NDN is a maternally imprinted gene consistently expressed in normal ovarian epithelium, is dramatically downregulated in the majority of ovarian cancers. Little or no NDN expression could be detected in 73% of 351 epithelial ovarian cancers. NDN was also downregulated in 10 ovarian cancer cell lines with total loss in 6 of 10. Re-expression of NDN decreased Bcl-2 levels and induced apoptosis, which significantly inhibited ovarian cancer cell growth in cell culture and in xenografts. In addition, re-expression of NDN inhibited cell migration by decreasing actin stress fiber and focal adhesion complex formation through deactivation of Src, FAK and RhoA. Loss of NDN expression in ovarian cancers could be attributed to LOH in 28% of 18 informative cases and to hypermethylation of CpG sites 1 and 2 of NDN promoter in 23% and 30% of 43 ovarian cancers, respectively. Promoter hypermethylation was also found in 5 of 10 ovarian cancer cell lines. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored NDN expression in 4 of 7 cell lines with enhanced promoter methylation levels. These observations support the conclusion that NDN is an imprinted tumor suppressor gene which affects cancer cell motility, invasion and growth and that its loss of function in ovarian cancer can be caused by both genetic and epigenetic mechanisms.

Glioblastoma (GBM) is the most lethal and aggressive adult brain tumor, requiring the development of efficacious therapeutics. Towards this goal, we screened five genetically distinct patient-derived brain-tumor initiating cell lines (BTIC) with a unique collection of small molecule epigenetic modulators from the Structural Genomics Consortium (SGC). We identified multiple hits that inhibited the growth of BTICs in vitro, and further evaluated the therapeutic potential of EZH2 and HDAC inhibitors due to the high relevance of these targets for GBM. We found that the novel SAM-competitive EZH2 inhibitor UNC1999 exhibited low micromolar cytotoxicity in vitro on a diverse collection of BTIC lines, synergized with dexamethasone (DEX) and suppressed tumor growth in vivo in combination with DEX. In addition, a unique brain-penetrant class I HDAC inhibitor exhibited cytotoxicity in vitro on a panel of BTIC lines and extended survival in combination with TMZ in an orthotopic BTIC model in vivo. Finally, a combination of EZH2 and HDAC inhibitors demonstrated synergy in vitro by augmenting apoptosis and increasing DNA damage. Our findings identify key epigenetic modulators in GBM that regulate BTIC growth and survival and highlight promising combination therapies.

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and β-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials.

No abstract available

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism bof reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples.

Anti-angiogenesis therapy has shown clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance rapidly emerges. Thus, approaches to overcome such resistance are needed. We developed the setting of adaptive resistance to anti-VEGF therapy, and performed a series of in vivo experiments in both immune competent and nude mouse models. Given the pro-angiogenic properties of tumor-associated macrophages (TAMs) and the dominant role of CSF1R in macrophage function, we added CSF1R inhibitors following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with a CSF1R inhibitor (AC708) after anti-VEGF antibody resistance had little to no measurable tumor burden upon completion of the experiment while those that did not receive a CSF1R inhibitor still had abundant tumor. To mimic clinically used regimens, mice were also treated with anti-VEGF antibody and paclitaxel until resistance emerged, and then a CSF1R inhibitor was added. The addition of a CSF1R inhibitor restored response to anti-angiogenesis therapy, resulting in 83% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone. Collectively, our data demonstrate that the addition of a CSF1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects.

High-grade serous ovarian carcinoma (HGSOC), the most common and aggressive subtype of epithelial ovarian cancer, is characterized by TP53 mutations and genetic instability. Using miRNA profiling analysis, we found that miR-145, a p53 regulated miRNA, was frequently down-regulated in HGSOC. miR-145 down-regulation was further validated in a large cohort of HGSOCs by qPCR. Overexpression of miR-145 in ovarian cancer cells significantly suppressed proliferation, migration and invasion in vitro and inhibited tumor growth and metastasis in vivo. Metadherin (MTDH) was subsequently identified as a direct target of miR-145, and was found to be significantly up-regulated in HGSOC. Furthermore, overexpression of MTDH rescued the inhibitory effects of miR-145 in ovarian cancer cells. Finally, we found that high level of MTDH expression correlated with poor prognosis of HGSOC. Therefore, lack of suppression of MTDH by miR-145 when p53 is dysfunctional leads to increased tumor growth and metastasis of HGSOC. Our study established a new link between p53, miR-145 and MTDH in the regulation of tumor growth and metastasis in HGSOC.

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

The S phase kinase-associated protein 1 (Skp1), an adaptor protein of the Skp1-Cul1-F-box protein complex, binds the ubiquitin E3 ligase Skp2 and is critical to its biological functions. Targeting of Skp1 by a small compound 6-O-angeloylplenolin (6-OAP) results in dissociation and degradation of Skp2 and mitotic arrest of lung cancer cells. Here, by using a proteome microarray containing 16,368 proteins and a biotinylated 6-OAP, we identified 99 proteins that could bind 6-OAP, with Skp1 and STAT3 sitting at the central position of the 6-OAP interactome. 6-OAP formed hydrogen bonds with Ser611/Ser613/Arg609 at the SH2 domain of STAT3 and inhibited the constitutive and interleukin-6-induced phosphorylated STAT3 (pSTAT3), leading to inhibitory effects on lung cancer cells and suppression of Skp2 transcription. STAT3 was overexpressed in tumor samples compared to counterpart normal lung tissues and was inversely associated with prognosis of the patients. 6-OAP inhibited tumor growth in SCID mice intravenously injected with lung cancer cells, and downregulated both STAT3 and Skp2 in tumor samples. Given that 6-OAP is a Skp1 inhibitor, our data suggest that this compound may target Skp1 and STAT3 to suppress Skp2, augmenting its anti-lung cancer activity.

Ovarian cancer is the most fatal gynecologic cancer with poor prognosis. Etiological factors underlying ovarian cancer genesis and progression are poorly understood. Previously, we have shown that JNK-associated Leucine zipper Protein (JLP), promotes oncogenic signaling. Investigating the role of JLP in ovarian cancer, our present study indicates that JLP is overexpressed in ovarian cancer tissue and ovarian cancer cells. Transient overexpression of JLP promotes proliferation and invasive migration of ovarian cancer cells. In addition, ectopic expression of JLP confers long-term survival and clonogenic potential to normal fallopian tube-derived epithelial cells. Coimmunoprecipitation and colocalization analyses demonstrate the in vivo interaction of JLP and JNK, which is stimulated by lysophosphatidic acid (LPA), an oncogenic lipid growth factor in ovarian cancer. We also show that LPA stimulates the translocation of JLP-JNK complex to the perinuclear region of SKOV3-ip cells. JLP-knockdown using shRNA abrogates LPA-stimulated activation of JNK as well as LPA-stimulated proliferation and invasive migration of SKOV3-ip cells. Studies using ovarian cancer xenograft mouse model indicate that the mice bearing JLP-silenced xenografts exhibits reduced tumor volume. Analysis of the xenograft tumor tissues indicate a reduction in the levels of JLP, JNK, phosphorylated-JNK, c-Jun and phosphorylated-c-Jun in JLP-silenced xenografts, thereby correlating the attenuated JLP-JNK signaling node with suppressed tumor growth. Thus, our results identify a critical role for JLP-signaling axis in ovarian cancer and provide evidence that targeting this signaling node could provide a new avenue for therapy.