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Cancer Research 67, 1927, March 1, 2007. doi: 10.1158/0008-5472.CAN-06-2687
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Inactivation of the Candidate Tumor Suppressor Par-4 in Endometrial Cancer

Gema Moreno-Bueno1, Pablo J. Fernandez-Marcos2, Manuel Collado2, Mercedes J. Tendero1, Socorro M. Rodriguez-Pinilla1, Isabel Garcia-Cao2, David Hardisson3, Maria T. Diaz-Meco4, Jorge Moscat4, Manuel Serrano2 and Jose Palacios1

1 Breast and Gynecological Cancer Group, 2 Tumor Suppression Group, Spanish National Cancer Center (CNIO); 3 Department of Pathology, University Hospital "La Paz"; and 4 Center of Molecular Biology "Severo Ochoa" (CSIC-UAM), Madrid, Spain

Requests for reprints: Manuel Serrano or Jose Palacios, Spanish National Cancer Center (CNIO), 3 Melchor Fernandez Almagro Street, Madrid E-28029, Spain. Phone: 34-91-732-8032; Fax: 34-91-732-8028; E-mail: mserrano{at}cnio.es or jose.palacios.sspa{at}juntadeandalucia.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Recently, it has been shown that mice deficient in the proapoptotic protein prostate apoptosis response 4 (Par-4) are specifically prone to develop endometrial carcinomas. Based on this, we have examined here the possible role of Par-4 as a tumor suppressor gene in human endometrial cancer. Using cDNA arrays, quantitative reverse transcription-PCR, and immunohistochemistry, we detected Par-4 down-regulation in ~40% of endometrial carcinomas. This alteration was not associated with phosphatase and tensin homologue (PTEN), K-RAS, or ß-catenin mutations, but was more frequent among tumors showing microsatellite instability (MSI) or among tumors that were estrogen receptor positive. Mutational analysis of the complete coding sequence of Par-4 in endometrial cancer cell lines (n = 6) and carcinomas (n = 69) detected a mutation in a single carcinoma, which was localized in exon 3 [Arg (CGA) 189 (TGA) Stop]. Interestingly, Par-4 promoter hypermethylation was detected in 32% of the tumors in association with low levels of Par-4 protein and was more common in MSI-positive carcinomas. Par-4 promoter hypermethylation and silencing was also detected in endometrial cancer cell lines SKUT1B and AN3CA, and reexpression was achieved by treatment with the demethylating agent 5'-aza-2'-deoxycytidine. Together, these data show that Par-4 is a relevant tumor suppressor gene in human endometrial carcinogenesis. [Cancer Res 2007;67(5):1927–34]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Endometrial carcinoma is the most common malignant gynecologic tumor in women (1, 2). From a clinicopathologic and epidemiologic point of view, endometrial carcinomas are subdivided in two different categories (3). Type I endometrial carcinomas are prototypically well-differentiated endometrioid endometrial carcinomas (EEC) that usually develop in premenopausal and perimenopausal women, are associated with estrogen stimulation, and coexist with, or are preceded by, atypical endometrial hyperplasia. A special case within this group is formed by poorly differentiated endometrioid tumors, which could constitute a separate class of cancers (4). On the other hand, type II tumors are nonendometrioid endometrial carcinomas (papillary serous and clear cell carcinomas) that occur in older women, are unrelated to estrogen exposure, and develop from atrophic endometrium through the so-called endometrial intraepithelial carcinoma. From a molecular point of view, K-RAS, PTEN, and ß-catenin mutations and microsatellite instability (MSI) are implicated in the initiation of EECs, whereas p53 mutations occur in most nonendometrioid carcinomas (3).

Prostate apoptosis response 4 (Par-4 or PAWR) gene was identified in a differential screen for proapoptotic genes in prostate carcinoma cell lines (5). Par-4 gene maps to chromosome 12q21, a region frequently deleted in some malignancies (6), and encodes a protein (38 kDa) containing a leucine zipper domain in the COOH-terminal region, which interacts with a variety of proteins, including the atypical protein kinases {zeta}PKC and {lambda}PKC and the tumor suppressor Wilms' tumor 1 (7–9). Among the mechanisms by which Par-4 triggers apoptosis, the best established one, supported by studies in genetically modified mice, is through inhibition of the {zeta}/{lambda}PKCs and the ensuing down-modulation of nuclear factor {kappa}B (NF{kappa}B) and its prosurvival effectors, such as X-linked inhibitor of apoptosis (XIAP). Specifically, Par-4–deficient mice present hyperactivation of NF{kappa}B and lymphoproliferative phenotypes, including enhanced T-cell–mediated liver damage during hepatitis, whereas {zeta}PKC-deficient mice present decreased NF{kappa}B activity, impaired development of lymphoid secondary organs, and lower sensitivity to T-cell–mediated hepatitis (10–14). More recently, it has been proposed that Par-4 activity is negatively regulated by Akt, which binds and phosphorylates Par-4, thus preventing the proapoptotic actions of Par-4 (15, 16).

Previous studies point to a link between Par-4 down-regulation and cancer. In particular, down-regulation of Par-4 occurs during cellular neoplastic transformation by the Ras oncogene through the Raf/mitogen-activated protein kinase-extracellular signal–regulated kinase (ERK) kinase/ERK pathway (17–19), and reduction or absence of Par-4 protein has been reported in renal cancer (20). However, beyond these suggestive lines of evidence, the involvement of Par-4 in human cancer has remained largely unexplored. Recently, we have shown that Par-4–null mice develop spontaneous prostate neoplasias and endometrial carcinomas, thus implicating Par-4 in the development of tumors in hormone-dependent tissues (14). Based on these data, we have focused here on the analysis of Par-4 in human endometrial carcinomas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Human samples. For cDNA array studies, we used 24 EECs that had been previously pathologically and molecularly characterized (21). In addition, we analyzed 16 normal cycling endometria that were obtained from patients ranging in age from 26 to 45 years and undergoing hysterectomy for the treatment of small intramural leiomyomata. Samples were obtained from the mid-fundus. All women had a clinical history of normal cycles of 28 ± 2 days and none had received hormone treatment in the previous year. Endometria were dated according to morphologic criteria, and in all patients the morphology of the endometrium was normal and correlated well with the days of the clinical cycle. Seven patients had late proliferative (histologic dating 10–14 days) endometrium and nine had early secretory (histologic dating 15–19 days) endometrium. Sections of frozen samples were split for RNA isolation and confirmatory histology by H&E staining.

For immunohistochemical analysis, we used 100 endometrioid endometrial carcinomas (EEC) obtained from surgical specimens from University Hospital "La Paz," Madrid. This cohort has previously been screened for PTEN, K-RAS, and ß-catenin mutations and for MSI (21–23). In addition, 16 normal endometrial samples were obtained from University Hospital "La Paz" (see above).

Mouse samples. The estrous cycle stage of 6-week-old C57BL6/J wild-type (WT) females was determined as described elsewhere (24). Two hours after determination of the estrous cycle stage, two mice at every stage were sacrificed. One whole uterine horn was frozen in liquid nitrogen for its subsequent analysis by immunoblotting; the other uterine horn was fixed in 10% buffered formalin, embedded in paraffin wax, sectioned at 4 µm, and stained with anti–Par-4 (see below).

Cell culture. Endometrial carcinoma cell lines (AN3CA, HEC1B, Ishikawa, KLE, SKUT1, and SKUT1B) were obtained from American Type Culture Collection5 (Rockville, MD) and were cultured according to their recommendations. With the exception of KLE, all the other cell lines were MSI positive. For colony formation assays, cells were plated at 60% to 80% confluency and, 1 day later, transfected with 8 µg of empty pCDNA3.1 (Invitrogen, Grand Island, NY) or pCDNA3.1-WTrPar4 containing full-length rat Par-4 and Fugene (Roche, Nutley, NJ) following the manufacturer's instructions. One day later, cells were trypsinized, counted, and 105 cells were plated in triplicate in plates of 10-cm diameter. The following day, the appropriate amount of G418 (Sigma, St. Louis, MO) was added (previously determined for each cell line). Medium was replaced twice every week until colonies were apparent (at least 2 weeks after plating to allow for selection). After 2 weeks, colonies were fixed with formaldehyde and stained with Giemsa. Colonies >1 mm were counted in the three plates per condition.

A stock solution (10 mmol/L) of 5-aza-2'deoxycytidine (5-aza-CdR, Sigma) was prepared in PBS (pH 6.5). For demethylation experiments, cells were plated at a density of 1 x 105 per 100-mm dish and treated the following day. Medium and 5-aza-CdR were changed every 24 h. 5-Aza-CdR was added to the different cells in the appropriate concentration (2.5–5 µmol/L).

cDNA arrays. Endometrial samples were hybridized against a Universal Reference RNA pool (Stratagene, Cedar Creek, TX) in cDNA arrays containing 6,386 genes represented by 9,726 clones (CNIO Oncochip), essentially as described before (21).

Quantitative reverse transcription-PCR. To validate cDNA array data, Par-4 expression was assessed by quantitative real-time PCR (TaqMan) using the ABI PRISM 7700 Sequence Detection System instrument and software (Applied Biosystems, Foster City, CA). Each reaction was done in triplicate from two cDNA dilutions. TaqMan reactions for target and internal control genes were done in separate tubes. The comparative threshold cycle (Ct) method was used to calculate the amplification factor as specified by the manufacturer. The internal standard ß2-microglobulin (Applied Biosystems) was used to normalize variations in the quantities of input cDNA. The amount of target and endogenous reference was determined from a standard curve for each experimental sample. The sequence of oligonucleotides and TaqMan probes used for the analysis of Par-4 were obtained using the Assays-by-Design (SM) File Builder program (Applied Biosystems). Each analysis was done in triplicate. Ratios of x-fold expression in the case of array data, as well as quantitative reverse transcription-PCR (RT-PCR) data, are expressed in the following manner: for ratios ≥1, as the direct ratio; for ratios <1, as the negative value of the inverse of the ratio.

Immunohistochemical analysis. Immunohistochemistry was done on human tissue microarray sections. Representative areas of EECs were carefully selected on H&E-stained sections and marked on individual paraffin blocks. Two-millimeter-diameter tissue cores were obtained from each specimen. The tissue cores were precisely arrayed in a paraffin block using a tissue microarray workstation (Beecher Instruments, Silver Spring, MD) as previously described (25). The tissue microarray included normal adjacent tissue (myometrium) for each tumor sample to evaluate variations in tissue fixation between samples. In addition, 16 normal samples, which represent the complete cycling endometrium, were also included. A H&E-stained section was reviewed to confirm the presence of morphologically representative areas of the original lesions. Tissue microarray blocks were sectioned at 4 µm and mounted on charged poly-L-lysine–coated slides. The sections were deparaffinized in xylene and rehydrated through a graded alcohol series to distilled water. The slides were subjected to antigen retrieval by microwave exposure in 10 mmol/L citrate buffer (pH 6.0) in a 750-W oven for 30 min. The slides were then cooled to room temperature and washed in PBS. Endogenous peroxidase activity was blocked by the incubation of the slides in hydrogen peroxide and methanol. The immunohistochemical analysis was done using the following antibodies: anti–Par-4 (Abcam, Cambridge, United Kingdom), anti-NF{kappa}B p65 (Santa Cruz Biotechnology, Santa Cruz, CA), anti–estrogen receptor (ER; SP1, MD Biosciences, St. Paul, MN), anti–progesterone receptor (PR; PgR, Dako, Glostrup, Denmark), anti–Ki-67 (MIB-1, Dako), and anti-XIAP (BD Biosciences, Franklin Lakes, NJ). The antibodies were detected by standard indirect immunoperoxidase procedures (LSAB, DakoCytomation, Glostrup, Denmark). Diaminobenzidine was used as a chromogen and light hematoxylin was used as a counterstain. Internal positive control for staining consisted of normal endometrium, interspersed inflammatory cells, and/or endothelium. In negative controls, the primary antibodies were omitted. Expression of selected makers was determined by two of the investigators (J.P. and S.M.R.). The percentage of stained cells was scored for ER, PR, Ki-67, XIAP, NF{kappa}B, and Par-4 in the two cores of each tumor and the mean value was used as the final score. For categorical analysis, a case was considered positive when the appropriate staining was found in >10% of cells for ER and PR. Cases were considered to have low expression of Ki-67, XIAP, NF{kappa}B, and Par-4 when staining was seen in ≤10% of cells.

The immunohistochemical analysis of mouse samples was done using uteri from 6-week-old WT female mice at every stage of the estrus cycle. In addition, one uterus from a Par-4–null female was added as a negative control. Samples were subjected to antigen retrieval in 10 mmol/L citrate buffer (pH 6.0) for 2 min in a pressure cooker. Immunohistochemical analysis was done with an anti–Par-4 polyclonal antibody (Abcam). The antibodies were detected by standard indirect immunoperoxidase procedures (LSAB, DakoCytomation).

Immunoblotting. Total protein extracts from endometrial cancer cell lines and whole mouse endometrium were obtained using radioimmunoprecipitation assay buffer supplemented with protease inhibitors (phenyl-methyl-sulfonyl fluoride, leupeptin, and pepstatin) and phosphatases (NaF and orthovanadate), and total protein was measured by a colorimetric detection assay (bicinchoninic acid protein assay; Pierce, Rockford, IL). Equal amounts of protein lysates (10–15 µg) were separated by SDS-PAGE on 10% to 12% gels and electrotransferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). Blots were blocked for 1 h at room temperature and then incubated overnight at 4°C with a primary antibody for Par-4 (Abcam) or actin (Sigma). Blots were rinsed with TBS/Tween 20 and incubated with peroxidase-conjugated secondary antibody (antimouse and antirabbit immunoglobulin G-horseradish peroxidase; Dako) for 45 min at room temperature. Protein expression was detected with enhanced chemiluminescence Western blotting reagents (Amersham Biosciences, Piscataway, NJ).

Par-4 mutational analysis. DNA was isolated from the whole tumoral sample by the standard phenol/chloroform extraction and ethanol precipitation procedure. The complete Par-4 coding sequence was screened for mutations by PCR/denaturing high-performance liquid chromatography/sequencing. PCR primer sequences are listed in Supplementary Table S1. Primers were designed using the OLIGO 4.0 program (National Biosciences, Inc., Plymouth, MN). PCR amplification was done in a 50-µL reaction volume containing 0.5 µmol/L of each oligonucleotide, 1.5 mmol/L MgCl2, 200 mmol/L deoxynucleotide triphosphate, 0.5 unit of Taq polymerase, and 50 ng of DNA. PCR conditions were as follows: one cycle of denaturation at 95°C for 5 min followed by 35 to 40 amplification cycles (denaturation, 95°C for 1 min; annealing, 53–56°C for 1 min; extension, 70°C for 1 min), and a final elongation cycle at 70°C for 10 min.

Following PCR, DNA samples were denatured at 95°C for 10 min and cooled slowly from 80°C to 25°C, decreasing at 1°C steps every 1 to 2 min in a thermal cycler, to promote heteroduplex formation, and analyzed by denaturing high-performance liquid chromatography (DHPLC 3500HT Transgenomic). Cases with abnormal migration patterns were sequenced using the ABI Prism dRhodamine dye terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems) and the ABI Prism 377 DNA Sequencer (Perkin-Elmer Biosystems). Sequencing was done in both directions with the primer used for PCR. All mutated cases were verified by repeated PCR/denaturing high-performance liquid chromatography/sequencing.

Par-4 promoter hypermethylation analysis. Methylation-specific PCR was done on bisulfite-treated DNA, as described elsewhere (26). Methylation-specific PCR primers were designed using the MethPrimer program6 to amplify a fragment of the CpG island localized between nucleotides 48 and 1,774, upstream the transcription start site of Par-4 gene (AF541923). The primer sequences are listed in Supplementary Table S1. PCR reactions were done in a 20-µL reaction volume containing 1 µmol/L of each oligonucleotide, 2 mmol/L MgCl2, 200 mmol/L deoxynucleotide triphosphate, 0.5 unit of Taq polymerase, and 50 to 100 ng of DNA. PCR conditions were as follows: one cycle of denaturation at 95°C for 5 min followed by 30 amplification cycles (denaturation, 95°C for 1 min; annealing, 52–55°C for 1 min; extension, 70°C for 1 min), and a final elongation cycle at 70°C for 10 min. To verify the results, a number of representative methylated and unmethylated products were sequenced.

Statistical analysis. Fisher's exact test was used to determine the statistical significance of the relationships between the immunohistochemical and clinicopathologic and genetic variables. The SPSS 13.0 program for Windows (SPSS, Inc., Chicago, IL) was used for this analysis. All P values were two sided.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Par-4 expression in normal endometrium. The normal human endometrium undergoes important morphologic and functional changes through the menstrual cycle under the control of ovarian hormones. We compared, using cDNA microarrays, the expression profiles of normal proliferative endometrium (n = 4), normal secretory endometrium (n = 4), and EECs (n = 24). The expression levels of Par-4 expression in normal proliferative endometrium were ~3-fold higher than in normal secretory endometrium, and, in turn, the levels of Par-4 were ~2-fold higher in normal secretory endometrium than in EEC (Fig. 1, left ; see Supplementary Table S2 for the individual values in each sample). These data were confirmed independently by quantitative RT-PCR (Fig. 1, right; see Supplementary Table S2 for the individual values in each sample).


Figure 1
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  		Figure 1. Par-4 expression in normal and cancer endometrium. Mean fold expression in endometrial samples of Par-4 by cDNA arrays (left) and quantitative RT-PCR (right). Values correspond to normal endometrium samples at the proliferative stage (NPE; n = 4) or at the secretory stage (NSE; n = 4) and to EECs (n = 24). Values are relative to the Universal Reference RNA pool (Stratagene), in the case of cDNA array data (left), or to the levels of a control RNA, which in this case was ß2-microglobulin (right). Ratios of x-fold expression are represented in the following manner: for ratios ≥1, as the direct ratio; for ratios <1, as the negative value of the inverse.

 
To further study Par-4 expression during the menstrual cycle, we did immunohistochemical analysis of normal endometrial samples corresponding to the proliferative phase (n = 7) and the secretory phase (n = 9). Supporting the above mRNA expression data, Par-4 protein was abundant during the proliferative phase with strong signals in epithelial and stromal cells (Fig. 2A ). In the case of epithelial cells, the location was mostly cytoplasmic, whereas in stromal cells, Par-4 was present both in the cytoplasm and in the nucleus. In contrast, the levels of Par-4 were clearly diminished during the secretory phase, both in epithelial and stromal cells (Fig. 2B). In accordance with the expression profile of Par-4 in the proliferative and secretory phases of the menstrual cycle, Par-4 levels changed similarly during the estral cycle of female mice. Specifically, Par-4 levels were higher in the proliferative phases of the murine estral cycle (proestrus and estrus) compared with the secretory phases (metaestrus and diestrus), both by immunohistochemistry and immunoblot analysis (Fig. 2C–E). It is worth mentioning that, in the case of murine endometrial stroma, the signal observed corresponded to infiltrating leukocytes, and this signal was not specific for Par-4 as deduced from the analysis of endometrial samples from Par-4–null mice (data not shown). The murine endometrial epithelia, both luminal and glandular, were strongly positive for Par-4, and this signal was specific as deduced from its complete absence in samples from Par-4–null mice (data not shown).


Figure 2
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  		Figure 2. Immunohistochemical analysis of Par-4 in normal endometrium. Representative examples of human normal proliferative endometrium (A; inset, higher magnification), human normal secretory endometrium (B), murine normal estrus (C), and murine normal diestrus (D). E, left, immunoblot analysis of Par-4 during the mouse endometrial cycle. Each lane corresponds to the analysis of the uterus of a single mouse. Right, quantification of the protein levels per stage of the endometrial cycle in a total of two uteri. Columns, average; bars, SD. The murine proestrus and estrus phases are conceptually similar to the human proliferative phase, and metaestrus and diestrus to the secretory phase. Bar, 200 µm.

 
The preferential expression of Par-4 during the proliferative phase of the endometrium was not anticipated, taking into account the reported proapoptotic role of Par-4 (see Introduction) and the low apoptotic rate characteristic of the proliferative endometrium, in contrast with the high apoptotic rate detected in late secretory and menstruating endometrium (27). Interestingly, a similar situation has been found for the proapoptotic protein PTEN, the levels of which are also higher during the proliferative phase of the endometrium (28). It has been proposed that Par-4, through its interaction with Akt, can be maintained in a nonfunctional state unable to exert apoptosis (15, 16). In this regard, it is relevant to mention that immunostaining against active Akt (phospho-Akt) also shows higher levels during the proliferative phase (29).

Par-4 expression in endometrial carcinomas. To corroborate the data mentioned above on the low levels of expression of Par-4 in EECs, we examined the levels of Par-4 in a series of endometrial cancer cell lines (n = 6), both by quantitative RT-PCR to measure mRNA levels and by immunoblot for protein levels. Cell lines KLE, Ishikawa, HEC-1B, and SKUT-1 presented detectable levels of Par-4 mRNA and protein, but, interestingly, no expression was detected in SKUT-1B and AN3CA (Fig. 3A and B ). We wondered whether endometrial cancer cell lines, either expressing endogenous Par-4 or not, were sensitive to the actions of Par-4 overexpression. Colony formation assays indicated that ectopic expression of Par-4 was able to decrease the proliferative potential of all the endometrial cancer cell lines (Fig. 3C).


Figure 3
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  		Figure 3. Par-4 expression in endometrial carcinoma cell lines. A, levels of Par-4 mRNA measured by quantitative RT-PCR. All values are relative to the levels of ß2-microglobulin. B, protein expression of Par-4 in different endometrial carcinoma cell lines. WT and Par-4–null mouse embryonic fibroblasts (MEFs) are included (rightmost lanes) as controls. C, antiproliferative effect of Par-4 overexpression in endometrial carcinoma cell lines. Cells were transfected with a plasmid expressing Par-4 and, 2 wks after, it was scored for the number of colonies. *, P < 0.05; **, P < 0.005 (Student's t test).

 
Based on the above data, we extended our analysis of Par-4 to a series of primary EECs (n = 100) that were analyzed by immunohistochemistry against Par-4. This series included 11 cases of poorly differentiated (G3) endometrial carcinomas. A summary of immunohistochemical and molecular features of this cohort of human EECs is presented in Supplementary Table S3. Tumors were classified as Par-4 negative when Par-4 was undetectable or minimally expressed (<10% of cells positive for Par-4). In total, ~40% of all the EECs were negative for Par-4 (Fig. 4A–C shows representative examples of Par-4–positive and Par-4–negative EECs). A similar percentage of Par-4 negative tumors was found within the subset of poorly differentiated EECs (5 of 11, 46%). Remarkably, those tumors that were Par-4 positive presented two patterns of Par-4 subcellular localization. The large majority (90%) presented a clear and apparently exclusive cytoplasmic localization (see example in Fig. 4A), whereas the rest of the Par-4–positive tumors (10%) had cytoplasmic and nuclear Par-4 localization (see example in Fig. 4B).


Figure 4
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  		Figure 4. Analysis of Par-4 in endometrial carcinomas. Representative examples of endometrial carcinomas positive for Par-4 and corresponding to carcinomas with mostly or exclusive cytoplasmic localization (A) or with both cytoplasmic and nuclear localization (B). C, example of endometrial carcinoma negative for Par-4 staining. D, identification of a nonsense mutation in the same Par-4–negative carcinoma shown in (C). Bar, 200 µm.

 
We examined whether Par-4 levels, as detected by immunohistochemistry, were associated with other molecular features of relevance (Table 1 ). Interestingly, we observed a reciprocal association between Par-4 and the ER, which was also noticeable, although not statistically significant, in the case of the PR. Recently, it has been reported that Par-4 can directly bind the androgen receptor acting as a positive transcriptional cofactor (30). Based on this and our current observations, it is tempting to speculate that Par-4 could mediate some of the actions of the ER and PR, and, consequently, the loss of Par-4 could be acting as an alternative to the loss of ER or PR in EECs.


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  		Table 1. Relationship between Par-4 expression and molecular features in EECs

 
We next asked whether Par-4 expression in EECs was related with those molecular alterations that are common in this histologic subtype, such as PTEN, KRAS, and ß-catenin mutations. Indeed, some of these pathways have been implicated in the regulation of Par-4 expression or function. For example, oncogenic Ras promotes a potent reduction of Par-4 expression (17–19). In addition, Par-4 has been proposed to be inhibited by Akt (15, 16) and, in this sense, inactivation of Par-4 could be redundant with the inactivation of PTEN. In our series of EECs, PTEN, KRAS, and ß-catenin mutations were found in 31%, 16.9%, and 10% of EEC, respectively (Supplementary Table S3). However, no association was observed between mutations of these genes and Par-4 (Table 1). It should be mentioned that some of these alterations, even if acting on the same pathways, could act cooperatively with the loss of Par-4 and not simply as alternative ways to disrupt a common molecular route. For example, mutations in PIK3CA often occur concomitantly in EECs, suggesting that they behave additively or synergistically (31).

Finally, the most striking association was found between Par-4 loss and MSI (Table 1). This is a suggestive association that merits further investigation and that could be, in part, related to a higher frequency of methylation of Par-4 in MSI tumors (see below).

Genetic and epigenetic inactivation of Par-4 in endometrial cancer. We completed our analysis of Par-4 in EECs by carrying out a detailed analysis of the genetic and epigenetic alterations that could underlie the frequent loss of Par-4 in this type of cancers. MSI is a frequent alteration in endometrial cancer that could also drive the occurrence of mutations, particularly at repetitive sequences, as it is the case for PTEN or TCF-4 (32, 33). In this regard, it is worth mentioning that exon 2 of Par-4 presents three tracks of C6 and four tracks of G5, which constitute typical hotspots for mutation in MSI tumors. We carried out a mutational analysis of the complete Par-4 coding sequence by PCR/denaturing high-performance liquid chromatography/sequencing analysis in the endometrial cancer cell lines studied before (n = 6; see Fig. 3) and in EECs (n = 69). The analysis revealed a single mutation in one EEC. The mutation consisted in a nonsense change in exon 3 of the gene [Arg (CGA) 189 -> (TGA) Stop; Fig. 4D], thus deleting the COOH-terminal half of Par-4 that contains a leucine zipper domain known to be required for interaction with the {zeta}/{lambda}PKCs and possess apoptotic activity (7, 34). Immunohistochemical analysis showed that this tumor did not have Par-4 expression (Fig. 4C). Therefore, mutations in Par-4 gene do not seem to be a frequent molecular alteration in EECs regardless of the presence or absence of MSI. However, the existence of one nonsense mutation that eliminates an important apoptotic domain of Par-4 is a valuable testimony of the tumor-suppressive role of Par-4 in this type of cancer.

Allelic losses of the Par-4 genomic region seem to be also uncommon in endometrial cancer because we did not detect this alteration by multiplex PCR consisting in specific multiplex amplification of Par-4 gene in the six endometrial cancer cell lines tested (data not shown).

Taking into account that genetic alterations did not explain changes in Par-4 expression in endometrial cancer, we next evaluated the possible role of Par-4 promoter hypermethylation in endometrial cancer cell lines and tumor samples. Par-4 promoter hypermethylation was detected in two endometrial cancer cell lines, SKUT1B and AN3CA (Fig. 5A and B ), with absence of Par-4 expression (see Fig. 3A and B). In addition, Par-4 promoter hypermethylation was detected in 19 of 59 endometrial tumors (32.2%; see Supplementary Table S3 and examples in Fig. 5C, tumors 16 and 59). The relatively low proportion of methylated DNA versus unmethylated DNA is not surprising in EECs, considering the abundant stroma that is characteristic of these tumors (see examples in Fig. 4). Importantly, there was a statistically significant association between Par-4 promoter hypermethylation and low levels of Par-4 protein (Table 1). This association, although statistically significant, is not absolute (e.g., there are Par-4–positive tumors that also show methylation of Par-4); this is a situation common in this type of analyses and it is most likely a reflection of the clonal heterogeneity of cancer. The EEC that had a nonsense mutation in Par-4 exon 3 also showed promoter hypermethylation, which could be consistent with methylation of one allele and point mutation of the other allele. Finally, it is worth mentioning that the subset of poorly differentiated EECs showed a similar incidence of Par-4 promoter hypermethylation (4 of 11, 36%).


Figure 5
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  		Figure 5. Aberrant methylation of the Par-4 promoter in endometrial carcinomas and cell lines. A, Par-4 promoter hypermethylation in endometrial cancer cell lines (U, unmethylated; M, methylated; IVD, in vitro methylated DNA; C, H2O used as negative control). B, direct sequencing of Par-4 promoter fragment (–1,053 to –973 nucleotides upstream of the transcription initiation site) amplified in AN3CA cells after bisulfite treatment. WT promoter sequence is shown on top (arrows, methylated cytosines; nonmethylated cytosines are converted into thymidines after bisulfite treatment). C, evidence of promoter hypermethylation in endometrial carcinomas. Tumors labeled with numbers 16 and 59 show evidence of promoter methylation. Endometrial carcinomas usually have an important stromal component, hence the abundance of nonmethylated promoter. D, reexpression of Par-4 mRNA measured by quantitative RT-PCR after treatment of endometrial cell lines (KLE and SKUT1 were used as control) with 5-aza-CdR (see Materials and Methods). All values are relative to the levels of ß2-microglobulin.

 
To verify the functional significance of Par-4 promoter hypermethylation as a cause of Par-4 down-regulation, we treated cells with or without methylation of Par-4 with the demethylating agent 5-aza-CdR. After 5-aza-CdR-treatment, we studied Par-4 mRNA expression by quantitative RT-PCR in treated and nontreated cells. No significant change of Par-4 mRNA level was observed in two control cell lines, KLE and SKUT1, which do not have methylated Par-4. In contrast, Par-4 mRNA was reexpressed after the demethylating treatment in cells with methylated Par-4 (i.e., SKUT1B and AN3CA), thus suggesting that the down-regulation of Par-4 mRNA was associated with methylation of the gene promoter (Fig. 5D).

Epigenetic mechanisms that result in aberrant gene expression are prominent features of many cancer types, and promoter hypermethylation is one of the main epigenetic mechanisms of gene silencing. In endometrial cancer, frequently methylated genes include RASSF1A, E-cadherin, HLTF, APC, hMLH1, and TIMP3 (35). Of special relevance is promoter hypermethylation of hMLH1 that produces the MSI phenotype in sporadic endometrial carcinomas. The frequency of methylation of the above-mentioned genes ranges between 20% and 50%, with the prominent exception of RASSF1A, which is methylated in ~80% of the EECs (35). In this context, the finding of Par-4 hypermethylation in ~32% of the EECs ranks Par-4 among the most frequently methylated genes in this type of malignancy. A connection between oncogenic signaling and Par-4 methylation has previously been proposed based on in vitro assays in which overexpression of oncogenic Ras rapidly triggers the methylation of Par-4 promoter (36). It is conceivable that oncogenic signaling, by Ras or other oncogenes, could explain, in part, the high frequency of methylation of Par-4 in endometrial cancers.

Finally, we found that Par-4 promoter hypermethylation in endometrial carcinomas was significantly more frequent in tumors with MSI (9 of 15, 60%) than in MSI-negative tumors (10 of 37, 27.0%; P = 0.05). Although we do not have an explanation for this association, it has previously been reported that promoter hypermethylation of certain genes, such as APC, also occurs more frequently among MSI-positive endometrial carcinomas (22, 37).

Concluding remarks. A role for Par-4 in endometrial cancer had previously been proposed based on the susceptibility of Par-4–deficient mice to develop endometrial cancer (14). Here, we extend this concept to human endometrial cancer. We describe that Par-4 expression is regulated along the normal hormonal cycle. More importantly, we report, for the first time, that Par-4 expression is frequently down-regulated in endometrial cancer due to promoter hypermethylation and, occasionally, by point mutation. The frequency of promoter hypermethylation ranks Par-4 among the most frequently methylated genes in this malignancy. Together, our data show that Par-4 is a tumor suppressor gene that plays a relevant role in human endometrial cancer.


   Acknowledgments
 
Grant support: Formación de Personal Investigador Fellowship from the Spanish Ministry of Education and Science (P.J. Fernandez-Marcos); Spanish Ministry of Education and Science (MEC) grant SAF2005-03018 and the European Union (INTACT, PROTEOMAGE; M. Serrano); and Fundación La Marató TV3 grant TV3/47 (J. Palacios).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Carolina Sanchez-Estavez for excellent technical collaboration and the CNIO Biotechnology Units (Immunohistochemistry and Comparative Pathology).


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

G. Moreno-Bueno and M. Collado are investigators of the Ramón y Cajal Program (2004) from the Spanish Ministry of Education and Science (MEC). P.J. Fernandez-Marcos is a predoctoral fellow of the MEC.

5 http://www.atcc.org. Back

6 http://www.urogene.org/methprimer. Back

Received 7/20/06. Revised 12/14/06. Accepted 12/27/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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