Oberg, Ann L.

Abstract Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited oncogenesis. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

Abstract Background: Critically shortened telomeres contribute to chromosomal instability and neoplastic transformation and are associated with early death of patients with certain cancer types. Shorter leukocyte telomere length (LTL) has been associated with higher risk for pancreatic ductal adenocarcinoma (PDAC) and might be associated also with survival of patients with PDAC. We investigated the association between treatment-naïve LTL and overall survival of patients with incident PDAC. Methods: The study included 642 consecutively enrolled PDAC patients in the Mayo Clinic Biospecimen Resource for Pancreas Research. Blood samples were obtained at the time of diagnosis, before the start of cancer treatment, from which LTL was assayed by qRT-PCR. LTL was first modeled as a continuous variable (per-interquartile range decrease in LTL) and then as a categorized variable (short, medium, long). Multivariable-adjusted HRs and 95% confidence intervals (CI) were calculated for overall mortality using Cox proportional hazard models. Results: Shorter treatment-naïve LTL was associated with higher mortality among patients with PDAC (HRcontinuous = 1.13, 95% CI: 1.01–1.28, P = 0.03; HRshortest vs. longest LTL = 1.29, 95% CI: 1.05–1.59, Ptrend = 0.01). There was a difference in the association between LTL and overall mortality by tumor stage at diagnosis; resectable tumors (HRcontinuous = 0.91; 95% CI: 0.73–1.12), locally advanced tumors (HRcontinuous = 1.29; 95% CI: 1.07–1.56), and metastatic tumors (HRcontinuous = 1.17; 95% CI: 0.96–1.42), Pinteraction = 0.04. Conclusion: Shorter treatment-naïve LTL is associated with poorer overall survival of patients with incident PDAC. Impact: Peripheral blood LTL might be a prognostic marker for PDAC.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed too late for effective therapy. The classic strategy for early detection biomarker advancement consists of initial retrospective phases of discovery and validation with tissue samples taken from individuals diagnosed with disease, compared with controls. Using this approach, we previously reported the discovery of a blood biomarker panel consisting of thrombospondin-2 (THBS2) and CA19-9 that together could discriminate resectable stage I and IIa PDAC as well as stages III and IV PDAC, with c-statistic values in the range of 0.96 to 0.97 in two phase II studies. We now report that in two studies of blood samples prospectively collected from 1 to 15 years prior to a PDAC diagnosis (Mayo Clinic and PLCO cohorts), THBS2 and/or CA19-9 failed to discriminate cases from healthy controls at the AUC = 0.8 needed. We conclude that PDAC progression may be heterogeneous and for some individuals can be more rapid than generally appreciated. It is important that PDAC early-detection studies incorporate high-risk, prospective prediagnostic cohorts into discovery and validation studies. Prevention Relevance: A blood biomarker panel of THBS2 and CA19-9 detects early stages of pancreatic ductal adenocarcinoma at diagnosis, but not when tested across a population up to 1 year earlier. Our findings suggest serial sampling over time, using prospectively collected samples for biomarker discovery, and more frequent screening of high-risk individuals.

Abstract Background: Whether circulating polyunsaturated fatty acid (PUFA) levels are associated with pancreatic cancer risk is uncertain. Mendelian randomization (MR) represents a study design using genetic instruments to better characterize the relationship between exposure and outcome. Methods: We utilized data from genome-wide association studies within the Pancreatic Cancer Cohort Consortium and Pancreatic Cancer Case–Control Consortium, involving approximately 9,269 cases and 12,530 controls of European descent, to evaluate associations between pancreatic cancer risk and genetically predicted plasma n-6 PUFA levels. Conventional MR analyses were performed using individual-level and summary-level data. Results: Using genetic instruments, we did not find evidence of associations between genetically predicted plasma n-6 PUFA levels and pancreatic cancer risk [estimates per one SD increase in each PUFA-specific weighted genetic score using summary statistics: linoleic acid odds ratio (OR) = 1.00, 95% confidence interval (CI) = 0.98–1.02; arachidonic acid OR = 1.00, 95% CI = 0.99–1.01; and dihomo-gamma-linolenic acid OR = 0.95, 95% CI = 0.87–1.02]. The OR estimates remained virtually unchanged after adjustment for covariates, using individual-level data or summary statistics, or stratification by age and sex. Conclusions: Our results suggest that variations of genetically determined plasma n-6 PUFA levels are not associated with pancreatic cancer risk. Impact: These results suggest that modifying n-6 PUFA levels through food sources or supplementation may not influence risk of pancreatic cancer.

Abstract About 50% of high-grade serous ovarian cancers (HGSOC) have defects in homologous recombination (HR) DNA repair. Intriguingly, HR-defective tumors are highly susceptible to poly(ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy. Mutations in BRCA1/2 or epigenetic silencing of BRCA1 via promoter hypermethylation contributes to ~30% of the HR defects, but other genetic alterations that cause HR deficiency in HGSOC remain unclear. To identify novel genes that may regulate HR, we studied Zinc Finger CCCH-Type Containing 18 (ZC3H18), encoded by ZC3H18. Notably, ZC3H18 lies within 16q24.2, a region with frequent copy number loss in HGSOC. Here we demonstrate that BRCA1 is transcriptionally activated by a previously unknown function of ZC3H18. We show that ZC3H18 interacts with an E2F site in the BRCA1 promoter where it facilitates recruitment of E2F4 to an adjacent E2F site to promote BRCA1 transcription. Consistent with the role of ZC3H18 in promoting BRCA1 expression, ZC3H18 depletion induces BRCA1 promoter methylation, reduces BRCA1 expression, disrupts HR, and sensitizes ovarian cancer cells to PARP inhibitors and platinum agents. Moreover, in patient-derived xenografts and primary HGSOC tumors, ZC3H18 mRNA levels were positively correlated with BRCA1 mRNA levels, further supporting the role of ZC3H18 in regulating BRCA1. Together, these findings suggest that loss and/or reduced ZC3H18 expression may help identify HGSOC patients most likely to benefit from PARP inhibitor and platinum-based therapies. Summary of the Unpublished Data/Significance/Innovation: These studies are innovative on several fronts. First, although BRCA1 methylation is a frequent event that mediates tumorigenesis and therapeutic responses in HGSOC, how BRCA1 methylation is regulated remains unknown. These studies will be the first to provide a mechanistic basis for how BRCA1 is methylated in HGSOC. Second, our studies are the first to show a role for ZC3H18 in any cancer. Third, we show that ZC3H18 regulates HR and that its depletion sensitizes ovarian cancer cells to PARP inhibitors and platinum therapy. Thus, these studies will provide important basis for developing ZC3H18 loss/decreased abundance as a novel biomarker for ovarian cancers that may respond to PARP inhibitors and platinum agents. Citation Format: Arun Kanakkanthara, Catherine J. Huntoon, Xiaonan Hou, Minzhi Zhang, Ethan P Heinzen, Ann L. Oberg, John S. Weroha, Scott H. Kaufmann, Larry M. Karnitz. Loss of ZC3H18 disrupts homologous recombination repair and sensitizes ovarian cancer cells to PARP inhibitors and DNA cross-linking agents [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A33.

Blood is a readily accessible biofluid containing a plethora of important proteins, nucleic acids, and metabolites that can be used as clinical diagnostic tools in diseases, including cancer. Like the on-going efforts for cancer biomarker discovery using the liquid biopsy detection of circulating cell-free and cell-based tumor nucleic acids, the circulatory proteome has been underexplored for clinical cancer biomarker applications. A comprehensive proteome analysis of human serum/plasma with high-quality data and compelling interpretation can potentially provide opportunities for understanding disease mechanisms, although several challenges will have to be met. Serum/plasma proteome biomarkers are present in very low abundance, and there is high complexity involved due to the heterogeneity of cancers, for which there is a compelling need to develop sensitive and specific proteomic technologies and analytical platforms. To date, liquid chromatography mass spectrometry (LC-MS)-based quantitative proteomics has been a dominant analytical workflow to discover new potential cancer biomarkers in serum/plasma. This review will summarize the opportunities of serum proteomics for clinical applications; the challenges in the discovery of novel biomarkers in serum/plasma; and current proteomic strategies in cancer research for the application of serum/plasma proteomics for clinical prognostic, predictive, and diagnostic applications, as well as for monitoring minimal residual disease after treatments. We will highlight some of the recent advances in MS-based proteomics technologies with appropriate sample collection, processing uniformity, study design, and data analysis, focusing on how these integrated workflows can identify novel potential cancer biomarkers for clinical applications.

Abstract Registry-based epidemiologic studies suggest associations between chronic inflammatory intestinal diseases and pancreatic ductal adenocarcinoma (PDAC). As genetic susceptibility contributes to a large proportion of chronic inflammatory intestinal diseases, we hypothesize that the genomic regions surrounding established genome-wide associated variants for these chronic inflammatory diseases are associated with PDAC. We examined the association between PDAC and genomic regions (±500 kb) surrounding established common susceptibility variants for ulcerative colitis, Crohn's disease, inflammatory bowel disease, celiac disease, chronic pancreatitis, and primary sclerosing cholangitis. We analyzed summary statistics from genome-wide association studies data for 8,384 cases and 11,955 controls of European descent from two large consortium studies using the summary data-based adaptive rank truncated product method to examine the overall association of combined genomic regions for each inflammatory disease group. Combined genomic susceptibility regions for ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis were associated with PDAC at P values < 0.05 (0.0040, 0.0057, 0.011, and 3.4 × 10−6, respectively). After excluding the 20 PDAC susceptibility regions (±500 kb) previously identified by GWAS, the genomic regions for ulcerative colitis, Crohn disease, and inflammatory bowel disease remained associated with PDAC (P = 0.0029, 0.0057, and 0.0098, respectively). Genomic regions for celiac disease (P = 0.22) and primary sclerosing cholangitis (P = 0.078) were not associated with PDAC. Our results support the hypothesis that genomic regions surrounding variants associated with inflammatory intestinal diseases, particularly, ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis are associated with PDAC. Significance: The joint effects of common variants in genomic regions containing susceptibility loci for inflammatory bowel disease and chronic pancreatitis are associated with PDAC and may provide insights to understanding pancreatic cancer etiology.

Abstract Background: Obesity and diabetes are major modifiable risk factors for pancreatic cancer. Interactions between genetic variants and diabetes/obesity have not previously been comprehensively investigated in pancreatic cancer at the genome-wide level. Methods: We conducted a gene–environment interaction (GxE) analysis including 8,255 cases and 11,900 controls from four pancreatic cancer genome-wide association study (GWAS) datasets (Pancreatic Cancer Cohort Consortium I–III and Pancreatic Cancer Case Control Consortium). Obesity (body mass index ≥30 kg/m2) and diabetes (duration ≥3 years) were the environmental variables of interest. Approximately 870,000 SNPs (minor allele frequency ≥0.005, genotyped in at least one dataset) were analyzed. Case–control (CC), case-only (CO), and joint-effect test methods were used for SNP-level GxE analysis. As a complementary approach, gene-based GxE analysis was also performed. Age, sex, study site, and principal components accounting for population substructure were included as covariates. Meta-analysis was applied to combine individual GWAS summary statistics. Results: No genome-wide significant interactions (departures from a log-additive odds model) with diabetes or obesity were detected at the SNP level by the CC or CO approaches. The joint-effect test detected numerous genome-wide significant GxE signals in the GWAS main effects top hit regions, but the significance diminished after adjusting for the GWAS top hits. In the gene-based analysis, a significant interaction of diabetes with variants in the FAM63A (family with sequence similarity 63 member A) gene (significance threshold P < 1.25 × 10−6) was observed in the meta-analysis (PGxE = 1.2 ×10−6, PJoint = 4.2 ×10−7). Conclusions: This analysis did not find significant GxE interactions at the SNP level but found one significant interaction with diabetes at the gene level. A larger sample size might unveil additional genetic factors via GxE scans. Impact: This study may contribute to discovering the mechanism of diabetes-associated pancreatic cancer.

Abstract Background: Leukocyte telomere length (LTL) has been associated with risk of multiple cancers, but its association with pancreatic ductal adenocarcinoma (PDAC) is unclear. We therefore investigated the association between peripheral blood LTL and PDAC risk, and examined effect modification by candidate SNPs previously reported to be associated with variation in LTL. Methods: A case–control study of 1,460 PDAC cases and 1,459 frequency-matched controls was performed using biospecimens and data from the Mayo Clinic Biospecimen Resource for Pancreas Research. Quantitative PCR was used to measure LTL and categorized into tertiles based on sex-specific control distribution. Eleven telomere-related SNPs also were genotyped. Logistic regression was used to calculate ORs and 95% confidence intervals (CI). Results: Shorter peripheral blood LTL was associated with a higher risk of PDAC (ORT1vsT3 = 1.26, 95% CI = 1.03–1.54, Ptrend = 0.02; ORcontinuous = 1.14, 95% CI = 1.02–1.28), but the association was restricted to cases with treatment-naïve blood samples (ORT1vsT3 = 1.51, 95% CI = 1.16–1.96, Ptrend = 0.002; ORcontinuous = 1.25, 95% CI = 1.08–1.45) and not cases whose blood samples were collected after initiation of cancer therapy (ORT1vsT3 = 1.10, 95% CI = 0.87–1.39, Ptrend = 0.42; ORcontinuous = 1.08, 95% CI = 0.94–1.23). Three SNPs (TERC-rs10936599, ACYP2-rs11125529, and TERC-rs1317082) were each associated with interindividual variation in LTL among controls, but there was no evidence of effect modification by these SNPs. Conclusions: Treatment-naïve short LTL is associated with a higher risk of PDAC, and the association does not differ by germline variation in the candidate telomere-related SNPs examined. Impact: Peripheral blood LTL might serve as a molecular marker for risk modeling to identify persons at high risk of PDAC.

5546 Background: CHFR, an E3 ubiquitin ligase that regulates Aurora A and Pololike Kinase 1, plays a critical role in the cellular response to mitotic stress. In particular, cells containing CHFR arrest in G2 when microtubule dynamics are disrupted. Conversely, low CHFR expression is associated with hypersensitivity to mitotic spindle poisons in breast cancer cell lines. Despite the extensive use of taxanes in front line treatment of epithelial ovarian cancer (EOC), little is known about CHFR expression in this disease. We examined CHFR expression and its relationship with clinical characteristics and outcome in women with EOC. Methods: Pretreatment EOC samples from women enrolled in the Mayo Clinic Biospecimen Resource for Ovarian Cancer Research who underwent initial surgical debulking between 1999-2009 were stained in triplicate with anti-CHFR antibodies. Samples were scored in a blinded fashion for CHFR expression. Associations between CHFR staining and histology, grade, and stage were assessed using chi-squared tests; associations between CHFR and overall survival (OS) or time to disease recurrence (TTR) were assessed using Cox proportional hazards models. Results: Samples from 354 women were stained; median age at diagnosis was 60.5 (range 21-93). At a median follow-up of 67 months, 221 women (62%) had died. Across all patients, moderate/strong CHFR staining was associated with poor OS (HR = 1.39, 95% CI: 1.07-1.81, p = 0.015). Moreover, moderate/strong CHFR expression was strongly associated with serous histology (p < 0.0001), high grade (p < 0.0001) and advanced stage (p = 0.0002). To assess whether the association between CHFR expression and OS was related to taxane sensitivity, a subgroup analysis was performed in the subset of patients (N=131) with high-grade serous EOC who received initial platinum/taxane chemotherapy. Moderate/strong CHFR expression failed to correlate with OS (HR=0.91, 95% CI: 0.57-1.45, p = 0.68) or TTR from the start of chemotherapy (HR = 0.85, 95% CI: 0.55-1.31, p = 0.55) in this subset. Conclusions: CHFR expression is associated with poor OS and adverse clinical characteristics in patients with EOC but does not appear to be related to taxane sensitivity in high-grade serous tumors.