A Novel 3DNA® Nanocarrier effectively delivers payloads to pancreatic tumors.

INTRODUCTION: Standard-of-care systemic chemotherapies for pancreatic ductal adenocarcinoma (PDAC) currently have limited clinical benefits, in addition to causing adverse side effects in many patients. One factor known to contribute to the poor chemotherapy response is the poor drug diffusion into PDAC tumors. Novel treatment methods are therefore drastically needed to improve targeted delivery of treatments. Here, we evaluated the efficacy of the 3DNA® Nanocarrier (3DNA) platform to direct delivery of therapeutics to PDAC tumors in vivo. MATERIALS AND METHODS: A panel of PDAC cell lines and a patient tissue microarray were screened for established tumor-specific proteins to identify targeting moieties for active targeting of the 3DNA. NRG mice with or without orthotopic MIA PaCa-2-luciferase PDAC tumors were treated intraperitoneally with 100 µl of fluorescently labeled 3DNA. RESULTS: Folic acid and transferrin receptors were significantly elevated in PDAC compared to normal pancreas. Accordingly, both folic acid- and transferrin-conjugated 3DNA treatments significantly increased delivery of 3DNA specifically to tumors in comparison to unconjugated 3DNA treatment. In the absence of tumors, there was an increased clearance of both folic acid-conjugated 3DNA and unconjugated 3DNA, compared to the clearance rate in tumor-bearing mice. Lastly, delivery of siLuciferase by folic acid-conjugated 3DNA in an orthotopic model of luciferase-expressing PDAC showed significant and prolonged suppression of luciferase protein expression and activity. CONCLUSION: Our study progresses the 3DNA technology as a reliable and effective treatment delivery platform for targeted therapeutic approaches in PDAC.

The RNA-Binding Protein HuR Posttranscriptionally Regulates the Protumorigenic Activator YAP1 in Pancreatic Ductal Adenocarcinoma.

Yes-associated protein 1 (YAP1) is indispensable for the development of mutant KRAS-driven pancreatic ductal adenocarcinoma (PDAC). High YAP1 mRNA is a prognostic marker for worse overall survival in patient samples; however, the regulatory mechanisms that mediate its overexpression are not well understood. YAP1 genetic alterations are rare in PDAC, suggesting that its dysregulation is likely not due to genetic events. HuR is an RNA-binding protein whose inhibition impacts many cancer-associated pathways, including the "conserved YAP1 signature" as demonstrated by gene set enrichment analysis. Screening publicly available and internal ribonucleoprotein immunoprecipitation (RNP-IP) RNA sequencing (RNA-Seq) data sets, we discovered that YAP1 is a high-confidence target, which was validated in vitro with independent RNP-IPs and 3' untranslated region (UTR) binding assays. In accordance with our RNA sequencing analysis, transient inhibition (e.g., small interfering RNA [siRNA] and small-molecular inhibition) and CRISPR knockout of HuR significantly reduced expression of YAP1 and its transcriptional targets. We used these data to develop a HuR activity signature (HAS), in which high expression predicts significantly worse overall and disease-free survival in patient samples. Importantly, the signature strongly correlates with YAP1 mRNA expression. These findings highlight a novel mechanism of YAP1 regulation, which may explain how tumor cells maintain YAP1 mRNA expression at dynamic times during pancreatic tumorigenesis.

HuR Plays a Role in Double-Strand Break Repair in Pancreatic Cancer Cells and Regulates Functional BRCA1-Associated-Ring-Domain-1(BARD1) Isoforms.

Human Antigen R (HuR/ELAVL1) is known to regulate stability of mRNAs involved in pancreatic ductal adenocarcinoma (PDAC) cell survival. Although several HuR targets are established, it is likely that many remain currently unknown. Here, we identified BARD1 mRNA as a novel target of HuR. Silencing HuR caused a >70% decrease in homologous recombination repair (HRR) efficiency as measured by the double-strand break repair (pDR-GFP reporter) assay. HuR-bound mRNAs extracted from RNP-immunoprecipitation and probed on a microarray, revealed a subset of HRR genes as putative HuR targets, including the BRCA1-Associated-Ring-Domain-1 (BARD1) (p < 0.005). BARD1 genetic alterations are infrequent in PDAC, and its context-dependent upregulation is poorly understood. Genetic silencing (siRNA and CRISPR knock-out) and pharmacological targeting of HuR inhibited both full length (FL) BARD1 and its functional isoforms (α, δ, Φ). Silencing BARD1 sensitized cells to olaparib and oxaliplatin; caused G2-M cell cycle arrest; and increased DNA-damage while decreasing HRR efficiency in cells. Exogenous overexpression of BARD1 in HuR-deficient cells partially rescued the HRR dysfunction, independent of an HuR pro-oncogenic function. Collectively, our findings demonstrate for the first time that BARD1 is a bona fide HuR target, which serves as an important regulatory point of the transient DNA-repair response in PDAC cells.

The FDA-Approved Anthelmintic Pyrvinium Pamoate Inhibits Pancreatic Cancer Cells in Nutrient-Depleted Conditions by Targeting the Mitochondria.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal aggressive cancer, in part due to elements of the microenvironment (hypoxia, hypoglycemia) that cause metabolic network alterations. The FDA-approved antihelminthic pyrvinium pamoate (PP) has previously been shown to cause PDAC cell death, although the mechanism has not been fully determined. We demonstrated that PP effectively inhibited PDAC cell viability with nanomolar IC50 values (9-93 nmol/L) against a panel of PDAC, patient-derived, and murine organoid cell lines. In vivo, we demonstrated that PP inhibited PDAC xenograft tumor growth with both intraperitoneal (IP; P < 0.0001) and oral administration (PO; P = 0.0023) of human-grade drug. Metabolomic and phosphoproteomic data identified that PP potently inhibited PDAC mitochondrial pathways including oxidative phosphorylation and fatty acid metabolism. As PP treatment reduced oxidative phosphorylation (P < 0.001), leading to an increase in glycolysis (P < 0.001), PP was 16.2-fold more effective in hypoglycemic conditions similar to those seen in PDAC tumors. RNA sequencing demonstrated that PP caused a decrease in mitochondrial RNA expression, an effect that was not observed with established mitochondrial inhibitors rotenone and oligomycin. Mechanistically, we determined that PP selectively bound mitochondrial G-quadruplexes and inhibited mitochondrial RNA transcription in a G-quadruplex-dependent manner. This subsequently led to a 90% reduction in mitochondrial encoded gene expression. We are preparing to evaluate the efficacy of PP in PDAC in an IRB-approved window-of-opportunity trial (IND:144822).

Effect of Hypercapnia, an Element of Obstructive Respiratory Disorder, on Pancreatic Cancer Chemoresistance and Progression.

BACKGROUND: Chronic obstructive respiratory disorders (ORDs) are linked to increased rates of cancer-related deaths. Little is known about the effects of hypercapnia (elevated CO2) on development of pancreatic ductal adenocarcinoma (PDAC) and drug resistance. STUDY DESIGN: Two PDAC cell lines were exposed to normocapnic (5% CO2) and hypercapnic (continuous/intermittent 10% CO2) conditions, physiologically similar to patients with active ORD. Cells were assessed for proliferation rate, colony formation, and chemo-/radiotherapeutic efficacy. In a retrospective clinical study design, patients with PDAC who had undergone pancreatic resection between 2002 and 2014 were reviewed. Active smokers were excluded to remove possible smoking-related protumorigenic influence. Clinical data, pathologic findings, and survival end points were recorded. Kaplan-Meier and Cox regression analyses were performed. RESULTS: Exposure to hypercapnia resulted in increased colony formation and proliferation rates in vitro in both cell lines (MIA-PaCa-2: 111% increase and Panc-1: 114% increase; p < 0.05). Hypercapnia exposure induced a 2.5-fold increase in oxaliplatin resistance (p < 0.05) in both cell lines and increased resistance to ionizing radiation in MIA-PaCa-2 cells (p < 0.05). Five hundred and seventy-eight patients were included (52% were male, median age was 68.7 years [interquartile range 60.6 to 76.8 years]). Cox regression analysis, assessing TNM staging, age, sex, and ORD status, identified ORD as an independent risk factor for both overall survival (hazard ratio 1.64; 95% CI, 1.2 to 2.3; p < 0.05) and disease-free survival (hazard ratio 1.68; 95% CI, 1.06 to 2.67). CONCLUSIONS: PDAC cells exposed to hypercapnic environments, which is common in patients with ORD, showed tumor proliferation, radioresistance, and chemoresistance. Patients with a history of ORD had a worse overall prognosis, suggesting that hypercapnic conditions play a role in the development and progression of PDAC and stressing the need for patient-tailored care.

Abemaciclib Is Effective Against Pancreatic Cancer Cells and Synergizes with HuR and YAP1 Inhibition.

Mutation or promoter hypermethylation of CDKN2A is found in over 90% of pancreatic ductal adenocarcinomas (PDAC) and leads to loss of function of cell-cycle inhibitors p16 (INK4A) and p14 (ARF) resulting in unchecked proliferation. The CDK4/6 inhibitor, abemaciclib, has nanomolar IC50s in PDAC cell lines and decreases growth through inhibition of phospho-Rb (pRb), G1 cell-cycle arrest, apoptosis, and the senescent phenotype detected with ß-galactosidase staining and relevant mRNA elevations. Daily abemaciclib treatments in mouse PDAC xenograft studies were safe and demonstrated a 3.2-fold decrease in tumor volume compared with no treatment (P < 0.0001) accompanying a decrease in both pRb and Ki67. We determined that inhibitors of HuR (ELAVL1), a prosurvival mRNA stability factor that regulates cyclin D1, and an inhibitor of Yes-Associated Protein 1 (YAP1), a pro-oncogenic, transcriptional coactivator important for CDK6 and cyclin D1, were both synergistic with abemaciclib. Accordingly, siRNA oligonucleotides targeted against HuR, YAP1, and their common target cyclin D1, validated the synergy studies. In addition, we have seen increased sensitivity to abemaciclib in a PDAC cell line that harbors a loss of the ELAVL1 gene via CRISP-Cas9 technology. As an in vitro model for resistance, we investigated the effects of long-term abemaciclib exposure. PDAC cells chronically cultured with abemaciclib displayed a reduction in cellular growth rates (GR) and coresistance to gemcitabine and 5-fluorouracil (5-FU), but not to HuR or YAP1 inhibitors as compared with no treatment controls. We believe that our data provide compelling preclinical evidence for an abemaciclib combination-based clinical trial in patients with PDAC. IMPLICATIONS: Our data suggest that abemaciclib may be therapeutically relevant for the treatment in PDAC, especially as part of a combination regimen inhibiting YAP1 or HuR.

Evaluation of Post-transcriptional Gene Regulation in Pancreatic Cancer Cells: Studying RNA Binding Proteins and Their mRNA Targets.

Post-transcriptional regulation of gene expression through interaction between RNA binding proteins (RBPs) and target mRNAs have gained considerable interest over the last decade. Altered expression of RBPs as detected in pancreatic ductal adenocarcinoma (PDAC) cells alters mRNA processing, and in turn, the entire transcriptome and proteome. Thus, this gene regulatory mechanism can regulate important pro-oncogenic signaling pathways (e.g., TP53, WEE1, and c-MYC) in PDAC cells. Ribonucleoprotein immunoprecipitation assays (RNP-IP or RIP) are a modified immunoprecipitation method to study physical interactions between RBPs and their mRNA targets. As a first step to explore RBP interactomes and define novel therapeutic targets and dysregulated pathways in disease, RIPs are a sensitive and established molecular biology technique used to isolate and differentiate bound transcripts to RBPs in a variety of experimental conditions. This chapter describes an up-to-date, detailed protocol for performing this assay in mammalian cytoplasmic extracts (i.e., PDAC cells), and reviews current methods to validate target binding sites such as electrophoretic mobility shift assay (EMSA) and cross-linking immunoprecipitation polymerase chain reaction (CLIP-PCR).

Elevated HuR in Pancreas Promotes a Pancreatitis-Like Inflammatory Microenvironment That Facilitates Tumor Development.

Human antigen R (ELAVL1; HuR) is perhaps the best-characterized RNA-binding protein. Through its overexpression in various tumor types, HuR promotes posttranscriptional regulation of target genes in multiple core signaling pathways associated with tumor progression. The role of HuR overexpression in pancreatic tumorigenesis is unknown and led us to explore the consequences of HuR overexpression using a novel transgenic mouse model that has a >2-fold elevation of pancreatic HuR expression. Histologically, HuR-overexpressing pancreas displays a fibroinflammatory response and other pathological features characteristic of chronic pancreatitis. This pathology is reflected in changes in the pancreatic gene expression profile due, in part, to genes whose expression changes as a consequence of direct binding of their respective mRNAs to HuR. Older mice develop pancreatic steatosis and severe glucose intolerance. Elevated HuR cooperated with mutant K-rasG12D to result in a 3.4-fold increase in pancreatic ductal adenocarcinoma (PDAC) incidence compared to PDAC presence in K-rasG12D alone. These findings implicate HuR as a facilitator of pancreatic tumorigenesis, especially in the setting of inflammation, and a novel therapeutic target for pancreatitis treatment.