Members of the endoplasmic reticulum membrane protein complex (EMC) family can affect various cellular processes, including protein transport, endoplasmic reticulum stress, and lipid homeostasis [19]. EMC in yeast can recruit protein maturation factors in the ER lumen and participate in the insertion of transmembrane domains through the synergistic translocation of Sec61 [20] and can cooperated with molecular chaperones to assist the correct folding of newly generated peptides [21]. EMC2, a member of EMC, can bind to the molecular chaperone heat shock protein HSP90 to promote the correct insertion and folding of the protein into the endoplasmic reticulum [22]. At present, the researches on the function and molecular mechanism of EMC2 in tumors are still very limited, only bioinformatics analysis reported that high expression of EMC2 is closely related to the poor prognosis of bladder urothelial carcinoma, breast cancer and uveal melanoma [23]. In this study, we uncovered a unique biological mechanism which orchestrates intracellular cholesterol biosynthesis. We unraveled the role of EMC2 in cholesterol synthetic dysfunction and ferroptosis suppression with bioinformatic analysis of TCGA dataset, protein mass spectrum (MS) and further accurate experiments. Mechanistically, we clarified that EMC2 could prevent FDFT1 from endoplasmic reticulum related degradation (ERAD) by controlling its protein quality in the endoplasmic reticulum. All these findings may offer a novel theoretical basis for exploiting novel anti-tumor drugs targeting EMC2 for TNBC.
The TNBC cell lines, MDA-MB-231, BT-20, and MDA-MB-468 used in this study were obtained from the Shanghai Jiaotong University affiliated Renji Hospital and tested for mycoplasma contamination. MDA-MB-231 and MDA-MB-468 were cultured in Leibovitz Medium (L15, Gibco) containing 10% Fetal Bovine Serum (FBS, Gibco) and 1% penicillin and streptomycin (Gibco). BT-20 was cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 10% Fetal Bovine Serum (FBS, Gibco) and 1% penicillin and streptomycin (Gibco). All the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO.
Antibodies and corresponding applications used in this study were shown in Supplementary Table 1. Matrigel was purchased from Corning. Polybrene and puromycin from Sigma-Aldrich. Cholesterol (C3045) from Sigma-Aldrich. MG132(S1748) from Beyotime. CHX (HY-12320), MCD (HY-101461), Eer I (HY-110078) from Med Chem Express.
The small interfering RNA (siRNA), knockdown and control lentiviruses (designated as shEMC2 and shNC) were purchased from Genepharma (Shanghai, China). The target sequences were listed in Supplementary Table 2. The overexpression or control lentiviruses (designated as oeEMC2, oeFDFT1, oeNC, and HA-Ub) were purchased from Obio Technology Co. (Shanghai, China). The construction reports were affiliated with the supplementary information.
The siRNAs and plasmids were transfected into the TNBC cells at a final concentration of 20 nM with the jetPRIME DNA & siRNA transfection reagent (Polyplus, #114-15) according to the manufacturer's instructions. The cells were harvested for the following experiment 24-72 h after transfection.
The stable cell lines were obtained by infection with the lentivirus and puromycin selection (10 μg/ml). The efficiency of stable cell line selection was examined by real-time PCR and western blotting.
Cell suspensions (3-5 × 10/well) were seeded in the 96-well culture plates and incubated or treated with the reagent. 10% CCK8 solution (Shanghai Coryea Biotechnology Co, KS301) was added into each well and cells were incubated for another 1 h and then measured by a microplate reader (BioTek) at 450 nm.
Cell suspensions (2-5 × 10/well) were seeded in the 6-well culture plates and incubated or treated with the reagent. After 2 weeks and the single colonies reached more than 50 cell, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Then cells were stained with 0.1% crystal violet for 1 h and washed. The colonies were counted under a microscope (Leica).
In the transwell migration assay, 2 × 10 cells were seeded into the upper compartment of the transwell chamber (Corning) in serum-free DMEM. For cell invasion assay, 2 × 10 cells were seeded into the matrigel upper chamber in serum-free DMEM. DMEM with 10% FBS was added in the lower chamber. After 24 h of incubation, the cells were fixed with 4%PFA and stained with 0.1% crystal violet. The cells were counted under a microscope (Leica).
Tumors and adjacent normal tissues of RENJI cohort 1,2, and 3 were obtained from patients with breast cancer who performed surgery at Shanghai Renji Hospital. RENJI cohort 1 contains 37 tumor tissues and 26 adjacent normal tissues for RNA extraction. RENJI cohort 2 contains 49 formalin-fixed paraffin-embedded human triple negative breast cancer specimens with survival information. RENJI cohort 3 contains 57 formalin-fixed paraffin-embedded human breast cancer specimens. None of the patients in these cohorts received chemotherapy before surgery.
The IHC staining of paraffin-embedded tissues was performed and scored as standard procedure. In brief, tumor samples were fixed in formalin, embedded in paraffin, and sectioned at 4 μm thickness. These sections were then subjected to IHC staining. Representative images were randomly taken at the mentioned magnification using a Leica DM2500 microscope. The IHC score was calculated according to the positive cell percentage calculated by the average calculation of two pathologists. The antibody application concentration of IHC was listed in the supplementary Table 1.
Total RNA was isolated using Simply P total RNA extraction kit (BioFlux, BSC52S1) following the manufacturer's instructions. The purity and concentration of RNA were measured with a UV spectrophotometer (Thermo Scientific NanoDrop, 2000C). The RNA was reverse transcripted with the HiScript-TS 2 × PCR Mix (Vazyme, RA103). Real-time PCR analysis was performed with the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711) and LightCycler 480 System (Roche). The reverse transcription and real-time PCR conditions were according to the manufacturer's protocol. The sequences of primers were listed in the Supplementary Table 3.
Samples were lysed in fresh RIPA Lysis Buffer (Epizyme, PC102) with protease inhibitor cocktail 100X (Sigma) and phosphatase inhibitor cocktail 100X (Sigma) according to the manufacturer's protocol. Then the protein concentrations were determined by the BCA protein assay (Beyotime, P0010). The Lysates were boiled for 10 min with the loading buffer (denaturing, reducing,5×, Epizyme, LT101). Western blot analysis used 10% polyacrylamide gel (Epizyme, PG212) and transferred it onto a PVDF transfer membrane (Millipore, IPVH00010). Then the blot membranes were incubated with corresponding antibodies and the bands were visualized by Enhanced chemiluminescence (ECL) (Millipore, WBKLS0500) and ChemiDoc Touching Imaging System (Bio-rad). The blot bands were quantified by Image Lab software (Version 6.0.1, Bio-rad). During quantification, β-actin and Calnexin were used for internal reference. The antibodies and applications were listed in the supplementary Table 1.
Cells were fixed with 4% paraformaldehyde (PFA) and incubated with permeabilizing solution (Sigma, Triton X-100). Then cells were incubated with corresponding antibodies and fluorescent images were captured using a Leica DM2500 microscope. The antibodies and applications were listed in the supplementary Table 1. The confocal imaging analysis was carried out by "Image J" software (National Institutes of Health, NIH).
The co-immunoprecipitation was performed with a classic magnetic protein A/G IP/Co-IP Kit (Epizyme, YJ201) according to the protocol. In brief, cells were lysed and incubated with corresponding antibodies at 4 °C overnight. Then, the lysis was incubated with the magnetic protein A/G at 4 °C overnight. Finally, the lysates were boiled for 10 mins with the loading buffer and for the next experiments. The antibodies and applications were listed in the supplementary Table 1.
The female five-week-aged BALB/c nude mice were obtained from Shanghai Charles River Animal Co., Ltd. Mice, were grouped into six animals randomly and housed in a specific pathogen-free (SPF) environment in the animal center of Renji Hospital. BT-20 cells (1 × 10/100 μl) and MDA-MB-231 cells (1 × 10/100 μl) were injected subcutaneously into the right flank of the mice to establish a breast cancer xenograft model. The tumor was calculated every 5 days after the injection as follows: Tumor volume (V) = tumor longer diameter (a) × tumor shorter diameter (b) /2. When the experiment ended, or the tumor volume reached 1500 mm, the mice were sacrificed, and the tumors were isolated for further experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University.
The free cholesterol levels of TNBC cells were determined using the free cholesterol assay kit (Applygen Technologies, E1016) following the protocol. In brief, cell samples were washed with PBS twice and then lysed and collected in microtubes. The samples and standard cholesterol with different concentrations were mixed and incubated with working solutions provided by the manufacturer for 20 min at room temperature and then measured by a microplate reader (BioTek) at 550 nm.
The level of Fe was detected using the iron assay kit (Sigma-Aldrich, MAK025) according to the operating protocol of the manufacturer. In brief, cell samples were homogenized in Iron Assay buffer and centrifuged at 16,000 × g for 10 min at 4 °C. The test samples and iron standards incubated for 30 min at 25 °C protected from light. Then Iron Probe was added to each well and mixed and incubated for 60 min at 25 °C protected from light. Finally, the ferrous iron levels were measured by a microplate reader (BioTek) at 593 nm.
The level of lipid ROS was evaluated using the C11-BODIPY (Thermo Fisher, D-3861). In brief, different groups of cells were incubated with 20 μM C11-BODIPY for 30 min and protected from the light. Then cells were washed and resuspended in the PBS, detected with a flow cytometer (BD Biosciences, LSRFortessa X-20). The relative ROS level was calculated with the FITC fluorescence intensity.
The level of GSH/GSSG ratio was measured by the GSH and GSSG assay kit (Beyotime, S0053) according to the manufacturer's procedure. In brief, cell samples were lysed, then put into liquid nitrogen and then at 37 °C twice. The samples were centrifuged at 10,000 g for 10 minsat 4 °C. The supernatant samples and standard GSH were mixed with wording buffer and incubated for 5 min at 25 °C. Then NADPH solution was added and the total GSH absorbances were measured by a microplate reader (BioTek) at 412 nm. Special reagents were then used to clear GSH and GSSG absorbances were measured by a microplate reader (BioTek) at 412 nm. The GSH/GSSG ration was calculated according to the protocol.
The label-free relative quantitative proteomics analyses for EMC2-associated proteins were performed at Instrumental Analysis Center at Shanghai Jiaotong University. The proteomics was operated with the Easy nLC1200/Q-Exactive plus mass spectrometer (Thermo Scientific). The raw data were searched against the UniProt-Human database.
The LC-MS analysis was performed at Shanghai Luming Biological Technology Co., Ltd. The proteomics was operated with the Ultimate 3000 RSLCnano/Q-Exactive plus mass spectrometer (Thermo Scientific). The raw data were searched against the UniProt-Human database.
Cells were transfected with combinations of plasmids and siRNA, including HA-ubiquitin plasmids. Before harvest, cells were treated with 10 μg/ml MG132 for 6 h. Then cells were used for the following research to detect the ubiquitination level of target proteins.
The extraction of ER was performed by the ER enrichment kit (Novus Biologicals, NBP2-29482) according to the protocol. In brief, cell samples were homogenized with 1×Isosmotic Homogenization Buffer followed by 100× PIC and centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was centrifuged at 12,000 × g for 15 min at 4 °C. Then the supernatant was centrifuged at 90,000 × g (Beckman Avanti J30I centrifuge with JS24 rotor) for 60 min at 4 °C. The pellet was collected as the total endoplasmic reticulum fraction for the following experiments.
Cells were fixed with 4% PFA and washed with PBS. Then cells were incubated with 0.05 mg/ml Filipin III (Sigma, SAE0087) for 1 h. Immunofluorescence images were captured using a Leica DM2500 microscope.
The aggresome was detected by PROTEOSTAT Aggresome Detection Kit (Enzo, ENZ-51035) according to the protocol. In brief, cells samples were washed with PBS twice and fixed with 4% PFA for 30 min at room temperature. Then, samples were incubated with a permeabilizing solution (Sigma, Triton X-100) and washed with PBS twice. Then, the Dual Detection Reagent was added and incubated for 30 min at room temperature, protected from light. Finally, the immunofluorescence images were captured using a Leica DM2500 microscope. The calculation was measured by "Image J" software (National Institutes of Health, NIH).
The cancer genome atlas (TCGA) datasets were obtained by the R package "TCGAbiolinks". Counting data is converted to transcripts per million (TPM) and normalized log2 (TPM + 1) while keeping clinical information intact. The Gene Expression Omnibus (GEO) datasets were obtained from the following website: https://www.ncbi.nlm.nih.gov/gds/?term=GSE. Only cancer tissue was included. The 27 cholesterol biosynthesis genes from the REACTOME pathway were obtained from the following website: https://reactome.org/content/detail/R-HSA-191273. Then we performed consensus analysis using the R package ConsensusClusterPlus (v1.54.0), with number of clusters set to two, repeating 100 times by extracting 80% of the total samples, and setting clusterAlg = "hc" and innerLinkage = 'ward.D2'.The differential expressed genes (DEGs) analysis was performed with R package Limma (v3.40.2) using the two clusters mentioned before. In the data from TCGA or GEO, we analyzed the adjusted P-values to correct for false positive results. In cholesterol biosynthesis candidate genes filter, 1432 upregulated mRNAs were selected (Log FC > 0.5, P value < 0.001, average expression >5) from TCGA Triple negative breast cancer, 1216 upregulated mRNAs were selected (Log FC > 0.25, P value < 0.1, average expression >7) from GSE106977, 1144 upregulated mRNAs were selected (Log FC > 0.25, P value < 0.001, average expression >5) from GSE76250, and 457 upregulated mRNAs were selected (Log FC > 0.8, P value < 0.05, average expression >5) from GSE38959. The candidate cholesterol biosynthesis-related genes were first selected by the overlapping of the DEGs in the four datasets.
Statistical analyses were performed using R-3.6.3 and GraphPad Prism 9.5.1. The experiments were repeated at least three times with similar results. All data were presented as the means ± SDs. Two-sided unpaired Student's t tests, one way ANOVA tests, chi-square tests, Kaplan-Meier analysis, and log-rank tests were used to evaluate the data according to the figure legends. Differences were listed in the figures as detailed numbers unless P < 0.001. All p values were two-sided unless otherwise specified.