Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy

Rôle du facteur de transcription Spi-1/PU.1 dans la progression leucémique (dérégulation des réseaux transcriptionnnels et inhibition de l'apoptose)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

The Spi1/PU.1 transcription factor accelerates replication fork progression by increasing PP1 phosphatase in leukemia

International audienc

Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1

The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells

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

<div><p>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. <i>In vivo</i>, 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.</p></div

p53 reprograms human caPSCs towards quiescence.

<p>caPSCs were treated with Nutlin-3a, Nutlin-3b (control) or PD332991 for 48h (C, G) or 72h (A,B, F). (A) Cells were immunostained for Ki67 and nuclei were stained with DAPI. Bar graph represents the percentage of nuclei positive for the Ki67 antigen. At least 100 cells per condition were counted. Values are plotted as mean +SD of at least 2 experiments. ***, p<0.001; **, p<0.01; *, p<0.05 by two-way ANOVA. (B) Representative images of caPSCs stained with BODIPY 493/503 for detection of neutral lipids. (C) Acta2 mRNA levels were quantified by RT-qPCR. Values were normalized to Rplp0 mRNA levels and are represented as fold change relative to Nutlin-3b treated cells. Bars indicate mean +SD of at least 3 experiments. ***, p<0.001; **, p<0.01; *, p<0.05 by one-way ANOVA. (D-E) caPSCs were treated with Nutlin-3a or Nutlin-3b for 72h. Nutlin-3a was removed from treated caPSCs and cells were cultured for an additional 72h with (+) or without (-) Nutlin-3a. (D) Representative images of cells stained with BODIPY 493/503 (E) Acta2 mRNA levels were quantified and represented as described in C. (F) Representative images of cells stained with BODIPY 493/503. (G) Acta2 mRNA levels were quantified and represented as described in C. Scale bars, 25 μM.</p

Cancer-associated pancreatic stellate cells express functional p53.

<p>(A) Immunoblot for p53 and p21 from primary caPSCs treated with Nutlin-3a (+) or its inactive enantiomer Nutlin-3b (-) for 48h. β-Actin serves as a loading control. (B) p21 and Mdm2 mRNA levels were quantified by real-time qPCR (RT-qPCR) in caPSCs treated with Nutlin-3a or Nutlin-3b for 48h. Values were normalized to Rplp0 mRNA levels and are represented as fold change relative to Nutlin-3b treated cells. Bars indicate mean +SD of at least 3 experiments. ***, p<0.001; **, p<0.01; *, p<0.05 by One-way ANOVA.</p

Stromal p53 activation reverses caPSC activation and reduces pancreatic desmoplasia in vivo.

<p>(A) Tumor bearing mice were treated with vehicle or RG7112 (200 mg/kg) and were sacrificed. Epithelial cells (EPCAM+) and fibroblasts (PDGFRα+) were isolated from dissociated tumors by FACS. p21 and Mdm2 mRNA levels were quantified by RT-qPCR and normalized to Rplp0 mRNA. Bars represent mean +SD of 3 different mice. *, p<0.05 by two-way ANOVA. (B-C) Mice transplanted with KPC cells were treated for 15 days with Vehicle or RG7112 (200 mg/kg) starting day 8 post-transplantation. Tumors were harvested and fixed in formalin. FFPE sections were subjected to (B) IHC using an αSMA antibody and (C) Masson’s Trichrome staining. Representative images are shown on the left and quantification on the right. At least eight 15X fields were quantified using Inform 2.1 software and mean values for each tumor are plotted on the graph. *, p<0.05 by Student’s test. Scale bar, 50 μM. (D) Metascape analysis was performed on RG7112 downregulated and upregulated genes (fold change >1.4 or <0.7, adjusted p<0.05). Six of the twenty most significant canonical pathways are shown and -log(pval) are indicated. (E) Heatmap representing selected genes from the RNA-seq analysis. Data are represented as log2 fold change, RG7112 versus Vehicle.</p