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Elucidating the earliest pathogenic steps in cancer development is fundamental to improving its early detection and prevention. Ovarian high-grade serous carcinoma (HGSC), a highly aggressive cancer, mostly originates from the fallopian tube epithelium through a precursor stage, serous tubal intraepithelial carcinoma (STIC). In this study, we performed spatial transcriptomic analysis to compare STICs, carcinoma, and their matched normal fallopian tube epithelium. Several differentially expressed genes in STICs and carcinomas were involved in cancer metabolism and detected in a larger independent transcriptomic dataset of ovarian HGSCs. Among these, insulin-like growth factor binding protein-2 (IGFBP2) was found to undergo DNA hypomethylation and to be increased at the protein level in STICs. Pyrosequencing revealed an association of IGFBP2 expression with the methylation state of its proximal enhancer, and 5-azacytidine treatment increased IGFBP2 expression. In postmenopausal fallopian tubes, where most STICs are detected, IGFBP2 immunoreactivity was detected in all 38 proliferatively active STICs but was undetectable in morphologically normal tubal epithelia, including those with TP53 mutations. In premenopausal fallopian tubes, IGFBP2 expression was limited to the secretory epithelium at the proliferative phase, and estradiol treatment increased IGFBP2 expression levels. IGFBP2 knockdown suppressed the growth of IGFBP2-expressing tubal epithelial cells via inactivation of the AKT pathway. Taken together, demethylation of the proximal enhancer of IGFBP2 drives tumor development by maintaining the increased IGFBP2 required for proliferation in an otherwise estrogen-deprived, proliferation-quiescent, and postmenopausal tubal microenvironment. Significance: Molecular studies of the earliest precursor lesions of ovarian cancer reveal a role of IGFBP2 in propelling tumor initiation, providing new insights into ovarian cancer development.

Amplification and overexpression of the MYC oncogene in tumor cells, including ovarian cancer cells, correlates with poor responses to chemotherapy. As MYC is not directly targetable, we have analyzed molecular pathways downstream of MYC to identify potential therapeutic targets. Here we report that ovarian cancer cells overexpressing glutaminase (GLS), a target of MYC and a key enzyme in glutaminolysis, are intrinsically resistant to platinum-based chemotherapy and are enriched with intracellular antioxidant glutathione. Deprivation of glutamine by glutamine-withdrawal, GLS knockdown, or exposure to the GLS inhibitor CB-839 resulted in robust induction of reactive oxygen species in high GLS-expressing but not in low GLS-expressing ovarian cancer cells. Treatment with CB-839 rendered GLShigh cells vulnerable to the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, and prolonged survival in tumor-bearing mice. These findings suggest consideration of applying a combined therapy of GLS inhibitor and PARP inhibitor to treat chemoresistant ovarian cancers, especially those with high GLS expression. Significance: Targeting glutaminase disturbs redox homeostasis and nucleotide synthesis and causes replication stress in cancer cells, representing an exploitable vulnerability for the development of effective therapeutics.

The evolution of chemoresistance is a fundamental characteristic of cancer that ultimately hampers its clinical management. However, it may be possible to improve patient outcomes significantly by a better understanding of resistance mechanisms, which cancers rely upon during the evolution to an untreatable state. Here we report an essential role of the stem cell reprogramming factor, PBX1, in mediating chemoresistance in ovarian carcinomas. In the clinical setting, high levels of PBX1 expression correlated with shorter survival in post-chemotherapy ovarian cancer patients. In tumor cells with low endogenous levels of PBX1, its enforced expression promoted cancer stem cell-like phenotypes, including most notably an increase in resistance to platinum-based therapy used most commonly for treating this disease. Conversely, silencing PBX1 in platinum-resistant cells that overexpressed PBX1 sensitized them to platinum treatment and reduced their stem-like properties. An analysis of published genome-wide chromatin immunoprecipitation data indicated that PBX1 binds directly to promoters of genes involved in stem cell maintenance and the response to tissue injury. We confirmed direct regulation of one of these genes, STAT3, demonstrating that the PBX1 binding motif at its promoter acted to positively regulate STAT3 transcription. We further demonstrated that a STAT3/JAK2 inhibitor could potently sensitize platinum-resistant cells to carboplatin and suppress their growth in vivo. Our findings offer a mechanistic rationale to target the PBX1/STAT3 axis to antagonize a key mechanism of chemoresistance in ovarian cancers and possibly other human cancers. Cancer Res; 76(21); 6351–61. ©2016 AACR.

Overexpression or amplification of the RSF1 gene has been associated with poor prognosis in various human cancers, including ovarian cancer. In previous work, RSF1 was identified as an amplified gene that facilitated the development of paclitaxel-resistant ovarian cancer. In the present study, we further demonstrated that RSF1 expression inversely correlated with paclitaxel response in patients with ovarian cancer and the mouse xenograft model. In addition, RSF1-overexpressing paclitaxel-resistant ovarian cancer cell lines were found to express elevated levels of genes regulated by NF-κB, including some involved with the evasion of apoptosis (CFLAR, XIAP, BCL2, and BCL2L1) and inflammation (PTGS2). In addition, ectopic expression of RSF1 using Tet-off inducible SKOV3 cells significantly enhanced NF-κB–dependent gene expression and transcriptional activation of NF-κB. An RSF1 knockdown using short hairpin RNAs suppressed these same pathways. Moreover, pretreatment with NF-κB inhibitors or downregulation of NF-κB–regulated gene expression considerably enhanced paclitaxel sensitivity in RSF1-overexpressing OVCAR3 and/or RSF1-induced SKOV3 cells. A coimmunoprecipitation assay revealed that RSF1 interacts with NF-κB and CREB-binding protein, a ubiquitous coactivator for NF-κB. Recruitment of RSF1 to the NF-κB binding element in the PTGS2 and XIAP promoters was demonstrated by the chromatin immunoprecipitation assay. Furthermore, hSNF2H, a well-known binding partner of RSF1, was partially involved in the interaction between RSF1 and NF-κB. Taken together, these data suggest that RSF1 may function as a coactivator for NF-κB, consequently augmenting expression of genes necessary for the development of chemoresistance in ovarian cancer cells. Cancer Res; 74(8); 2258–69. ©2014 AACR.

Introduction: Ovarian and endometrial cancers are, respectively, the most lethal and most frequent female reproductive tract malignancies. Currently, there are no effective screening methodologies or protocols for these cancers. We hypothesized that the use of ultra-deep, targeted gene sequencing on a highly specific and gynecologic cancer-focused analyte source, namely, uterine lavage fluid, could detect somatic mutations associated with these cancers. Using these highly sensitive NGS-based assays, we demonstrated both (1) the ability to detect tumor-specific mutations in uterine lavage from women even with microscopic stage IA ovarian and endometrial cancers and (2) the existence of highly prevalent populations of cancer driver gene mutated cells in uterine tissue from women without cancer. Methods: In total, and following written informed consent, samples were prospectively collected and analyzed from two institutions from more than 250 women at the time of their surgery. Sequencing was performed using both Ion Torrent and Illumina-based sequencing and additional orthogonal mutation validation was performed using dPCR and Sanger sequencing. Results: Targeted sequencing of cellular and cell free DNA isolated from uterine lavage samples was performed on all samples in a blinded fashion. Histopathologic diagnosis and sample decoding, revealed that lavage mutations were present in nearly all women with ovarian and endometrial cancer, even those with microscopic stage IA disease. These mutations were demonstrated to be tumor-specific by sequencing their respective tumors. Notably, we also identified mutations in approximately half of all women without any evidence of cancer. To define the possible origin of these mutations in normal tissue, we analyzed uterine tissue from women without cancer. Two hundred biopsy samples were obtained from 24 uterine samples following hysterectomy for non-cancerous conditions. Remarkably, sequencing revealed relatively high allele fraction cancer-associated mutations in more than half of these samples. The overwhelming majority of samples harbored low frequency mutations. Conclusions: Our studies demonstrate the ability to detect with very high sensitivity, tumor-specific somatic mutations in women with ovarian and endometrial cancers; suggesting the future possibility of earlier diagnosis and better outcomes. At the same time, our studies again highlight caveats regarding NGS-based results for cancer screening. Finally, the identification of a previously unknown, highly prevalent, cancer-driver mutation landscape in apparently normal uterine tissue opens the door onto new areas of biologic exploration for understanding tumorigenesis and the protective mechanisms which inhibit cancer development. Citation Format: Deep S. Pandya, Shannon Tomita, Olga Camacho, Sabina Swierczek, Catalina Camacho, Kelsey Engelman, Stephanie Polukort, Maria Mercedes Padron, Jordan RoseFigura, Jon Irish, Linus Chuang, Vaagn Andikyan, Samantha Cohen, Paul Fiedler, Steven Sieber, Ie-Ming Shih, Jean-Noel Billaud, Boris Reva, Robert Sebra, Peter Dottino, John A. Martignetti. Development of a targeted liquid biopsy for early gynecologic cancer detection leads to discovery of a highly prevalent genomic landscape of cancer driver gene mutations in uterine tissue from women without cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2729.

NAC1 is a transcriptional corepressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer–binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant-negative or siRNA strategies reduced cell retraction and abscission during late-stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin-binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biologic and clinical implications. Cancer Res; 72(16); 4085–96. ©2012 AACR.

NOTCH3 gene amplification plays an important role in the progression of many ovarian and breast cancers, but the targets of NOTCH3 signaling are unclear. Here, we report the use of an integrated systems biology approach to identify direct target genes for NOTCH3. Transcriptome analysis showed that suppression of NOTCH signaling in ovarian and breast cancer cells led to downregulation of genes in pathways involved in cell-cycle regulation and nucleotide metabolism. Chromatin immunoprecipitation (ChIP)-on-chip analysis defined promoter target sequences, including a new CSL binding motif (N1) in addition to the canonical CSL binding motif, that were occupied by the NOTCH3/CSL transcription complex. Integration of transcriptome and ChIP-on-chip data showed that the ChIP target genes overlapped significantly with the NOTCH-regulated transcriptome in ovarian cancer cells. From the set of genes identified, we showed that the mitotic apparatus organizing protein DLGAP5 (HURP/DLG7) was a critical target. Both the N1 motif and the canonical CSL binding motif were essential to activate DLGAP5 transcription. DLGAP5 silencing in cancer cells suppressed tumorigenicity and inhibited cellular proliferation by arresting the cell cycle at the G2–M phase. In contrast, enforced expression of DLGAP5 partially counteracted the growth inhibitory effects of a pharmacologic or RNA interference–mediated NOTCH inhibition in cancer cells. Our findings define direct target genes of NOTCH3 and highlight the role of DLGAP5 in mediating the function of NOTCH3. Cancer Res; 72(9); 2294–303. ©2012 AACR.

Pre-B-cell leukemia homeobox-1 (PBX1), a transcriptional factor and downstream effector of Notch signaling pathway, plays pivotal roles in a wide spectrum of tumors, as well as developmental, inflammatory, autoimmune, and neurodegenerative disorders. Exploiting the crystal structure of the Pbx1-DNA complex, we developed a novel small-molecular inhibitor T417 that can directly block Pbx1-binding to DNA, unlike other existing compounds that interfere with protein-protein interactions. When T417 docks into the hydrophobic pocket of Pbx1 protein, the small molecule can dampen PBX1 transcription activity by hindering its binding to the promoter regions of PBX1 downstream target genes. Intriguingly, the amount of PBX1 expression in cells can dictate its response to T417. Increment of PBX1 expression is found in the high-grade serous carcinoma (HGSC) and carboplatin-resistant (CR) cells, and its expression is generally low in normal tissues as compared to the transformed tissues, making it such an arguably unique therapeutic target in ovarian cancer cells. This expression pattern illustrates the very minimal toxicity of T417 on normal tissues and organs in animal models, while it imposed in vitro and in vivo detrimental effects toward HGSC and CR cells. Besides, T417 holds synergistic cytotoxic effects with DNA damage-related drugs including PARP inhibitor and platinum-based drug. As PBX1 was shown to participate in maintaining cancer stem cell (CSC)-like phenotypes and promoting resistance to antitumor drugs, T417 is able to hammer out the stemness traits of CR cells to revert to a differentiated status through tacking PBX1 signaling cascade. The novel PBX1-targeting compound selectively interferes with PBX1-binding to DNA, which potentially points to powerful therapeutics and broad applications for the treatment of different human malignancies and stem cell therapy. Citation Format: Yao-An Shen, Jin Jung, Yohan Suryo Rahmanto, Licia Selleri, Ie-Ming Shih, Chi-Mu Chuang, Tian-Li Wang. A novel small-molecule compound targeting PBX1-DNA interaction impedes cancer cell survival and carboplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1966.

Somatic mutations in ARID1A, a SWI/SNF chromatin remodeling gene, are prevalent in human cancer and represent an emerging mechanism by which a molecular genetic alteration reprograms epigenomes and propels tumorigenesis. Through comprehensive sequencing of chromatin immunoprecipitation and chromatin accessibility, we found chromatin binding regions for ARID1A/BRG1-containing SWI/SNF remodeling complexes, which were enriched at enhancers and corresponded to euchromatin state. ARID1A deletion caused global re-distribution of BRG1-containing complexes in chromatin. Integrative analyses of ATAC-seq, ChIP-seq, and transcriptome data obtained from endometrial epithelium and human endometrioid carcinomas identified high-confidence ARID1A-regulated genes that participate in myriad key tumor and developmental signaling circuitry tissue. Deletion of Arid1a was found to inactivate the TGF-β pathway and to accelerate tumor progression from pre-cancerous lesions to endometrioid carcinomas. This study provides compelling evidence that Arid1a loss is required for developing endometrial cancer from its precursor. Collectively, targeting epigenomic modifications may hold promise for suppressing invasive behaviors of neoplasms carrying ARID1A mutations. Citation Format: Yohan Suryo Rahmanto, Xu Shi, Wenjing Shen, Tsutomu Miyamoto, Yu Yu, Xi Chen, Meng-Horng Lee, Vivek Singh, Michele I. Vitolo, Denis Wirtz, Ronny Drapkin, Jianhua Xuan, Ie-Ming Shih, Tian-Li Wang. Inactivation of ARID1A-SWI/SNF complex alters chromatin compactness at enhancer regions and affects transcription of key tumor signaling circuitry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2493.

Ovarian serous carcinoma, the most common and lethal type of ovarian cancer, is thought to develop from two distinct molecular pathways. High-grade (HG) serous carcinomas contain frequent TP53 mutations, whereas low-grade (LG) carcinomas arise from serous borderline tumors (SBT) and harbor mutations in KRAS/BRAF/ERBB2 pathway. However, the molecular alterations involved in the progression from SBT to LG carcinoma remain unknown. In addition, the extent of deletion of tumor suppressors in ovarian serous carcinomas has not been well studied. To further address these two issues, we assessed DNA copy number changes among affinity-purified tumor cells from 37 ovarian serous neoplasms including SBT, LG, and HG tumors using high-density 250K single nucleotide polymorphism arrays. Chromosomal instability index as measured by changes in DNA copy number was significantly higher in HG than in LG serous carcinomas. Hemizygous ch1p36 deletion was common in LG serous carcinomas but was rarely seen in SBT. This region contains several candidate tumor suppressors including miR-34a. In contrast, in HG serous carcinomas, significant numbers of amplifications and deletions, including homozygous deletions, were identified. Among homozygous deletions, loci containing Rb1, CDKN2A/B, CSMD1, and DOCK4 were most common, being present in 10.6%, 6.4%, 6.4%, and 4.3%, respectively, in independent 47 affinity-purified HG serous carcinomas. Except for the CDKN2A/B region, these homozygous deletions were not present in either SBT or LG tumors. Our study provides a genome-wide homozygous deletion profile in HG serous carcinomas, which can serve as a molecular foundation to study tumor suppressors in ovarian cancer. [Cancer Res 2009;69(9):4036–42]

Notch3 gene amplification has recently been identified in ovarian cancer but the Notch3 effectors that are involved in the development of ovarian cancer remain elusive. In this study, we have identified Pbx1, a proto-oncogene in hematopoietic malignancy, as a Notch3 target gene. Pbx1 expression is transcriptionally regulated by Notch3 activation, and Notch3/CSL protein complex directly binds to the Pbx1 promoter segment harboring the CSL-binding sequence. The growth-inhibitory effect of γ-secretase inhibitor could be partially reversed by ectopic Pbx1 expression. Furthermore, functional studies by Pbx1 short hairpin RNA knockdown show that Pbx1 is essential for cell proliferation and tumorigenicity. Taken together, the above findings indicate that Pbx1 is a direct Notch3-regulated gene that mediates the survival signal of Notch3 in ovarian cancer. [Cancer Res 2008;68(21):8852–60]

The Notch signaling pathway represents a critical component in the molecular circuits that control cell fate during development. Aberrant activation of this pathway contributes to tumorigenesis. The role of Notch in human cancer has been highlighted recently by the presence of activating mutations and amplification of Notch genes in human cancer and by the demonstration that genes in the Notch signaling pathway could be potential therapeutic targets. It has become clear that one of the major therapeutic targets in the Notch pathway are the Notch receptors, in which γ-secretase inhibitors prevent the generation of the oncogenic (intracellular) domain of Notch molecules and suppress the Notch activity. This review article summarizes the biological roles of Notch molecules in cancer development with special emphasis on the promise and challenges in applying γ-secretase inhibitors as a new line of targeted therapeutic agents. [Cancer Res 2007;67(5):1879–82]

Gene amplification is one of the common mechanisms that activate oncogenes. In this study, we used single nucleotide polymorphism array to analyze genome-wide DNA copy number alterations in 31 high-grade ovarian serous carcinomas, the most lethal gynecologic neoplastic disease in women. We identified an amplicon at 19p13.12 in 6 of 31 (19.5%) ovarian high-grade serous carcinomas. This amplification was validated by digital karyotyping, quantitative real-time PCR, and dual-color fluorescence in situ hybridization (FISH) analysis. Comprehensive mRNA expression analysis of all 34 genes within the minimal amplicon identified Notch3 as the gene that showed most significant overexpression in amplified tumors compared with nonamplified tumors. Furthermore, Notch3 DNA copy number is positively correlated with Notch3 protein expression based on parallel immunohistochemistry and FISH studies in 111 high-grade tumors. Inactivation of Notch3 by both γ-secretase inhibitor and Notch3-specific small interfering RNA suppressed cell proliferation and induced apoptosis in the cell lines that overexpressed Notch3 but not in those with minimal amount of Notch3 expression. These results indicate that Notch3 is required for proliferation and survival of Notch3-amplified tumors and inactivation of Notch3 can be a potential therapeutic approach for ovarian carcinomas. (Cancer Res 2006; 66(12): 6312-8)

Activation of mitogen-activated protein kinase (MAPK) occurs in response to various growth stimulating signals and as a result of activating mutations of the upstream regulators, KRAS and BRAF, which can be found in many types of human cancer. To investigate the roles of MAPK activation in tumors harboring KRAS or BRAF mutations, we inactivated MAPK in ovarian tumor cells using CI-1040, a compound that selectively inhibits MAPK kinase, an upstream regulator of MAPK and thus prevents MAPK activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated tumor cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. Long serial analysis of gene expression identified several differentially expressed genes in CI-1040-treated MPSC1 cells harboring an activating mutation in BRAF (V599L). The most striking changes were down-regulation of cyclin D1, COBRA1, and transglutaminase-2 and up-regulation of tumor necrosis factor–related apoptosis-induced ligand, thrombospondin-1, optineurin, and palladin. These patterns of gene expression were validated in other CI-1040-treated tumor cells based on quantitative PCR. Constitutive expression of cyclin D1 partially reversed the growth inhibitory effect of CI-1040 in MPSC1 cells. Our findings indicate that an activated MAPK pathway is critical in tumor growth and survival of ovarian tumors with KRAS or BRAF mutations and suggest that the CI-1040 induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian tumors.

Molecular genetic changes that are associated with the initiating stage of tumor development are important in tumorigenesis. Ovarian serous borderline tumors (SBTs), putative precursors of low-grade serous carcinomas, are among the few human neoplasms with a high frequency of activating mutations in BRAF and KRAS genes. However, it remains unclear as to how these mutations contribute to tumor progression. To address this issue, we compared the mutational status of BRAF and KRAS in both SBTs and the adjacent epithelium from cystadenomas, the presumed precursor of SBTs. We found that three of eight SBTs contained mutant BRAF, and four SBTs contained mutant KRAS. All specimens with mutant BRAF harbored wild-type KRAS and vice versa. Thus, seven (88%) of eight SBTs contained either BRAF or KRAS mutations. The same mutations detected in SBTs were also identified in the cystadenoma epithelium adjacent to the SBTs in six (86%) of seven informative cases. As compared to SBTs, the cystadenoma epithelium, like ovarian surface epithelium, lacks cytological atypia. Our findings provide cogent evidence that mutations of BRAF and KRAS occur in the epithelium of cystadenomas adjacent to SBTs and strongly suggest that they are very early events in tumorigenesis, preceding the development of SBT.

Clinical trials targeting the immune tumor microenvironment (TME) in epithelial ovarian cancer (EOC) typically have included patients with heavily pre-treated advanced disease and demonstrated only marginal efficacy. A better understanding of how the EOC TME evolves with progression from primary to recurrent disease may inform future immunotherapy trials. Here, we evaluate the immune TME in primary and recurrent EOC using tissue microarrays. Our cohort included matched primary and recurrent tumors from 17 patients, and additional non-matched primary tumors from 20 patients and recurrent tumors from 15 patients. We stained for CD8, FOXP3 (regulatory T cells (Tregs)), CD68 (tumor associated macrophages (TAMs)), programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) by immunohistochemistry to interrogate the immune composition of the TME. Tregs increased in recurrent tumors compared to primary tumors (8.0 vs 14.2/HPF, p=0.0210). Higher TAM density was associated with higher levels of Treg and CD8+ T cell infiltrates in recurrent tumors (p=0.001 and p Citation Format: Laureen S. Ojalvo, Elizabeth D. Thompson, Tian-Li Wang, Alan K. Meeker, Ie-Ming Shih, Amanda N. Fader, Ashley Cimino-Mathews, Leisha A. Emens. Profiling the immune tumor microenvironment in primary and recurrent epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3991. doi:10.1158/1538-7445.AM2017-3991

Loss of function mutation of the epigenetic modulator ARID1A, a component of SWI/SNF chromatin remodeling complex, occurs in a number of cancers most notably ovarian. ARID1A is mutated in up to 60% and 30% of clear cell and endometrioid ovarian cancers, respectively. However, there currently is no effective therapy that targets ARID1A-deficient human cancers. Here we show ARID1A-deficient ovarian cancer cells are selectively sensitive to inhibition of EZH2's methyltransferase activity. EZH2 is the catalytic subunit of the polycomb repressor complex 2, which represses gene expression by generating tri-methylation epigenetic mark on lysine 27 of histone H3 (H3K27Me3). Utilizing a library of small molecules against epigenetic targets, we identified an EZH2 small molecule inhibitor that selectively inhibited the growth of ARID1A-deficient ovarian cancer cells, while exhibiting no effects in ARID1A wild-type cells. Genome-wide gene expression profile revealed that there is significant overlap of genes altered by ARID1A restoration and EZH2 methyltransferase inhibition. Specifically, the tumor suppressor and inhibitor of PI3K/AKT signaling, PIK3IP1, is regulated by both ARID1A and EZH2. Chromatin immunoprecipitation assays confirmed the occupancy of ARID1A and EZH2 at the PIK3IP1 promoter. In an intraperitoneal (IP) xenograft mouse model, EZH2 inhibitor GSK126 treatment resulted in a significant decrease in the number of IP tumor nodules. Moreover, in an independent orthotopic intra-bursal xenograft model using ARID1A wildtype and deficient cells, the EZH2 inhibitor GSK126 only suppressed the growth of ARID1A-deficient tumors. Notably, the observed effects correlated with a decrease in expression of H3K27Me3, pAKT and Ki67, a cell proliferation marker, and an increase in PIK3IP1. These data indicate loss of ARID1A and gain of EZH2 could act in epigenetic concert to promote cancer phenotypes. Taken together, these findings imply that inhibition of the methyltransferase EZH2 represents a novel therapeutic strategy for ARID1A-deficient cancers. Citation Format: Benjamin Bitler, Azat Garipov, Ie Ming Shih, Rugang Zhang. Targeting EZH2 methyltransferase activity: A novel therapeutic approach to ARID1A-deficient cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B30.

A 250K single-nucleotide polymorphism array was used to study subchromosomal alterations in oral squamous cell carcinoma (OSCC). The most frequent amplification was found at 7p11.2 in 9 of 29 (31%) oral cancer patients. Minimal genomic mapping verified a unique amplicon spanning from 54.6 to 55.3 Mb on chromosome 7, which contains SEC61G and epidermal growth factor receptor (EGFR). Results from fluorescence in situ hybridization, transcriptome, and immunohistochemistry analyses indicated that the expression level of EGFR, but not of SEC61G, was up-regulated and tightly correlated with DNA copy number in 7p11.2 amplified tumors. Among the members of the erbB family, EGFR (HER1) was found to be the most frequently amplified and highly expressed gene in both human and mouse oral tumors (P < 0.01). Genes for downstream effectors of EGFR, including KRAS, mitogen-activated protein kinase 1, and CCND1, were also found amplified or mutated, which resulted in activation of EGFR signaling in 55% of OSCC patients. Head and neck squamous cancer cells with different EGFR expression levels showed differential sensitivity to antitumor effects of AG1478, a potent EGFR inhibitor. AG1478-induced EGFR inactivation significantly suppressed tumor development and progression in a mouse oral cancer model. Our data suggest that EGFR signaling is important in oral cancer development and that anti-EGFR therapy would benefit patients who carry the 7p11.2 amplicon in their tumors. [Cancer Res 2009;69(6):2568–76]

Cancer mortality and morbidity are primarily related to recurrent tumors, and characterization of recurrence-associated genes should illuminate fundamental properties of tumor progression and provide new therapeutic targets. We have previously identified NAC-1, a member of the BTB/POZ gene family and a transcription repressor, as a gene associated with recurrent ovarian carcinomas after chemotherapy. We further showed that homodimerization of NAC-1 proteins is essential for tumor growth and survival. In this study, we applied serial analysis of gene expression and identified growth arrest and DNA-damage–inducible 45-γ interacting protein (Gadd45GIP1) as one of the downstream genes negatively regulated by NAC-1. NAC-1 knockdown in both SKOV3 and HeLa cells that expressed abundant endogenous NAC-1 induced Gadd45GIP1 expression transcriptionally; on the other hand, engineered expression of NAC-1 in NAC-1–negative RK3E and HEK293 cells suppressed endogenous Gadd45GIP1 expression. In NAC-1–expressing tumor cells, induction of dominant negative NAC-1 conferred a growth-inhibitory effect that can be partially reversed by Gadd45GIP1 knockdown. Induced Gadd45GIP1 expression resulted in growth arrest in SKOV3 and HeLa cells both in vitro and in vivo. In summary, NAC-1 contributes to tumor growth and survival by at least inhibiting Gadd45GIP1 expression, which has a tumor suppressor effect in cancer cells. [Cancer Res 2007;67(17):8058–64]

The ubiquitin-proteasome system (UPS) mediates targeted protein degradation. Notably, the UPS determines levels of key checkpoint proteins controlling apoptosis and proliferation by controlling protein half-life. Herein, we show that ovarian carcinoma manifests an overstressed UPS by comparison with normal tissues by accumulation of ubiquitinated proteins despite elevated proteasome levels. Elevated levels of total ubiquitinated proteins and 19S and 20S proteasome subunits are evident in both low-grade and high-grade ovarian carcinoma tissues relative to benign ovarian tumors and in ovarian carcinoma cell lines relative to immortalized surface epithelium. We find that ovarian carcinoma cell lines exhibit greater sensitivity to apoptosis in response to proteasome inhibitors than immortalized ovarian surface epithelial cells. This sensitivity correlates with increased cellular proliferation rate and UPS stress rather than absolute proteasome levels. Proteasomal inhibition in vitro induces cell cycle arrest and the accumulation of p21 and p27 and triggers apoptosis via activation of caspase-3. Furthermore, treatment with the licensed proteasome inhibitor PS-341 slows the growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice. In sum, elevated proliferation and metabolic rate resulting from malignant transformation of the epithelium stresses the UPS and renders ovarian carcinoma more sensitive to apoptosis in response to proteasomal inhibition. (Cancer Res 2006; 66(7): 3754-63)

Apolipoprotein E (ApoE) has been recently identified as a potential tumor-associated marker in ovarian cancer by serial analysis of gene expression. ApoE has long been known to play a key role in lipid transport, and its specific isoforms may participate in atherosclerogenesis. However, its role in human cancer is not known. In this study, apoE expression was frequently detected in ovarian serous carcinomas, the most common and lethal type of ovarian cancer. It was not detected in serous borderline tumors and normal ovarian surface epithelium. Inhibition of apoE expression using an apoE-specific siRNA led to G2 cell cycle arrest and apoptosis in an apoE-expressing ovarian cancer cell line, OVCAR3, but not in apoE-negative cell lines. Furthermore, the phenotype of apoE siRNA–treated OVCAR3 cells was reversed by expressing engineered mutant apoE with introduced silent mutations in the siRNA target sequence. Expression of apoE in nuclei was significantly associated with a better survival in patients who presented peritoneal effusion at the time of diagnosis (5-year follow-up, P = 0.004). This study suggests a new role of apoE in cancer as apoE expression is important for the proliferation and survival in apoE-expressing ovarian cancer cells.

Tumor-released DNA in blood represents a promising biomarker for cancer detection. It has been postulated that tumor necrosis causes release of DNA of varying sizes, which contrasts apoptosis in normal tissue that releases smaller and more uniform DNA fragments. To test the hypothesis that increased DNA integrity, i.e., a longer DNA strand, is a tumor-associated marker in plasma, we determined the genomic DNA integrity index in plasma DNA using real-time PCR assays. A DNA integrity index and DNA concentration in plasma were determined in 61 patients with gynecological and breast cancers and 65 female patients without neoplastic diseases. We found that the area under the receiver-operating characteristic curve for DNA integrity index was 0.911 for cancer versus nonneoplastic patients. Given 100% specificity, the highest sensitivity achieved in detecting the cancer group was 62% (95% confidence interval = 0.50-0.74) at the index cutoff of 0.59. Fifty percent of stage I cancers had a DNA integrity index above this cutoff. All 11 patients with benign adnexal masses that clinically can be confused with malignant gynecological neoplasms demonstrated DNA integrity index0.59. Our findings suggest that increased DNA integrity in plasma DNA is associated with cancer, and measurement of DNA integrity may provide a simple and inexpensive measure for cancer detection.

Background: Rsf-1, a chromatin remodeling factor that forms an ISWI chromatin-remodeling complex with SNF2H, has been implicated in the oncogenesis of a variety of human cancers, yet its functions in promoting tumor development remains unclear. In this study, we found Rsf-1 interacted with hRap1, a telomere binding protein involved in telomere length regulation. We aimed at characterizing the interaction between Rsf-1 and hRap1 and exploring the effect of Rsf-1 overexpression on telomere length in ovarian cancer cells. Methods: Protein microarray and co-immunoprecipitation were employed to screen and validate Rsf-1 interaction proteins. Deletion mutants of both Rsf-1 and hRap1 were constructed to map the binding domains responsible for their interaction. To determine whether RSF complex was enriched in the telomeric regions, we performed chromatin immunoprecipitation (ChIP) -qPCR in two ovarian cancer cell lines (OVCAR3 with high RSF-1 gene amplification and Rsf-1 inducible SKOV3) with antibodies against Rsf-1 and SNF2H. A ChIP-sequencing dataset performed with Rsf-1 and SNF2H antibodies using OVCAR3 cells was also examined for enrichment of telomeric sequences. To investigate the effect of Rsf-1 overexpression on telomere length, we evaluated by qPCR the telomere length in two Rsf-1 inducible cell lines (SKOV3 and BRK). Results: We identified hRap1, a telomere binding protein, as an interaction partner of Rsf-1 by protein microarray and co-immunoprecipitation. Protein interaction domain mapping revealed that C-terminal domain of hRAP1 and PHD domains and arginine-rich region of RSF1 were critical for this interaction. Both ChIP-qPCR and ChIP-sequencing demonstrated that Rsf-1 and SNF2H were enriched in the telomeres, suggesting that RSF complex was recruited by hRAP1 to the telomeres. Induction of RSF1 expression resulted in shortened telomere length in two different cell lines. The results suggest that RSF complex may be involved in the telomere length regulation mediated by hRAP1. Conclusions: Rsf-1 interacted with hRap1 through the PHD domain and the arginine-rich region. RSF complex bound telomeres, probably through the interaction with hRap1. Rsf-1 overexpression led to telomere length shortening, which may contribute to genomic instability in cancer cells. Citation Format: Ren-Chin Wu, Bin Guan, Yusuke Kobayashi, Tian-Li Wang, Ie-Ming Shih. Rsf-1, a chromatin remodeling protein, interacts with shelterin protein hRap1 and induces telomere shortening. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A63.

Leiomyosarcoma (LMS) is the most common uterine sarcoma, but is a rare cancer. The genetic events related to LMS metastasis are unknown to date. The aim of the present study was to compare the global gene expression patterns of primary uterine LMS and LMS metastases in order to identify potential biological targets. Gene expression profiles of 13 primary uterine LMS and 15 LMS metastases were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated primary from metastatic LMS samples. We identified 203 unique probes that were significantly differentially expressed in the two tumor groups by greater than 2-fold with 1% FDR cutoff using one-way ANOVA with Benjamini-Hochberg correction, of which 94 and 109 were overexpressed in primary and metastatic LMS, respectively. Genes overexpressed in primary uterine LMS included OSTN, NLGN4X, NLGN1, SLITRK4, MASP1, XRN2, ASS1, RORB, HRASLS and TSPAN7. Genes overexpressed in LMS metastases included FOLR3, TDO2, CRYM, GJA1, TSPAN10, THBS1, SGK1, SHMT1, EGR2 and AGT. Results for selected genes were validated by quantitative real-time PCR and immunohistochemistry. We present the first study in which gene expression profiling was shown to distinguish between primary uterine LMS and LMS metastases. The molecular signatures unique to primary and metastatic LMS may aid in understanding tumor progression in this cancer and in providing a molecular basis for prognostic studies and therapeutic target discovery. Citation Format: Ben Davidson, Vera Maria Abeler, Mette Førsund, Arild Holth, Ie-Ming Shih, Gunnar B. Kristensen, Li Chen, Yanqin Yang, Tian-Li Wang. Gene expression signatures differentiate primary and metastatic uterine leiomyosarcoma. [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 401. doi:10.1158/1538-7445.AM2013-401

ARID1A encoding BAF250a is a recently identified tumor suppressor involved in chromatin remodeling. Inactivating somatic mutations occur in a wide variety of human cancers including ovarian carcinoma. Interestingly, ARID1A mutations significantly co-occur with either PIK3CA or PTEN mutations in human neoplastic tissues, suggesting a molecular collaboration of ARID1A and PI3K/PTEN pathways in tumor development. We tested this possibility in Arid1a knockout mice (n=10) and Arid1a/Pten double knockout mice (n= 12), in which Arid1a and Pten can be conditionally deleted in the mouse ovarian surface epithelium (OSE) by intra-ovarian bursal injection of Ad-Cre virus. Arid1a knockout mice did not develop discernable histopathological changes 12 months after Ad-Cre administration in OSE, a finding similar to Pten knockout mice as reported previously. In contrast, Arid1a/Pten double knockout mice developed ovarian tumors in 40% mice 6 months after Ad-Cre virus injection. The tumors effaced ovarian tissues, disseminated intraperitoneally and developed ascites. None of the tumors expressed detectable ARID1A and Pten immunoreactivity, while fibroblastic cells and OSE cells from control ovaries are positive for both markers. Although all the tumors appear poorly differentiated, 80% tumors were positive for cytokeratin 8, suggestive of an epithelial origin. The remaining 60% mice without grossly visible tumors showed hyperplasia of OSE in Ad-Cre treated ovaries but not in non-injected controls. The hyperplasia of OSE developed as early as 2 months after Ad-Cre administration. In summary, our results demonstrate that mutation of Arid1a requires a second hit to initiate tumorigenesis. Collaboration between Arid1a inactivation and PI3K/Pten pathway aberration is sufficient to induce OSE hyperplasia and to develop poorly differentiated carcinomas. Citation Format: Bin Guan, Tian-Li Wang, Ie-Ming Shih. Arid1a loss in collaboration with PI3K pathway activation leads to ovarian tumorigenesis in mouse. [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 LB-259. doi:10.1158/1538-7445.AM2013-LB-259

Nucleus accumbens-associated protein 1 (NAC1) is a member of the BTB (Bric-a-Brac Tramtrack Broad complex) family of proteins. Recent studies have demonstrated NAC1 expression is essential for sustaining cancer cell proliferation and migration, but its molecular mechanisms remain elusive. In this report, we show that NAC1 is an actin monomer binding protein with a Kd of 0.78 μM, and the BTB domain is the actin-binding domain. Disrupting NAC1 protein complex function using a dominant-negative construct or RNA-interference resulted in reduced traction and incomplete abscission during late stage cytokinesis leading to multi-nucleation of cancer cells. We examined the phenotype of Nac1 knockout mice, and discovered the loss in expression of the Nac1 protein led to a patterning defect of the vertebral skeletal axis. Nac1-null mice were correlated with morphological transformation of the sixth lumbar vertebra into a sacral identity; a decreased total number of pre-sacral vertebrae elements from 26 in wild type mice to 25. Taken together, we explored the role of NAC1 as an actin binding protein essential for the cytokinesis of cancer cells, and its effects on patterning of the murine vertebral axis. These findings advance our knowledge of this physiologically-relevant protein in the field of mammalian development and cancer biology. 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 LB-115. doi:1538-7445.AM2012-LB-115

Most uterine smooth muscle tumors can be broadly subdivided into two groups: benign leiomyoma (including variant subtypes) and leiomyosarcoma based on cytologic atypia, mitotic activity and coagulative tumor cell necrosis. A small number defies classification into unequivocally benign and unequivocally malignant categories. These tumors, classified as “atypical leiomyoma” and “STUMP” (Smooth muscle Tumors of Uncertain Malignant Potential), generally behave in a benign fashion. Although p53 overexpression, as determined by immunohistochemical staining, has been reported in the atypical group and the leiomyosarcomas, it is uncertain whether they harbor TP53 mutations. In addition, a recent study has reported mutations in the mediator complex 12 (MED12) gene in the majority of benign uterine leiomyomas. We therefore performed a mutational analysis of uterine smooth muscle tumors to determine their TP53 and MED12 mutation status, in order to better understand their molecular genetic characteristics. A total of 9 uterine conventional leiomyomas, 27 atypical leiomyomas and 12 leiomyosarcomas from 40 patients were analyzed. DNA samples were sequenced and TP53 (from exons 4 to 9) and MED12 (exon 2) mutations were analyzed. Clinico-pathological data were collected. TP53 mutations were detected in 17 (63%) of 27 atypical leiomyomas and 8 (67%) of 12 leiomyosarcomas. MED12 mutations were identified in 6 (67%) of 9 conventional leiomyomas, 7 (26%) of 27 atypical leiomyomas and 1 (8%) of 12 leiomyosarcomas. Follow-up was available in 30 patients. None of the patients with atypical leiomyoma had recurrence or died of her disease (average follow-up 23 months). Seven patients with leiomyosarcoma, 4 of whom had TP53 mutations, died of their disease (average follow-up 42 months). The preliminary findings of this study indicate that the presence of TP53 mutations in uterine smooth muscle tumors is not sufficient to drive malignant behavior. Moreover, based on the presence of a MED12 mutation in one case of leiomyosarcoma, we postulate that at least some leiomyosarcomas develop from benign leiomyomas. Future correlated molecular genetic and clinicopathologic studies with a larger number of cases and long-term follow-up information are necessary to confirm this important finding. 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 5536. doi:1538-7445.AM2012-5536

NAC1 is a nuclear factor belonging to the BTB gene family. The BTB domain of NAC1 is responsible for its homodimerization and interaction with other proteins to form a complex that plays an important role in maintaining the pluripotency of ES cells and in the development of chemo-resistance in ovarian cancer cells. Elucidating the molecular mechanisms underlying how NAC1 contributes to drug resistance is fundamental to improve clinical outcome in recurrent post-chemotherapy ovarian cancer patients. The purpose of this study was to determine whether autophagy, a conserved response to several types of stresses including therapeutic stress, was involved in NAC1- mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused a robust activation of autophagy in NAC1-expressing ovarian cancer cell lines including A2780, OVCAR3, and SKOV3, based on the alteration of LC3-II and p62 by Western blot analysis, and the presence of GFP-LC3 puncta by microscopic observation. We further demonstrated that knockdown of NAC1 expression by RNA interference or inactivation of NAC1 by inducing the expression of NAC1 deletion mutant that contains only the BTB domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity as measured by MTT viability assay. Moreover, we observed that inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis, as evidenced by increases in Annexing V staining and in the levels of pro-apoptotic proteins, caspase-3 and PARP. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was mediated through suppression of autophagy, we assessed the effects of autophagy inhibitors, 3-MA and chloroquine, on the cytotoxicity of cisplatin. Treatment of cancer cells with 3-MA and chloroquine also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagic response indeed renders the NAC1-expressing ovarian cancer cells more sensitive to cisplatin. The regulation of autophagy by NAC1 appeared to be mediated via HMGB1 (high mobility group box1), as NAC1 could regulate the expression, translocation and release of HMGB1, which is known to activate autophagy by disrupting the interaction between beclin 1 and bcl-2. The results of our study not only revealed a new mechanism governing cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1-mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer or other types of malignancies. Animal studies are in progress to evaluate the impact of NAC1 on antitumor efficacy of cisplatin in tumor xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2877. doi:10.1158/1538-7445.AM2011-2877

Somatic mutation of TP53 is the most common molecular genetic alteration in high grade serous carcinoma (HGSC) of the ovary, occurring in more than 95% of cases. As serous tubal intraepithelial carcinomas (STICs) have been proposed to be the most likely precursor of HGSC, we undertook a study to determine whether STICs harbor TP53 mutations as well. Mutations of TP53 have been reported in STICs and associated HGSC but that study was limited to 5 cases. We therefore performed TP53 mutational analysis in a larger series of STICs and concurrent HGSCs and correlated the mutational status with p53 immunoreactivity. Formalin-fixed paraffin-embedded tissue specimens were obtained from 18 HGSCs with concurrent STICs; 8 of them contained two discrete STICs. Approximately 1,000 cells from STIC and normal-appearing fallopian tubal epithelium were laser-capture microdissected, while HGSCs were manually microdissected. Genomic DNA was extracted, PCR amplified and sequenced. TP53 mutations were analyzed from exons 4 to 8. Immunohistochemistry (IHC) for p53 was performed in all the cases. TP53 mutations were detected in 15 (83.3%) of 18 HGSCs. Importantly, TP53 mutations were not detected in the corresponding FTE samples from the same patients, confirming they are somatic mutations in HGSCs. STIC and associated HGSC shared identical TP53 mutations in 15 (93.8%) of 16 patients including 7 who had two STICs. The discordant case showed TP53 mutation in the HGSC but not in two separate concurrent STICs. By IHC, all the cases demonstrated the same pattern of p53 immunoreactivity (either all positive or completely negative) except the case with wild-type TP53 in the STIC and mutant TP53 in the HGSC. By IHC the STIC was p53 negative and the HGSC p53 positive. The three TP53 wild-type HGSCs and their associated STICs exhibited undetectable nuclear p53 by IHC. In contrast, four p53-negative HGSCs contained mutant TP53 with either a deletion or an insertion mutation. Our findings provide cogent evidence that TP53 mutations occur in most STICs, and that both STIC and concurrent HGSC share the identical TP53 mutations in the majority of cases. Future molecular genetic studies are necessary to delineate the clonal relationship and tumor progression from STIC to HGSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2011-221

Genomic amplification is a frequent molecular genetic alteration in many cancer types that results in upregulation and thus activation of oncogenes located in the amplified regions. However, the mechanism by which amplified genes are upregulated is poorly understood. We hypothesized that epigenetic regulation of gene expression was important for activation in addition to the increased DNA copy number for genes within amplified genomic loci. To test this hypothesis, we used ovarian cancer as an example because it is a highly lethal disease and, more importantly, ovarian cancer cells are characterized by a high level of genomic instability as reflected by widespread DNA copy number changes. We analyzed the data from chromatin immunoprecipitation array (Chip-chip) and single nucleotide polymorphism (SNP) array to determine the correlation between specific histone modifications and genome-wide DNA copy number alterations in OVCAR3 ovarian cancer cells and ovarian surface epithelial cell culture. Among 8 histone marks examined, representing both heterochromatic and euchromatic chromatin, we identified the strongest correlation between increased DNA copy number and the active histone marks, histone H4 acetylation at lysine 5 (p-value 7.8×10^-6; FDR 5.2×10^-5) and lysine 12 (p-value 7.6×10^-5; FDR 2.6×10^-4). In contrast, there was no significant correlation between DNA amplification and repressive histone marks including histone H3 methylation at lysines 9 and 27. These correlations were validated by quantitative real-time PCR of ChIP samples at confirmed amplified regions on chromosomes 11 and 19, which harbor the RSF1 and AKT2 oncogenes. Furthermore, all genes within the amplicons were overexpressed in the OVCAR3 cell line harboring the amplicons as compared to a normal ovarian surface epithelial cell line. By using ovarian cancer as a disease model, our results indicate that an amplicon-associated specific histone modification pattern facilitates gene expression and suggest that both genetic (DNA copy number gain) and epigenetic (active histone marks) events contribute to upregulation of amplified genes which may ultimately contribute to the development of human cancer. 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 4871.

PBX1 has been initially identified as a proto-oncogene in human leukemia in a form of the E2A-PBX1 fusion protein. PBX1 is currently known as a regulator of self renewal of stem cells which contributes to maintenance of a “stemness” phenotype mainly through TGF-ß signaling. PBX1 was recently shown to be elevated in ovarian cancer and its locus was found to be frequently amplified in many cancer types including ovarian carcinoma. In order to further understand the clinicopathological and prognostic significance of PBX1 in ovarian carcinoma, we investigated the gene expression of PBX1 in patients with ovarian high-grade serous carcinoma (HGSC). PBX1 mRNA levels were analyzed in a panel of 78 ascites from patients with HGSC (including 36 primaries and 42 recurrences) by quantitative RT-PCR. Moreover, we performed immunohistochemical staining for PBX1 in 25 pairs of matched primary and recurrent HGSCs from the same patients. The results were correlated with clinicopathological data. Kaplan-Meier analysis demonstrated higher PBX1 levels in ascites from patients with recurrent/chemoresistant HGSC and an association with worse overall survival (p=0.026) as well as disease-free survival (p=0.024). Patients’ effusions with high PBX1 levels (based on median cut-off, n=22) had a mean overall survival of 26 months vs. 39 months for patients whose effusions showed low PBX1 expression (n=20). Patients’ effusions with high PBX1 expression (n=21) also had a mean progression-free survival of 4 months vs. 10 months for patients whose effusions showed low PBX1 levels (n=20). Furthermore, we found more intense PBX1 immunoreactivity in recurrent HGSCs than in matched primaries (Paired t test, p=0.024). Finally, PBX1 expression in primary HGSCs did not demonstrate any significant correlation with patients’ clinical outcome. This study is the first one demonstrating that PBX1 expression correlates with recurrent disease and is associated with worse clinical outcome in patients who develop recurrent HGSC. Future studies will aim at determining the usefulness of PBX1 immunoreactivity as a biomarker to predict patient treatment response and whether it can be a potential molecule for drug-targeted treatment in patients with recurrent HGSC. 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-50.

Telomere shortening is one of the main genetic manifestations in human cancer which can contribute to initial chromosomal instability and ultimately tumor progression. The purpose of this study was to determine if changes in telomere length occur in serous tubal intraepithelial carcinoma (STIC), a putative precursor of “ovarian” high-grade serous carcinoma (HGSC). 22 STICs from 15 patients with concurrent HGSCs were analyzed by performing telomere-specific FISH and p53 immunofluorescence, to assist in identifying STICs. Telomere length in STICs was compared to HGSCs using normal fallopian tube epithelium (NFT), and stromal cells as controls. Telomeres length of these HGSCs was compared to 134 HGSCs, serving as a validation set. Immunostaining for Ki67, a proliferation index, and H2AX, a marker of DNA damage, was also performed. We found that STICs had the shortest telomeres, as 18 (82%) STICs had short telomeres, while 2 (9%) showed no change, and 2 (9%) had long telomeres compared to the NFT (Table 1). The majority of HGSCs also displayed shorter telomeres than the associated NFT. However, among 12 paired HGSCs and STICs, 7 HGSCs showed a significant increase in telomere length, 1 showed a decrease in length and 4 did not show any change when compared to their matched STICs. These differences between NFT and STICs, and between STICs and HGSCs were significant (p< 0.05). Finally, we found a weak correlation between telomere length in STICs and the percentage of H2AX positive cells (R2= 0.1021). In conclusion, the finding of shortened telomeres, which have been shown to be one of the earliest molecular changes in carcinogenesis, in the vast majority of STICs provides further support to the proposal that STICs are precursors of HGSC and opens new areas of research in elucidating the early events of ovarian high-grade serous carcinogenesis.Table 1:The relative telomere length in NFT, STIC, and HGSC in the same patients (SD, Standard Deviation; NA, Not Available).PatientSTICNFTSTICHGSCP-value STICP-value STICP-value HGSC nonoMeanSDMeanSDMeanSDcompared to NFTcompared to HGSCcompared to NFTKi67 (%)yH2AX (%)1Tl9.93.76.519.71.51.70.35670.174< 0.00019002T28.65.94.32.64.03.60.00060.7130.000660502T38.65.94.93.74.03.60.00510.3440.000640603T417.55.13.32.5NANA< 0.0001NANA503T517.55.16.83.3NANA< 0.0001NANA200 T622.07.87.04.514.26.6< 0.00010.0001< 0.000160100 T722.07.83.02.114.26.6< 0.00010.0001< 0.0001601005TS9.24.43.94.66.53.4< 0.00010.01570.010110906T93.92.236.16.144.011.9< 0.00010.002< 0.000120807T108.23.43.73.73.44.1< 0.00010.767< 0.0001301008Til14.712.70.80.95.83.9< 0.00010.0001< 0.0001801009T1217.210.19.05.48.04.60.00020.443< 0.000180NA9T1317.210.19.05.28.04.60.00020.443< 0.000180NA10T143.22.80.81.6NANA0.0001NANA1010010T153.22.81.51.6NANA0.0055NANA5010010T163.22.80.00.1NANA< 0.0001NANA504011T172.82.02.63.328.523.00.77750.0001 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-343.