Deletion of the mRNA stability factor ELAVL1 (HuR) in pancreatic cancer cells disrupts the tumor microenvironment integrity.

Stromal cells promote extensive fibrosis in pancreatic ductal adenocarcinoma (PDAC), which is associated with poor prognosis and therapeutic resistance. We report here for the first time that loss of the RNA-binding protein human antigen R (HuR, ELAVL1) in PDAC cells leads to reprogramming of the tumor microenvironment. In multiple in vivo models, CRISPR deletion of ELAVL1 in PDAC cells resulted in a decrease of collagen deposition, accompanied by a decrease of stromal markers (i.e. podoplanin, α-smooth muscle actin, desmin). RNA-sequencing data showed that HuR plays a role in cell-cell communication. Accordingly, cytokine arrays identified that HuR regulates the secretion of signaling molecules involved in stromal activation and extracellular matrix organization [i.e. platelet-derived growth factor AA (PDGFAA) and pentraxin 3]. Ribonucleoprotein immunoprecipitation analysis and transcription inhibition studies validated PDGFA mRNA as a novel HuR target. These data suggest that tumor-intrinsic HuR supports extrinsic activation of the stroma to produce collagen and desmoplasia through regulating signaling molecules (e.g. PDGFAA). HuR-deficient PDAC in vivo tumors with an altered tumor microenvironment are more sensitive to the standard of care gemcitabine, as compared to HuR-proficient tumors. Taken together, we identified a novel role of tumor-intrinsic HuR in its ability to modify the surrounding tumor microenvironment and regulate PDGFAA.

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.

Igs as Substrates for Transglutaminase 2: Implications for Autoantibody Production in Celiac Disease.

Autoantibodies specific for the enzyme transglutaminase 2 (TG2) are a hallmark of the gluten-sensitive enteropathy celiac disease. Production of the Abs is strictly dependent on exposure to dietary gluten proteins, thus raising the question how a foreign Ag (gluten) can induce an autoimmune response. It has been suggested that TG2-reactive B cells are activated by gluten-reactive T cells following receptor-mediated uptake of TG2-gluten complexes. In this study, we propose a revised model that is based on the ability of the BCR to serve as a substrate to TG2 and become cross-linked to gluten-derived peptides. We show that TG2-specific IgD molecules are preferred in the reaction and that binding of TG2 via a common epitope targeted by cells using the IgH variable gene segment (IGHV)5-51 results in more efficient cross-linking. Based on these findings we hypothesize that IgD-expressing B cells using IGHV5-51 are preferentially activated, and we suggest that this property can explain the previously reported low number of somatic mutations as well as the overrepresentation of IGHV5-51 among TG2-specific plasma cells in the celiac lesion. The model also couples gluten peptide uptake by TG2-reactive B cells directly to peptide deamidation, which is necessary for the activation of gluten-reactive T cells. It thereby provides a link between gluten deamidation, T cell activation, and the production of TG2-specific Abs. These are all key events in the development of celiac disease, and by connecting them the model may explain why the same enzyme that catalyzes gluten deamidation is also an autoantigen, something that is hardly coincidental.

Long-lived plasma cells from human small intestine biopsies secrete immunoglobulins for many weeks in vitro.

To understand the biology of Ab-secreting cells in the human small intestine, we examined Ab production of intestinal biopsies kept in culture. We found sustained IgA and IgM secretion as well as viable IgA- or IgM-secreting cells after >4 wk of culture. The Ab-secreting cells were nonproliferating and expressing CD27 and CD138, thus having a typical plasma cell phenotype. Culturing of biopsies without tissue disruption gave the highest Ab production and plasma cell survival suggesting that the environment regulates plasma cell longevity. Cytokine profiling of the biopsy cultures demonstrated a sustained presence of IL-6 and APRIL. Blocking of the activity of endogenous APRIL and IL-6 with BCMA-Fc and anti-human IL-6 Ab demonstrated that both these factors were essential for plasma cell survival and Ab secretion in the biopsy cultures. This study demonstrates that the human small intestine harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.

Anti-idiotypic response in mice expressing human autoantibodies.

Celiac disease is an autoimmune illness characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals. The immune response is based on both cellular and humoral components, although the former seem to be more important in the pathogenesis. The autoantibody response is directed at the enzyme tissue transglutaminase, tTG or TG2, which possibly play a role in the onset of the disease. In this study we sought to develop an animal model in which to analyze the immunological regulation and significance of anti-TG2 antibodies, by expressing specific human single-chain antibody fragments in mice using adeno-associated virus vectors. Upon vector injection in the skeletal muscles, high and persistent systemic levels of anti-TG2 antibodies were obtained. Mice injected with vectors encoding antibodies also recognizing rodent TG2, also developed a strong anti-idiotypic response. This finding raises the question of whether an anti-idiotypic response to anti-TG2 antibodies is a factor associated with celiac disease.

Characterizing monoclonal antibody epitopes by filtered gene fragment phage display.

In the present paper, we describe a novel approach to map monoclonal antibody epitopes, using three new monoclonal antibodies that recognize h-TG2 (human transglutaminase 2) as an example. The target gene was fragmented and cloned upstream of an antibiotic-resistance gene, in the vector pPAO2, to select for in-frame polypeptides. After removal of the antibiotic-resistance gene by Cre/Lox recombination, an antigen fragment phage display library was created and selected against specific monoclonal antibodies. Using the h-TG2 fragment library, we were able to identify epitopes. This technique can also be broadly applied to the study of protein-protein interactions.