Oakley, F. | Search bioRxiv

bioRxiv2018

Regulation of checkpoint kinase signalling and tumorigenesis by the NF-κB regulated gene, CLSPN

Inhibition of the tumour promoting activities of NF-{kappa}B by cell signalling pathways has been proposed as a natural mechanism to limit the development of cancer. However, there has been a lack of evidence for these effects in vivo. Here we report that RelAT505A mice, where a CHK1 targeted Thr505 phosphosite is mutated to alanine, display earlier onset of MYC driven lymphoma than wild type littermates. We describe a positive feedback loop in which the NF-{kappa}B subunits RelA and c-Rel, in a manner dependent upon RelA Thr505 phosphorylation, drive the expression of the ATR checkpoint kinase regulator Claspin in response to DNA replication stress in cancer cells. This in turn is required for maintenance of CHK1 activity. Loss of a single allele of the Clspn gene in mice is sufficient to drive earlier tumorigenesis and low levels of CLSPN mRNA expression are associated with worse survival in some forms of human cancer. We propose that loss of this pathway early in tumorigenesis promotes cancer development through increased genomic instability. However, in malignant cancer cells it can help promote their addiction to the checkpoint kinase signalling required for the maintenance of genomic integrity. Importantly, disruption of this pathway leads to resistance of cells to treatment with CHK1 inhibitors. Claspin expression could therefore act as a biomarker for responsiveness of patients to CHK1 inhibitors and provide a potential pathway for the development of tumour resistance.

cancer biology↗

bioRxiv2018

A novel bioreactor technology for modelling fibrosis in human and rodent precision-cut liver slices.

Summary boxO_LIWhat is already known about this subject?\nO_LICurrently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.\nC_LIO_LIPreclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.\nC_LIO_LIPrecision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.\nC_LI\nC_LIO_LIWhat are the new findings?\nO_LIWe have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.\nC_LIO_LIBioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.\nC_LIO_LIAdministration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.\nC_LI\nC_LIO_LIHow might it impact on clinical practice in the foreseeable future?\nO_LIThe extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.\nC_LIO_LIBioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.\nC_LI\nC_LI\n\nAbstractO_ST_ABSObjectiveC_ST_ABSPrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.\n\nDesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGF{beta}1 and PDGF{beta}{beta} stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and Smooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.\n\nResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGF{beta}1 and PDGF{beta}{beta} stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGF{beta}1 and PDGF{beta}{beta} stimulated PCLS and fibrotic PCLS.\n\nConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.

cell biology↗

bioRxiv2017

Activation of NF-κB by a novel, CDK4-regulated, nucleolar stress response pathway

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-{kappa}B transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-{kappa}B also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-{kappa}B pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-{kappa}B signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-{kappa}B induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-{kappa}B pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-{kappa}B pathway. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-{kappa}B in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-{kappa}B nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

cell biology↗