Modified gateway system for double shRNA expression and Cre/lox based gene expression

<p>Abstract</p> <p>Background</p> <p>The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations.</p> <p>Results</p> <p>Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines.</p> <p>Conclusion</p> <p>Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.</p

Whole genomes define concordance of matched primary, xenograft, and organoid models of pancreas cancer

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among solid malignancies and improved therapeutic strategies are needed to improve outcomes. Patient-derived xenografts (PDX) and patient-derived organoids (PDO) serve as promising tools to identify new drugs with therapeutic potential in PDAC. For these preclinical disease models to be effective, they should both recapitulate the molecular heterogeneity of PDAC and validate patient-specific therapeutic sensitivities. To date however, deep characterization of the molecular heterogeneity of PDAC PDX and PDO models and comparison with matched human tumour remains largely unaddressed at the whole genome level. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumours and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 'trios' of matched primary tumour, PDX, and PDO. We developed a pipeline to score concordance between PDAC models and their paired human tumours for genomic events, including mutations, structural variations, and copy number variations. Tumour-model comparisons of mutations displayed single-gene concordance across major PDAC driver genes, but relatively poor agreement across the greater mutational load. Genome-wide and chromosome-centric analysis of structural variation (SV) events highlights previously unrecognized concordance across chromosomes that demonstrate clustered SV events. We found that polyploidy presented a major challenge when assessing copy number changes; however, ploidy-corrected copy number states suggest good agreement between donor-model pairs. Collectively, our investigations highlight that while PDXs and PDOs may serve as tractable and transplantable systems for probing the molecular properties of PDAC, these models may best serve selective analyses across different levels of genomic complexity

Identification and Validation of Novel Oncogenes and Tumor Suppressors in Pancreatic Ductal Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is driven by somatic genetic alterations. Functional studies are essential to classify PDAC alterations as either drivers or passengers of this disease. In this work I hypothesized that integrative genomics and functional studies will identify and validate novel drivers of pancreatic carcinogenesis. In the first study I uncovered common and differential roles of cyclin Ds in PDAC. Results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling in PDAC cell lines. Furthermore, CCND1 plays a role in PDAC cell migration. In the second study I identified CCDC68 as a putative tumor suppressor gene in PDAC. Loss of CCDC68 resulted in increased in vitro proliferation and in vivo tumorigenicity of PDAC cell lines associated with amplified MAPK signaling. Loss of CCDC68 function in PDAC was documented through copy number loss and the expression of novel CCDC68Δ69-114 variant devoid of tumor suppressive function. Truncated in-frame protein CCDC68Δ69-114 was shown to be a result of exon skipping in 30% of patients harboring donor splice variant rs.1344011. I also report that the expressed ratios of CCDC68 wt/Δ69-114 variants fluctuates among different patients suggesting that the tumor suppressive function of CCDC68 might be regulated by a critical balance between two isoforms. The third study explored the molecular mechanism(s) of CCDC68 tumor suppression in PDAC. To this end, the biology between tumor suppressive CCDC68wt and non-tumor suppressive CCDC68Δ69-114 variant was compared in H6c7 and 293T cells. Analysis revealed that both isoforms associate with centrosomes in interphase, disappear in mitosis and interact with mother centriole protein CEP170. However, loss of 69-114 amino acids in CCDC68 variant significantly decreased its protein stability further shown to be associated with loss of the lysine acetylation site K94. These findings suggest that the CCDC68Δ69-114 variant is not a stable protein and also point to the significance of lysine acetylation regulating the tumor suppressive properties of CCDC68 protein. Overall, this dissertation reports cyclin D1, cyclin D3 and CCDC68 as genetic alterations playing significant roles in pancreatic carcinogenesis.Ph

Identification and Validation of Novel Oncogenes and Tumor Suppressors in Pancreatic Ductal Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is driven by somatic genetic alterations. Functional studies are essential to classify PDAC alterations as either drivers or passengers of this disease. In this work I hypothesized that integrative genomics and functional studies will identify and validate novel drivers of pancreatic carcinogenesis. In the first study I uncovered common and differential roles of cyclin Ds in PDAC. Results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling in PDAC cell lines. Furthermore, CCND1 plays a role in PDAC cell migration. In the second study I identified CCDC68 as a putative tumor suppressor gene in PDAC. Loss of CCDC68 resulted in increased in vitro proliferation and in vivo tumorigenicity of PDAC cell lines associated with amplified MAPK signaling. Loss of CCDC68 function in PDAC was documented through copy number loss and the expression of novel CCDC68Δ69-114 variant devoid of tumor suppressive function. Truncated in-frame protein CCDC68Δ69-114 was shown to be a result of exon skipping in 30% of patients harboring donor splice variant rs.1344011. I also report that the expressed ratios of CCDC68 wt/Δ69-114 variants fluctuates among different patients suggesting that the tumor suppressive function of CCDC68 might be regulated by a critical balance between two isoforms. The third study explored the molecular mechanism(s) of CCDC68 tumor suppression in PDAC. To this end, the biology between tumor suppressive CCDC68wt and non-tumor suppressive CCDC68Δ69-114 variant was compared in H6c7 and 293T cells. Analysis revealed that both isoforms associate with centrosomes in interphase, disappear in mitosis and interact with mother centriole protein CEP170. However, loss of 69-114 amino acids in CCDC68 variant significantly decreased its protein stability further shown to be associated with loss of the lysine acetylation site K94. These findings suggest that the CCDC68Δ69-114 variant is not a stable protein and also point to the significance of lysine acetylation regulating the tumor suppressive properties of CCDC68 protein. Overall, this dissertation reports cyclin D1, cyclin D3 and CCDC68 as genetic alterations playing significant roles in pancreatic carcinogenesis.Ph

Modified gateway system for double shRNA expression and Cre/lox based gene expression

Abstract Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies

β-catenin mediates growth defects induced by centrosome loss in a subset of APC mutant colorectal cancer independently of p53.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway

Differential roles of cyclin D1 and D3 in pancreatic ductal adenocarcinoma

Abstract Background The cyclin D1 (CCND1) and cyclin D3 (CCND3) are frequently co-overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here we examine their differential roles in PDAC. Results CCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p Conclusions Our results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis.</p

Differential roles of cyclin D1 and D3 in pancreatic ductal adenocarcinoma

Abstract Background The cyclin D1 (CCND1) and cyclin D3 (CCND3) are frequently co-overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here we examine their differential roles in PDAC. Results CCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p < 0.005). In contrast, focal adhesion/actin cytoskeleton, MAPK and NF B signaling appeared to characterize the target genes and their interacting proteins in CCND1 suppressed PANC1 cells. Conclusions Our results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis

Centrosome loss represses normal HCOs growth in a p53-dependent manner.

A) Control and p53 KO normal HCOs were grown from single adult stem cells in the presence of DMSO or 0.5 μM centrinone B for 14 days. Organoids were then fixed, stained with DAPI to label DNA (blue) and phalloidin to label actin (red) and imaged. Merged maximum intensity projections of images are shown. B) The areas of individual organoids were quantified in the merged maximum intensity projections (MIPs) of images from (A) (n = 3) and presented in the graph as values relative to the DMSO control where every dot represents an organoid (***PC) Tide analysis of p53 gene indel efficiency in the normal HCO p53 KO line. The algorithm provides the R2 value as a goodness-of-fit measure and calculates the statistical significance for each indel. Red represents significant indels (PD) Representative maximum intensity projections of p53 immunofluorescence staining in control and p53 KO normal HCOs. E) Normal human organoids were extracted from Matrigel and lysed. p53 and the loading control (GAPDH) protein levels were assessed using western blot analysis. F) Quantification of (E). Protein levels from three independent experiments were quantified and presented in the graph (n = 3, **PG) High-resolution maximum intensity projection images of CEP192 (centrosome) and DNA (DAPI) immunofluorescence staining in selected normal HCOs from (A). H) The number of CEP192 foci and nuclear objects was assessed in independent z-sections spanning three (DMSO) or five (centrinone) independent organoids. The foci to nucleus ratio was determined for each organoid (*P<0.05). Scale bars are (A) 500 μm, (D) 25 μm and (G) 10 μm.</p

Centrosome loss-induced organoid growth defect/death is β-catenin dependent in CSC-406 cancer line.

A) APC mutational status in the three cancer HCOs obtained via whole exome sequencing data. B) CSC-406 cancer organoids were grown from an equal number of single cells in the presence of DMSO or the indicated concentrations of XAV939 with DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI (blue) and phalloidin (green) and imaged. Merged maximum intensity projections of representative images are shown. C) Percentage of surviving organoids in different conditions from (B) was quantified and presented in the graph (n = 3, *PD) The areas of individual organoids from B were quantified in the merged maximum intensity projection images and the relative values are presented in the graph, every dot represents an organoid (n = 3, *PE) CSC-406 cancer organoids were treated with DMSO or 20 μM XAV939 for three days, extracted from Matrigel and lysed. b-catenin and the loading control (GAPDH) protein levels were assessed using western blot analysis. The same set of Western blots was used to generate Fig 4E. F) Protein levels from three independent experiments were quantified and presented in the graph (n = 3, ***PG) CSC-406 cancer organoids were grown from an equal number of single cells in the presence of DMSO or 3 μM ICG-001 and were treated with DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI (blue) and phalloidin (red) and imaged. Merged maximum intensity projections of representative images are shown. H) Percentage of surviving organoids in different conditions from (G) was quantified and presented in the graph (n = 3, ***PI) The areas of individual organoids from (G) were quantified in the merged maximum intensity projection images and the relative values are presented in the graph, every dot represents an organoid (n = 3, ***P<0.001, ns = non-significant, One-way ANOVA with Bonferroni post-hoc). Scale bars are (B and G) 500 μm.</p