Estrogen-Induced Hypermethylation Silencing of RPS2 and TMEM177 Inhibits Energy Metabolism and Reduces the Survival of CRC Cells

Abstract

Estrogen (E2, 17β estradiol) is recognized for its regulatory role in numerous genes associated with energy metabolism and for its ability to disrupt mitochondrial function in various cancer types. However, the influence of E2 on the metabolism of colorectal cancer (CRC) cells remains largely unexplored. In this study, we examined how E2 affects mitochondrial function and energy production in CRC cells, utilizing two distinct CRC cell lines, HCT-116 and SW480. Cell viability, mitochondrial function, and the expression of several genes involved in oxidative phosphorylation (OXPHOS) were assessed in estrogen receptor α (ERα)-expressing and ERα-silenced cells treated with increasing concentrations of E2 for 48 h. Our results indicated that the cytotoxicity of E2 against CRC cells is mediated by the E2/ERα complex, which induces disturbances in mitochondrial function and the OXPHOS pathway. Furthermore, we identified two novel targets, RPS2 and TMEM177, which displayed overexpression, hypomethylation, and a negative association with ERα expression in CRC tissue. E2 treatment in CRC cells reduced the expression of both targets through promoter hypermethylation. Treatment with 5-Aza-2-deoxycytidine increased the expression of RPS2 and TMEM177. This epigenetic effect disrupts the mitochondrial membrane potential (MMP), resulting in decreased activity of the OXPHOS pathway and inhibition of CRC cell growth. Knockdown of RPS2 or TMEM177 in CRC cells resulted in anti-cancer effects and disruption of MMP and OXPHOS. These findings suggest that E2 exerts ERα-dependent epigenetic reprogramming that leads to significant mitochondria-related anti-growth effects in CRC.

Keywords: colorectal cancer; estrogen; estrogen receptors; metabolism; mitochondria; oxidative phosphorylation.

Figure 1

Effect of E2 treatment on colorectal cancer (CRC) cell survival. (a) HCT-116 cells were stained with crystal violet (CV) to measure cell proliferation following treatment with increasing concentrations of E2 for 48 h. (b) HCT-116 cell survival was measured using (3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) following treatment with increasing concentrations of E2 for 48 h. (c) HCT-116 cells were stained using trypan blue and counted under the microscope following ethanol, vehicle, and E2 treatment for 24 and 48 h. (d) Real-Time Cell Analysis (RTCA) was used to measure real-time HCT-116 cell proliferation for up to 120 h. Data are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test unless otherwise indicated. * p-value < 0.05, and ** p-value < 0.01.

Figure 2

E2-ERα signaling modulates glucose metabolism. (a) 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG) uptake level was measured using flow cytometry in HCT-116 cells treated with increasing concentrations of E2 for 48 h. (b) Reactive Oxygen Species (ROS) generation was measured by the 2′,7′-Dichlorofluorescin diacetate (DCFDA) assay in HCT-116 cells treated with increasing concentrations of E2 for 48 h. (c) Estrogen receptor α (ERα) and estrogen receptor β (ERβ) expression was assessed using a Western blot with E2 treatment in HCT-116 cells. (d) The effect of the propyl pyrazole triol (PPT), selective ERα agonist, 24 and 48 h treatment on HCT-116 proliferation. (e) A flowchart representing genes positively or negatively correlated with ESR1 gene expression in CRC patients. Data are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test unless otherwise indicated. ns Not significant, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 and **** p-value < 0.0001.

Figure 3

ERα silencing diminishes the effect of E2 on ATP production in HCT-116 cells. (a) ERα knockdown was confirmed using a Western blot. Representative blots are shown; corresponding β-actin loading controls and densitometry (normalized to β-actin) are provided. (b) Cellular ROS generation was measured by DCFDA assay following treatment with 20 nM E2 with/without siERα in HCT-116 cells. (c) An energy map was used to represent general Seahorse XF ATP rate assay results following treatment with 20 nM E2 with/without siERα in HCT-116 cells. (d) The kinetic profile of oxygen consumption (OCR) and extracellular acidification (ECAR) was measured using Seahorse XF ATP rate assay following E2 treatment. (e) Net ATP production rate from mitochondrial and glycolytic ATP production following E2, PPT, and siERα treatments. Data are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test unless otherwise indicated. ns Not significant, * p-value < 0.05, and ** p-value < 0.01.

Figure 4

E2 signaling modulates mitochondrial function in HCT-116 cells. (a) The depolarization of MMP was examined at 20X magnification following treatment of ERα-silenced HCT-116 cells with 20 nM E2. Increased mitochondrial permeability is indicated by red color and dye accumulation in the cytoplasm is indicated by green color. (b) Quantitative analysis of MMP at 590 nm. (c) Correlation between ESR1 mRNA expression and eight mitochondrial markers in CRC patients. Red line is the line of best fit (Regression Line) (d) Protein expression of mitochondrial markers in E2-treated HCT-116 cells by Western blotting. Representative blots are shown; corresponding β-actin loading controls and densitometry as bar graph (normalized to β-actin) are provided. Data are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test unless otherwise indicated. ns Not significant, * p-value < 0.05, and *** p-value < 0.001.

Figure 5

Identification of potential epigenetic targets for E2 in CRC. (a) A flowchart representing genes with the potential to be targeted by E2 signaling in CRC cells. (b) RPS2 and TMEM177 promoter methylation profile in CRC versus normal tissue. (c) RPS2 and TMEM177 mRNA expression in CRC versus normal tissue. (d) RPS2 and TMEM177 methylation levels following 10 and 20 nM E2 or 5-Aza-2-deoxycytidine (Aza) treatment in HCT-116 cells. (e) RPS2 and TMEM177 mRNA expression following 10 and 20 nM E2 or Aza treatment in HCT-116 cells. (f) RPS2 and TMEM177 protein expression following 20 nM E2 with or without siESR1 treatment in HCT-116 cells. Representative blots are shown; corresponding β-actin loading controls and densitometry (normalized to β-actin) are provided. Data are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test unless otherwise indicated. ** p-value < 0.01, *** p-value < 0.001 and **** p-value < 0.0001.

Figure 6

RPS2 and TMEM177 enhance cell viability and sustain mitochondrial function in HCT-116 cells. (a) Cell morphology, CV, and apoptosis were evaluated in HCT-116 cells treated with 30 pmol of siRPS2 or siTMEM177. (b) Depolarization of MMP was observed after silencing RPS2 and TMEM177 in HCT-116 cells, 20X magnification. Increased mitochondrial permeability as indicated by red color, green color indicates cytoplasmic dye accumulation (c) Quantitative analysis of the MMP at 590 nm. (d) ROS generation in RPS2- and TMEM177-silenced HCT-116 cells as measured by the DCFDA assay. Data are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test unless otherwise indicated. ** p-value < 0.01 and *** p-value < 0.001.