LIPG‐promoted lipid storage mediates adaptation to oxidative stress in breast cancer

Figures

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  Figure 1. LIPG overexpression in MCF‐7 cells results in intracellular lipid storage. MCF‐7 cells were transfected with a LIPG construct (LIPG‐OE) or an empty vector (EV) and incubated for 48 h with (a) 800 μg HDL, (b) 800 μg PC‐OA in serum‐free DMEM, (c) 800 μg oleic acid (OA) complexed to bovine serum albumin and (d) no substrate. Intracellular triacylglyceride (TAG) levels were quantified with the triglyceride quantification assay kit. PLIN2 mRNA levels were analysed by qPCR. Lipid droplets were visualized with Bodipy 493/503 staining (green). Nuclei were stained with DAPI (blue). The bars represent the mean ±SEM and pictures are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, unpaired two‐tailed Student's t‐test. [Color figure can be viewed at wileyonlinelibrary.com]

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  Figure 2. Upregulation of LIPG in NeuT‐induced senescence contributes to lipid storage. (a) Representative Western blot showing expression of the NeuT oncogene in MCF‐7/NeuT cells treated with doxycycline (+dox) or untreated (−dox) at different time points. (b) Immunofluorescence illustrating senescence‐associated morphological changes and co‐induction of the reporter EGFP (green) upon dox treatment. The actin cytoskeleton was stained with rhodamine phalloidin (red); nuclei were stained with DAPI (blue); scale bar: 50 μm. (c) qPCR showing LIPG mRNA expression in MCF‐7/NeuT cells incubated with/without dox during the indicated time periods. Data represent the mean fold change in mRNA relative to time point 0 h ± SEM (n = 3). (d) Representative Western blot showing the 68 kDa LIPG protein and its 40 kDa cleavage product in supernatants of MCF‐7/NeuT cells (6d −/+ dox) incubated with heparin and densitometric quantification of the 68 kDa LIPG band from Western blots signals of three independent experiments (see also Fig. S3, Supporting Information). (e) Quantification of cellular triacylglycerides (TAG) in MCF‐7/NeuT cells cultivated for 7d with/without dox (time point of high LIPG…

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  Figure 3. LIPG becomes upregulated upon cellular stress‐mediated inhibition of de novo fatty acid synthesis (FAS). (a) Schematic illustration of the initial steps of de novo FAS, regulation of acetyl‐CoA carboxylase (ACC) by AMPK, and pharmacological inhibition by TOFA and cerulenin. (b) qPCR analysis of LIPG mRNA expression in MCF‐7 cells incubated for 24 h with 6 μM TOFA (n = 4). (c) Representative Western blot showing protein levels of TXNRD1 in MCF‐7/NeuT cells incubated with/without dox at the indicated time points. (d) TBARS assay showing levels of ROS in cellular extracts of MCF‐7/NeuT cells incubated 7d with/without dox (n = 3). (e) Representative Western blots showing the phosphorylation status of AMPK in MCF‐7/NeuT cells (6d with/without dox) and densitometric quantification of the ratios of phospho‐AMPK (p‐AMPK) and total‐AMPK (t‐AMPK) to β‐actin from Western blot signals of three independent experiments. (f) Representative Western blot showing the phosphorylation status of ACC in MCF‐7/NeuT cells (6d with/without dox) and densitometric quantification of the ratios of phospho‐ACC (p‐ACC) and total‐ACC (t‐ACC) to calnexin from Western blot signals of three independent…

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  Figure 4. Upregulation of LIPG by CoCl2 contributes to lipid storage and adaptation to oxidative stress and is abrogated by ROS scavengers. (a) Representative Western blot showing AMPK phosphorylation in MCF‐7 cells exposed to different concentrations of CoCl2 for 24 h and densitometric quantification of the ratio (phospho‐AMPK/total‐AMPK) from Western blot signals of three independent experiments. (b) Representative Western blot showing ACC phosphorylation in MCF‐7 cells exposed to 0.89 mM of CoCl2 for 24 h and densitometric quantification of the ratios (p‐ACC and t‐ACC to calnexin) from Western blot signals of three independent experiments. (c) qPCR analysis showing LIPG, PLIN2 and TXNRD1 mRNA levels in MCF‐7 cells treated for 24 h with the indicated concentrations of CoCl2. (d) Quantification of triacylglycerides (TAG) in MCF‐7 cells exposed for 24 h to the indicated concentrations of CoCl2 and visualization of lipid droplets by BODIPY 493/503 staining (green) in 0.89 mM CoCl2‐treated and untreated (FM) MCF‐7 cells; red: Rhodamine phalloidin staining of the actin cytoskeleton; blue: DAPI. Scale bars: 20 μm. (e) Visualization of lipid droplets in MCF‐7 cells exposed to 0.89 mM CoCl2 for…

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  Figure 5. Expression of LIPG in human breast cancer and association with metastasis‐free survival. (a) Box plots showing the distribution of LIPG mRNA expression in 18 breast cancer datasets, including node‐negative, untreated cohorts as well as cohorts of patients who had received adjuvant (tamoxifen (TAM) only), hormonal and/or chemotherapy and/or both and/or unknown treatment (adjuvant*) and neoadjuvant therapy. (b) Correlation of Affymetrix and qPCR results with Pearson correlation r = 0.9623, p < 0.001. (c) Kaplan–Meier plots showing association of high LIPG expressing tumours (4 out of 200 tumours using the cut‐off log2 ≥ 98th percentile) with shorter metastasis free survival (MFS) in the Mainz cohort (GSE11121) (left) and in the combined cohort of untreated patients (16 out of 824 tumours, consisting of GSE11121, GSE2034, GSE5327, GSE6532 and GSE7390) (right). p*: p‐Value of the permutation test; p: p‐Value from the log‐rank test. (d) top: Representative images of LIPG IHC of 259 node‐negative breast carcinomas of the tissue microarray (TMA); scale bars: 100 μm; (d) bottom, left: barplot showing LIPG protein expression distribution in the TMA; (d) bottom, middle: Kaplan–Meier plot…