Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

ROBO2 is a stroma suppressor gene in the pancreas and acts via TGF-β signalling.

Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1Cre;Robo2F/F) show increased activation of Robo1+ myofibroblasts and induction of TGF-β and Wnt pathways. During pancreatitis, Pdx1Cre;Robo2F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-β inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2low;ROBO1high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-β inhibitors or other stroma /immune modulating agents

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Proteogenomic analysis identifies a novel human SHANK3 Isoform

© 2015 by the authors; licensee MDPI, Basel, Switzerland. Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC) protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions