ApoE is under the control the PPARα/HIF1α interplay in breast CSCs.

<p>(A) ApoE mRNA qPCR analysis in N−/T-MS (samples 1–5) and in MCF10/MCF7-MS. (B) ApoE mRNA qPCR analysis in TAF supernantants (10%, 24 h), TNFα (0.75 ng/mL, 24 h)-exposed MCF7-MS. ApoE mRNA qPCR analysis in (C) pV/pPPARα (48 h)-transfected MCF7-MS and MCF10-MS, and in (D) HIF1 vector (48 h) or SCR/siHIF1 (72 h)-transfected MCF7-MS. Data are expressed as mean ±S.D., n = 3, *<i>p</i><0.05, ^#<i>p</i><0.01, ^§<i>p</i><0.005, ANOVA test. n.s.: not significant.</p

Beta-Catenin/HuR Post-Transcriptional Machinery Governs Cancer Stem Cell Features in Response to Hypoxia

<div><p>Hypoxia has been long-time acknowledged as major cancer-promoting microenvironment. In such an energy-restrictive condition, post-transcriptional mechanisms gain importance over the energy-expensive gene transcription machinery. Here we show that the onset of hypoxia-induced cancer stem cell features requires the beta-catenin-dependent post-transcriptional up-regulation of CA9 and SNAI2 gene expression. In response to hypoxia, beta-catenin moves from the plasma membrane to the cytoplasm where it binds and stabilizes SNAI2 and CA9 mRNAs, in cooperation with the mRNA stabilizing protein HuR. We also provide evidence that the post-transcriptional activity of cytoplasmic beta-catenin operates under normoxia in basal-like/triple-negative breast cancer cells, where the beta-catenin knockdown suppresses the stem cell phenotype <i>in vitro</i> and tumor growth <i>in vivo</i>. In such cells, we unravel the generalized involvement of the beta-catenin-driven machinery in the stabilization of EGF-induced mRNAs, including the cancer stem cell regulator IL6. Our study highlights the crucial role of post-transcriptional mechanisms in the maintenance/acquisition of cancer stem cell features and suggests that the hindrance of cytoplasmic beta-catenin function may represent an unprecedented strategy for targeting breast cancer stem/basal-like cells.</p> </div

Peroxisome Proliferator Activated Receptor-α/Hypoxia Inducible Factor-1α Interplay Sustains Carbonic Anhydrase IX and Apoliprotein E Expression in Breast Cancer Stem Cells

<div><h3>Aims</h3><p>Cancer stem cell biology is tightly connected to the regulation of the pro-inflammatory cytokine network. The concept of cancer stem cells “inflammatory addiction” leads to envisage the potential role of anti-inflammatory molecules as new anti-cancer targets. Here we report on the relationship between nuclear receptors activity and the modulation of the pro-inflammatory phenotype in breast cancer stem cells.</p> <h3>Methods</h3><p>Breast cancer stem cells were expanded as mammospheres from normal and tumor human breast tissues and from tumorigenic (MCF7) and non tumorigenic (MCF10) human breast cell lines. Mammospheres were exposed to the supernatant of breast tumor and normal mammary gland tissue fibroblasts.</p> <h3>Results</h3><p>In mammospheres exposed to the breast tumor fibroblasts supernatant, autocrine tumor necrosis factor-α signalling engenders the functional interplay between peroxisome proliferator activated receptor-α and hypoxia inducible factor-1α (PPARα/HIF1α). The two proteins promote mammospheres formation and enhance each other expression via miRNA130b/miRNA17-5p-dependent mechanism which is antagonized by PPARγ. Further, the PPARα/HIF1α interplay regulates the expression of the pro-inflammatory cytokine interleukin-6, the hypoxia survival factor carbonic anhydrase IX and the plasma lipid carrier apolipoprotein E.</p> <h3>Conclusion</h3><p>Our data demonstrate the importance of exploring the role of nuclear receptors (PPARα/PPARγ) in the regulation of pro-inflammatory pathways, with the aim to thwart breast cancer stem cells functioning.</p> </div

Hypoxia induces CA9 and SNAI2 expression via HIF1-alpha dependent mRNA production and beta-catenin dependent stabilization.

<p><b>A</b>, HIF-1alpha transcriptional reporter (HRE-Luc) assay in MCF7 cells transfected with wild type beta-catenin (Beta-wt) under Nor/1%pO₂ conditions or in combination with HIF1-alpha (HIF1a) encoding vector; <b>B</b>, HRE-Luc assay in 1%pO₂-exposed ctrl/shBeta MCF7 cells; <b>C</b>, CA9-Luc and SNAI2-Luc assay in ctrl/Beta-wt transfected and in ctrl/shBeta MCF7 cells under Nor/1%pO₂ conditions; <b>D</b>, CA9 and SNAI2 mRNA stability assay following inhibition of Polymerase 2 transcriptional activity by actinomycin D (100ng/ml) in ctrl/shBeta MCF7 cells exposed to 1%pO₂; <b>E</b>, Schematic representation of the HIF1-alpha/beta-catenin interplay in breast cancer cells in response to hypoxia: HIF1-alpha promotes transcription and cytoplasmic beta-catenin enhances stabilization of SNAI2 and CA9 mRNAs; the negative effect of beta-catenin on HIF-1alpha-induced transcription is also depicted. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

Beta-catenin enhances the breast cancer stem cell phenotype in response to hypoxia independently of its nuclear transcriptional activity.

<p><b>A</b>, Western analysis (WB) of beta-catenin, SNAI2 and CA9 protein levels in Ctrl/shBeta MCF7 cells upon Nor/1%pO₂ conditions; <b>B</b>, MS forming assay in stable beta-catenin silenced (shBeta) MCF7 cells upon Nor/1%pO₂ conditions; <b>C</b>, Cytofluorimetric analysis of CD44^high/CD24^low stem/progenitor population in ctrl/shBeta MCF7 cells upon Nor/1%pO₂ conditions; <b>D</b>, Real-Time PCR analysis of ESR1 mRNA level in Ctrl/shBeta MCF7 cells upon Nor/1%pO₂ conditions; <b>E</b>, Immunofluorescence (IF) analysis of Beta-catenin in Nor/1%pO₂ MCF7 cells; <b>F</b>, WB analysis of beta-catenin in Nor/1%pO₂ MCF7 cells cytoplasmic and nuclear fractions (lamin B and beta-tubulin were used as fractionation controls); <b>G</b>, beta-catenin/TCF transcriptional reporter (TOPFLASH) assay in MCF7 cells and T-MS under Nor/1%pO₂. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

Inhibition of ApoE expression in T-MS.

<p>(A) PPARα mRNA qPCR analysis, (B) PPRELuc activity and (C) MS formation assay in siApoE (48 h)-transfected MCF7 cells, in normoxic and hypoxic condition; (D) ApoE CAIX, IL6, SLUG, KRT18 and ERα mRNA qPCR analysis in siApoE (48 h)-transfected MCF7-MS. (E) CAIXLuc, IL6Luc, SLUGLuc and ERαLuc activity in siApoE (48 h)-transfected MCF7-MS; ApoE mRNA qPCR analysis (F) and ApoE protein expression (G) in PGZ (20 µM, 24 h)-exposed MCF7-MS and T-MS (samples 19–20). Data are expressed as mean ±S.D., n = 3, *<i>p</i><0.05, ^#<i>p</i><0.01, ^§<i>p</i><0.005, ANOVA test. n.s.: not significant.</p

Beta-catenin maintains the stem/progenitor cell pool in normoxia, independently of its nuclear transcriptional activity.

<p><b>A</b>, CA9 and SNAI2 Real Time PCR analysis and CA9-luc and SNAI2-Luc promoter activity in adherent MCF7 cells and MCF7-MS; <b>B</b>, WB analysis of SNAI2 and CA9 in MCF7-MS; <b>C</b>, beta-catenin IF analysis in adherent MCF7 cells and MCF7-MS; <b>D</b>, TOPFLASH assay and WB analysis of beta-catenin in adherent MCF7 cells and MCF7-MS. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

Schematic representation of the data.

<p>TAF secreted TGFβ induces TNFα expression in breast CSCs. TNFα binds TNFR1 on breast CSCs and activates the PPARα/HIF1α interplay which up-regulates miR130b expression. The interplay is counterbalanced by PPARγ via miR17-5p up-regulation. In turn, the PPARα/HIF1α interplay regulates CAIX, ApoE, IL6 and SLUG expression.</p

PPARγ antagonizes the PPARα/HIF1α interplay and inhibits pro-inflammatory CSCs pathways.

<p>(A) PPARγ mRNA qPCR analysis in HIF1 vector (48 h) or SCR/siHIF1 (72 h)-transfected MCF7-MS. (B) PPARα and PPARγ mRNA qPCR analysis in adherent MCF10 <i>vs</i> MCF10-MS and in adherent MCF7 <i>vs</i> MCF7-MS; (C) HRELuc activity in hypoxic T-MS (samples 15–16) exposed to PPARγ agonist PGZ (20 µM, 24 h) (D) NF-κBLuc, IL6Luc, SLUGLuc activity in MDA-MB-231 breast cancer cells exposed to PGZ (20 µM, 24 h). Data are expressed as mean ±S.D., n = 3, *<i>p</i><0.05, ^#<i>p</i><0.01, ^§<i>p</i><0.005, ANOVA test. n.s.: not significant.</p

Autocrine TNFα loop in MS exposed to the TAF supernatant.

<p>TNFR1 (A), TNFα (B) and IL6 (C) real-time reverse transcriptase quantitative (qPCR) mRNA analysis in MCF7-MS, MCF10-MS, T-MS and N-MS (samples 1–5). (D) MS formation assay in TNFα (0.75 ng/mL), NAF and TAF supernatant (10% final concentration)-exposed MCF10 and MCF7 for 24 to 72 h; (E) TNFα and TGFβ ELISA test on TAF and NAF supernatants (samples 6–12); (F) TNFα mRNA qPCR analysis in TGFβ (1.0 ng/mL, 24 h)-exposed MCF10-MS and MCF7-MS (G) TNFα mRNA qPCR analysis in TAF supernatant (10%, 24 h)-exposed MCF10/MCF7-MS, and in N−/T-MS (samples 5–6, n = 2); (H) TAF supernatant-induced T-MS formation assay in presence/absence of TNFα inhibitory antibody (1.5 µg/mL, 24 h, sample 13). Data are expressed as mean ± Standard Deviation (S.D.), n = 3 unless otherwise specified, *<i>p</i><0.05, #<i>p</i><0.01, §<i>p</i><0.005, ANOVA test. n.s.: not significant.</p