Dicer Is Required for Maintenance of Adult Pancreatic Acinar Cell Identity and Plays a Role in Kras-Driven Pancreatic Neoplasia

The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted Dicer specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that Dicer is essential for the maintenance of acinar cell identity. Acinar cells lacking Dicer showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to Dicer gene dosage. Homozygous Dicer deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous Dicer deletion accelerated PanIN initiation, revealing complex roles for Dicer in the regulation of both normal and neoplastic pancreatic epithelial identity.     

Notch1 Is Not Required for Acinar-to-Ductal Metaplasia in a Model of Kras-Induced Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma is believed to arise from precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). Mouse models have demonstrated that targeted expression of activated K-ras to mature acinar cells in the pancreas induces the spontaneous development of PanIN lesions; implying acinar-to-ductal metaplasia (ADM) is a key event in this process. Recent studies suggest Notch signaling is a key regulator of ADM. To assess if Notch1 is required for K-ras driven ADM we employed both an in vivo mouse model and in vitro explant culture system, in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in acinar cells. Our results demonstrate that oncogenic K-ras is sufficient to drive ADM both in vitro and in vivo but that loss of Notch1 has a minimal effect on this process. Interestingly, while loss of Notch1 in vivo does not affect the severity of PanIN lesions observed, the overall numbers of lesions were greater in mice with deleted Notch1. This suggests Notch1 deletion renders acinar cells more susceptible to formation of K-ras-induced PanINs.     

Notch and Kras in pancreatic cancer: At the crossroads of mutation,differentiation and signaling

Activating mutations in the KRAS proto-oncogene occur almost ubiquitously in pancreatic ductal adenocarcinoma (PDAC) and in its putative precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Conditional expression of an activated Kras allele in the mouse pancreas produces a model that faithfully recapitulates PanIN formation and progression to PDAC. Importantly, although nearly every cell in the pancreata of these mice express activated Kras, only a very small minority of cells give rise to PanINs. How the transforming activity of Kras is constrained in the pancreas remains unknown, and the cell types from which PanINs and PDAC arise are similarly unknown. Here, we describe our recent results demonstrating that acinar cells are competent to form Kras-induced PanINs, and that active Notch signaling can synergize with Kras in PanIN initiation and progression. Further efforts to understand how Notch and Kras synergize, as well as experiments to determine how other pancreatic cell types contribute to PDAC development, should aid in the development of new therapies and early detection techniques that are desperately needed for this cancer.     

Common activation of canonical Wnt signaling in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.     

Iron homeostasis regulates the activity of the microRNA pathway through poly(C)-binding protein 2

MicroRNAs (miRNAs) control gene expression by promoting degradation or repressing translation of target mRNAs. The components of the miRNA pathway are subject to diverse modifications that can modulate the abundance and function of miRNAs. Iron is essential for fundamental metabolic processes, and its homeostasis is tightly regulated. Here we identified iron chelators as a class of activator of the miRNA pathway that could promote the processing of miRNA precursors. We show that cytosolic iron could regulate the activity of the miRNA pathway through poly(C)-binding protein 2 (PCBP2). PCBP2 is associated with Dicer and promotes the processing of miRNA precursors. Cytosolic iron could modulate the association between PCBP2 and Dicer, as well as the multimerization of PCBP2 and its ability to bind to miRNA precursors, which can alter the processing of miRNA precursors. Our findings reveal a role of iron homeostasis in the regulation of miRNA biogenesis.     

MUC1 enhances tumor progression and contributes toward immunosuppression in a mouse model of spontaneous pancreatic adenocarcinoma

MUC1, a membrane tethered mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic adenocarcinoma. However, the role of MUC1 in pancreatic cancer has been elusive, partly due to the lack of an appropriate model. We report the characterization of a novel mouse model that expresses human MUC1 as a self molecule (PDA.MUC1 mice). Pancreatic tumors arise in an appropriate MUC1-tolerant background within an immune-competent host. Significant enhancement in the development of pancreatic intraepithelial preneoplastic lesions and progression to adenocarcinoma is observed in PDA.MUC1 mice, possibly due to increased proliferation. Tumors from PDA.MUC1 mice express higher levels of cyclooxygenase-2 and IDO compared with PDA mice lacking MUC1, especially during early stages of tumor development. The increased proinflammatory milieu correlates with an increased percentage of regulatory T cells and myeloid suppressor cells in the pancreatic tumor and tumor draining lymph nodes. Data shows that during pancreatic cancer progression, MUC1-mediated mechanisms enhance the onset and progression of the disease, which in turn regulate the immune responses. Thus, the mouse model is ideally suited for testing novel chemopreventive and therapeutic strategies against pancreatic cancer.     

Genetic analyses reveal a requirement for Dicer1 in the mouse urogenital tract

Despite the increasing interest in other classes of small RNAs, microRNAs (miRNAs) remain the most widely investigated and have been shown to play a role in a number of different processes in mammals. Many studies investigating miRNA function focus on the processing enzyme Dicer1, which is an RNAseIII protein essential for the biogenesis of active miRNAs through its cleavage of precursor RNA molecules. General deletion of Dicer1 in the mouse confirms that miRNAs are essential for development because embryos lacking Dicer1 fail to reach the end of gastrulation. Here we investigate the role of Dicer1 in urogenital tract development. We utilised a conditional allele of the Dicer1 gene and two Cre-expressing lines, driven by HoxB7 and Amhr2, to investigate the effect of Dicer1 deletion on both male and female reproductive tract development. Data presented here highlight an essential role for Dicer1 in the correct morphogenesis and function of the female reproductive tract and confirm recent findings that suggest Dicer1 is required for female fertility. In addition, HoxB7:Cre-mediated deletion in ureteric bud derivatives leads to a spectrum of anomalies in both males and females, including hydronephrotic kidneys and kidney parenchymal cysts. Male reproductive tract development, however, remains largely unaffected in the absence of Dicer1. Thus, Dicer1 is required for development of the female reproductive tract and also normal kidney morphogenesis.     

MicroRNA-deficient NK cells exhibit decreased survival but enhanced function

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.     

An essential role for Dicer in adipocyte differentiation

Dicer is a cellular enzyme required for the processing of pre‐miRNA molecules into mature miRNA, and Dicer and miRNA biogenesis have been found to play important roles in a variety of physiologic processes. Recently, reports of alterations in miRNA expression levels in cultured pre‐adipogenic cell lines during differentiation and findings of differences between the miRNA expression signatures of white and brown adipose have suggested that miRNA molecules might regulate adipocyte differentiation and the formation of adipose tissue. However, direct evidence that miRNAs regulate adipogenesis is lacking. To determine if Dicer and mature miRNA govern adipocyte differentiation, we utilized primary cells isolated from mice bearing Dicer‐conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis. Our results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre‐adipocytes. Furthermore, the requirement for Dicer in adipocyte differentiation is not due to miRNA‐mediated alterations in cell proliferation, as deletion of the Ink4a locus and the prevention of premature cellular senescence normally induced in primary cells upon Dicer ablation fails to rescue adipogenic differentiation in fibroblasts and pre‐adipocytes. J. Cell. Biochem. 110: 812–816, 2010. © 2010 Wiley‐Liss, Inc.     

The regulatory roles of miRNA and methylation on oncogene and tumor suppressor gene expression in pancreatic cancer cells

Carcinogenesis is driven by an accumulation of mutations and genetic lesions, which leads to activation of oncogenes and inactivation of tumor suppressor genes. However, the molecular mechanisms by which the expression of these genes was regulated in pancreatic cancer remains unclear. In this study, we investigated the regulatory effects of microRNA and methylation on the expression of k-ras, TP53 and PTEN genes in pancreatic cancer cells. The protein and miRNA levels were measured by Western blotting and Northern blotting, respectively. Xenograft pancreatic tumor models were established by inoculating BxPC-1, Capan-2, and Panc-1 tumor cells into athymic nu/nu mice. A disparate level of KRAS, p53, PTEN, Dnmts, and Dicer 1 proteins as well as let-7i, miR-22, miR-143, and miR-29b miRNA was observed in BxPC-1, Capan-2, and Panc-1 cells. Knockdown of Dicer 1 expression in BxPC-3 and Panc-1 cells resulted in significant increases in KRAS, p53, PTEN, and Dnmts protein levels and significant decreases in miR-22, miR-143, let-7i, and miR-29b expression. Knockdown of Dicer 1 expression in Capan-2 cells significantly increased p53 and PTEN expression, while significantly decreased miR-22 and miR-143 expression, but had no effects on PTEN, Dnmts, let-7i, and miR-29b expression. Knockdown of Dicer 1 expression significantly inhibited xenograft BxPC-3 tumor growth, but promoted xenograft Panc-1 tumor growth. In contrast, knockdown of Dicer 1 expression had no effect on xenograft Capan-2 tumor growth. Our study suggested that different pancreatic cancer cell lines exhibited obvious discrepancies in gene expression profiles, implying that different molecular mechanisms are involved in the carcinogenesis of pancreatic cancer subclasses. Our study highlighted the importance of personalized therapy.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

Identification of MicroRNA Target Genes in Vivo

MicroRNAs (miRNAs) are short non-coding RNAs transcribed from intergenic or intronic sequences as long precursors that are sequentially processed by the endonucleases Drosha and Dicer into short double-stranded sequences. It is clear that miRNAs play essential roles in gene expression, development, and cell fate specification in animals. However, one of the barriers of miRNA research is how to find the target genes. In this study, we have developed a rapid and effective method to isolate miRNA target genes in vivo. MicroRNA was synthesized in vitro and labeled by biotin. After transfected into cells, the miRNA/mRNA complexes were isolated by streptavidin-coated magnetic beads. hsa-miR155 was taken as model to validate this method, which is a very important modulator in tumor development. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.     

Ductal cells of the pancreas

Ductal cells of the pancreas form the epithelial lining of the branched tubes that deliver enzymes produced by pancreatic acinar cells into the duodenum. In addition, these cells secrete bicarbonate that neutralizes stomach acidity. During development, epithelium of endodermal origin evaginates from the future duodenum area and invades the mesenchyme to form a complex branched network. All endocrine, acinar and ductal cells arise from common precursors in this epithelial structure. Adult ductal cells share some similarities with embryonic primitive ducts and may retain the ability to generate endocrine cells in the adult. Based on challenged pancreas regeneration experiments, the adult ductal cells have been proposed to be pancreatic stem cells but their role in normal endocrine cell turnover has recently been challenged. Manipulating their ability to give rise to endocrine cells may open new avenues in the treatment of diabetes and therefore they have recently been under scrutiny. In addition, in the main form of pancreatic cancer, pancreas adenocarcinoma, tumor cells share similarities with ductal cells. The secrets of an appropriate therapy for this deadly cancer may thus reside in the biology of ductal cells.     

Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin

MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.