p140Cap modulates the mevalonate pathway decreasing cell migration and enhancing drug sensitivity in breast cancer cells

Abstract

p140Cap is an adaptor protein involved in assembling multi-protein complexes regulating several cellular processes. p140Cap acts as a tumor suppressor in breast cancer (BC) and neuroblastoma patients, where its expression correlates with a better prognosis. The role of p140Cap in tumor metabolism remains largely unknown. Here we study the role of p140Cap in the modulation of the mevalonate (MVA) pathway in BC cells. The MVA pathway is responsible for the biosynthesis of cholesterol and non-sterol isoprenoids and is often deregulated in cancer. We found that both in vitro and in vivo, p140Cap cells and tumors show an increased flux through the MVA pathway by positively regulating the pace-maker enzyme of the MVA pathway, the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), via transcriptional and post-translational mechanisms. The higher cholesterol synthesis is paralleled with enhanced cholesterol efflux. Moreover, p140Cap promotes increased cholesterol localization in the plasma membrane and reduces lipid rafts-associated Rac1 signalling, impairing cell membrane fluidity and cell migration in a cholesterol-dependent manner. Finally, p140Cap BC cells exhibit decreased cell viability upon treatments with statins, alone or in combination with chemotherapeutic at low concentrations in a synergistic manner. Overall, our data highlight a new perspective point on tumor suppression in BC by establishing a previously uncharacterized role of the MVA pathway in p140Cap expressing tumors, thus paving the way to the use of p140Cap as a potent biomarker to stratify patients for better tuning therapeutic options.

Fig. 1. p140Cap increases the metabolic flux through the MVA pathway.

A List of Gene Ontology terms related to cholesterol and lipid metabolism that are enriched in a dataset of up-regulated genes in MDA-MB-231 p140Cap cells. B Schematic representation of MVA pathway with the main intermediates, end-products, and targeted enzymes. The de novo synthesis of cholesterol (C), geranylgeranyl diphosphate (GGPP) (D), and ubiquinone (UQ) (E) was measured in mock and p140Cap MDA-MB-231, SKBR3, and HEK293T cells by incubating with 1 µCi [³H] acetate for 24 h, recovering the lipids, and measuring the radioactivity by liquid scintillation. Unpaired test (**P < 0.001; ***P < 0.0001). Error bar: SEM. F, G, H Cholesterol, GGPP, and UQ synthesis in SKBR3 cells treated with the indicated concentration of the MVA pathway inhibitors Simvastatin (SIMV), Zoledronic Acid (ZA), and Squalestatin 1 (SQ). ANOVA test and Bonferroni post-test (***P < 0.0001), Error bar: SEM.

Fig. 2. p140Cap controls HMGCR through transcriptional and post-translational mechanisms.

A HMGCR activity in mock and p140Cap MDA-MB-231 and SKBR3 cells; 60 nCi [14 C] HMG-CoA was added to microsomal extracts. Mevalonolactone was recovered and quantified by liquid scintillation. B HMGCR activity in tumors derived from mock and p140Cap TUBO cells orthotopically injected in Balb/c mice. C Immunoblot of immunoprecipitated total HMGCR and pSer HMGCR from mock and p140Cap MDA-MB-231 and SKBR3 cells. The ER-resident protein Calreticulin (CRT) was used as a loading control of microsomal extracts. D Quantification of mRNA levels of genes involved in the mevalonate pathway in SKBR3 p140Cap cells relative to mock cells. E Analysis of SREBP2 activity by Dual Luciferase assay in HEK293T cells transiently transfected with the indicated vectors. F Protein levels of nuclear mature SREBP2 (mSREBP2) normalized on Lamin A/C at different time points after transfection of p140Cap in HEK293T cells (see the relative western blot in Supplementary Fig. 1I). G, H Immunoblot showing protein levels and ubiquitination of immunoprecipitated HMGCR in control cells (CTRL), following cholesterol loading (+chol) or depletion (+MβCD) experiments in MDA-MB-231 and SKBR3 cells. The ER-resident CRT protein was used as a loading control. I HMGCR ubiquitination levels in MDA-MB-231 through an E3 ligases activity assay. J, K Quantifications of loading depletion experiments. Cholesterol loading assays: cells were incubated with β-methyl cyclodextrin (MβCD) then fresh medium containing cholesterol/MβCD complexes was added. Cholesterol depletion assays: cells were incubated with MβCD. Cholesterol was measured using the Cholesterol Fluorimetric Assay kit and is expressed as µmol cholesterol/mg cell proteins. L Measurement of cholesterol levels in tumors in Balb/c mice derived from mock and p140Cap TUBO cells. M Immunoblot analysis of p140Cap in microsomal fractions and whole cell lysates of MDA-MB-231, TUBO and HEK293T cell lines. The ER-resident CRT protein was used as a loading control. From (A–L) Unpaired test (*P < 0.05; **P < 0.01; ***P < 0.001). Error bar: SEM.

Fig. 3. p140Cap increases cholesterol efflux through enhanced activity of ABCA1 and ABCG1.

A Cholesterol efflux in MDA-MB-231 and SKBR3 cells. After incubation with 1 µCi/ml with [³H] cholesterol, cells were washed and let grow in a fresh medium for 24 h. Media was collected, cholesterol was extracted and quantified by liquid scintillation. The amount of [³H] cholesterol effluxed in mock cells was considered 100. B Evaluation of ABCA1/ABCG1 activity: ABCA1 and ABCG1 were immunoprecipitated from plasma membrane vesicles and the ATPase activity was measured by a spectrophotometric assay. The absorbance was converted into μmol hydrolyzed phosphate/min/mg proteins. ATPase activity was expressed as a percentage towards mock cells. C, D Cholesterol synthesis measurements upon 1 uM Simvastatin (SIMV) treatment for 48 h. E, F Relative HMGCR activity in cells treated with 1 uM SIMV for 48 h. G, H Cholesterol efflux measured upon 1uM SIMV treatment for 48 h. From (A–H) % ANOVA and Bonferroni post-test (**P < 0.01; ***P < 0.001). Error bar: SEM.

Fig. 4. p140Cap decreases membrane fluidity impairing cell migration ability.

A, B Membrane cholesterol quantification in mock and p140Cap MDA-MB-231 and SKBR3 cells. C Cholesterol measurement of membranes derived from TUBO mock and p140Cap tumors. D, E Membrane fluidity was measured in cholesterol loading/depletion conditions using a membrane fluidity kit according to the manufacturer’s protocol. From (A–E) ANOVA and Bonferroni post-test (*P < 0.05; ***P < 0.001; Error bar: SEM). F Representative images of Wound healing migration assays. 1 × 10⁶ MDA-MB-231 cells were seeded in each well and allowed to reach confluence as a monolayer. The monolayer was scratched across the center of the well and gently washed to remove the detached cells. Cells were cholesterol loaded and allowed to migrate for 48 h. G Quantification of wound area. ANOVA and Bonferroni post-test (*P < 0.05; ***P < 0.001; Error bar: SEM).

Fig. 5. p140Cap downregulates Rac1 activity in lipid rafts.

A, B Mock and p140Cap MDA-MB-231 and SKBR3 cells were labeled with Alexa CTX-β and analyzed by flow cytometry. Quantification was performed considering fluorescence mean intensity for each sample. C Immunoblot of fractions recovered from a sucrose gradient ultracentrifuge of MDA-MB-231 cells. Triton-X 100 insoluble fractions were identified by flotillin-1, whereas TritonX-100 insoluble fractions are positive for CD71. D Rac1 Activation Assay performed on the lipid-raft enriched fraction (#4) of MDA-MB-231 cells. Representative immunoblot of the pull-down experiment with Rac1, WB of flotillin-1, and p140Cap. E Densitometric analysis of relative GTP-Rac1 levels, normalized on flotillin-1. In (A, B, E) ANOVA and Bonferroni post-test (*P < 0.05; ***P < 0.001; Error bar: SEM).

Fig. 6. p140Cap increases cell sensitivity to statins treatment.

A, B Fraction of viable cells upon simvastatin treatment. The absorbance of untreated cells was considered 100%; the results were expressed as a percentage of viable cells treated with SIMV vs. untreated cells. C, D, E Viability assay quantification of cells treated with chemotherapeutic agents or the combinational treatment (F, G, H) with chemotherapeutic agents and SIMV at the indicated concentrations. ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001). Error bar: SEM. I Combination index upon combined treatment of Doxorubicin and Simvastatin at indicated concentrations: CI < 1 indicates a synergistic effect. DRI50 is the dose reduction index. CI was calculated with the CalcuSyn software (www.biosoft.com/w/calcusyn.htm).

Fig. 7. Schematic representation of p140Cap effects in cholesterol metabolism of BC cells.

p140Cap expressing cells exhibit upregulated activity of HMGCR due to increased SREBP2 transcription factor activity and HMGCR protein stability. p140Cap associates to the ER compartment. Moreover, p140Cap cells show enhanced cholesterol efflux and increased cholesterol localization to the plasma membrane, leading to reduced membrane fluidity and cell migration ability. p140Cap also associates with lipid rafts, negatively regulating Rac1 activity. Finally, p140Cap expression decreases cell viability upon statin, conventional chemotherapeutic agents, and combined treatment.