Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10-5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression.

Osteopontin (OPN) is the most upregulated gene in primary central nervous system lymphoma (PCNSL) compared to non-CNS diffuse large B cell lymphoma (DLBCL). We show here that OPN is a key mediator of intracerebral tumor growth, invasion, and dissemination in CNS lymphoma, and that these effects depend upon activation of NF-κB. We further show that activation of NF-κB by OPN occurs through a unique mechanism in which intracellular OPN (iOPN) causes transcriptional downregulation of the NF-κB inhibitors, A20/TNFAIP3 and ABIN1/TNIP1, and secretory OPN (sOPN) promotes receptor-mediated activation of NF-κB. We also identify NF-κB-mediated induction of matrix metalloproteinase-8 (MMP-8) as a specific feature of OPN-mediated tissue invasion. These results implicate OPN as a candidate for development of targeted therapy for patients with PCNSL.

Ovarian cancer represents the most lethal tumor type among malignancies of the female reproductive system. Overall survival rates remain low. In this study, we identify the serine protease inhibitor Kazal type 1 (SPINK1) as a potential therapeutic target for a subset of ovarian cancers. We show that SPINK1 drives ovarian cancer cell proliferation through activation of epidermal growth factor receptor (EGFR) signaling, and that SPINK1 promotes resistance to anoikis through a distinct mechanism involving protease inhibition. In analyses of ovarian tumor specimens from a Mayo Clinic cohort of 490 patients, we further find that SPINK1 immunostaining represents an independent prognostic factor for poor survival, with the strongest association in patients with nonserous histological tumor subtypes (endometrioid, clear cell, and mucinous). This study provides novel insight into the fundamental processes underlying ovarian cancer progression, and also suggests new avenues for development of molecularly targeted therapies.

Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic.

Currently there is a lack of targeted therapies that lead to long-term attenuation or regression of disease in patients with advanced clear cell renal cell carcinoma (ccRCC). Our group has implemented a high-throughput genetic analysis coupled with a high-throughput proliferative screen in order to investigate the genetic contributions of a large cohort of overexpressed genes at the functional level in an effort to better understand factors involved in tumor initiation and progression. Patient gene array analysis identified transcripts that are consistently elevated in patient ccRCC as compared to matched normal renal tissues. This was followed by a high-throughput lentivirus screen, independently targeting 195 overexpressed transcripts identified in the gene array in four ccRCC cell lines. This revealed 31 'hits' that contribute to ccRCC cell proliferation. Many of the hits identified are not only presented in the context of ccRCC for the first time, but several have not been previously linked to cancer. We further characterize the function of a group of hits in tumor cell invasion. Taken together these findings reveal pathways that may be critical in ccRCC tumorigenicity, and identifies novel candidate factors that could serve as targets for therapeutic intervention or diagnostic/prognostic biomarkers for patients with advanced ccRCC.

Epithelial-mesenchymal transition (EMT) is characterized by loss of cell-cell junctions, polarity and epithelial markers, and in turn, acquisition of mesenchymal features and motility. Changes associated with this developmental process have been extensively implicated in breast cancer progression and metastasis. Matrix metalloproteinases (MMPs) have been identified as specific inducers of EMT in mammary epithelial cells. MMP-3 induces EMT associated with malignant transformation via a pathway dependent upon production of reactive oxygen species (ROS). While the process by which exposure to MMP-3 leads to induction of ROS has been extensively studied, exactly how the MMP-3-induced ROS stimulate EMT remains unknown. Here, we used profiling methods to identify MMP-3-induced transcriptional alterations in mouse mammary epithelial cells, finding common overlap with changes mediated by nuclear factor-κB (NF-κB) and found in advanced breast cancer. In cultured cells, we found that Snail, an ROS-dependent key mediator of MMP-3-induced changes, is regulated by NF-κB in response to MMP-3. More specifically, we found MMP-3 to cause binding of p65 and cRel NF-κB subunits to the Snail promoter, leading to its transcription. Our results identify a specific pathway by which MMPs induce EMT and malignant characteristics, and provide insight into potential therapeutic approaches to target MMP-associated breast cancers.

We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10-3). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.

We previously identified associations with ovarian cancer outcome at five genetic loci. To identify putatively causal genetic variants and target genes, we prioritized two ovarian outcome loci (1q22 and 19p12) for further study. Bioinformatic and functional genetic analyses indicated that MEF2D and ZNF100 are targets of candidate outcome variants at 1q22 and 19p12, respectively. At 19p12, the chromatin interaction of a putative regulatory element with the ZNF100 promoter region correlated with candidate outcome variants. At 1q22, putative regulatory elements enhanced MEF2D promoter activity and haplotypes containing candidate outcome variants modulated these effects. In a public dataset, MEF2D and ZNF100 expression were both associated with ovarian cancer progression-free or overall survival time. In an extended set of 6,162 epithelial ovarian cancer patients, we found that functional candidates at the 1q22 and 19p12 loci, as well as other regional variants, were nominally associated with patient outcome; however, no associations reached our threshold for statistical significance (p<1×10-5). Larger patient numbers will be needed to convincingly identify any true associations at these loci.

RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI), with adjustment for European ancestry. We conducted gene-level analyses using the Admixture Maximum Likelihood (AML) test and the Sequence-Kernel Association test for common and rare variants (SKAT-CR). Association analysis revealed top risk-associated SNP rs77027562 (OR (95% CI)= 1.39 (1.17-1.64), P=1.0x10-4) in ADAR3 and rs185455523 in SND1 (OR (95% CI)= 0.68 (0.56-0.83), P=2.0x10-4). When restricting to serous histology (n=6,500), the magnitude of association strengthened for rs185455523 (OR=0.60, P=1.0x10-4). Gene-level analyses revealed that variation in ADAR was associated (P<0.05) with EOC susceptibility, with PAML=0.022 and PSKAT-CR=0.020. Expression quantitative trait locus analysis in EOC tissue revealed significant associations (P<0.05) with ADAR expression for several SNPs in ADAR, including rs1127313 (G/A), a SNP in the 3' untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing.

Matrix metalloproteinases (MMPs) have been implicated in diverse roles in breast cancer development and progression. While many of the different MMPs expressed in breast cancer are produced by stromal cells MMP-9 is produced mainly by the tumor cells themselves. To date, the functional role of tumor cell-produced MMP-9 has remained unclear. Here, we show that human breast cancer cell-produced MMP-9 is specifically required for invasion in cell culture and for pulmonary metastasis in a mouse orthotopic model of basal-like breast cancer. We also find that tumor cell-produced MMP-9 promotes tumor vascularization with only modest impact on primary tumor growth, and that silencing of MMP-9 expression in tumor cells leads to an altered transcriptional program consistent with reversion to a less malignant phenotype. MMP-9 is most highly expressed in human basal-like and triple negative tumors, where our data suggest that it contributes to metastatic progression. Our results suggest that MMP9 may offer a target for anti-metastatic therapies for basal-like triple negative breast cancers, a poor prognosis subtype with few available molecularly targeted therapeutic options.

Genome-wide association studies have identified several common susceptibility alleles for epithelial ovarian cancer (EOC). To further understand EOC susceptibility, we examined previously ungenotyped candidate variants, including uncommon variants and those residing within known susceptibility loci.

Risk assessment and treatment choice remain a challenge in early non-small-cell lung cancer (NSCLC). Alternative splicing is an emerging source for diagnostic, prognostic and therapeutic tools. Here, we investigated the prognostic value of the actin cytoskeleton regulator hMENA and its isoforms, hMENA11a and hMENAΔv6, in early NSCLC. The epithelial hMENA11a isoform was expressed in NSCLC lines expressing E-CADHERIN and was alternatively expressed with hMENAΔv6. Enforced expression of hMENAΔv6 or hMENA11a increased or decreased the invasive ability of A549 cells, respectively. hMENA isoform expression was evaluated in 248 node-negative NSCLC. High pan-hMENA and low hMENA11a were the only independent predictors of shorter disease-free and cancer-specific survival, and low hMENA11a was an independent predictor of shorter overall survival, at multivariate analysis. Patients with low pan-hMENA/high hMENA11a expression fared significantly better (P≤0.0015) than any other subgroup. Such hybrid variable was incorporated with T-size and number of resected lymph nodes into a 3-class-risk stratification model, which strikingly discriminated between different risks of relapse, cancer-related death, and death. The model was externally validated in an independent dataset of 133 patients. Relative expression of hMENA splice isoforms is a powerful prognostic factor in early NSCLC, complementing clinical parameters to accurately predict individual patient risk.

MISIIR is a potential target for ovarian cancer (OC) therapy due to its tissue-specific pattern of expression. 3C23K is a novel therapeutic monoclonal anti-MISIIR antibody designed to recruit effector cells and promote cell death through ADCC (antibody dependent cell-mediated cytotoxicity). Our objective was to determine the tolerability and efficacy of 3C23K in OC patient-derived xenografts (PDX) and to identify factors affecting efficacy. Quantitative RT-PCR, immunohistochemistry (IHC), and flow cytometry were used to categorize MISIIR expression in established PDX models derived from primary OC patients. We selected two high expressing models and two low expressing models for in vivo testing. One xenograft model using an MISIIR over-expressing SKOV3ip cell line (Z3) was a positive control. The primary endpoint was change in tumor size. The secondary endpoint was final tumor mass. We observed no statistically significant differences between control and treated animals. The lack of response could be secondary to a number of variables including the lack of known biomarkers of response, the low membrane expression of MISIIR, and a limited ability of 3C23K to induce ADCC in PDX models. Further study is needed to determine the magnitude of ovarian cancer response to 3C23K and also if there is a threshold surface expression to predict response.

Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E -mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E -mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAFV600E -mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.

Gene fusions play a critical role in some cancers and can serve as important clinical targets. In epithelial ovarian cancer (EOC), the contribution of fusions, especially by histological type, is unclear. We therefore screened for recurrent fusions in a histologically diverse panel of 220 EOCs using RNA sequencing. The Pipeline for RNA-Sequencing Data Analysis (PRADA) was used to identify fusions and allow for comparison with The Cancer Genome Atlas (TCGA) tumors. Associations between fusions and clinical prognosis were evaluated using Cox proportional hazards regression models. Nine recurrent fusions, defined as occurring in two or more tumors, were observed. CRHR1-KANSL1 was the most frequently identified fusion, identified in 6 tumors (2.7% of all tumors). This fusion was not associated with survival; other recurrent fusions were too rare to warrant survival analyses. One recurrent in-frame fusion, UBAP1-TGM7, was unique to clear cell (CC) EOC tumors (in 10%, or 2 of 20 CC tumors). We found some evidence that CC tumors harbor more fusions on average than any other EOC histological type, including high-grade serous (HGS) tumors. CC tumors harbored a mean of 7.4 fusions (standard deviation [sd] = 7.4, N = 20), compared to HGS EOC tumors mean of 2.0 fusions (sd = 3.3, N = 141). Few fusion genes were detected in endometrioid tumors (mean = 0.24, sd = 0.74, N = 55) or mucinous tumors (mean = 0.25, sd = 0.5, N = 4) tumors. To conclude, we identify one fusion at 10% frequency in the CC EOC subtype, but find little evidence for common (> 5% frequency) recurrent fusion genes in EOC overall, or in HGS subtype-specific EOC tumors.

Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor.

Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer.

The homeobox A (HOXA) region of protein-coding genes impacts female reproductive system embryogenesis and ovarian carcinogenesis. The 5-prime end of HOXA includes three long non-coding RNAs (lncRNAs) (HOXA10-AS, HOXA11-AS, and HOTTIP) that are underexplored in epithelial ovarian cancer (EOC). We evaluated whether common genetic variants in these lncRNAs are associated with EOC risk and/or have functional roles in EOC development. Using genome-wide association study data from 1,201 serous EOC cases and 2,009 controls, an exonic variant within HOXA11-AS, rs17427875 (A>T), was marginally associated with reduced serous EOC risk (OR = 0.88 (95% CI: 0.78-1.01, p = 0.06). Functional studies of ectopic expression of HOXA11-AS minor allele T in EOC cells showed decreased survival, proliferation, migration, and invasion compared to common allele A expression. Additionally, stable expression of HOXA11-AS minor allele T reduced primary tumor growth in mouse xenograft models to a greater extent than common allele A. Furthermore, HOXA11-AS expression levels were significantly lower in human EOC tumors than normal ovarian tissues (p < 0.05), suggesting that HOXA11-AS has a tumor suppressor function in EOC which may be enhanced by the T allele. These findings demonstrate for the first time a role for HOXA11-AS in EOC with effects that could be modified by germline variants.

Breast cancer invasion involves the loss of cell-cell junctions and acquisition of an invasive, migratory phenotype, and breast cancer cells of the basal intrinsic subtype are more invasive and metastatic than breast cancer cells of other subtypes. ARHGEF11 is a RhoGEF that was previously shown to bind to the tight junction protein ZO-1 at perijunctional actomyosin ring (PJAR), a network of cortically organized actin and myosin filaments associated with junctional complexes that regulates cell-cell adhesion and polarization. We show here that ARHGEF11 shows splice isoform expression that differs according to the intrinsic subtype of breast cancer cells and that controls their invasive phenotype. Luminal subtype breast cancer cells express the isoform of ARHGEF11 lacking exon 38 (38-), which binds to ZO-1 at PJAR and is necessary for formation and maintenance of cell-cell junctions. Basal subtype breast cancer cells express the isoform of ARHGEF11 containing exon 38 (38+), which does not bind to ZO-1 and which drives cell migration and motility. Depletion of ARHGEF11 in basal subtype breast cancer cells is sufficient to alter cell morphology from a mesenchymal stellate form with extensive cell protrusions to a cobblestone-like epithelial form, and to suppress growth and survival both in vitro and in vivo. These findings show that the expression of the particular splice isoform of ARHGEF11 is critically linked to the malignant phenotype of breast cancer cells, identifying ARHGEF11 exon 38(+) as a biomarker and target for therapy of breast cancer.