Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

Evaluation of vitamin D biosynthesis and pathway target genes reveals UGT2A1/2 and EGFR polymorphisms associated with epithelial ovarian cancer in African American Women.

An association between genetic variants in the vitamin D receptor (VDR) gene and epithelial ovarian cancer (EOC) was previously reported in women of African ancestry (AA). We sought to examine associations between genetic variants in VDR and additional genes from vitamin D biosynthesis and pathway targets (EGFR, UGT1A, UGT2A1/2, UGT2B, CYP3A4/5, CYP2R1, CYP27B1, CYP24A1, CYP11A1, and GC). Genotyping was performed using the custom-designed 533,631 SNP Illumina OncoArray with imputation to the 1,000 Genomes Phase 3 v5 reference set in 755 EOC cases, including 537 high-grade serous (HGSOC), and 1,235 controls. All subjects are of African ancestry (AA). Logistic regression was performed to estimate odds ratios (OR) and 95% confidence intervals (CI). We further evaluated statistical significance of selected SNPs using the Bayesian False Discovery Probability (BFDP). A significant association with EOC was identified in the UGT2A1/2 region for the SNP rs10017134 (per allele OR = 1.4, 95% CI = 1.2-1.7, P = 1.2 × 10-6 , BFDP = 0.02); and an association with HGSOC was identified in the EGFR region for the SNP rs114972508 (per allele OR = 2.3, 95% CI = 1.6-3.4, P = 1.6 × 10-5 , BFDP = 0.29) and in the UGT2A1/2 region again for rs1017134 (per allele OR = 1.4, 95% CI = 1.2-1.7, P = 2.3 × 10-5 , BFDP = 0.23). Genetic variants in the EGFR and UGT2A1/2 may increase susceptibility of EOC in AA women. Future studies to validate these findings are warranted. Alterations in EGFR and UGT2A1/2 could perturb enzyme efficacy, proliferation in ovaries, impact and mark susceptibility to EOC.Includes NIHR and CRUK

Polygenic Risk Modelling for Prediction of Epithelial Ovarian Cancer Risk

Funder: Funding details are provided in the Supplementary MaterialAbstractPolygenic risk scores (PRS) for epithelial ovarian cancer (EOC) have the potential to improve risk stratification. Joint estimation of Single Nucleotide Polymorphism (SNP) effects in models could improve predictive performance over standard approaches of PRS construction. Here, we implemented computationally-efficient, penalized, logistic regression models (lasso, elastic net, stepwise) to individual level genotype data and a Bayesian framework with continuous shrinkage, “select and shrink for summary statistics” (S4), to summary level data for epithelial non-mucinous ovarian cancer risk prediction. We developed the models in a dataset consisting of 23,564 non-mucinous EOC cases and 40,138 controls participating in the Ovarian Cancer Association Consortium (OCAC) and validated the best models in three populations of different ancestries: prospective data from 198,101 women of European ancestry; 7,669 women of East Asian ancestry; 1,072 women of African ancestry, and in 18,915 BRCA1 and 12,337 BRCA2 pathogenic variant carriers of European ancestry. In the external validation data, the model with the strongest association for non-mucinous EOC risk derived from the OCAC model development data was the S4 model (27,240 SNPs) with odds ratios (OR) of 1.38(95%CI:1.28–1.48,AUC:0.588) per unit standard deviation, in women of European ancestry; 1.14(95%CI:1.08–1.19,AUC:0.538) in women of East Asian ancestry; 1.38(95%CI:1.21-1.58,AUC:0.593) in women of African ancestry; hazard ratios of 1.37(95%CI:1.30–1.44,AUC:0.592) in BRCA1 pathogenic variant carriers and 1.51(95%CI:1.36-1.67,AUC:0.624) in BRCA2 pathogenic variant carriers. Incorporation of the S4 PRS in risk prediction models for ovarian cancer may have clinical utility in ovarian cancer prevention programs.</jats:p

Fidelity of human ovarian cancer patient-derived xenografts in a partially humanized mouse model for preclinical testing of immunotherapies

Background Immune checkpoint blockers (ICBs) have been approved by the Food and Drug Administration to be used alone in front-line therapies or in combination with other regimens for certain advanced cancers. Since ICB only works in a subset of patients and has limited efficacy in treating ovarian cancer (OVC), developing preclinical models that help to understand which patients may derive benefit from ICB would be of tremendous benefit in OVC.Methods Here, we generated preclinical human OVC models from freshly resected tumors, which include six patient-derived xenografts (PDXs) from six different patient tumors, three transplantable OVC PD spheroid lines (PD-sphs), and 3 cell lines (PD-CLs). We tested the therapeutic combination of anti-PD1/CTLA4 antibodies with (1) autologous tumor-associated leukocytes (TALs) on the growth of PD-sphs in a coculture system in vitro, (2) with adoptively transferred autologous peripheral blood mononuclear cells or TALs in patient-derived OVC models using partially humanized mice, NSG-HHDxSGM3 (N-HSGM3).Results We show that PD-1 and CTLA-4 dual blockade when combined with autologous TALs effectively reduced PD-sph number in a co-culture system and led to regression of established PD-CLs and PDXs in the N-HSGM3 mice. Combinatorial PD-1 and CTLA-4 blockade increased the frequency and function of tumor-specific CD8 T cells. These CD8 T cells persisted in the tumor microenvironment, exhibited memory phenotype and protected animals from tumor growth on tumor rechallenge. Gene expression analysis of tumors resistant to dual PD1/CTLA4 blockade treatment identified upregulation of antigen processing and presentation pathways and downregulation of extracellular matrix organization genes.Conclusions These findings describe a novel platform for developing patient-derived preclinical tumor models suitable for rationally testing combinatorial ICB in the context of autologous tumor-reactive T cells. This platform can be further developed for testing additional targeted therapies relevant to OVC

Elevated expression of the serine-arginine protein kinase 1 gene in ovarian cancer and its role in Cisplatin cytotoxicity in vitro.

Alternatively spliced variants of several oncogenes and tumor suppressors have been shown to be important for their tumorigenicity. In the present study we have tested whether serine-arginine protein kinase 1 (SRPK1), a major regulator of splicing factors, is involved in ovarian cancer progression and plays a role in chemo-sensitivity. By Western blot analyses, SRPK1 protein was found to be overexpressed in 4 out of 6 ovarian cancer cell lines as compared with an immortalized ovarian surface epithelial cell line; and in 55% of ovarian tumor samples as compared with non-neoplastic ovarian tissue samples. Reduction of SRPK1 expression using small interfering RNA (siRNA) encoding small hairpin RNA in ovarian cancer cells led to (i) reduced cell proliferation rate, slower cell cycle progression and compromised anchorage-independent growth and migration ability in vitro, (ii) decreased level of phosphorylation of multiple serine-arginine proteins, and P44/42MAPK and AKT proteins, and (iii) enhanced sensitivity to cisplatin. Together, these results suggest that elevated SRPK1 expression may play a role in ovarian tumorigenesis and SRPK1 may be a potential target for ovarian cancer therapy

An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer

Abstract Background Pancreatic cancer is a deadly disease with a very low 5-year patient survival rate of 6–8%. The major challenges of eliminating pancreatic cancer are treatment resistance and stromal barriers to optimal drug access within the tumor. Therefore, effective molecular targeting drugs with high intra-tumor access and retention are urgently needed for managing this devastating disease in the clinic. Methods This study has used the following in vitro and in vivo techniques for the investigation of exceptional anticancer drug FL118’s efficacy in treatment of resistant pancreatic cancer: cell culture; immunoblotting analysis to test protein expression; DNA sub-G1 flow cytometry analyses to test cell death; MTT assay to test cell viability; pancreatic cancer stem cell assays (fluorescence microscopy tracing; matrigel assay; CD44-positive cell colony formation assay); human luciferase-labeled pancreatic tumor orthotopic animal model in vivo imaging; pancreatic cancer patient-derived xenograft (PDX) animal models; and toxicology studies with immune-competent BALB/cj mice and beagle dogs. Results Our studies found that FL118 alone preferentially killed cisplatin-resistant cancer cells, while a combination of FL118 with cisplatin synergistically killed resistant pancreatic cancer cells and reduced spheroid formation of treatment-resistant pancreatic cancer stem-like cells. Furthermore, using in vivo-imaging, we found that FL118 in combination with cisplatin strongly inhibited both drug-resistant pancreatic xenograft tumor growth and metastasis. In PDX model, we demonstrated that FL118 alone effectively eliminated PDX tumors, while FL118 in combination with gemcitabine eliminated PDX tumors that showed relative resistance (less sensitivity) to treatment with FL118. These FL118 efficacy results are consistent with our molecular-targeting data showing that FL118 inhibited the expression of multiple antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and ERCC6, a critical regulator of DNA repair, in treatment-resistant pancreatic stem-like cancer cells. Furthermore, FL118 toxicity studies in BALB/cj mice and beagle dogs indicated that FL118 exhibits favorable hematopoietic and biochemical toxicities. Conclusion Together, our studies suggest that FL118 is a promising anticancer drug for further clinical development to effectively treat drug-resistant pancreatic cancer alone or in combination with other pancreatic cancer chemotherapeutic drugs

SRPK1 is overexpressed in 55% of ovarian tumor samples.

<p>(A) Immunohistochemical staining for SRPK1 protein expression in normal and tumor ovary tissue sections. The cells staining positively for SRPK1 antibody are brown. For negative control, the host-IgG was used (not shown). E, epithelium; S, stroma. (B) Western blot analysis of SRPK1 protein in 21 ovarian tumor samples (T), and 9 non-neoplastic ovarian tissue samples (N). The normal and tumor samples in the lowest panel were cropped from two different blots for the purpose of presentation. The lower bands in the blot probed with anti-SRPK1 antibody are probably proteolytic fragments of SRPK1. (C) Box-Whisker plot with SRPK1/actin fold expression derived from densitometric analysis of the bands in Western blot analysis. Whiskers encompass all the tumors (n = 47) or the normal samples (n = 9), boxes contain 50% of data, and the center lines in the boxes show medians. Mean expression was compared by Student’s <i>t</i>-test (p = 0.03).</p

Phosphorylation patterns of SR-proteins and MAPK42/44 and AKT proteins are altered in SRPK1-knockdown SKOV3 cells.

<p>Western blot analysis was performed on lysates prepared from SKOV3-derived pSM2-EV cells and two stable SRPK1-knockdown clones. Antibodies were used to detect the phosphorylated SR proteins (mAB104), MAPK42/44 (Thr²⁰²/Tyr²⁰⁴), and AKT (Ser^473/Thr³⁰⁸) as well as total protein levels of MAPK42/44, and AKT.</p

Effects of SRPK1-knockdown on cell cycle progression in SKOV3 cells.

<p>(A) Cell cycle progression from G2/M-arrest. Cells at 80% confluence were arrested in G2/M phases with nocodazole (NOC, 600 ng/ml) for 12 hr, washed with phosphate buffered saline (PBS), and released into medium without drug. Cells were collected at the indicated times and subjected to propidium iodine staining and flow cytometric analysis. Representative data obtained from two independent experiments are shown. (B) Cell cycle progression from G0/G1-arrest. Cells at 100% confluence were incubated in medium without serum. Quiescent cells were activated with 10% FBS-containing medium. Cells were collected every 3 hr for flow cytometric analysis as described above. The percentage of cells in G1, S, and G2/M was determined using Modfit program and a histogram graph was generated using the WinList program (Verity Software House). Representative data obtained from two independent experiments are shown.</p