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ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-dependent downregulation of actomyosin (MLC-2) contractility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. Our findings implicate a RAR-β/MLC-2 pathway in peritumoural stromal remodelling and mechanosensory-driven activation of PSCs, and further suggest that mechanical reprogramming of PSCs with retinoic acid derivatives might be a viable alternative to stromal ablation strategies for the treatment of PDAC.

SUBMITTER: Chronopoulos A

PROVIDER: S-EPMC5023948 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion.

Chronopoulos Antonios A Robinson Benjamin B Sarper Muge M Cortes Ernesto E Auernheimer Vera V Lachowski Dariusz D Attwood Simon S García Rebeca R Ghassemi Saba S Fabry Ben B Del Río Hernández Armando A

Nature communications 20160907

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-dependent downregulation of actomyosin (MLC-2) contractility. We show ...[more]

PMID: 27600527

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Project description:BackgroundActivation of pancreatic stellate cells (PSCs) is a key cause of chronic pancreatitis (CP), while inhibition of transforming growth factor-β (TGF-β) signaling renders PSCs inactive. Inhibitory Smads (I-Smads) impede TGF-β intracellular signaling and may provide a way to alleviate CP. Thus, we aimed to investigate the molecular mechanism of I-Smads in CP animals and freshly-isolated PSCs.MethodsSixteen male C57BL/6 mice were randomly divided into two groups; a control group (treated with saline) and a CP group (treated with caerulein) for 6 weeks. Masson's staining was performed to identify fibrosis, and immunohistochemistry (IHC) was performed to measure the levels of Smad6 between the two groups. An improved method derived from internal digestion was used to isolate PSCs from male Sprague Dawley rats. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining were used to measure the messenger ribonucleic acid (mRNA) and protein levels of alpha-smooth muscle actin (α-SMA). Plasmids of I-Smads or SB431542 were transfected into freshly-isolated PSCs, and relative mRNA levels of marker genes were quantified by qRT-PCR. The two-tailed Student's t-test was performed to assess significance.ResultsThe Smad6 protein level was significantly higher in the pancreas tissue of CP mice compared to the control group. A large number of PSCs were isolated from rat pancreas using an improved isolating method and were confirmed by quiescent and active PSC markers including cluster differentiation antigen 133 (CD133), perilipin 2 (Plin2), α-SMA, Desmin, and collagen 1 (Col1). The mRNA levels of both Smad6 and Smad7 were down-regulated during freshly-isolated PSC activation. Over-expression of both Smad6 and Smad7 in freshly-isolated PSC reduced the mRNA level of α-SMA, glial fibrillary acidic protein (GFAP), Desmin, Col1, Col3, and fibronectin 1 (Fn1) significantly. SB431542 reduced the mRNA level of α-SMA, Col1, Col3, and Fn1 significantly in freshly-isolated PSCs.ConclusionsThis study demonstrated that CP promoted the expression of I-Smads, which suppressed the activation of freshly-isolated PSCs via a negative feedback loop.

Project description:Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells.

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ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion.

Publications

ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion.

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