Linkage of E2F1 transcriptional network and cell proliferation with respiratory chain activity in breast cancer cells

Abstract

Mitochondria are multifunctional organelles; they have been implicated in various aspects of tumorigenesis. In this study, we investigated a novel role of the basal electron transport chain (ETC) activity in cell proliferation by inhibiting mitochondrial replication and transcription (mtR/T) using pharmacological and genetic interventions, which depleted mitochondrial DNA/RNA, thereby inducing ETC deficiency. Interestingly, mtR/T inhibition did not decrease ATP levels despite deficiency in ETC activity in different cell types, including MDA-MB-231 breast cancer cells, but it severely impeded cell cycle progression, specifically progression during G2 and/or M phases in the cancer cells. Under these conditions, the expression of a group of cell cycle regulators was downregulated without affecting the growth signaling pathway. Further analysis suggested that the transcriptional network organized by E2F1 was significantly affected because of the downregulation of E2F1 in response to ETC deficiency, which eventually resulted in the suppression of cell proliferation. Thus, in this study, the E2F1-mediated ETC-dependent mechanism has emerged as the regulatory mechanism of cell cycle progression. In addition to E2F1, FOXM1 and BMYB were also downregulated, which contributed specifically to the defects in G2 and/or M phase progression. Thus, ETC-deficient cancer cells lost their growing ability, including their tumorigenic potential in vivo. ETC deficiency abolished the production of reactive oxygen species (ROS) from the mitochondria and a mitochondria-targeted antioxidant mimicked the deficiency, thereby suggesting that ETC activity signaled through ROS production. In conclusion, this novel coupling between ETC activity and cell cycle progression may be an important mechanism for coordinating cell proliferation and metabolism.

Keywords: Breast neoplasm; E2F1 transcription factor; cell cycle; electron transport; reactive oxygen species.

Figure 1

Cell cycle arrest induced in electron transport chain (ETC)‐deficient MDA‐MB‐231 (MDA) cells. Cells exposed to ethidium bromide (EtBr) (250 ng/mL) on the days indicated (MDA/ρ0) were analyzed. (a) mitochondrial DNA (mtDNA) relative to genomic DNA (18S) (mt/18S DNA) quantified using qPCR. (b) Levels of cytochrome b (Cyt.b) mRNA as analyzed by qRT‐PCR. (c) Mitochondrial membrane potential (ΔΨm) monitored with Mito‐ID. (d) ATP levels were determined using an ATP determination kit and normalized against the protein levels. (e) Cell proliferation in the presence (ρ0) and absence (normal) of EtBr. (f) Cell cycle distribution determined using DNA histograms obtained by flow cytometry. (g) Representative DNA histograms for normal (0 day) and ρ0 (6 days) cells. Bottom: Histograms did not change with the incubation of cells with EtBr (250 ng/mL) for 30 min. (h) Morphology of cells incubated with or without EtBr (250 ng/mL) for 5 days. Magnification: ×100. (i) Activation‐dependent phosphorylation of ERK1/2 as detected by western blot. The pair shown is for the phosphorylated (p) form and for the total protein. *P < 0.05 and **P < 0.01.

Figure 2

Downregulation of cell cycle regulators in electron transport chain (ETC)‐deficient MDA and T‐47D cells. Cell proliferation (a) and cell cycle distribution (b) determined as described in Figure 1(e, f) in ethidium bromide (EtBr)‐treated T‐47D human breast cancer cells. (c, d) MDA/ρ0 cells, as described in Figure 1, were analyzed on the days indicated. (c) Left: mRNA levels of cyclins (A2, B1, B2, D1 and E), BMYB and FOXM1 quantified using qRT‐PCR. Right: protein levels of the corresponding mRNA detected by western blot. GD was the loading control. (d) Left: mRNA levels of Cyt.b and E2F1–8 evaluated by qRT‐PCR. Right: Western blot analysis of E2F1 and 8. GD was the loading control. (e) mRNA levels of Cyt.b and E2F1–8 quantified by qRT‐PCR in T‐47D/ρ0 cells. *P < 0.05 and **P < 0.01. NS, not significant.

Figure 3

Downregulation of cell cycle regulators in E2F1‐knockdown cells. MDA cells were treated with 50 nM of ON‐TARGETplus Human E2F1 siRNA‐SMARTpool (E2F1) or non‐targeting Pool siRNA (NT). (a) Validation of E2F1 knockdown by western blotting in cells incubated with siRNA for 48 h. (b) mRNA levels of cyclins (A2, B1, B2, D1 and E), BMYB and FOXM1, which were analyzed together with that of E2F1 by qRT‐PCR. (c) Western blot analysis of proteins corresponding to the mRNA in (b). GD was the loading control. (d) Effects of E2F1 knockdown (7 days) on cell proliferation compared with those of E2F2 and E2F8. (e) Schematic representation of the electron transport chain (ETC)‐dependent cell cycle control mechanisms (see text). (f) Effects of double knockdown of E2F1 and FOXM1 (6 days) with the siRNA on cell proliferation. siRNA: N; NT, E; E2F1, F; FOXM1, E/F; E2F1+ FOXM1. (g) Cell cycle distribution under the knockdown of E2F1 alone or in combination with FOXM1 for 6 days. *P < 0.05 and **P < 0.01.

Figure 4

Downregulation of cell cycle regulators and cell proliferation in mitochondrial transcription factor A (TFAM)‐knockdown cells. TFAM‐knockdown MDA cells were established as described in Figure S3(a,b). Levels of mtDNA relative to genomic DNA (18S) (mt/18S DNA) (a) and Cyt.b mRNA (b), mitochondrial membrane potential (ΔΨm) (c), and ATP levels (d) were determined as described in Figure 1(a–d) after incubating the cells in the absence of Dox for 7 days (a) and 48 h (b–d). (e, f) mRNA levels of cyclins (A2, B1, B2, D1 and E) and FOXM1 (e), as well as those of Cyt.b and E2F1–8 (f) quantified by qRT‐PCR in cells where TFAM was knocked down by incubating in the absence of Dox (Dox [−]) for the days indicated. (g) Western blot analysis of representative proteins corresponding to the mRNA in (e) and (f). GD was the loading control. (h) Proliferation of cells with TFAM knockdown by incubating with Dox (−) for the days indicated. (i) Cell cycle distribution of control (0) and TFAM‐knockdown cells (Dox [–] 5 days) in the presence of 2‐CM (20 μM) (5 + 2‐CM), as described in Figure S3(c). *P < 0.05 and **P < 0.01. NS, not significant.

Figure 5

Suppression of anchorage‐independent cell growth and tumor growth in vivo by mitochondrial transcription factor A (TFAM) knockdown. (a) Anchorage‐independent cell growth of the TFAM short hairpin RNA (shRNA)‐expressing MDA cells, as described in Figure S3a,b. Cells grown in methylcellulose under Dox (−) conditions for 4 weeks were photographed (Right; scale bar: 300 μm) and the areas of colonies in images were quantified using ImageJ software (left). Values represent the mean ± SD. **P < 0.01. (b) TFAM shRNA‐expressing cells established from MDA/GFP cells were implanted into the mammary fat pads of SCID mice and the tumor size was monitored. Each data point represents the mean ± SD based on four or five xenografts. **P < 0.01.

Figure 6

Decrease in the intracellular reactive oxygen species (ROS) levels in electron transport chain (ETC)‐deficient cells and its significance in controlling cell proliferation. (a) ROS levels of normal and pseudo‐ρ0 cells determined using H₂DCFDA for flow cytometry. Left: Representative histogram showing the H₂DCFDA florescence intensity obtained from calcein‐labeled MDA/ρ0 cells (black [NegaCtr]): ethidium bromide (EtBr) treatment 0 days (unstained), blue: 0 days (stained), red: 3 days (stained). Right: The H₂DCFDA florescence intensity normalized against that of calcein blue in an individual cell was determined in at least 10 000 cells and plotted. (b) MDA cells were treated with 0.5 μM Mitq or CoQ for 24 h, and the ROS levels were determined, as described in (a). Left: Histogram (black [NegaCtr]): unstained control, blue (−): untreated and stained control, red (Mitq): Mitq‐treated and stained, green (CoQ): CoQ‐treated and stained. (c) mRNA levels of cyclins (A2, B1, B2, D1 and E), BMYB, FOXM1 and E2F1–8 in MDA cells treated with 0.5 μM of Mitq and CoQ for 24 h, which were quantified by qRT‐PCR. (d) Western blot analysis of representative cell cycle regulators with MDA cells treated as described in (c) for 48 h. GD was the loading control. (e) Proliferation of MDA cells treated with 0.2 and 0.5 μM Mitq, and 0.5 μM CoQ for 6 days. (f) Cell cycle distribution of MDA cells treated as described in (e) for 48 h. (g) MDA cells treated with 0.5 μM Mitq for 2 h were incubated with 100 μg/mL cycloheximide (CHX) for the times indicated. E2F1 expression levels were examined by conducting western blot analysis, quantified using Image J software, and the relative intensities are shown after normalization against GD. *P < 0.05 and **P < 0.01. NS, not significant.