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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.

Supplementary Figure 1 from Unraveling the Mysteries of PAX8 in Reproductive Tract Cancers

Supplementary Table 1, Figures 1-7 from 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 γ-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. [Cancer Res 2008;68(14):5716–23]

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.

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 anti a ngiogenic effects. The findings reveal a non–cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.

Worldwide, the number of new ovarian cancer cases approaches 300,000 with more than 180,000 deaths every year. The low survival-rate reflects the limitations of current therapies and highlights the importance of identifying new therapeutic targets. Despite significant recent efforts to identify novel vulnerabilities in ovarian cancer, none have led to effective durable therapies with improvement in overall survival. PAX8, a lineage-transcription factor, whose expression is a major molecular feature of ovarian carcinomas, represents a novel therapeutic target. Herein, we have identified SOX17 as a bona fide PAX8-interacting partner and elucidated the impact of this interaction on the development of ovarian cancer. Importantly, we found that PAX8 and SOX17 regulate tumor angiogenesis in vitro and in vivo. The role of PAX8 and SOX17 in the regulation of angiogenesis reveals a novel function for these factors in regulating the tumor microenvironment and highlight this pathway as a viable therapeutic target.

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 major problem in the treatment of ovarian cancer is the heterogeneity among ovarian tumors. DNA and RNA sequencing studies have demonstrated both intertumoral and intratumoral genetic variation. Despite efforts to elucidate common signaling pathways among various ovarian cancer subtypes, few have led to meaningful patient stratification or individualized targeted molecular therapies. A theoretical new approach to the treatment of ovarian cancer would be to target the signaling pathways that are essential to the development of the HGSOC progenitor cells, the secretory epithelium of the fallopian tube (FT). PAX8, a transcription factor that identifies and sustains high-grade serous ovarian carcinomas (HGSOC), is also the main regulator of FT development. We investigated whether blocking the ability of PAX8 to influence its gene targets, either by interrupting PAX8 protein-protein interactions or by inhibiting the products of PAX8-driven signaling, has the potential to eliminate the primary growth stimulus for high-grade serous tumors. Herein, we have identified SOX17 as a bona fide PAX8-interacting partner and elucidated their collaborative impact on HGSOC. We observed that PAX8 and SOX17 are master regulators of HGSOC identity, as both were found binding in super-enhancer regions regulating most of the same set of genes. Ontology analysis after PAX8 or SOX17 loss showed alteration in three main pathways: cell cycle, apoptosis, and angiogenesis. Most remarkably, we discovered that PAX8 and SOX17 regulate angiogenesis in vitro and in vivo by suppressing SERPINE1, which enables VEGFR2 signaling pathway. These results broaden our understanding of the roles of PAX8 and SOX17 in the regulation of angiogenesis and suggest novel therapeutic strategies that could overcome ovarian cancer heterogeneity and resistance. Citation Format: Daniele Chaves-Moreira, Marilyn A. Mitchell, Jessica Rendi, Robbin Nameki, Donita C. Brady, Simone Sidoli, Benjamin Garcia, Patrice J. Morin, Kate Lawrenson, Ronny Drapkin. PAX8 drives ovarian cancer angiogenesis through interaction with SOX17 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A37.

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 mullerian-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.

See related article by Sparks et al., [Cancer Res 1998;58:1130–4][1] . Visit the Cancer Research 75th Anniversary [timeline][2]. In the mid 1990s, the human genome project was well underway, but it would still be more than 5 years before the first draft of the human genome was published and more

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.

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7+ and CCR5+ fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARγ2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.

Tight junctions (TJs) play crucial roles in tissue homeostasis and inflammation through their roles in the control of paracellular transport and barrier function. There is evidence that these functions are compromised in older organisms, but the exact mechanisms leading to TJ deterioration are not well understood. Claudin proteins are a family of membrane proteins that constitute the structural barrier elements of TJs and therefore play a major role in their formation and function. Using immunohistochemistry and immunoblotting, we have studied the expression of six different claudin proteins (claudin-1, -2, -3, -4, -5, and -7) in three tissues (liver, kidney, and pancreas) of aging male and female mice. In general, we find an age-dependent decrease in the expression of several claudin proteins in all three tissues observed, although the exact changes are tissue specific. Our findings provide a possible basis for the decrease in tissue barrier function in older organisms.

The tight junction (TJ) proteins claudin-3 and claudin-4 have been reported to be differentially expressed in uterine serous papillary carcinoma (USPC), a rare form of endometrial cancer characterized by a particularly high recurrence rate and poor prognosis. Preclinical experiments suggest that increased expression of both TJ proteins may in part mediate the biologically aggressive phenotype of USPC. Our aim was to determine claudin-3 and claudin-4 expression in a large cohort of surgically staged patients with USPC and clear cell endometrial cancer (n=137), and to compare the expression pattern and prognostic relevance of both claudins with that seen in patients with endometrioid endometrial cancer (n=150). 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

Claudin proteins belong to a large family of transmembrane proteins essential to the formation and maintenance of tight junctions (TJs). In ovarian cancer, TJ protein claudin-4 is frequently overexpressed and may have roles in survival and invasion, but the molecular mechanisms underlying its regulation are poorly understood. In this report, we show that claudin-4 can be phosphorylated by protein kinase C (PKC) at Thr189 and Ser194 in ovarian cancer cells and overexpression of a claudin-4 mutant protein mimicking the phosphorylated state results in the disruption of the barrier function. Furthermore, upon phorbol ester-mediated PKC activation of OVCA433 cells, TJ strength is decreased and claudin-4 localization is altered. Analyses using PKC inhibitors and siRNA suggest that PKCepsilon, an isoform typically expressed in ovarian cancer cells, may be important in the TPA-mediated claudin-4 phosphorylation and weakening of the TJs. Furthermore, immunofluorescence studies showed that claudin-4 and PKCepsilon are co-localized at the TJs in these cells. The modulation of claudin-4 activity by PKCepsilon may not only provide a mechanism for disrupting TJ function in ovarian cancer, but may also be important in the regulation of TJ function in normal epithelial cells.

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.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

Recent studies have suggested an oncogenic role of the BTB/POZ-domain genes in hematopoietic malignancy. The aim of this study is to identify and characterize BTB/POZ-domain genes in the development of human epithelial cancers, i.e., carcinomas. In this study, we focused on ovarian carcinoma and analyzed gene expression levels using the serial analysis of gene expression (SAGE) data in all 130 deduced BTB/POZ genes. Our analysis reveals that NAC-1 is significantly overexpressed in ovarian serous carcinomas and several other types of carcinomas. Immunohistochemistry studies in ovarian serous carcinomas demonstrate that NAC-1 is localized in discrete nuclear bodies (tentatively named NAC-1 bodies), and the levels of NAC-1 expression correlate with tumor recurrence. Furthermore, intense NAC-1 immunoreactivity in primary tumors predicts early recurrence in ovarian cancer. Both coimmunoprecipitation and double immunofluorescence staining demonstrate that NAC-1 molecules homooligomerize through the BTB/POZ domain. Induced expression of the NAC-1 mutant containing only the BTB/POZ domain disrupts NAC-1 bodies, prevents tumor formation, and promotes tumor cell apoptosis in established tumors in a mouse xenograft model. Overexpression of full-length NAC-1 enhanced tumorigenicity of ovarian surface epithelial cells and NIH 3T3 cells in athymic nu / nu mice. In summary, NAC-1 is a tumor recurrence-associated gene with oncogenic potential, and the interaction between BTB/POZ domains of NAC-1 proteins is critical to form the discrete NAC-1 nuclear bodies and essential for tumor cell proliferation and survival.

Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.

Claudin proteins form a large family of integral membrane proteins crucial for tight junction formation and function. Our previous studies have revealed that claudin-3 and claudin-4 proteins are highly overexpressed in ovarian cancer. To clarify the roles of claudins in ovarian tumorigenesis, we have generated human ovarian surface epithelial (HOSE) cells constitutively expressing wild-type claudin-3 and claudin-4. Expression of these claudins in HOSE cells increased cell invasion and motility as measured by Boyden chamber assays and wound-healing experiments. Conversely, small interfering RNA (siRNA)–mediated knockdown of claudin-3 and claudin-4 expression in ovarian cancer cell lines reduced invasion. Claudin expression also increased cell survival in HOSE cells but did not significantly affect cell proliferation. Moreover, the claudin-expressing ovarian epithelial cells were found to have increased matrix metalloproteinase-2 (MMP-2) activity indicating that claudin-mediated increased invasion might be mediated through the activation of MMP proteins. However, siRNA inactivation of claudins in ovarian cancer cell lines did not have a significant effect on the high endogenous MMP-2 activity present in these cells, showing that malignant cells have alternative or additional pathways to fully activate MMP-2. Taken together, our results suggest that claudin overexpression may promote ovarian tumorigenesis and metastasis through increased invasion and survival of tumor cells.

Malignant pleural mesothelioma (MPM) is a fatal neoplasm with no acceptable curative approaches. We used serial analysis of gene expression (SAGE) to compare the gene expression pattern of a surgically resected MPM to the autologous normal mesothelium. Intelectin gene overexpression (>139-fold) was found in the tumor. Online SAGE datasets revealed intelectin to be consistently present in mesothelioma(s), ovarian cancer, and colon cancer. Intelectin mRNA expression was found by RT-PCR in 4 of 5 resected MPM tumors, and Intelectin protein expression was confirmed by immunohistochemistry in 28 of 53 MPM tumors, and in 4 of 4 mesothelioma cell lines studied by Western blot. A marked induction in intelectin gene expression was observed among human primary mesothelial cells as a consequence of crocidolite asbestos exposure and simian virus 40 infection. Intelectin overexpression in mesothelioma could have potential screening, and therapeutic implications.

The platinum compounds cisplatin and carboplatin are commonly used in cancer chemotherapy. However, tumors frequently develop resistance to these compounds, significantly decreasing their usefulness in the clinic. In the past few years, basic research has unraveled novel and unexpected mechanisms for the development of platinum resistance. For example, it has been reported that MUC1 expression and particularly the localization of its C-terminal subunit to the mitochondria may affect cisplatin resistance. Another recent finding suggests that cisplatin damage may activate DNA-dependent protein kinase (DNA-PK) to initiate a death signal that can be transmitted to neighboring cells through gap junctions, adding to a growing belief that the interactions of cancer cells with their surroundings may be important to the outcome of chemotherapy. While most clinical efforts have focused on identifying alternative regimens for drug-resistant cancer, it might be possible to exploit our knowledge of the mechanism of platinum resistance to specifically reverse resistance and increase platinum efficacy. The strategy of drug resistance reversal therapy (DRRT) may have significant impact on our approaches to the treatment and management of drug-resistant tumors.

In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.

A better understanding of changes in gene expression during ovarian tumorigenesis and the identification of specific tumor markers may lead to novel strategies for diagnosis and therapy for this disease. Using our serial analysis of gene expression (SAGE) data, as well as public SAGE databases that contained a total of 137 SAGE libraries representing a wide variety of normal and neoplastic tissues, we identified five novel SAGE tags specifically expressed in ovarian cancer. Database analysis, cloning and, sequencing of the corresponding expressed sequence tags revealed details about these transcripts that we named human ovarian cancer-specific transcripts (HOSTs). HOST1 was found to be identical to the gene encoding ovarian marker CA125 (MUC16). HOST2 is a novel gene containing multiple copies of retroviral-related sequences without an obvious open reading frame. HOST3 encodes the tight-junction protein claudin-16 (CLDN16). HOST4 encodes a poorly characterized proteoglycan link protein (LP), and HOST5 codes for a type II sodium-dependent phosphate transporter (SLC34A2). Except for MUC16, these genes have not previously been shown to be expressed in ovarian or other cancers. Northern blot analysis confirmed that HOST genes are rarely expressed in normal tissues or nonovarian cancers, but are frequently expressed in ovarian cancer-derived cell lines and primary tumors. Moreover, HOST genes are upregulated in all four major subtypes of ovarian cancer compared to cultivated ovarian surface epithelial cells, as concluded by real-time reverse transcription (RT)-PCR using a panel of microdissected ovarian tumors. The sodium-dependent phosphate transporter (HOST5/SLC34A2) expression was associated with increased differentiation in ovarian serous tumors. While the roles of HOSTs in ovarian malignant transformation remain unclear, we propose that HOSTs may represent alternative targets for diagnosis and therapy and of this deadly disease.

Background & Aims: Enterotoxigenic Bacteroides fragilis that secrete a zinc-dependent metalloprotease toxin termed the B. fragilis toxin (BFT) have been associated with acute diarrheal disease. BFT rapidly cleaves the extracellular domain of E-cadherin, leading to the complete degradation of the E-cadherin protein. E-cadherin is the primary intercellular adhesion protein of the zonula adherens, and its cytoplasmic domain associates with the nuclear signaling protein β-catenin. The goal of this study was to examine if BFT triggers β-catenin nuclear signaling in intestinal epithelial cells. Methods: Cell biologic and biochemical techniques were combined to address β-catenin nuclear signaling stimulated by BFT. Results: Loss of membrane-associated E-cadherin after BFT treatment of human colonic epithelial cells (HT29/C1 clone) triggers β-catenin nuclear localization within 3 hours. Subsequently, c- myc transcription and translation are induced and persistent cellular proliferation ensues, mediated in part by β-catenin/T-cell factor–dependent transcriptional activation. Cellular proliferation is stimulated by as little as 5 × 10 −10 mol/L BFT. Conclusions: To our knowledge, BFT is the first bacterial toxin reported to activate T-cell factor–dependent β-catenin nuclear signaling in intestinal epithelial cells. These results suggest that genetic evolution of this common colonic commensal has rendered an organism with the potential to contribute to oncogenic transformation in the colon.

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in human breast cancer. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes involved in lipid metabolism, including FAS. SREBP-1c expression is induced in liver and adipose tissue by insulin and by fasting/refeeding and is critical for nutritional regulation of lipogenic gene expression. In contrast, upregulation of fatty acid metabolism during in vitro transformation of human mammary epithelial cells and in breast cancer cells was driven by increased MAP kinase and PI 3-kinase signaling, which increased SREBP-1 levels. SREBP-1a was more abundant than SREBP-1c in many proliferative tissues and cultured cells and was thus a candidate to regulate lipogenesis for support of membrane synthesis during cell growth. We now show that SREBP-1c and FAS mRNA were both increased by H-ras transformation of MCF-10a breast epithelial cells and were both reduced by exposure of MCF-7 breast cancer cells to the MAP kinase inhibitor, PD98059, or the PI 3-kinase inhibitor, wortmannin, while SREBP-1a and SREBP-2 showed less variation. Similarly, the mRNA levels for FAS and SREBP-1c in a panel of primary human breast cancer samples showed much greater increases than did those for SREBP-1a and SREBP-2 and were significantly correlated with each other, suggesting coordinate regulation of SREBP-1c and FAS in clinical breast cancer. We conclude that regulation of FAS expression in breast cancer is achieved through modulation of SREBP-1c, similar to the regulation in liver and adipose tissue, although the upstream regulation of liopgenesis differs in these tissues.

A gene's expression pattern provides clues to its role in normal physiology and disease. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project (CGAP) uses serial analysis of gene expression (SAGE). Over 5 million transcript tags from more than 100 human cell types have been assembled. To enhance the utility of this data, the CGAP SAGE project created SAGE Genie, a web site for the analysis and presentation of SAGE data ( http://cgap.nci.nih.gov/ SAGE). SAGE Genie provides an automatic link between gene names and SAGE transcript levels, accounting for alternative transcription and many potential errors. These informatics advances provide a rapid and intuitive view of transcript expression in the human body or brain, displayed on the SAGE Anatomic Viewer. We report here an easily accessible view of nearly any gene's expression in a wide variety of malignant and normal tissues.

Since its discovery as a protein associated with the cytoplasmic region of E-cadherin, beta-catenin has been shown to perform two apparently unrelated functions: it has a crucial role in cell-cell adhesion in addition to a signaling role as a component of the Wnt/wg pathway. Wnt/wg signaling results in beta-catenin accumulation and transcriptional activation of specific target genes during development. It is now apparent that deregulation of beta-catenin signaling is an important event in the genesis of a number of malignancies, such as colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer. beta-catenin mutations appear to be a crucial step in the progression of a subset of these cancers, suggesting an important role in the control of cellular proliferation or cell death. The APC/beta-catenin pathway is highly regulated and includes players such as GSK3-beta, CBP, Groucho, Axin, Conductin, and TCF. c-MYC and cyclin D1 were recently identified as a key transcriptional targets of this pathway and additional targets are likely to emerge. Published 1999 John Wiley & Sons, Inc.

Inactivation of the adenomatous polyposis coli ( APC ) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by β-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of β-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of β-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or β-catenin.

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.

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)β function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3β 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-3β-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3β as a novel therapeutic target in ovarian tumorigenesis.

The I kappa B proteins bind to Rel/NF-kappa B transcription factors and modulate their activities. Although originally described only as cytoplasmic inhibitors of Rel/NF-kappa B transcription complexes, it is now clear that I kappa B proteins also have other functions.

Synthetic antisense oligonucleotides have been used to inhibit specific protein synthesis in vivo. Antisense oligonucleotides directed to kinesin heavy chain were injected into the vitreous of anesthetized rabbits in order to assess the effects on transport in the retinal ganglion cells whose axons form the optic nerve. The antisense oligonucleotide specifically inhibited retinal kinesin synthesis by 82 +/- 7% (n = 4). The rapid axonal transport of the membrane proteins into the optic nerve was concomitantly inhibited by 70 +/- 10% (n = 4). These results provide direct evidence for the specific role of kinesin in rapid anterograde transport in vivo and indicate the utility of antisense oligonucleotides to explore neuronal dynamics in a specific neuronal cell type in a living animal.

I kappa B proteins regulate Rel/NF-kappa B transcription complexes through a direct protein-protein interaction. In addition, we have previously shown that certain I kappa B proteins (I kappa B alpha and I kappa B gamma) can act as activators of transcription when fused to the DNA-binding domain of GAL4. We now show that a mutant chicken I kappa B alpha protein that cannot interact with Rel proteins in vitro did not activate transcription when fused to GAL4 in chicken embryo fibroblasts (CEF) and Saccharomyces cerevisiae, and did not inhibit growth in yeast; in contrast, an I kappa B alpha mutant that can still interact in vitro with Rel proteins activated transcription in both CEF and yeast and inhibited growth in yeast. In CEF, GAL4-I kappa B alpha mediated transcription activation was inhibited by co-transfection with an expression vector for a RelA (p65) protein that contained sequences needed for interaction with I kappa B alpha but that was deleted of its transcription activation domain. Therefore, it appears that GAL4-I kappa B alpha activates transcription by interacting with an endogenous Rel family protein in CEF. In contrast, the activation domain from I kappa B gamma behaved as a genuine acidic activator of transcription and did not inhibit growth when expressed in yeast. Since transcription activation and growth inhibition by GAL4-I kappa B alpha mutants in yeast correlated with their ability to interact with vertebrate Rel proteins, our results suggest that these activities of GAL4-I kappa B alpha are mediated through interaction with a Rel-like protein in yeast, which is important for cell growth.

The beneficial effects of caloric restriction in increasing longevity and forestalling age-related diseases are well known. Dietary restriction of methionine also renders similar benefits. We recently showed in a renal epithelial cell culture system that reduction of culture medium methionine by 80% resulted in altered tight junctional (TJ) claudin composition and also improved epithelial barrier function ( 51 ). In the current study, we examined the effect of dietary restriction of methionine on TJ barrier function in rat gastrointestinal tissue to see whether this phenomenon also holds true in a tissue model and for a different epithelial cell type. After 28 days on methionine-restricted (MR) diet, rats showed small but significant reductions in the plasma and (intracellular) colonocyte levels of methionine. Colon mucosal sheets from rats on the MR diet showed increased transepithelial electrical resistance with concomitant decrease in paracellular diffusion of14C-d-mannitol, suggesting improved barrier function relative to rats on control diet. This improved barrier function could not be explained by changes in colon crypt length or frequency. Neither was the colonocyte mitotic index nor the apoptotic frequency altered significantly. However, TJ composition/structure was being altered by the MR diet. RT-PCR and Western blot analysis showed an increase in the abundance of claudin-3 and an apparent change in the posttranslational modification of occludin, data reinforcing a paracellular barrier alteration. Overall, our data suggest that reduction in dietary intake of methionine results in improved epithelial barrier function by inducing altered TJ protein composition.

We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.

The p50 subunit of the NF-kappa B transcription complex is derived from the N-terminal half of a larger precursor protein, p105. Although a fair amount is known about functions located within the p50 sequences, less is known about the C-terminal half of p105. In this report, we have identified a potent transcription activation domain located in the C terminus of mouse p105. In addition, the I kappa B beta proteins chicken p40 and human MAD-3, proteins that are related to the p105 C terminus, strongly activated transcription in chicken cells and yeast when fused to GAL4 DNA-binding sequences. Furthermore, chicken p40 is primarily located in the nucleus of chicken cells when overexpressed from a retroviral vector. Our results suggest novel models for the function and regulation of NF-kappa B transcription complexes.

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7(+) and CCR5(+ )fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARgamma2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.

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.

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-29-deoxycytidine restored CLDN11 expression. Moreover, siRNAmediated 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.

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 down-regulated. 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.

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.

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.