Régulation de l'expression de la thrombospondine 1 et étude de son rôle dans la tumorigenèse

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The Wnt non-canonical signaling modulates cabazitaxel sensitivity in prostate cancer cells.

BACKGROUND:Despite new drugs, metastatic prostate cancer remains fatal. Growing interest in the latest approved cabazitaxel taxane drug has markedly increased due to the survival benefits conferred when used at an earlier stage of the disease, its promising new therapeutic combination and formulation, and its differential toxicity. Still cabazitaxel's mechanisms of resistance are poorly characterized. The goal of this study was thus to generate a new model of acquired resistance against cabazitaxel in order to unravel cabazitaxel's resistance mechanisms. METHODS:Du145 cells were cultured with increasing concentrations of cabazitaxel, docetaxel/ taxane control or placebo/age-matched control. Once resistance was reached, Epithelial-to-Mesenchymal Translation (EMT) was tested by cell morphology, cell migration, and E/M markers expression profile. Cell transcriptomics were determined by RNA sequencing; related pathways were identified using IPA, PANTHER or KEGG software. The Wnt pathway was analyzed by western blotting, pharmacological and knock-down studies. RESULTS:While age-matched Du145 cells were sensitive to both taxane drugs, docetaxel-resistant cells were only resistant to docetaxel and cabazitaxel-resistant cells showed a partial cross-resistance to both drugs concomitant to EMT. Using RNA-sequencing, the Wnt non-canonical pathway was identified as exclusively activated in cabazitaxel resistant cells while the Wnt canonical pathway was restricted to docetaxel-resistant cells. Cabazitaxel-resistant cells showed a minimal crossover in the Wnt-pathway-related genes linked to docetaxel resistance validating our unique model of acquired resistance to cabazitaxel. Pharmacological and western blot studies confirmed these findings and suggest the implication of the Tyrosine kinase Ror2 receptor in cabazitaxel resistant cells. Variation in Ror2 expression level altered the sensitivity of prostate cancer cells to both drugs identifying a possible new target for taxane resistance. CONCLUSION:Our study represents the first demonstration that while Wnt pathway seems to play an important role in taxanes resistance, Wnt effectors responsible for taxane specificity remain un-identified prompting the need for more studies

In vivo mechanisms by which tumors producing thrombospondin 1 bypass its inhibitory effects

Thrombospondin 1 (TSP1) is a multifunctional protein able to activate TGFβ and to inhibit angiogenesis in vivo. Although usually thought of as an inhibitor of tumor growth, TSP1 may sometimes be present at high levels during tumor progression, suggesting that tumors can eventually overcome their anti-tumor effects. Using a tet-repressible expression system, we demonstrate that murine TSP1 delayed the onset of tumor growth when produced in the tumor bed by rat fibrosarcoma tumor cells or by stromal fibroblasts coinjected with unmodified C6 glioma tumor cells. Yet upon prolonged exposure to TSP1, tumors came to grow at the same rate in the presence as in the absence of TSP1 and transplantation experiments showed that they had become insensitive to inhibition by TSP1 in both syngeneic and immune compromised hosts. Tumor resistance to TSP1 developed as a result of the in vivo outgrowth of pre-existing tumor cell variants that (1) secreted increased amounts of angiogenic factors that counterbalanced the inhibitory effect of TSP1 on neovascularization and (2) grew more efficiently in the presence of TSP1-activated TGFβ. These results indicate that prolonged and continuous local delivery of a single multifunctional angiogenesis inhibitor like TSP1 to fast-growing tumors can lead to tumor resistance in vivo by fostering the outgrowth of subpopulations that are a by-product of the genetic instability of the tumor cells themselves

The synthetic P18 peptide mimics the inflammation-related function of PEDF.

<p>(<b>A</b>) Migration of macrophages (Green) assessed by confocal microscopy, towards 3D CL1 spheroids (Red) treated with PEDF (10 nM), P18 peptide (10 nM) or control buffer for 24 hours. (<b>B</b>) Quantitative analyses using the two point analysis function (NIS-Elements AR 4.00.03, Carl Zeiss) and showing that as PEDF, P18 peptide significantly stimulates the migration of macrophages towards the spheroids. Error bars show SD of the mean, based on two independent experiments. Statistical analyses were performed using the Student’s t test, *: p < 0.05. (<b>C</b>) Nomarski representative images of RAW 264.7 cells treated with PEDF (10 nM), P18 peptide (10 nM) or control buffer indicative of significant changes in macrophages morphology under PEDF and P18 exposure (10 nM). (<b>D</b>) Total RNAs from RAW 264.7 (Top graphs) and BMDMs (tumor-bearing mice; bottom graphs) treated with or without PEDF and P18 were analyzed by qRT-PCR for iNOS, TNFa, and IL10 mRNAs. Results were normalized to S15 and are presented as relative fold change compared to expression levels in non-treated cells. Data points represent mean ± SD of triplicate samples from two independent experiments. Statistical analyses were performed using the Student’s t test, *: p < 0.05. (<b>E</b>) Representative Nomarski/confocal images of CL1-Ctrl PCa cells (Red)–macrophages co-cultures demonstrating that as PEDF, the P18 peptide (10 nM) induced the phagocytosis of CL1 cells <i>in vitro</i>.</p

Effect of Atglistatin on macrophages differentiation and phagocytic activity.

<p>(<b>A</b>) Quantification analyses of PCa cell phagocytosis in CL1-Ctrl/RAW 264.7 co-cultures treated with PEDF (10 nM), Atglistatin (40 μM) or PEDF/Atglistatin combination. Data points represent ROI mean intensity ± SD of triplicate samples per treatment condition from two independent experiments. (<b>B</b>) Quantification analyses of the «dendrite-like» structure length using the ImageJ software. Data points show the mean ± SD of triplicate samples per treatment condition from two independent experiments. Statistical analyses were performed using the Welch and Brown-Forsythe tests followed by the Games-Howell test, *: p < 0.05.</p

PEDF increases the tumoricidal activity of macrophages towards prostate cancer cells <i>in vitro</i>

<div><p>Background</p><p>Although inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment <i>in vitro</i>, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis.</p><p>Methods</p><p>RAW 264.7 and THP-1 cells, and BMDMs were grown <i>in vitro</i> as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry.</p><p>Results</p><p>We found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects were observed with P18 PEDF-derived peptide.</p><p>Conclusions</p><p>These data established that modulating the molecular interactions between macrophages and PCa cells using PEDF may be a promising strategy for PCa treatment.</p></div

PEDF induces the phagocytosis of tumor cells through an apoptosis-dependent mechanism and possibly via the production of Superoxide radicals.

<p>(<b>A, Left</b>) Representative images of CL1-Ctrl PCa cells (Red) and RAW 264.7 macrophages treated with buffer control, PEDF (10 nM), ZVAD (40 μM), or combination. (<b>A, Right</b>) Quantification of tumor cell phagocytosis (ROI mean intensity) averaged from two different experiments. Data were represented using a boxplot graph showing the median, inter-quartile range, upper and lower quartiles, and whiskers. Statistical analysis was performed using the Student's t test. *: P<0.05. (<b>B, Left</b>) Cell cycle distribution of CL1 cells exposed to PEDF (10 nM). Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*p < 0.05). (<b>B, Right</b>) Representative images of fragmented DNA (Green) in CL1 cells treated with PEDF (10 nM) or control showing that PEDF has no direct tumoricidal effect on tumor cells. Nuclei were visualized by DAPI (blue) staining. (<b>C, Left</b>) Representative images of fragmented DNA (Green) in CL1 cells treated with RAW 264.7 CM or CM-PEDF. Nuclei were visualized by DAPI (blue) staining. (<b>C, Right</b>) Apoptosis quantification measured by counting the number of apoptotic cells (Green) over the total amount of cells (>100 cells). Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*p < 0.05). (<b>D</b>) Quantification of Superoxide production by RAW 264.7 macrophages treated ± PEDF (10nM). Data points represent mean ± SD of quadruplicate samples from two independent experiments; *: p < 0.05.</p

Effect of α-CD47 and Angiostatin combination on the phagocytosis of CL1 cells.

<p>Quantification analyses of PCa cell phagocytosis in CL1-Ctrl/RAW 264.7 co-cultures treated with α-CD47 (100 ng/μl) or Angiostatin (10 nM) alone or in combination. Data points represent ROI mean intensity ± SD of triplicate samples per treatment condition from two independent experiments. Statistical analyses were performed using the Welch and Brown-Forsythe tests followed by the Games-Howell test, *: p < 0.05.</p