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Globally, ovarian cancer is the 6th most common malignancy in developed countries, responsible for 100,300 new cases and 64,500 deaths annually (Jemal et al., 2011). Approximately 90% of ovarian cancers arise within the ovarian surface epithelium (OSE) or the fallopian tube surface epithelium; the remainder of ovarian malignancies develops from other ovarian tissues (sex cord-stromal, germ cell, or mixed cell tumors). The overall prognosis for epithelial ovarian carcinoma (EOC) is poor: Diagnosis is typically late-stage due to the lack of effective screening methods and vague presenting symptoms, with 5-year survival at 40% for stage III and 20% for stage IV patients (Heintz et al., 2006). Despite excellent initial activity, the standard treatment consisting of cytoreductive surgery followed by platinumand taxane-based chemotherapy often fails with a recurrence rate of over 80% in stage III and IV disease. Therefore, novel therapeutic approaches are needed to improve the outcomes in this population. Research efforts have yielded insight into the etiology, signaling mechanisms, and progression of ovarian cancer, yet much remains poorly understood. Physiologically, steroid hormones are intimately involved in ovulation, reproduction, and function of normal OSE cells. There is growing evidence that estrogen, progesterone, and other hormones also play a role in the development and progression of ovarian cancer (Leung & Choi, 2007). “Incessant ovulation” with repetitive injury and repair of OSE and subsequent cumulative DNA damage is one of the hypothesized risk factors for ovarian cancer (Fathalla, 1971), yet this does not explain the occurrence of the majority of ovarian carcinomas well after the reproductive years (Berek & Hacker, 2010). Interestingly, although oral contraceptives (OCs), increasing parity, and prolonged breastfeeding all decrease cumulative risk (Gwinn et al., 1990; Risch et al., 1994), progestin-only contraceptives offer just as much protective benefit as estrogen-containing OCs without prohibiting ovulation (Risch, 1998). Another hypothesis for the development of ovarian cancer, the “gonadotropin hypothesis,” stipulates that gonadotropins contribute to ovarian carcinogenesis through follicle stimulating hormone (FSH)and luteinizing hormone (LH)-mediated excess stimulation of ovarian tissue. This hypothesis is consistent with the protective effect of OCs, and the observation that the majority of cases of epithelial ovarian cancer develop postmenopausally after a surge in gonadotropin levels. In vitro, gonadotropins such as FSH activate mitogenic pathways and stimulate ovarian epithelial cell proliferation (Choi et al., 2002). In addition to gonadotropins, excess androgens and estrogen have also been linked to the progression and possibly development of ovarian cancer. In vivo treatment of mice with

Purpose: Mucinous ovarian carcinomas have a distinct clinical pattern compared with other subtypes of ovarian carcinoma. Here, we evaluated (i) stage-specific clinical significance of mucinous ovarian carcinomas in a large cohort and (ii) the functional role of Src kinase in preclinical models of mucinous ovarian carcinoma. Experimental Design: A total of 1,302 ovarian cancer patients including 122 (9.4%) cases of mucinous carcinoma were evaluated for survival analyses. Biological effects of Src kinase inhibition were tested using dasatinib-based therapy in a novel orthotopic mucinous ovarian cancer model (RMUG-S-ip2). Results: Patients with advanced-stage mucinous ovarian cancer had significantly worse survival than those with serous histology: median overall survival, 1.67 versus 3.41 years, P = 0.002; median survival time after recurrence of 0.53 versus 1.66 years, P < 0.0001. Among multiple ovarian cancer cell lines, RMUG-S-ip2 mucinous ovarian cancer cells showed the highest Src kinase activity. Moreover, oxaliplatin treatment induced phosphorylation of Src kinase. This induced activity by oxaliplatin therapy was inhibited by concurrent administration of dasatinib. Targeting Src with dasatinib in vivo showed significant antitumor effects in the RMUG-S-ip2 model but not in the serous ovarian carcinoma (SKOV3-TR) model. Combination therapy of oxaliplatin with dasatinib further showed significant effects on reducing cell viability, increasing apoptosis, and in vivo antitumor effects in the RMUG-S-ip2 model. Conclusions: Our results suggest that poor survival of women with mucinous ovarian carcinoma is associated with resistance to cytotoxic therapy. Targeting Src kinase with a combination of dasatinib and oxaliplatin may be an attractive approach for this disease. Clin Cancer Res; 17(16); 5367–78. ©2011 AACR.

Notch and Hedgehog activate cell-cycle progression of adult and cancer stem cells. Notch is activated by DLL and Jag presents on neighboring cells. We investigated the effects of density of the Notch-activating ligand, Jag-1, and targeting Gli-1, in activation of division of paclitaxel/taxol-resistant, (PTX Res) ovarian cancer cells SKOV3 (SKOV3). We used the specific gamma-presenilin inhibitor, DAPT, to identify the specificity of activating signals for Notch-1 and created 'butterfly-duplex-3548-Gli-1-inhibitory RNA' (i-Gli-1.RNA) to inhibit cell division. To accurately quantify kinetics of division, the expression of CD44 and CD24 was determined in each gated population of divided cells. CD44 High proliferated when activated by Jag-1 Low and poorly when activated by Jag-1 High. DAPT inhibited proliferation of cells activated by Jag-1 Low, and increased proliferation of cells activated by Jag-1 High. Only 5-10% of cells activated by Jag-1 High and Jag-1 Low divided fast, polynomial, and symmetric. i-Gli-1.RNA eliminated more than 50% of the small CD44 High/CD24 Neg cells in divisions 3 and 4. This effect appeared specific compared with cells transfected with negative control siRNA. i-Gli-1.RNA had no effect on large CD44 High/CD24 Neg cells, but inhibited the population of CD44 High/CD24 Low cells. Expansion of CD44 High inversely correlated with Jag-1 density on activating autologous tumor and fibrosarcoma cells. Created i-RNAs may decrease the resting CSC pool. Notch and Gli-1 signals play an important role in proliferation/division and survival of cancer stem cells. Targeting Notch-1 through its enhancer Gl-1, should be significant for novel treatments to eliminate taxol-resistant cancer stem cells (CSC). i.Gli-1 RNA should be more effective if used together with Taxol.

A polylinker with rare restriction sites was introduced into a mini-Tn5 derivative. These sites include M.XbaI-DpnI (TCTAGATCTAGA), which is rare in most bacterial genomes, SwaI (ATTTAAAT) and PacI (TTAATTAA), which are rare in G+C-rich genomes, NotI (GCGGCCGC) and SfiI (GGCCN5GGCC), which are rare in A+T-rich genomes, and BlnI (CCTAGG), SpeI (ACTAGT), and XbaI (TCTAGA), which are rare in the genomes of many gram-negative bacteria. This Tn5(pfm) (pulsed-field mapping) transposon carries resistance to chloramphenicol and kanamycin to allow selection in a wide variety of background genomes. This Tn5(pfm) was integrated randomly into the Salmonella typhimurium and Serratia marcescens genomes. Integration of the new rare SwaI, PacI, BlnI, SpeI, and XbaI sites was assayed by restriction digestion and pulsed-field gel electrophoresis. Tn5(pfm) constructs could be valuable tools for pulsed-field mapping of gram-negative bacterial genomes by assisting in the production of physical maps and restriction fragment catalogs. For the first applications of a Tn5(pfm), we bisected five of the six largest BlnI fragments in the S. typhimurium genome, bisected the linearized 90-kb pSLT plasmid, and used Tn5(pfm) and Tn10 to trisect the largest BlnI fragment.

The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to different growth signals and is frequently deregulated in cancer. There are three Raf kinases-A-Raf, B-Raf, and C-Raf; however, only B-Raf is frequently mutated in various cancers. The most common B-Raf mutation involves a substitution of a glutamic acid residue to a valine moiety at codon 600. Subsequently, the MAPK pathway is constitutively activated, even in the absence of any growth signals. Although early attempts to target Ras have not yielded any viable drug candidates, many novel compounds inhibiting the activities of Raf and MEK have been developed and investigated in clinical trials in recent years. The first MEK inhibitor (CI-1040) lacked efficacy in clinical trials, but its low toxicity has encouraged the search for novel compounds with enhanced target potency to inhibit MAPK activation at low nanomolar concentrations. In this review, we will discuss new patents or patent applications related to inhibitors of the Ras/Raf/MEK/ERK pathway.

The single nucleotide polymorphism (SNP) array was originally developed to determine the genotypes of a study population for linkage analysis or individual genetic variation analysis. Over the last few years, the number of SNP loci that can be evaluated in a single assay has increased from approximately 1500 to more than 500,000, covering all 22 autosomes and the X chromosome. Because the hybridization signal of each oligonucleotide on the SNP array depends on the amount of target DNA, various statistic algorithms have been developed to estimate the copy number of each SNP locus. Several studies have demonstrated the utility of the SNP array analysis in the detection of gene copy number aberrations in tumor DNA. In this review, we discuss the use of the SNP array analysis in determining tumor aberrations in gene copy numbers, the challenge of using tumor samples in the analysis, and improvements in software for copy number measurement using SNP arrays. The unique ability of the SNP array to determine both the genotype and copy number has uncovered novel DNA amplification events that involve only a single allele.

Ovarian cancer is complex disease composed of different histological grades and types. However, the underlying molecular mechanisms involved in the development of different phenotypes remain largely unknown. Epidemiological studies identified multiple exogenous and endogenous risk factors for ovarian cancer development. Among them, an inflammatory stromal microenvironment seems to play a critical role in the initiation of the disease. The interaction between such a microenvironment, genetic polymorphisms, and different epithelial components such as endosalpingiosis, endometriosis, and ovarian inclusion cyst in the ovarian cortex may induce different genetic changes identified in the epithelial component of different histological types of ovarian tumors. Genetic studies on different histological grades and types provide insight into the pathogenetic pathways for the development of different disease phenotypes. However, the link between all these genetic changes and the etiological factors remains to be established.

A 2-tier system that classifies ovarian serous carcinoma (OSC) as low grade or high grade is gaining acceptance. Women with low-grade OSC generally have higher 5-year survival rates than do women with high-grade OSC. We examined the expression of various markers to further understand the molecular differences between low-grade and high-grade OSCs: the potential therapeutic targets or prognostic markers Her-2/neu, estrogen receptor, and progesterone receptor (PR); the metastasis-associated markers cyclin D1 (BCL1), E-cadherin, matrix metalloproteinase (MMP) 2, and MMP-9; and the cell proliferation-associated markers BCL1, Ki-67 antigen (Ki-67), and p53. For this immunohistochemical analysis, we used paraffin-embedded specimens from 47 patients with advanced-stage low-grade OSC and from 49 patients with advanced-stage high-grade OSC. Our results showed that low-grade tumors expressed significantly higher levels of estrogen receptor, PR, and E-cadherin than did high-grade tumors, suggesting the involvement of gonadal steroid hormones, especially in the pathogenesis of low-grade OSC; the PR positivity was also observed in the stromal component of these low-grade tumors. On the other hand, high-grade tumors trended toward increased expression of MMP-9, BCL1, p53, and Ki-67, and robust MMP-9 positivity was observed in the stromal component of these high-grade tumors. These differences may lead to the development of different therapeutic strategies for women with either the low-grade or the high-grade form of OSC.

Purpose To identify markers that can predict overall survival in patients with high-grade advanced stage serous adenocarcinomas. Patients and Methods Oligonucleotide array comparative genomic hybridization (aCGH) was performed on 42 microdissected high-grade serous ovarian tumor samples. aCGH segments were obtained and a prediction Cox model was built and validated by the standard leave one out analysis. Both DNA and mRNA copy numbers of selected genes located on the candidate aCGH segments were determined by quantitative polymerase chain reaction (qPCR) and quantitative reverse transcriptase PCR (qRT-PCR) analyses. The gene that showed the highest correlation was further validated on an independent set of specimens and was selected for further functional studies. Results Two chromosomal regions, 4p16.3 and 5q31-5q35.3, exhibited the strongest correlation with overall survival (P < .01). From the 5q31 region, fibroblast growth factor 1 (FGF-1) was selected for further validation study. FGF-1 mRNA copy number was significantly correlated with DNA copy number and protein expression levels (P = .021 and < .001), and both FGF-1 mRNA and protein levels were significantly associated with overall survival (P = .018 and .042). This association was validated for protein expression on an independent set of 81 samples, significant to P = .006. Further studies showed significant correlation between FGF-1 protein expression and CD31+ staining in the tumor stroma (P = .024). Finally, both cancer cells and endothelial cells treated with exogenous FGF-1 showed a significant increase in cell motility and survival. Conclusion Amplification of FGF-1 at 5q31 in ovarian cancer tissues leads to increased angiogenesis, and autocrine stimulation of cancer cells, which may result in poorer overall survival in patents with high-grade advanced stage serous ovarian cancer.

Survivin, a member of the inhibitor of apoptosis protein family, is implicated in the dysregulation of apoptosis in human cancers. Survivin and survivin-deltaEx3, one of its two alternatively spliced isoforms, confer anti-apoptotic activities in human tumours, while survivin-2B antagonizes such anti-apoptotic properties. The current study was undertaken to examine the mRNA expression of survivin isoforms and their correlation with clinical staging and outcome in 20 medulloblastoma (MB) tumours, three MB cell lines and normal brain tissues (a foetal and an adult cerebellum) by densitometry scanning of 32p-dCTP incorporated reverse transcription polymerase chain reaction (RT-PCR) products and quantitative real-time PCR. Our results showed that the normal adult brain only expressed low levels of survivin-deltaEx3 mRNA, while the foetal brain expressed all three isoforms, with wild-type survivin as the dominant transcript. All three survivin isoforms were detected in all the MB cell lines and tumours analysed. Immunohistochemical staining also demonstrated survivin protein expressions in all five paraffin-embedded MBs, with predominant nuclear localization. Although overexpressions of survivin were not associated with the presence of metastatic MB or tumour histological subtypes, elevated expressions of survivin-deltaEx3 were significantly associated with progressive/recurrent tumours (P-value = 0.024). Our data demonstrated that overexpression of survivin mRNA is a common feature in MBs, may contribute to their anti-apoptosis properties and clinical behaviours, and predicts a poor clinical outcome, independent of clinical staging or tumour histology.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy. Although most patients respond to chemotherapy, relapse often occurs as chemoresistance disease. We previously reported the tumor-promoting role of Microfibrillar-Associated Protein 5 (MFAP5) in ovarian cancer. Follow up studies suggested a pro-angiogenic and chemo-protective role of MFAP5. Based on these findings, the present study aims at delineating the mechanism by which stromal MFAP5 modulates tumor angiogenesis and chemoresistance, and evaluating the potential of targeting MFAP5 as therapy for ovarian cancer. Immunostaining of HGSOC tissue sections revealed a significant correlation between stromal MFAP5 expression and intratumoral microvessel density. Further endothelial cell motility and tube formation assays on human endothelial cells demonstrated the pro-angiogenic roles of exogenous MFAP5. Furthermore, anti-αVβ3 integrin antibody and cell permeant calcium chelator, BAPTA, attenuated MFAP5's effects, suggesting that MFAP5 exerts its biological activity on endothelial cells through the αVβ3 integrin receptor and calcium signaling. In vivo studies showed that extensive tubular network was formed in the matrigel mixed with MFAP5 injected into mice (p In addition, analysis on stromal gene expression profiles revealed that stromal MFAP5 expression is significantly increased in the group of patients with shorter progression-free survival when compared to those with longer progression-free survival. Further cell survival assays showed that pretreating to HGSOC cell lines with recombinant MFAP5 confers resistance to ovarian cancer cells towards paclitaxel treatment. RNA-Seq analysis was performed to delineate the underlying mechanism of such induced chemoresistance. To conclude, stromal MFAP5 contributes to ovarian tumor angiogenesis and chemoresistance. Hence, targeting stromal MFAP5 could be a potential therapy for ovarian cancer treatment. Citation Format: Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Anil K. Sood, Michael J. Birrer, Samuel C. Mok. CAF-derived MFAP5 promotes tumor angiogenesis and confers paclitaxel resistance in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4184. doi:10.1158/1538-7445.AM2015-4184

Background: Uterine papillary serous carcinoma (UPSC) comprises less than 10% of all endometrial cancers but accounts for a disproportionately high number of deaths. Few prospective studies focus solely on this aggressive subtype, making it difficult to determine optimal treatment strategies. The purpose of this study was therefore to identify novel therapeutic targets that could aid in the management of UPSC. Methods: Clinical and RNA sequencing data from The Cancer Genome Atlas (TCGA) was used to identify genes that were differentially expressed between endometrioid endometrial carcinoma (EEC) and UPSC, which also showed a significant association with overall patient survival duration in UPSC patients. Ubiquitin C-terminal hydrolase L1 (UCHL1) upregulation and correlation with survival was validated by immunohistochemical staining of an independent cohort of paraffin samples. UCHL1 was silenced by siRNA transfection and lentiviral shRNA transduction in UPSC cell lines ARK1, ARK2 and HEC50 to evaluate the role of UCHL1 on cell proliferation, migration and cell cycle progression. RPPA data from TCGA was used to identify proteins whose expression correlated with UCHL1 expression. Interaction between UCHL1 and cyclin B1 was determined by co-immunoprecipitation. The effect of UCHL1 silencing on cyclin B1 levels was evaluated by Western blot analysis, and the effect on cell cycle progression was determined by flow cytometry. Results: UCHL1 mRNA and protein expression levels were significantly higher in in UPSC compared to EEC. Kaplan-Meier analysis and cox regression of the TCGA data set and an independent cohort of patient samples revealed that UCHL1 expression correlated significantly with poorer overall survival in UPSC, especially in late-stage patients. siRNA-mediated silencing did not affect cell migration, but reduced the proliferation rates of ARK1, ARK2 and HEC50 in vitro. UCHL1 RNA expression was positively correlated with cyclin B1 protein levels in the TCGA data set. Accordingly, UCHL1 silencing reduced cyclin B1 protein but not mRNA expression levels in ARK1, ARK2 and HEC50. Cell cycle analysis of ARK1, ARK2 and HEC50 after UCHL1 silencing showed a reduction in the percentage of cells in the S and G2/M phases, with or without cell synchronization. Conclusion: These findings indicate that UCHL1 contributes to the aggressiveness of UPSC by stabilizing cyclin B1 and increasing cell proliferation, suggesting that it may be a therapeutic target for UPSC. Citation Format: Suet Ying Kwan, Samuel C. Mok, Kwong-Kwok Wong, Rosemarie E. Schmandt, Karen H. Lu. Ubiquitin C-terminal hydrolase L1 (UCHL1) modulates uterine papillary serous cancer progression through interaction with cyclin B1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3559. doi:10.1158/1538-7445.AM2015-3559

Objective: PAX2 is recognized as a significant factor in the development of urogenital system. It is expressed in multiple tumors and is essential in tumor cell survival. This study aimed to investigate the effect, expression, methylation of PAX2 in endometrial cancer, and the possible regulative mechanism of DNA methylation related PAX2 expression. Methods: PAX2 expression in endometrial cancer, hyperplasia and normal tissues was detected by immunohistochemistry, and its expression in cell lines was measured by real-time PCR, western blot and immunocytochemistry. The role of PAX2 on cell viability and mobility was measured by CCK-8, invasion and migration assays. Bisulfite sequencing and MassARRAY were used to quantify the DNA methylation level of PAX2 promoter in endometrial cells and tissues. Deletion analysis of PAX2 promoter was performed by luciferase reporter gene to analyze its promoter activity. Transcription factors (TFs) combined with PAX2 promoter were forecasted by TF search database and the regulatory function of these TFs were measured by detecting the expression of PAX2 mRNA and protein following the knock down of these TFs. Results: PAX2 was over-expressed in endometrial cancer tissues compared to hyperplasia and normal endometrium. Compare to normal endometrial epithelial cells, endometrial cancer cell lines exhibited moderate to high PAX2 expression. Knocking down PAX2 by siRNA reduced cell viability, invasion and migration, while PAX2 over-expression exhibited opposite role. The methylation level of PAX2 promoter was higher than normal counterparts in both cancer tissues and cells, which was positively related to PAX2 expression. After 5-Aza-CdR treatment, the PAX2 mRNA and protein was down-regulated along with the methylation level of PAX2 promoter. Deletion analysis confirmed that a repressive transcriptional regulatory region of PAX2 promoter coincided with the hypermethylated region found in MassARRAY analysis. The binding sites of myeloid zinc finger 1 (MZF1) and the early growth responsive gene 1(EGR-1) were found in the repressive transcriptional regulatory region of PAX2 promoter. Knocking down MZF1 or EGR-1 upregulated both the mRNA and protein level of PAX2 after 5-Aza-CdR treatment, which indicated that MZF1 and EGR-1 may act as repressive transcription factors in the DNA methylation related PAX2 transcription. Conclusion: PAX2 is involved in carcinogenesis of endometrial cancer by stimulating cell growth and promoting cell motility. Overexpression of PAX2 in endometrial cancer may be regulated by promoter hypermethylation via repressive transcription factors such as MZF1 or EGR-1. Citation Format: Nan Jia, Jieyu Wang, Qing Li, Xiang Tao, Keqin Hua, Yinhua Yu, Jan P. Baak, Kwong Kwok Wong, Weiwei Feng. Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2015-4768

Ovarian cancer is the most lethal gynecologic malignancy in the US. While major research efforts have been dedicated to identify disease related genes and mutations in cancer cells, the role of the stromal tumor microenvironment in disease progression is relatively unexplored. We previously reported that while ovarian cancer cells have minimal response to TGF-beta, TGF-beta induced secretory proteins from cancer associated fibroblasts (CAFs) promote ovarian cancer motility and invasion potential. The present study seeks to further evaluate the involvement of TGF-beta signaling in CAFs in the progression of ovarian cancer and the therapeutic potential of targeting such signaling in cancer treatment. By comparing the gene expression profiles of laser-microdissected ovarian tumor stromal tissues from high-grade serous ovarian cancer (HGSOC) patients with short and long survival, a differentially expressed gene signature was identified. Using the IPA platform, signaling pathways and upstream regulators activated in CAFs from patients with short survival were identified. TGF-beta signaling was found to be significantly activated (p = 2.47E-03), while the ligand TGF-beta 1 was identified as the most significant upstream regulator (Z-score = 5.189, p = 8.85E-29), suggested the involvement of CAF TGF-beta signaling in poor patient survival. Further immunohistochemistry and survival analyses revealed the clinical relevance of multiple TGF-beta regulated stromal genes, including VCAN, MFAP5 and COMP in poor disease prognosis. Subsequently, cell culture studies were performed to evaluate the effects of TGF-beta in ovarian cancer progression. While exogenous TGF-beta did not affect proliferation, motility and invasion potential of ovarian cancer cells directly, TGF-beta promoted ovarian cancer cells motility and invasion potential in a co-culture system of cancer cells and CAFs. Similarly, treatment of exogenous TGF-beta-induced CAF secretory proteins promoted ovarian cancer progression, suggested the presence of CAFs is required for TGF-beta mediated ovarian cancer progression and targeting such TGF-beta signaling in CAFs could be a novel modality of ovarian cancer treatment. To reprogram CAFs by targeting its TGF-beta signaling, multiple approaches were tested in co-culture systems. Using either siRNAs or small molecule inhibitors, TGF-beta signaling in CAFs was effectively suppressed and inhibited ovarian cancer progression was observed. Based on our current findings, animal studies will be performed to further evaluate the efficacy of targeting the CAF TGF-beta signaling pathway in ovarian cancer treatment. This study presents the paradigm which activation of TGF-beta signaling in ovarian CAFs is significantly associated to the poor clinical outcomes. Targeting such pathway activation in CAFs could effectively suppress tumor progression and thereby presented a novel approach in cancer treatment. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Reprogramming the TGF-beta signaling in cancer-associated fibroblasts inhibits ovarian cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5066. doi:10.1158/1538-7445.AM2015-5066

The chromatin remodeling SWI/SNF complex is mutated in 20% of all cancers and ARID1A is the most frequently mutated subunit. However, the tumor suppressive functions of ARID1A are not fully understood and no feasible therapeutic strategies are available for ARID1A-mutant cancers. Recent studies found that loss of ARID1A is associated with increased phosphorylation of AKT. We found that from a study that analyzed data from Project Achilles, a broad shRNA screening project, PIK3CA is the number 2 gene essential for survival of ARID1A-mutant cell lines compared to ARID1A-wildtype cell lines (P = 7.37 × 10-6, FDR < 0.001). Based on these findings, we hypothesized that the PI3K pathway is a potential therapeutic target in ARID1A-mutant cancers. We analyzed the Cancer Genome Atlas (TCGA) datasets and found that mutations in the PI3K pathway co-occur with ARID1A mutations. In addition, the number of co-existing PI3K pathway mutations in the same sample is higher when ARID1A is mutated. We knocked down PIK3CA in ARID1A-wildtype cells (RMG1 and OVCAR3) and ARID1A-mutant cells (OVAS and HCH-1). We found that proliferation was impaired more profoundly in ARID1A-mutant cells. Interestingly, HCH-1 cells are wildtype in PIK3CA, PTEN, PIK3R1 and KRAS, but are still sensitive to PIK3CA depletion. For an unbiased approach, we analyzed the Genomics of Drug Sensitivity in Cancer datasets, which contain drug responses of a large cancer cell line panel to 138 anti-cancer drugs. We compared the drug responses of 49 cell lines harboring inactivating ARID1A-mutations with 266 ARID1A-wildtype cell lines. We found that the presence of inactivating ARID1A mutations is highly associated with sensitivity to mTOR inhibitor AZD8055 (ranked 2nd, P = 2.00 × 10-3) and AKT inhibitor MK2206 (ranked 4th, P = 7.98 × 10-3). This association is still significant for MK2206 when we removed cell lines with PIK3CA, KRAS, PTEN, PIK3R1 and TSC1 alterations (P = 1.32 × 10-2). Finally, we investigated how ARID1A loss can directly increase PI3K/mTOR activity. Using microarray analysis, we found that knockdown of ARID1A up-regulated MYC and MYC target genes, including SLC7A5, an amino-acid transporter required for mTOR activation. Analysis of TCGA datasets showed that MYC amplification and ARID1A mutations are mutual exclusive events, suggesting that overexpression of MYC and loss of ARID1A may converge on the same pathway. In conclusion, we found that ARID1A-mutant cells are highly sensitive to PI3K/mTOR inhibition. Although ARID1A mutations frequently co-occur with PI3K pathway mutations, it is not the sole explanation of this specific sensitivity. ARID1A loss may increase mTOR signaling through MYC target gene SLC7A5. However, increase in PI3K/mTOR activity maybe a long term effect of ARID1A loss. Together, our data identified PI3K/mTOR signaling is essential for survival of ARID1A-mutant cancers and PI3K/mTOR inhibitors can be used as therapeutic strategies. Citation Format: Suet-Yan Kwan, Daisy I. Izaguirre, Xuanjin Cheng, Suet-Ying Kwan, Yvonne TM Tsang, Hoi-Shan Kwan, Kwong-Kwok Wong. The PI3K/mTOR pathway is a potential therapeutic target in cancers with ARID1A mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4697. doi:10.1158/1538-7445.AM2015-4697

Ovarian cancer is the fifth most common form of cancer in women in the United States. It is a complex disease composed of different histological grades and histological types. Most of epithelial ovarian cancer cases are detected at an advanced stage. Patients usually respond to primary treatment with surgery and chemotherapy. However, the disease usually recurs and is ultimately fatal. So far, a satisfactory screening procedure and regime to treat the recurrence disease are not available. High-throughput genomic analyses have the potential to change the detection and the treatment of ovarian neoplasms. They can help diagnose subtypes of disease and predict patient survival. New diagnostic and prognostic markers for ovarian cancer are emerging. One day, profiling may influence treatment decisions, informing both which patients should receive chemotherapy and what type of chemotherapeutic agents should be employed. As greater numbers of tumor samples are analyzed, the power of these profiling studies will increase, raising the possibility that novel molecular targets and less toxic therapies will be identified. These powerful techniques hold the potential to unravel the genetic origins of ovarian cancer. Hopefully, this will translate into earlier diagnosis and better patient outcome from disease.

Mobilization of tumor-reactive CD8 + T cells remains the major challenge of cancer immunotherapy. Knowing how and when the T cell response expands and differentiates after antigen stimulation would make a significant contribution to the development of tumor vaccines. In the current study, we used CFSE-based cell sorting and cDNA microarray to identify the gene expression profile of adjacent generations of T cells after PHA stimulation. Early-divided generations of T cells responded to stimulation by activating cell cycle and surviving gene pathways, while late generations of T cells had more dramatic changes in transcription of cytokine genes. Reconstruction of biochemical pathways, activated in both early and late generations of T cells, also confirmed the impact of division in focal-adhesion kinases. Because most tumors are infiltrated by lymphocytes, our studies indicate a novel approach to identify 'systemic biological responses' of T cells, which could determine the design, and optimization of effective tumor vaccines.

Mobilization of tumor-reactive CD8+ T cells remains the major challenge of cancer immunotherapy. Knowing how and when the T cell response expands and differentiates after antigen stimulation would make a significant contribution to the development of tumor vaccines. In the current study, we used CFSE-based cell sorting and cDNA microarray to identify the gene expression profile of adjacent generations of T cells after PHA stimulation. Early-divided generations of T cells responded to stimulation by activating cell cycle and surviving gene pathways, while late generations of T cells had more dramatic changes in transcription of cytokine genes. Reconstruction of biochemical pathways, activated in both early and late generations of T cells, also confirmed the impact of division in focal-adhesion kinases. Because most tumors are infiltrated by lymphocytes, our studies indicate a novel approach to identify 'systemic biological responses' of T cells, which could determine the design, and optimization of effective tumor vaccines.

Ovarian cancer is the fifth most common form of cancer in women in the United States. It is a complex disease composed of different histological grades and histological types. Most of epithelial ovarian cancer cases are detected at an advanced stage. Patients usually respond to primary treatment with surgery and chemotherapy. However, the disease usually recurs and is ultimately fatal. So far, a satisfactory screening procedure and regime to treat the recurrence disease are not available. High‐throughput genomic analyses have the potential to change the detection and the treatment of ovarian neoplasms. They can help diagnose subtypes of disease and predict patient survival. New diagnostic and prognostic markers for ovarian cancer are emerging. One day, profiling may influence treatment decisions, informing both which patients should receive chemotherapy and what type of chemotherapeutic agents should be employed. As greater numbers of tumor samples are analyzed, the power of these profiling studies will increase, raising the possibility that novel molecular targets and less toxic therapies will be identified. These powerful techniques hold the potential to unravel the genetic origins of ovarian cancer. Hopefully, this will translate into earlier diagnosis and better patient outcome from disease.

Using single nucleotide polymorphic (SNP) allele arrays, we analyzed 28 pediatric gliomas consisting of 14 high-grade gliomas and 14 low-grade gliomas. Most of the low-grade gliomas had no detectable loss of heterozygosity (LOH) in any of the 11,562 SNP loci; exceptions were two gangliogliomas (3q and 9p), one astrocytoma (6q), and two subependymal giant cell astrocytomas (16p and 21q). On the other hand, all high-grade gliomas had various degrees of LOH affecting 52 to 2,168 SNP loci on various chromosomes. LOH occurred most frequently in regions located at 4q (54%), 6q (46%), 9p (38%), 10q (38%), 11p (38%), 12 (38%), 13q (69%), 14q (54%), 17 (38%), 18p (46%), and 19q (38%). We also detected amplifications of epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor α (PDGFRα) in a few of the 13 cases of glioblastoma multiforme analyzed. Interestingly, the amplified EGFR and PDGFRα were located within regions of LOH. SNP loci with LOH and copy number changes were validated by sequencing and quantitative PCR, respectively. Our results indicate that, in some pediatric glioblastoma multiforme, one allele each of EGFR and PDGFRα was lost but the remaining allele was amplified. This may represent a new molecular mechanism underlying tumor progression. (Cancer Res 2006; 66(23): 11172-8)

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.

Purpose: Patients with ovarian clear cell adenocarcinoma generally have a poor response to combination chemotherapy and have overall poorer prognosis than patients with other histologic types of ovarian cancer. Genetic changes in this group of cancer have not been thoroughly explored. Identification of these changes may provide us new therapeutic targets to treat this disease. Experimental Design: Genomic and expression array analyses were applied on 30 clear cell ovarian cancer cases and 19 serous cases using a 10,816-element cDNA microarray platform. Further validation and clinical correlation studies were done on differentially expressed genes that are related to chemoresistance. Results: Based on array analyses, 12 genes showed a significant increase in DNA and mRNA copy number and 5 genes showed a significant decrease in DNA and RNA copy number in clear cell tumors compared with those in the serous type. One of the genes was ABCF2, which belongs to the ATP-binding cassette gene superfamily and has been shown to amplify in other tumor types. Validation studies were done using real-time quantitative PCR and immunohistochemistry. The results showed significantly higher ABCF2 DNA and mRNA copy number and protein levels in clear cell cases compared with those in serous cases. Furthermore, in 20 clear cell cases with chemoresponse data available, ABCF2 cytoplasmic staining was significantly higher in nonresponders than that in the responders (60.0% versus 28.5%; P = 0.0002). Conclusions: These data suggest that ABCF2 protein may be a prognostic marker for ovarian clear cell ovarian adenocarcinoma.

Objective. (1) To identify and (2) validate genes that are up-regulated in ovarian cancer, and (3) to investigate whether the activity of a candidate gene, creatine kinase B (CKB) is elevated in pre-operative sera from ovarian cancer patients compared to patients with benign pelvic masses and normal controls. Methods. MICROMAX cDNA microarray system and RNA derived from pooled ovarian cancer cell lines and normal ovary surface epithelial cells (HOSE) were used to identify differentially expressed genes. Using RNA from both cell lines and from tissue obtained through laser capture microdissection (LCM), we performed quantitative PCR in order to validate up-regulation of one of these genes, creatine kinase B (CKB). Using a commercially available enzyme assay, CKB activity was measured in pre-operative serum samples obtained from 45 ovarian cancer patients, 49 patients with a benign pelvic mass, as well as 37 normal controls. Statistical analysis was preformed using an unpaired Student's t test. Results. Microarray technology revealed that CKB gene expression had a cancer to HOSE ratio of 18. RNA levels of CKB, measured by real-time PCR, were elevated a mean (and standard error) of 36-fold (8.4) in cancer cell lines compared with HOSE cells and 22.75-fold (10.45) in microdissected ovarian cancer epithelial cells compared with normal ovarian epithelial cells. In serum, the mean (±standard error) of CKB enzyme activity in cancer cases was 24.7 U/L units (±5.1) compared to 9.6 U/L (±1.6) for benign mass cases ( P = 0.0088) and to 8.5 U/L (1.7) for normal controls ( P = 0.0096). Conclusions. Microarray technology offers a method to identify tumor biomarkers with potential clinical usefulness. Our data indicated that CKB gene expression is up-regulated in ovarian cancer cells in vitro and in vivo and that CKB enzyme activity is significantly elevated in sera from ovarian cancer patients, including those with stage I disease. These findings suggest a potential role for CKB as a marker for early diagnosis.

The epithelial cell adhesion molecule (Ep-CAM) exhibited an ovarian cancer:normal human ovarian surface epithelium ratio of 444. For validation studies, real-time quantitative PCR analysis and immunohistochemistry were performed in normal and malignant ovarian epithelial cell lines and tissues. To evaluate the potential of the Ep-CAM autoantibody as a tumor marker, we examined the amount of Ep-CAM autoantibody in serum samples obtained from ovarian cancer patients and normal controls by an ELISA. Real-time quantitative PCR analysis revealed significant overexpression of Ep-CAM mRNA in cancer cell lines (P0.001) and microdissected cancer tissues (P0.05), compared with that in cultured normal human ovarian surface epithelium and microdissected germinal epithelium, respectively. Immunolocalization of the Ep-CAM autoantibody showed that the sera of ovarian cancer patients expressed higher levels of Ep-CAM autoantibody than benign tumor patients and normal controls (P0.05). The levels of Ep-CAM autoantibody found were as follows: 0.132 in 52 patients with ovarian cancer, 0.098 in 26 cases with benign gynecologic disease, and 0.090 in 26 normal women. This investigation has shown that the Ep-CAM autoantibody was found to be associated with ovarian cancer and suggested that future research assessing its clinical usefulness would be worthwhile.

To develop a sensitive cDNA microarray based uterotrophic assay, ovarectomized mice were treated with a low dose of 17-β-estradiol (0.2 μg/kg/day) over a three-day period. The average increases in uterine weight were 13%, 23% and 70% after treatment at day 1, day 2 and day 3 respectively. Twenty-four hours after each treatment, uteri were dissected for total RNA extraction and gene expression profiles were assayed with a mouse cDNA microarray containing more than 5000 cDNA elements. From the analysis, we were able to detect 72 genes that were induced more than 2-fold 24 hours after the ovarectomized mice were treated a single dose of 17-β-estradiol. 49 of these genes form a tight cluster when analyzed by the software OmniVizPro™ based on their temporal expression profiles. The number of genes induced more than two-fold increases to more than 200 after the ovarectomized mice were treated with 17-β-estradiol once a day for 1 or 2 more days. These inducible genes include both known and unknown genes. Identified known genes are involved in cell division, transcription activation, stress response, oncogene, and other biochemical activities. These results suggest that gene expression profiles can be used as an alternative endpoint for uterotrophic assay. Further analysis and exploitation of this set of genes will allow us to develop a more sensitive and specific assay for the detection of estrogenic chemical as well as the understanding of the signaling pathway elicited by 17-β-estradiol.

Ovarian cancer is the most lethal gynecologic malignancy. Although anti-angiogenic therapies look promising, no major strides have been made to improve ovarian cancer patient survival over the past decade. Therefore, finding new molecular targets that can improve therapeutic benefit from anti-angiogenic strategies are of paramount importance. Microfibrillar-Associated Protein 5 (MFAP5) was previously reported to be associated with prognosis and promoted ovarian cancer progression. In ovarian tumor samples, we identified a positive correlation between stromal MFAP5 and CD31 expressions which suggested a pro-angiogenic role of MFAP5. This study aims at delineating the mechanism of stromal MFAP5 in modulating tumor angiogenesis and investigating the potential of targeting MFAP5 as an anti-angiogenic therapy for ovarian cancer treatment. Increased motility and tube formation were observed in the presence of exogenous MFAP5 in both human microvascular endothelial cell 1 (HMEC-1) and telomerase immortalized microvascular endothelial cells (TIME). Besides, both anti-αVβ3 integrin antibody and cell permeant calcium chelator, BAPTA, attenuated MFAP5 stimulated motility. These suggested that MFAP5 exerts its biological activity through the αVβ3 integrin receptor and calcium signal transduction. In vivo, extensive CD34 positive tubular network was formed in the matrigel mixed with recombinant MFAP5 injected subcutaneously into mice. Further analysis using the Leica Metamorph software confirmed that there were significantly increases in the number of nodes, total tube area, number of tube segment and branch points in MFAP5 containing matrigel compared to control (p Finally, microarray analysis on endothelial cells invaded into Matrigel plug, identified a set of motility promoting genes that were associated with calcium signaling. Among them, upregulation of Lipoma-Peferred Partner (LPP), an actin cytoskeleton protein, was validated by qRT-PCR and western blot analyses. Silencing LPP by siRNA abolished MFAP5 stimulated motility in both HMEC-1 and TIME. Further western blot analyses showed that MFAP5 modulated endothelial cell motility and invasion potential through calcium dependent ERK/CREB/LPP signaling pathways. To conclude, stromal MFAP5 acts on αVβ3 integrin receptor and modulates endothelial cell behavior through calcium signaling. It contributes to ovarian tumor angiogenesis and tumor progression. Hence, targeting stromal MFAP5 could be a potential anti-angiogenic therapy for ovarian cancer treatment. Citation Format: Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Anil K. Sood, Michael J. Birrer, Samuel C. Mok. CAF-derived MFAP5 promotes ovarian tumor angiogenesis through calcium dependent LPP signaling pathway. [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 1029. doi:10.1158/1538-7445.AM2014-1029

Ovarian cancer is the most lethal gynecologic malignancy in the United States. While the tumor microenvironment has been shown to play important roles in cancer pathogenesis, large-scale transcriptome profiles of the stromal component of ovarian tumors and the identification of stromal prognostic markers and therapeutic targets are lacking. This study seeks to identify secreted stromal factors and evaluate their clinical significance. Transcriptome profiling and clustering analysis on microdissected ovarian cancer associated fibroblasts (CAFs) samples from 83 patients revealed a heterogeneous gene expression pattern. Since CAFs can be derived from mesenchymal stem cells (MSCs), we compared transcriptome profiles of MSC samples obtained from healthy individuals with those from CAFs. Results showed that CAF expression profiles could be classified into two major subtypes. The MSC subtype, which has gene expression pattern highly resembling to that of undifferentiated MSCs, is significantly associated with poorer patient survival when compared to the non-MSC subtype. Further analyses identified two expression clusters within the MSC subtype: 1) the CAF-C cluster which expressed high level of MSC chondrogenic differentiation associated genes including CSPG2, SFRP2 and COMP, and 2) the CAF-O cluster which expressed high level of osteogenic differentiation associated genes including RUNX2 and BGN. Survival analysis showed that patients with the CAF-O expression subtype had overall survival rates comparable to those with the non-MSC subtypes, while the CAF-C subtype is associated with worst clinical outcomes. Pathway analyses revealed strong interactions among these stromal genes. Among them, SFRP2, which has been shown to inhibit osteogenic differentiation of MSCs by inhibiting Wnt signaling and subsequently suppress RUNX2 expression, was selected for further validation studies. Stromal SFRP2 and RUNX2 protein expression levels were evaluated by immunohistochemistry on paraffin tissue sections from 94 patients. Results showed that high levels of stromal SFRP2 were significantly associated with poor patient survival (p < 0.001), while high levels of stromal RUNX2 were significantly associated with improved patient survival (p < 0.001). A significant inverse correlation between SFRP2 and RUNX2 expression levels was also observed (r = -0.279, p = 0.037), suggested that the interactions among these genes may generate different landscapes in the tumor microenvironment, which modulate MSC differentiation and subsequently cancer cell aggressiveness. This study presents a new paradigm of which osteogenic-like and chondrogenic-like CAF signatures are associated with better and poorer patient survival respectively. Understanding the relationship between the expression patterns of stromal factors and MSC differentiation would provide us with new insights into ovarian cancer pathogenesis. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Identification and characterization of stromal factors with clinical significance in the ovarian tumor microenvironment. [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 4799. doi:10.1158/1538-7445.AM2014-4799

Patients with ovarian serous cancer are usually younger and survive longer than patients with high-grade serous cancer. Unfortunately, most low-grade serous cancer patients eventually die of the disease because recurrent low-grade serous carcinoma is relative chemoresistant. So far, the known mutations identified in low-grade ovarian serous carcinomas and their putative precursor ovarian serous borderline tumors are BRAF and KRAS mutations. BRAF and KRAS mutations together have been identified in approximately 60% of serous borderline tumors and early stage low-grade serous carcinomas. However, BRAF mutation is rare in advanced-stage low-grade serous carcinomas. To further investigate the molecular pathogenesis of low-grade serous carcinoma, mutation analyses of 409 cancer related genes were performed in 21 low-grade ovarian serous carcinoma, 8 ovarian serous borderline tumors and one ovarian cystadenoma using next generation sequencer. A total of nineteen missense mutations were identified in 20 samples but ten samples had no detectable mutations. BRAF mutation was detected in 3 serous borderline tumors and one low-grade serous carcinoma. KRAS mutations were detected in three low-grade serous carcinomas. Other than BRAF and KRAS mutations, we also detected other prevalent mutations: ATRX mutations were detected in 3 low-grade ovarian serous carcinomas and a cystadenoma; KMT2C mutations were detected in 3 low-grade ovarian serous carcinomas; ATM mutations were detected in 2 low-grade ovarian serous carcinomas. Interestingly, both ATRX and MLL3 genes are involved in the chromatin remodeling. Moreover, both ATRX and ATM are involved in DNA damaging response. Thus, the pathogenesis of low-grade serous carcinoma may involve de-regulation of chromatin remodeling pathway and DNA damaging response. Further analysis of a large cohort of low-grade serous carcinomas for these prevalent mutations is ongoing. Citation Format: Yvonne TM Tsang, Daisy Izaguirre, Suet-Yan Kwan, Samuel C. Mok, David Gershenson, Kwong-Kwok Wong. Ovarian low grade serous cancer: Mutation analysis with a comprehensive 409 cancer gene panel. [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 1529. doi:10.1158/1538-7445.AM2014-1529

Using the MICROMAX™ cDNA microarray system, we were able to identify genes that are differentially overexpressed in ovarian cancer. A total of 30 putative genes, which are differentially overexpressed in ovarian cancer cell lines, were identified. The differential expression of some of these genes was further confirmed by real-time RT-PCR. Using this strategy, we have identified genes that either overexpress in all cancer cell lines or in only some cancer cell lines. Further characterization of these genes will allow them to be exploited in diagnosis, prognosis, anticancer therapy, and molecular classification of ovarian cancer.

The purpose of this project is to provide fundamental knowledge on environment stress response of subsurface bacteria and a radiation-resistant bacterium (Deinococcus radiodurans). This information will be particularly useful in the development of successful bioremediation strategies. These organisms represent two phylogenetically distinct groups of soil bacteria, each of which has specific features of interest for bioremediation. The subsurface bacteria, Sphingomonas spp (Savannah River Site), have unique degradative capabilities of organic waste. The study of subsurface bacteria will serve as a model for bioremediation using indigenous bacteria. D. radiodurans exhibits high resistance to external stress such as ionizing radiation and organic solvent. The study of D. radiodurans will serve as a model for the use of genetically engineered bacteria for bioremediation.

As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome. The average insert size is 17 kb. Sequences were obtained from both ends of each clone in this set. A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank. This resource of clones is available from the Salmonella Genome Stock Center. A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes. Fully 30% of the S. typhimurium sequences have no close homologs in the GenBank database.

Deinococcus radiodurans R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase ( katA ) and superoxide dismutase ( sodA ) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.

Pulsed-field gel electrophoresis (PFGE) has allowed the separation of DNA fragments of five million base pairs or more, which is about the same size or greater than most bacterial genomes. When a bacterial genome is cut into a few discrete fragments, then PFGE is an ideal tool for the construction of physical maps (see also Chapters 25 and 26).

'To provide increased knowledge of stress responses of indigenous microbes at contaminated sites as well as using microbes as molecular probes for monitoring the performance and effectiveness of bioremediation, the authors propose to identify the stress-inducible genes and promoters from two soil bacteria, Deinococcus radiodurans and Sphingomonas F199. These organisms represent two phylogenetically distinct groups of soil bacteria, each of which has specific features of interest for bioremediation. D. radiodurans exhibits high resistance to external stress; F199 is a deep subsurface (Savannah River Site) organism with unique degradative capabilities. Research Statement To realize the full potential of bioremediation, an understanding of microbial community and individual bacterial responses to the stresses encountered at contaminated sites is needed. Knowledge about genetic responses of soil and subsurface bacteria to environmental stresses, which include low nutrients, low oxygen, and mixed pollutants, will allow extrapolation of basic principles to field applications either using indigenous bacteria or genetically engineered microorganisms. Defining bacterial responses to those stresses presents an opportunity for improving bioremediation strategies, and should contribute to environmental management and restoration actions that would reduce the cost and time required to achieve DOE''s cleanup goals.'

Ovarian cancer is the most common cause of gynecologic malignancy related mortality in women. The combination of tumor reductive surgery and chemotherapy is the main therapeutic strategy for ovarian cancer. However, especially in the most of advanced cases, complete site reduction is impossible to achieve. In those cases, the sensitivity to chemotherapy strongly affects their prognoses. MicroRNAs (miRNAs), which are approximately 22-nucleotide non-coding RNAs, regulate protein expression by translational inhibition and degradation of their target mRNAs. The expression of miRNAs is dysregulated in various types of cancers, suggesting that miRNAs are involved in carcinogenesis and cancer progression. The purpose of present work was to identify miRNAs which are related to chemo-sensitivity in ovarian cancer. Using Next Generation Sequencing (NGS), miRNA profiling was performed in 38 advanced high-grade serous ovarian cancers (HGSOCs). RNAs including miRNAs were procured from the epithelial component of the laser microdissected tumor tissue removed from patients undergoing primary surgery without neo-adjuvant chemotherapy. A total of 1935 miRNAs and miRNA variants were detected by NGS and normalized expression data of each miRNAs and miRNA variants were used for further analyses. Comparing the copy numbers of each miRNAs between chemo-refractory group (patients with disease progression in the period of first line chemotherapy, n=7) and chemo-sensitive group (patients with disease progression in more than 6 months after completion of first line chemotherapy, n=13) by Mann-Whitney U test, 77 miRNAs and miRNA variants were found to be dysregulated with significant differences (p Our results strongly suggest that mir-625-3p might be a prognostic marker that can be used to predict chemo-sensitivity in patients with HGSOC. Further characterization of mir-625-3p as a therapeutic target for HGSOC is in progress. Citation Format: Tetsushi Tsuruga, Chi Lam Au Yeung, Cecilia S. Leung, Tsz-Lun Yeung, Kwong K. Wong, Rosemarie Schmandt, Ngai Na Co, Karen H. Lu, Samuel Mok. Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing. [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 1939. doi:10.1158/1538-7445.AM2013-1939

Uterine papillary serous carcinoma (UPSC) comprises less than 10% of all endometrial cancers but accounts for a disproportionately high number of deaths. Due to its rarity, few prospective studies have been conducted which focus on this aggressive subtype, and it has been difficult to establish optimal treatment strategies. With poor survival rates and high recurrence rates, the identification of novel therapeutic targets would aid in the management of UPSCs. RNAseq data from The Cancer Genome Atlas (TCGA) was analyzed using Significant Analysis of Microarrays to identify genes that were differentially expressed between 81 cases of grade 2 endometrioid endometrial carcinoma (EEC) and 41 cases of UPSC. A gene of interest was then selected based on its subcellular location, correlation with survival and normal tissue specificity. The findings were validated by immunohistochemical staining of UPSC paraffin sections. All patient samples were obtained and stored in accordance with human subject research protocols approved by the Institutional Review Board. All paraffin sections were reviewed by a pathologist. By comparing the RNAseq profiles of UPSCs and EECs, we identified 242 genes that were differentially expressed (q Citation Format: Suet Ying Kwan, Kwong-Kwok Wong, Rosemarie E. Schmandt, Karen H. Lu. UCHL1 as a potential therapeutic target in uterine papillary serous carcinoma. [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 4401. doi:10.1158/1538-7445.AM2013-4401

Ovarian cancer treatment consists of surgery followed by a chemotherapy combination entailing a platinum agent (cisplatin/carboplatin) and a taxane (paclitaxel). While 70-80% of patients initially respond to the standard treatment, the majority will relapse, and of these patients, 70-90% die because of drug resistance. Therefore, identifying markers that can predict therapeutic response and/or therapeutic targets is of great interest. To identify genes involved in chemoresistance, a microarray study of an ovarian cancer cell line (A2780) treated with varying doses of cisplatin and at varying time points was conducted. This study showed GDF15 had the highest fold-induction by cisplatin treatment. GDF15 induction by cisplatin was further validated by RT-PCR and Western blot analysis in the A2780 and 12 other cell lines with varying sensitivity/resistance to cisplatin. To further test whether GDF15 plays a role in cisplatin resistance, we knocked down its expression by esiRNA. GDF15 knockdown revealed an increase in the cisplatin IC50, suggesting that GDF15 sensitizes ovarian cancer cells to cisplatin. To determine whether GDF15 was also induced in vivo we injected mice intraperitoneally with either A2780 (cisplatin sensitive) or RMG1 (cisplatin resistant) cells. Once the tumors developed, the mice were treated with either 2.5 mg/kg or 5 mg/kg cisplatin and both the tumors and sera collected 24 and 48 hours after treatment. ELISA results on the serum showed an increase in GDF15 in mice after cisplatin treatment. Although there was no correlation between the two cisplatin doses used and GDF15 there was a correlation between GDF15 and tumor weight. Thus, our results suggest that GDF15 is induced by cisplatin both in vitro and in vivo and has a potential role as a serum biomarker to predict chemotherapeutic response. Citation Format: Daisy I. Izaguirre, Suet Yan Kwan, Zhifei Zu, Yvonne T. Tsang-Lee, Kwong-Kwok Wong. GDF15 as a serum marker of cisplatin response in ovarian cancer cells. [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 3474. doi:10.1158/1538-7445.AM2013-3474

We have developed a novel method to clone and sequence minute quantities of DNA. The method was applied to sequence a 180 kb plasmid pNL1. The first step was the production of a size distributed population of DNA molecules that were derived from the 180 kb plasmid pNL1. The first step was accomplished by a random synthesis reaction using Klenow fragment and random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN6, 5'-GTAATACGACTCACTATAGGGCNNNNNN-3'. In the second step, Klenow-synthesized molecules were amplified by PCR using T7 primer (5'-GTAATACGACTCACTATAGGGC-3'). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules with sizes ranging from 28 bp to >23 kb which were detectable on an agarose gel. The Klenow-synthesized molecules were then used as templates for standard PCR with T7 primer. PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for cloning and sequencing. From the same Klenow-synthesized molecules, we were also able to generate PCR products with sizes up to 23 kb by long range PCR. A total 232.5 kb sequences were obtained from 593 plasmid clones and over twenty putative genes were identified. Sequences from these 593 clones were assembled into 62 contigs and 99 individual sequence fragments with a total unique sequence of 86.3 kb.

Microbial ecosystems are characterized by genetic and taxonomic diversity. Investigations of microbial diversity have gained benefits from the development of various typing techniques to distinguish strains more precisely [1, 10, 14, 19]. These techniques may be divided into two categories, those based on phenotypic and those based on genotypic characters. Typing techniques based on phenotype include isozyme electrophoresis, whole-cell protein profiling [17], sugar metabolism profiling, total fatty acids profiling [6], phage typing [7], and various immunoblotting techniques [10, 12]. Typing techniques based on genotype include DNA-DNA hybridization, restriction enzyme analysis, RFLP, ribotyping [1], plasmid profiling [4, 5] and DNA fingerprinting by Arbitrarily Primed PCR (APPCR) [23, 24], RAPDs (Random Amplified Polymorphic DNA) [29], or rep-PCR [20]. APPCR is the subject of this chapter.

e15583 Background: Epithelial ovarian cancer (OC) is a molecularly and clinically heterogeneous disease, yet treatment is the same for all subtypes. Type I carcinomas, including low grade serous, mucinous, clear cell, and endometrioid OCs, are relatively chemoresistant. In contrast, Type II or high grade serous OCs are more aggressive yet responsive to chemotherapy. Molecularly, 96% of high grade serous OCs harbor p53 mutations, whereas Type I carcinomas are more frequently p53 wild-type. Nutlin-3 is a novel therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis. We therefore sought to validate nutlin-3 as a therapeutic compound for p53 wild-type OCs. Methods: Two low grade serous, 2 clear cell, 2 endometrioid, 3 mucinous, and 4 high grade serous OC cell lines were treated with varying concentrations of nutlin-3, and proliferation assays were performed. A p53-null cell line was used as a negative control. The cells were then treated with nutlin-3 at their IC50s, and qRT-PCR and Western blot (WB) quantified MDM2, p53, and p21 expression at 24, 48, and 72 hours after treatment. The cell lines were sequenced for p53 mutations in exons 5-8. Results: Responses to nutlin-3 were variable and dependent on p53 mutation status. Low grade and clear cell lines were most sensitive to nutlin-3, endometrioid type were intermediately sensitive, and mucinous were resistant. Two of the four high grade cell lines were sensitive to nutlin-3, both of which were p53 wild-type. Endometrioid and low grade serous cell lines possessing heterozygous p53 mutations were still more sensitive than the p53-null cell line. On qRT-PCR and WB, levels of p53 were decreased after nutlin-3 treatment and were absent in the p53-null cell line. MDM2 and p21 were initially upregulated in sensitive cell lines, but returned to near-baseline levels after 72 hours. In the more resistant cell lines, p21 induction was reduced or absent. Conclusions: Nutlin-3 is a novel potential therapeutic agent for p53 wild-type ovarian carcinoma, and is dependent on an intact p53 pathway with p21 upregulation. Even OC cell lines with heterozygous p53 mutation responded modestly to nutlin-3 treatment. Further investigation of nutlin-3 treatment in ovarian cancer is warranted.

Tumor microenvironment is crucial for tumor progression. Cancer associated fibroblasts (CAFs) support tumor growth and invasion by actively secrete different proteins into the extracellular matrix. Using transcriptome profiling analysis on microdissected normal and malignant ovarian tumor tissues, we obtained a CAF specific gene signature. One of the identified genes, versican, showed a 15fold increase in expression in the fibroblastic stromal component of the tumor tissue. This study seeks to evaluate the clinical significance of stromal versican over-expression, to delineate the role of versican in ovarian cancer progression and to understand the regulatory mechanism of versican. Clinical significance of stromal versican expression was evaluated by Kaplan Meier analysis (N=102). Results suggested that high versican expression in cancer stroma is significantly associated with poor overall and progression free survival in high-grade serous ovarian cancer (HGSC) patients (p 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 1507. doi:1538-7445.AM2012-1507

Using transcriptome profiling analysis, we identified a gene signature in the fibroblastic stromal component of high grade serous ovarian tumor tissue. One of the most differentially expressed proteins was microfibrillar associated protein 5 (MFAP5). This study aims at validating the differential expression patterns of MFAP5 in ovarian cancer stroma, evaluating the clinical significance of stromal MFAP5 over-expression and delineating the functional role and mechanism of MFAP5 in modulating angiogenesis and tumor progression. Quantitative RT-PCR analysis was first performed on RNA isolated from microdissected normal and malignant ovarian tissue samples and immunolocalization of MFAP5 protein was performed on paraffin tissue sections (n=102) to validate differential stromal MFAP5 expression. Both demonstrated significant higher levels of MFAP5 expression in the fibroblastic stromal component of cancer tissue samples. Kaplan Meier analysis demonstrated a significant correlation between stromal MFAP5 expression and poor overall patient survival (p 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 4270. doi:1538-7445.AM2012-4270

Despite the fact that ovarian epithelial ovarian cancer is a molecularly and histologically heterogeneous disease, standard treatment is the same for all subtypes. Most high-grade serous ovarian carcinomas (OCs) are responsive to initial platinum/taxane chemotherapy, but low-grade serous OCs and clear cell OCs are relatively chemoresistant with limited treatment options upon recurrence, and most patients succumb to the disease. It is therefore of interest to exploit the molecular differences between high-grade serous OCs and other histological types in order to develop novel therapeutics. Over 90% of high-grade serous OCs harbor p53 mutations, whereas low-grade serous OCs are p53 wild-type but more frequently contain KRAS and BRAF mutations. Similarly, ovarian clear cell carcinomas are p53 wild-type but more frequently contain PI3K mutations. Nutlin-3 functions via inhibition of MDM2, thereby activating wild-type p53 and inducing apoptosis. We therefore sought to validate nutlin-3 as a potential novel therapeutic compound for p53 wild-type ovarian carcinomas. Two low-grade serous OC cell lines (HOC-7, MPSC-1), two clear cell OC cell lines (TOV21G, OVAS), and one high-grade serous OC p53-null cell line (SKOV3) were treated with varying concentrations of nutlin-3 (0-70 µm) for 72 hours, and cellular proliferation assays were performed. The three serous OC cell lines were then treated with nutlin-3 (5 µm), and quantitative reaction real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were employed to quantify MDM2, p53, and p21 expression at 24, 48, and 72 hours after treatment. The MPSC-1 cell line was then sequenced for p53 mutations in exons 5, 6, 7, and 8. Nutlin-3 inhibited cellular proliferation in a dose-dependent manner in the clear cell and low-grade serous cell lines, but not the p53-null high-grade serous cell line, as expected. Both PI3K-mutant clear cell ovarian cancer cell lines exhibited decreased growth in response to nutlin-3, but to a lesser extent than the low-grade serous cell lines. HOC-7, which bears a KRAS mutation, was more sensitive to inhibition than BRAF-mutated MPSC-1. Sequencing for p53 mutation in MPSC-1 surprisingly revealed a heterozygous p53 40 bp deletion in exon 5, which accounted for the reduced sensitivity of MPSC-1 to nutlin-3. On qRT-PCR and WB, levels of p53 were slightly decreased after nutlin-3 treatment and were absent in SKOV3. MDM2 and p21 were initially upregulated, but returned to near-baseline levels after 72 hours. Nutlin-3 is a novel potential therapeutic agent for low-grade serous ovarian carcinoma, and is dependent on an intact p53 pathway. It appears to function via upregulation of p21 with subsequent cell cycle arrest. Further studies characterizing response to nutlin-3 according to tumor mutation status are underway. 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 2784. doi:1538-7445.AM2012-2784

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to identify conditionally expressed genes in prokaryotes. Differential gene expression in Salmonella typhimurium LT2 in response to peroxide treatment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typhimurium infection. To identify genes inducible by hydrogen peroxide stress, total RNA from peroxide-treated and untreated bacterial cultures were RAP fingerprinted with six different arbitrarily selected primers. A 435-base RAP product that was differentially amplified by RAP using the reverse sequencing primer was cloned and sequenced. Northern blot analysis confirmed that the RNA corresponding to this clone, RSP435, was induced when bacteria were treated with hydrogen peroxide. The RNA was not induced in an oxyR1 mutant that constitutively expressed a subset of hydrogen peroxide-inducible genes. Using pulsed-field gel electrophoresis and dot blot hybridization to an array of induced Mud-P22 integrations, the gene corresponding to RSP435 was mapped to two places, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in different parts of the genome. Identification, cloning, and mapping of the conditionally expressed RSP435 cDNA were performed entirely by physical means, demonstrating that the strategy should complement genetic methods for many prokaryotic or archaebacterial systems and should be applicable to organisms in which genetic methods are difficult to perform or have not yet been developed.