Rapamycin regulates stearoyl CoA desaturase 1 expression in breast cancer

Abstract

Mammalian target of rapamycin (mTOR) signaling is a central regulator of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, making mTOR a promising therapeutic target. In clinical trials, rapamycin analogs have shown modest response rates for most cancer types, including breast cancer. Therefore, there is an urgent need to better understand the mechanism of action of rapamycin to improve patient selection and to monitor pathway inhibition. To identify novel pharmacodynamic markers of rapamycin activity, we carried out transcriptional profiling of total and polysome-associated RNA in three breast cancer cell lines representing different subtypes. In all three cell lines, we found that rapamycin significantly decreased polysome-associated mRNA for stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Activators of mTOR increased SCD1 protein expression, whereas rapamycin, LY294002, and BEZ235 decreased SCD1 protein expression. Rapamycin decreased total SCD1 RNA expression without inducing a significant decline in its relative polysomal recruitment (polysome/total ratio). Rapamycin did not alter SCD1 mRNA stability. Instead, rapamycin inhibited SCD1 promoter activity and decreased expression of mature transcription factor sterol regulatory element binding protein 1 (SREBP1). Eukaryotic initiation factor 4E (eIF4E) small interfering RNA (siRNA) decreased both SCD1 and SREBP1 expression, suggesting that SCD1 may be regulated through the mTOR/eIF4E-binding protein 1 axis. Furthermore, SCD1 siRNA knockdown inhibited breast cancer cell growth, whereas overexpression increased growth. Taken together these findings show that rapamycin decreases SCD1 expression, establishing an important link between cell signaling and cancer cell fatty acid synthesis and growth.

Figure 1

Rapamycin decreases SCD1 expression. A, MDA-MB-468, MCF7 and BT-474 cells were treated with 100 nM rapamycin or 0.01% DMSO for 24 hours. Polysomal RNA was separated by sucrose gradient centrifugation. Total polysomal RNA was extracted and hybridized to Affymetrix Human Genome U133 Plus 2.0 chips. The RNA expression in the rapamycin-treated samples was compared to that of untreated total and polysomal RNA samples using Student’s t-test. The gene of interest was considered significant in each cell line if it met a false discovery rate of 20%. All comparisons that met this cut-off are demarcated by an asterisk (*). Data are represented as means ± standard error of the means (SEMs). B, Q-PCR analysis was performed to quantitatively assess total RNA, monosomal and polysomal fractions in MDA-MB-468 and MCF7 cells treated with rapamycin vs. vehicle for 24 hours. Actin was used as the endogenous control. RNA expression in rapamycin treated and untreated samples were compared by using Student’s t-test. Data are represented as means ± range (min-max). C, Northern blot analysis for SCD1 and actin was performed on total RNA isolated from MDA-MB-468 and MCF7 cells grown in either rapamycin or vehicle for 24 and 96 hours. D, To study the effect of rapamycin on SCD1 in vivo, MDA-MB-468 or MCF7 xenografts were treated with either rapamycin or vehicle for 1 day or 3 weeks. Tumor volumes at day 22 are shown as means ± SEMs. Vehicle vs. rapamycin groups were compared using Student’s t-test. (left upper panel). Protein lysates prepared from three xenografts were printed on RPPA slides and probed with P-S6RP (Ser 240/244) antibody. Relative P-S6RP expression in rapamycin treated and untreated groups were compared by using Student’s t-test. Data are shown as means ± SEMs (right upper panel). Total RNA from three tumor samples from each group was evaluated using Q-PCR to assess SCD1 and actin expression (lower panels). Data are represented as means ± SEMs.

Figure 2

SCD1 expression is inhibited by PI3K/mTOR inhibitors and increased by insulin signaling. A. MDA-MB-468, MCF7 and BT-474 cells were treated with 100 nM rapamycin or DMSO for 24, 48, 72, and 96 hours, and 10% SDS-PAGE and western blotting using SCD1 and actin antibody were performed (upper panels). MCF7 cells were treated with various concentrations of rapamycin for 96 hours. Western blotting was performed using SCD1 and actin antibody (lower panel). B, MCF7 cells were incubated overnight in serum-free media. Cells were then cultured for 8 hours in one of the following conditions: no treatment, medium containing 10 μg/ml of insulin, or 100 ng/ml of IGF-1. 10% SDS-PAGE and western blotting using SCD1 antibody and actin were performed (left panel). The experiments were replicated in triplicate and quantified according to a relative expression of SCD1/β Actin. Top band was used for quantification. Relative SCD1 expression in the treatment groups was compared to that of the no treatment group using Student’s t-test. Data are represented as means ± SEMs (middle panel). After overnight serum starvation, cells were cultured for 8 hours in one of the following conditions: no treatment, or medium containing 25 or 100 ng/ml of IGF-1 in the absence or presence of pre-treatment with 100 nM rapamycin (right panel). 10% SDS-PAGE and western blotting using SCD1 antibody and actin were performed. C. MCF7 cells were transfected with control and constitutively active Akt (CA-Akt) plasmids. Sixty hours later, serum-free media was added and cells were incubated for an additional 36 hours. Western blotting was performed for SCD1, Akt, phospho-S6 ribosomal protein (Ser 240/244) and actin (right panel). D, (Left panel) MDA-MB-468 cells were cultured for 24 hours with no treatment, DMSO, 100 nM rapamycin, and 50 μM LY294002. Western blotting was performed using SCD1 and actin antibody. S6K1 and phospho-S6K1 (Thr 389) were used to confirm inhibition of the mTOR pathway. (Middle panel) MDA-MB-468 cells were cultured with DMSO, 1, 10 or 100 nM BEZ235. Lysates were collected at 6 hours (for P-Akt and Akt) or 24 hours (for SCD1 and actin). Western blotting was performed using P-Akt (Thr 308), Akt, SCD1 and actin antibody. (Right panel) MDA-MB-468 cells were cultured with DMSO, 1, 10 or 100 nM BEZ235. Lysates were collected at 6 hours and western blotting was performed using P-Akt (Ser 473), Akt and actin antibody.

Figure 3

Rapamycin regulates SCD1 promoter activity, but not SCD1 mRNA stability. A, MDA-MB-468 cells were treated with 100 nM rapamycin or vehicle at 12 time points spanning 24 hours. All samples were incubated with actinomycin D at a final concentration of 5 μg/ml. No treatment was used at the 0 hour time points, which were the designated controls for each study. Total RNA was extracted from each sample and analyzed via northern blotting. Quantitation of the northern blot was shown below. B, Western blot analyses in MCF7 cells were used to study the effects of rapamycin (100 nM) on the mevinolin-based (5 μM) induction of SCD1 in medium supplemented with lipid-deficient serum. These results were replicated in triplicate. C, Dual luciferase assays were performed after co-transfecting MCF7 cells with SCD1 promoter reporter (hSCD1-Luc pGL3) and control (pRL) plasmids. Results from triplicate experiments show a repression of the SCD1 promoter by rapamycin.

Figure 4

Rapamycin regulates expression of mature SREBP1. A, MCF7 and MDA-MB-468 cells were treated with 100 nM rapamycin or vehicle for 24 and 96 hours. 8% SDS-PAGE and western blotting using SREBP1 and actin antibodies were performed. Each SREBP1 blot has a precursor band (P), and a smaller mature band (M). These results were confirmed in triplicate experiments. Nuclear protein extracts from MCF7 (B) and MDA-MB-468 (C) cells treated with 100 nM rapamycin or vehicle for 1 and 4 days were assayed for specific transcription factor-DNA binding activity. All experiments were replicated in triplicate and quantified in comparison to vehicle using Student’s t-test. Data are represented as means ± SEMs. D, MDA-MB-468 cells were treated with 100 nM rapamycin for 96 hours. SDS-PAGE and western blotting using ACC, FAS, SCD1, SREBP1 and actin antibodies were performed.

Figure 5

eIF4E knockdown decreases SCD1 and SREBP1 expression and SCD1 promoter activity. A, MDA-MB-468 cells were transfected with siRNA for mTOR and eIF4E. After 72 hours, western blotting with SCD1, mTOR, and eIF4E and actin was performed. These results were confirmed in triplicate experiments. B, MDA-MB-468 cells were transfected with control siRNA or two separate sequences of eIF4E siRNA. After 72 hours, western blotting with SCD1, eIF4E and actin was performed. C, MDA-MB-468 cells were transfected with single or pool siRNA for S6K1 and 72 h later western blotting was performed using S6K1, Akt, P-Akt (Thr 308), P-Akt (Ser 473), SCD1, SREBP1 and actin antibody (left panel). MDA-MB-468 cells were transfected with siRNA for eIF4E and S6K1. After 72 hours, western blotting with SCD1, SREBP1, eIF4E and S6K1 were performed (right panel). These results were confirmed in triplicate experiments. D, MCF7 cells were first transfected with siRNA for eIF4E. Dual luciferase assays were then performed after co-transfecting with SCD1 promoter reporter (hSCD1-Luc pGL3) and control (pRL) plasmids. Treatment with and without rapamycin served as control. These results reflect an average of three independent experiments performed in triplicate (left panel). MDA-MB-468, MCF7 and BT-474 cells were transfected with vector, 4E-BP1 WT or 4E-BP1 5A plasmids. SCD1 promoter reporter (hSCD1-Luc pGL3) and control (pRL-TK) plasmids were co-transfected and 96 h later dual luciferase assays were performed. Vector transfection served as control. Analysis was done by using one-way ANOVA and Tukey post hoc test (right panel). This experiment was repeated three times in triplicates. Bars represent SEM.

Figure 6

SCD1 regulates breast cancer cell growth. A, Left panel shows baseline SCD1 protein expression in 15 breast cancer cell lines. Western blotting was performed using SCD1 and actin antibody. SCD1 and actin bands were quantified and the average of three independent experiments was shown as relative SCD1/actin expression in the right panel. The same chart also shows half maximal inhibitory concentration (IC₅₀) values of breast cancer cell lines. Cells were treated with increasing concentrations of rapamycin for five days and IC₅₀ was determined on the basis of the dose-response curves using sulforhodamine B (SRB) assay. B, MCF7 cells (left upper panel) MDA-MB-468 cells (right upper panel) were transfected with single or pool control siRNA and SCD1 siRNA. Cells were grown in medium supplemented with lipid deficient serum and cell growth analysis was then performed with readings recorded after 96 hours of treatment by SRB assay. Cells treated with and without rapamycin are the designated controls. These results reflect experiments performed in triplicate. Western blotting with SCD1 and actin were performed on the treated cells of both MCF7 and MDA-MB-468 cells to confirm adequate suppression of SCD1 by siRNA after 96 hours. MCF7 cells (left lower panel) or BT474 cells (right lower panel) were transfected with single control siRNA, SCD1 siRNA or a SCD1 siRNA #2. Cells were grown in regular medium and cell growth analysis was then performed with readings recorded after 96 hours of treatment by SRB assay. SCD1 siRNA treated cells were compared with control siRNA treated cells using Student’s t-test. These results are representative of two independent experiments. C, MCF7 (left), MDA-MB-468 (middle) or BT474 cells (right panel) were transfected with single control siRNA, SCD1 siRNA or a SCD1 siRNA #2. Cells were grown in regular medium and analyzed for cell cycle profile after 96 hours. Percentage of cells in G0-G1 was compared using Student’s t-test. D, MDA-MB-231 SCD1 over-expressing stable cells were grown alongside control in media supplemented with lipid deficient serum. Cell growth analysis was then performed with readings recorded after 96 hours of growth. These results reflect experiments performed in triplicate (left panel). Western blotting with SCD1 and actin were performed on MDA-MB-231 over-expressing stable cells alongside their respective control to confirm expression of the transfected myc-tagged SCD1 protein (right panel).