The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17.

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.

Unraveling the Mysteries of PAX8 in Reproductive Tract Cancers.

Paired Box 8 (PAX8) is a lineage-specific transcription factor that has essential roles during embryogenesis and tumorigenesis. The importance of PAX8 in the development of the reproductive system is highlighted by abnormalities observed upon the loss or mutation of this PAX family member. In cancer, PAX8 expression is deregulated in a key set of neoplasms, including those arising from the Müllerian ducts. The roles of PAX8 in oncogenesis are diverse and include epigenetic remodeling, stimulation of proliferation, inhibition of apoptosis, and regulation of angiogenesis. PAX8 can interact with different protein partners during cancer progression and may exhibit significant function-altering alternative splicing. Moreover, expression of PAX8 in cancer can also serve as a biomarker for diagnostic and prognostic purposes. In this review, we focus on the roles of PAX8 in cancers of the reproductive system. Understanding the diverse mechanisms of action of PAX8 in development and oncogenesis may identify new vulnerabilities in malignancies that currently lack effective therapies.

Genetically-defined ovarian cancer mouse models.

Epithelial ovarian cancer (EOC), the deadliest of gynaecological cancers, is a disease that remains difficult to detect early and treat efficiently. A significant challenge for researchers in the field is that the aetiology of EOC and the molecular pathways important for its development are poorly understood. Moreover, precursor lesions have not been readily identifiable, making the mechanisms of EOC progression difficult to delineate. In order to address these issues, several genetically-defined ovarian mouse models have been generated in the past 15 years. However, because of the recent suggestion that most EOCs may not originate from the ovarian surface 'epithelium', but from other tissues of the female genital tract, some models may need to be re-evaluated within this new paradigm. In this review, we examine several genetically-defined EOC models and discuss how the new paradigm may explain some of the features of these models. A better understanding of the strengths and limitations of the current EOC mouse models will undoubtedly allow us to utilize these tools to better understand the biology of the disease and develop new approaches for EOC prevention, detection, and treatment.

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.

Novel MicroRNA Reporter Uncovers Repression of Let-7 by GSK-3ß.

Several members of the let-7 microRNA family are downregulated in ovarian and other cancers. They are thought to act as tumor suppressors by lowering growth-promoting and anti-apoptotic proteins. In order to measure cellular let-7 levels systematically, we have developed a highly sensitive let-7 reporter assay system based on the expression of a chimeric mRNA that contains the luciferase coding region and a 3'-untranslated region (UTR) bearing two let-7-binding sites. In cells expressing the reporter construct, termed pmirGLO-let7, luciferase activity was high when let-7 was absent, while luciferase activity was low when let-7 levels were elevated. The ovarian cancer cell lines BG-1 and UCI-101 were transfected with the let-7 reporter and surveyed with a library of kinase inhibitors in order to identify pathways affecting let-7 activity. Among the inhibitors causing changes in endogenous let-7 abundance, the lowering of glycogen synthase kinase 3 (GSK-3)ß function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3ß increased both mature and primary-let-7 levels in BG-1 cells, and decreased BG-1 cell survival. Further studies identified p53 as a downstream effector of the GSK-3ß-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3ß as a novel therapeutic target in ovarian tumorigenesis.

Non-steroidal anti-inflammatory drugs decrease E2F1 expression and inhibit cell growth in ovarian cancer cells.

Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs) drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1.

The growth response to androgen receptor signaling in ERα-negative human breast cells is dependent on p21 and mediated by MAPK activation.

INTRODUCTION: Although a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells. METHODS: To create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells. RESULTS: We found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21. CONCLUSIONS: These studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy.

Gene expression and pathway analysis of ovarian cancer cells selected for resistance to cisplatin, paclitaxel, or doxorubicin.

BACKGROUND: Resistance to current chemotherapeutic agents is a major cause of therapy failure in ovarian cancer patients, but the exact mechanisms leading to the development of drug resistance remain unclear. METHODS: To better understand mechanisms of drug resistance, and possibly identify novel targets for therapy, we generated a series of drug resistant ovarian cancer cell lines through repeated exposure to three chemotherapeutic drugs (cisplatin, doxorubicin, or paclitaxel), and identified changes in gene expression patterns using Illumina whole-genome expression microarrays. Validation of selected genes was performed by RT-PCR and immunoblotting. Pathway enrichment analysis using the KEGG, GO, and Reactome databases was performed to identify pathways that may be important in each drug resistance phenotype. RESULTS: A total of 845 genes (p < 0.01) were found altered in at least one drug resistance phenotype when compared to the parental, drug sensitive cell line. Focusing on each resistance phenotype individually, we identified 460, 366, and 337 genes significantly altered in cells resistant to cisplatin, doxorubicin, and paclitaxel, respectively. Of the 845 genes found altered, only 62 genes were simultaneously altered in all three resistance phenotypes. Using pathway analysis, we found many pathways enriched for each resistance phenotype, but some dominant pathways emerged. The dominant pathways included signaling from the cell surface and cell movement for cisplatin resistance, proteasome regulation and steroid biosynthesis for doxorubicin resistance, and control of translation and oxidative stress for paclitaxel resistance. CONCLUSIONS: Ovarian cancer cells develop drug resistance through different pathways depending on the drug used in the generation of chemoresistance. A better understanding of these mechanisms may lead to the development of novel strategies to circumvent the problem of drug resistance.

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion.

BACKGROUND: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC. METHODOLOGY/PRINCIPAL FINDINGS: A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration. CONCLUSION/SIGNIFICANCE: Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.

Loss of Klotho during melanoma progression leads to increased filamin cleavage, increased Wnt5A expression, and enhanced melanoma cell motility.

We have previously shown that Wnt5A-mediated signaling can promote melanoma metastasis. It has been shown that Wnt signaling is antagonized by the protein Klotho, which has been implicated in aging. We show here that in melanoma cells, expressions of Wnt5A and Klotho are inversely correlated. In the presence of recombinant Klotho (rKlotho), we show that Wnt5A internalization and signaling is decreased in high Wnt5A-expressing cells. Moreover, in the presence of rKlotho, we observe an increase in Wnt5A remaining in the medium, coincident with an increase in sialidase activity, and decrease in syndecan expression. These effects can be inhibited using a sialidase inhibitor. In addition to its effects on Wnt5A internalization, we also demonstrate that Klotho decreases melanoma cell invasive potential by a second mechanism that involves the inhibition of calpain and a resultant decrease in filamin cleavage, which we demonstrate is critical for melanoma cell motility.

MicroRNAs in ovarian carcinomas.

The molecular mechanisms involved in epithelial ovarian cancer initiation and progression are just beginning to be elucidated. In particular, it has become evident that microRNAs (miRNAs or miRs), a class of molecules that post-transcriptionally regulate gene expression, play a major role in ovarian tumorigenesis. Several microRNA profiling studies have identified changes in microRNA patterns that take place during ovarian cancer development. While most deregulated microRNAs are down-regulated in cancer, and may therefore act as tumor suppressors, others are elevated and may represent novel oncogenes in this disease. A number of microRNAs identified as aberrantly expressed in ovarian carcinoma have been shown to have important functional roles in cancer development and may therefore represent targets for therapy. In addition, some of the microRNA patterns may have prognostic significance. The identification of functional targets represents a major hurdle in our understanding of microRNA function in ovarian carcinoma, but significant progress is being made. It is hoped that a better understanding of the microRNA expression and roles in ovarian cancer may provide new avenues for the detection, diagnosis, and therapy of this deadly disease.

Silencing of claudin-11 is associated with increased invasiveness of gastric cancer cells.

BACKGROUND: Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11 (CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region. METHODOLOGY/PRINCIPAL FINDINGS: Levels of CLDN11 methylation and mRNA expression were measured in primary gastric cancer tissues, noncancerous gastric mucosae, and cell lines of gastric origin using quantitative methylation-specific PCR (qMSP) and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2'-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness. CONCLUSIONS/SIGNIFICANCE: These data suggest that hypermethylation of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.

The claudins.

The claudin multigene family encodes tetraspan membrane proteins that are crucial structural and functional components of tight junctions, which have important roles in regulating paracellular permeability and maintaining cell polarity in epithelial and endothelial cell sheets. In mammals, the claudin family consists of 24 members, which exhibit complex tissue-specific patterns of expression. The extracellular loops of claudins from adjacent cells interact with each other to seal the cellular sheet and regulate paracellular transport between the luminal and basolateral spaces. The claudins interact with multiple proteins and are intimately involved in signal transduction to and from the tight junction. Several claudin mouse knockout models have been generated and the diversity of phenotypes observed clearly demonstrates their important roles in the maintenance of tissue integrity in various organs. In addition, mutation of some claudin genes has been causatively associated with human diseases and claudin genes have been found to be deregulated in various cancers. The mechanisms of claudin regulation and their exact roles in normal physiology and disease are being elucidated, but much work remains to be done. The next several years are likely to witness an explosion in our understanding of these proteins, which may, in turn, provide new approaches for the targeted therapy of various diseases.

Possible angiogenic roles for claudin-4 in ovarian cancer.

Claudin proteins are frequently overexpressed in various tumors such as breast, prostate and ovarian cancer. While their functions in cancer have not been completely elucidated, roles in survival, adhesion and invasion have been suggested. In order to clarify the roles of claudins in ovarian cancer, we have performed gene expression profiling of ovarian surface epithelial cells overexpressing claudin-4 and compared the expression patterns to the parental, non-expressing cells. Claudin-4 expression leads to the differential expression of several genes, including many that have previously been implicated in angiogenesis. In particular, angiogenic cytokines, such as IL-8, were found elevated while genes of the angiostatic interferon pathway were found downregulated. In vitro assays show that claudin-4-expressing cells produce factors that can stimulate angiogenesis as measured by tube formation and migration in HUVEC cells. In addition, an in vivo mouse dorsal skinfold assay confirms that cells expressing claudin-4 secrete factors that can mediate angiogenesis in the dorsal skin of mice. Our data suggest a novel function for claudin-4 in cancer and provide an additional rationale for its common overexpression in human tumors.

Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer.

BACKGROUND: The absence of highly sensitive and specific serum biomarkers makes mass screening for ovarian cancer impossible. The claudin proteins are frequently overexpressed in ovarian cancers, but their potential as prognostic, diagnostic, or detection markers remains unclear. Here, we have explored the possible use of these proteins as screening biomarkers for ovarian cancer detection. METHODS: Claudin protein shedding from cells was examined by immunoblotting of conditioned culture media. The presence of claudins in exosomes released from ovarian cancer cells was demonstrated by sucrose gradient separation and immunogold electron microscopy experiments. Claudin-4-containing exosomes in the plasma of ovarian cancer patients were evaluated in a pilot panel of 63 ovarian cancer patients and 50 healthy volunteers. The CA125 marker was also assessed in these samples and compared with claudin-4 positivity. RESULTS: We show that full-length claudins can be shed from ovarian cancer cells in culture and found in the media as part of small lipid vesicles known as exosomes. Moreover, 32 of 63 plasma samples from ovarian cancer patients exhibited the presence of claudin-4-containing exosomes. In contrast, only one of 50 samples from individuals without cancer exhibited claudin-4-positive exosomes. In our small panel, at a specificity of 98%, the claudin-4 and CA125 tests had sensitivities of 51% and 71%, respectively. The two tests did not appear to be independent and were strongly correlated. CONCLUSION: Our work shows for the first time that claudin-4 can be released from ovarian cancer cells and can be detected in the peripheral circulation of ovarian cancer patients. The development of sensitive assays for the detection of claudin-4 in blood will be crucial in determining whether this approach can be useful, alone or in combination with other screening methods, for the detection of ovarian cancer.

Serial analysis of gene expression reveals differential expression between endometriosis and normal endometrium. Possible roles for AXL and SHC1 in the pathogenesis of endometriosis.

BACKGROUND: Endometriosis is a clinical condition that affects up to 10% of the women of reproductive age. Endometriosis is characterized by the presence of endometrial tissues outside the uterine cavity and can lead to chronic pelvic pain, infertility and, in some cases, to ovarian cancer. METHODS: In order to better understand the pathogenesis of endometriosis, we have used Serial Analysis of Gene Expression (SAGE) to identify genes differentially in this disease by studying three endometriotic tissues and a normal endometrium sample. Promising candidates (AXL, SHC1, ACTN4, PI3KCA, p-AKT, p-mTOR, and p-ERK) were independently validated by immunohistochemistry in additional normal and endometriotic tissues. RESULTS: We identified several genes differentially expressed between endometriosis and normal endometrium. IGF2, ACTN4, AXL, and SHC1 were among the most upregulated genes. Comparison of the endometriosis gene expression profiles with the gene expression patterns observed in normal human tissues allowed the identification of endometriosis-specific genes, which included several members of the MMP family (MMP1,2,3,10,11,14). Immunohistochemical analysis of several candidates confirmed the SAGE findings, and suggested the involvement of the PI3K-Akt and MAPK signaling pathways in endometriosis. CONCLUSION: In human endometriosis, the PI3K-Akt and MAPK signaling pathways may be activated via overexpression of AXL and SHC1, respectively. These genes, as well as others identified as differentially expressed in this study, may be useful for the development of novel strategies for the detection and/or therapy of endometriosis.

Jagged-1 and Notch3 juxtacrine loop regulates ovarian tumor growth and adhesion.

Notch3 gene amplification and pathway activation have been reported in ovarian serous carcinoma. However, the primary Notch3 ligand that initiates signal transduction in ovarian cancer remains unclear. In this report, we identify Jagged-1 as the highest expressed Notch ligand in ovarian tumor cells as well as in peritoneal mesothelial cells that are in direct contact with disseminated ovarian cancer cells. Cell-cell adhesion and cellular proliferation were reduced in Notch3-expressing ovarian cancer cells that were cocultured with Jagged-1 knockdown mesothelial and tumor feeder cells. Interaction of Notch3-expressing ovarian cancer cells with Jagged-1-expressing feeder cells activated the promoter activity of candidate Notch3 target genes, and this activity was attenuated by Notch3 siRNA. Constitutive expression of the Notch3 intracellular domain significantly suppressed the Jagged-1 shRNA-mediated growth inhibitory effect. In Notch3-expressing ovarian cancer cells, Jagged-1-stimulating peptides enhanced cellular proliferation, which was suppressed by gamma-secretase inhibitor and Notch3 siRNA. Taken together, our results show that Jagged-1 is the primary Notch3 ligand in ovarian carcinoma and Jagged-1/Notch3 interaction constitutes a juxtacrine loop promoting proliferation and dissemination of ovarian cancer cells within the intraperitoneal cavity.

MicroRNA expression and identification of putative miRNA targets in ovarian cancer.

BACKGROUND: MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of cancers, and these genes are thought to function as both tumor suppressors and oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: Using microRNA microarrays, we identify several miRNAs aberrantly expressed in human ovarian cancer tissues and cell lines. miR-221 stands out as a highly elevated miRNA in ovarian cancer, while miR-21 and several members of the let-7 family are found downregulated. Public databases were used to reveal potential targets for the highly differentially expressed miRNAs. In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels. Interestingly, there was little overlap between the predicted and the experimental targets or pathways, or between experimental targets/pathways obtained using different cell lines, highlighting the complexity of miRNA target selection. CONCLUSION/SIGNIFICANCE: Our results identify several differentially expressed miRNAs in ovarian cancer and identify potential target transcripts that may be regulated by these miRNAs. These miRNAs and their targets may have important roles in the initiation and development of ovarian cancer.

Claudin-3 and claudin-4 expression in serous papillary, clear-cell, and endometrioid endometrial cancer.

OBJECTIVE: Tight junction (TJ) proteins claudin-3 and claudin-4 may be differentially expressed in uterine serous papillary carcinoma (USPC), a rare form of endometrial cancer characterized by a particularly poor prognosis. Our aim was to determine the expression pattern and prognostic relevance of claudin-3 and claudin-4 in a large cohort of endometrial cancer patients of diverse histological type and stage. METHODS: Claudin-3 and claudin-4 expression was studied in a cohort of 287 patients with endometrial cancer including 137 cases of USPC or clear-cell histology using immunohistochemistry. Patients were completely surgically staged. Outcome data is available on all 287 patients. RESULTS: The rate of claudin-3 and claudin-4 expression was significantly higher in USPC and clear-cell endometrial cancer compared to endometrioid endometrial cancer (claudin-3: 78% and 61% versus 38%, p<.0001; claudin-4: 56% and 44% versus 9%, p<.0001). Furthermore, expression of both TJ proteins was significantly associated with poor clinical outcome (claudin-3, DFS RR 1.70, p=.0087, OS RR 1.62, p=.0247; claudin-4, DFS RR 2.66, p<0.0001, and OS RR 2.50, p<0.0001). However, both markers did not maintain prognostic independence in multivariate analyses, as their expression was tightly associated with more advanced disease stages (p<.0001 for both), and higher nuclear grade (p<.0001 for both). CONCLUSION: These clinical observations confirm the hypothesis based on preclinical evidence that increased expression of claudin-3 and claudin-4 may contribute to the aggressive phenotype of endometrial cancer of serous papillary or clear-cell histology and suggest their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.