Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype

Antitumor effects of somatostatin.

International audienceSince its discovery three decades ago as an inhibitor of GH release from the pituitary gland, somatostatin has attracted much attention because of its functional role in the regulation of a wide variety of physiological functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. In addition to its negative role in the control of endocrine and exocrine secretions, somatostatin and analogs also exert inhibitory effects on the proliferation and survival of normal and tumor cells. Over the past 15 years, studies have begun to reveal some of the molecular mechanisms underlying the antitumor activity of somatostatin. This review covers the present knowledge in the antitumor effect of somatostatin and analogs and discusses the perspectives of novel clinical strategies based on somatostatin receptor sst2 gene transfer therapy

βArrestin-1 and Mcl-1 modulate self-renewal growth of cancer stem-like side-population cells in non-small cell lung cancer.

Side population (SP) cells have been reported to have properties of cancer stem-like cells (CSCs) in non-small cell lung carcinoma (NSCLC), yet their molecular features have not been fully elucidated. Here we show that, NSCLC-SP cells were enriched in G(0)/G-(1) phase of cell cycle, had higher aldehyde dehydrogenase activity as well as higher clonogenic and self-renewing ability compared to main population (MP) cells. Interestingly, SP cells were also able to trans-differentiate into angiogenic tubules in vitro and were highly tumorigenic as compared to MP cells. SP-derived tumors demonstrated the intratumoral heterogeneity comprising of both SP and MP cells, suggesting the self-renewal and differentiation ability of SP cells are manifested in vivo as well. βArrestin-1 (βArr1) is involved in the progression of various cancers including NSCLCs and we find that depletion of βArr1 significantly blocked the SP phenotype; whereas depletion of βArr2 had relatively minor effects. Ectopic expression of βArr1 resulted in increased SP frequency and ABCG2 expression while abrogation of βArr1 expression suppressed the self-renewal growth and expansion of A549 cells. Anti-apoptotic protein Mcl-1 is known to be one of the key regulators of self-renewal of tissue stem cells and is thought to contribute to survival of NSCLC cells. Our experiments show that higher levels of Mcl-1 were expressed in SP cells compared to MP cells at both transcriptional and translational levels. In addition, Obatoclax, a pharmacological inhibitor of Mcl-1, could effectively prevent the self-renewal of both EGFR-inhibitor sensitive and resistant NSCLC cells. In conclusion, our findings suggest that βArr1 and Mcl-1 are involved in the self-renewal and expansion of NSCLC-CSCs and are potential targets for anti-cancer therapy

Characterization of SP cells by flow cytometry.

<p>(<b>A</b>) FACS analysis on single cell suspension of human NSCLC cell lines stained with Hoechst 33342 dye showing SP cells. SP cells are enclosed within the area demarcated in pink. Fumitremorgin C inhibited the efflux of the dye and caused the disappearance of SP cells. The frequency of SP cells is represented. (<b>B, C</b>) Cell cycle analysis using area-histogram parameter for the blue emission of Hoechst 33342 as described in the text. The profile shown in B is the representative for all the four cell lines. (<b>D</b>) Aldefluor assay on Hoechst 33342 stained H1975 and H1650 cells. The base line fluorescence was established by inhibiting ALDH activity with DEAB (Left) to generate the gate to identify ALDH-Hi cells in total as well as the SP and MP cells that have not been incubated with DEAB (three right panels). All ALDH-Hi cells are enclosed within the area demarcated in pink. The fold difference in frequency of ALDH-Hi cells between SP and MP cells is plotted.</p

NSCLC-SP cells have cancer stem cell-like properties <i>in vitro</i>.

<p>(<b>A</b>) Growth curve on 10,000 H1650-SP or MP cells were generated using MTT cell proliferation assay in regular growth medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055982#pone.0055982-Singh1" target="_blank">[22]</a>. (<b>B</b>) SP or MP cells from H1650 cell line were plated in serum free medium supplemented with EGF and bFGF for 10 days. The average (±SD) number of spheres from 1000 cells is plotted. (<b>C</b>) H1650-SP cells resulted in larger and more colonies compared to MP cells when plated at the density of 1000 cells per 60 mm plate. (<b>D</b>) Cell viability was assayed after 21 days of plating via MTT assay. (<b>E</b>) SP or MP cells were plated on Matrigel and grown in endothelial growth medium (Lonza) overnight. SP cells gave rise to angiogenic like tubules effectively. (<b>F</b>) Real time qPCR analysis on SP and MP cells performed for <i>CD31</i> mRNA expression. (<b>G</b>) CD31 expression on angiogenic tubules as visualized by immunofluorescence.</p

Mcl-1 regulates the self-renewal of SP cell.

<p>(<b>A, B</b>) Relative expression of Mcl-1 was examined at mRNA and protein levels for indicated cell lines by RT-PCR and western blot analysis. (<b>C</b>) Structure of Obataoclax and its treatment schedule is represented. (<b>D</b>) H1650-SP cells were sorted and plated for self-renewal assay in the presence or absence of Obatoclax at indicated concentration. Average number of spheres generated per well from 1000 cells is plotted (mean±SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. (<b>E</b>) SP cells were sorted from erlotinib resistant H1975 cell line and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated per well from 1000 cells is plotted (mean±SD) and (<b>F</b>) phase contrast microscopy images of the spheres in presence or absence of drugs are presented. (<b>G</b>) Western-blot analysis of βArr1 and βArr2 in Obatoclax treated cells. Inhibition of Mcl-1 did not affect the expression of βArr1 and βArr2 in any of the cell lines tested.</p

Stromally Derived Lysyl Oxidase Promotes Metastasis of Transforming Growth Factor-β–Deficient Mouse Mammary Carcinomas

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR