Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer

Abstract

Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors.

Keywords: HDAC inhibitor; breast cancer; carnitine palmitoyl transferase-1A; tumor metabolism; tumor specific target.

Figure 1. Protein expression, localization and transferase activity of CPT1A in MCF7 breast cancer cells compared to MCF12F control cells

A. Western blot analysis of CPT1A from nuclear extracts of MCF7 and MCF12F cells. β-actin protein level was shown as normalizer. B. RT-PCR analysis of CPT1A isoforms (CPT1Av1 and CPT1Av2) expression in SK-BR-3 and MDA-MB-231 cells. 327bp was the expected size for the variant 1 amplicon, the classical form of CPTI-A. As shown only the CPTI-Av2 was detected in SK-BR3 and MDA-MB-231 cells (amplicon size: 163 bp). C. Immunostaining of HA was performed in MCF-7 and MCF12F cells transfected with empty plasmid (CTRL) or CPT1Av2. D. Transfection efficiency was evaluated by RT-PCR after 72 hrs. Amplification for β2-microglobulin was performed as control E. Spectrophotometrical assay was performed to analyze CPT activity from nuclear and cytoplasm extracts of MCF-7 and MCF12F transfected cells. Histograms show the relative percent activity obtained by Ellman's reagent at 412 ηm, from three independent experiments.

Figure 2. Expression levels of the two CPT1A isoforms (CPT1Av1 and CPT1Av2) in 48h silenced MCF-7

A. Cells were transfected with siRNA matching both the two variants (siRNAall), the transcript variant 2 (siRNA₂) or scrambled sequence (NC). β2-microglobulin house-keeping gene was amplified to normalize template input. Results are representative of three independent experiments. B. Real time RT-PCR analysis. Data are expressed after normalizing to β2-microglobulin levels (endogenous control). Results are mean values (± standard deviations) of triplicate from two independent experiments. *P value < 0.01. C. CPT1A immunostaining in MCF-7 cells 48h after silencing with siRNAs matching the variant 1 (siRNA₁), the variant 2 (siRNA₂) or targeting both the two CPT1A variants (siRNA_all). Images are representative of three independent experiments. D. A minimum of 100 cells per sample were observed using optical microscope at 20X magnification lens. The results (means values ± standard deviations) were given as percentage of CPT1A positively-stained nuclei per total cell nuclei number. *P= 0.03, siRNA₂ or siRNA_all-transfected versus mock transfected (NC).

Figure 3

A. Cell viability after CPT1Av2 silencing in MCF-7 (A1), SK-BR-3 (A2) and MDA-MB-231 (A3) cells. Dye exclusion test (trypan blue staining) performed in cells transfected for 72h with siRNA2 at different concentrations. Decreased viability was significant at 70 nM CPT1A2 siRNAs. The cell viability on SK-BR-3 and MDA-MB-231 and the apoptotic assay were performed at 70 nM siRNAs concentration. Results are mean values ± standard deviations of three independent experiments. *P= 0.05, siRNA2 versus NC. **P<0.001, siRNA2 versus NC. B. Apoptosis detection with Annexin V (FITC) and propidium iodide (red) staining (40X magn.) The cells seeded in 4 wells/chamberslides were transfected for 72h with scrambled sequence (NC) or CPT1A siRNA2. Silenced cells showed early (Ann+, PI-) and late (Ann+, PI+) apoptotic events (arrows), as compared with un-treated cells. C. HOECHST DNA staining. Images show representative results of three independent experiments. Arrows indicates apoptotic figures and nuclear fragmentation. 5mM sodium butyrate was added in culture 48h after silencing (72h full incubation time), or alone for 24h. A particular of a nucleus is shown in the upper right side. Original magnification 100X. D. The results of HOECHST staining obtained from three independent experiments are given as percentage of apoptotic figures per total cell nuclei number. Asterisks indicates statistical significant differences (P≤0.05) in treated cells versus scrambled control (NC). Butyrate alone was able to induce apoptotic cell death also in MCF-12F control cells. E. CASPASE-9 expression in protein extracts from CPT1Av2 silenced (siRNA₂) or/and treated with 5mM sodium-butyrate (NaB) MCF-7 cells versus scrambled control (NC).

Figure 4. HDAC activity and HDAC1 expression in protein extracts from CPT1Av2 silenced (siRNA2) or/and treated with 5mM sodium-butyrate (NaB) MCF-7 cells

A. Colorimetric assay was performed to analyze HDAC activity on nuclear extracts of MCF-7 cells. Histograms show the optical densities (O.D.) (means ± standard deviations) obtained by ELISA plate reader at 405 nm from three independent experiments. *P≤0.03, siRNA₂ or butyrate-treated versus mock transfected cells (NC). ^#P=0.1, siRNA2 and butyrate co-treated versus single treatments. B. Western blot analysis for the different classes (I, II and III) of HDAC proteins was performed on protein extracts from CPT1Av2-silenced (siRNA₂) and/or butyrate-treated (NaB) MCF-7 cells. β-actin or Sp1 levels are reported as normalizer of the extracts.

Figure 5. The molecular complex CPT1-Is2/HDAC1 displays an higher stability compared with the other complex formed with CPT1-Is1

A. Resulting complexes of CPT1-Is1/HDAC and CPT1-Is2/HDAC B. as obtained by molecular docking conducted by PatchDock and refined by FireDock. The CPT1 proteins are reported as gold ribbon, the residues that are different in the two isoforms (residues 746-772 for CPT1-Is1 and 746-755 for CPT1-Is2) are red colored, the protein surface is also reported as semitransparent and cyan colored. HDAC-like protein (1c3r) is reported as coral ribbon; the active site, identified as the residues in contact with the substrate [20], are depicted in green C. Root mean square deviations (RMSD) of CPT1A from its starting position in the complex with HDAC1. Rototranslational motions were removed by fitting the positions of HDAC atoms to their coordinates in the initial structure.

Figure 6. Acetyl-histone H4 expression levels in protein extracts from CPT1Av2 silenced (siRNA2) or/and treated with 5mM sodium-butyrate (NaB) MCF-7 cells

A. Western blot analysis of acetyl histone H4 protein was performed in acid protein extracts from CPT1A2 silenced (48h) and/or butyrate treated (24h) MCF-7 cells. Optical densities (O.D.) indicate mean values ± standard deviations of three independent experiments as described in materials and methods.* P ≤0.02, siRNA₂ or butyrate-treated versus mock transfected cells (NC). ^#P=0.08, siRNA2 and butyrate co-treated versus single treatments. B. Immunostaining of acetyl histone H4 (Ac-H4) was performed on MCF-7 cells transfected for 48h with scrambled sequence (NC) or siRNAs against CPT1Av2 (siRNA2), or treated with sodium butyrate (NaB) for 24h. Apoptotic bodies were evident in siRNA2 transfected cells (arrow).

Figure 7. Expression analysis of CPT1Av2 silencing target genes

A. Array image of Oligogene array cancer pathway finder performed on mRNAs extracted from CPT1A2 silenced or mock treated (NC) MCF-7 cells. B. The mRNA expression folds (y axis) were obtained comparing the mRNA levels detected in siRNA₂ treated MCF-7 cells (48h) versus mRNA levels in mock-transfected cells (NC), as mean of triplicate experiments. Only the expression fold ≥ 1,5 was relevant and included in the histogram (P ≤ 0.05).