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. 2021 Oct 6;163(1):bqab212. doi: 10.1210/endocr/bqab212

TXNIP Links Anticipatory Unfolded Protein Response to Estrogen Reprogramming Glucose Metabolism in Breast Cancer Cells

Yuanzhong Wang 1, Shiuan Chen 1,✉
PMCID: PMC8570585  PMID: 34614512

Abstract

Estrogen and estrogen receptor (ER) play a fundamental role in breast cancer. To support the rapid proliferation of ER+ breast cancer cells, estrogen increases glucose uptake and reprograms glucose metabolism. Meanwhile, estrogen/ER activates the anticipatory unfolded protein response (UPR) preparing cancer cells for the increased protein production required for subsequent cell proliferation. Here, we report that thioredoxin-interacting protein (TXNIP) is an important regulator of glucose metabolism in ER+ breast cancer cells, and estrogen/ER increases glucose uptake and reprograms glucose metabolism via activating anticipatory UPR and subsequently repressing TXNIP expression. In 2 widely used ER+ breast cancer cell lines, MCF7 and T47D, we showed that MCF7 cells express high TXNIP levels and exhibit mitochondrial oxidative phosphorylation (OXPHOS) phenotype, while T47D cells express low TXNIP levels and display aerobic glycolysis (Warburg effect) phenotype. Knockdown of TXNIP promoted glucose uptake and Warburg effect, while forced overexpression of TXNIP inhibited glucose uptake and Warburg effect. We further showed that estrogen represses TXNIP expression and activates UPR sensor inositol-requiring enzyme 1 (IRE1) via ER in the breast cancer cells, and IRE1 activity is required for estrogen suppression of TXNIP expression and estrogen-induced cell proliferation. Our study suggests that TXNIP is involved in estrogen-induced glucose uptake and metabolic reprogramming in ER+ breast cancer cells and links anticipatory UPR to estrogen reprogramming glucose metabolism.

Keywords: estrogen, breast cancer, glucose metabolism, TXNIP, unfolded protein response

Approximately 70% of breast cancers express estrogen receptor (ER, which includes ERα and ERβ, ER refers to ERα in this paper unless otherwise indicated). Estrogen and ERα play a fundamental role in breast cancer. After binding to ligand estrogen, ER translocates into the nucleus and regulates gene transcription to drive breast cancer cell growth, proliferation, and survival. Estrogen/ER can also crosstalk with growth factor receptors (such as epidermal growth factor receptor [EGFR] and insulin-like growth factor receptor [IGFR]) in the cell membrane and cytoplasm and activate multiple downstream kinase pathways to promote breast cancer progression (1).

Metabolic reprogramming is a hallmark of cancer (2). Cellular energy source adenosine triphosphate (ATP) is mainly produced via glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS, together with the Krebs cycle, is much more efficient in ATP production than glycolysis (36 ATP vs 2 ATP). Normal cells rely primarily on OXPHOS for energy production, while most cancer cells use glycolysis as a mean of energy production regardless of oxygen availability, which is known as Warburg effect. Although it is less efficient in ATP production, the Warburg effect provides substrates for anabolism to synthesize macromolecules (ie, nucleic acids, lipids, and proteins) to adapt the rapid growth of cancer cells (3). Therefore, increased lactate production through glycolysis is now accepted as a prime example of metabolic reprogramming and an emerging hallmark of cancer cells (4). Despite this, not all cancer cells exhibit Warburg effect (5). It remains elusive why some cancer cells exhibit the OXPHOS phenotype while others are of the Warburg effect type. It is known that estrogen increases glucose uptake and reprograms glucose metabolism to promote tumor progression in breast cancer (6). However, the underlying mechanisms are still not fully understood.

The unfolded protein response (UPR) is a cellular stress response related to endoplasmic reticulum (EnR) stress and plays important roles in many physiological and pathological processes. UPR mainly consists of 3 sensors: protein kinase RNA-like EnR kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). Activation of these sensors of the UPR contributes to EnR homeostasis by reducing EnR protein loading and inducing expression of chaperones that increase protein folding capacity. Mild EnR stress response or UPR is protective, but hyperactive UPR converts it from cytoprotective to cytotoxic (7). Emerging evidence indicates that estrogen, acting via ERα, induces rapid anticipatory activation of UPR in ER+ breast cancer, preparing cancer cells for the increased protein production required for subsequent estrogen-induced cell proliferation (8-11).

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein with wide-ranging functions impacting energy metabolism (12). It was first discovered to be upregulated by vitamin D3 in HL-60 cells (13). Later, studies found that TXNIP interacts with and inhibits thioredoxin (14), and thus plays an important role in maintaining redox homeostasis. It has been reported that TXNIP inhibits glucose uptake and breakdown by reducing glucose transporter 1 (Glut1) expression, inhibiting hypoxia-inducible factor 1 (HIF1) activity and enhancing phosphatase and tensin homolog (PTEN) activity (12). Although TXNIP acts as an important regulator of glucose metabolism in many types of cells, it was still unclear whether TXNIP is involved in estrogen-induced glucose uptake and metabolic reprogramming in ER+ breast cancer.

In the current study, we investigated estrogen reprogramming glucose metabolism in ER+ breast cancer cells. Our results showed that TXNIP is involved in estrogen-induced glucose uptake and metabolic reprogramming, and it links anticipatory UPR to estrogen reprogramming glucose metabolism.

Materials and Methods

Drugs and Inhibitors

The following compounds and reagents were used in this study: 17β-estradiol (Sigma), ICI 182,780 (Tocris Bioscience), GSK2656157 (Calbiochem), 4μ8C (Calbiochem), KIRA6 (Calbiochem).

Cell Culture

MCF7 and T47D cells were cultured in the MEM medium containing 10% fetal bovine serum (FBS), 2mM L-glutamine, 1mM sodium pyruvate and 1% nonessential amino acids. HCC1428 cells were cultured in the IMEM medium with 10% FBS. To evaluate the effect of estrogen, the cells were switched to their respective phenol red-free MEM or IMEM medium with 10% charcoal:dextran-stripped (CD) FBS for 2 days for hormone deprivation before 17β-estradiol (E2) treatment.

Organoid Generation and Culture

COH-GS4 patient-derived xenografts (PDXs) were generated as previously described (15). COH-GS4 organoids were generated from COH-GS4 PDX. For generation of organoids, we followed the Basic Protocol #1 described in the paper by DeRose Y et al (16). Briefly, a PDX fragment was processed into organoids as described in the protocol, and the organoids were seeded on the top of matrix gel of the wells in a 12- or 6-well plate, with the addition of modified M87 medium (1 mL/well for 12-well plates or 2 mL/well for 6-well plates). For investigation of the E2 effect, the organoids were switched to culture in phenol red-free M87 medium without adding E2, and the FBS used for the medium preparation was switched to CD FBS.

Small Interfering RNA Transfection

Small interfering RNA (siRNA) transfection was performed using RNAi max (Invitrogen, Carlsbad, USA), and the reverse transfection method was used as previously described (17). Briefly, cells were trypsinized and diluted in the medium at 1 × 105 cells/mL. For each 6-cm dish, 10 μL RNAi max transfection agent and 15 μL 10μM siRNA against the interest gene or siRNA negative control were diluted in 250 μL OPTI-MEM I medium, respectively. After being mixed and incubated for 10 minutes, the mixtures of siRNA and transfection agent were dispersed into 6-cm dishes. Cell suspensions (1 × 105 cells/mL) were overlaid onto the transfection complexes at 4 mL cells/dish. TXNIP siRNA (sc-44943), ERα siRNA (sc-29305), PERK siRNA (sc-36213), IRE1 siRNA (sc-40705), ATF6 siRNA (sc-37699) and XBP1 siRNA (sc-38627) were purchased from Santa Cruz.

Quantitative Reverse Transcription–Polymerase Chain Reaction

T47D cells were hormone-stripped for 2 days and then treated with increasing doses of E2 for 48 hours before harvesting for quantitative reverse transcription–polymerase chain reaction (RT-qPCR). RNA extraction and RT-qPCR was performed as previously described (18). The primers used in the real-time PCR were as follows: for TXNIP, 5′-CAGCCAACAGGTGAGAATGA-3′ and 5′-TTGAAGGATGTTCCCAGAGG-3′; for β-actin, 5′-CACCAACTGGGACGACAT-3′ and 5′-GCACAGCCTGGATAGCAAC-3′.

Western Blotting

Cells were lysed in RIPA buffer on ice for 10 minutes, and then sonicated for 30 seconds. After centrifugation, the supernatants were collected and mixed with 2 × SDS sample buffer and boiled for 5 minutes. Protein concentration was quantified using the BioRad Protein Assay. Western blotting was performed as previously described (19). The following antibodies were used: the rabbit polyclonal ERα antibodies (Santa Cruz Biotechnology Cat# sc-543, RRID:AB_631471; https://antibodyregistry.org/search.php?q=AB_631471) and the mouse monoclonal antibodies to ERα (Santa Cruz Biotechnology Cat# sc-8002, RRID:AB_627558; https://antibodyregistry.org/search.php?q=AB_627558) and β-actin (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA); the rabbit monoclonal antibodies to TXNIP (Cell Signaling Technology Cat#14715, RRID:AB_2714178), SGK3 (Cell Signaling Technology Cat# 8573, RRID:AB_10949896), BiP (Cell Signaling Technology Cat# 3177, RRID:AB_2119845), IRE1 (Cell Signaling Technology Cat# 3294, RRID:AB_823545), XBP1s (Cell Signaling Technology Cat# 12782, RRID:AB_2687943), PERK (Cell Signaling Technology Cat# 5683, RRID:AB_10841299), eIF2α (Cell Signaling Technology Cat# 5324, RRID:AB_10692650), phospho-eIF2α (Ser51) (Cell Signaling Technology Cat# 3398, RRID:AB_2096481), ATF4 (Cell Signaling Technology Cat# 11815, RRID:AB_2616025), ATF6 (Cell Signaling Technology Cat# 65880, RRID:AB_2799696), MLX (Cell Signaling Technology Cat# 85570, RRID:AB_2800058), FoxO1 (Cell Signaling Technology Cat# 2880, RRID:AB_2106495), NDRG1 (Cell Signaling Technology Cat# 9408, RRID:AB_11140640) and GAPDH (Cell Signaling Technology Cat# 2118, RRID:AB_561053), and mouse CHOP monoclonal antibody (Cell Signaling Technology Cat# 2895, RRID:AB_2089254) were purchased from Cell Signaling Technology (Danvers, USA).

Metabolite Profile Analysis

For cell metabolite profile analysis, the cells were cultured in 6-well plates or 35-mm dishes. After culture for the indicated time in each experiment, the cultured medium was collected for metabolite measurement, and the cells were harvested for protein concentration measurement. For cell metabolite measurement, the cultured medium was spun down to remove cell debris, and 0.8 mL supernatant was used to measure glucose, glutamine, and lactate concentrations by the Nova Biomedical BioProfile 100 or 400. The fresh medium without culture of any cells, which was put in the same cell culture incubator, served as the control. The analysis was as followed: glucose uptake = glucose in the uncultured medium (mM) – glucose in the cultured medium (mM); glutamine uptake = glutamine in the uncultured medium (mM) – glutamine in the cultured medium (mM); lactate production = lactate in the cultured medium (mM) – lactate in the uncultured medium (mM). All the nutrient uptake and lactate production values were normalized to protein concentration of the cultured cells.

Generation of TXNIP-Inducible Cell Lines

TXNIP-inducible cell lines T47D/tetON/TXNIP and MCF7/tetON/TXNIP were generated using the Retro-X-Tet-On Advanced inducible expression system (Clontech Laboratories, Inc) according to the manufacturer’s instructions. In brief, 293T cells cultured in 10-cm dishes were transiently cotransfected with pCMV-GP (gag-pol–expressing vector), a retroviral expression vector (pRetroX-Tet-On Advanced vector for the expression of the tetracycline-controlled transactivator rtTA-Advanced or pRetroX-Tight-Pur-TXNIP vector for the expression of TXNIP under the inducible response promoter, PTight), and an envelope (env)-expressing vector (pVSV-G) using Lipofectamine 2000 (Invitrogen). At 5 hours posttransfection, the culture medium containing DNA and Lipofectamine 2000 was replaced with 8 mL of fresh complete medium, and the cells were continuously cultured for an additional 48 hours before the crude viral supernatant was collected. The virus supernatant was briefly centrifuged and aliquoted, either used immediately or stored at −70 °C until use.

T47D and MCF7 cells were seeded in 35-mm dishes at a density to produce about 50% confluence 12 to 18 hours after seeding. To generate T47D/TetOn and MCF7/TetOn cells that constitutively express the tetracycline-controlled transactivator rtTA-Advanced, the T47D and MCF7 cells in the 35-mm dishes were infected with the RetroX-Tet-On Advanced virus stock, respectively. In brief, the medium was removed, 1 mL of virus stock was added to each well, and polybrene was added to a final concentration of 4 μg/mL. Twenty-four hours after infection, cells were cultured in the fresh complete medium containing 800 μg/mL G418 for about 2 weeks. The selective medium was replaced every 2 to 3 days. After G418 selection for 2 weeks, the G418-resistant clones were pooled and were further infected with the RetroX-Tight-Pur-TXNIP virus stock. Twenty-four hours after infection, the cells were cultured in the medium containing 2 μg/mL puromycin. The stable inducible cell lines were established after puromycin selection for about 2 weeks.

MTT Assay

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to measure cell viability. At the indicated time, the medium in each well of 24-well plate was removed from cells and replaced with 0.5 mL fresh phenol red-free medium containing 0.5 mg/mL MTT. After incubation at 37 °C for 1 hour, the medium was discarded and 0.5 mL dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan dye trapped in the living cells. One hundred microliters of the supernatant was then transferred into a 96-well plate and read in a SpectraMax M5 plate reader (Molecular Devices) at A570.

Cell Viability Assay Using Violet Staining

The cells were seeded into 24-well plates and cultured in normal or growth medium or hormone-depleted medium with the relevant treatments as indicated in each experiment. Every 3 days, the cells were replaced with fresh medium and under the same treatment until the time as indicated. The cells were washed with warm phosphate-buffered saline once before fixation and staining. For fixation and staining, we followed the protocol as described in the reference (20).

Statistical Analysis

Data from MTT and metabolite profile analysis were from 3 independent experiments, and are expressed as means ± SD. The Student t test was used to analyze the statistical significance. A P value of <0.05 was considered statistically significant.

Results

ER+ Breast Cancer Cell Lines Exhibit Different Metabolic Phenotypes

MCF7 and T47D are 2 widely used ER+ breast cancer cell lines. Both cell lines depend on estrogen for growth (Fig. 1A and 1B). To investigate the effect of estrogen on their metabolism, we performed a metabolite profile analysis after MCF7 and T47D cells were hormone-stripped for 48 hours and then treated with or without 10nM E2. The uptake of glucose and/or glutamine was increased upon E2 treatment in these 2 cell lines (Fig. 1C and 1D). In comparison, much less glucose was consumed in MCF7 cells than in T47D cells under the same culture conditions (Fig. 1E). Moreover, the ratio of lactate production to glucose consumption was much lower in MCF7 than in T47D cells, while glutamine uptake was more dramatically increased in response to E2 treatment in MCF7 cells compared with T47D cells (Fig. 1C and 1D). Our results indicate that T47D cells are more aerobic glycolysis (Warburg effect) type while MCF7 cells are more OXPHOS type regarding glucose metabolism, and that estrogen regulates glucose metabolism in these 2 ER+ breast cancer cell lines with bioenergetic differences.

Figure 1.

Figure 1.

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Estrogen reprograms metabolism in ER+ breast cancer cells. A, B, Effect of estrogen on the proliferation of T47D and MCF7 cells. T47D cells (A) and MCF7 (B) were seeded into 24-well plates after being hormone-stripped for 2 days and continued to culture in hormone-depleted medium. After overnight culture, the cells were treated with DMSO or 10nM E2. Cell proliferation was measured by the MTT assay at the indicated time after E2 treatment. *P < 0.05 vs DMSO. C, D, Effect of estrogen on nutrient uptake and lactate production in T47D and MCF7 cells. T47D cells (C) and MCF7 cells (D) were seeded into 35-mm dishes and cultured in hormone-depleted medium after being hormone-stripped for 48 hours. The cells were treated with DMSO or 10nM E2 for 29 hours. The cell cultured media were collected for metabolite measurement as described in “Materials and Methods,” and the cells were harvested for protein concentration measurement. The values for uptake of nutrients and lactate production were normalized to the protein concentration. a, P < 0.05 vs DMSO for Gluc; b, P < 0.05 vs DMSO for Lac; c, P < 0.05 vs DMSO for Gln; n.s, not statistically significantly vs DMSO for Gln. E, The same amount of T47D cells and MCF7 cells were seeded into 35-mm dishes and cultured in hormone-depleted medium after being hormone-stripped for 48 hours. The cells were treated with DMSO or 10nM E2 for 29 hours. The cell cultured medium were collected for glucose uptake, and the cells were harvested for protein concentration measurement. The values for uptake of glucose were normalized to the protein concentration. *P < 0.05.

TXNIP Represses Glucose Uptake and Warburg Effect in ER+ Breast Cancer Cells

TXNIP has been reported to be a negative regulator of cellular glucose uptake in a variety of cells (12). When comparing TXNIP expression between MCF7 and T47D cells, we found that MCF7 cells had much higher levels of TXNIP than T47D cells under the same culture conditions (Fig. 2A), which agrees with our observation that MCF7 cells take up much less glucose than T47D cells. Moreover, the level of TXNIP in T47D cells was greatly reduced upon the addition of E2 to hormone-depleted medium, while TXNIP level (at a high basal level) in MCF7 cells was reduced to a much less degree under the same treatment (Fig. 2B).

Figure 2.

Figure 2.

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TXNIP regulates glucose uptake and metabolism in ER+ breast cancer cells. A, Western blot analysis of TXNIP expression in T47D and MCF7 cells cultured in their normal growth medium. B, T47D and MCF7 cells were hormone-deprived for 2 days, and then treated with or without 10nM E2 for 48 hours before harvesting for Western blot analysis. Abbreviation: HD, hormone deprivation. C, D, MCF7 cells were reversely transfected with siRNA negative control or TXNIP siRNA. At 72 hours posttransfection, the cells were harvested for Western blot analysis (C), and the cell cultured medium was collected for metabolite measurements (D). The uptake of nutrients and lactate production were normalized to the protein concentration. *P < 0.05 vs SiNC; n.s, not statistically significantly. E-H, T47DtetON/TXNIP cells (E, F) and MCF7tetON/TXNIP (G, H) were cultured in normal growth medium and added with or without 100 ng/mL doxycycline for 8 hours, then were replaced with fresh medium in the presence or absence of doxycycline for 19 hours. The cells were harvested for Western blot analysis (E, G), and the cultured medium was collected for metabolite measurement (F, H). The uptake of nutrients and lactate production were normalized to the protein concentration. a, P < 0.05 vs w/o DOX for Gluc; b, P < 0.05 vs DMSO for Lac; n.s, P > 0.05 vs DMSO for Gln.

To demonstrate that TXNIP controls glucose uptake and metabolism in ER+ breast cancer cells, we first knocked down TXNIP in MCF7 cells (Fig. 2C). Depletion of TXNIP in MCF7 cells significantly increased glucose uptake and lactate production while it had little effect on glutamine uptake (Fig. 2D), supporting that TXNIP specifically represses glucose uptake and lactate generation in MCF7 cells. To provide more evidence, we generated TXNIP-inducible cell lines designated T47D/tetON/TXNIP and MCF7/tetON/TXNIP. Induction of TXNIP expression by doxycycline in T47D/tetON/TXNIP cells markedly suppressed glucose uptake and lactate production (Fig. 2E and 2F), confirming that TXNIP inhibits glucose uptake and glycolysis. In MCF7/tetON/TXNIP cells which have high basal levels of TXNIP, induction of TXNIP could further suppress glucose uptake and lactate production (Fig. 2G and 2H).

TXNIP Is Involved in E2-Mediated Warburg Effect and Cell Proliferation

As shown earlier in Fig. 1C, E2 promoted the Warburg effect in T47D cells. We tested whether TXNIP is involved in the E2-induced Warburg effect. To this end, we knocked down TXNIP in T47D cells and then treated with or without E2. Depletion of TXNIP significantly increased glucose uptake and glycolysis in the absence of estrogen in T47D cells (Fig. 3A and 3B), further confirming that TXNIP suppresses glucose uptake and Warburg effect. E2 increased glucose uptake in T47D cells transfected with siRNA negative control, but not in the cells transfected with TXNIP siRNA. E2-induced lactate production was also largely compromised in the TXNIP-depleted T47D cells, supporting that TXNIP is involved in estrogen-mediated Warburg effect. We further examined the effect of TXNIP overexpression on estrogen-mediated glucose metabolism. As shown in Fig. 3C and 3D, induction of TXNIP expression totally blocked estrogen-induced glucose uptake and glycolysis. Given that glucose is an important nutrient for cell proliferation and TXNIP is involved in estrogen-induced glucose uptake and metabolism reprogramming, we tested whether TXNIP is involved in estrogen-induced cell proliferation. Both MTT and colony survival assays showed that induction of TXNIP significantly suppressed E2-induced cell proliferation (Fig. 3E and 3F).

Figure 3.

Figure 3.

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TXNIP is involved in estrogen-mediated Warburg effect and cell proliferation. A, B, T47D cells were cultured in hormone-depleted medium and transfected with siRNA negative control or TXNIP siRNA. Twenty-four hours after transfection, the cells were treated with DMSO or 10nM E2 for 48 hours. The cells were harvested for Western blot analysis (A) and the culture medium was collected for metabolite profile analysis (B). The glucose uptake and lactate production were normalized to protein concentration. a, P < 0.05 vs siNC of DMSO group; n.s., P > 0.05 vs siNC of E2 group. C, D, T47DtetON/TXNIP cells were cultured in hormone-depleted medium in the presence or absence of 100 ng/mL doxycycline for 24 hours. Then the cells were replaced with fresh hormone-depleted medium in the presence or absence of doxycycline and treated with DMSO or 10nM E2 for 31 hours. The cells were harvested for Western blot analysis (C), and the cultured medium was collected for metabolite profile analysis (D). The glucose uptake and lactate production were normalized to the protein concentration. a, P < 0.05 vs w/o DOX of DMSO group; b, P < 0.05 vs w/o DOX of E2 group. E, F, T47DtetON/TXNIP cells were seeded into 24-well plates after being hormone-stripped for 2 days and continued to culture in hormone-depleted medium. The cells were added with or without 100ng/mL doxycycline. After overnight culture, the cells were treated with DMSO or 10nM E2. Cell proliferation was measured by the MTT assay at the indicated time after E2 treatment (E). At day 6, the cells were fixed and stained with crystal violet (F). Images were taken using a scanner. *P < 0.05.

Estrogen Represses TXNIP Expression via ERα in Breast Cancer Cells

Several studies have showed that estrogen reduces TXNIP expression in vitro and in vivo (21-25). We proposed that this could be a mechanism for estrogen-induced glucose uptake and metabolic reprogramming in ER+ breast cancer cells. To confirm that estrogen suppresses TXNIP expression in ER+ breast cancer cells, T47D and HCC1428 cells were hormone-stripped for 2 days and then treated with E2. TXNIP expression was repressed in dose- and time dependent manners (Fig. 4A and 4B). Moreover, selective ER degrader ICI182,780 blocks estrogen-induced TXNIP downregulation in both T47D and HCC1428 cell lines (Fig. 4C), supporting that E2 suppressed TXNIP expression via ERα. Knockdown of ERα also dramatically increased TXNIP expression (Fig. 4D). Using the organoids generated from a PDX line that was derived from an advanced ER+ breast cancer patient (15), we found that E2 reduced TXNIP expression, and ICI182,780 increased TXNIP expression in 3D culture (Fig. 4E-4G). RT-qPCR results showed that E2 dose-dependently decreased Txnip mRNA levels in T47D cells (Fig. 4H), which is consistent with the notion that estrogen represses TXNIP expression at the transcriptional level (21-25). To rule out that E2 represses TXNIP expression via inducing its proteasomal degradation, we tested whether proteasomal inhibitor MG132 can block E2-induced TXNIP downregulation. As shown in Fig. 4I, MG132 could not attenuate E2-induced TXNIP downregulation. In contrast, as it is well established that E2 induces ERα proteasomal degradation, Fig. 4I showed that MG132 blocked E2-induced ERα downregulation.

Figure 4.

Figure 4.

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Estrogen suppresses TXNIP expression via ERα. A, T47D and HCC1428 cells were hormone-stripped for 2 days and then treated with different doses of E2 for 48 hours before being harvested for Western blot analysis. SGK3 is an estrogen-induced protein serving as the positive control. B, T47D cells were hormone-stripped for 2 days and then treated with 10nM E2 for the indicated time before harvested for Western blot analysis. C, Western blot analysis of TXNIP expression in T47D and HCC1428 cells after treatment with or without 10nM E2 alone or plus 100nM ICI182,780 for 48 hours. D, HCC1428 cells were reversely transfected with siRNA negative control or ERα siRNA. At 72 hours posttransfection, the cells were harvested for Western blot analysis. E, Light microscopy of the organoids generated from COH-GS4 PDXs. F, Western blot analysis of COH-GS4 organoids treated with or without 10nM E2 for 4 days. G, Western blot analysis of COH-GS4 organoids treated with or without 100nM ICI182,780 for 4 days. H, T47D cells were hormone-stripped for 2 days and then treated with increasing doses of E2 for 48 hours before being harvested for RT-qPCR. The relative TXNIP mRNA levels were normalized to β-actin. I, T47D cells were hormone-stripped for 2 days and then treated with DMSO, 10nM E2 alone or plus 10μM MG132, and 10μM MG132 for 24 hours before being harvested for Western blot analysis.

IRE1 Activity Is Required for Estrogen Suppression of TXNIP Expression

Although estrogen suppressing TXNIP expression has been reported (21-25), the underlying mechanisms have not been revealed. Estrogen activates anticipatory UPR preparing breast cancer cells for the increased protein production required for subsequent cell proliferation (8-11). We confirmed that estrogen activates the IRE1 and ATF6 branches of anticipatory UPR in T47D cells (Fig. 5A). Interestingly, estrogen slightly suppressed PERK branch, suggesting that estrogen divergently regulates 3 branches of UPR. Given that UPR and estrogen signaling might interplay, we examined the effects of UPR on E2 signaling. Strikingly, E2 suppression of TXNIP expression in T47D cells was completely blocked by 2 different types of IRE1 inhibitors: 4µ8C, which inhibits IRE1 kinase activity by blocking substrate access to IRE1; and KIRA6, which inhibits IRE1 RNase by breaking its oligomers (Fig. 5B and 5C) (26, 27). In contrast, PERK inhibitor GSK2656157 failed to block estrogen suppression of TXNIP expression. Similar results were obtained in HCC1428 cells (Fig. 5D), suggesting that the observation is not restricted to one cell line. Requirement of IRE1 for estrogen-mediated suppression of TXNIP expression was further confirmed with genetic depletion of IRE1 by siRNA in T47D cells (Fig. 5E). Since XBP1 is the most important downstream target of IRE1, we tested whether XBP1 is involved in estrogen suppression of TXNIP expression. Knockdown of XBP1 did not attenuate estrogen repression of TXNIP expression (Fig. 5E), suggesting that estrogen repression of TXNIP expression is IRE1 dependent but XBP1 independent. To reveal the underlying mechanism, we also checked the expression levels of ChREBP, MLX, FoxO1, all of which are important regulators of TXNIP expression. However, none of the single molecule changes was sufficient to explain estrogen repressing TXNIP expression via IRE1 (Fig. 5B-5D).

Figure 5.

Figure 5.

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IRE1 activity is required for estrogen suppression of TXNIP expression. A, T47D cells were hormone-stripped for 2 days and then treated with or without 10nM E2 or 10nM E2 plus 100nM ICI182,780. After 48 hours, the cells were harvested for Western blot analysis to examine the effect of estrogen on UPR (right panel). Left panel is the diagram of 3 branches of UPR. B, C, T47D cells were hormone-stripped for 2 days and then treated with or without 10nM E2 alone or plus 1µM GSK2656157, 20µM 4µ8C, or 1µM KIRA6 for 31 hours, respectively. The cells were harvested for Western blot analysis. (D) HCC1428 cells were hormone-stripped for 2 days and then treated with 10nM E2 alone or plus 1µM GSK2656157 or 20µM 4µ8C for 31 hours. Cells were harvested for Western blot analysis. E, T47D cells were cultured in hormone-depleted medium and transfected with siRNA negative control, PERK siRNA, IRE1 siRNA, or ATF6 siRNA. At 24 hours posttransfection, the cells were treated with DMSO or 10nM E2. After 48 hours E2 treatment, the cells were harvested for Western blot analysis.

IRE1 Activity Is Required for Estrogen-Induced Glucose Uptake and Cell Proliferation

Since IRE1 is involved in estrogen-mediated TXNIP suppression, we further investigated whether IRE1 affects estrogen-induced glucose uptake and metabolic reprogramming. Inhibition of IRE1 by 4µ8C or KIRA6 had little effect on glucose uptake of T47D cells in the absence of E2, but significantly suppressed E2-induced glucose uptake (Fig. 6A), suggesting that IRE1 activity is required for E2-induced glucose uptake. Inhibition of IRE1 increased lactate production in the absence of E2, but decreased E2-induced lactate production. Interestingly, inhibition of PERK by GSK2656157 dramatically increased glucose uptake and lactate production in the absence of E2, while it moderately enhanced E2-induced glucose uptake and lactate production, indicating that PERK branch of UPR suppresses glucose uptake and Warburg effect. Consistent with the effect of small chemical inhibitors in glucose metabolism, IRE1 siRNA attenuated estrogen-induced glucose uptake while PERK siRNA promoted E2-induced glucose uptake and lactate production (Fig. 6B and 6C). In contrast, knockdown of activating transcription factor 6 (ATF6) had little effect on E2-induced glucose uptake and lactate production. The data suggest that IRE1 promotes estrogen-induced glucose uptake while the PERK branch suppresses glucose uptake and glycolysis.

Figure 6.

Figure 6.

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IRE1 activity is required for estrogen-induced glucose uptake and cell proliferation. A, T47D cells were hormone-stripped for 3 days and then treated with or without 10nM E2 alone or plus 1µM GSK2656157, 20µM 4µ8C, or 1µM KIRA6 for 30 hours. The cultured medium was collected for metabolite profile analysis. The glucose uptake and lactate production were normalized to the protein concentration. a, P < 0.05 vs DMSO; b, P < 0.05 vs E2; n.s, P > 0.05 vs DMSO. B, C, T47D cells were cultured in hormone-depleted medium and transfected with siRNA negative control, PERK siRNA, IRE1 siRNA, or ATF6 siRNA. At 24 hours posttransfection, the cells were treated with DMSO or 10nM E2. After 48 hours, the cells were harvested for Western blot analysis (B), and the cultured medium was collected for metabolite profile analysis (C). The glucose uptake and lactate production were normalized to protein concentration. a, P < 0.05 vs siNC of DMSO group; b, P < 0.05 vs siPERK of DMSO group; c, P < 0.05 vs siIRE1 of DMSO group; d, P < 0.05 vs siATF6 of DMSO group; n.s, P > 0.05 vs siIRE1 of DMSO group. D, E, Effect of GSK2656157 and 4µ8C on cell viability of T47D cells in the absence or presence of E2. T47D cells were seeded into 24-well plates after being hormone-stripped for 2 days and continued to culture in hormone-depleted medium. After overnight, the cells were treated with DMSO or 10nM E2. Cell proliferation was measured by the MTT assay at the indicated time after E2 treatment (D). At day 6, the cells were fixed and stained with crystal violet (E). Images were taken using a scanner.

We then examined the effect of UPR on estrogen-induced cell proliferation. Inhibition of IRE1 dramatically suppressed estrogen-induced cell proliferation while inhibition of PERK had little effect on estrogen-induced cell proliferation (Fig. 6D and 6E), implying that the IRE1 branch is important for estrogen-induced cell proliferation in ER+ breast cancer cells.

TXNIP Level Is Associated With Clinical Outcome of ER+ Breast Cancer Patients

It has been reported that TXNIP is a tumor suppressor, and its levels are associated with cancer stages and clinical outcome in many cancers, including breast cancer (28-31). Here we specifically focused on ER+ breast cancer patients and analyzed the correlation between TXNIP level and patient prognosis by using the public available database (http://kmplot.com/analysis/). The Kaplan–Meier plotter of 2561 ER+ breast cancer patients showed that high Txnip mRNA levels have high relapse-free survival (upper quartile survival: 59 months for low-expression cohort vs 97.3 months for high-expression cohort) (Fig. 7A). The patients with high expression of TXNIP also have higher overall survival according to the data from 720 ER+ patients in the database (Fig. 7B). These data indicate that TXNIP expression level is associated with clinical outcome of ER+ breast cancer patients.

Figure 7.

Figure 7.

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Txnip mRNA level is associated with the clinical outcome of ER+ breast cancer patient. A, The correlation between Txnip mRNA levels and the relapse-free survival in 2561 ER+ breast cancer patients in the database. B, The correlation between Txnip levels and the overall survival in 720 ER+ breast cancer patients in the database. The KM plotter database website: http://kmplot.com/analysis/.

Discussion

Warburg effect is a characteristic of cancer. Despite it, not all the tumors exhibit Warburg effect. In this study, 2 widely used breast cancer cell lines, MCF7 and T47D, exhibited 2 different metabolic phenotypes in glucose metabolism upon estrogen treatment (OXPHOS phenotype for MCF7 vs glycolysis phenotype for T47D), although they both are ER+ and rely on estrogen for growth. Our study is consistent with the previous study showing bioenergetic difference between these cell lines (32). We found that the TXNIP is differentially expressed in these 2 cell lines, and its levels are inversely correlated with glucose uptake. Moreover, E2 differentially regulates glucose metabolism in these 2 cell lines. E2 promotes the Warburg effect in T47D cells but not in MCF7 cells. Interestingly, E2 more effectively promotes glutamine uptake in MCF7 cells than in T47D cells. Like glucose, glutamine is also an important source of energy. It is the most common source for anaplerosis and produces ATP through the Krebs cycle and OXPHOS (33). Since MCF7 cells are more OXPHOS type, they consume less glucose but rely more on glutamine, which is also supported by our previous study showing that MCF7-derived cells can still largely proliferate in the absence of glucose, but completely stop proliferation in the absence of glutamine (34).

A previous study showed that estrogen modulates metabolic pathway adaption to the available glucose (35). Our study suggests that TXNIP plays a pivotal role in glucose uptake of ER+ breast cancer cells, and estrogen regulation of TXNIP expression might be a delicate mechanism for estrogen-induced glucose uptake and metabolic reprogramming in ER+ breast cancer cells, since TXNIP negatively regulates glucose uptake and Warburg effect, and estrogen reduces TXNIP expression level. In comparison to MCF7 cells, T47D cells consumed more glucose under the same culture condition and exhibited more glycolysis type. Not surprisingly, E2 downregulated TXNIP expression more obviously in T47D cells than in MCF7 cells. More studies are needed to test whether TXNIP level determines glucose metabolic phenotype in ER+ breast cancer.

The UPR is mediated by at least 3 well-conserved stress sensors: PERK, IRE1, and ATF6. Three sensors give rise to 3 separate branches of the response, all of which aim to alleviate the burden of misfolded proteins and to ensure successful EnR protein homeostasis (36). Among the 3 sensors, PERK and IRE1 share sequence and structural similarity in the EnR luminal domain (37). It is widely believed that in breast cancer cells, estrogen signaling activates all 3 arms of the UPR which is known as the anticipatory UPR (9-11), and IRE1/XBP1 signaling is intimately linked to ERα signaling in luminal breast cancer (38, 39). Estrogen/ERα signaling activates IRE1 branch of anticipatory UPR through a phospholipase C γ–mediated opening of EnR IP3R calcium channels in breast cancer cells (11). A recent study has shown that Ire1a (the gene coding IRE1) is a direct transcriptional target of ligand-activated ERα in hematopoietic progenitor cells (40). In this study, we found that E2/ERα signaling divergently regulates the UPR pathways in ER+ breast cancer cells (activating IRE1 and ATF6 branches while slightly inhibiting the PERK branch). Not surprisingly, some studies have also shown that estrogen represses the PERK branch of UPR (41-43). Sustaining activation of the PERK branch has been reported to mediate estrogen-induced apoptosis in some ER+ breast cancer cells (44, 45). Moreover, the other sex hormone androgen has been reported to divergently regulate the UPR pathways in a similar way in androgen-dependent prostate cancer cells (46).

Estrogens play a fundamental role in the physiology of the reproductive, cardiovascular, skeletal, and central nervous systems. Although several studies have revealed that estrogen represses TXNIP expression (21-25), the mechanisms of estrogen suppressing TXNIP expression have not been resolved. Here we found that IRE1 is required for estrogen suppression of TXNIP expression in ER+ breast cancer cells since inhibition of IRE1 by small compounds or genetic knockdown blocked estrogen suppressing TXNIP expression. This seems to be contradictory with the previous findings that EnR stress upregulates TXNIP expression, and IRE1 induces TXNIP expression in INS-1 cells (47). However, we need to keep in mind that estrogen/ERα signaling activates anticipatory UPR or mild EnR stress, which anticipates future requirements for increased protein folding capacity, and results in promoting rapid growth of cancer cells. In contrast to anticipatory UPR, moderate and severe EnR stress halts cell proliferation in order to restore cellular homeostasis (48). In INS-1 cells, IRE1 under the terminal EnR stress elevates TXNIP expression to activate the NLRP3 inflammasome causing programmed cell death. In contrast, estrogen activates anticipatory UPR, which is required for cell proliferation, a finding that is supported by our results that inhibition of IRE1 blocks estrogen-induced cell proliferation of ER+ breast cancer cells. After activation of IRE1 through anticipated UPR by estrogen, ER+ breast cancer cells reduce TXNIP expression, thus increasing glucose uptake and glycolysis to prepare for rapid growth in response to estrogen. Therefore, estrogen/ERα-IRE1-TXNIP axis may be an important mechanism for breast cancer cells to proliferate in response to estrogen. It is worth mentioning that estrogen regulation of the PERK branch might also contribute to estrogen reprogramming glucose metabolism since we found that estrogen slightly represses PERK signaling and PERK suppresses glucose uptake and glycolysis. It is still needed to reveal how IRE1 is involved in estrogen-induced TXNIP suppression.

In summary, TXNIP is an important negative regulator of glucose uptake and Warburg effect in ER+ breast cancer cells, and estrogen/ER represses TXNIP expression via anticipatory UPR. Through estrogen/ERα-IRE1-mediated TXNIP suppression, breast cancer cells increase glucose uptake and glycolysis to facilitate their rapid proliferation. Clinically, lower TXNIP expression is linked to worse clinical outcome of ER+ breast cancer.

Acknowledgments

We would like to thank Dr. Mei Kong and Dr. Ying Yang from UC Irvine for the help with metabolite profile analysis.

Financial Support: This work was supported by National Institutes of Health (NIH) grant CA44735 (to S.C.), U01ES026137 (S.C.), and the Lester M. and Irene C. Finkelstein Chair endowment (S.C.). The Analytical Cytometry Core, Bioinformatics core, Electron Microscope Core, Light Microscope Core, and Integrative Genomics core were supported by the National Cancer Institute of the NIH under award P30CA33572.

Author Contributions: Y.W. and S.C. designed research; Y.W. performed research; Y.W. and S.C. contributed new reagents/analytic tools; Y.W. analyzed data; and Y.W. and S.C. wrote the paper.

Glossary

Abbreviations

ATF6

activating transcription factor 6

ATP

adenosine triphosphate

CD

charcoal:dextran-stripped

DMSO

dimethyl sulfoxide

E2

17β-estradiol

EnR

endoplasmic reticulum

ER

estrogen receptor (α)

FBS

fetal bovine serum

IRE1

inositol-requiring enzyme 1

OXPHOS

oxidative phosphorylation

PDX

patient-derived xenograft

PERK

protein kinase RNA-like endoplasmic reticulum kinase

RT-qPCR

quantitative reverse transcription–polymerase chain reaction

siRNA

small interfering RNA

TXNIP

thioredoxin-interacting protein

UPR

unfolded protein response

Additional Information

Disclosure Summary: The authors have nothing to disclose.

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

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Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

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