Project description:Cyclooxygenase-2 (COX-2) is upregulated in pancreatic ductal adenocarcinomas (PDAC). However, how COX-2 promotes PDAC development is unclear. While previous studies have evaluated the efficacy of COX-2 inhibition via the use of non steroidal anti-inflammatory drugs (NSAIDs) or the COX-2 inhibitor celecoxib in PDAC models, none have addressed the cell intrinsic vs. microenvironment roles of COX-2 in modulating PDAC initiation and progression. We tested the cell intrinsic role of COX-2 in PDAC progression, using both loss-of-function and gain-of-function approaches. Cox-2 deletion in Pdx1+ pancreatic progenitor cells significantly delays the development of PDAC in mice with K-ras activation and Pten haploinsufficiency. Conversely, COX-2 over-expression promotes early onset and progression of PDAC in the K-ras mouse model. Loss of PTEN function is a critical factor in determining lethal PDAC onset and overall survival. Mechanistically, COX-2 over-expression increases P-AKT levels in the precursor lesions of Pdx1+;K-rasG12D/+;Ptenlox/+ mice in the absence of Pten LOH. In contrast, Cox-2 deletion in the same setting diminishes P-AKT levels and delays cancer progression. These data suggest an important cell intrinsic role for COX-2 in tumor initiation and progression through activation of the PI3K/AKT pathway. PDAC that is independent of intrinsic COX-2 expression eventually develops with decreased FKBP5 and increased GRP78 expression, two alternate pathways leading to AKT activation. Together, these results support a cell intrinsic role for COX-2 in PDAC development and suggest that, while anti-COX-2 therapy may delay the development and progression of PDAC, mechanisms known to increase chemoresistance through AKT activation must also be overcome. Murine mutants with pancreatic specific loss of Pten (Pten +/-) and K-ras activation (K-rasG12D) and either COX-2 over-expression (Cox-2 COE) or knockout (Cox-2 KO) under regulation of the Pdx-1 promoter developed pancreatic ductal adenocarcinoma. RNA was extracted from pancreatic tumors from individual mutants with pathology thought to closely mimic the human disease. Pancreatic tissue was subject to RNA extraction and hybridization on Affymetrix cDNA microarrays.
Project description:COX-2 is upregulated in pancreatic ductal adenocarcinomas (PDAC). However, how COX-2 promotes PDAC development is unclear. While previous studies have evaluated the efficacy of COX-2 inhibition via the use of nonsteroidal anti-inflammatory drugs (NSAID) or the COX-2 inhibitor celecoxib in PDAC models, none have addressed the cell intrinsic versus microenvironment roles of COX-2 in modulating PDAC initiation and progression. We tested the cell intrinsic role of COX-2 in PDAC progression using both loss-of-function and gain-of-function approaches. Cox-2 deletion in Pdx1+ pancreatic progenitor cells significantly delays the development of PDAC in mice with K-ras activation and Pten haploinsufficiency. Conversely, COX-2 overexpression promotes early onset and progression of PDAC in the K-ras mouse model. Loss of PTEN function is a critical factor in determining lethal PDAC onset and overall survival. Mechanistically, COX-2 overexpression increases p-AKT levels in the precursor lesions of Pdx1(+); K-ras(G12D)(/+); Pten(lox)(/+) mice in the absence of Pten LOH. In contrast, Cox-2 deletion in the same setting diminishes p-AKT levels and delays cancer progression. These data suggest an important cell intrinsic role for COX-2 in tumor initiation and progression through activation of the PI3K/AKT pathway. PDAC that is independent of intrinsic COX-2 expression eventually develops with decreased FKBP5 and increased GRP78 expression, two alternate pathways leading to AKT activation. Together, these results support a cell intrinsic role for COX-2 in PDAC development and suggest that while anti-COX-2 therapy may delay the development and progression of PDAC, mechanisms known to increase chemoresistance through AKT activation must also be overcome.
Project description:We found that BAP1 (BRCA1 Associated Protein-1) shows loss of heterozygosity in over 25% of pancreatic cancer patients and functions as tumor suppressor. Conditional deletion of Bap1 in murine pancreas led to genomic instability, accumulation of DNA damage, and an inflammatory response that evolved to pancreatitis with full penetrance. Concomitant expression of oncogenic KrasG12D led to malignant transformation and development of invasive and metastatic pancreatic cancer. At the molecular level, BAP1 maintains the integrity of the exocrine pancreas by regulating genomic stability and its loss confers sensitivity to radio- and platinum-based therapies. Overall design: Murine cell lines were estabished from Bap1 null pancreata expressing KrasG12D and analyzed by ChIP-seq for H3K4me1, H3K4me3, H3K27me3, H3K27ac, H2AK119ub, and H2BK120ub.
Project description:Epidemiological and experimental data implicate cutaneous human papillomavirus infection as co-factor in the development of cutaneous squamous cell carcinomas (cSCCs), particularly in immunocompromised organ transplant recipients (OTRs). Herein, we established and characterized a skin cancer model, in which Mus musculus papillomavirus 1 (MmuPV1) infection caused cSCCs in cyclosporine A (CsA)-treated mice, even in the absence of UV light. Development of cSCCs and their precursors were observed in 70% of MmuPV1-infected, CsA-treated mice on back as well as on tail skin. Immunosuppression by systemic CsA, but not UV-B irradiation, was a prerequisite, as immunocompetent or UV-B-irradiated mice did not develop skin malignancies after infection. In the virus-driven cSCCs the MmuPV1-E6/E7 oncogenes were abundantly expressed, and transcriptional activity and productive infection demonstrated. MmuPV1 infection induced the expression of phosphorylated H2AX, but not degradation of proapoptotic BAK in the cSCCs. Transfer of primary cells, established from a MmuPV1-induced cSCC from back skin, into athymic nude mice gave rise to secondary cSCCs, which lacked viral DNA, demonstrating that maintenance of the malignant phenotype was virus independent. This papillomavirus-induced skin cancer model opens future investigations into viral involvement, pathogenesis, and cancer surveillance, aiming at understanding and controlling the high incidence of skin cancer in OTRs.
Project description:Although pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in >90% of primary pancreatic cancers, with absent or low levels in early precursor lesions.Here, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis.HMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r(2) = 0.93; p < 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1.Our studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.
Project description:Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (?20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (?15%); and (iii) no visible papillomas and viral clearance (?65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.
Project description:Studies of a hybrid zone between two house mouse subspecies (Mus musculus musculus and M. m. domesticus) along with studies using laboratory crosses reveal a large role for the X chromosome and multiple autosomal regions in reproductive isolation as a consequence of disrupted epistasis in hybrids. One limitation of previous work has been that most of the identified genomic regions have been large. The goal here is to detect and characterize precise genomic regions underlying reproductive isolation. We surveyed 1401 markers evenly spaced across the genome in 679 mice collected from two different transects. Comparisons between transects provide a means for identifying common patterns that likely reflect intrinsic incompatibilities. We used a genomic cline approach to identify patterns that correspond to epistasis. From both transects, we identified contiguous regions on the X chromosome in which markers were inferred to be involved in epistatic interactions. We then searched for autosomal regions showing the same patterns and found they constitute about 5% of autosomal markers. We discovered substantial overlap between these candidate regions underlying reproductive isolation and QTL for hybrid sterility identified in laboratory crosses. Analysis of gene content in these regions suggests a key role for several mechanisms, including the regulation of transcription, sexual conflict and sexual selection operating at both the postmating prezygotic and postzygotic stages of reproductive isolation. Taken together, these results indicate that speciation in two recently diverged (c. 0.5 Ma) house mouse subspecies is complex, involving many genes dispersed throughout the genome and associated with distinct functions.
Project description:Genes that are differentially expressed in pancreatic cancers and under epigenetic regulation are of considerable biological and therapeutic interest. We used global gene expression profiling and epigenetic treatment of pancreatic cell lines including pancreatic cancer cell lines, pancreatic cancer-associated fibroblasts, and cell lines derived from nonneoplastic pancreata. We examined expression and epigenetic alterations of cyclooxygenase-1 (COX-1) and COX-2 in pancreatic cancers and normal pancreas and performed proliferation, knockdown, and coculture experiments to understand the role of stromal sources of prostaglandins for pancreatic cancers. We identify COX-1 as a gene under epigenetic regulation in pancreatic cancers. We find that COX-1 expression is absent in many pancreatic cancer cells and some of these cancers also lack COX-2 expression. Suspecting that such cancers must rely on exogenous sources of prostaglandins, we show that pancreatic cancer stromal cells, such as fibroblasts expressing COX-1 and COX-2, are a likely source of prostaglandins for pancreatic cancer cells deficient in COX. Knocking down the prostaglandin transporter multidrug resistance-associated protein-4 in fibroblasts suppresses the proliferation of cocultured pancreatic cancer cells lacking COX. Pancreatic cancers that lack COX can use exogenous sources of prostaglandins. Blocking multidrug resistance-associated protein-4 may be a useful therapeutic strategy to deplete COX-deficient pancreatic cancers of prostaglandins.
Project description:Liver fibrosis is a major pathological feature of chronic liver diseases, including liver cancer. MicroRNAs (miRNAs), small noncoding RNAs, regulate gene expression posttranscriptionally and play important roles in various kinds of diseases; however, miRNA-associated hepatic fibrogenesis and its acting mechanisms are poorly investigated. Therefore, we performed an miRNA microarray in the fibrotic livers of Mus musculus treated with carbon-tetrachloride (CCl₄) and analyzed the biological functions engaged by the target genes of differentially-expressed miRNAs through gene ontology (GO) and in-depth pathway enrichment analysis. Herein, we found that four miRNAs were upregulated and four miRNAs were downregulated more than two-fold in CCl₄-treated livers compared to a control liver. Eight miRNAs were predicted to target a total of 4079 genes. GO analysis revealed that those target genes were located in various cellular compartments, including cytoplasm, nucleolus and cell surface, and they were involved in protein-protein or protein-DNA bindings, which influence the signal transductions and gene transcription. Furthermore, pathway enrichment analysis demonstrated that the 72 subspecialized signaling pathways were associated with CCl₄-induced liver fibrosis and were mostly classified into metabolic function-related pathways. These results suggest that CCl₄ induces liver fibrosis by disrupting the metabolic pathways. In conclusion, we presented several miRNAs and their biological processes that might be important in the progression of liver fibrosis; these findings help increase the understanding of liver fibrogenesis and provide novel ideas for further studies of the role of miRNAs in liver fibrosis.
Project description:The house mouse, Mus musculus, is one of the most ubiquitous invasive species worldwide and in Australia is particularly common and widespread, but where it originally came from is still unknown. Here we investigated this origin through a phylogeographic analysis of mitochondrial DNA sequences (D-loop) comparing mouse populations from Australia with those from the likely regional source area in Western Europe. Our results agree with human historical associations, showing a strong link between Australia and the British Isles. This outcome is of intrinsic and applied interest and helps to validate the colonization history of mice as a proxy for human settlement history.