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. 2013 Jun 18;110(25):10258-63.
doi: 10.1073/pnas.1222404110. Epub 2013 Jun 3.

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

Hanna Zirath  1 , Anna Frenzel, Ganna Oliynyk, Lova Segerström, Ulrica K Westermark, Karin Larsson, Matilda Munksgaard Persson, Kjell Hultenby, Janne Lehtiö, Christer Einvik, Sven Påhlman, Per Kogner, Per-Johan Jakobsson, Marie Arsenian Henriksson
Affiliations

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

Hanna Zirath et al. Proc Natl Acad Sci U S A. .

Abstract

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.

Keywords: cancer therapy; fatty acid oxidation; mitochondria; oxidative phosphorylation; small molecule.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
10058-F4 interferes with the MYCN/Max interaction. (A) Proximity ligation assay (PLA) on MYCN/Max interaction after 6 h treatment with 75 µM 10058-F4 in KCN69n cells (***P < 0.0001, mean ± SD, n = 5). (Left) Green signals indicate close proximity between MYCN and Max. (Center) Immunofluorescence of MYCN expression from the same cell sections. (Right) Merged PLA and MYCN signals. (Scale bar, 20 µm.) (B) Quantification of the PLA in A. (C) Coimmunoprecipitation (IP) of Max/c-MYC and Max/MYCN from cell extracts treated in vitro with 100 μM 10058-F4. (D) Western blot analysis of MYCN expression in MNA NB cell lines treated for 48 h with 10058-F4 at indicated concentrations. (E) EMSAs after treatment in culture with 10058-F4 for 48 h at indicated concentrations. Specific antibodies (Ab) were used to identify DNA-bound c-Myc/Max and MYCN/Max protein complexes, indicated with arrows to the left.
Fig. 2.
Fig. 2.
10058-F4 targets MNA NB cells and induces neuronal differentiation and therapeutic effects in vivo in MNA NB. (A) Change in the percentage of NB cells in the G1 phase of the cell cycle compared with untreated control after treatment with indicated concentrations of 10058-F4 for 24 h (mean ± SD, n = 3). (B) 10058-F4–induced cell death in MNA and non-MNA NB cell lines at indicated concentrations as assessed by the percentage of cells with subG1 DNA content (mean ± SD, n = 3, 72 h). BE(2) indicates SK-N-BE(2) cells. (C) Morphological differentiation of SK-N-BE(2) cells in response to 10058-F4 (60 µM), nerve growth factor (NGF, 50 ng/mL), or in combination for 15 d. (Scale bar, 50 µm.) (D) Western blot shows TrkA expression after 15 d treatment with 10058-F4. (E) Kaplan-Meyer survival of 10058-F4–treated TH-MYCN mice. Two animals per treatment group were homozygous for the MYCN transgene, and the rest were heterozygous. The median number of days in treatment was 11 (n = 27) for control and 21 (n = 9) for 10058-F4–treated animals (P = 0.0303).
Fig. 3.
Fig. 3.
MYC inhibition results in lipid accumulation. (A) Oil Red O staining of SK-N-BE(2) cells treated with 10058-F4 (60 µM) for 3 d. (B) Transmission electron microscopic pictures of SK-N-BE(2) cells treated with 10058-F4 (60 µM) for 8 d. Arrows indicate lipid droplets (N = nucleus). (Scale bar, 1 µm.) (C and D) Immunoblot of MYCN (C) and Oil Red O staining (D) 4 d after transfection of SK-N-BE(2) cells with scrambled (shSCR) or anti-MYCN (shMYCN) shRNA. (E and F) Immunoblot of MYCN (E) and Oil Red O staining (F) in response to JQ1 (2.5 µM) after 48 h and 3 d, respectively. (G) Oil Red O staining of rat embryonic fibroblasts with different c-myc status after treatment with 10058-F4 (100 µM) for 7 d. (Scale bars, 20 µm in all panels unless specified otherwise.)
Fig. 4.
Fig. 4.
Downstream MYC signaling pathways are targeted by 10058-F4. (A–C) Quantitative LC-MS/MS–based proteomic analysis using SK-N-BE(2) cells treated for 20 h with 10058-F4 (60 µM), or after 24 h induction of retrovirally transduced SK-N-BE(2) cells with anti-MYCN shRNA. Ingenuity software and PANTHER classification were used for data analysis and predictions. (A) GO analysis shows significantly affected biological processes. (B) The most significantly affected canonical cell signaling pathways in the two different conditions as indicated.
Fig. 5.
Fig. 5.
Lipid accumulation occurs after inhibition of oxidative phosphorylation or β-oxidation and mitochondrial structure is perturbed by 10058-F4. (A) Number of down-regulated metabolism-related proteins of indicated categories, according to quantitative LC-MS/MS proteomic data (cutoff ratio: 0.667) from the two experimental conditions. (B) Schematic figure shows catalytic steps in metabolic pathways affected by 10058-F4, according to LC-MS/MS proteomic analysis data. Red arrow lines indicate down-regulation of enzymes involved in regulating the respective metabolic steps. (C) Oil Red O staining of lipid accumulation in SK-N-BE(2) cells in response to 3 d treatment with inhibitors of metabolic pathways as indicated. (Scale bar, 20 µm.) (D) Oil red O staining of SK-N-BE(2) cells treated with 10058-F4 in combination with TOFA or cerulenin for 3 d. (Scale bar, 20 μM). (E) Electron microscopic pictures of SK-N-BE(2) cells treated with 10058-F4 (50 µM) for 8 d. Arrows indicate affected mitochondria in the 10058-F4-treated cells. N = nucleus. (Scale bar, 1 µm.)
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