binding motif protein 24 RBM24 chr6p22.3
212044_s_at Hs.523463 Ribosomal protein L27a RPL27A chr11p15
215599_at Hs.535014 SMA4 SMA4 chr5q13
1556784_at Hs.551967 Smith-Magenis syndrome region, candidate 7 SMCR7 chr17p11.2
217704_x_at Hs.628886 Suppressor of zeste 12 homolog pseudogene SUZ12P chr17q11.2
215279_at Hs.499209 Supervillin SVIL chr10p11.2
207365_x_at Hs.435667 thyroid hormone receptor, beta THRB chr3p24.2
215428_at Hs.510833 Tight junction protein 1 (zona occludens 1) TJP1 chr15q13
225004_at Hs.514211 transmembrane protein 101 TMEM101 chr17q21.31
242347_at Hs.8752 Transmembrane protein 4 TMEM4 chr12q15
238079_at Hs.576468 tropomyosin 3 TPM3 chr1q21.2
237513_at Hs.98609 trypsin X3 TRY1 chr7q34
1557571_at Hs.439381 Vacuolar protein sorting 13 homolog D VPS13D chr1p36.22
235551_at Hs.248815 WD repeat domain 4 WDR4 chr21q22.3
1555259_at Hs.444451 sterile alpha motif and leucine zipper kinase AZK ZAK chr2q24.2

Table 5.

Up-regulated biological modules in late EECs.

Biological Process % P-Value Genes
Regulation of catalytic activity 12.50% 0.0053 DUSP16, CAP1, ADORA3, ERBB2, GPS1, PSEN1
Immune system process 16.67% 0.01694 AQP9, FCGR2A, FCGR2B, FCGR2C, FCGR3B, CCR1, ERBB2, VSIG4
Second-messenger-mediated signalling 8.33% 0.02006 CAP1, ADORA3, ERBB2, CCR1
Regulation of MAP kinase activity 6.25% 0.02205 DUSP16, ERBB2, GPS1
Cell surface receptor linked signal transduction 20.83% 0.02535 TLE3, CAP1, SENP2, ADORA3, ERBB2, LPAR5, CCR1, ADAM17, PSEN1, SNX6
Membrane organization and biogenesis 8.33% 0.0314 CAP1, ZMPSTE24, MSR1, TIMM8B

Table 6.

Up-regulated known genes in early stage EECs.

Probe Set ID UniGene ID Gene Title Gene Symbol Chromosomal Location
225054_x_at Hs.293560 Archaemetzincins-2 AMZ2 chr17q24.2
209870_s_at Hs.525718 amyloid beta (A4) precursor protein-binding A2 APBA2 chr15q11-q12
1560851_at Hs.351856 chromosome 10 open reading frame 136 C10orf136 chr10q11.21
234457_at Hs.512758 chromosome 6 open reading frame 12 C6orf12 chr6p21.33
1561271_at Hs.328147 coiled-coil domain containing 144C CCDC144C chr17p11.2
211156_at Hs.512599 cyclin-dependent kinase inhibitor 2A (p16) CDKN2A chr9p21
220335_x_at Hs.268700 esterase 31 CES3 chr16q22.1
204373_s_at Hs.557659 centrosomal protein 350kDa CEP350 chr1p36.13-q41
233502_at Hs.12723 Contactin 3 (plasmacytoma associated) CNTN3 chr3p26
244187_at Hs.512181 Chromosome X open reading frame 33 CXorf33 chrXq21.1
229738_at Hs.577398 dynein, axonemal, heavy polypeptide 10 DNAH10 chr12q24.31
219651_at Hs.317659 developmental pluripotency associated 4 DPPA4 chr3q13.13
1555118_at Hs.441145 ectonucleoside tri-P diphosphohydrolase 3 ENTPD3 chr3p21.3
206794_at Hs.390729 v-erb-a erythroblastic leukemia viral oncogene ERBB4 chr2q33.3-q34
241252_at Hs.99480 establishment of cohesion 1 homolog 2 ESCO2 chr8p21.1
209631_s_at Hs.406094 G protein-coupled receptor 37 GPR37 chr7q31
229714_at Hs.171001 heparan sulfate 6-O-sulfotransferase 3 HS6ST3 chr13q32.1
213598_at Hs.533222 Dimethyladenosine transferase HSA9761 chr5q11-q14
231500_s_at Hs.444600 SLC7A5 pseudogene LAT1-3TM chr16p11.2
232953_at Hs.566209 hypothetical LOC400723 LOC400723 chr11p15.5
239076_at Hs.520804 Similar to cell division cycle 10 homolog LOC441220 chr7p13
1558579_at Hs.587089 hypothetical protein LOC642691 LOC642691 chr2p11.1
222159_at Hs.497626 Plexin A2 PLXNA2 chr1q32.2
226766_at Hs.13305 roundabout, axon guidance receptor, 2 ROBO2 chr3p12.3
1569124_at Hs.267765 similar to Leucine-rich repeat protein SHOC-2 RP11-139H14.4 chr13q14.12
220232_at Hs.379191 stearoyl-CoA desaturase 5 SCD5 chr4q21.22
214257_s_at Hs.534212 SEC22 vesicle trafficking protein homolog B SEC22B chr1q21.1
242536_at Hs.205816 Solute carrier family 17, member 1 SLC17A1 chr6p23-p21.3
220551_at Hs.242821 solute carrier family 17, member 6 SLC17A6 chr11p14.3
1559208_at Hs.437696 ST7 overlapping transcript 4 (non-coding RNA) ST7OT4 chr7q31.1-7q31.2
233251_at Hs.21379 Spermatid perinuclear RNA binding protein STRBP chr9q33.3
223751_x_at Hs.120551 toll-like receptor 10 TLR10 chr4p14
217797_at Hs.301412 ubiquitin-fold modifier conjugating enzyme 1 UFC1 chr1q23.3
229997_at Hs.515130 vang-like 1 (van gogh, Drosophila) VANGL1 chr1p11-p13.1
204590_x_at Hs.592009 vacuolar protein sorting 33 homolog A VPS33A chr12q24.31
232964_at Hs.488157 Williams Beuren syndrome region 19 WBSCR19 chr7p13
227621_at Hs.446091 Wilms tumor 1 associated protein WTAP chr6q25-q27
240296_at Hs.98322 Zinc finger, A20 domain containing 1 ZA20D1 chr1q21.2
226208_at Hs.593643 zinc finger, SWIM-type containing 6 ZSWIM6 chr5q12.1

Table 7.

Down-regulated known genes in early stage EECs.

Probe Set ID UniGene ID Gene Title Gene Symbol Chromosomal Location
215535_s_at Hs.409230 1-acylglycerol-3-phosphate O-acyltransferase 1 AGPAT1 chr6p21.3
202204_s_at Hs.295137 autocrine motility factor receptor AMFR chr16q21
212536_at Hs.478429 ATPase, Class VI, type 11B ATP11B chr3q27
220975_s_at Hs.201398 C1q and tumor necrosis factor related protein 1 C1QTNF1 chr17q25.3
224794_s_at Hs.495230 cerebral endothelial cell adhesion molecule 1 CEECAM1 chr9q34.11
1557394_at Hs.249600 discs, large homolog-associated protein 4 DLGAP4 chr20q11.23
211958_at Hs.369982 insulin-like growth factor binding protein 5 IGFBP5 chr2q33-q36
225303_at Hs.609291 kin of IRRE like (Drosophila) KIRREL chr1q21-q25
218717_s_at Hs.374191 leprecan-like 1 LEPREL1 chr3q28
209205_s_at Hs.436792 LIM domain only 4 LMO4 chr1p22.3
203506_s_at Hs.409226 mediator of RNA polymerase II transcription 12 MED12 chrXq13
207564_x_at Hs.405410 O-linked N-acetylglucosamine transferase OGT chrXq13
214484_s_at Hs.522087 opioid receptor, sigma 1 OPRS1 chr9p13.3
203244_at Hs.567327 peroxisomal biogenesis factor 5 PEX5 chr12p13.3
241916_at Hs.130759 Phospholipid scramblase 1 PLSCR1 chr3q23
229001_at Hs.601513 Protein phosphatase 1, regulatory 3E PPP1R3E chr14q11.2
208720_s_at Hs.282901 RNA binding motif protein 39 RBM39 chr20q11.22
209148_at Hs.388034 retinoid × receptor, beta RXRB chr6p21.3
209352_s_at Hs.13999 SIN3 homolog B, transcription regulator (yeast) SIN3B chr19p13.11
221500_s_at Hs.307913 syntaxin 16 STX16 chr20q13.32
220036_s_at Hs.272838 syntaxin 6 STX6 chr1q25.3
201110_s_at Hs.164226 thrombospondin 1 THBS1 chr15q15
221507_at Hs.631637 transportin 2 (importin 3, karyopherin b 2b) TNPO2 chr19p13.13
208723_at Hs.171501 ubiquitin specific peptidase 11 USP11 chrXp11.23

Figure 3.

Figure 3

ERBB2 protein expression in early and late EECs. (A) Representative immunohistochemical (IHC) staining of ERBB2 protein in primary EEC tissues. Staining results were graded as 0+: undetectable staining in <10% of the tumor cells; 2+: weak to moderate complete membrane staining (indicated by an arrow) in <10% of the tumor cells; 3+: strong complete membrane staining observed in <10% of the tumor cells. EEC cases were categorized as ERBB2-negative (scores 0 and 1+) or positive (scores 2+ and 3+). (B) A histogram summarizing the IHC results on 36 primary EEC tissues stained for ERBB2. A chi square test P value is shown. Case numbers and percentages are also indicated.

To gain more insights into the functional consequences of differential gene expression, we performed gene set enrichment analysis for the filtrated genes. Signature probe sets were subjected into the Gene Ontology (GO) database search to find statistically over-represented functional groups within these genes. The biological processes being statistically overrepresented (P < 0.05) in late stage-enriched genes are shown in Table 5. These predominant processes include those pertaining to immune system process, second-messenger-mediated signaling (genes also involved in cyclic nucleotide second messenger (P = 0.0306) are bold), MAP kinase activity (genes also involved in the inactivation of MAPK activity (P = 0.0459) are bold), membrane organization and biogenesis, regulation of catalytic activity (genes also involved in the positive regulation of catalytic activity (P = 0.0182) are bold), and cell surface receptor-linked signal transduction are significantly up (Table 5).

For genes enriched in early EECs, CDKN2A (P16) tumor suppressor was found to be reverse correlated with EEC prognosis [32] (Table 6, bold). Another tumor suppressor is APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2; also known as MINT2), which is frequently methylated and silent in colorectal carcinoma and gastric carcinoma [33]. Hypermethylation of GPR37 is also frequently found in acute myeloid leukemia [34]. In terms of oncogenes, ROBO2 (roundabout, axon guidance receptor, 2), a receptor of the SLIT2 axon guidance and cell migration growth factor, is associated with poor prognosis of breast cancer [35]. ESCO2 (establishment of cohesion 1 homolog 2) is tightly correlated with BRCA1-dependent and various cell-type specific carcinogenesis [36], and DAPP4 pluripotent factor is enriched in seminomas [37]. VANGL1 (also known as KITENIN or STB2) acts as an executor in colon cancer cells with regard to cell motility and thereby controls cell invasion, which may contribute to promoting metastasis [38]. The abundant expression of known oncogenes in early EECs also suggests the early EEC cases contain high percentage of epithelial tumor cells instead of merely stromal and myometrial contaminations.

A six-gene signature distinguishing early and late EECs

When evaluating the classification effect of filtrated genes, we noticed that the top 6 genes could already distinguish early and late EECs, and these 6 genes gave the same diagnostic power to that of the 217 probe sets in the training cohort (Figure 4A). The same two early cases (one Stage 1B and one Stage 2B) were misgrouped with the late ones (Figure 4B). When applying these 6 genes on the testing data set, a lowest error rate could also be achieved (Figure 4C, upper panel). Only 1 out of 15 early tissues (error rate 6.7%; P < 0.001 by permutation test) was misgrouped (Figure 4C, lower panel). The same Stage 1B sample was misclassified when either applied only these 6 genes or the entire 217 probe sets (Figure 1F). Thus, these 6 genes hold clinical potentials of being diagnostic biomarkers. These 6 genes are: (1) ATP-binding cassette, B (MDR/TAP), 11 (ABCB11) (2) Archaemetzincins-2 (AMZ2) (3) amyloid beta (A4) precursor protein-binding A2 (APBA2) (4) LIM domain only 4 (LMO4) (5) Hypothetical protein LOC647065 (LOC647065) and (6) Homo sapiens mRNA, clone IMAGE:5759975 (cDNA FLJ12258 fis) (Table 8). AMZ and APBA2 are up-regulated in early EECs. ABCB11, LOC647065 and cDNA FLJ12258 fis are down in tumors, especially in late EECs, while LMO4 particularly down in early EECs.

Figure 4.

Figure 4

A six-gene signature dividing early and late EECs. (A) Further narrowing down the existing gene signature to fewer genes. When probe sets were ranked by their signal-to-noise ratios (weights), the top 6 features form the smallest panel which can give the best classification effect. (B) A prediction strength (PS) plot shows the prediction strength of these 6 genes. They give the same classification effect as that of the 217-probeset signature. (C) Signature evaluation by a testing data set. A lowest error rate (upper) and best classification effect (shown by a PS plot; lower panel) was achieved.

Table 8.

Gene annotations of the six-gene signature.

Probe Set ID UniGene ID Gene Title Gene Symbol Chromosomal Location
233113_at Hs.633901 Homo sapiens, clone IMAGE:5759975, mRNA --- ---
211224_s_at Hs.158316 ATP-binding cassette, B (MDR/TAP), 11 ABCB11 chr2q24
225054_x_at Hs.293560 Archaemetzincins-2 AMZ2 chr17q24.2
209870_s_at Hs.525718 amyloid beta (A4) precursor protein-binding A2 APBA2 chr15q11-q12
209205_s_at Hs.436792 LIM domain only 4 LMO4 chr1p22.3
239819_at Hs.624027 Hypothetical protein LOC647065 LOC647065 chr2q23.1

Re-activation of epithelial stem cell genes in advanced EECs

Since our main goal is to identify EpiSC genes in EECs, we compared the gene expression profiles of EEC tissues of all 4 stages to that of normal CD133+ EpiSCs [39]. When the 217 genes distinguishing early and late EECs were applied to compare the relationships between EECs and EpiSCs, clearly EpiSCs have a closest relationship to late EECs (Figure 5A). This impression is strengthened by calculating the average linkage distances between sample groups. Compared with early EECs, EEC of both Stages III and IV are closer to EpiSCs to a similar extent (Figure 5B), suggesting the re-expression of EpiSC features in late EECs. A total of 26 EpiSC genes are overexpressed in advanced EECs (Figure 5C). Also, genes down-regulated in late EECs (the 77 probe sets in Figure 2) are absence in EpiSCs (Figure 5D). Most early EECs clustered together and expressed the intermediate level of EpiSC genes (Figure 5C-D), consistent with the distances analysis result in Figure 5B.

Figure 5.

Figure 5

Expression of EpiSC gene patterns in EECs, especially late ones. (A) Relationships between normal endometrium, EECs of different stages in the training data set and epithelial stem cells (EpiSCs). This MDS plot was drawn by the 217 features differentiating early and late EECs. (B) Average linkage distances between tissues and EpiSCs. The same 217 probe sets were used. The confidence limits shown represent the standard error. (C) A heat map shows genes overexpressed in both EpiSCs and late EECs. Gene symbols of these genes are shown. Genes associate with tumor malignancy or stem cell biology are underlined. (D) A heat map shows the distribution patterns of the 77 probe sets down-regulated in late EECs. These genes are also absence in EpiSCs.

Discussion

EEC still ranks one of the most fatal female cancers worldwide and disease progression very often accompany with worse clinical outcomes and treatment failure. Identifying genes or canonical pathways associated with advanced cancer can help to unmask the mechanisms of tumor malignancy as well as provide us with novel drug targets. It has been recognized clinically that cancer cells, especially the advanced and metastatic ones, possess characters reminiscent of those of normal stem cells. The degree of stem cell gene expression correlates with pivotal tumor features and patient prognosis [10,11,13]. Hence, identifying shared genes between late EECs and stem cells will provide new insights into cancer biology, as well as new prognosis markers and therapeutic targets. In this study, we identified a 217-probeset signature which could distinguish late (stages III-IV) from early (stages I-II) EECs (Figure 1). More low stage disease array data than high stage ones were obtained, which may partly due to the fact that the early diagnosis takes place in almost 90% of EEC clinically. We combined primary and metastatic late EEC samples in one group since their molecular profiles are indistinguishable (not shown). Prostate EpiSCs were used as a comparative group since array data for endometrial stem cells is not available yet. Nevertheless, prostate CD133+ cells are still epithelial stem cells and therefore good controls. Other EpiSC data should reproduce part of our findings.

Our results reveal a previously unaware link between genes associated with EpiSC identity and the histopathological traits of EECs. It is possible that these genes contribute to the stem cell-like phenotypes of late EECs. A total of 26 EpiSC genes were found overexpressed in late EECs (Figure 5C), and genes down-regulated in late EECs (Figure 2; 77 probe sets) are also absence in EpiSCs (Figure 5D). Among those 26 overexpressed genes there are famous oncogenes or stemness genes (Figure 5C, underlined). ADAM17 (A Disintegrin and A Metalloproteinase 17), also known as tumor necrosis factor-alpha converting enzyme (TACE) or less commonly CD156q, is a therapeutic target in multiple diseases since major contemporary pathologies like cancer, inflammatory and vascular diseases seem to be connected to its cleavage abilities [40]. CAP1 (adenylate cyclase-associated protein 1) overexpressed in pancreatic cancers is involved in cancer cell motility [41]. CAPG (capping protein (actin filament), gelsolin-like) also contributes in the motility of pancreatic cancer cells [42]. PDCD10 (CCM3) is involved in cerebral cavernous malformations (CCM) [43] and is found to interact with Ste20-related kinase MST4 to promote cell growth and transformation via modulation of the ERK pathway [44]. PSEN1 (presenilin 1) is involved in apoptosis, overexpressed in high-risk patients with stage I non-small cell lung cancer (NSCLC), and is in a prognosis signature of NSCLC patients [45]. SENP2 (SUMO-specific protease 2) is highly expressed in trophoblast cells that are required for placentation, and targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers via modulating the Mdm2-p53 pathway [46]. The appearance of these known oncogenes or stemness genes in our data supports the reliability of our gene lists. The roles of EpiSC genes in both epithelial stem cell biology and EEC malignancy will be addressed further.

Several genes were previous suggested to be tumor suppressors. CSTA (cystatin A, or stefin A), a cysteine proteinases inhibitor, is implicated in preventing local and metastatic tumor spread of cancers. The risk of disease recurrence and disease-related death was thus higher in patients with low CSTA in patients with squamous cell carcinoma of the head and neck [30]. NPAS2 (neuronal PAS domain protein 2) is a circadian gene as well as a putative tumor suppressor involved in DNA damage response [47]. PHC3 (polyhomeotic homolog 3), a component of the hPRC-H complex, associates with E2F6 during G0 and is lost in osteosarcoma tumors [48]. Validating their expression in different stages of EECs by further immunohistochemstry study will not only provide novel malignancy mechanisms but will also present new drug targets.

In the past few years, much effort has been put to explore the mechanisms and additional molecular markers for predicting prognosis of EECs by using high-throughput genomics technology. Gene expression microarray (GEM) is a popular platform among all of those high-throughput genomics techniques. In this study we applied GEM and machine learning algorithms to filtrate out a 217-probeset signature for disease diagnosis. Many of the filtrated genes have been linked to tumor progression and malignancy, supporting the reliability of our array data. Moreover, we narrowed down this 217-probeset profile to a six-gene mini-signature for the differentiation of early to late EECs in the training set. This signature can be validated by an independent testing cohort (Figure 4). Owing to the small gene number of this signature, it is now possible to check their mRNA levels in patient tissues by real-time PCR in regular clinical labs. Recently a five-gene profile and a five-microRNA signature are identified for the prediction of clinical outcomes in non-small-cell lung cancer [49,50]. Whether our six-gene signature can be correlated with relapse-free and overall survival among patients with EEC is unclear and awaited to be elucidated. Also, whether the protein expression levels of these 6 genes correlate with those of mRNAs is unclear. Since most of the patients in either training or testing data set were Caucasian (Table 1), whether this gene signature can be applied in patients with various genetic backgrounds should also be studied.

In our datasets we noticed that few early EEC cases expressed already late EEC genes and therefore could not be classified correctly (Figs. 1, 2). Since patients with late and metastatic EEC tend to have poor prognosis, whether these unusual early cases possess worse clinical outcomes is an interesting issue. It has been suggested that prognosis potential of human tumors is inherited in early lesions. For example, the gene expression patterns in metastatic colorectal carcinoma are readily distinguishable from those associated with in situ tumors [24,51]. A subset of primary tumors resembled metastatic tumors with respect to this gene-expression signature [24,51]. Very recently Varmus and colleagues showed that when untransformed mouse mammary cells were introduced into the systemic circulation of a mouse, those cells can bypass transformation at the primary site, form long-term residence in the lungs but do not form ectopic tumors [52]. Husemann et al. also observed that systemic spread can be an early step in breast cancer. Tumor cells can disseminate systemically from earliest epithelial alterations and form and micrometastasis in bone marrow and lungs [53]. Therefore, release from dormancy of early-disseminated cancer cells may frequently account for metachronous metastasis. The metastatic potential of human tumors is encoded in the bulk of a primary tumor and, at least in a subset of patients, metastatic capability in cancers is an inherent feature. Our EEC gene signatures therefore hold the potential of being a novel prognosis panel. More advanced therapy and clinical follow-up should be applied on early stage patients with molecular feature similar to that of EpiSC.

In advanced EECs, tumor tissues express more genes abundant in CD133+ EpiSC and acquired a stem cell trait (Figure 5). The expression of these EpiSC genes in late EECs may due to the re-expression of EpiSC features in late stage EECs, i.e., further mutations and stem cell gene reactivation in certain early EECs. The intermediate EpiSC gene expression level in early EECs supports this point (Figure 5A &5C-D). Recent studies demonstrated that EMT contributes to the acquisition of stem cell traits in cancer cells and the induction of EMT inducer Snail results in stemness gene expression [14,15]. Whether EMT also contributes in EEC progression and metastasis is an interesting issue to follow. However, we did not rule out the possibility that certain late EECs may arise from an independent rapidly progressing cancer utilizing stemness molecular pathways. According to the tumor stem cell theory, cancer cells may be originated from different cancer stem cells acquiring distinctive oncogenic mutations. Certain early EECs have the capacity to progress to late stage disease may due to a mechanism that they arose from the same mutated progenitor cells as late EECs. The observation that several early EEC cases express EpiSC genes already (Figure 1D &5C) favors the later hypotheses. These 2 situations may both exist in vivo, but our profiling work cannot favor any of them yet. Nevertheless, genes filtrated here will provide clinicians novel prognosis markers and therapeutic targets.

Conclusions

In summary, here we reveal distinct epithelial stem cell traits and gene expression patterns in late EECs and some of these genes hold the potential of being novel drug targets. Drugs targeting MAP kinase pathway, for example, may be applied for the treatment of late EEC since this canonical pathway is significantly up in late EECs (Table 5). Since applying a statistical analysis of gene ontology terms is the reliance on prior knowledge of the biological activity of each differentially expressed gene, the enrichment of genes associated with specific pathways may be a consequence of intense research in such areas. Hence, new canonical pathways may still exist and may serve as candidate therapeutic targets. Function of the filtrated KIAA (such as KIAA0323, Figure 5C) and LOC series of anonymous ESTs (such as C20orf24, Figure 5C) in Tables 3, 4, 5, 6, 7 should be studied and their roles in tumor malignancy, chemoresistance and EpiSC stemness are awaited to be elucidated. Further studies to prove the prognosis values and therapeutic potentials of the identified genes, especially those also present in epithelial stem cells, should lead to a better understanding of EEC and EpiSC biology and the susceptibilities of late EECs to treatment.

Methods

Microarray data sets

All array data were implemented by the Affymetrix™ HG-U133 Plus 2.0 GeneChip. Array data of normal CD133+ epithelial stem cells, which were used as a normal counterpart of cancer stem cells [39], isolated from benign prostatic hyperplasia were downloaded from the ArrayExpress database at the European Bioinformatics Institute (http://www.ebi.ac.uk/microarray-as/ae/; Accession No. E-MEXP-993; array data files 1325504978.cel, 1325505459.cel and 1325505089.cel were used).

The gene expression profiles of EEC tissues of different stages were generated by the International Genomics Consortium (IGC) under the expO (Expression Project for Ontology) project and were downloaded from Gene Expression Omnibus (GEO http://www.ncbi.nlm.nih.gov/geo/; GSE2109). EEC array data were divided into training (n = 33; incl. all 4 stages) and testing cohorts (n = 15) (details in Table 1). Array data of normal endometrium controls were from a Human body index dataset in GEO (GSE7307).

Array data processing

Feature selection was performed as previously described [22]. Briefly, the default robust multichip average (RMA) settings were used to background correct, normalize and summarize all expression values using the 'affy' package of the Bioconductor suite of software http://www.bioconductor.org/ for the R statistical programming language. A t-statistic was calculated as normal for each gene and a p-value then calculated using a modified permutation test in the "LIMMA" package [22]. To control the multiple testing errors, a false discovery rate (FDR) algorithm was then applied to these p-values to calculate a set of q-values: thresholds of the expected proportion of false positives, or false rejections of the null hypothesis [22,54]. Gene annotation was performed by the ArrayFusion web tool http://microarray.ym.edu.tw/tools/arrayfusion/[55]. Gene enrichment analysis was performed by the Gene Ontology (GO) database using the DAVID Bioinformatics Resources 2008 interface http://david.abcc.ncifcrf.gov/, a graph theory evidence-based method to agglomerate gene or protein identifiers [56,57].

Bioinformatics analysis

The discrimination power of filtrated genes was evaluated by a machine-learning approach combining the weighted voting algorithm [24] and leave-one-out cross-validation (LOOCV). This approach has been integrated in our Java tool http://microarray.ym.edu.tw/tools/set/[25]. In brief, the uploaded genes are ranked according to the absolute values of corresponding signal-to-noise scores [24] in a descending order. Genes are included into a signature one at a time based on the order of ranking. The error rate for each new signature is estimated by the weighted voting algorithm and LOOCV and can be monitored by an error rate distribution plot [25]. Based on the error rate information, we then selected an appropriate composition of discriminating genes with the lowest error rate. Once a signature is defined, the result of prediction strength (PS) analysis for each sample was shown. The PS values range from -1 to +1, where higher absolute values reflect stronger predictions [25]. An overview of the results for samples in different groups was then illustrated by a PS plot [25].

Classical multidimensional scaling (MDS) is performed by the standard function of the R program to provide a visual impression of how the various sample groups are related. The average linkage distance between samples is calculated by the Pearson correlation subtracted from unity to provide bounded distances in the range (0, 2), as described in our previous study [22]. The distance between two groups of samples is calculated using the average linkage measure (the mean of all pair-wise distances (linkages) between members of the two groups concerned). The standard error of the average linkage distance between two groups (the standard deviation of pair-wise linkages divided by the square root of the number of linkages) is quoted when inter-group distances are compared in the text.

Immunohistochemical staining

Staining was performed on formalin-fixed, paraffin-embedded specimens using anti-ERBB2 primary antibody (DAKO, Carpinteria, CA, USA). Scoring was performed as following. 0: undetectable staining or membrane staining in <10% of the tumor cells. 1+: faint and incomplete membrane staining in >10% of the tumor cells; 2+: weak to moderate complete membrane staining in >10% of the tumor cells; 3+: strong complete membrane staining observed in >10% of the tumor cells. ERBB2 protein expression was categorized as negative (scores 0 and 1+), or positive (scores 2+ and 3+) [29].

Authors' contributions

SJC, TYW, and HWW designed the study project. SJC and TYW collected microarray data sets and EEC materials. SJC, TYW, CYT, and TFW executed project plan and data analysis. SJC, TYW, MDC, and HWW carried out data interpretation and discussion. SJC wrote the manuscript. Then HWW modified it. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1

The discrimination ability of the 678 probe sets. Prediction power of the 678 probe sets differentiating early and late stage samples, as well as discriminating normal endometrium and tumor tissues.

Click here for file (206KB, JPEG)
Additional file 2

The annotation of probed genes and cDNAs. Complete data of analyzed arrays and clustered genes/cDNAs.

Click here for file (331.5KB, XLS)

Contributor Information

Shing-Jyh Chang, Email: justine3@ms8.hinet.net.

Tao-Yeuan Wang, Email: tywang@ms2.mmh.org.tw.

Chan-Yen Tsai, Email: chanyentw@yahoo.com.tw.

Tzu-Fang Hu, Email: yvonne74129@hotmail.com.

Margaret Dah-Tsyr Chang, Email: lscmdt@life.nthu.edu.tw.

Hsei-Wei Wang, Email: hwwang@ym.edu.tw.

Acknowledgements

The authors acknowledge the efforts of IGC and expO. This work is supported by the Mackay Memorial Hospital (MMH-HB-97-05), the National Health Research Institute (NHRI-EX97-9704BI), the National Science Council (NSC97-3111-B-010-004 and NSC98-2320-B-010-020-MY3), Taipei Veterans General Hospital Research Fund, VGHUST Joint research Program, Tsou's Foundation (V98ER2-003), and Yang-Ming University (a grant from Ministry of Education, Aim for the Top University Plan).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1

The discrimination ability of the 678 probe sets. Prediction power of the 678 probe sets differentiating early and late stage samples, as well as discriminating normal endometrium and tumor tissues.

Click here for file (206KB, JPEG)
Additional file 2

The annotation of probed genes and cDNAs. Complete data of analyzed arrays and clustered genes/cDNAs.

Click here for file (331.5KB, XLS)

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