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

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

Involvement of the beta-catenin-driven machinery in the stabilization of EGF-induced mRNAs.

<p><b>A</b>, <b>B</b>, Fluidigm® Real-Time PCR analysis of EGFR pathway in ctrl/shBeta MDA-MB-468 and MDA-MB-231 cells. The assay distinguishes mature-mRNA and immature (pre-mRNA) levels; <b>C</b>, IL6 mRNA stability assay following Polymerase 2 transcriptional activity inhibition by actinomycin D (100ng/ml) in 1%pO₂ exposed ctrl/shBeta MCF7 and MDA-MB-468 cells; <b>D</b>, Quantitative IL6 mRNA immunoprecipitation assay with mouse IgG/beta-catenin antibody in 1%pO₂ T-MS (Real-Time PCR) and normoxic MDA-MB-468 cells (Standard RT-PCR); <b>E</b>, Real-Time PCR of IL6 mRNA levels in PRF and 40S cytoplasmic fractions in Nor/1%pO₂ MCF7, MDA-MB-468 and MDA-MB-231 cells; <b>F</b>, IL6 promoter-driven luciferase (IL6-Luc) assay in Ctrl, Beta-wt and 1%pO₂ exposed shBeta MCF7 cells. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

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

Beta-catenin stabilizes CA9 and SNAI2 mRNAs through direct binding and facilitating the shift of HuR/mRNA complexes to the ribosomal compartment.

<p><b>A</b>, CA9 and SNAI2 3’UTR-luciferase reporter (CA9-3’UTR-Luc and SNAI2-3’UTR-Luc) assay in ctrl/shBeta MCF7 cells exposed to Nor/1%pO₂ conditions; <b>B</b>, CA9 and SNAI2 3’UTR-luciferase reporter assay in ctrl/shBeta MDA-MB-468 cells; Schematic diagram of the CA9 or SNAI2 3’UTR luciferase encoding vectors carrying either CA9 or SNAI2 3’UTR sequences inserted between the luciferase coding sequence and the polyadenylation site; <b>C</b>, Quantitative CA9 and SNAI2 mRNA immunoprecipitation assay with mouse IgG/beta-catenin antibody in 1%pO₂ MCF7 cells, MDA-MB-468 and MDA-MB-231 cells; <b>D</b>, WB analysis of cytoplasm pre-ribosomal (PRF) and 40S ribosomal (40S) cytoplasmic fractions of ctrl/shBeta MCF7 upon exposure to Nor/1%pO₂ conditions; total beta-catenin protein levels are reported in Figure 2A and S5A; E, Real-Time PCR analysis of CA9 mRNA levels in PRF/40S cytoplasmic fractions of 1%pO₂ exposed ctrl/shBeta MCF7, MDA-MB-468 and MDA-MB-231 cells; <b>F</b>, WB of beta-catenin and HuR protein levels in ctrl/beta-wt and in ctrl/shBeta MCF7 cells; <b>G</b>, WB analysis of HuR in PRF and 40S cytoplasmic fractions of ctrl/shBeta MCF7 cells exposed to Nor/1%pO₂; note that total HuR protein levels of Nor/1%pO₂ MCF7 cells refer to Figure S5D; H, Schematic representation of the cytoplasmic beta-catenin/HuR post-transcriptional machinery in the regulation of CA9 and SNAI2 mRNAs, via stabilization, direct binding and shuttling to the ribosomal compartment. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

In normoxic basal-like breast cancer cells, cytoplasmic beta-catenin promotes stem cell features <i>in vitro</i> and tumor growth <i>in vivo</i> via constitutive stabilization of CA9 and SNAI2 mRNAs.

<p><b>A</b>, Beta-catenin IF analysis in luminal MCF7 cells and in basal-like MDA-MB-468 and MDA-MB-231 cells; <b>B</b>, Cytofluorimetric analysis of the CD44^high/CD24^low population in ctrl/shBeta MDA-MB-468 and MDA-MB-231 cells; <b>C</b>, Ctrl/shBeta MDA-MB-468 10-weeks mammary fat pad xenograft assay (n=5, each group); representative pictures of xenograft tissues hematoxylin-eosin staining are include; <b>D</b>, WB analysis of CA9, SNAI2 and beta-catenin protein levels in ctrl/shBeta normoxic MDA-MB-468 and MDA-MB-231 cells; <b>E</b>, CA9 and SNAI2 mRNA stability assay following inhibition of Polymerase 2 transcriptional activity by actinomycin D (100ng/ml) in Ctrl/shBeta MDA-MB-468 and MDA-MB-231 cells; <b>F</b>, Four weeks growth curve of ctrl/shSNAI2 MDA-MB-231 subcutaneous xenograft assay (n=6, each group); <b>G</b>, Hematoxylin-eosin, ESR1 and CDH1 immunohistochemical stainings in xenograft tissue sections. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-3

Y as untreated cells. c. 10 nmol/L cetuximab treated cells. Filopodi are evident. Insert: microvilli reduction is evident. d. 10 nmol/L cetuximab plus 10 nM EGF treated cells. Filipodi and some vesicles are evident. Insert: microvilli reduction is evident. e. 1 μmol/L gefitinib treated cells. Some vesicles are evident. Insert: microvilli reduction is evident. f. 1 μmol/L gefitinib plus 10 nM EGF treated cells. Lamellipodi, some vesicles and weak contacts with nearby cells are evident. Insert: microvilli reduction is evident.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-1

<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-4

Li are present. b. EGF treated cells. A lot of vesicles are evident. Insert: a small number of microvilli are present. c. 10 nmol/L cetuximab treated cells. The microvilli diminish in number and lose their erect position. Insert: microvilli make contacts forming a star morphology. d. 10 nmol/L cetuximab plus 10 nM EGF treated cells. Same morphology as 10 nmol/L cetuximab treated cells. e. 1 μmol/L gefitinib treated cells. The microvilli diminish in number and lose their erect position. Insert: microvilli make contacts forming a star morphology. f. 1 μmol/L gefitinib plus 10 nM EGF treated cells. Same morphology as 1 μmol/L gefitinib treated cells.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p