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Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs) are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell cycle arrest, and senescence. There is growing evidence that stromal p53 also exerts anti-tumor activity by paracrine mechanisms, though a role for stromal p53 in PDAC has not yet been described. Here, we demonstrate that activation of stromal p53 exerts anti-tumor effects in PDAC. We show that primary cancer-associated PSCs (caPSCs) isolated from human PDAC express wild-type p53, which can be activated by the Mdm2 antagonist Nutlin-3a. Our work reveals that p53 acts as a major regulator of PSC activation and as a modulator of PDAC fibrosis. In vitro, p53 activation by Nutlin-3a induces profound transcriptional changes, which reprogram activated PSCs to quiescence. Using immunofluorescence and lipidomics, we have also found that p53 activation induces lipid droplet accumulation in both normal and tumor-associated fibroblasts, revealing a previously undescribed role for p53 in lipid storage. In vivo, treatment of tumor-bearing mice with the clinical form of Nutlin-3a induces stromal p53 activation, reverses caPSCs activation, and decreases fibrosis. All together our work uncovers new functions for stromal p53 in PDAC.

SUBMITTER: Saison-Ridinger M

PROVIDER: S-EPMC5718507 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

Saison-Ridinger Maya M DelGiorno Kathleen E KE Zhang Tejia T Kraus Annabelle A French Randall R Jaquish Dawn D Tsui Crystal C Erikson Galina G Spike Benjamin T BT Shokhirev Maxim N MN Liddle Christopher C Yu Ruth T RT Downes Michael M Evans Ronald M RM Saghatelian Alan A Lowy Andrew M AM Wahl Geoffrey M GM

PloS one 20171206 12

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs) are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell ...[more]

PMID: 29211796

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Project description:BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been shown to associate with a better outcome. However, the underlying mechanisms of this benefit remain unclear. Metformin has been found to modulate the activity of stellate cells in other disease settings. In this study, we examine the effect of metformin on PSC activity, fibrosis and inflammation in PDACs.Methods/resultsIn overweight, diabetic PDAC patients and pre-clinical mouse models, treatment with metformin reduced levels of tumor extracellular matrix (ECM) components, in particular hyaluronan (HA). In vitro, we found that metformin reduced TGF-ß signaling and the production of HA and collagen-I in cultured PSCs. Furthermore, we found that metformin alleviates tumor inflammation by reducing the expression of inflammatory cytokines including IL-1? as well as infiltration and M2 polarization of tumor-associated macrophages (TAMs) in vitro and in vivo. These effects on macrophages in vitro appear to be associated with a modulation of the AMPK/STAT3 pathway by metformin. Finally, we found in our preclinical models that the alleviation of desmoplasia by metformin was associated with a reduction in ECM remodeling, epithelial-to-mesenchymal transition (EMT) and ultimately systemic metastasis.ConclusionMetformin alleviates the fibro-inflammatory microenvironment in obese/diabetic individuals with pancreatic cancer by reprogramming PSCs and TAMs, which correlates with reduced disease progression. Metformin should be tested/explored as part of the treatment strategy in overweight diabetic PDAC patients.

Project description:The complex network of biochemical and biophysical cues in the pancreatic desmoplasia not only presents challenges to the fundamental understanding of tumor progression, but also hinders the development of therapeutic strategies against pancreatic cancer. Residing in the desmoplasia, pancreatic stellate cells (PSCs) are the major stromal cells affecting the growth and metastasis of pancreatic cancer cells by means of paracrine effects and extracellular matrix protein deposition. PSCs remain in a quiescent/dormant state until they are 'activated' by various environmental cues. While the mechanisms of PSC activation are increasingly being described in literature, the influence of matrix stiffness on PSC activation is largely unexplored. To test the hypothesis that matrix stiffness affects myofibroblastic activation of PSCs, we have prepared cell-laden hydrogels capable of being dynamically stiffened through an enzymatic reaction. The stiffening of the microenvironment was created by using a peptide linker with additional tyrosine residues, which were susceptible to tyrosinase-mediated crosslinking. Tyrosinase catalyzes the oxidation of tyrosine into dihydroxyphenylalanine (DOPA), DOPA quinone, and finally into DOPA dimer. The formation of DOPA dimer led to additional crosslinks and thus stiffening the cell-laden hydrogel. In addition to systematically studying the various parameters relevant to the enzymatic reaction and hydrogel stiffening, we also designed experiments to probe the influence of dynamic matrix stiffening on cell fate. Protease-sensitive peptides were used to crosslink hydrogels, whereas integrin-binding ligands (e.g., RGD motif) were immobilized in the network to afford cell-matrix interaction. PSC-laden hydrogels were placed in media containing tyrosinase for 6h to achieve in situ gel stiffening. We found that PSCs encapsulated and cultured in a stiffened matrix expressed higher levels of αSMA and hypoxia-inducible factor 1α (HIF-1α), suggestive of a myofibroblastic phenotype. This hydrogel platform offers a facile means of in situ stiffening of cell-laden matrices and should be valuable for probing cell fate process dictated by dynamic matrix stiffness.Statement of significanceHydrogels with spatial-temporal controls over crosslinking kinetics (i.e., dynamic hydrogel) are increasingly being developed for studying mechanobiology in 3D. The general principle of designing dynamic hydrogel is to perform cell encapsulation within a hydrogel network that allows for postgelation modification in gel crosslinking density. The enzyme-mediated in situ gel stiffening is innovative because of the specificity and efficiency of enzymatic reaction. Although tyrosinase has been used for hydrogel crosslinking and in situ cell encapsulation, to the best of our knowledge tyrosinase-mediated DOPA formation has not been explored for in situ stiffening of cell-laden hydrogels. Furthermore, the current work provides a gradual matrix stiffening strategy that may more closely mimic the process of tumor development.

Dataset Information

Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

Publications

Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

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