Inhibition of glycolysis represses the growth and alleviates the endoplasmic reticulum stress of breast cancer cells by regulating TMTC3

2.1 Bioinformatics analysis

In order to sort the candidate gene for our study, we downloaded the data from the datasets GSE110960 (effect of restricted glycolysis on gene expression in MCF-7 cells) and GSE111204 (effect of restricted glycolysis on gene expression in MDA-MB-231 cells) from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/).

The common differentially expressed genes (DEGs) were sorted using GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/), and a Venn diagram was subsequently drawn to identify the common DEGs using Venny online software (version 2.1.0, http://bioinfogp.cnb.csic.es/tools/venny/).

2.2 Clinical sample collection

To determine the role of TMTC3 played in BC, 30 paired cancer (defined as “Tumor”) and adjacent tissues (defined as “Normal”) were collected from the Department of Pathology in China−Japan Union Hospital of Jilin University, all of which were harvested from patients admitted from February 2019 to January 2020. All tissues collected were rinsed with saline (IN9000, Solarbio Lifesciences, China) and snap frozen in liquid nitrogen as appropriate.

2.3 Cell culture

All cell lines and the materials used for cell culture were ordered from Procell (Wuhan, China, https://www.procell.com.cn/) unless specified otherwise. Human breast epithelial cell MCF-10A (CL-0525) and BC cell lines MCF-7 (CL-0149), MDA-MB-231 (CL-0150), MDA-MB-436 (CL-0383), and SK-BR-3 (CL-0211) were ordered and cultured as needed.

MCF-10A cells were maintained in Dulbecco’s modified Eagle’s medium/F12 medium (PM150312) blended with 5% horse serum (164215), 20 ng/mL of epidermal growth factor (P00033, Solarbio Lifesciences, China), 0.5 μg/mL of hydrocortisone (G8450, Solarbio Lifesciences, China), 10 μg/mL of insulin (I8830, Solarbio Lifesciences, China), 1% non-essential amino acid (PB180424), and 1% penicillin−streptomycin (PB180120). MCF-7 cell was cultured in minimum essential medium (PM150140) with 0.01 mg/mL of insulin. Leibovitz’s L-15 medium (PM151010) was used to ensure the growth of MDA-MB-231 and MDA-MB-436 cells. SK-BR-3 cell was grown in McCoy’s 5A medium (PM150710). All media for BC cells were supplemented with 10% fetal bovine serum (164210) and 1% penicillin−streptomycin.

All cells were finally incubated in the Sanyo MCO-18AIC(UV) CO₂ incubator (SA-MC018, Marshall Scientific, LLC., Hampton, NH, USA) at 37°C and 5% CO₂. As TMTC3 was lowly expressed in MCF-7 and MDA-MB-231 cells, these two cells were used for subsequent studies.

2.4 Cell treatment and transfection

For cell treatment, both MCF-7 and MDA-MB-231 cells were cultured in the following manner: cells in the control group were cultured normally with no supplementation, while those in the glucose and 2-deoxyglucose groups were cultured in the medium with the supplementation of glucose (10 mM, G8150, Solarbio Lifesciences, China) and 2-deoxyglucose (50 mM, D8930, Solarbio Lifesciences, China), respectively [24].

For transfection, TMTC3 overexpression plasmid was constructed based on the vector pcDNA 3.1 (V790-20, Invitrogen, Carlsbad, CA, USA), and the empty pcDNA 3.1 was used as the negative control (NC).

BC cells MCF-7 and MDA-MB-231 (1 × 10⁶ cells/well) were first seeded in a six-well plate with complete medium at 37°C and 5% CO₂ to be 90% confluent at the time point of transfection. Then, the transfection on both MCF-7 and MDA-MB-231 cells were successfully performed with the help of lipofectamine 2000 transfection reagent (11668-030, Invitrogen, USA) as per the protocols of the manufacturer. After 48 h, all cells were harvested for subsequent studies.

2.5 Lactic acid production determination assay

All procedures of lactic acid production assay were repeated as illustrated previously [25]. The concentration of lactic acid in the harvested lysates was determined using a lactate assay kit (D799851, Sangon Biotech, Shanghai, China) in accordance with the protocols of the manufacturer. MCF-7 and MDA-MB-231 cells (5 × 10⁵ cells/well) with or without transfection were cultured in six-well plates and resuspended in extraction buffer I, followed by lysis at a power of 300 W for 3  min in total (3  s of ultrasonication, at an interval of 7 s) using an XC-II D ultrasonic cell crusher (Nanjing Ningkai Instrument Co., Ltd, Nanjing, China). The lysates were harvested after centrifugation at 12,000 × g at 4°C for 10 min in a 5810 Centrifuge (Eppendorf, Hamburg, Germany). A volume of 0.8 mL of the supernatant was collected and added with 0.15 mL of extraction buffer II, followed by another centrifugation at 12,000 × g at 4°C for 10 min. The absorbance at 570 nm was monitored by Varioskan^™ LUX multimode microplate reader (VLBLATD2; ThermoFisher Scientific, Waltham, MA, USA).

2.6 Cell-Counting Kit-8 (CCK-8) assay

MCF-7 and MDA-MB-231 cells (2 × 10³ cells/well) with different intervention were cultured within 96-well plates at 37°C and 5% CO₂ for 24 and 48 h, and 10 μL of CCK-8 solution (CA1210; Solarbio Lifesciences, China) was added into each well of the plates for a further 4-h incubation in the incubator. The optical density (OD) value was measured using Varioskan^™ LUX multimode microplate reader at an absorbance of 450 nm.

Then, the cell viability was calculated using the following formula:

The OD_dosage represented the OD value of the wells containing transfected and treated cells and CCK-8 reagent, OD_blank symbolized the OD value of the wells with the medium and CCK-8 reagent only, and OD_{no dosage} was the OD value of the wells to which untransfected or untreated cells were added.

2.7 Colony formation assay

Following the intervention as needed, MCF-7 and MDA-MB-231 cells (1 × 10³ cells/well) were seeded in six-well plates at 37°C and 5% CO₂ for 14 days, and the colonies formed were fixed with 4% paraformaldehyde (P1110; Solarbio Lifesciences, China) for 15 min and stained with crystal violet (C8470; Solarbio Lifesciences, China) for 30 min. All colonies formed were then observed and photographed using a digital camera (OM-D E-M5 Mark III; Olympus, Tokyo, Japan). The colony formation rates of cells in each group were calculated by the software SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA, USA).

2.8 Flow cytometry assay

We collected 1 × 10⁶ transfected MCF-7 and MDA-MB-231 cells, washed with phosphate buffered saline (P1010; Solarbio Lifesciences, China) and suspended using 1 mL of binding buffer (1×), followed by centrifugation at 300 × g in a 5810 Centrifuge. The supernatant was abandoned subsequently. All cells were then resuspended with 1 mL of binding buffer (1×), and the density was adjusted to 1 × 10⁶ cells/mL. A volume of 100 μL of cells suspension was added into each tube, and 5 μL of Annexin V-FITC was also added. A gentle blend was performed at room temperature (RT) for 5 min avoiding light, after which 5 μL of propidium iodide (PI) was added into the tube and all cells were additionally incubated at RT for 5 min in the dark. The apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit (CA1020, Solarbio Lifesciences, China) using a S1000EON flow cytometer (Stratedigm, Inc., San Jose, CA, USA), and all data were analyzed using Kaluza C Analysis Software 1.12 (Beckman Coulter, Indianapolis, IN, USA).

2.9 RNA isolation and reverse-transcription quantitative PCR

Total RNA in tissues (both tumor and normal) and cells (BC cells and human breast epithelial cell MCF-10A) was isolated using a total RNA extraction reagent (R1100; Solarbio Lifesciences, China) as guided by the manufacturer, and all isolated RNA samples were preserved in −80°C. The measurement on the isolated RNA was conducted in the NanoDrop^™ lite spectrophotometer (ND-LITE; ThermoFisher Scientific, USA). All procedures of PCR were then performed by a one-Step RT-PCR kit (T2210; Solarbio Lifesciences, China) and operated in CFX384 Touch real-time PCR system (Bio-Rad, Hercules, CA, USA) under the following detailed conditions: 50°C for 20 min for reverse transcription, 95°C for 3 min for pre-denaturation, followed by 40 cycles of 95°C for 15 s for denaturation, and 60°C for 25 s for final extension.

Relative expressions were quantified using the 2^−ΔΔCT method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference [26]. The sequences of primers are listed in Table 1.

2.10 Western blot

Relative protein expression was calculated using western blot as confirmed in a previous study [27]. All materials used were the products of Elabsciences (Wuhan, China) unless specified. Total protein in transfected MCF-7 and MDA-MB-231 cells was lysed and extracted using radio-immunoprecipitation assay lysis buffer (E-BC-R327), and the concentration was quantified using bicinchoninic acid (BCA) method with a BCA protein kit (E-BC-K318). A volume of 20 μL of protein lysates was electrophoresed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (E-IR-R305) and quickly transferred into polyvinylidene fluoride membrane (E-BC-R266). Then the membrane was blocked by 5% skimmed milk at RT for 2 h and incubated in primary and secondary antibodies (the condition for the incubation with the primary antibodies was 4°C overnight and that for secondary antibodies was RT for 1 h).

For subsequent procedures of visualization, the membrane was rinsed using tris-buffer saline tween (E-BC-R335) three times, and was visualized using an enhanced chemiluminescent substrate detection kit (E-BC-R347) within an imaging system (Tanon 5200CE, Tanon, Shanghai, China), and gray values of each band were calculated by ImageJ 5.0 (Bio-Rad, USA).

The primary antibodies (Abcam, Cambridge, UK) used here were those against Caspase-12 (ab62484, 42 kDa), C/EBP homologous protein (CHOP, ab11419, 31 kDa), glucose-regulated protein 78 (GRP78, ab21685, 75 kDa), B-cell lymphoma-2 (Bcl-2, ab182858, 26 kDa), Bcl-2 associated X (Bax, ab32503, 21 kDa), and housekeeping control GAPDH (ab8245, 36 kDa), and the secondary antibodies were the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (E-AB-1003) and HRP-conjugated goat anti-mouse IgG (E-AB-1001). All antibodies (both primary and secondary) were diluted to a ratio of 1:2,000 for our current study.

2.11 Statistical analyses

All statistical analyses were implemented with Graphpad Prism 8.0 (GraphPad, Inc., La Jolla, CA, USA), where all data were the indication of three independent tests and expressed as mean ± standard deviation (SD). Statistical significance, which was determined with one-way analysis of variance followed by Bonferroni post hoc test and paired t test as appropriate, was defined when p-value was lower than 0.05.

1. Ethics statement: The ethics committee of China−Japan Union Hospital of Jilin University has carefully reviewed and approved the conduction of our study (endorse number: 2021-KYLL-030008). Meanwhile, all recruited patients have signed the informed consent in written form and agreed to the usage of their tissues in our study.

3.1 TMTC3 was lowly expressed in BC tissue and cell

To sort the candidate gene for our study, we first downloaded the datasets of GSE110960 (effect of restricted glycolysis on gene expression in MCF-7 cells) and GSE111204 (effect of restricted glycolysis on gene expression in MDA-MB-231 cells) from GEO. A Venn diagram was subsequently drawn to sort the common DEGs, 12 of which were then identified, including TMTC3 (Figure 1a). TMTC3 contributes to the O-mannosylation of E-cadherin, and dysregulation of glycosylation is one of the important mechanisms leading to tumor heterogeneity; in addition, TMTC3 is involved in the regulation of ERS, and ERS affected the malignant growth and progression of tumors. Thus, TMTC3 was used for our studies.

Figure 1

TMTC3 was lowly expressed in BC tissue and cell. (a) Dataset GSE110960 (effect of restricted glycolysis on gene expression in MCF-7 cells) and GSE111204 (effect of restricted glycolysis on gene expression in MDA-MB-231 cells) were used to sort the candidate gene for our study, which was available from GEO (https://www.ncbi.nlm.nih.gov/geo/). A Venn diagram was drawn based on the results. (b) Relative TMTC3 expression in BC (tumor) and adjacent (normal) tissue was calculated via reverse-transcription quantitative PCR (n = 30 for each group). GAPDH was used as the internal control. (c) Relative TMTC3 expression in BC cells (MCF-7, MDA-MB-231, MDA-MB-436, and SK-BR-3) and breast epithelial cell MCF-10A was quantified with reverse-transcription quantitative PCR. GAPDH was used as the internal control. All data were expressed as mean ± SD, which was indicative of three independent tests. ^*** p < 0.001, vs MCF-10A. TMTC3: transmembrane O-mannosyltransferase targeting cadherins 3; BC: breast cancer.

Then we measured TMTC3 expression in both BC and adjacent tissue, and a low TMTC3 expression was evidenced in BC tissue (Figure 1b, p < 0.001). Likewise, we also detected the expression of TMTC3 in BC cells and breast epithelial cell MCF-10A, and the result showed that TMTC3 expression was lower in BC cells (MCF-7, MDA-MB-231, MDA-MB-436, SK-BR-3) than that in MCF-10A cells (Figure 1c, p < 0.001). Among these BC cells, TMTC3 expression in MCF-7 and MDA-MB-231 cells was lower than other BC cells, and thus these two cells were used for our subsequent studies.

3.2 Glycolysis promotion represses TMTC3 expression yet enhances lactic acid production and growth of BC cell, while inhibition elicited contrary results

For subsequent procedures, we cultured both MCF-7 and MDA-MB-231 cells in their respective media supplemented with glycose or 2-deoxyglucose, which have been suggested to promote or repress glycolysis [24]. In the beginning, we measured TMTC3 expression in BC cells after the cells were cultured in the media supplemented with glycose or 2-deoxyglucose, and it was found that the expression of TMTC3 was repressed following the supplementation of glycose, whereas 2-deoxyglucose supplementation elevated TMTC3 expression in BC cells (Figure 2a, p < 0.01).

Figure 2

Glycolysis promotion represses TMTC3 expression yet enhances the lactic acid production and growth of BC cell, while the inhibition did contrarily. (a) The effects of glucose (which promoted glycolysis) and 2-deoxyglucose (which repressed glycolysis) on TMTC3 expression in BC cells MCF-7 and MDA-MB-231 were confirmed with reverse-transcription quantitative PCR. GAPDH was used as internal control. (b–e) The effects of glucose and 2-deoxyglucose on lactic acid production (b), viability (c), proliferation (d), and apoptosis (e) of BC cells MCF-7 and MDA-MB-231 were evaluated via lactic acid production, CCK-8, colony formation and flow cytometry assays, respectively. All data were expressed as mean ± SD, which was indicative of three independent tests. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, vs control. OD: optical density.

Lactic acid was one of the end-products of aerobic glycolysis, which had both the capability to change the tumor microenvironment and an impact on cancer-associated cells [28]. It was also demonstrated that after the supplementation of glycose, the concentration of lactic acid in BC cells was evidently raised, while the additional supplementation of 2-deoxyglucose reduced the concentration of lactic acid (Figure 2b, p < 0.001). When it comes to viability (Figure 2c), proliferation (Figure 2d), and apoptosis (Figure 2e), it was discovered that the supplementation of glycose raised the viability and colony formation rate yet reduced apoptosis in BC cells (Figure 2c–e, P < 0.05); however, contrary results were displayed in the BC cells when the culture medium was added with 2-deoxyglucose. That is, the supplementation of 2-deoxyglucose decreased the viability at 24 and 48 h and colony formation rate yet increased apoptosis in BC cells (Figure 2c–e, p < 0.01). The aforementioned results suggested that promoting glycolysis using glucose represses TMTC3 expression yet enhances lactic acid production and growth in BC cells, whereas inhibiting glycolysis via 2-deoxyglucose increased TMTC3 expression yet repressed the growth of BC cells.

To additionally confirm the mechanisms, we measured the expressions of both ER stress- and apoptosis-related factors in BC cells, as evidenced by the interaction among ER stress, apoptosis, and glycolysis [29,30]. All ER stress-related factors, including Caspase-12, CHOP, and GRP78, and apoptosis-related factor Bcl-2 were promoted but another apoptosis-related factor Bax was suppressed in BC cells cultured in the medium with glucose (Figure 3a–f, p < 0.01). However, in those cells maintained in the medium with 2-deoxyglucose, the decreased levels on ER stress-related factors, including Caspase-12, CHOP, and GRP78, and apoptosis-related factor Bcl-2 were exhibited yet that of apoptosis-related factor Bax was increased (Figure 3a–f, p < 0.05).

Figure 3

Glycolysis posed a regulatory effect on the expressions of factors related to both ER stress and apoptosis in BC cell. (a–f) The effects of glucose and 2-deoxyglucose on ER stress- and apoptosis-related factors (Caspase-12 (b), CHOP (c), GRP78 (d), Bcl-2 (e), and Bax (f)) in BC cells MCF-7 and MDA-MB-231 were unveiled with Western blot. GAPDH was used as internal control. All data were expressed as mean ± SD, which was indicative of three independent tests. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, vs control. ER: endoplasmic reticulum; CHOP: C/EBP homologous protein; GRP78: glucose-regulated protein 78; Bax: Bcl-2 associated X; Bcl-2: B-cell lymphoma-2.

3.3 Overexpressed TMTC3 abrogated the effects of glycolysis in BC cells

To further confirm the interaction between glycolysis and TMTC3 in BC cells, we transfected TMTC3 overexpression plasmid into BC cells, which were subsequently cultured in the medium supplemented with glycose. The successful transfection was evidenced by the increased TMTC3 expression in BC cells which have been maintained in the medium with glycose (Figure 4a, p < 0.001). No evident effect on the lactic acid concentration in BC cells was displayed in BC cells following the transfection of TMTC3 overexpression plasmid (Figure 4b), whereas it was clear that TMTC3 overexpression decreased the viability and colony formation yet enhanced the apoptosis of BC cells cultured with glycose (Figure 4c–e, p < 0.01). Herein, we concluded that overexpressed TMTC3 could have abrogated the effects of glycolysis promotion on the malignant behaviors of BC cells.

Figure 4

Overexpressed TMTC3 abrogated the effects of glycolysis. (a) The effects of glucose and TMTC3 on TMTC3 expression in BC cells MCF-7 and MDA-MB-231 were unveiled in accordance with the results of reverse-transcription quantitative PCR. GAPDH was used as internal control. (b–e) The effects of glucose and TMTC3 on the lactic acid production (b), viability (c), proliferation (d), and apoptosis (e) of BC cells MCF-7 and MDA-MB-231 were evaluated via lactic acid production, CCK-8, colony formation and flow cytometry assays, respectively. All data were expressed as mean ± SD, which was indicative of three independent tests. ^** p < 0.01, ^*** p < 0.001, vs glucose + NC. NC: negative control.

As for the ER stress- and apoptosis-related factors, TMTC3 overexpression diminished the effects of glycose on promoting Caspase-12, CHOP, GRP78, and Bcl-2 yet repressing Bax in BC cells (Figure 5a–f, p < 0.001). The results thus suggested that TMTC3 overexpression reversed the regulatory effects of glycolysis on the ER stress- and apoptosis-related factors in BC cells.

Figure 5

Both TMTC3 and glycolysis posed regulatory effects on the expressions of both ER stress- and apoptosis-related factors in BC cell. (a–f) The effects of glucose and TMTC3 on ER stress- and apoptosis-related factors (Caspase-12 (b), CHOP (c), GRP78 (d), Bcl-2 (e), and Bax (f)) in BC cells MCF-7 and MDA-MB-231 were unveiled with Western blot. GAPDH was used as internal control. All data were expressed as mean ± SD, which was indicative of three independent tests. ^*** p < 0.001, vs glucose + NC.