Search

author, Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis^1, ^2, ^3, ^4. It represents an attractive therapeutic target^5, ^6 in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy^7. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis^8, ^9. In HIF-1_α knockdown DLD-1 colon cancer cells (DLD-1^HIF-kd), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1_α (DLD-1^HIF-wt). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1^HIF-kd but not DLD-1^HIF-wt cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-_KB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1^HIF-kd but not DLD-1^HIF-wt xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1α may be most effective when IL-8 is simultaneously targeted.

Interleukin-18 (IL-18), a member of the IL-1 family, is known to play a relevant role in rheumatoid arthritis (RA) physiopathology mainly by promoting the inflammatory response. The aim of this work was to investigate the possible implication of two single-nucleotide polymorphisms (SNPs) [−607 A/C (rs1946518) and −137 G/C (rs187238)] within the IL-18-promoter region in RA predisposition and clinical course. A total of 362 unrelated RA patients and 339 healthy controls were genotyped using a real-time polymerase chain reaction (PCR) method for the −607 A/C SNP and a sequence-specific PCR method (PCR-SSP) for the −137 G/C polymorphism. No statistically significant differences were observed for both −607 and −137 IL-18-promoter polymorphisms between RA patients and controls, considering either allelic or genotypic frequencies. In addition, no association was found with the haplotypes inferred by the two polymorphisms and RA susceptibility. This was also the case when RA patients were stratified according to sex, age at the onset of the disease, rheumatoid factor status, and extraarticular manifestations. Our data suggest that −607 A/C (rs1946518) and −137 G/C (rs187238) polymorphisms within the IL-18-promoter region do not play a major role in RA predisposition.

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P0.05), but again, EDN1 secretion was unchanged (P0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P0.05), but MLC produced less progesterone in the presence of ActPBMC (P0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

Mullerian Inhibiting Substance (MIS), a 140-kDa homodimer glycoprotein member of the TGF-β superfamily of biological-response modifiers, causes regression of the Mullerian ducts in developing male embryos. MIS also can induce growth arrest and apoptosis in ovarian and cervical cancer cell lines. The embryonic progenitor of the ovarian and cervical epithelium is the coelomic epithelium, the same tissue that regresses under the direction of MIS in the male. The endometrium and uterus also arise from the coelomic epithelium and the Mullerian ducts. Here, we show that both normal human endometrium and endometrial cancers express the receptor for MIS and that MIS can inhibit the proliferation of a number of human endometrial cancer cell lines that express the MIS type II receptor. In the representative endometrial cancer cell line AN3CA, MIS affects the expression of key cell-cycle regulatory proteins. This work broadens the scope of tumors that MIS can potentially control and, by elucidating the MIS signaling pathway, identifies other potential avenues for intervention.

Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of beta3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and beta3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.

The aim of this work was to investigate the possible influence of the recently described CT60 A/G dimorphism of the CTLA4 (cytotoxic T-lymphocyte antigen 4) gene in the susceptibility to two different autoimmune inflammatory intestinal disorders, inflammatory bowel disease (IBD) and celiac disease. We analyzed a case-control cohort composed of 528 Spanish patients with IBD (284 with Crohn disease and 244 with ulcerative colitis) and 454 unrelated healthy individuals, and additionally a group of 90 celiac disease families. CT60 genotyping was performed with a TaqMan 5' allelic discrimination assay. After comparing patients with IBD with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CTLA4/CT60 dimorphism. In addition, by means of familial and case-control analysis, no evidence for a statistically significant association was observed between CTLA4/CT60 and celiac disease susceptibility. Therefore, our results suggest that the CTLA4/CT60 polymorphism does not play a major role in inflammatory intestinal disorders.

In the present work, we investigate the possible effect of a CAn microsatellite polymorphism in the nuclear factor kappa B1 (NFKB1) gene on predisposition to celiac disease (CD). Seventy-eight Spanish families with CD were genotyped using a polymerase chain reaction (PCR)-fluorescent method, and the transmission patterns of different CAn alleles were analysed. Furthermore, in order to type the CAn polymorphism more accurately, samples between 126 and 144 bp were cloned and sequenced. A trend of association with the 132-bp allele was found (P = 0.02). This allele was more frequently transmitted to affected sibs, although the results of statistical tests were not significant after correction for multiple comparisons. After sequencing, we found that the 132-, 138- and 142-bp alleles had two As at the end of the CA microsatellite, with the other alleles presenting the described pattern (NCB1 nucleotide U60337) for the microsatellite repeats. These results suggest that the NFKB1 CAn microsatellite does not play a major role in CD susceptibility. In addition, a more detailed molecular characterization of the CA microsatellite is described.

Apoptosis, a form of cell death responsible for eliminating cells from the body that are damaged, senescent, potentially harmful, or no longer useful, plays a significant role in almost all aspects of ovarian development and function in vertebrate and invertebrate species. Through the years, studies of laboratory animal models and human tissues have begun to assimilate a molecular blueprint of the hormones, genes, and pathways that modulate apoptosis in ovarian germ cells and somatic cells, leading to the prospects of developing new treatments for improving reproductive health and fertility in women. In addition, significant progress has been made in elucidating a central, and perhaps somewhat surprising, role for the physiological cell death program of apoptosis in mediating pathological ovarian damage caused by exposure of women to a host of environmental, occupational, and clinical insults. However, a considerable number of the observations published to date with respect to the regulation of apoptosis in the ovary are based on correlative gene expression analyses or on the provision of single agents to isolated ovarian cells or follicles cultured in vitro. We know comparatively little of the hormones, genes, and pathways that are functionally relevant to the modulation of ovarian cell death in vivo, or of how these hormones, genes, and pathways interact to coordinate things such as granulosa cell apoptosis during follicle atresia and oocyte depletion during fetal and postnatal life. These concepts and a broad overview of the apoptosis program and its control are presented in this chapter.

Cables, a cyclin-dependent kinase (cdk) interacting protein, has recently been identified and mapped to human chromosome 18q11. Cables appears to be primarily involved in cell cycle regulation and cell proliferation. Overexpression of Cables in Hela and other cell lines inhibits cell proliferation and tumor formation. We hypothesize that loss of Cables expression is associated with ovarian cancer. To test our hypothesis, we examined Cables expression in the four most common subtypes of ovarian carcinomas: serous, endometrioid, mucinous, and clear cell. In addition, mucinous and serous borderline tumors were also included. Loss of Cables expression was observed at high frequency in ovarian serous (11 of 14 cases, 79%) and endometrioid (5 of 10 cases, 50%) carcinomas. In contrast, strong Cables staining was detected in all clear cell carcinomas (10 cases) and mucinous tumors (5 carcinomas and 5 borderline tumors). The majority of serous borderline tumors (11 of 14 cases, 79%) showed positive Cables staining, with the rest showing focal loss of Cables expression. Furthermore, RT-PCR revealed the lack of Cables mRNA in a human ovarian cancer xenograft. No correlation was noted between loss of Cables and histologic grade, tumor stage, and survival. In conclusion, our results indicate that loss of Cables is common in ovarian serous and endometrioid carcinomas and imply that Cables may be involved in the pathogenesis of these two types of ovarian carcinomas.

Objective The aim of this study was to analyze the role of the major histocompatibility complex class I chain-related gene A (MICA) transmembrane polymorphism in celiac disease (CD) susceptibility. Methods Sixty-one celiac Spanish families were genotyped for MICA transmembrane polymorphism by a polymerase chain reaction method combined with fluorescent technology. A transmission disequilibrium test was performed to investigate the preferential transmission of MICA alleles to the affected offspring. Results The MICA A5.1 allele was shown to be significantly transmitted to the affected siblings. This association was independent of the CD-predisposing DQ2 haplotype. Additionally, we classified our celiac families into typical and atypical groups as we found a significant association with MICA A5.1 in typical celiac families. There was also an association tendency with atypical families. Conclusions Our data suggest that the MICA A5.1 allele is associated with CD development independently of DQ2-extended haplotype and clinical forms of CD.

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F2α (PGF2α) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways (e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.

Fas ligand (FasL) is implicated as a mediator of luteolysis. However, a gap exists in our understanding of the Fas-mediated signaling mechanisms that are involved in either the loss of progesterone production or the structural regression of the corpus luteum. In the present study we investigated the acute and chronic effects of FasL with respect to activation of cytokine/stress-induced signaling pathways and apoptosis in bovine steroidogenic cells. More specifically, we investigated soluble FasL (sFasL)-activated production of ceramide, a second messenger of the sphingomyelin pathway, and activation of p38(MAPK), a member of the MAPK family. sFasL activated the sphingomyelin pathway, as evidenced by a 2-fold increase (P0.05) in the production of ceramide. Pretreatment with imipramine (50 micro M), an inhibitor of acid sphingomyelinase activity, attenuated (75%; P0.05) sFasL-induced ceramide production, suggesting that the increase in ceramide was partially the result of acid sphingomyelinase-mediated hydrolysis of sphingomyelin. Treatment of luteal cells with sFasL or a cell-permeable ceramide analog (C8) for 24-48 h resulted in a significant increase (P0.05) in apoptosis. Western blot analysis revealed that sFasL had little effect on the activation of p38(MAPK) in primary bovine luteal steroidogenic cells. Furthermore, pretreatment with the p38(MAPK) inhibitor SB203580 failed (P0.05) to inhibit sFasL- or C8-induced death. Although sFasL did not alter basal progesterone levels detected in the culture medium, C8 caused a significant increase (P0.05) in progesterone concentrations within the medium. Collectively, these data suggest that the role of FasL in luteolysis may be to activate the stress-induced sphingomyelin pathway that, in turn, serves as a mediator of apoptosis.

Ovarian cancers are thought to result from the accumulation of multiple genetic aberrations that transform ovarian and/or fallopian tube surface epithelial cells, allowing for their abnormal growth, proliferation and metastasis. We carried out genome-wide copy-number analysis using array comparative genomic hybridization on a panel of mouse ovarian cancer (OVCA) cell lines previously established in our laboratory. We identified a recurrent focal amplification on mouse chromosomal region 2qB, which contains the LIM homeodomain containing transcription factor 1B (Lmx1b) gene. LMX1B is not expressed in normal human ovary, but is expressed in many human OVCA cell lines and primary tumors. High expression of LMX1B correlates with poor outcome. To clarify the role of LMX1B in ovarian carcinogenesis, we transduced LMX1B into a panel of mouse and human OVCA cell lines and demonstrated that LMX1B strongly promotes migration of cancer cells in culture and accelerates xenograft growth in nude mice. Conversely, knockdown of LMX1B in a human cell line with endogenous high expression of LMX1B inhibits cell migration in vitro and tumor growth in vivo. Microarray analysis of cells overexpressing LMX1B identified NF-κB pathway as a potential mediator of tumor progression and subsequent treatment of NF-κB inhibitor decreased the migratory capacity of these cells. Thus, our data demonstrate that LMX1B functions as an oncogene in OVCA pathogenesis. Citation Format: Gayatry Mohapatra, Lei He, Lankai Guo, Vinod Vathipadiekal, Petra Sergent, Whitfield Growdon, Bo Rueda, David Engler, Sandra Orsulic, Michael Birrer. Identification of LMX1B as a novel oncogene in human ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1534. doi:10.1158/1538-7445.AM2014-1534

Purpose:Uterineserouscarcinoma(USC)isanaggressivesubtypeofendometrialcancerthatcommonly harbors HER2 gene amplification. We investigated the effectiveness of HER2 inhibition using lapatinib and trastuzumab in vitro and in xenografts derived from USC cell lines and USC patient-derived xenografts. Experimental Design: Immunohistochemistry and FISH were performed to assess HER2 expression in 42 primary USC specimens. ARK1, ARK2, and SPEC2 cell lines were treated with trastuzumab or lapatinib. Cohorts of mice harboring xenografts derived from ARK2 and SPEC2 cell lines and EnCa1 and EnCa2 primaryhumanUSCsamplesweretreatedwitheithervehicle,trastuzumab,lapatinib,orthecombinationof trastuzumab and lapatinib. Acute and chronic posttreatment tumor samples were assessed for downstream signaling alterations and examined for apoptosis and proliferation. Results: HER2 gene amplification (24%) correlated significantly with HER2 protein overexpression (55%). All models were impervious to single-agent trastuzumab treatment. Lapatinib decreased in vitro proliferation of all cell lines and in vivo growth of HER2-amplified xenografts (ARK2, EnCa1). In addition, dual therapy with trastuzumab and lapatinib resulted in significant antitumor activity only in ARK2 and EnCa1 tumors. Dual HER2 therapy induced on target alteration of downstream MAPK and PI3K pathway mediators only in HER2-amplified models, and was associated with increased apoptosis and decreased proliferation. Conclusions: Although trastuzumab alone did not impact USC growth, dual anti-HER2 therapy with lapatinib led to improved inhibition of tumor growth in HER2-amplified USC and may be a promising avenue for future investigation. Clin Cancer Res; 20(24); 6517–28. � 2014 AACR.

Objective Toll-like receptors (TLRs) are integral parts of the innate immune system and have been implicated in complications of pregnancy. The longitudinal expression of TLRs on dendritic cells in the maternal circulation during uncomplicated pregnancies is unknown. The objective of this study was to prospectively evaluate TLRs 1-9 as expressed on dendritic cells in the maternal circulation at defined intervals throughout pregnancy and postpartum. Study Design This was a prospective cohort of 30 pregnant women with uncomplicated pregnancies and 30 nonpregnant controls. TLRs and cytokine expression was measured in unstimulated dendritic cells at 4 defined intervals during pregnancy and postpartum. Basal expression of TLRs and cytokines was measured by multicolor flow cytometry. The percent-positive dendritic cells for each TLRs were compared with both nonpregnant and postpartum levels with multivariate linear regression. Results TLRs 1, 7, and 9 were elevated compared with nonpregnant controls with persistent elevation of TLR 1 and interleukin-12 (IL-12) into the postpartum period. Concordantly, levels of IL-6, IL-12, interferon alpha, and tumor necrosis factor alpha increased during pregnancy and returned to levels similar to nonpregnant controls during the postpartum period. The elevated levels of TLR 1 and IL-12 were persistent postpartum, challenging notions that immunologic changes during pregnancy resolve after the prototypical postpartum period. Conclusion Normal pregnancy is associated with time-dependent changes in TLR expression compared with nonpregnant controls; these findings may help elucidate immunologic dysfunction in complicated pregnancies.

The aim of this study was to characterize endometrial cancer in women 40 years of age and younger, with special attention toward body-mass index (BMI).A retrospective review of women age 40 and under with endometrial cancer was performed. Patients were identified via tumor registry data as well as a search of pathology department diagnoses over the dates 1980-1998. Data were abstracted regarding tumor grade and histology, stage, treatment, smoking, use of oral contraceptives, BMI, medical and family history, parity, and survival. Data were also collected with regard to uterine conservation and pregnancies following endometrial cancer diagnoses.Ninety-five patients were identified. The age range was 24-40 years (median 37) with BMI ranging from 17.5 to 63.6 (median 28.4). Forty-eight patients (52%) were not obese, with BMI30. Seventy-six patients (80%) had stage I disease and 60 patients (63%) had grade 1 disease. All but 4 patients had endometrioid histology. Women with BMI25 were more likely to have advanced disease (P = 0.04) and more likely to have high-risk histology (P = 0.02). Of the 4 patients with high-risk histology (clear cell or serous papillary), all had BMI25. Twelve patients were treated medically rather than surgically, and 4 patients achieved pregnancy, with 5 live births.Women under 40 who are not obese are at higher risk of both advanced disease and high-risk histology. Further study at the molecular and genetic level is ongoing in our laboratory to determine whether the mechanism of disease is different in slender woman.

Current evidence suggests that stress-induced apoptosis is mediated through the activation of the mitogen-activated protein kinase (MAPK) signaling cascade. We hypothesize that stress-related signaling events documented in other cell lines may also occur in the corpus luteum. To test this, cultured bovine luteal cells were exposed to UV irradiation and harvested at different intervals (0, 30, 120, 240 and 360 min) for analysis of protein or apoptotic cell death. In response to UV treatment cellular levels of phosphorylated p38MAPK and jun-n-terminal kinase (JNK) were increased within 30 min and remained elevated over controls for the duration of the experiment. In contrast, the levels of the phosphorylated forms of p42MAPK and p44MAPK were dramatically reduced. The changes in MAPK signaling were similar to those observed in response to tumor necrosis factor alpha, a cytokine implicated in luteal regression. The UV-induced changes in MAPK phosphorylation were associated with an increase in caspase 3 activity and apoptotic cell death. Taken together, these data demonstrate that stress-induced signaling events in the corpus luteum are similar to those observed in unrelated cell types. Thus, stress-related signaling events may play a role in luteal regression.

Our previous studies demonstrated that endothelin-1 (ET-1), a 21-amino acid vasoconstrictor peptide, has a paracrine regulatory role in bovine corpus luteum (CL). The peptide is produced within the gland where it inhibits progesterone production by acting via the selective type A endothelin (ETA) receptors. The present study was designed to characterize ETA receptor gene expression in different ovarian cell types and its hormonal regulation. ETA receptor messenger RNA (mRNA) levels were high in follicular cells as well as in CL during luteal regression. At this latter stage, high ETA receptor expression concurred with low prostaglandin F2α receptor mRNA. The ETA receptor gene was expressed by all three major cell populations of the bovine CL; i.e. small and large luteal cells, as well as in luteal endothelial cells. Among these various cell populations, the highest ETA receptor mRNA levels were found in endothelial cells. cAMP elevating agents, forskolin and LH, suppressed ETA receptor mRNA expression in luteinized theca cells (LTC). This inhibition was dose dependent and was evident already after 24 h of incubation. In luteinized granulosa cells (LGC), 10 and 100 ng/ml of insulin-like growth factor I and insulin (only at a concentration of 2000 ng/ml) markedly decreased ETA receptor mRNA levels. In both LGC and LTC there was an inverse relationship between ETA receptor gene expression and progesterone production; insulin (in LGC) and forskolin (in LTC) enhanced progesterone production while inhibiting ETA receptor mRNA levels. Our findings may therefore suggest that, during early stages of luteinization when peak levels of both LH and insulin-like growth factor I exist, the expression of ETA receptors in the gland are suppressed. This study demonstrates physiologically relevant regulatory mechanisms controlling ETA receptor gene expression and further supports the inhibitory role of ET-1 in CL function.

Ovarian cancer is the 5th most common cancer among women, in which epithelial ovarian cancer (EOC) is the most common type, accounting for 90% of cases. Currently, the standard treatment for ovarian cancer is surgical debulking followed by chemotherapy. Although initially EOC patients are highly responsive to standard platinum-derived chemotherapy, the majority of cancer patients will eventually relapse and experience chemoresistance. In spite of numerous efforts to improve EOC treatment, the five-year overall survival rate is still disappointing at 30%. It is therefore necessary to examine the fundamental causes of treatment resistance and use this information to design new therapeutic strategies. Recently, the concept of tumor-initiating cells and their supporting tumor microenvironment has been proposed as potential targets for cancer treatment due to their observed chemoresistance and ability to repopulate tumors. These cells are proposed to reside deep within tumors, potentially in oxygen-deprived environments. We recently observed that a light-based treatment modality known as photodynamic therapy (PDT) may be uniquely suited to targeting and eliminating this cellular population. PDT applies a light activated drug (photosensitizer) to selectively target and kill cancer cells through generation of reactive oxygen species (ROS), making it an attractive alternative cancer treatment modality with the potential to overcome adaptive therapeutic resistance. Characteristics of PDT, including the direct destruction of cellular organelles, selective tumor accumulation, and photochemistry-driven tumor killing mechanism give PDT advantages over traditional chemotherapy and radiotherapy. We have found that a lysosome targeted photosensitizer known as EtNBS specifically localizes and accumulates into the acidic and hypoxic tumor microenvironment in 3D cultures to successfully kill otherwise unresponsive cellular populations. EtNBS-mediated PDT is thus a potential modality in treating tumor initiating cells. In addition, the quantitative and label-free optical coherence tomography (OCT) imaging modality has recently been demonstrated to provide real time information regarding tumor tissue structure and therapeutic dynamics, making it a potential tool to study tumors and their associated change during the course of tumor development and treatment. Here, we utilized a 3D in vitro metastatic ovarian cancer culture model, with an oxygen sensor and OCT imaging as a combinational platform to study tumor initiating cells and the effects of their associated tumor microenvironment on therapeutic response under standard and EtNBS-PDT treatment. By mapping tumor response with this integrated platform, this work represents a mechanism- based targeted approach that will hopefully enable the identification of cellular targets to overcome disease relapse and adaptive chemoresistance. Citation Format: Hsin-I Hung, Oliver J. Klein, Yookyung Jung, Kashmira S. Kulkarni, Bo R. Rueda, Rosemary Foster, Conor L. Evans. Mapping, targeting, and eliminating therapeutically unresponsive ovarian cancer with high content imaging and photodynamic therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B44.

One of the most challenging aspects of cancer treatment is intratumoral heterogeneity (ITH). However it is been extremely difficult to monitor the extent to which individual clonal populations contribute to and affect tumor behavior because of the inability to culture tumor cells from patients in vitro without clonal selection under standard culture conditions, which lead to a loss in initial heterogeneity. Using a medium developed by Dr. Tan Ince that allows primary tumor cells to undergo continuous population doublings with minimal, if any, selection for or against specific clones, we generated 80 clonal populations from a Clear Cell Carcinoma (CCC) patient sample and monitored the extent of phenotypic and genomic heterogeneity among 12 of the clonal populations. These clones showed striking heterogeneity in copy number alterations as well as morphologic and phenotypic heterogeneity in vitro in assays such as anoikis, growth in soft agar, population doubling time, and growth in 3D reconstituted basement membrane cultures. To examine correlations between in vitro properties and tumor formation in vivo, we tagged the clones with Gaussia-Luciferase (Gluc), a secreted luciferase, which allowed us to monitor tumor burden over time using a blood-based assay. Based on tumor growth at 10 weeks, only two clonal populations were able to grow in the peritoneum when injected alone, one of which (#31) showed a much more rapid growth rate than the other; however #31 was still limited in its tumorigenic capacity, generating only ascitic tumor populations. To address whether functional crosstalk between clones affects tumor progression, we injected mixtures of the clones. Interestingly, mixtures of the 12 clones resulted in the generation of more aggressive in vivo phenotypes, e.g. the formation of solid peritoneal tumors and metastasis to lungs and brain, behaviors not detected with any individual clones. These results suggest that there is cooperativity between the clones to enhance growth and induce metastasis. The study has provided evidence that cell lines generated from individual clones vary significantly in their functional activities in vitro and in vivo. Moreover, by studying the single cell clones, we have found evidence for the existence of interclonal crosstalk during tumor progression and are currently studying the nature and impact of such cross talks on tumor progression and metastasis. This abstract is also presented as Poster A57. Citation Format: Suha Naffar-Abu Amara, Laura M. Selfors, Marit Krohn, Tan A. Ince, Bo R. Rueda, Rosemary Foster, Gordon B. Mills, Joan S. Brugge. Cellular heterogeneity of ovarian carcinoma cells and its impact on tumor behavior. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR09.

5578 Background: Ovarian cancer (OvCa) is the most lethal gynecologic malignancy in the United States. Chemotherapy is effective but seldom curative, mainly due to the development of chemoresistant recurrent disease. Our current research investigates the efficacy of inhibiting the Notch pathway with a gamma-secretase inhibitor (GSI), MRK-003, in an OvCa xenograft model as a single agent therapy and in combination with standard chemotherapy. Methods: Mice bearing xenografts derived from clinically platinum sensitive human ovarian serous carcinomas were treated with GSI or vehicle, or with either vehicle, GSI alone, paclitaxel and carboplatinum (T/C) alone, or the combination of GSI and T/C. In addition, mice bearing xenografts derived from patients with clinically platinum resistant disease were given GSI with or without paclitaxel. Gene transcript levels of several factors in the Notch pathway were analyzed using RT-PCR. Notch1 and Notch3 protein levels were evaluated by western blotting. The Wilcoxon rank-sum test was used to assess significance between the different treatment groups. Results: Expression of Notch1 and Notch3 was highly variable across all analyzed OvCa samples. Treatment with GSI alone significantly decreased tumor growth in 3 of 4 platinum sensitive ovarian tumors (all p < 0.05), as well as in 1 of 3 platinum resistant tumors (p = 0.04). Furthermore, the combination of GSI and paclitaxel was significantly more effective than GSI alone and paclitaxel alone in all platinum resistant ovarian tumors (all p < 0.05). The addition of GSI did not alter the effect of T/C in platinum sensitive tumors. Although the response of each tumor to GSI did not correlate with its endogenous level of Notch expression, 2 of the 3 tumors resistant to paclitaxel but sensitive to the combination of GSI and paclitaxel showed elevated Notch activity by RT-PCR. Conclusions: Inhibition of the Notch signaling cascade with a gamma-secretase inhibitor reduces tumor growth in vivo, most notably in combination with paclitaxel in a platinum resistant setting. These promising findings underscore the need for further investigation of the preclinical and clinical effectiveness of Notch inhibitors in OvCa.

e13564 Background: Alterations in the PI3K pathway are highly prevalent in endometrial cancer due to PIK3CA mutation and loss of PTEN. Given these data, we investigated the anti-tumor activity of the PI3K inhibitor NVP-BKM120 (BKM) as a single agent and in combination with standard cytotoxic chemotherapy in a human primary endometrial xenograft model. Methods: NOD/SCID mice bearing xenografts of primary human tumors with and without PIK3CA gene mutations were randomly divided into two- and four-arm cohorts with equivalent tumor volumes. Three single agent experiments tested the effectiveness of NVP-BKM120 against two endometrioid (PIK3CA wild type and H1047L mutant) and one carcinosarcoma (PIK3CA R88Q mutant) endometrial cancer. Three four arm experiments tested NVP-BKM120 alone and in combination with paclitaxel and carboplatin (P/C) in two endometrioid tumors (both R88Q) and one carcinosarcoma (no PIK3CA mutation detected). Following in vivo study, tumors from the NVP-BKM120 , P/C, P/C+ NVP-BKM120 and vehicle treated mice were processed for determination of PI3K/AKT/mTOR pathway activation. Wilcoxan rank sum analysis was utilized to compare tumor growth across all treatment experiments. Results: In endometrioid single agent experiments, NVP-BKM120 resulted in tumor growth suppression starting at days 5-10 compared to the linear growth observed in vehicle treated tumors (p

In many human cancers, the tumor suppressor, p27kip1 (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization; lack of nuclear p27 causes aberrant cell cycle progression and cytoplasmic p27 can mediate cell migration/metastasis. We previously showed that estrogen (E2) induces Skp2-dependent degradation of p27 and cell proliferation in primary endometrial epithelial cells (EECs) and endometrial carcinoma (ECA) cell lines (e.g., ECC-1) suggesting a pathogenic mechanism for type I endometrial carcinoma (ECA), an estrogen (E2)-linked cancer. The current studies show that treatment of ECC-1 cells with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) increase protein levels of p27, increase p27 half-life by 6 hours and inhibit cell proliferation (IC50 10μM). Two of five SKP2E3LIs, designated as C2 and C20 specifically increase p27 in the nucleus while decreasing p27 in the cytoplasm in both ECC-1 cells and primary ECA cells. In addition, C2 and C20 prevent both E2-induced proliferation and degradation of nuclear p27. These data suggest that Skp2E3LIs can function in the nucleus to prevent E2-induced degradation of p27 in primary ECA cells to regain growth control by directly blocking p27 ubiquitylation and subsequent degradation. Skp2E3LIs enable a chemical biology approach to understanding the functional significance of the ubiquitin pathway in p27-mediated cell cycle regulation and importantly, are the first specific proteasome inhibitors that pharmacologically target the binding interaction between the E3ligase, SCF-Skp2/Cks1 and p27 to prevent its ubiquitylation and subsequent degradation. These targeted inhibitors represent a major therapeutic advancement over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of p27. Citation Format: Savvas C. Pavlides, Kuang-Tzu Huang, Lily Wu, Bo R. Rueda, Stephanie V. Blank, Khushbakhat R. Mittal, Timothy Cardozo, Leslie I. Gold. Inhibitors of Skp2 E3ligase stabilize nuclear p27kip1 for regain of growth regulation in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2013-3410

Introduction: In female embryos, the reproductive organs arise from the Mullerian ducts and associated epithelium; these structures regress under the influence of MIS produced by male embryos. The MIS type II receptor (MISIIR) can mediate MIS-induced growth inhibition of malignant ovarian and cervical cells. We propose that endometrial cancers will express the MISIIR and MIS will inhibit endometrial cancer growth. Methods: Human samples were obtained under IRB approval (2002-P-001324). Rat tissue was collected under institutional protocol 1999-N-00138-Rats. Cells lines were purchased from American Tissue and Cell Culture. Tissue and cell samples were analyzed by Western, PCR, growth inhibition assays, and FACS. Results: Western detected MISIIR protein in adult rat uterus; reverse transcriptase PCR detected MISIIR RNA in normal and malignant human endometrium. AN3CA and KLE, two human endometrial cancer cell lines, expressed MISIIR. MIS inhibited AN3CA and KLE cell growth 67.3% (p < 0.0001, n = 4) and 52.5% (p = 0.0041, n = 3), respectively. MIS-treated AN3CA cells demonstrated G1 arrest, increased Caspase 3 cleavage indicating apoptosis, increased levels of cell cycle proteins p107 and p130 and transcription factor E2F3, and decreased levels of transcription factor E2F1. Conclusions: MISIIR is expressed in normal endometrium and in human endometrial cancer. MIS inhibits the growth of two human endometrial cancer cell lines, induces cell cycle arrest and apoptosis in AN3CA cells, and affects proteins important to the control of cell cycle progression. In the future, treatment targets for MIS may broaden to include advanced endometrial cancer

A complementary DNA clone encoding a functional receptor for prostaglandin F2 alpha (PGF2 alpha) has been isolated from an ovine large luteal cell complementary DNA library (prepared from day 10 mid-luteal phase RNA). This receptor, which has been designated FP, consists of 362 amino acids (M(r) = 40,982) and is a member of the family of G protein-coupled receptors. Radioligand binding studies with membranes prepared from transfected COS cells demonstrated specific 17-[3H]phenyl-trinor-PGF2 alpha binding that was displaced by prostanoids in the order of 17-phenyl-trinor-PGF2 alphaPGF2 alphafluprostenolPGD2PGE28-epi PGF2 alpha. Xenopus laevis oocytes injected with RNA encoding the ovine FP receptor responded to 17-phenyl-trinor-PGF2 alpha with increased membrane chloride conductance in calcium-free medium. Northern blot analysis with RNA from day 10 corpus luteum showed a major band of approximately 6.1 kilobases. On day 14, when luteolysis usually starts, the abundance of this 6.1-kilobase band was variable between individual ewes, and on day 16, when luteolysis is underway, the message was uniformly less abundant. This variability appeared to correlate with circulating progesterone. Thus, when the progesterone level was high (days 10 and 14 depending on whether luteolysis had started), the amount of FP receptor message was high, whereas when the progesterone level was low or falling (day 16), the amount of FP receptor message decreased. We have cloned an ovine FP receptor whose expression confers appropriate functional activity in COS cells and Xenopus oocytes. Furthermore, the level of messenger RNA encoding the FP receptor is high in the midluteal phase ovine corpus luteum and decreases during luteolysis.

Endometrial cancer is the most common gynecologic malignancy and the majority of patients are cured with surgery alone. A substantial subset of patients, however, present with high grade uterine papillary serous carcinoma (UPSC) and these patients account for a disproportionate degree of the recurrence, chemoresistance and death associated with endometrial cancer. Novel treatment strategies incorporating molecularly targeted therapies would be of great value in effective clinical management of these tumors. HER2 (ErbB2) hyperactivity has been associated with 10-20% of UPSC, though efforts to target this signature with trastuzumab have yielded no response in a recent prospective trial. In breast cancer, HER2/HER3 (ErbB2/ErbB3) dimerization has been associated with trastuzumab resistance. The expression profile of HER3 in UPSC is currently unknown. The objective of this investigation was to characterize HER2 and HER3 expression in a UPSC series and correlate these findings with clinical outcomes including survival. Under an IRB approved protocol, we identified 33 patients who underwent initial surgical staging at our institution between 2000 and 2009 with archival formalin fixed, paraffin embedded blocks and complete clinical follow up data. These tumor samples were subjected to immunohistochemistry (IHC) for HER2 and HER3 as well as fluorescent in situ hybridization (FISH) to assess the amplification status of the HER2 gene. Non-parametric tests were utilized to test correlation between detected levels of HER2 or HER3 protein expression and HER2 gene amplification and survival analysis was performed using the Kaplan-Meier method. HER2 gene amplification was observed in 34% of the cohort while HER2 protein over-expression, defined as 2+ or 3+ staining, was found in 59% of samples tested. HER2 gene amplification was associated with high HER2 protein expression (p < 0.05). Moreover, HER2 gene amplification was significantly associated with a decreased overall survival (p < 0.05). Finally, HER3 over-expression, defined as 2+ or 3+ staining, was observed in 52% of the entire cohort and HER3 protein levels exhibited a significant inverse relationship to HER2 protein expression and HER2 gene amplification (p < 0.05). These findings suggest that HER2 and HER3 signaling may function independently of one another in UPSC. The observed alterations in HER2 and HER3 expression levels constitute clinically relevant and prevalent signatures in UPSC that merit further investigation to determine the potential benefits of therapies targeting the epidermal growth receptor pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4556. doi:1538-7445.AM2012-4556

Recurrent ovarian cancer is believed to be due, in part, to the presence of cancer stem cells that escape the cytotoxic effects of standard chemotherapeutics and lead to relapse. Results from our laboratory and others suggest that human ovarian tumors contain rare populations of cells that are capable of initiating new tumor formation in vivo. Our objective was to analyze the effect of platinum based chemotherapy on these tumor initiating cell populations in a genetically defined mouse model of ovarian cancer. Our studies focused on a mouse ovarian cancer cell line (T2) with defined mutations in TP53, c-MYC and AKT that readily forms aggressive tumors following subcutaneous injection into non-diabetic/severe combined immunodeficient (NOD-SCID) mice. We used flow cytometry to evaluate the T2 induced tumors for expression of stem cell antigen-1 (Sca1), a known marker of murine stem cells, and CD133, an antigen which has been shown to enrich for tumor initiating cells derived from human ovarian tumors. FACS based isolation of Sca1+ and CD133+ T2 tumor-derived cells followed by injection into NOD/SCID mice assessed their relative tumor initiating capacity. Mice bearing T2-derived tumors were treated with either vehicle or paclitaxel and carboplatinum (T/C) to evaluate the effect of T/C on the Sca1+ and CD133+ tumor cell population. We consistently observed that Sca1+ and the CD133 + cells comprised 3-7% and 0.1-0.4 % of the total cell population in T2-derived tumors, respectively. Following FACS based isolation and injection into NOD/SCID mice, Sca1+ and CD133 + cells were able to form tumors much more rapidly and at lower cell numbers than their Sca1- and CD133- counterparts, suggesting that the Sca1+ and CD133+ populations are enriched in tumor initiating cells. Although, administration of T/C resulted in a significant decrease in tumor volume relative to vehicle treated tumors, we detected no significant difference in the levels of Sca1+ and CD133+ cells in tumors treated with T/C compared to vehicle controls. Furthermore, cell cycle analysis of the Sca1+ and CD133+ populations determined that these cells were enriched in the quiescent G0/G1 phase of the cell cycle. Our results provide evidence that the T2-derived Sca1+ and CD133+ tumor initiating cells are relatively quiescent, persist following standard platinum based chemotherapy and likely contribute to disease recurrence in ovarian cancer. While the specific role of these cells in post-chemotherapy tumor recurrence needs to be evaluated, our findings suggest that future studies should be directed towards targeting these cell populations which likely contribute to the resurgence of the ovarian cancer and thereby reduce the overall survival rate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3474. doi:1538-7445.AM2012-3474

The high mortality associated with ovarian cancer is attributed to the lack of any reliable early detection method, unknown pathogenesis of the disease, and the development of recurrent and chemoresistant tumors. Current efforts have focused on the identification of therapeutics that may be used independently or in combination with current chemotherapeutic regimens to reduce tumor volume. To date, limited research has focused on preventing or delaying disease recurrence. The Hedgehog (Hh) signal transduction pathway is inactive in most adult cells. Malignant activation of the Hh pathway through the signaling protein Smoothened (Smo) occurs in a broad range of cancers, including ovarian. IPI-926 is a potent orally delivered small molecule that targets the Hh pathway by inhibiting Smo. Recent studies from our laboratory provide evidence that IPI-926 slows serous ovarian cancer growth in a primary human tumor xenograft model. More importantly, IPI-926 delays the resurgence of tumor growth typically observed after cytoreduction with paclitaxel and carboplatinum (T/C) treatment. Our current objective was to assess whether this effect of IPI-926 required that the Smo inhibitor be administered during a critical window following T/C treatment to prevent the resurgence of tumor growth. To test our hypothesis, mice bearing human ovarian cancer xenografts were treated with vehicle or T/C. T/C treatment was withdrawn following significant reduction (30-50%) in tumor volume. The original vehicle treated cohort was divided into 2 arms which then either received IPI-926 or continued on vehicle for the duration of the experiment. Mice in the T/C cohort were divided into 3 groups. Group 1 received vehicle alone (T/C-Vehicle), group 2 received IPI-926 immediately following the last T/C dose (T/C-IPI-926) and group 3 received no vehicle or IPI treatment for 14 days following the last T/C dose (Window). Group 3 was then maintained on IPI-926 treatment starting at day 15 post-T/C withdrawal. The withdrawal of T/C led to a dramatic increase in tumor volume in the T/C-Vehicle group. As previously observed, tumor growth inhibition was maintained in mice receiving IPI-926 immediately following the cessation of T/C treatment. In contrast, delaying the administration of IPI-926 following T/C prevented the suppression of tumor growth as evidenced by the increased tumor volume. Our data suggest that blocking Hh pathway activity immediately following chemotherapy maintains and prolongs the inhibitory effect of chemotherapy on ovarian tumor growth. The absence of an IPI-926-mediated inhibition of tumor resurgence following a 14-day delay in treatment supports the concept that there is a critical period for tumor re-establishment. Our data, along with those of others, suggest that the early stages of ovarian tumor re-growth may be dependent on Hh pathway signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3285. doi:1538-7445.AM2012-3285

Background: Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, basal cell, small cell lung, pancreatic, prostate and ovarian. This investigation was designed to determine if inhibition of this pathway could inhibit serous ovarian cancer growth. Methodology: We utilized an in vivo pre-clinical model of serous ovarian cancer to characterize the anti-tumor activity of Hh pathway inhibitors cyclopamine and a clinically applicable derivative, IPI-926. Primary human serous ovarian tumor tissue was used to generate tumor xenografts in mice that were subsequently treated with cyclopamine or IPI-926. Principal Findings: Both compounds demonstrated significant anti-tumor activity as single agents. When IPI-926 was used in combination with paclitaxel and carboplatinum (T/C), no synergistic effect was observed, though sustained treatment with IPI-926 after cessation of T/C continued to suppress tumor growth. Hh pathway activity was analyzed by RT-PCR to assess changes in Gli1 transcript levels. A single dose of IPI-926 inhibited mouse stromal Gli1 transcript levels at 24 hours with unchanged human intra-tumor Gli1 levels. Chronic IPI-926 therapy for 21 days, however, inhibited Hh signaling in both mouse stromal and human tumor cells. Expression data from the micro-dissected stroma in human serous ovarian tumors confirmed the presence of Gli1 transcript and a significant association between elevated Gli1 transcript levels and worsened survival. Conclusions/Significance: IPI-926 treatment inhibits serous tumor growth suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may hold promise as a novel therapeutic target, especially in the maintenance setting.

Background Pre-clinical studies have demonstrated that natural and synthetic histone deacetylase (HDAC) inhibitors can impede the in vitro and in vivo growth of cell lines from a variety of gynecologic and other malignancies. We investigated the anti-tumor activity of panobinostat (LBH589) both in vitro and in vivo as either a single agent or in combination with conventional cytotoxic chemotherapy using patient-derived xenograft (PDX) models of primary serous ovarian tumors.

Aim Pre-B cell colony-enhancing factor (PBEF) is intricately involved in inflammation and fibrosis, functional polymorphisms of PBEF have been previously shown to influence PBEF expression and pulmonary damage. Systemic sclerosis (SSc) is a disease in which inflammation, fibrosis and pulmonary deterioration are prominent hallmarks. Therefore the authors here investigate the role of the PBEF -1001T>G and PBEF -1543C>T polymorphisms in the genetic predisposition to SSc susceptibility and pulmonary involvement. Patients and methods The authors genotyped DNA from 2737 SSc patients and 1913 matched healthy controls, both from eight different ethnic populations. Genotyping was performed using custom Taqman 59 allelic discrimination assays. In addition, PBEF serum expression levels were measured by ELISA and correlated with genotypes. Results In two separate populations and in a meta-analysis, the combined PBEF -1543CC -1001TT genotype , hence carrying no minor alleles, was found associated with SSc susceptibility (p=0.009 OR 1.20 (95% CI 1.05 to 1.37)). In addition, these subjects showed an increased decline in forced vital capacity over 15 years follow-up (p=0.02) (HR 1.64, 95% CI 1.02 to 2.64) and a higher PBEF serum concentration (p PBEF -1001TT were at lower risk for PAH development within 15 years of disease onset compared to the carriers with genotypes PBEF -1001GG and PBEF -1001TG (p Conclusions This data identify PBEF as a novel candidate gene that influences SSc susceptibility, pulmonary function and the development of PAH.

Like other solid tumors, endometrial tumors have been shown to contain a subset of tumor initiating cells although little is known about how these rare sub-fractions are regulated. Our primary objective was to analyze potential epigenetic regulation of such a tumor initiating cell population in human endometrial cancer cells. To accomplish this, we demonstrated by flow cytometry that primary endometrial tumors contain CD133+ cells. To assess their tumor initiating capacity, serially transplanted endometrial human tumor explants generated in NOD/SCID mice were harvested, enzymatically dissociated, depleted of H-2Kd+ mouse cells and sorted via flow cytometry to generate relatively pure (> 98.8 %) CD133+ and CD133− fractions. These positive and negative fractions were serially diluted and subcutaneously injected into immunocompromised mice. The CD133+ fractions had a significantly increased capacity for tumor formation relative to the CD133− fractions and this difference was more pronounced as the number of injected cells decreased. Interestingly, the level of CD133+ tumor cells appeared to be enriched following serial transplantation as evidenced by flow cytometric and immunohistochemical analyses. It has been proposed that methylation may play a role in regulation of tumor initiating cells. To investigate this possibility in endometrial cancer, we isolated DNA from serially transplanted tumors and analyzed the methylation status of CpG islands located upstream of the CD133 transcription start site. This region was shown to be a target of methylation, which led us to determine whether changing the methylation status would alter CD133 expression in endometrial cancer cells. We treated 4 individual human endometrial cancer cell lines with either vehicle or 5 μM 5-aza-2′-deoxyctidine (5-aza-DC), for 72 hours, and evaluated post-treatment levels of CD133 expression by RT-PCR and flow cytometry. In the tested cell lines, CD133 mRNA levels, as measured by RT-PCR, were increased following treatment with 5-aza-DC suggesting that methylation of the CD133 promoter was suppressing its expression. To extend this finding, we analyzed the percentage of CD133 expressing cells in either vehicle treated or 5-aza-DC by flow cytometry. Consistent with the RT-PCR results, the frequency of CD133-expressing cells was increased in 3 of the 4 cell lines following treatment with 5-aza-DC. It is not clear however, if the 5-aza-DC mediated demethylation and subsequent shift in the percentage of CD133+ cells correlates with a shift in the frequency of cells that have increased tumor initiating capacity. Nevertheless, CD133 expression in human endometrial cancer cells is regulated at least in part by methylation and altering this may cause these tumor cells to become more sensitive to standard chemotherapy regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4302.

We have been developing tools to reproducibly correlate the expression of multidrug-resistance (MDR)-associated genes with response to chemotherapy in primary cancers. This study reports the results of a novel MDR gene expression analysis of primary serous carcinoma of the ovary utilizing a TaqMan Low Density Array (TLDA) chip which includes 380 previously characterized multidrug resistance (MDR)-associated genes that were initially identified in cultured cancer cells. Primary tumor samples from 133 patients from 4 sites were studied. All patients were subsequently treated with standard chemotherapy and had known clinical outcome. A 13 gene signature was identified whose expression added statistical power to the risk status of the patients based on standard clinical parameters (age, CA125, and success of surgical debulking) (log-rank statistic p=0.02). When subsets of patients with defined clinical risk were studied, we found that a subset of clinically high risk patients that had low expression of the 13 gene signature had a better outcome than would be predicted by purely clinical criteria. Similarly, clinically low risk patients with high expression of the 13 gene signature had a poorer prognosis. Since the mechanism of action of the 13 MDR genes involved in the signature are well-understood, it might be possible to devise therapeutic strategies to some of these targets with the goal of improving clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-51.