Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3' UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies, emphasizing particular subgroups, were relatively small. Therefore, we comprehensively evaluated ovarian and breast cancer risks as well as clinical outcome associated with rs61764370.

Paclitaxel is a mainstay of treatment for many solid tumors, and frequently, clinical outcome is influenced by paclitaxel sensitivity. Despite this, our understanding of the molecular basis of paclitaxel response is incomplete. Recently, it has been shown that microRNAs (miRNAs) influence messenger RNA (mRNA) transcriptional control and can contribute to human carcinogenesis. In the present study, our objective was to identify miRNAs associated with cancer cell line response to paclitaxel and to evaluate these miRNAs as therapeutic targets to increase paclitaxel sensitivity. We measured the expression of 335 unique miRNAs in 40 human cancer cell lines selected from the NCI panel. We then integrated miRNA expression data with publicly available paclitaxel-sensitivity (GI₅₀) data for each of the 40 cell lines to identify miRNAs associated with paclitaxel sensitivity. Ovarian cancer cell lines with differential miRNA expression and paclitaxel sensitivity were transiently transfected with miRNA precursors and inhibitors, and the effects on in vitro cell paclitaxel sensitivity were evaluated. Pearson's correlation identified 2 miRNAs (miR-367 and miR-30a-5p) associated with the NCI40 cell line in vitro paclitaxel response (P<0.0003). Ovarian cancer cells were selected based on the association between paclitaxel sensitivity and miR-367/miR-30a-5p expression. Overexpression of miR-367 in the paclitaxel-sensitive cells [PA1; IC₅₀, 1.69 nM, high miR-367 (2.997), low miR-30a-5p (-0.323)] further increased paclitaxel sensitivity, whereas miR-367 depletion decreased paclitaxel sensitivity. In contrast, overexpression and depletion of miR-30a-5p in the paclitaxel-resistant cells [OVCAR4; IC₅₀, 17.8 nM, low miR-367 (-0.640), high miR-30a-5p (3.270)] decreased and increased paclitaxel sensitivity, respectively. We identified and successfully targeted miRNAs associated with human cancer cell line response to paclitaxel. Our strategy of integrating in vitro miRNA expression and drug sensitivity data may not only aid in the characterization of determinants of drug response but also in the identification of novel therapeutic targets to increase activity of existing therapeutics.

Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.

Disruption in circadian gene expression, whether due to genetic variation or environmental factors (e.g., light at night, shiftwork), is associated with increased incidence of breast, prostate, gastrointestinal and hematologic cancers and gliomas. Circadian genes are highly expressed in the ovaries where they regulate ovulation; circadian disruption is associated with several ovarian cancer risk factors (e.g., endometriosis). However, no studies have examined variation in germline circadian genes as predictors of ovarian cancer risk and invasiveness. The goal of the current study was to examine single nucleotide polymorphisms (SNPs) in circadian genes BMAL1, CRY2, CSNK1E, NPAS2, PER3, REV1 and TIMELESS and downstream transcription factors KLF10 and SENP3 as predictors of risk of epithelial ovarian cancer (EOC) and histopathologic subtypes. The study included a test set of 3,761 EOC cases and 2,722 controls and a validation set of 44,308 samples including 18,174 (10,316 serous) cases and 26,134 controls from 43 studies participating in the Ovarian Cancer Association Consortium (OCAC). Analysis of genotype data from 36 genotyped SNPs and 4600 imputed SNPs indicated that the most significant association was rs117104877 in BMAL1 (OR = 0.79, 95% CI = 0.68-0.90, p = 5.59 × 10-4]. Functional analysis revealed a significant down regulation of BMAL1 expression following cMYC overexpression and increasing transformation in ovarian surface epithelial (OSE) cells as well as alternative splicing of BMAL1 exons in ovarian and granulosa cells. These results suggest that variation in circadian genes, and specifically BMAL1, may be associated with risk of ovarian cancer, likely through disruption of hormonal pathways.

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10(-5)). For three cis-eQTL associations (P<1.4 × 10(-3), FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6 × 10(-10) for risk variants (P<10(-4)) within 10 kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC.

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10(-20)), ER-negative BC (P=1.1 × 10(-13)), BRCA1-associated BC (P=7.7 × 10(-16)) and triple negative BC (P-diff=2 × 10(-5)). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10(-3)) and ABHD8 (P<2 × 10(-3)). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3'-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations.

Sclerosing adenosis (SA), found in ¼ of benign breast disease (BBD) biopsies, is a histological feature characterized by lobulocentric proliferation of acini and stromal fibrosis and confers a two-fold increase in breast cancer risk compared to women in the general population. We evaluated a NanoString-based gene expression assay to model breast cancer risk using RNA derived from formalin-fixed, paraffin-embedded (FFPE) biopsies with SA.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX) or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID) mice from Epstein-Barr virus (EBV)-infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab) to 5.6% (n = 160 with rituximab), and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

Copy number variants (CNVs) have been implicated in many complex diseases. We examined whether inherited CNVs were associated with overall survival among women with invasive epithelial ovarian cancer. Germline DNA from 1,056 cases (494 deceased, average of 3.7 years follow-up) was interrogated with the Illumina 610 quad genome-wide array containing, after quality control exclusions, 581,903 single nucleotide polymorphisms (SNPs) and 17,917 CNV probes. Comprehensive analysis capitalized upon the strengths of three complementary approaches to CNV classification. First, to identify small CNVs, single markers were evaluated and, where associated with survival, consecutive markers were combined. Two chromosomal regions were associated with survival using this approach (14q31.3 rs2274736 p = 1.59 × 10(-6), p = 0.001; 22q13.31 rs2285164 p = 4.01 × 10(-5), p = 0.009), but were not significant after multiple testing correction. Second, to identify large CNVs, genome-wide segmentation was conducted to characterize chromosomal gains and losses, and association with survival was evaluated by segment. Four regions were associated with survival (1q21.3 loss p = 0.005, 5p14.1 loss p = 0.004, 9p23 loss p = 0.002, and 15q22.31 gain p = 0.002); however, again, after correcting for multiple testing, no regions were statistically significant, and none were in common with the single marker approach. Finally, to evaluate associations with general amounts of copy number changes across the genome, we estimated CNV burden based on genome-wide numbers of gains and losses; no associations with survival were observed (p > 0.40). Although CNVs that were not well-covered by the Illumina 610 quad array merit investigation, these data suggest no association between inherited CNVs and survival after ovarian cancer.

Obesity has a strong genetic influence, with some variants showing stronger associations among women than men. Women are also more likely to distribute weight in the abdomen following menopause. We investigated whether genetic loci link with obesity-related phenotypes differently by menopausal status.

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

Recent studies have suggested the importance of HLA genes in determining immune responses following rubella vaccine. The telomeric class III region of the HLA complex harbors several genes, including lymphotoxin alpha (LTA), tumor necrosis factor (TNF) and leukocyte specific transcript -1 (LST1) genes, located between the class I B and class II DRB1 loci. Apart from HLA, little is known about the effect of this extended genetic region on HLA haplotypic backgrounds as applied to immune responses.

One difficult question facing researchers is how to prioritize SNPs detected from genetic association studies for functional studies. Often a list of the top M SNPs is determined based on solely the p-value from an association analysis, where M is determined by financial/time constraints. For many studies of complex diseases, multiple analyses have been completed and integrating these multiple sets of results may be difficult. One may also wish to incorporate biological knowledge, such as whether the SNP is in the exon of a gene or a regulatory region, into the selection of markers to follow-up. In this manuscript, we propose a Bayesian latent variable model (BLVM) for incorporating "features" about a SNP to estimate a latent "quality score", with SNPs prioritized based on the posterior probability distribution of the rankings of these quality scores. We illustrate the method using data from an ovarian cancer genome-wide association study (GWAS). In addition to the application of the BLVM to the ovarian GWAS, we applied the BLVM to simulated data which mimics the setting involving the prioritization of markers across multiple GWAS for related diseases/traits. The top ranked SNP by BLVM for the ovarian GWAS, ranked 2(nd) and 7(th) based on p-values from analyses of all invasive and invasive serous cases. The top SNP based on serous case analysis p-value (which ranked 197(th) for invasive case analysis), was ranked 8(th) based on the posterior probability of being in the top 5 markers (0.13). In summary, the application of the BLVM allows for the systematic integration of multiple SNP "features" for the prioritization of loci for fine-mapping or functional studies, taking into account the uncertainty in ranking.

Genome-wide association studies (GWAS) have so far reported 12 loci associated with serous epithelial ovarian cancer (EOC) risk. We hypothesized that some of these loci function through nearby transcription factor (TF) genes and that putative target genes of these TFs as identified by coexpression may also be enriched for additional EOC risk associations.

Percent mammographic density (PD) estimates the proportion of stromal, fat, and epithelial breast tissues on the mammogram image. Adjusted for age and body mass index (BMI), PD is one of the strongest risk factors for breast cancer. Inherited factors are hypothesized to explain between 30 and 60% of the variance in this trait. However, previously identified common genetic variants account for less than 6% of the variance in PD, leaving much of the genetic contribution to this trait unexplained. We performed the first study to examine whether germline copy number variation (CNV) are associated with PD. Two genome-wide association studies (GWAS) of percent density conducted on the Illumina 660W-Quad were used to identify and replicate the association between candidate CNVs and PD: the Minnesota Breast Cancer Family Study (MBCFS) and controls from the Mayo Venous Thromboembolism (Mayo VTE) Case-Control Study, with 585 and 328 women, respectively. Linear models were utilized to examine the association of each probe with PD, adjusted for age, menopausal status and BMI. Segmentation was subsequently performed on the probe-level test statistics to identify candidate CNV regions that were associated with PD.

A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

The mechanisms of immune response are structured within a highly complex regulatory system. Genetic associations with variation in the immune response to rubella vaccine have typically been assessed one locus at a time. We simultaneously assessed the associations between 726 SNPs tagging 84 candidate immune response genes and rubella-specific antibody levels. Blood samples were obtained from 714 school-aged children who had received two doses of MMR vaccine. Associations between rubella-specific antibody levels and 726 candidate tagSNPs were assessed both one SNP at a time and in a variety of multigenic analyses.