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Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7− cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7–β-catenin–Tp63–GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential “persister cancer cell” phenotype.Significance:Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.

N6-Methyladenosine (m6A) is the most abundant modification of mammalian mRNAs. RNA methylation fine tunes RNA stability and translation, altering cell fate. The fat mass- and obesity-associated protein (FTO) is an m6A demethylase with oncogenic properties in leukemia. Here, we show that FTO expression is suppressed in ovarian tumors and cancer stem cells (CSC). FTO inhibited the self-renewal of ovarian CSC and suppressed tumorigenesis in vivo, both of which required FTO demethylase activity. Integrative RNA sequencing and m6A mapping analysis revealed significant transcriptomic changes associated with FTO overexpression and m6A loss involving stem cell signaling, RNA transcription, and mRNA splicing pathways. By reducing m6A levels at the 3′UTR and the mRNA stability of two phosphodiesterase genes (PDE1C and PDE4B), FTO augmented second messenger 3′, 5′-cyclic adenosine monophosphate (cAMP) signaling and suppressed stemness features of ovarian cancer cells. Our results reveal a previously unappreciated tumor suppressor function of FTO in ovarian CSC mediated through inhibition of cAMP signaling. Significance: A new tumor suppressor function of the RNA demethylase FTO implicates m6A RNA modifications in the regulation of cyclic AMP signaling involved in stemness and tumor initiation.

PURPOSE Molecular cancer subtyping is an important tool in predicting prognosis and developing novel precision medicine approaches. We developed a novel platform-independent gene expression–based classification system for molecular subtyping of patients with high-grade serous ovarian carcinoma (HGSOC). METHODS Unprocessed exon array (569 tumor and nine normal) and RNA sequencing (RNA-seq; 376 tumor) HGSOC data sets, with clinical annotations, were downloaded from the Genomic Data Commons portal. Sample clustering was performed by non-negative matrix factorization by using isoform-level expression estimates. The association between the subtypes and overall survival was evaluated by Cox proportional hazards regression model after adjusting for the covariates. A novel classification system was developed for HGSOC molecular subtyping. Robustness and generalizability of the gene signatures were validated using independent microarray and RNA-seq data sets. RESULTS Sample clustering recaptured the four known The Cancer Genome Atlas molecular subtypes but switched the subtype for 22% of the cases, which resulted in significant ( P = .006) survival differences among the refined subgroups. After adjusting for covariate effects, the mesenchymal subgroup was found to be at an increased hazard for death compared with the immunoreactive subgroup. Both gene- and isoform-level signatures achieved more than 92% prediction accuracy when tested on independent samples profiled on the exon array platform. When the classifier was applied to RNA-seq data, the subtyping calls agreed with the predictions made from exon array data for 95% of the 279 samples profiled by both platforms. CONCLUSION Isoform-level expression analysis successfully stratifies patients with HGSOC into groups with differing prognosis and has led to the development of robust, platform-independent gene signatures for HGSOC molecular subtyping. The association of the refined The Cancer Genome Atlas HGSOC subtypes with overall survival, independent of covariates, enhances the clinical annotation of the HGSOC cohort.

The lack of precision therapies combined with limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain/blood-tumor barriers have contributed to glioblastoma (GBM) being one of the most difficult cancers to effectively treat. We have developed a novel precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference (RNAi)-based spherical nucleic acids (SNAs). In this study, we used SNAs consisting of gold nanoparticle cores covalently conjugated with radially oriented and densely packed siRNA oligonucleotides. Based upon previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in non-human primates, and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event-level (NOAEL), followed by tumor resection. Safety assessment revealed no significant treatment-related toxicities. Inductively coupled plasma mass spectrometry, X-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold (Au) enrichment observed in the tumor-associated endothelium, macrophages and tumor cells. NU-0129 uptake into glioma cells correlated with significant reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of NU-0129-treated recurrent vs. matched primary (i.e., untreated) tumor. Our results establish SNA nanoconjugates as a brain-penetrant precision medicine approach for the systemic treatment of GBM.

Defining traits of platinum tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum tolerant cells and tumors were identified by using in vitro and in vivo ovarian cancer (OC) models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were found to be enriched in ALDH (+) cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared to parental cells. Additionally, platinum-tolerant cells and tumors highly expressed the Wnt receptor, Frizzled 7 (FZD7). FZD7 knock down improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7(+) from FZD7(-) cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protects cells from chemotherapy-induced oxidative stress. FZD7(+) platinum-tolerant OC cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63 and glutathione metabolism gene sets were strongly correlated in the OC Tumor Cancer Genome Atlas (TCGA) database and in human OC specimens residual after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial stem cell features, characterized by FZD7 expression and dependent on FZD7-β-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum tolerant cancer cells and provide new insight into a potential “persister cancer cell” phenotype.

Recent work has highlighted the tumor microenvironment as a central player in cancer. In particular, interactions between tumor and immune cells may help drive the development of brain tumors such as glioblastoma multiforme (GBM). Despite significant research into the molecular classification of glioblastoma, few studies have characterized in a comprehensive manner the immune infiltrate in situ and within different GBM subtypes.In this study, we use an unbiased, automated immunohistochemistry-based approach to determine the immune phenotype of the four GBM subtypes (classical, mesenchymal, neural and proneural) in a cohort of 98 patients. Tissue Micro Arrays (TMA) were stained for CD20 (B lymphocytes), CD5, CD3, CD4, CD8 (T lymphocytes), CD68 (microglia), and CD163 (bone marrow derived macrophages) antibodies. Using automated image analysis, the percentage of each immune population was calculated with respect to the total tumor cells. Mesenchymal GBMs displayed the highest percentage of microglia, macrophage, and lymphocyte infiltration. CD68+ and CD163+ cells were the most abundant cell populations in all four GBM subtypes, and a higher percentage of CD163+ cells was associated with a worse prognosis. We also compared our results to the relative composition of immune cell type infiltration (using RNA-seq data) across TCGA GBM tumors and validated our results obtained with immunohistochemistry with an external cohort and a different method. The results of this study offer a comprehensive analysis of the distribution and the infiltration of the immune components across the four commonly described GBM subgroups, setting the basis for a more detailed patient classification and new insights that may be used to better apply or design immunotherapies for GBM.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-β cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

Glioblastoma multiforme remains the deadliest malignant brain tumor, with glioma stem cells (GSC) contributing to treatment resistance and tumor recurrence. We have identified MAPK-interacting kinases (MNK) as potential targets for the GSC population in glioblastoma multiforme. Isoform-level subtyping using The Cancer Genome Atlas revealed that both MNK genes (MKNK1 and MKNK2) are upregulated in mesenchymal glioblastoma multiforme as compared with other subtypes. Expression of MKNK1 is associated with increased glioma grade and correlated with the mesenchymal GSC marker, CD44, and coexpression of MKNK1 and CD44 predicts poor survival in glioblastoma multiforme. In established and patient-derived cell lines, pharmacologic MNK inhibition using LY2801653 (merestinib) inhibited phosphorylation of the eukaryotic translation initiation factor 4E, a crucial effector for MNK-induced mRNA translation in cancer cells and a marker of transformation. Importantly, merestinib inhibited growth of GSCs grown as neurospheres as determined by extreme limiting dilution analysis. When the effects of merestinib were assessed in vivo using an intracranial xenograft mouse model, improved overall survival was observed in merestinib-treated mice. Taken together, these data provide strong preclinical evidence that pharmacologic MNK inhibition targets mesenchymal glioblastoma multiforme and its GSC population. Implications: These findings raise the possibility of MNK inhibition as a viable therapeutic approach to target the mesenchymal subtype of glioblastoma multiforme. Mol Cancer Res; 14(10); 984–93. ©2016 AACR.

Stratification of cancer patients into different molecular groups is essential for developing targeted therapies. High-throughput technologies, such as microarrays and next-generation sequencing, have been extensively used for generating multi-omics data. Indeed, The Cancer Genome Atlas (TCGA) consortium, one of the most comprehensive and popular databases of cancer, has been accumulating large volumes of invaluable data for more than 30 cancer types, offering unprecedented opportunity to attain new insights in cancer biology. Despite the technological advances, analyzing, integrating and translating the gene signatures across different platforms remains a computationally challenging task. Here, we developed a novel computational framework that integrates genomic and clinical data to stratify cancer patients into different molecular subgroups and predict clinically applicable phenotypes, such as survival. Application of this user-friendly framework derives platform-independent isoform-level gene signatures that can be translated from high-dimensional platforms (e.g., RNA-Seq) to clinically adaptable low-dimensional molecular assays (e.g., RT-PCR) for prognostic stratification. We applied the pipeline on two TCGA lung cancer datasets—Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LUSC). Using independent test data, we achieved about 93% (LUAD) and 98% (LUSC) classification accuracy using less than 70 isoform-level gene signature. The proposed informatics platform is applicable to other cancer types in TCGA data portal. Citation Format: Segun Jung, Yingtao Bi, Ramana V. Davuluri. Informatics framework for clustering and deriving gene signatures for prognostic stratification of cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-47.

6025 Background: Platinum resistant ovarian cancer (PROC) remains a disease of high need. Immune checkpoint inhibitors (ICI) have modest activity. We hypothesized that priming with a hypomethylating agent (HMA) guadecitabine (G) will improve the anti-tumor activity of ICI in PROC by enhancing tumor cell recognition by CD8+ T cells. Methods: This open-label phase II study used a Simon’s two-stage design. Eligible patients (pts) had recurrent PROC; ECOG PS of 0-1; normal end organ function; and measurable disease. Up to 5 prior cytotoxic regimens were allowed. Treatment consisted of G 30mg/m2 sq D1-4 and pembrolizumab (P) 200mg iv D5. Each cycle was 21 days. The primary endpoint was response rate (RR). Secondary endpoints were progression-free survival (PFS), clinical benefit rate (CBR), and toxicity assessment. Translational endpoints were LINE1 methylation in PBMCs, global tumor methylation, and immune endpoints. Tumor biopsies were obtained at baseline and after 2 cycles. If 2 patients experienced clinical benefit in stage I [n = 16], enrollment proceeded to stage II. The null hypothesis was rejected for ≥ 6 responses in 35 evaluable patients. Results: 48 pts were enrolled, 43 were treated, and 33 were evaluable for response. Histology was serous (35), endometrioid (2), clear cell (3) and other (3). Median age was 63 (range 40-88) and median number of prior regimens was 4 [range 1-8]. Two PRs were recorded in the first stage, allowing second stage of enrollment. Overall, there were 2 PRs (RR = 6.6%) and 16 pts had stable disease (SD) [48%]. The clinical benefit rate (PR + SD > 3 months) was 27%. One patient continued treatment for > 2 yrs. Grade 3-4 related toxicities were neutropenia [20], lymphopenia, (9), anemia (2), neutropenic fever (1), rash (1), and others (8). There were 13 grade 3-4 SAEs and 4 grade 5 SAEs, assessed as being unrelated to treatment. LINE1 was hypomethylated in PBMCs D5 vs. D1 (n = 21, p = 0.001). Epic arrays measured global tumor methylation, with 39579 CpG sites (0.05%) being differentially methylated (C2D5 vs. C1D1, n = 11, paired t-test; p < 0.01). Main pathways affected included endosomal transport, K+ transport, cathecolamine secretion, etc. PDL1 staining in archival tissue showed tumor staining > 0 in 16 of 35 and tumor/stroma interface staining > 0 in 20 of 35 specimens. Antigen-specific cytotoxic T cell activity was increased in CD8+ cells from ascites (C2D5 vs. C1D1). Conclusions: G+P has modest anti-tumor activity in patients with PROC, but some patients experienced prolonged disease stabilization. Biomarkers of response are being investigated. Clinical trial information: NCT02901899.

The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call “entrapping”. Our results support a key role for uc.339 in lung cancer.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

Oncogenic mutations in two isocitrate dehydrogenase (IDH)-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico and wet-bench analyses indicate that non-mutated IDH1 mRNA and protein are commonly overexpressed in primary GBMs. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs the survival of animal subjects bearing patient-derived xenografts (PDXs). On a molecular level, diminished IDH1 activity results in reduced α-ketoglutarate (αKG) and NADPH production, paralleled by deficient carbon flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common metabolic adaptation by GBMs to support macromolecular synthesis, aggressive growth, and therapy resistance.

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice’s brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients’ PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

IntroductionBreast cancer being a multifaceted disease constitutes a wide spectrum of histological and molecular variability in tumors. However, the task for the identification of these variances is complicated by the interplay between inherited genetic and epigenetic aberrations. Therefore, this study provides an extrapolate outlook to the sinister partnership between DNA methylation and single-nucleotide polymorphisms (SNPs) in relevance to the identification of prognostic markers in breast cancer. The effect of these SNPs on methylation is defined as methylation quantitative trait loci (meQTL).Materialsand MethodsWe developed a novel method to identify prognostic gene signatures for breast cancer by integrating genomic and epigenomic data. This is based on the hypothesis that multiple sources of evidence pointing to the same gene or pathway are likely to lead to reduced false positives. We also apply random resampling to reduce overfitting noise by dividing samples into training and testing data sets. Specifically, the common samples between Illumina 450 DNA methylation, Affymetrix SNP array, and clinical data sets obtained from the Cancer Genome Atlas (TCGA) for breast invasive carcinoma (BRCA) were randomly divided into training and test models. An intensive statistical analysis based on log-rank test and Cox proportional hazard model has established a significant association between differential methylation and the stratification of breast cancer patients into high- and low-risk groups, respectively.ResultsThe comprehensive assessment based on the conjoint effect of CpG–SNP pair has guided in delaminating the breast cancer patients into the high- and low-risk groups. In particular, the most significant association was found with respect to cg05370838–rs2230576, cg00956490–rs940453, and cg11340537–rs2640785 CpG–SNP pairs. These CpG–SNP pairs were strongly associated with differential expression of ADAM8, CREB5, and EXPH5 genes, respectively. Besides, the exclusive effect of SNPs such as rs10101376, rs140679, and rs1538146 also hold significant prognostic determinant.ConclusionsThus, the analysis based on DNA methylation and SNPs have resulted in the identification of novel susceptible loci that hold prognostic relevance in breast cancer.

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR–17–5p, and miR–20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Author(s): Macyszyn, Luke; Akbari, Hamed; Pisapia, Jared M; Da, Xiao; Attiah, Mark; Pigrish, Vadim; Bi, Yingtao; Pal, Sharmistha; Davuluri, Ramana V; Roccograndi, Laura; Dahmane, Nadia; Martinez-Lage, Maria; Biros, George; Wolf, Ronald L; Bilello, Michel; O'Rourke, Donald M; Davatzikos, Christos | Abstract: BackgroundMRI characteristics of brain gliomas have been used to predict clinical outcome and molecular tumor characteristics. However, previously reported imaging biomarkers have not been sufficiently accurate or reproducible to enter routine clinical practice and often rely on relatively simple MRI measures. The current study leverages advanced image analysis and machine learning algorithms to identify complex and reproducible imaging patterns predictive of overall survival and molecular subtype in glioblastoma (GB).MethodsOne hundred five patients with GB were first used to extract approximately 60 diverse features from preoperative multiparametric MRIs. These imaging features were used by a machine learning algorithm to derive imaging predictors of patient survival and molecular subtype. Cross-validation ensured generalizability of these predictors to new patients. Subsequently, the predictors were evaluated in a prospective cohort of 29 new patients.ResultsSurvival curves yielded a hazard ratio of 10.64 for predicted long versus short survivors. The overall, 3-way (long/medium/short survival) accuracy in the prospective cohort approached 80%. Classification of patients into the 4 molecular subtypes of GB achieved 76% accuracy.ConclusionsBy employing machine learning techniques, we were able to demonstrate that imaging patterns are highly predictive of patient survival. Additionally, we found that GB subtypes have distinctive imaging phenotypes. These results reveal that when imaging markers related to infiltration, cell density, microvascularity, and blood-brain barrier compromise are integrated via advanced pattern analysis methods, they form very accurate predictive biomarkers. These predictive markers used solely preoperative images, hence they can significantly augment diagnosis and treatment of GB patients.

Characterization of molecular aberrations in ovarian serous cystadenocarcinoma (OVSC) is highly crucial for developing precision medicine approaches. While gene-level analysis of gene expression profiles of The Cancer Genome Atlas (TCGA) OVSC samples revealed four molecular subgroups (differentiated, proliferative, mesenchymal, and immunoreactive), the association of the derived subtypes with the clinical and pathologic features, such as stage, cytoreduction, and chemotherapy, is not statistically significant. Considering the fact that the majority of human genes produce multiple transcript-variants that could be involved in different functional pathways, we reanalyzed the TCGA exon-array expression profiles at the transcript variant or isoform level by following the novel informatics methodology we developed and successfully applied for glioblastoma (Pal et al., Nucleic Acids Res 2014). Non-negative matrix factorization (NMF) clustering of TCGA ovarian serous adenocarcinoma samples, based on isoform-level gene-expression profiles, recaptured the four known molecular subgroups but switched the subtype for 26% of the samples, resulting in significant (p=0.009) survival differences among the refined subgroups. The survival analysis was further extended to multivariate Cox proportional hazard model considering the different subgroups, and the clinical parameters as the predictor variables. Patients classified under mesenchymal group (HR = 1.44; 95% CI, 0.99-2.08; P < 0.05) and in stage III (HR = 2.39, 95%of CI, 1.25-4.56, P < 0.05), IV (HR= 3.16; 95% of CI, 1.58-6.32, P < 0.05) were identified to be at high risk or decreased survival, while those receiving intraperitoneal (IP) chemotherapy (HR, 0.63; 95% of CI, 0.38-1.03, P < 0.1) were associated with longer survival. We are in the process of translating the isoform-level gene signature into clinically adaptable molecular subtyping assay and validating on an independent patient cohort, with potential implications for developing robust diagnostic assays for OVSC patient stratification. Citation Format: Arunima Shilpi, Brandon L. Seagle, Manoj Kandpal, Shohreh Shahabi, Ramana V. Davuluri. Identification of prognostic molecular subtypes of ovarian serous cystadenocarcinoma by isoform-level gene expression analysis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B74.

Metastasis contributes to the vast majority of cancer-related mortalities. Metastatic tumors secrete factors that systemically affect various organs leading to dysfunction and accelerated death. More than 80% of metastatic cancer patients experience a progressive and debilitating loss of muscle mass and function by a process known as cachexia. Cachectic patients suffer deterioration of diaphragm and cardiac muscles and often die prematurely due to respiratory and cardiac failure. The prognosis for these patients is further diminished by the fact that they are often too weak to tolerate standard doses of anti-cancer treatments. Cachexia is therefore an important determinant of therapeutic response, outcome and patient survival in advanced cancer patients. Although systemic metabolic derangements and chronic inflammation predominate in cachexia, the underlying molecular mechanisms driving its development are not well understood. Therefore, insights into the specific interventions that could either reverse or prevent cachexia are expected to improve treatment outcome, survival and quality of life in cancer patients. In this study, we identified a metal ion transporter, ZIP14 that was upregulated in cachectic muscles from five independent metastatic models, as well as in metastatic cancer patients. We find that TNF-alpha and TGF-beta cytokines upregulate ZIP14 in muscles, which in turn results in the accumulation of intracellular zinc. Increased zinc influx in muscle cells is associated with the degradation of myofibrillar proteins that contributes to muscle atrophy and muscle mass loss. Importantly, germline ablation or muscle-specific depletion of Zip14 markedly inhibits cancer-induced muscle wasting. Our study demonstrates a novel function of ZIP14 in muscles as a mediator of cachexia in advanced cancers. Insights from this study can be used to develop therapeutic strategies to prevent cachexia, and improve the survival and quality of life in metastatic cancer patients. Citation Format: Anup Biswas, Gang Wang, Wanchao Ma, Courtney Coker, Kurenai Tanji, Manoj Kandpal, Ramana Davuluri, Swarnali Acharyya. Zinc transporter, ZIP14, as a mediator of systemic muscle wasting in metastatic cancers [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 5732.

It has been proposed that by silencing tumor suppressor genes, DNA methylation plays an important function in cancer initiation and progression. DNA hypomethylating agents are being developed as anti-cancer agents. However, this approach causes unrestricted hypomethylation, which could potentially activate tumor-promoting mechanisms. Therefore a strategy capable of inducing hypomethylation of selected genes is needed. Towards this goal, we designed a strategy to induce demethylation of a coordinated gene program by using Ten-eleven translocation (TET1) dioxygenase, which converts 5 -methylcytosine to 5-hydroxymethylcytosine. We constructed a fusion gene TET1-DBD containing the catalytic domain of TET1 and the DNA binding domain of Snail, the transcription factor that regulates epithelial-to-mesenchymal transition (EMT). We hypothesized that this fusion gene will bind to promoters targeted by Snail, induce hypomethylation, reactivate gene expression, and block EMT. To test this hypothesis, we used TET1-DBD and measured induction of EMT in an ovarian (SKOV3) and breast cancer (MCF10A) model driven by TGF-β. Genome-wide DNA methylation was assessed by Reduced Representation Bisulfite Sequencing (RRBS) to measure the downstream targets of TET1-DBD. TET1-DBD increased mRNA and protein expression levels of E-cadherin in transfected cells and prevented TGF-β induced expression of fibronectin and vimentin in MCF10A and SKOV3 cells. RRBS analysis demonstrated hypomethylation of genes targeted by Snail-DBD. Our results suggest that TET1-DBD selectively binds to and hypomethylates promoters of genes targeted by Snail, resulting in inhibition of the EMT program. We propose a novel approach to induce selective promoter hypomethylation and to block key oncogenic processes by using TET1 fused to a transcription factor “carrier”. Citation Format: HAO HUANG, Qing Yu, Maooj Kandpal, Ramana V. Davuluri, Daniela Matei. Pathway-directed DNA demethylation as an epithelial to mesenchymal targeting strategy [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 1396.

Resistance to TGF-beta is frequently observed in ovarian cancer, and disrupted TGF-beta/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-beta/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-beta/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P

Posttranslationally modified histones and DNA hypermethylation frequently interplay to deregulate gene expression in cancer. We report that acute myeloid leukemia (AML) with an aberrant histone methyltransferase, the mixed lineage leukemia partial tandem duplication (MLL-PTD), exhibits increased global DNA methylation versus AML with MLL-wildtype (MLL-WT; P = .02). Among the differentially methylated genes, the SLC5A8 tumor suppressor gene (TSG) was more frequently hypermethylated (P = .003). In MLL-PTD+ cell lines having SLC5A8 promoter hypermethylation, incubation with decitabine activated SLC5A8 expression. Ectopic SLC5A8 expression enhanced histones H3 and H4 acetylation in response to the histone deacetylase inhibitor, valproate, consistent with the encoded protein—SMCT1—short-chain fatty acid transport function. In addition, enhanced cell death was observed in SMCT1-expressing MLL-PTD+ AML cells treated with valproate. Within the majority of MLL-PTD AML is a mechanism in which DNA hypermethylation silences a TSG that, together with MLL-PTD, can contribute further to aberrant chromatin remodeling and altered gene expression.

We present the implementation of an application using caGrid, which is the service-oriented Grid software infrastructure of the NCI cancer Biomedical Informatics Grid (caBIG™), to support design and analysis of custom microarray experiments in the study of epigenetic alterations in cancer. The design and execution of these experiments requires synthesis of information from multiple data types and datasets. In our implementation, each data source is implemented as a caGrid Data Service, and analytical resources are wrapped as caGrid Analytical Services. This service-based implementation has several advantages. A backend resource can be modified or upgraded, without needing to change other components in the application. A remote resource can be added easily, since resources are not required to be collected in a centralized infrastructure.

Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Global patterns of hypermethylation are tumor-type specific and nonrandom. The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression. Using restriction landmark genomic scanning on samples derived from tissue culture and in vivo models of T cell lymphomas, we found that MYC overexpression gave rise to a specific signature of CpG island hypermethylation. This signature reflected gene transcription profiles and was detected only in advanced stages of disease. The further inactivation of the Pten, p53, and E2f2 tumor suppressors in MYC-induced lymphomas resulted in distinct and diagnostic CpG island methylation signatures. Our data suggest that tumor-specific DNA methylation in lymphomas arises as a result of the selection of rare DNA methylation events during the course of tumor development. This selection appears to be driven by the genetic configuration of tumor cells, providing experimental evidence for a causal role of DNA hypermethylation in tumor progression and an explanation for the tremendous epigenetic heterogeneity observed in the evolution of human cancers. The ability to predict genome-wide epigenetic silencing based on relatively few genetic alterations will allow for a more complete classification of tumors and understanding of tumor cell biology., Author Summary Genetic and epigenetic alterations of the genome are common features of cancers. The relationship between these two types of alterations, however, remains unclear. One type of epigenetic modification—DNA methylation in promoter sequences of genes—is of particular interest, since tumor cells have different patterns of promoter methylation than normal cells. Previous studies on human tumor samples have suggested a link between genetic alterations and the induction of aberrant DNA methylation; however, this link has been difficult to rigorously assess because of the incredible genetic heterogeneity found in human cancer. In this study, a mouse model of T cell lymphoma was used to explore the relationship between genetic and epigenetic modifications experienced by tumor cells. By introducing defined genetic changes into preneoplastic T cells of mice, such as the overexpression of the MYC oncogene and the ablation of tumor suppressor genes, we could carefully evaluate how these genetic changes impacted promoter methylation profiles during development of lymphomas in vivo. We found that the introduction of different genetic insults resulted in unique and diagnostic profiles of promoter methylation. Understanding how these methylation signatures contribute to tumor progression could eventually have diagnostic, prognostic, and therapeutic value for human cancers.

In search of tumor suppressor genes in papillary thyroid carcinoma (PTC), we previously used gene expression profiling to identify genes underexpressed in tumor compared with paired unaffected tissue. While searching for loss of heterozygosity (LOH) in genomic regions harboring candidate tumor suppressor genes, we detected LOH in a approximately 20 kb region around marker D9S176. Several ESTs flanking D9S176 were underexpressed in PTC tumors, and for one of the ESTs, downregulation was highly associated with the activating BRAF mutation V600E, the most common genetic lesion in PTC. A novel gene, NAMA, (noncoding RNA associated with MAP kinase pathway and growth arrest) containing the affected EST was cloned and characterized. NAMA is weakly expressed in several human tissues, and the spliced forms are primarily detected in testis. Several characteristics of NAMA suggest that it is a nonprotein coding but functional RNA; it has no long open reading frames (ORFs); the exons exhibit low sequence identity in the evolutionarily conserved regions; it is inducible by knockdown of BRAF, inhibition of the MAP kinase pathway, growth arrest and DNA damage in cancer cell lines. We suggest that NAMA is a noncoding RNA associated with growth arrest.

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) α signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERα target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERα signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERα targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERα target genes, providing new direction for research in this classical signaling pathway.

CpG island methylation is an epigenetic alteration that contributes to tumorigenesis by transcriptional inactivation of genes. Little is known about the overall levels of CpG island methylation in chronic lymphocytic leukemia (CLL). To provide a baseline estimate of global aberrant methylation and identify target sequences for additional investigation, we performed Restriction Landmark Genomic Scanning on 10 CLL samples. Two methylation-sensitive landmark enzymes were used (NotI and AscI), allowing assessment of over 3000 CpG islands in each sample. Tumor-derived Restriction Landmark Genomic Scanning profiles were compared with profiles from CD19-selected B cells from normal volunteers and matched normal neutrophils from 4 CLL patients. We found 2.5–8.1% (mean 4.8%) of the CpG islands in CLL samples were aberrantly methylated compared with controls, and the methylation events had a nonrandom distribution (P < 0.0001). Furthermore, we identified 193 aberrantly methylated sequences, of which 93% have CpG island characteristics and 90% have homology to genes or expressed sequences. One such gene, the G protein-coupled metabotropic glutamate receptor 7 (GRM7), possibly inhibits cyclic AMP signaling in the induction of apoptosis. Bisulfite sequencing of GRM7 confirmed extensive CpG island methylation, and treatment with 5-aza-2′-deoxycytidine (decitabine) resulted in up-regulated expression of several genes in vitro with concurrent cellular depletion of DNMT1 protein. Our dual-enzyme global methylation study shows that CLL is characterized by widespread nonrandom CpG island methylation similar to other tumors and provides a panel of novel methylation targets that can be used in larger studies designed to assess impact on disease progression and survival.

The p53 tumor suppressor promotes cell cycle arrest or apoptosis in response to stress. Previous work suggests that the promyelocytic leukemia gene (PML) can act upstream of p53 to enhance transcription of p53 targets by recruiting p53 to nuclear bodies (NBs). We show that PML is itself a p53 target gene that also acts downstream of p53 to potentiate its antiproliferative effects. Hence, p53 is required for PML induction in response to oncogenes and DNA damaging chemotherapeutics. Furthermore, the PML gene contains p53 binding sites that confer p53 responsiveness to a heterologous reporter and can bind p53 in vitro and in vivo. Finally, cells lacking PML show a reduced propensity to undergo senescence or apoptosis in response to p53 activation, despite the induction of several p53 target genes. These results identify an additional element of PML regulation and establish PML as a mediator of p53 tumor suppressor functions.

Hirschsprung disease (HSCR) is a common congenital disorder characterized by aganglionosis of the gut. The seemingly unrelated multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Yet, germline mutations in the RET proto-oncogene are associated with both MEN 2 and HSCR. In the former, gain-of-function mutations in a limited set of codons is found, whereas, in the latter, loss-of-function mutations are found. However, germline RET mutation is associated with only 3% of a population-based series of isolated HSCR, and little is known about susceptibility to sporadic MTC. We have found previously that specific haplotypes comprising RET coding single-nucleotide polymorphisms (SNPs) comprising exon 2 SNP A45A were strongly associated with HSCR, whereas haplotypes associated with exon 14 SNP S836S were associated with MTC. In this study, we describe three novel intron 1 SNPs, and, together with the coding SNP haplotypes, the data suggest the presence of distinct ancestral haplotypes for HSCR and sporadic MTC in linkage disequilibrium with a putative founding susceptibility locus/loci. The data are consistent with the presence of a very ancient, low-penetrance founder locus approximately 20-30 kb upstream of SNP A45A, but the failure of the SNPs to span the locus presents challenges in modeling mode of transmission or ancestry. We postulate that this founding locus is germane to both isolated HSCR and MTC but also that different mutations in this locus would predispose to one or the other.

Glioblastoma multiforme (GBM) is notoriously resistant to therapy, and the development of a durable cure will require the identification of broadly relevant regulators of GBM cell tumorigenicity and survival. Here, we identify Sprouty2 (SPRY2), a known regulator of receptor tyrosine kinases (RTK), as one such regulator. SPRY2 knockdown reduced proliferation and anchorage-independent growth in GBM cells and slowed xenograft tumor growth in mice. SPRY2 knockdown also promoted cell death in response to coinhibition of the epidermal growth factor receptor (EGFR) and the c-MET receptor in GBM cells, an effect that involved regulation of the ability of the p38 mitogen-activated protein kinase (MAPK) to drive cell death in response to inhibitors. Analysis of data from clinical tumor specimens further demonstrated that SPRY2 protein is definitively expressed in GBM tissue, that SPRY2 expression is elevated in GBM tumors expressing EGFR variant III (EGFRvIII), and that elevated SPRY2 mRNA expression portends reduced GBM patient survival. Overall, these results identify SPRY2 and the pathways it regulates as novel candidate biomarkers and therapeutic targets in GBM. Implications: SPRY2, counter to its roles in other cancer settings, promotes glioma cell and tumor growth and cellular resistance to targeted inhibitors of oncogenic RTKs, thus making SPRY2 and the cell signaling processes it regulates potential novel therapeutic targets in glioma. Mol Cancer Res; 13(8); 1227–37. ©2015 AACR.

Significance Despite measurable advances in cancer treatment, patients with glioblastoma multiforme (GBM) typically survive only 12–14 mo because of tumor recurrence. Tumor hypoxia has been associated with chemoresistance. We hypothesized that the pronounced repression of microRNA-218 ( miR-218 ) observed in samples from patients with highly hypoxic and necrotic GBM contributes to this recurrent phenotype. We demonstrate here that mice harboring intracranial tumors with increased miR-218 expression exhibit significantly reduced tumor burden and increased survival when challenged with the chemotherapeutic agent temozolomide. Moreover, low miR-218 levels increase the expression of multiple components of the receptor tyrosine kinase signaling pathway, which promote the activation of hypoxia-inducible factor. The identification of the molecular bases for miR-218 -mediated chemoresistance should promote the development of targeted therapies.

The ecotropic virus integration site 1 (EVI1) transcription factor is associated with human myeloid malignancy of poor prognosis and is overexpressed in 8-10% of adult AML and strikingly up to 27% of pediatric MLL-rearranged leukemias. For the first time, we report comprehensive genomewide EVI1 binding and whole transcriptome gene deregulation in leukemic cells using a combination of ChIP-Seq and RNA-Seq expression profiling. We found disruption of terminal myeloid differentiation and cell cycle regulation to be prominent in EVI-induced leukemogenesis. Specifically, we identified EVI1 directly binds to and downregulates the master myeloid differentiation gene Cebpe and several of its downstream gene targets critical for terminal myeloid differentiation. We also found EVI1 binds to and downregulates Serpinb2 as well as numerous genes involved in the Jak-Stat signaling pathway. Finally, we identified decreased expression of several ATP-dependent P2X purinoreceptors genes involved in apoptosis mechanisms. These findings provide a foundation for future study of potential therapeutic gene targets for EVI1-induced leukemia.

Context Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown. Objective To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL. Design, setting, and patients CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)-associated protein kinase 70 kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples. Main outcome measures Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation. Results In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02). Conclusions A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.

Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility. To test this, we analyzed SNPs known to be associated with BC risk, in silico and in vitro, for their ability to modify miRNA binding sites and miRNA gene regulation and referred to these as target SNPs. We identified rs1982073-TGFB1 and rs1799782-XRCC1 as target SNPs, whose alleles could modulate gene expression by differential interaction with miR-187 and miR-138, respectively. Genome-wide bioinformatics analysis predicted ∼64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA::mRNA duplexes by >90%. To assess whether target SNPs are implicated in BC susceptibility, we conducted a case-control population study and observed that germline occurrence of rs799917-BRCA1 and rs334348-TGFR1 significantly varies among populations with different risks of developing BC. Luciferase activity of target SNPs, allelic variants, and protein levels in cancer cell lines with different genotypes showed differential regulation of target genes following overexpression of the two interacting miRNAs (miR-638 and miR-628-5p). Therefore, we propose that transcribed target SNPs alter miRNA gene regulation and, consequently, protein expression, contributing to the likelihood of cancer susceptibility, by a novel mechanism of subtle gene regulation. Cancer Res; 70(7); 2789–98

Biomedical informatics encompasses a set of disciplines focused on developing, implementing, and perfecting the use of informatics and computational tools in biomedical research and clinical care. In this volume, we focus on a number of areas crucial to the establishment of state-of-the-art informatics methods and systems to support cancer research. We provide motivation for undertaking such developments and deployments, a quick overview of the field, and hopes for the impact on cancer treatment and survival in this introduction.

Epigenetic alterations, including gain or loss of DNA methylation, are a hallmark of nearly every malignancy. Changes in DNA methylation can impact expression of cancer-related genes including apoptosis regulators and tumor suppressors. Because such epigenetic changes are reversible, they are being aggressively investigated as potential therapeutic targets. Here we use the Eμ-TCL1 transgenic mouse model of chronic lymphocytic leukemia (CLL) to determine the timing and patterns of aberrant DNA methylation, and to investigate the mechanisms that lead to aberrant DNA methylation. We show that CLL cells from Eμ-TCL1 mice at various stages recapitulate epigenetic alterations seen in human CLL. Aberrant methylation of promoter sequences is observed as early as 3 months of age in these animals, well before disease onset. Abnormally methylated promoter regions include binding sites for the transcription factor FOXD3. We show that loss of Foxd3 expression due to an NF-κB p50/p50:HDAC1 repressor complex occurs in TCL1-positive B cells before methylation. Therefore, specific transcriptional repression is an early event leading to epigenetic silencing of target genes in murine and human CLL. These results provide strong rationale for the development of strategies to target NF-κB components in CLL and potentially other B-cell malignancies.

In this review we present some recent advances in understanding the roles of non-coding RNAs, including microRNAs and ultraconserved genes, in colorectal cancer and the way these advances can be translated for better cure of patients. MicroRNAs are a class of small RNAs that do not code for proteins and yet function as gene regulators. The deregulation of microRNA expression is involved in the initiation, progression, and dissemination of any type of human tumor. The underlying mechanisms of microRNA deregulation in human cancers are just starting to be understood. Germline and somatic mutations in microRNAs or polymorphisms in the messenger protein-coding RNAs targeted by microRNAs may also contribute to the tumor phenotype. Profiling microRNAs by various methods has identified signatures associated with the diagnosis, staging, progression, and prognosis of human colorectal cancers. Consequently, miRNAs have potential as diagnostic biomarkers and therapeutic targets in colorectal cancers. Ultraconserved genes represent a recently identified class of transcripts, mainly non-coding, that are highly conserved during evolution and can regulate miRNAs by direct interaction. Fingerprints of ultraconserved genes expression can classify cancers, including colorectal cancers, and ultraconserved genes may be involved in metastasis. Thus, having a clearer understanding of the mechanisms involved in the deregulation of non-coding RNAs in colorectal cancers is expected to contribute greatly to the development of new microRNA-based strategies for the diagnosis and treatment of patients.

SHP2, a Src-homology 2 domain (SH2)-containing tyrosine phosphatase encoded by PTPN11, is a ubiquitously expressed cytoplasmic enzyme that regulates events downstream of several growth factor and cytokine receptors involved in survival, pro-migratory and differentiation signaling. In the normal developing nervous system, SHP2 is involved in neural stem cell self renewal. Dysregulation of SHP2 function has been implicated in tumorigenesis and characterized as an activated downstream regulator of oncogenes in gastric carcinoma, anaplastic large-cell lymphoma, breast cancer and glioblastoma. In the context of glioblastoma, we have shown that SHP2, and specifically its phosphatase activity, is directly involved in EGFRvIII-induced oncogenesis. Our present research aims at understanding the function of SHP2 within different glioma cell types as well as the four characterized subtypes of glioblastoma (GBM). Genomic analysis of SHP2 expression using the TCGA GBM cohort shows differential expression among the recently characterized subtypes of GBMs, with higher SHP2 mRNA expression in the classical subtype. Additionally, SHP2 is preferentially expressed in glioma stem cells versus differentiated cells isolated from the same patients. Furthermore, our current analysis shows that reduction of SHP2 expression in glioma stem cells affects the expression of genes linked to stemness. SHP2 PTPase function may therefore be a link between normal stem cell proliferation and gliomagenesis induced by EGFR/RTK activated signaling. Citation Format: Laura Roccograndi, Yingtao Bi, Ramana Davuluri, Nadia Dahmane, Donald M. O'Rourke. SHP2 expression and function in different subtypes of glioblastoma. [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 2247. doi:10.1158/1538-7445.AM2015-2247

Stratification of cancer patients into different molecular groups is essential for developing targeted therapies. High-throughput technologies, such as microarrays and next-generation sequencing, have been extensively used for generating multi-omics data. Indeed, The Cancer Genome Atlas (TCGA) consortium, one of the most comprehensive and popular databases of cancer, has been accumulating large volumes of invaluable data for more than 30 cancer types, offering unprecedented opportunity to attain new insights in cancer biology. Despite the technological advances, analyzing, integrating and translating the gene signatures across different platforms remains a computationally challenging task. Here, we developed a novel computational framework that integrates genomic and clinical data to stratify cancer patients into different molecular subgroups and predict clinically applicable phenotypes, such as survival. Application of this user-friendly framework derives platform-independent isoform-level gene signatures that can be translated from high-dimensional platforms (e.g., RNA-Seq) to clinically adaptable low-dimensional molecular assays (e.g., RT-PCR) for prognostic stratification. We applied the pipeline on two TCGA lung cancer datasets_Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LUSC). Using independent test data, we achieved about 93% (LUAD) and 98% (LUSC) classification accuracy using less than 70 isoform-level gene signature. The proposed informatics platform is applicable to other cancer types in TCGA data portal. Note: This abstract was not presented at the meeting. Citation Format: Segun Jung, Yingtao Bi, Ramana V. Davuluri. Informatics framework for clustering and deriving gene signatures for prognostic stratification of cancer patients. [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 2185. doi:10.1158/1538-7445.AM2015-2185

Introduction: The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with metastases and nearly all will succumb to disease within 6-12 months of clinical presentation (Hidalgo, 2010). Recently, p120catenin (p120) was identified as a “cancer candidate gene” in sleeping beauty-induced PDAC and p120 loss is associated with poor patient survival (Mann et al, 2012). P120 is critical in stabilizing E-cadherin (E-cad) at the adherens junctions. In this study, we wished to unravel new roles for p120 in the context of PDAC initiation and progression (epithelial-mesenchymal transition or EMT), as well as metastasis. Results: We have introduced a floxed p120 allele into the Pdx1cre;LSL-KrasG12D;R26YFP (KCY) PDAC mouse model. Mice with homozygous p120 deletion in the context of a LSL-KrasG12D allele (KCYp120fl/fl) are not viable. However, mice with heterozygous p120 loss (KCYp120fl/wt) are born according to Mendelian ratios. At 20 weeks of age, KCYp120fl/wt mice show a remarkable acceleration of the Kras-driven phenotype. KCYp120fl/wt mice (n=21) harbor the entire spectrum of precursor lesions, including PanINs (1-3), mucinous cystic neoplasms (MCN) and intraductal papillary mucinous neoplasms (IPMN) and metastatic PDAC. The phenotype in control KCYp120wt/wt mice (n=12) was restricted to early PanINs. We next asked whether the second allele of p120 is lost during metastatic dissemination. We demonstrated that the remaining p120 allele is expressed in liver metastases, indicating that p120 loss-of-heterozygosity did not occur. Of note, in liver metastases, p120 co-localizes with E-cad, thereby indicating that a single p120 allele is sufficient to stabilize E-cad. This raised the question of whether one p120 allele is required to establish epithelial integrity at the metastatic site. In order to address this mechanistically, and since KCYp120fl/fl are not viable, we performed three-dimensional (3D) culture experiments with pancreatic cells isolated from non-recombined LSL-KrasG12D/+;p120wt/wt;R26YFP (KYp120wt/wt), KYp120fl/wt and KYp120fl/fl mice. Cells were Cre-recombined in vitro. In 3D culture, KYp120wt/wt cells form round multicellular cysts. Monoallelic p120 loss only disrupts the symmetry of cysts while biallelic loss completely prevents cells from forming organized, cyctic structures. We next injected these cell lines orthotopically into the pancreata of immunodeficient mice. KYp120wt/wt cells form PanIN-like structures. Interestingly, KYp120fl/wt establish large cysts reminiscent of IPMN/MCN lesions. In the cysts, p120 is localized at the plasma membrane with E-cad. However, the invasive fronts of tumors show significantly less p120 expression. Finally, KYp120fl/fl cells form poorly differentiated tumors invading into the surrounding stroma. Given the fact that p120 stabilizes E-cad and that E-cad is critical in EMT and MET, we injected the KYp120wt/wt, KYp120fl/wt and KYp120fl/fl cell lines directly into the portal veins of these mice. KYp120fl/wt cells colonize the liver and retain the remaining p120 allele and display membranous E-cad localization. By contrast, although KYp120fl/fl cells are able to generate invasive primary pancreatic tumors when being injected orthotopically, these cells fail to colonize the liver. Conclusions: Our findings demonstrate that monoallelic loss of p120 accelerates Kras-driven PDAC formation. However, one allele of p120 is required to establish epithelial integrity and metastases. Taken together, these results for the first time underscore the importance of p120-mediated EMT plasticity in order to complete the metastatic cascade in vivo. Furthermore, therapeutic strategies to prevent EMT alone may not be sufficient in light of our results but rather require novel approaches to target EMT plasticity so as to enhance survival in metastatic PDAC. This abstract is also presented as Poster A63. Citation Format: Maximilian Reichert, Basil S. Bakir, Christopher M. Hahn, Gregory P. Botta, Andrew D. Rhim, Robert H. Vonderheide, Albert B. Reynolds, Yingtao Bi, Ramana Davuluri, Burcu Saka, N. Volkan Adsay, Anil K. Rustgi. p120 catenin mediated epithelial-to-mesenchymal plasticity determines the metastatic potential of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR05.

Purpose: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. Experimental Design: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. Conclusion: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease.

The cancer microenvironment and interaction between cancer and stromal cells play critical roles in tumor development and progression. The molecular features of cancer stroma are less well understood than those of cancer cells. Cancer-associated stromal fibroblasts are the predominant component of stroma associated with colon cancer and its functions remain unclear. Fibroblast cell cultures were established from metastatic colon cancer in liver, liver away from the metastatic lesions, and skin from three patients with metastatic colorectal cancer. We generated expression profiles of cancer-associated fibroblasts using oligochip arrays and compared them to those of uninvolved fibroblasts. The conditioned media from the cancer-associated fibroblast cultures enhanced proliferation of colon cancer cell line HCT116 to a greater extent than cultures from uninvolved fibroblasts. In microarray expression analysis, cancer-associated fibroblasts clustered tightly into one group and skin fibroblasts into another. Approximately 170 of 22,000 genes were up-regulated in cancer-associated fibroblasts (fold change > 2, P < 0.05) as compared to skin fibroblasts, including many genes encoding cell adhesion molecules, growth factors, and COX2. By immunohistochemistry in-vivo, we confirmed COX2 and TGFB2 expression in cancer-associated fibroblasts in metastatic colon cancer. The distinct molecular expression profiles of cancer-associated fibroblasts in colon cancer metastasis support the notion that these fibroblasts form a favorable microenvironment for cancer cells.

Translating the molecular signatures from classification strategies into robust diagnostic assays is essential for cancer therapy guidance and prognosis determination. To develop a clinically applicable molecular stratification assay for Glioblastoma multiforme (GBM) with significant prognostic value, we designed a novel classification system based on isoform-level gene expression profiles; an avenue unexplored for diagnostic and prognostic use. Using isoform-level expression clustering of The Cancer Genome Atlas (TCGA) samples, we identified four GBM subgroups with significant (p=0.0103) survival differences. A four-class classifier, built with 121 transcript-variants, assigns GBM patients’ molecular subtype with 92% accuracy. The classifier was translated to a high-throughput RT-qPCR assay and validated on an independent cohort of 206 glioblastoma samples. We found that proneural patients have the worst prognosis except for younger patients ( Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A26. Citation Format: Yingtao Bi, Sharmistha Pal, Luke Macyszyn, Louise C. Showe, Donald O'Rourke, Ramana V. Davuluri. Translation of isoform-level gene signature into robust diagnostic assay for glioblastoma subtyping. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A26.

Abstract: Translating the molecular signatures from classification strategies into robust diagnostic assays is essential for cancer therapy guidance and prognosis determination. To develop a clinically applicable molecular stratification assay for Glioblastoma multiforme (GBM) with significant prognostic value, we designed a novel classification system based on isoform-level gene expression profiles; an avenue unexplored for diagnostic and prognostic use. Using isoform-level expression clustering of The Cancer Genome Atlas (TCGA) samples, we identified four GBM subgroups with significant (p=0.0103) survival differences. A four-class classifier, built with 121 transcript-variants, assigns GBM patients' molecular subtype with 92% accuracy. The classifier was translated to a high-throughput RT-qPCR assay and validated on an independent cohort of 206 glioblastoma samples. We found that proneural patients have the worst prognosis except for younger patients ( Citation Format: Yingtao Bi, Sharmistha Pal, Luke Macyszyn, Donald O'Rourke, Ramana Davuluri. Translation of isoform-level gene signature into robust diagnostic assay for glioblastoma subtyping. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C06.

Glioblastomamultiforme (GBM) is one of the most deadly cancers in adult and even with aggressive therapy prognosis is poor with a median survival of about 15 months. Recent large scale genomic studies have shed light on the molecular mechanisms of cancer and shown the existence of distinct molecular sub-groups of GBM whose identification is the key to develop specific therapeutic regimen for each GBM group. Since transcriptome studies, by us and others, have discovered that majority of genes produce multiple protein isoforms, which could be involved in multiple functional pathways, we hypothesized that the isoform-level expression profiling will generate better classification to identify themolecular sub-groups of GBM. To test this hypothesis, we performed isoform-level analysis of the exon array expression data for GBM patient samples from the TCGA data portal, and discovered that isoform-level analysis identifies 2.5 fold more differentially expressed transcript variants than differentially expressed genes captured by gene-level analysis, indicating that isoform-level expression profiling is more sensitive in identifying molecular changes among GBM patients. Next, we applied consensus non-negative matrix factorization (NMF) clustering method, based on isoform-level expression ofmost variable isoforms and effectively grouped the GBM samples into 4 sub-groups with significant (p=0.0103) survival differences between the groups. In contrast, though clustering based on gene-level expression produced four homogenous groups there was no significant survival difference among the sub-groups. Based on the prognostic value of the molecular sub-groups, our goal was to build a classifier that can assign each GBM patient a molecular sub-group.We compared the prediction accuracy of a gene based vs an isoform based classifier to identify sub-group and found that isoform based classifier is a better predictor (85%vs90%). Using the Random forest feature selection we have build a classifier based on the expression of 147 isoforms that is ∼91% accurate and have developed a high-throughput RT-qPCR assay to measure the expression of these discriminatory isoforms. We have successfully validated the classifier to identify the molecular sub-group in an independent cohort of GBM patient samples from the Human Brain Tumor Tissue bank at University of Pennsylvania.Our study has led to the development of a classification assay for GBM patient sub-grouping, which can be useful in developing targeted drug therapy for each sub-group, and suggests that isoform based expression analysis can lead to better molecular classification of cancer, a requirement for the quest of personalized therapy. Citation Format: Sharmistha Pal, Yingtao Bi, Lukasz Macyszyn, Louise C. Showe, Donald M. O'Rourke, Ramana V. Davuluri. Molecular classification of glioblastoma multiforme using isoform-level gene expression signatures. [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 5597. doi:10.1158/1538-7445.AM2013-5597 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Background: Lung cancer is the leading cause of cancer related mortality, with NSCLC the most prevalent form. Despite advances in treatment options (minimally invasive surgery, CT-guided radiation, novel chemotherapy regimens, targeted therapeutics) prognosis remains dismal. Most research to date has focused on cancer cell intrinsic mutagenesis (K-ras, p53, EGFR, EML4-ALK fusion), however recent studies have begun to recognize that cross talk between tumor epithelial cells and intratumoral stromal cells contributes to tumor progression. Unfortunately, little is known about the contribution or role of the stroma in NSCLC. Here, we present the first comprehensive analysis of specific individual stromal components of human NSCLC, which has led to the identification of stromal-specific activated pro-tumorigenic genes of prognostic and therapeutic potential. Materials and Methods: Surgically resected Stage I/II adenocarcinoma and matched adjacent non-neoplastic lung tissue were mechanically dissociated within 1hour of resection. Cell populations marked with specific antibodies were sorted to homogeneity by flow cytometry. RNA was isolated to construct cDNA libraries, which were deep sequenced using the Illumina HiSeq 2000. Validation of differentially regulated genes was carried out by RT-PCR and immunostaining. Mouse models of K-rasG12D and p53flox/flox derived NSCLC together with a bone marrow transplantation approach were used to determine the functional role of activated genes, and a NSCLC tissue microarray was used to ascertain their prognostic value. Results: Flow cytometry analysis revealed a 4-fold increase in the number of intratumoral hematopoietic cells, compared with adjacent non-neoplastic lung tissue. Deep RNA sequencing of two predominant myeloid specific subpopulations, CD11b+CD33+ monocytic myeloid cells and CD11b+CD33- polymorphonuclear neutrophils, revealed 783 and 938 differentially regulated genes respectively when comparing tumor vs. adjacent lung-derived cells. Many genes encoded secreted proteins such as OPN, TSP1, CCL7, and MMP12, suggesting activation of aberrant autocrine and paracrine regulatory pathways likely to support stromal-mediated tumor progression. We will present data showing the prognostic and therapeutic potential of these genes in NSCLC. Conclusions: This study is based on an emerging paradigm that activated intratumoral stromal cells play a fundamental role in promoting tumor growth. Using a unique and novel approach of quantitative analysis of individual stromal components from freshly procured NSCLC tumor tissue, we have identified differentially regulated genes previously undiscovered in traditional whole tumor profiling approaches. The activated stromal genes constitute novel therapeutic targets against lung cancer, either alone or in combination with treatments that target cancer cells. Citation Format: Anna Durrans, Dingcheng Gao, Ravi Gupta, Seongho Ryu, Navneet Narula, Kari R. Fischer, Hyejin Choi, Brendon M. Stiles, Abu Nasar, Ramana V. Davuluri, Nasser K. Altorki, Vivek Mittal. Analysis of the reprogrammed bone marrow-derived stroma in lung cancer unravels potential targets for prognosis and therapy. [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 4972. doi:10.1158/1538-7445.AM2013-4972

Glioblastoma multiforme (GBM) is the most common primary central nervous system tumor, accounting for nearly 80% of the 22,000 malignant brain tumors diagnosed annually in the United States. Patients who receive a diagnosis of glioblastoma have a dismal prognosis and median survival of 12-14 months following the standard therapy. Therefore, there is a need for improved molecular classification of GBM to identify highly sensitive and specific biomarkers and therapeutic targets. Recent genome wide transcriptome studies, by us and others, have discovered that one gene gives rise to multiple protein isoforms, which could be involved in multiple functional pathways. Based on this model, we hypothesize that the isoform-level expression profiling will lead to significantly improved classification of molecular sub-types and identification of more specific isoform based regulatory modules that are disrupted in each GBM molecular sub-type. Using the GBM data sets from TCGA data portal, we identified 2903 genes, which differ significantly in expression between the normal brain and GBM. In contrast, similar analysis using transcript-level expression showed that the expression of 7639 transcripts (correspond to 4401 genes) was significantly altered in GBMs, suggesting that measuring isoform/mRNA variant expression is more sensitive to identify molecular changes in GBM. Furthermore, we identified 51 genes (e.g. RTN3, DCLK2, PKM2, ACTN1), mostly associated with cellular assembly and organization, whose transcript variants show opposite expression pattern, an information lost in a gene centric approach. Although GBMs have been grouped into four subtypes (Proneural, Neural, Classical, Mesenchymal) based on gene expression and mutation profiling, these sub-types do not show any survival differences. Since we observed higher expression differences at isoform-level, we investigated whether isoform-level expression estimates can improve the grouping in terms of disease prognosis. We observed that isoform level clustering always generated a better survival grouping and effectively grouped the 173 core samples into 4 sub-groups with significant (p=0.024) survival differences between the groups. Interestingly, we identified a subgroup, consisting of selected proneural samples, which exhibits higher survival and is characterized by isoform specific overexpression of oligodendroglial markers including Olig1, Olig2, Sox 4, Sox6, Sox8, and Sox10. Gene level expression fails to identify Olig1, Sox6 and Sox10 as significantly misregulated in this subgroup. Similarly, we identify an isoform specific network in another subgroup that could confer resistance to EGFR tyrosine kinase inhibitor, erlotinib. Our analyses suggests that the quest for genomics based personalized medicine requires molecular classification and systematic understanding of the gene regulatory networks at the isoform level. 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 2990. doi:1538-7445.AM2012-2990

Refractory to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in aberrant expression of downstream target genes in the disease. We hypothesized that aberrant expression of TGF-β/SMAD4 targets are mediated through epigenetic mechanism and also contribute to resistance to TGF-β meditated growth inhibition. Our previous report using chromatin immunoprecipitation microarray (ChIP-chip) identified FBXO32 as one of SMAD4 targets in immortalized ovarian surface epithelial cell (IOSE) (Qin et al., BMC Syst Biol, 17: 73, 2009). In the present study, we investigated the mechanism conferring FBXO32 down-regulation, its clinical significance, and its function in ovarian cancer. Our result showed that expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines (HeyC2, SKOV3, CP70, A2708, MCP2, MCP3) using real time RT-PCR. Promoter methylation of FBXO32 was seen in ovarian cancer cell lines, HeyC2 and SKOV3, that display constitutive TGF-β/SMAD4 signaling. Moreover, our finding that epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggested that epigenetic regulation of FBXO32 in ovarian cancer may be a common event. Re-expression of FBXO32 markedly impeded proliferation of a platinum-resistant ovarian cancer cell lines, HeyC2 and CP70 (colony number: HeyC2: 19.33 ± 3.06 vs 1 ± 0, P< 0.005; CP70: 46.5 ± 6.36 vs 1.5 ± 0.7, P< 0.001) by using colony formation assay, due to increased apoptosis of the cells (CP70; apoptotic cell%, control: 2.55 ± 0.17; FBXO32: 10.72 ± 1.07, P 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 3072.