Expression of PGK1 in Breast Cancers Alters Their Sensitivity to Ferroptosis Induction via Metabolic Reprogramming

Expression of PGK1 in Breast Cancers Alters Their Sensitivity to Ferroptosis Induction via Metabolic Reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of PGK1 in Breast Cancers Alters Their Sensitivity to Ferroptosis Induction via Metabolic Reprogramming Felix Oyelami, Andrew Shinkle, Chrispus Ngule, Folake Oyelami, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7323200/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Therapeutic resistance and recurrence are among the major contributors to poor outcomes for patients with breast cancer. Induction of ferroptosis, a form of cellular death characterized by toxic lipid peroxide overload, has emerged as a promising therapeutic strategy against breast cancers including triple-negative breast cancer(TNBC). Nevertheless, certain types of cancer are impervious to induction of ferroptosis and the underlying mechanisms remain incompletely clear. In this study, we show that phosphoglycerate kinase 1 (PGK1), an important enzyme in glycolysis, is highly expressed in breast tumors, and the elevated levels of PKG expression correlate with advanced tumor stages, poor prognosis and ferroptosis insensitivity, particularly in TNBCs. Using genetic or pharmacological inhibition, we demonstrate that knockdown or inhibition of PGK1 enhances ferroptosis sensitivity in both TNBC and luminal breast cancer cell lines. We further demonstrate that depletion of PGK1 destabilizes glutathione peroxidase 4 (GPX4), an anti-ferroptotic defense peroxidase, thereby disturbing cellular redox homeostasis and promoting lipid peroxidation. Moreover, targeting PGK1 disrupts glycolytic metabolism and sensitizes breast cancer cells to ferroptosis induction in tumor cells subjected to glucose deprivation or treated with glycolytic inhibitors. In orthotopic TNBC models, loss of tumoral PGK1 augments the action of the ferroptosis inducer, imidazole ketone erastin (IKE), in inhibiting tumor growth and metastasis, and enhances CD8 + T cell-mediated anti-tumor immunity. These results indicate that PGK1 has a critical role in modulating breast cancer invulnerability to induction of ferroptosis, implying that this kinase may be exploited as a therapeutic target to sensitize breast cancers, especially, TNBC, to ferroptosis inducers. PGK1 ferroptosis GPX4 breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message PGK1 overexpression contributes to ferroptosis resistance in aggressive breast cancers, including triple-negative breast cancer. Genetic or pharmacological inhibition of PGK1 sensitizes tumor cells to ferroptosis induction and suppresses tumor growth in vivo. PGK1 targeting enhances anti-tumor immune responses, indicating potential synergy with immune checkpoint blockade. Combining PGK1 inhibition with ferroptosis inducers offers a promising therapeutic approach for treatment-resistant breast cancers. Introduction Therapy resistance and tumor recurrence are the main causes of the mortality of breast cancer patients [ 1 ], [ 2 ]. Increasing evidence has shown that metabolic reprogramming enables cancer cells to survive in hostile microenvironments, sustain growth and proliferation, and eventually escape death [ 3 ], [ 4 ], [ 5 ]. Thus, targeting the key metabolic components to promote cancer cell death has been considered a promising strategy for the treatment of aggressive cancers including lethal breast cancer subtypes such as triple-negative breast cancer (TNBC) [ 6 ], [ 7 ], [ 8 ]. Ferroptosis, a non-apoptotic and iron-dependent form of programmed cell death, is characterized by an overload of lipid peroxidation and loss of redox balance. In recent years, activating ferroptotic cell death for the treatment of various cancers has been under extensive investigation. Here, we report that phosphoglycerate kinase 1 (PGK1), an essential glycolytic enzyme [ 9 ], [ 10 ], [ 11 ], is highly expressed in breast cancers including TNBC and contributes to cellular insensitivity to ferroptosis induction. We show both in vitro and in vivo that depleting PGK1 by RNA interference or inhibiting the activity of this kinase by its small molecule inhibitor can render tumor cell sensitivity to induction of ferroptotic cell death, and this sensitization is mediated through metabolic reprogramming. In ferroptosis-sensitive cells, PGK1 loss leads to Pyruvate dehydrogenase (PDH) downregulation, restricting pyruvate entry into the Tricarboxylic Acid (TCA) cycle and causing a compensatory shift toward glycolysis. This inefficient adaptation increases metabolic stress, extracellular acidification, and lipid peroxidation, ultimately promoting ferroptosis. Conversely, in cells that do not exhibit ferroptosis sensitization, PDH is upregulated, maintaining oxidative metabolism and redox homeostasis, hampering ferroptotic susceptibility. The results of this study imply that targeting PGK1 in combination with a ferroptosis inducer may be exploited as a potentially novel approach to the treatment of ferroptosis-insensitive breast cancer and likely other malignant tumors. Materials and Methods Reagents. Erastin (MedChemExpress, NJ, USA, #HY-15763), Imidazole ketone erastin (MedChemExpress, NJ, USA, #HY-114481), NG52 (MedChemExpress, NJ, USA, #HY-15154, ), Ferrostatin (MedChemExpress, NJ, USA, #HY-100579), Laemli lysis buffer, RIPA lysis buffer, Complete protease inhibitor (Roche, MH, GERMANY, # 0469331001), Dimethyl Sulfoxide (DMSO) (VWR, PA, USA, #0231), Triton X-100 (SIGMA ALDRICH MO, USA, #T8787, RRID: AB_2629483) Aquabluer solution (BocaScientific, Wahington, MA, USA, #6015, RRID: AB_12345678), Necrostatin (Selleck, TX, USA, #S8037), Liproxstatin (Selleck, TX, USA, #S7699), Bovine serum albumin (Santa Cruz Biotechnology, TX, USA, #sc-2323A), N-Acetyl cysteine (SIGMA ALDRICH MO, USA, #A9164), Z-VAD FMK (Selleck, TX, USA, #S7023), mirVana™ MiRNA isolation kit (Thermofisher Scientific, VL, Lithuania, # AM1561). DAPI (Thermofisher Scientific, VL, Lithuania, # P36941, RRID: AB_2629482) jetPRIME Transfection Reagent (Polyplus-satorius, IL, FRANCE), PGK1 siRNA (Dharmacon, CA, United Kingdom, #5230), PGK1 siRNA (Santa Cruz Biotechnology, TX, USA, #sc-36215), Crystal violet (SIGMA ALDRICH MO, USA, #C3886, RRID: SCR_015456), MojoSort™ Mouse CD8 T cell Isolation Kit (Biolegend, CA, USA, #480008) PGK1 shRNA (m) Lentiviral Particles (Santa Cruz Biotechnology, TX, USA, # sc-36216-V) Antibodies. PGK1 (Santa Cruz Biotechnology, TX, USA, #sc-130335, RRID:AB_2165228), PGK1 (NOVUS biological, CO, USA, #NBP2-19784, RRID:AB_2786860), ACSL4 (Santa Cruz Biotechnology, TX, USA, #sc-365230, RRID:AB_10847863), GPX4 (Santa Cruz Biotechnology, TX, USA, #sc-166437, RRID:AB_2279252), FSP1 (AMID) (Santa Cruz Biotechnology, TX, USA, #sc-376594, RRID:AB_11149443), BMAL1 (Santa Cruz Biotechnology, TX, USA, #sc-365645, RRID:AB_RRID:AB_10842165), Pyruvate Dehydrogenase (Cell Signaling Technology, MA, USA, #2784, RRID:AB_2061989), PKM1/2 (Cell Signaling Technology, MA, USA, #3186, RRID:AB 2162606), PKM2 (Cell Signaling Technology, MA, USA, #3186, RRID:AB 1904096),b-Actin (Cell Signaling Technology, MA, USA, #3700, RRID:AB_2242334), LDHA (Cell Signaling Technology, MA, USA, #3582, RRID:AB_10694487), Ferritin (Santa Cruz Biotechnology, TX, USA, #sc-376594, RRID:AB_11149443), CD71 (Santa Cruz Biotechnology, TX, USA, #sc-32272, RRID:AB_627832), Cyclohexamide (Cell Signaling Technology, MA, USA, #2112), MG132 (Cell Signaling Technology, MA, USA, #2194), CD25 (Cell Signaling Technology, MA, USA, #39475, RRID:AB_2799050), Granzyme B (Cell Signaling Technology, MA, USA, #17215, RRID:AB_10694683). Bioinformatics Analysis To investigate the expression and clinical relevance of PGK1 in breast cancer, publicly available datasets such as RNA seq data from the TCGA, along with GEO datasets, were retrieved using cBioPortal (RRID: SCR_014555) for transcriptomic profiling and copy number variation analysis. Expression patterns of PGK1 in the luminal, HER2-positive, basal and triple-negative breast cancer (TNBC) subtypes were compared to normal breast tissues. Clinical outcome data, including Overall Survival (OS), Post-Progression survival (PPS), and Relapse-Free Survival (RFS), were obtained from the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR) using the KM Plotter[ 12 ][ 13 ]. To determine PGK1 protein expression levels, UALCAN (RRID: SCR_018826) was used to analyze Clinical Proteomic Tumor Analysis Consortium (CPTAC) data, allowing for stage-specific and subtype-specific comparisons between breast tumors and normal tissues. Breast cancer patients were stratified into high and low PGK1 expression groups, then the prognostic significance of PGK1 was investigated using the Kaplan-Meier Plotter (RRID: SCR_018753) which compared the overall survival (OS) and Relapse-free survival (RFS) between the groups. Additionally, ferroptosis-related gene signatures were examined using datasets from GEO to explore the association between PGK1 expression and ferroptosis resistance. Cell culture MCF7 (RRID: CVCL_0031), MDAMB231 (RRID: CVCL_0062), BT549 (RRID: CVCL_1092), MDAMB468 (RRID: CVCL_0419) and cells were acquired from ATCC (ATCC, VA, USA). HCC70 (RRID: CVCL_1258), T47D (RRID: CVCL_0553), HCC1187 (RRID: CVCL_1248) and MDAMB453 (RRID: CVCL_0418) were kind gifts from Dr Wu Yadi. For in vitro experiments, Cells were cultured in RPMI, DMEM-F 12 or High-glucose DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and incubated at 36.7 o C with 5% C02. For drug treatment, 5,000 cells were treated with various concentrations of Erastin (0, 0.5, 1, 2, 4, 10, 20, 40, 80 µM) for up to 72 hours. To interfere with ferroptosis and other cell death mechanisms, cells were treated with Erastin alone or in combination with NG52 (5µM) cell death inhibitors such as ferrostatin (20µM), Liproxstatin (1µM), Necrostatin (20µM), Zvad-FMK (100µM), NACC (20mM) as described by Ghoochani et.al, 2021[ 14 ]. Western blotting Cells were collected into cold Phosphate-buffered saline, spun down and protein was extracted in Laemli or RIPA buffer supplemented with protease inhibitor. Protein concentration was evaluated using a Bicinchoninic Acid (BCA) assay kit (thermos Scientific, #23228, Waltham, MA, USA). Cell lysates were run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 volts for 2 hours. Proteins were transferred using an immune-blot PVDF membrane in a transfer buffer at 100 volts for 80 minutes). Membranes were blocked in 5% defatted milk in PBST for one hour and blots were incubated overnight with appropriate primary antibody at 4 o C. Secondary antibody was incubated at room temperature for 1 hour. PBST buffer was used to wash the membranes for 20 minutes before and after incubation in Horse-radish Peroxidase (HRP)- secondary antibody. Protein expression levels were visualized using the ChemiDOC™ MP imaging system (BioRad, Hercules, CA, USA) Quantitative Real-time PCR RNA was extracted from the cell pellets using an Invitrogen mirVana™ MiRNA isolation kit (Thermofisher Scientific, VL, Lithuania, # AM1561). First-strand cDNA was produced from 2µg of total RNA using Invitrogen SuperScript™ IV First-strand Synthesis System (Thermofisher Scientific, VL, Lithuania, #18091050), and qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Seahorse Analysis The extracellular Acidification Rate (ECAR) was evaluated by the Seahorse Bioscience Extracellular Flux Analyzer (XF 96 Agilent, RRID: SCR_019545). MCF7, MDAMB231 and MDAMB468 cells were seeded in 96-well seahorse plates at densities of 5000, 15,000 and 20,000 per well respectively in 200⎧L of DMEM and allowed to attach overnight. Next, Cells were treated according to experimental conditions and incubated for an additional 48 hours. Before the assay, cells were washed twice and incubated in a seahorse assay medium at 37°C in a non-CO₂ incubator for 1 hour to equilibrate. The assay was performed following the standard Glycolysis stress test protocol with consecutive injections of glucose oligomycin, and 2-deoxyglucose (2-DG). ECAR was measured at baseline level and following each injection to assess glycolytic flux. The data obtained were normalized to the total protein content per well as determined by the BCA protein assay and subsequently analyzed using Seahorse Wave software (RRID: SCR_014526). Immunofluorescence Assay Tumor cells (2 x10 5 cells/mL) were seeded on sterile round coverslips that were placed in a 24-well plate and treated following the experimental procedure. The cells were then fixed with 300 µL of cold methanol (-10 o C) for 5 mins, air dried and permeabilized with 0.25% Triton X-100 in PBS for 10 mins. Next, the cells were blocked with Blocking Solution (2% Bovine Serum Albumin + 0.1% Triton X-100 in PBS) for 1 hr at room temperature, incubated overnight at 4 0 C with primary antibodies diluted in blocking solution according to the manufacturer’s instructions. Subsequent steps were performed protected from light. The cells were washed three times with 1X PBS, then incubated with secondary antibodies diluted 1:500 in Blocking Solution for 1 hr at room temperature. Cells were washed with 1X PBS for 5 mins, incubated in DAPI (300 nM) at room temperature for 5 mins and washed for another 5 mins with 1X PBS. Slides were mounted on carrying glass and stored overnight at 4°C in dark conditions. For tissues, Formalin-fixed paraffin-embedded (FFPE) tumor resections from Balb/c mice were processed by the biospecimens core facility of the University of Kentucky per the approved Institutional Review Board (IRB) protocol). Immunohistochemistry was performed following the procedures as previously described by[ 15 ]. Protein localization was visualized with a fluorescent microscope at specified exposure times. Cell viability assay Aquabluer redox indicator for cell viability was used to evaluate cell viability. Briefly, 5 × 10 4 control or PGK1-depleted (siRNA-transfected or NG52-treated) cells were seeded into 100µl of growth medium per well in 96-well plates and then treated with Erastin alone or in combination with Fer-1 (20µM), Liprox (1µM) Nec-1 (20µM), Z-vad-FMK (100µM), n-acetyl cysteine (20mM) and cultured for 72 hours. Afterwards, cells were cultured in 100ul of reconstituted Aquabluer solution, incubated for four hours and the absorbance was measured at 450 nm by a microplate reader to evaluate cell viability. For all drug treatments, compounds were dissolved in DMSO and further diluted in cell culture medium. The final concentration of DMSO in all experiments did not exceed 0.5% (v/v) , a level reported to be non-cytotoxic in breast cancer cell lines such as MCF7 and MDAMB231. All control groups were treated with an equivalent volume of DMSO alone to serve as baseline for normalization of viability or response. Colony formation assay 200 control or PGK-depleted MCF7, MDAMB468 and 4T1 cells were seeded in a 12-well flat bottom plate and allowed to adhere overnight. Then, the cells were treated with DMSO as controls or 1.25µM of Erastin for 14 days. Afterwards, the cells were fixed in 4% paraformaldehyde for 14 minutes, stained with crystal violet solution for 15 minutes, rinsed with double distilled water and air-dried at room temperature for later visualization of the colonies. Cell transfection MCF7 and MDAMB231 cells were transfected with 800nm of PGK1 siRNA using jetPRIME Transfection Reagent. Following transfection, cells were incubated at 37 o C for 72 hours before experimental use. L2T lentiviral particles were transfected into 4T1 cells at a concentration of 40µg/ml with Polybrene (10µg/ml) overnight, then cells were allowed to recover for 3 days and expanded before experimental use. Stable PGK1 knock-down cell lines were generated by transfecting 10µl of mouse PGK1 lentiviral shRNA particles with 10µg/ml of puromycin into wild-type 4T1 cells. Stable PGK1 knock-down clones were then selected with puromycin and expanded before experimental use. Animal Studies Animals used in this investigation were treated humanely according to the recommendations set by the American Veterinary Medical Association, and the Institutional Animal Care and Use Committee of the University of Kentucky approved all the test protocols (protocol number IBC-24-4) 20 BALB/c ( RRID: IMSR_JAX:000651 ) mice were equally orthotopically injected with 1x10 6 L2T-transfected Wild-type or PGK1 knockdown 4T1 cells in the fifth mammary fat pad and tumor size was measured every four days weekly. After tumor size reached 80-100mm 3 , mice were randomly separated into two groups comprising 5 mice each. Mice were then intraperitoneally treated with either vehicle (5% DMSO, 4% PEG, 4%Tween-80 in saline) or Imidazole ketone Erastin (40mg/kg) once every day for 14 days. Isolation of Cytotoxic T Cells from Spleen Mice were orthotopically inoculated with 4T1 cells, and the tumors were allowed to grow for 21 days under the aforementioned treatment conditions. Next, the mice were euthanized, and their spleens were harvested. The MojoSort™ Mouse CD8 T cell Isolation Kit was used to isolate cytotoxic killer cells from the spleens of mice from each treatment group. This kit utilizes positive selection to isolate CD8 T cells from other immune and stroma cells. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the University of Kentucky (protocol code IBC-24-4 JMY; Date approved :01.28.2025). In accordance with our approved Institutional Animal Care and Use Committee (IACUC) protocol, animals bearing tumors were monitored closely, and mice were euthanized when tumor volume reached 1 cm in diameter or earlier if signs of discomfort or distress were observed. This limit is consistent with ethical guidelines to minimize animal suffering and has been explicitly described in our approved protocol (IBC-24-489). We confirm that in all experimental cohorts, the maximal tumor size of 1 cm in diameter was not exceeded. All animals were euthanized promptly upon reaching this limit or upon displaying any signs of distress, in strict compliance with our IACUC-approved humane endpoints. Statistical analysis Statistical analysis was performed using GraphPad Prism version 10.4.1 (GraphPad Software, CA, USA). All experimental studies comprised at least three independent experiments represented as mean ± SD. T- test was used for two groups or Analysis of Variance (ANOVA) for multiple groups. Significance was defined as p-value < 0.05 between experimental and control groups. Results 3.1 High PGK1 expression in breast Cancer is associated with poor prognosis and ferroptosis insensitivity. We observed that PGK1 was expressed at different levels among subtypes of breast cancer cell lines, even within similar categories of breast cancers (Fig. 1A). Using Xena browser, we analyzed the comprehensive datasets from the Cancer Genome Atlas Program (TCGA). In the TCGA samples of which the majority are primary tumor tissues, PGK1 expression was higher in breast tumor tissues than in their normal counterparts. In the 1097 samples, we observed that the expressions of the PGK1 gene (transcripts per million) were at least 1.5-fold higher (p < 0.05) in the human primary tumors than in normal breast tissue (Fig. 1B). As breast cancer is highly heterogeneous and its prognosis varies among different subtypes of the disease, we performed bioinformatic analysis of PGK1 expression in different subtypes of breast cancer. We grouped PGK1 expression in TCGA pan -cancer datasets into luminal, HER2, triple-negative tumors and normal breast tissues. The dataset, comprising 833 samples, has 566 luminal, 37 HER2 positive, 116 triple-negative, and 114 normal tissue samples. Figure 1C shows that PGK1 expression was highly elevated in TNBCs and HER2 samples as compared to other subclasses of breast cancer. Also, we observed a progressive increase of PGK1 expression as tumors advanced, with the highest PGK1 expression detected in stage IV cancers (Fig. 1D). Using the Kaplan Meier plotter, we stratified the breast cancer samples from 4929 patients into two groups based on PGK1 expression level: high expression group and low expression group. The patients with high PGK1-expressing tumors had lower survival than the patients with low PGK1-expressing tumors (Fig. 1E). Notably, in the datasets from the Gene Expression Omnibus database that were categorized as ferroptosis-sensitive or ferroptosis-resistant based on the immunophenotyping of 30 tumors, PGK1 expression was significantly upregulated in the ferroptosis-resistant tumors (Fig. 1F). These bioinformatic analyses inform that higher PGK1 gene expression is associated with poorer prognosis, advanced-stage breast cancer and ferroptosis insensitivity, particularly in TNBC and HER2-positive tumors. 3.2 Expression of PGK1 in breast cancers modulates their response to induction of ferroptosis. As we observed the heterogenous effects of PGK1 expression on several signaling including GPX4, a regulator of ferroptosis, in breast cancer cells (Fig. 2A), we evaluated the effect of PGK1 expression on ferroptosis activity in breast cancer cells by testing the response of both TNBC cell lines (MDAMB231, MDAMB468 and BT549) and luminal cancer cell lines (MCF7 and T47D) to erastin, a small molecule inducer of ferroptosis. Table I and Fig. 2B show that TNBC lines MDAMB231, MDAMB468 and BT549 were more sensitive to induction of ferroptosis than the luminal cancer cell lines MCF7 and T47D. We next treated those cell lines with a series of concentrations of NG52, a small molecule inhibitor of PGK1, and observed that IC 50 values of NG52 in the luminal breast cancer cell lines MCF7 and T47D cell lines were 42µM and 75µM, higher than that in the TNBC cells (5µM, 17µM and 27µM for MDAMB231, HCC1187, and MDAMB453 respectively) (Fig. 2C; Table II), suggesting that TNBC cells are more vulnerable to PGK1 inhibition that the luminal breast cancer cells. The differing responses of HCC70 and HCC1187 compared to other TNBC cell lines may reflect the known heterogeneity in ferroptosis sensitivity among TNBC subtypes. Recent studies, including Yang et al., 2023 [ 16 ], have shown that TNBC cells vary widely in their metabolic dependencies and resistance to ferroptosis, due to differences in glutathione metabolism, GPX4 expression, and oxidative stress adaptation. This stresses the importance of considering intragroup heterogeneity when designing ferroptosis-based interventions. To determine the effect of PGK1 on ferroptosis activity, we measured and compared the ferroptosis markers in tumor cells with or without depletion or inhibition of PGK1. We found that, while Acyl-CoA synthetase long-chain family member 4 (ACSL4), an enzyme involved in regulating fatty acid metabolism and a driver of ferroptosis, was downregulated in the PGK1-depleted MCF7 cells but unchanged in the PGK1-depleted MDAMB231. GPX4, an anti-ferroptosis peroxidase, was downregulated in both PGK1-depleted MCF7 and MDAMB231cells ( Fig. 2D), suggesting that loss of PGK1 induces a pro-ferroptotic shift through the GPX4 pathway in both of the cell lines. We also show that PGK1 depletion relocalized GPX4 to the nucleus (Supp Fig. 1A). PGK1 does not seem to affect the transcription of GPX4 in TNBCs, as GPX4 mRNA levels were comparable between the control and the PGK1-depleted MDAMB231 cells but not MCF7 (Fig. 2E). Consistently, depletion of PGK1 sensitized both triple-negative and luminal human breast cancers to the erastin-induced cell death (Supp Fig. 1B, 1C). Notably, ~ 40% more ferroptotic cell death was observed in MDAMB468 (Fig. 2F). and MDAMB453 cells treated with the PGK1 inhibitor NG52 as compared with erastin alone, but only 20% more ferroptotic cell death was seen in the luminal MCF7 and T47D cells subjected to NG52 treatment (Fig. 2G). To further demonstrate the role of PGK1 in modulating ferroptosis, we generated the MDAMB231 (human TNBC) and 4T1 (murine TNBC) cells subjected to stable knockdown of PGK1 (Supp Fig. 1D) and treated them with the ferroptosis inducer erastin in the presence or absence of ferroptosis inhibitors (ferrostatin-1, liproxstatin) or apoptosis inhibitor (ZVAD-FMK). These experiments showed that only the ferroptosis inhibitors but not the apoptosis inhibitor prevented cell death, indicating ferroptosis as the primary cell death mechanism in the PGK1-depleted cells (Supp Fig. 1E, 1F). These results demonstrate that PGK1 can modulate ferroptosis in breast cancer cells via degrading GPX4, with TNBC cells showing greater sensitivity to PGK1 depletion. 3.3 Metabolic reprogramming is associated with the PGK1-regulated ferroptosis in breast cancer. Since PGK1 is a key glycolytic regulator, we queried whether PGK1 modulates ferroptosis via altering glucose metabolism. We found that with increasing glucose concentrations, ferroptotic activity increased moderately (Fig. 3A). Treatment of both luminal and TNBC cell lines with 2DG, a glycolytic inhibitor, enhanced their sensitivity to the effect of the ferroptosis inducer erastin (Fig. 3B). To determine whether modulation of ferroptosis by PGK1 results from its effect on metabolic signaling, we analyzed its downstream targets in the control and PGK1-depleted cells under ferroptosis-inducing conditions. We found a consistent downregulation of pyruvate dehydrogenase (PDH), but not Lactate dehydrogenase A (LDHA) and Pyruvate kinase M1/2 (PKM1/2), in the PGK1-depleted MDAMB468 and MCF7 cells, and both of which exhibited increased sensitivity to induction of ferroptosis (Fig. 3D, 3E,). In contrast, MDAMB231 cells, which did not show increased sensitivity to ferroptosis following PGK1 depletion, displayed an upregulation of PDH expression under the same conditions (Fig. 3C, 2G). This discrepancy might account for the difference in sensitivity to ferroptosis induction among those tumor cells. In addition, although PGK1 has a key role in glycolysis, we observed an increased ECAR in the PGK1-depleted MDAMB468 cells (Fig. 3F), likely a consequence of a compensatory shift toward glycolysis due to PDH downregulation, limiting pyruvate oxidation, and this inefficient adaptation might trigger metabolic stress that enhances ferroptosis sensitivity. Conversely, the PGK1-depleted MDAMB231 cells, which are ferroptosis-insensitive, upregulated PDH to maintain oxidative metabolism and prevent further sensitization (Fig. 3G). To further analyze the role of PDH in PGK1-regulated ferroptosis, we treated the PGK1-depleted MDAMB231 cells with hypoxia or oligomycin, an inhibitor of Adenosine triphosphate (ATP) synthase. We show that PDH was substantially down-regulated (Fig. 4A) and the sensitivity to ferroptosis was significantly enhanced in those treated cells as compared with the control cells (Fig. 4B). Similarly, inhibiting PDH in the PGK1-depleted T47D, MCF7, MDAMB453 and HCC70 cells sensitized them to the erastin-induced cell death (Fig. 4C). Notably, 30-hour PDH inhibition produced a cell death level nearly comparable to 72-hour ferroptosis induction in the PGK1-depleted cells (Fig. 4C), suggesting PDH upregulation as a possible resistance mechanism. These results imply that PGK1 depletion can sensitize certain subtypes of breast cancer to ferroptosis induction by disrupting metabolic balance, and this sensitization correlates with PDH downregulation. 3.4 Targeting PGK1-mediated ferroptosis enhances anti-tumor immunity. To assess the importance of the PGK1-regulated ferroptosis in viv o, we inoculated the murine TNBC 4T1 cells with or without depletion of PGK1, followed by treatment with either vehicle or imidazole ketone erastin (IKE), a ferroptosis inducer (Fig. 5A). These experiments demonstrated that IKE significantly slowed tumor growth, with a more pronounced reduction in tumor volume observed in mice bearing PGK1-depleted tumors in those with tumors expressing PGK1 (Fig. 5C–5E). Furthermore, IKE treatment completely abrogated lung metastasis in mice bearing the PGK1-depleted 4T1 tumors, whereas mice with control tumors exhibited metastatic progression (Fig. 6A, 6B). Also, we observed a progressive decrease of the spleen size in the mice of the treatment groups, with the following extent: control 4T1-inoculated (vehicle-treated) > control 4T1-inoculated (IKE-treated) > PGK1-depleted 4T1-inoculated (vehicle-treated) > PGK1-depleted 4T1-inoculated (IKE-treated) (Fig. 6C, 6D). Western blot analysis of the T cells isolated from those spleens showed a corresponding increase in the expression of Granzyme B, a key cytolytic enzyme, and CD25, a T cell activation marker, following the same pattern as above (Fig. 6E). These observations imply that PGK1 depletion in combination with IKE treatment can enhance T cell activation. To further evaluate T cell functionality, we co-cultured CD8 + T cells from the spleens of the mice with 4T1 tumor cells, followed by cytotoxicity assay. Figure 6F shows that CD8 + T cells from the mice bearing PGK1-depleted 4T1 cells and receiving IKE treatment had a significantly enhanced tumor cell killing ability than CD8 + T cells from the mice bearing control 4T1cells (Fig. 6F), indicating that targeting PGK1 in combination with ferroptosis induction can strengthen the cytotoxic T lymphocyte-mediated antitumor immunity. Additionally, immunohistochemical analysis of tumor sections showed a progressive reduction in GPX4 expression across the treatment groups: GPX4 expression was high in the control 4T1-inoculated (vehicle-treated) tumors, moderately reduced in the control 4T1-inoculated (IKE-treated) tumors, further decreased in the PGK1-depleted 4T1-inoculated (vehicle-treated) tumors, and completely absent in the PGK1-depleted 4T1-inoculated (IKE-treated) tumors (Fig. 6G). Discussion PGK1 was selected for this study due to its elevated expression in aggressive breast cancer subtypes and its association with poor prognosis, as revealed in TCGA and GEO datasets. While prior studies have implicated PGK1 in promoting breast cancer progression [ 17 ], [ 18 ], the role of PGK1 in modulating ferroptosis, an important form of cell death, has not been well elucidated. Our findings demonstrate that PGK1 inhibition not only destabilizes the ferroptosis regulator GPX4 but also reprograms pyruvate metabolism by modulating PDH levels, ultimately influencing the cellular susceptibility to ferroptosis under normoxic and hypoxic conditions. These insights uncover previously uncharacterized function of PGK1 in regulating ferroptosis resistance, offering a novel metabolic target for therapeutic intervention in breast cancer. We show that PGK1 is highly expressed in advanced stages of breast cancers, especially in HER2 + and TNBC, and that PGK1 expression appears to be causally associated with tumor sensitivity to ferroptosis induction. We further show that modulation of ferroptosis by PGK1 is associated with metabolic reprogramming, and that ferroptosis-resistant breast cancers have high expression of PGK1, which has a critical role in controlling GPX4, thereby contributing to cellular sensitivity to ferroptosis induction. Importantly, our study indicates that the effect of tumoral PGK1 on ferroptosis induction may vary among the subtype of breast cancers, with TNBCs showing greater vulnerability to ferroptosis following PGK1 depletion as compared to the luminal subtypes. While multiple studies have implicated PGK1 overexpression in oncogenic activities such as tumor progression and therapy resistance [ 19 ], [ 20 ], others have argued for its anti-tumorigenic as was the case in Lewis lung cancer[ 21 ]. However, how PGK1 metabolically reprograms tumor cells to survive under ferroptotic conditions remains to be fully elucidated. There are studies showing the metabolic underpinnings of ferroptosis including fatty acid metabolism, iron handling, mevalonate pathway, and thiol metabolism on lipid peroxidation, a catalyst for ferroptosis [ 22 ], [ 23 ], [ 24 ], [ 25 ]. Here, we demonstrate that PGK1 has a role in regulating the levels of ferroptosis markers ACSL4, FSP1, Ferritin, and GPX4, the evidence supporting the ferroptosis-modulating function of PGK1. Contradictory to a previous study showing that pyruvate dehydrogenase enhances the autooxidation of dihydrolipoamide, promoting ferroptosis in human fibrosarcoma cells[ 26 ], we show that PGK1 modulates ferroptosis susceptibility via its downstream target PDH. This discrepancy might be due to cancer-specific roles of PDH in ferroptosis response. Notwithstanding, we demonstrate that PGK1 promotes ferroptosis resistance in the TNBC MDAMB231 cells by regulating PDH, which prevents an inefficient compensatory glycolytic shift under ferroptotic conditions. This stabilization reduces metabolic stress, extracellular acidification, and lipid peroxidation, ultimately inhibiting ferroptosis. In addition, PGK1 depletion disrupts metabolic balance, with the glycolytic shift, contributing to ferroptosis susceptibility in certain breast cancer subtypes. Of note, we observed that TNBC MDAMB231 cells were more sensitive to ferroptosis under normoxia compared to hypoxic conditions, a phenomenon that is linked to the differential expression of PDH under these circumstances. Elevated PDH expression under normoxic conditions enhance oxidative phosphorylation, resulting in elevated lipid peroxidation and mitochondrial ROS, hallmarks known to promote ferroptosis [ 27 ], [ 28 ], [ 29 ]. In contrast, during hypoxic conditions or after chemical inhibition of ATP synthase, PDH is significantly attenuated, reducing mitochondrial activity and a shift towards the glycolytic phenotype. Hypoxia-induced metabolic reprogramming may limit ferroptosis susceptibility by maintaining redox balance and attenuating oxidative stress. Therefore, the oxygen-mediated regulation of PDH seems to modulate the metabolic response and ferroptosis sensitivity in MDAMB231, highlighting a mechanistic basis for their differential response to ferroptosis induction under varying oxygen tensions. It is likely that PGK1 and PDH are differentially regulated by oxygen availability. Under normoxic conditions, PGK1 facilitates pyruvate production and glycolytic flux, maintaining PDH depression and mitochondrial metabolism. However, hypoxia already limits oxidative phosphorylation, and combined with PGK1 depletion further restricts glycolytic output and pyruvate supply. This metabolic stress possibly induces PDH suppression through feedback mechanisms, likely through upregulated phosphorylation by hypoxia-induced pyruvate dehydrogenase kinases (PDKs) that ultimately inactivate PDH [ 30 ]. Thus, this two-way impairment of mitochondrial metabolism underlies the observed PDH downregulation and may explain reduced sensitivity to ferroptosis under hypoxia. To summarize our findings in the context of metabolic regulation and ferroptosis, we propose a schematic model (Supp Fig. 2A) that illustrates how PGK1 and PDH influence ferroptosis sensitivity under normoxic and hypoxic conditions. This diagram integrates our experimental data with established metabolic pathways to provide a unified mechanistic framework. New insights have revealed that ferroptosis resistance not only directly contributes to malignant phenotypes of breast cancer but may also modulate the immunosuppressive features of the TME through its interaction with immune cells [ 25 ], [ 31 ], [ 32 ], [ 33 ]. In this study, we show the immunomodulatory potential of targeting PGK1 in combination with ferroptosis induction. Although we observed that IKE treatment shrinks tumor in immune-competent BALB/c mice, the effect of PGK1 depletion during IKE treatment is striking, which shows the abrogation of both micro and macroscopic tumor metastasis to the lungs. Also, we observed a reduction in the spleen size and a corresponding increase in T cell activation markers such as Granzyme B and CD25, suggesting that this combined treatment not only promotes ferroptosis but also improves anti-tumor immunity. It remains to be determined whether the reduced spleen size indicates decreased tumor-induced immunosuppression or a reduction in tumor-associated immune cell populations. However, CD8⁺ T cells extracted from IKE-treated mice bearing the PGK1-depleted tumors exhibited enhanced cytotoxicity when cocultured with 4T1 murine TNBCs (Fig. 6F), suggesting an augmented T cell-mediated anti-tumor response. Beyond its role in glycolysis and immune modulation, PGK1 is also linked with cellular stress pathways through the AMPK–mTOR axis. This axis integrates energy sensing (via AMPK) and anabolic signaling (via mTOR), both of which are essential for regulating autophagy and tumor cell survival. Therapeutic combinations such as metformin (an AMPK activator) and rapamycin (an mTOR inhibitor) have demonstrated efficacy in TNBC models [ 34 ], [ 35 ], [ 36 ]. Given the link between ferroptosis and metabolic stress, co-targeting PGK1 with modulators of the AMPK–mTOR pathway may intensify oxidative damage and enhance ferroptotic cell death. Thus, these strategies might help overcome therapy resistance and improve treatment outcomes in aggressive breast cancers. Recent efforts have led to the development of promising small-molecule PGK1 inhibitors. Notably, CBR-470-1 was reported to inhibit PGK1 enzymatic activity, attenuating glycolytic flux and shrinking tumor growth in preclinical models [ 37 ]. In a similar trend, Ilicicolin H, a fungal metabolite and a dual inhibitor of PGK1 and mitochondrial complex III, disrupted tumor energy metabolism and exhibited anticancer effects[ 38 ]. The proof-of-concept experiments using these agents suggest that PGK1 is a druggable target, and their emerging profiles support the feasibility of combining PGK1 inhibition with ferroptosis inducers to enhance metabolic vulnerability in breast cancers. Further investigation of these compounds in the context of ferroptosis sensitivity may broaden therapeutic strategies, especially in metabolically active and therapy-resistant subtypes of malignancies such as TNBC. Our Study identifies PGK1 as a mediator of ferroptosis resistance and provides a rationale for targeting PGK1 in conjunction with ferroptosis induction as a potential therapeutic strategy for aggressive breast cancers such as TNBC. By simultaneously inhibiting tumor growth and enhancing anti-tumor immune responses, this approach might offer a multifaceted attack on cancer cells for improving the effectiveness of cancer immunotherapy such as immune checkpoint inhibitors. Abbreviations 2-DG 2-Deoxy-D-glucose ACSL4 Acyl-CoA synthetase long-chain family member 4 AMPK AMP-activated protein kinase ANOVA Analysis of Variance ATP Adenosine triphosphate BCA Bicinchoninic Acid CD25 Cluster of Differentiation 25 CD71 Cluster of Differentiation 71 DAPI 4′,6-diamidino-2-phenylindole DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl Sulfoxide ECAR Extracellular Acidification Rate FBS Fetal Bovine Serum FFPE Formalin-Fixed Paraffin-Embedded GEO Gene Expression Omnibus GPX4 Glutathione Peroxidase 4 HRP Horseradish Peroxidase IC50 Half maximal inhibitory concentration IKE Imidazole Ketone Erastin KM Kaplan-Meier LDHA Lactate Dehydrogenase A OS Overall Survival PBS Phosphate Buffered Saline PBST PBS with Tween 20 PDH Pyruvate Dehydrogenase PGK1 Phosphoglycerate Kinase 1 PPS Post-Progression Survival PVDF Polyvinylidene Fluoride RFS Relapse-Free Survival RNA Ribonucleic Acid RPMI Roswell Park Memorial Institute medium SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulfateâ“Polyacrylamide Gel Electrophoresis TCA Tricarboxylic Acid TCGA The Cancer Genome Atlas TME Tumor Microenvironment TNBC Triple-Negative Breast Cancer cDNA Complementary DNA mTOR Mechanistic Target of Rapamycin qRT-PCR Quantitative Real-Time Polymerase Chain Reaction shRNA Short Hairpin RNA siRNA Small Interfering RNA Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of Data and Materials The data generated in this study are publicly available in Gene Expression Omnibus (GEO) at GSE148297. Competing interests The authors declare that they have no competing interests" in this section. Clinical Trial Number Not applicable The authors declare that they have no competing interests" in this section. Funding This investigation was self-funded by the Yang Lab. Author Contribution FO performed some bioinformatic investigation into PGK1 expression across a. cancer subtypes, and b. tumor staging. She also analyzed the raw data from the PDH depletion experiments using statistical tools. TO conducted a bioinformatic analysis of the prognostic significance of PGK1 expression in relation to post-progression survival, relapse-free survival, and overall survival in patients with luminal and HER2-positive breast cancer. OO conducted bioinformatic investigation into the differences between sensitive and ferroptosis-resistant cell lines using GEO datasets. He also investigated the Protein and gene expression of PGK1 across various tissues and their corresponding tumors. AA brought in his methods for evaluating different protein expressions of PGK1 downstream targets. He also reviewed the draft of this manuscript. CN and AS worked in the curation of Data as well as their validation for this research work. XR and J Y supervised and sourced for funds and resources to carry out this research work. At the time of this work, Oluwafunminiyi Obaleye, Amos Akinyemi, Chrispus Ngule, Andrew Shinkle, Xingcong Ren, and Jin-Ming Yang were affiliated with the Department of Toxicology and Cancer Biology, University of Kentucky, USA. Folake Oyelami was affiliated with the Department of Microbiology, University of Ilorin, Nigeria. Tijesunimi Oyetunde was an independent bioinformatics collaborator based in Manitoba, Canada. References “Breast Cancer Statistics | How Common Is Breast Cancer? | American Cancer Society.” Accessed: Dec. 10, 2024. [Online]. Available: https://www.cancer.org/cancer/types/breast-cancer/about/how-common-is-breast-cancer.html R. L. Siegel, A. 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Zheng et al., “PGK1 inhibitor CBR-470-1 protects neuronal cells from MPP+,” Aging (Albany NY), vol. 12, no. 13, pp. 13388–13399, 2020, doi: 10.18632/AGING.103443. M. Li, A. Zhang, X. Qi, R. Yu, and J. Li, “A novel inhibitor of PGK1 suppresses the aerobic glycolysis and proliferation of hepatocellular carcinoma,” Biomed Pharmacother, vol. 158, Feb. 2023, doi: 10.1016/j.biopha.2022.114115. Tables Table I: IC50 values of Breast cancer cell lines to erastin treatment after 72hrs Cell line Category IC50 ( μ M ± SEM) MDAMB231 Triple-Negative 4.8 BT549 Triple-Negative 6.0 MDAMB468 Triple-Negative 9.0 MDAMB453 Triple-Negative 22.0 HCC1187 Triple-Negative 41.0 HCC70 Triple-Negative >60.0 T47D Luminal >60.0 MCF7 Luminal >60.0 Table I: Viability measurement of human triple-negative breast cancers MDAMB231, BT549, MDAMB468, MDAMB453, HCC1187, and luminal cancers T47D and MCF7 cell lines treated with increasing doses of erastin for 72 hours. Data are represented as the mean ± SD (n=6). Table II: IC50 values of Breast cancer cell lines to NG52 treatment after 72hrs Cell line Category IC50 ( μ M ± SEM) MDAMB231 Triple-Negative 5.0 BT549 Triple-Negative 40.0 MDAMB468 Triple-Negative 35.0 MDAMB453 Triple-Negative 27.0 HCC1187 Triple-Negative 17.0 HCC70 Triple-Negative 40.0 T47D Luminal 42.0 MCF7 Luminal 75.0 Table II: Viability measurement of human triple-negative breast cancers MDAMB231, BT549, MDAMB468, MDAMB453, HCC1187, and luminal cancers T47D and MCF7 cell lines treated with increasing doses of PGK1 inhibitor NG52 for 72hours. Data are represented as the mean ± SD (n=6) Supplementary Files Suppfig1.docx Supplementary Fig. 1. PGK1 Depletion and Ferroptosis Sensitivity in Breast Cancer Cells. A. Representative images of control and PGK1-depleted MCF7 cell line and their quantification. Expressions of PGK1 and GPX4 are shown. Colony formation assay of MCF7 cells (B.) and 4T1 cells C. treated with vehicle, erastin (1.25 ì M), NG52(5 ì M) or their combination. D. Representative images of control and stable PGK1-depleted 4T1 and MDAMB231 cell line. Cell viability analysis of E. human MDAMB231 and F. murine 4T1 carcinomas after ferroptosis induction in combination with cell death inhibitors ZVAD-FMK, Liproxstatin, Ferrostatin and N-acetyl-cysteine (NACC). Mean ± SD; one-way ANOVA, ns: not significant, *P< 0.05, **P<0.01, ***P<0.001, ****P<0.0001. Suppfig2.docx Supplementary Fig. 2. PGK1 regulates ferroptosis sensitivity and patient prognosis in breast cancer. A. Schematic model illustrating how PGK1 regulates ferroptosis sensitivity through glycolysis-dependent mitochondrial metabolism and lipid peroxidation. PGK1 inhibition sensitizes cells to ferroptosis by impairing redox homeostasis and shifting metabolic flux. B. Cell viability analysis in MDAMB231 cells following erastin treatment, PGK1 silencing (siPGK1), or combination treatment. (C–E) Kaplan–Meier survival curves showing the association of PGK1 expression with clinical outcomes in breast cancer patients. C. High PGK1 expression correlates with shorter post-progression survival (p = 0.003). D. PGK1 expression is associated with significantly reduced Relapse-Free Survival in the overall breast cancer cohort (p < 1e-16). Subtype-specific analysis shows that high PGK1 expression is significantly associated with poor Overall Survival (OS) in E. luminal breast cancer (p = 0.0031), but not in F. HER2-enriched breast cancer (p = 0.36). or G. Triple-Negative breast cancer (p = 0.2). Data source: Kaplan–Meier Plotter (Győrffy et al., Comput. Struct. Biotechnol. J. 2021), integrating GEO Affymetrix microarray and TCGA RNA-seq cohorts with OS, RFS, and DFS endpoints.[12]. Mean ± SD; one-way ANOVA, ns: not significant, *P< 0.05, **P<0.01, ***P<0.001, ****P<0.0001 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 03 Sep, 2025 Reviewers invited by journal 03 Sep, 2025 Editor assigned by journal 21 Aug, 2025 First submitted to journal 16 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7323200","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509659885,"identity":"6d541daa-c262-405c-9439-5c1f2dc81e3c","order_by":0,"name":"Felix Oyelami","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBAC9gbmhgMQFhAnVDAYgNgS+LTwHGCEamEGaTlDpBYGuBbGNmK0sDc2HuZhsEnsZ2Z+uuHhvMPGBgeYD97mwaeF52DDwRkMaYkzm9nMbiRuO2xmcIAt2RqfFnuJxIYDHxgOJ244zADWYmNwgMdMGq8t8g8bDiQAtew/zP7tRuIckBb+b/i1SDBCbWHmAdrSAHIYDxt+LTyJQL8YpBnPOMxTdiPhWLqx5GE2Y8s5+LSwHz78mafCRra/vX3bzR811oZ9x5sf3niDRwsEGDA4NsDYCocJKocAezhLvgG3qlEwCkbBKBiZAADRSlJLVxlcmQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5194-1968","institution":"University of Kentucky","correspondingAuthor":true,"prefix":"","firstName":"Felix","middleName":"","lastName":"Oyelami","suffix":""},{"id":509659886,"identity":"7216badf-df4f-40fd-97e6-8861a48cebb8","order_by":1,"name":"Andrew Shinkle","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Shinkle","suffix":""},{"id":509659887,"identity":"116045bd-0e71-4bee-b3fc-1ac8ba86c159","order_by":2,"name":"Chrispus Ngule","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Chrispus","middleName":"","lastName":"Ngule","suffix":""},{"id":509659888,"identity":"22d0ebb0-ad13-40bb-bd50-70d7ec8eeb97","order_by":3,"name":"Folake Oyelami","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Folake","middleName":"","lastName":"Oyelami","suffix":""},{"id":509659889,"identity":"08c16c06-7d40-4ca2-a778-64fe64a8dbaa","order_by":4,"name":"Oluwafunminiyi Obaleye","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Oluwafunminiyi","middleName":"","lastName":"Obaleye","suffix":""},{"id":509659890,"identity":"98ad6933-69f8-4bea-b7bb-a897f6d0b54d","order_by":5,"name":"Akinyemi Amos","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Akinyemi","middleName":"","lastName":"Amos","suffix":""},{"id":509659891,"identity":"329a8935-27b6-44db-9eec-a1b0b0ac7b76","order_by":6,"name":"Tijesunimi Oyetunde","email":"","orcid":"","institution":"Brandon University","correspondingAuthor":false,"prefix":"","firstName":"Tijesunimi","middleName":"","lastName":"Oyetunde","suffix":""},{"id":509659892,"identity":"4c2d6fc4-4269-46c0-9186-737bfb7a86a1","order_by":7,"name":"Xingcong Ren","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Xingcong","middleName":"","lastName":"Ren","suffix":""},{"id":509659893,"identity":"74211405-1b65-49c0-93d3-2199237a46ee","order_by":8,"name":"Jin-Ming Yang","email":"","orcid":"https://orcid.org/0000-0002-8703-0493","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Jin-Ming","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-08-08 03:59:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7323200/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7323200/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90985715,"identity":"ac962e0e-132c-42ad-9728-bc6a9e9008fe","added_by":"auto","created_at":"2025-09-10 09:54:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1432080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh PGK1 expression is associated with poor prognosis and ferroptosis resistance in breast cancer patients. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eWestern blotting of PGK1 in TNBCs and non-TNBC cell lines. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eGene expression levels of PGK1 in normal versus breast cancer primary tumors in TCGA datasets. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eGene expression levels of PGK1 in normal, luminal, HER2 and TNBC tissues in TCGA dataset samples. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. PGK1 expression across different stages of breast cancer.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e E\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Effect of PGK1 expression on overall survival of patients with breast cancer, as analyzed using Kaplan Meier Plotter. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eAbundance profile for PGK1 across sensitive vs ferroptosis-resistant cancers in a GEO dataset. Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001. Note: The data shown in this panel (1A) were obtained from an independent repeat of the experiment to ensure result integrity and to avoid redundancy with Figure 2A.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/20c5b585c9b8f637457cf627.png"},{"id":90983326,"identity":"0074bd34-bb0e-482c-ac3a-85c62ad1aeaa","added_by":"auto","created_at":"2025-09-10 09:38:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2032568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepletion of PGK1 Sensitizes Breast Cancer to Ferroptosis. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western blotting of ferroptosis biomarkers and PGK1 signaling in TNBCs and non-TNBC cell lines. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Dose-response curve of the ferroptosis inducer erastin in breast cancer cells. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Dose-response curve of the PGK1 inhibitor NG52 in breast cancer cells. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western Blotting of ferroptosis biomarkers, PGK1 and its downstream targets in control and PGK1-depleted MCF7 and MDAMB231 carcinomas. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Expression of GPX4 mRNA in MCF7 and MDAMB231 cells with or without the depletion of PGK1. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Colony formation assay. MDAMB468 colonies after treatment with vehicle, erastin (1.25\u003c/em\u003eì\u003cem\u003eM), NG52(5\u003c/em\u003eì\u003cem\u003eM) or their combination. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eG. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ePercentage viability analysis of MDAMB468, MDAMB453, HCC70, T47D, MCF7 and MDAMB231 cell lines after treatment with vehicle, Erastin, NG52 or their combination for 72hrs. Mean ± SD; t-test, one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, **P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/5b4dbc49d632f10b8e648f2c.png"},{"id":90982764,"identity":"b299685c-e492-43c5-bca3-716cae2dfda5","added_by":"auto","created_at":"2025-09-10 09:30:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2164167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic changes associated with PGK1-modulated ferroptosis\u003c/strong\u003e. \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Effect of glucose deprivation on viability in breast cancer cell lines during ferroptosis induction. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eEffect of glycolysis inhibition on viability in breast cancer cell lines during ferroptosis induction. Western blotting image of ferroptosis biomarkers, PGK1 expression and its downstream targets after treatment with DMSO, NG52, Erastin or their combination in \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e MDAMB231, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e MCF7 and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eMDAMB468. Sea horse assay- ECAR analysis in \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e MDAMB468 and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eG.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e MDAMB231 cell lines after treatment with vehicle, Erastin, NG52 or their combination. Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/6d34091be0d3f1a39f34ca91.png"},{"id":90984748,"identity":"c1ca09e4-9d6b-4ff9-adb2-ede65adfac68","added_by":"auto","created_at":"2025-09-10 09:46:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1324427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of metabolic alteration on the sensitivity of breast cancer cells to induction of ferroptosis. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western blotting of ferroptosis biomarkers, PGK1 expression and its downstream targets in MDAMB231 after treatment with DMSO, NG52, Erastin or their combination under normoxic or hypoxic conditions for 72hrs. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eCell viability analysis of MDAMB231 cell line after treatment with vehicle, Erastin, NG52 or their combination under normoxic and hypoxic conditions. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003eCell viability analysis of control and PGK1-depleted T47D, MCF7, MDAMB453 and HCC70 cell lines following oligomycin treatment under ferroptosis-inducing conditions for 30hrs. The data shown in this panel (4A) were obtained from an independent repeat of the experiment to ensure result integrity and to avoid redundancy with Figure 3C. Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/d5e3f08fd36e00e69477de6b.png"},{"id":90983341,"identity":"11c01afa-0a0b-4667-a163-76953b47eb77","added_by":"auto","created_at":"2025-09-10 09:38:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2663891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting of PGK1 enhances ferroptosis in orthotopic mouse tumor model.\u003c/strong\u003e\u0026nbsp; \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Orthotopic mouse tumor model.\u0026nbsp; Mice were orthotopically implanted with luciferase-transfected 4T1 murine carcinoma cells (1x 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e6 \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ecells/ injection, on the 5\u003c/em\u003e\u003csup\u003e\u003cem\u003eth\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e mammary fat pad, n=5) with or without the depletion of PGK1. On day seven following inoculation, mice were given Imidazole ketone erastin (40mg/kg) or vehicle intraperitoneally once every day for 14 days to induce ferroptosis. Tumor growth was constantly monitored with the Lago imaging system. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Tumor growth rates from Balb/c mice inoculated with control or PGK1-depleted L2T-4T1 carcinomas, which were treated with IKE (40mg/kg) or vehicle. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e luminescence intensity of tumor size from mice inoculated with control or PGK1-depleted L2T-4T1 carcinomas and treated with IKE (40mg/kg) or vehicle. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp; Representative image of tumors collected from mice inoculated with control or PGK1-depleted L2T-4T1 carcinomas and treated with IKE (40mg/kg) or vehicle, and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e their quantitation analysis. Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/e33ca72aff6c56edee127db3.png"},{"id":90982791,"identity":"b89a86f3-c277-4ce7-99a6-440eeaf8aa6f","added_by":"auto","created_at":"2025-09-10 09:30:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2647049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting of\u003c/strong\u003e \u003cstrong\u003ePGK1 strengthens antitumor immunity in mouse tumor model.\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Luminescence intensity of L2T-transfected 4T1 carcinoma metastasis in a 24-well organ array plate and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Quantitation analyses of Fig. 6A. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative image of the spleens collected from mice inoculated with control or PGK1-depleted L2T-4T1 carcinomas and treated with IKE (40mg/kg) or vehicle, and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e The quantitation analysis of the spleen size. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eWestern blotting of activation markers. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e cytotoxic effects of CD8 T cells that were extracted from mice inoculated with control or PGK1-depleted L2T-4T1 carcinomas and treated with IKE (40mg/kg) or vehicle. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eG. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRepresentative images of tissue section from mice inoculated with control or PGK1-depleted L2T-4T1 carcinomas and treated with IKE (40mg/kg) or vehicle. Expressions, and quantification of \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. GPX4 and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eI.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Caspase-3 are shown. Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/673ea613e499aedc90af19bc.png"},{"id":91148917,"identity":"bf146e4b-0424-4c49-a39e-88cd225392d1","added_by":"auto","created_at":"2025-09-12 06:46:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17099509,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/354f45cd-d89a-4bdb-8a62-501a55dff8db.pdf"},{"id":90982793,"identity":"4e107713-33c2-45fa-a7f1-fb6d9be5811b","added_by":"auto","created_at":"2025-09-10 09:30:14","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":611930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1. PGK1 Depletion and Ferroptosis Sensitivity in Breast Cancer Cells.\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative images of control and PGK1-depleted MCF7 cell line and their quantification. Expressions of PGK1 and GPX4 are shown. Colony formation assay of MCF7 cells (B.) and 4T1 cells \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003etreated with vehicle, erastin (1.25\u003c/em\u003eì\u003cem\u003eM), NG52(5\u003c/em\u003eì\u003cem\u003eM) or their combination. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRepresentative images of control and stable PGK1-depleted 4T1 and MDAMB231 cell line. Cell viability analysis of \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e human MDAMB231 and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e murine 4T1 carcinomas after ferroptosis induction in combination with cell death inhibitors ZVAD-FMK, Liproxstatin, Ferrostatin and N-acetyl-cysteine (NACC). Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Suppfig1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/71fe206112038211da100160.docx"},{"id":90984751,"identity":"7af98a7d-e76e-4a56-97f1-0fa1cb08df48","added_by":"auto","created_at":"2025-09-10 09:46:13","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":431675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2. PGK1 regulates ferroptosis sensitivity and patient prognosis in breast cancer.\u003c/strong\u003e\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Schematic model illustrating how PGK1 regulates ferroptosis sensitivity through glycolysis-dependent mitochondrial metabolism and lipid peroxidation. PGK1 inhibition sensitizes cells to ferroptosis by impairing redox homeostasis and shifting metabolic flux. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Cell viability analysis in MDAMB231 cells following erastin treatment, PGK1 silencing (siPGK1), or combination treatment. (C–E) Kaplan–Meier survival curves showing the association of PGK1 expression with clinical outcomes in breast cancer patients. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e High PGK1 expression correlates with shorter post-progression survival (p = 0.003). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e PGK1 expression is associated with significantly reduced Relapse-Free Survival in the overall breast cancer cohort (p \u0026lt; 1e-16). Subtype-specific analysis shows that high PGK1 expression is significantly associated with poor Overall Survival (OS) in \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e luminal breast cancer (p = 0.0031), but not in \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e HER2-enriched breast cancer (p = 0.36). or \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eG.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Triple-Negative breast cancer (p = 0.2). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eData source:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eKaplan–Meier Plotter (Győrffy et al., Comput. Struct. Biotechnol. J. 2021), integrating GEO Affymetrix microarray and TCGA RNA-seq cohorts with OS, RFS, and DFS endpoints.[12]. Mean ± SD; one-way ANOVA, ns: not significant, *P\u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Suppfig2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7323200/v1/b718fc424de85850424fa254.docx"}],"financialInterests":"","formattedTitle":"Expression of PGK1 in Breast Cancers Alters Their Sensitivity to Ferroptosis Induction via Metabolic Reprogramming","fulltext":[{"header":"Key message ","content":"\u003col\u003e\n \u003cli\u003e PGK1 overexpression contributes to ferroptosis resistance in aggressive breast cancers, including triple-negative breast cancer.\u003c/li\u003e\n \u003cli\u003eGenetic or pharmacological inhibition of PGK1 sensitizes tumor cells to ferroptosis induction and suppresses tumor growth in vivo.\u003c/li\u003e\n \u003cli\u003ePGK1 targeting enhances anti-tumor immune responses, indicating potential synergy with immune checkpoint blockade.\u003c/li\u003e\n \u003cli\u003eCombining PGK1 inhibition with ferroptosis inducers offers a promising therapeutic approach for treatment-resistant breast cancers.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eTherapy resistance and tumor recurrence are the main causes of the mortality of breast cancer patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Increasing evidence has shown that metabolic reprogramming enables cancer cells to survive in hostile microenvironments, sustain growth and proliferation, and eventually escape death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, targeting the key metabolic components to promote cancer cell death has been considered a promising strategy for the treatment of aggressive cancers including lethal breast cancer subtypes such as triple-negative breast cancer (TNBC) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ferroptosis, a non-apoptotic and iron-dependent form of programmed cell death, is characterized by an overload of lipid peroxidation and loss of redox balance. In recent years, activating ferroptotic cell death for the treatment of various cancers has been under extensive investigation. Here, we report that phosphoglycerate kinase 1 (PGK1), an essential glycolytic enzyme [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], is highly expressed in breast cancers including TNBC and contributes to cellular insensitivity to ferroptosis induction. We show both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e that depleting PGK1 by RNA interference or inhibiting the activity of this kinase by its small molecule inhibitor can render tumor cell sensitivity to induction of ferroptotic cell death, and this sensitization is mediated through metabolic reprogramming. In ferroptosis-sensitive cells, PGK1 loss leads to Pyruvate dehydrogenase (PDH) downregulation, restricting pyruvate entry into the Tricarboxylic Acid (TCA) cycle and causing a compensatory shift toward glycolysis. This inefficient adaptation increases metabolic stress, extracellular acidification, and lipid peroxidation, ultimately promoting ferroptosis. Conversely, in cells that do not exhibit ferroptosis sensitization, PDH is upregulated, maintaining oxidative metabolism and redox homeostasis, hampering ferroptotic susceptibility. The results of this study imply that targeting PGK1 in combination with a ferroptosis inducer may be exploited as a potentially novel approach to the treatment of ferroptosis-insensitive breast cancer and likely other malignant tumors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eReagents.\u003c/b\u003e Erastin (MedChemExpress, NJ, USA, #HY-15763), Imidazole ketone erastin (MedChemExpress, NJ, USA, #HY-114481), NG52 (MedChemExpress, NJ, USA, #HY-15154, ), Ferrostatin (MedChemExpress, NJ, USA, #HY-100579), Laemli lysis buffer, RIPA lysis buffer, Complete protease inhibitor (Roche, MH, GERMANY, # 0469331001), Dimethyl Sulfoxide (DMSO) (VWR, PA, USA, #0231), Triton X-100 (SIGMA ALDRICH MO, USA, #T8787, RRID: AB_2629483) Aquabluer solution (BocaScientific, Wahington, MA, USA, #6015, RRID: AB_12345678), Necrostatin (Selleck, TX, USA, #S8037), Liproxstatin (Selleck, TX, USA, #S7699), Bovine serum albumin (Santa Cruz Biotechnology, TX, USA, #sc-2323A), N-Acetyl cysteine (SIGMA ALDRICH MO, USA, #A9164), Z-VAD FMK (Selleck, TX, USA, #S7023), mirVana\u0026trade; MiRNA isolation kit (Thermofisher Scientific, VL, Lithuania, # AM1561). DAPI (Thermofisher Scientific, VL, Lithuania, # P36941, RRID: AB_2629482) jetPRIME Transfection Reagent (Polyplus-satorius, IL, FRANCE), PGK1 siRNA (Dharmacon, CA, United Kingdom, #5230), PGK1 siRNA (Santa Cruz Biotechnology, TX, USA, #sc-36215), Crystal violet (SIGMA ALDRICH MO, USA, #C3886, RRID: SCR_015456), MojoSort\u0026trade; Mouse CD8 T cell Isolation Kit (Biolegend, CA, USA, #480008) PGK1 shRNA (m) Lentiviral Particles (Santa Cruz Biotechnology, TX, USA, # sc-36216-V)\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibodies.\u003c/b\u003e PGK1 (Santa Cruz Biotechnology, TX, USA, #sc-130335, RRID:AB_2165228), PGK1 (NOVUS biological, CO, USA, #NBP2-19784, RRID:AB_2786860), ACSL4 (Santa Cruz Biotechnology, TX, USA, #sc-365230, RRID:AB_10847863), GPX4 (Santa Cruz Biotechnology, TX, USA, #sc-166437, RRID:AB_2279252), FSP1 (AMID) (Santa Cruz Biotechnology, TX, USA, #sc-376594, RRID:AB_11149443), BMAL1 (Santa Cruz Biotechnology, TX, USA, #sc-365645, RRID:AB_RRID:AB_10842165), Pyruvate Dehydrogenase (Cell Signaling Technology, MA, USA, #2784, RRID:AB_2061989), PKM1/2 (Cell Signaling Technology, MA, USA, #3186, RRID:AB 2162606), PKM2 (Cell Signaling Technology, MA, USA, #3186, RRID:AB 1904096),b-Actin (Cell Signaling Technology, MA, USA, #3700, RRID:AB_2242334), LDHA (Cell Signaling Technology, MA, USA, #3582, RRID:AB_10694487), Ferritin (Santa Cruz Biotechnology, TX, USA, #sc-376594, RRID:AB_11149443), CD71 (Santa Cruz Biotechnology, TX, USA, #sc-32272, RRID:AB_627832), Cyclohexamide (Cell Signaling Technology, MA, USA, #2112), MG132 (Cell Signaling Technology, MA, USA, #2194), CD25 (Cell Signaling Technology, MA, USA, #39475, RRID:AB_2799050), Granzyme B (Cell Signaling Technology, MA, USA, #17215, RRID:AB_10694683).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBioinformatics Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the expression and clinical relevance of PGK1 in breast cancer, publicly available datasets such as RNA seq data from the TCGA, along with GEO datasets, were retrieved using cBioPortal (RRID: SCR_014555) for transcriptomic profiling and copy number variation analysis. Expression patterns of PGK1 in the luminal, HER2-positive, basal and triple-negative breast cancer (TNBC) subtypes were compared to normal breast tissues. Clinical outcome data, including Overall Survival (OS), Post-Progression survival (PPS), and Relapse-Free Survival (RFS), were obtained from the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR) using the KM Plotter[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To determine PGK1 protein expression levels, UALCAN (RRID: SCR_018826) was used to analyze Clinical Proteomic Tumor Analysis Consortium (CPTAC) data, allowing for stage-specific and subtype-specific comparisons between breast tumors and normal tissues. Breast cancer patients were stratified into high and low PGK1 expression groups, then the prognostic significance of PGK1 was investigated using the Kaplan-Meier Plotter (RRID: SCR_018753) which compared the overall survival (OS) and Relapse-free survival (RFS) between the groups. Additionally, ferroptosis-related gene signatures were examined using datasets from GEO to explore the association between PGK1 expression and ferroptosis resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMCF7 (RRID: CVCL_0031), MDAMB231 (RRID: CVCL_0062), BT549 (RRID: CVCL_1092), MDAMB468 (RRID: CVCL_0419) and cells were acquired from ATCC (ATCC, VA, USA). HCC70 (RRID: CVCL_1258), T47D (RRID: CVCL_0553), HCC1187 (RRID: CVCL_1248) and MDAMB453 (RRID: CVCL_0418) were kind gifts from Dr Wu Yadi. For in vitro experiments, Cells were cultured in RPMI, DMEM-F 12 or High-glucose DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and incubated at 36.7\u003csup\u003eo\u003c/sup\u003eC with 5% C02. For drug treatment, 5,000 cells were treated with various concentrations of Erastin (0, 0.5, 1, 2, 4, 10, 20, 40, 80 \u0026micro;M) for up to 72 hours. To interfere with ferroptosis and other cell death mechanisms, cells were treated with Erastin alone or in combination with NG52 (5\u0026micro;M) cell death inhibitors such as ferrostatin (20\u0026micro;M), Liproxstatin (1\u0026micro;M), Necrostatin (20\u0026micro;M), Zvad-FMK (100\u0026micro;M), NACC (20mM) as described by Ghoochani et.al, 2021[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were collected into cold Phosphate-buffered saline, spun down and protein was extracted in Laemli or RIPA buffer supplemented with protease inhibitor. Protein concentration was evaluated using a Bicinchoninic Acid (BCA) assay kit (thermos Scientific, #23228, Waltham, MA, USA). Cell lysates were run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 volts for 2 hours. Proteins were transferred using an immune-blot PVDF membrane in a transfer buffer at 100 volts for 80 minutes). Membranes were blocked in 5% defatted milk in PBST for one hour and blots were incubated overnight with appropriate primary antibody at 4\u003csup\u003eo\u003c/sup\u003eC. Secondary antibody was incubated at room temperature for 1 hour. PBST buffer was used to wash the membranes for 20 minutes before and after incubation in Horse-radish Peroxidase (HRP)- secondary antibody. Protein expression levels were visualized using the ChemiDOC\u0026trade; MP imaging system (BioRad, Hercules, CA, USA)\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative Real-time PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA was extracted from the cell pellets using an Invitrogen mirVana\u0026trade; MiRNA isolation kit (Thermofisher Scientific, VL, Lithuania, # AM1561). First-strand cDNA was produced from 2\u0026micro;g of total RNA using Invitrogen SuperScript\u0026trade; IV First-strand Synthesis System (Thermofisher Scientific, VL, Lithuania, #18091050), and qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeahorse Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe extracellular Acidification Rate (ECAR) was evaluated by the Seahorse Bioscience Extracellular Flux Analyzer (XF 96 Agilent, RRID: SCR_019545). MCF7, MDAMB231 and MDAMB468 cells were seeded in 96-well seahorse plates at densities of 5000, 15,000 and 20,000 per well respectively in 200⎧L of DMEM and allowed to attach overnight. Next, Cells were treated according to experimental conditions and incubated for an additional 48 hours. Before the assay, cells were washed twice and incubated in a seahorse assay medium at 37\u0026deg;C in a non-CO₂ incubator for 1 hour to equilibrate. The assay was performed following the standard Glycolysis stress test protocol with consecutive injections of glucose oligomycin, and 2-deoxyglucose (2-DG). ECAR was measured at baseline level and following each injection to assess glycolytic flux. The data obtained were normalized to the total protein content per well as determined by the BCA protein assay and subsequently analyzed using Seahorse Wave software (RRID: SCR_014526).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTumor cells (2 x10\u003csup\u003e5\u003c/sup\u003e cells/mL) were seeded on sterile round coverslips that were placed in a 24-well plate and treated following the experimental procedure. The cells were then fixed with 300 \u0026micro;L of cold methanol (-10\u003csup\u003eo\u003c/sup\u003eC) for 5 mins, air dried and permeabilized with 0.25% Triton X-100 in PBS for 10 mins. Next, the cells were blocked with Blocking Solution (2% Bovine Serum Albumin\u0026thinsp;+\u0026thinsp;0.1% Triton X-100 in PBS) for 1 hr at room temperature, incubated overnight at 4\u003csup\u003e0\u003c/sup\u003eC with primary antibodies diluted in blocking solution according to the manufacturer\u0026rsquo;s instructions. Subsequent steps were performed protected from light. The cells were washed three times with 1X PBS, then incubated with secondary antibodies diluted 1:500 in Blocking Solution for 1 hr at room temperature. Cells were washed with 1X PBS for 5 mins, incubated in DAPI (300 nM) at room temperature for 5 mins and washed for another 5 mins with 1X PBS. Slides were mounted on carrying glass and stored overnight at 4\u0026deg;C in dark conditions. For tissues, Formalin-fixed paraffin-embedded (FFPE) tumor resections from \u003cem\u003eBalb/c\u003c/em\u003e mice were processed by the biospecimens core facility of the University of Kentucky per the approved Institutional Review Board (IRB) protocol). Immunohistochemistry was performed following the procedures as previously described by[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Protein localization was visualized with a fluorescent microscope at specified exposure times.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAquabluer redox indicator for cell viability was used to evaluate cell viability. Briefly, 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e control or PGK1-depleted (siRNA-transfected or NG52-treated) cells were seeded into 100\u0026micro;l of growth medium per well in 96-well plates and then treated with Erastin alone or in combination with Fer-1 (20\u0026micro;M), Liprox (1\u0026micro;M) Nec-1 (20\u0026micro;M), Z-vad-FMK (100\u0026micro;M), n-acetyl cysteine (20mM) and cultured for 72 hours. Afterwards, cells were cultured in 100ul of reconstituted Aquabluer solution, incubated for four hours and the absorbance was measured at 450 nm by a microplate reader to evaluate cell viability. For all drug treatments, compounds were dissolved in DMSO and further diluted in cell culture medium. The final concentration of DMSO in all experiments did not exceed \u003cb\u003e0.5% (v/v)\u003c/b\u003e, a level reported to be non-cytotoxic in breast cancer cell lines such as MCF7 and MDAMB231. All control groups were treated with an equivalent volume of DMSO alone to serve as baseline for normalization of viability or response.\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony formation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e200 control or PGK-depleted MCF7, MDAMB468 and 4T1 cells were seeded in a 12-well flat bottom plate and allowed to adhere overnight. Then, the cells were treated with DMSO as controls or 1.25\u0026micro;M of Erastin for 14 days. Afterwards, the cells were fixed in 4% paraformaldehyde for 14 minutes, stained with crystal violet solution for 15 minutes, rinsed with double distilled water and air-dried at room temperature for later visualization of the colonies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell transfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMCF7 and MDAMB231 cells were transfected with 800nm of PGK1 siRNA using jetPRIME Transfection Reagent. Following transfection, cells were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 72 hours before experimental use. L2T lentiviral particles were transfected into 4T1 cells at a concentration of 40\u0026micro;g/ml with Polybrene (10\u0026micro;g/ml) overnight, then cells were allowed to recover for 3 days and expanded before experimental use. Stable PGK1 knock-down cell lines were generated by transfecting 10\u0026micro;l of mouse PGK1 lentiviral shRNA particles with 10\u0026micro;g/ml of puromycin into wild-type 4T1 cells. Stable PGK1 knock-down clones were then selected with puromycin and expanded before experimental use.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal Studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnimals used in this investigation were treated humanely according to the recommendations set by the American Veterinary Medical Association, and the Institutional Animal Care and Use Committee of the University of Kentucky approved all the test protocols (protocol number IBC-24-4) 20 \u003cem\u003eBALB/c (\u003c/em\u003eRRID: IMSR_JAX:000651\u003cem\u003e)\u003c/em\u003e mice were equally orthotopically injected with 1x10\u003csup\u003e6\u003c/sup\u003e L2T-transfected Wild-type or PGK1 knockdown 4T1 cells in the fifth mammary fat pad and tumor size was measured every four days weekly. After tumor size reached 80-100mm\u003csup\u003e3\u003c/sup\u003e, mice were randomly separated into two groups comprising 5 mice each. Mice were then intraperitoneally treated with either vehicle (5% DMSO, 4% PEG, 4%Tween-80 in saline) or Imidazole ketone Erastin (40mg/kg) once every day for 14 days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of Cytotoxic T Cells from Spleen\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice were orthotopically inoculated with 4T1 cells, and the tumors were allowed to grow for 21 days under the aforementioned treatment conditions. Next, the mice were euthanized, and their spleens were harvested. The MojoSort\u0026trade; Mouse CD8 T cell Isolation Kit was used to isolate cytotoxic killer cells from the spleens of mice from each treatment group. This kit utilizes positive selection to isolate CD8 T cells from other immune and stroma cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInstitutional Review Board Statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the University of Kentucky (protocol code IBC-24-4 JMY; Date approved :01.28.2025).\u003c/p\u003e\u003cp\u003eIn accordance with our approved Institutional Animal Care and Use Committee (IACUC) protocol, animals bearing tumors were monitored closely, and mice were euthanized when tumor volume reached 1 cm in diameter or earlier if signs of discomfort or distress were observed. This limit is consistent with ethical guidelines to minimize animal suffering and has been explicitly described in our approved protocol (IBC-24-489). We confirm that in all experimental cohorts, the maximal tumor size of 1 cm in diameter was not exceeded. All animals were euthanized promptly upon reaching this limit or upon displaying any signs of distress, in strict compliance with our IACUC-approved humane endpoints.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism version 10.4.1 (GraphPad Software, CA, USA). All experimental studies comprised at least three independent experiments represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003cem\u003eT-\u003c/em\u003etest was used for two groups or Analysis of Variance (ANOVA) for multiple groups. Significance was defined as p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 between experimental and control groups.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 High PGK1 expression in breast Cancer is associated with poor prognosis and ferroptosis insensitivity.\u003c/h2\u003e\n \u003cp\u003eWe observed that PGK1 was expressed at different levels among subtypes of breast cancer cell lines, even within similar categories of breast cancers (Fig. 1A). Using Xena browser, we analyzed the comprehensive datasets from the Cancer Genome Atlas Program (TCGA). In the TCGA samples of which the majority are primary tumor tissues, PGK1 expression was higher in breast tumor tissues than in their normal counterparts. In the 1097 samples, we observed that the expressions of the PGK1 gene (transcripts per million) were at least 1.5-fold higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the human primary tumors than in normal breast tissue (Fig. 1B). As breast cancer is highly heterogeneous and its prognosis varies among different subtypes of the disease, we performed bioinformatic analysis of PGK1 expression in different subtypes of breast cancer. We grouped PGK1 expression in TCGA \u003cem\u003epan\u003c/em\u003e-cancer datasets into luminal, HER2, triple-negative tumors and normal breast tissues. The dataset, comprising 833 samples, has 566 luminal, 37 HER2 positive, 116 triple-negative, and 114 normal tissue samples. Figure 1C shows that PGK1 expression was highly elevated in TNBCs and HER2 samples as compared to other subclasses of breast cancer. Also, we observed a progressive increase of PGK1 expression as tumors advanced, with the highest PGK1 expression detected in stage IV cancers (Fig. 1D). Using the Kaplan Meier plotter, we stratified the breast cancer samples from 4929 patients into two groups based on PGK1 expression level: high expression group and low expression group. The patients with high PGK1-expressing tumors had lower survival than the patients with low PGK1-expressing tumors (Fig. 1E). Notably, in the datasets from the Gene Expression Omnibus database that were categorized as ferroptosis-sensitive or ferroptosis-resistant based on the immunophenotyping of 30 tumors, PGK1 expression was significantly upregulated in the ferroptosis-resistant tumors (Fig. 1F). These bioinformatic analyses inform that higher PGK1 gene expression is associated with poorer prognosis, advanced-stage breast cancer and ferroptosis insensitivity, particularly in TNBC and HER2-positive tumors.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Expression of PGK1 in breast cancers modulates their response to induction of ferroptosis.\u003c/h2\u003e\n \u003cp\u003eAs we observed the heterogenous effects of PGK1 expression on several signaling including GPX4, a regulator of ferroptosis, in breast cancer cells (Fig.\u0026nbsp;2A), we evaluated the effect of PGK1 expression on ferroptosis activity in breast cancer cells by testing the response of both TNBC cell lines (MDAMB231, MDAMB468 and BT549) and luminal cancer cell lines (MCF7 and T47D) to erastin, a small molecule inducer of ferroptosis. Table I and Fig.\u0026nbsp;2B show that TNBC lines MDAMB231, MDAMB468 and BT549 were more sensitive to induction of ferroptosis than the luminal cancer cell lines MCF7 and T47D. We next treated those cell lines with a series of concentrations of NG52, a small molecule inhibitor of PGK1, and observed that IC\u003csub\u003e50\u003c/sub\u003e values of NG52 in the luminal breast cancer cell lines MCF7 and T47D cell lines were 42\u0026micro;M and 75\u0026micro;M, higher than that in the TNBC cells (5\u0026micro;M, 17\u0026micro;M and 27\u0026micro;M for MDAMB231, HCC1187, and MDAMB453 respectively) (Fig. 2C; Table II), suggesting that TNBC cells are more vulnerable to PGK1 inhibition that the luminal breast cancer cells. The differing responses of HCC70 and HCC1187 compared to other TNBC cell lines may reflect the known heterogeneity in ferroptosis sensitivity among TNBC subtypes. Recent studies, including Yang et al., 2023 [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e], have shown that TNBC cells vary widely in their metabolic dependencies and resistance to ferroptosis, due to differences in glutathione metabolism, GPX4 expression, and oxidative stress adaptation. This stresses the importance of considering intragroup heterogeneity when designing ferroptosis-based interventions.\u003c/p\u003e\n \u003cp\u003eTo determine the effect of PGK1 on ferroptosis activity, we measured and compared the ferroptosis markers in tumor cells with or without depletion or inhibition of PGK1. We found that, while Acyl-CoA synthetase long-chain family member 4 (ACSL4), an enzyme involved in regulating fatty acid metabolism and a driver of ferroptosis, was downregulated in the PGK1-depleted MCF7 cells but unchanged in the PGK1-depleted MDAMB231. GPX4, an anti-ferroptosis peroxidase, was downregulated in both PGK1-depleted MCF7 and MDAMB231cells \u003cstrong\u003e(\u003c/strong\u003eFig. 2D), suggesting that loss of PGK1 induces a pro-ferroptotic shift through the GPX4 pathway in both of the cell lines. We also show that PGK1 depletion relocalized GPX4 to the nucleus (Supp Fig. 1A). PGK1 does not seem to affect the transcription of GPX4 in TNBCs, as GPX4 mRNA levels were comparable between the control and the PGK1-depleted MDAMB231 cells but not MCF7 (Fig. 2E). Consistently, depletion of PGK1 sensitized both triple-negative and luminal human breast cancers to the erastin-induced cell death (Supp Fig. 1B, 1C). Notably, ~\u0026thinsp;40% more ferroptotic cell death was observed in MDAMB468 (Fig. 2F). and MDAMB453 cells treated with the PGK1 inhibitor NG52 as compared with erastin alone, but only 20% more ferroptotic cell death was seen in the luminal MCF7 and T47D cells subjected to NG52 treatment (Fig. 2G). To further demonstrate the role of PGK1 in modulating ferroptosis, we generated the MDAMB231 (human TNBC) and 4T1 (murine TNBC) cells subjected to stable knockdown of PGK1 (Supp Fig. 1D) and treated them with the ferroptosis inducer erastin in the presence or absence of ferroptosis inhibitors (ferrostatin-1, liproxstatin) or apoptosis inhibitor (ZVAD-FMK). These experiments showed that only the ferroptosis inhibitors but not the apoptosis inhibitor prevented cell death, indicating ferroptosis as the primary cell death mechanism in the PGK1-depleted cells (Supp Fig. 1E, 1F). These results demonstrate that PGK1 can modulate ferroptosis in breast cancer cells via degrading GPX4, with TNBC cells showing greater sensitivity to PGK1 depletion.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Metabolic reprogramming is associated with the PGK1-regulated ferroptosis in breast cancer.\u003c/h2\u003e\n \u003cp\u003eSince PGK1 is a key glycolytic regulator, we queried whether PGK1 modulates ferroptosis via altering glucose metabolism. We found that with increasing glucose concentrations, ferroptotic activity increased moderately (Fig.\u0026nbsp;3A). Treatment of both luminal and TNBC cell lines with 2DG, a glycolytic inhibitor, enhanced their sensitivity to the effect of the ferroptosis inducer erastin (Fig.\u0026nbsp;3B). To determine whether modulation of ferroptosis by PGK1 results from its effect on metabolic signaling, we analyzed its downstream targets in the control and PGK1-depleted cells under ferroptosis-inducing conditions. We found a consistent downregulation of pyruvate dehydrogenase (PDH), but not Lactate dehydrogenase A (LDHA) and Pyruvate kinase M1/2 (PKM1/2), in the PGK1-depleted MDAMB468 and MCF7 cells, and both of which exhibited increased sensitivity to induction of ferroptosis (Fig.\u0026nbsp;3D, 3E,). In contrast, MDAMB231 cells, which did not show increased sensitivity to ferroptosis following PGK1 depletion, displayed an upregulation of PDH expression under the same conditions (Fig.\u0026nbsp;3C, 2G). This discrepancy might account for the difference in sensitivity to ferroptosis induction among those tumor cells. In addition, although PGK1 has a key role in glycolysis, we observed an increased ECAR in the PGK1-depleted MDAMB468 cells (Fig.\u0026nbsp;3F), likely a consequence of a compensatory shift toward glycolysis due to PDH downregulation, limiting pyruvate oxidation, and this inefficient adaptation might trigger metabolic stress that enhances ferroptosis sensitivity. Conversely, the PGK1-depleted MDAMB231 cells, which are ferroptosis-insensitive, upregulated PDH to maintain oxidative metabolism and prevent further sensitization (Fig.\u0026nbsp;3G).\u003c/p\u003e\n \u003cp\u003eTo further analyze the role of PDH in PGK1-regulated ferroptosis, we treated the PGK1-depleted MDAMB231 cells with hypoxia or oligomycin, an inhibitor of Adenosine triphosphate (ATP) synthase. We show that PDH was substantially down-regulated (Fig. 4A) and the sensitivity to ferroptosis was significantly enhanced in those treated cells as compared with the control cells (Fig. 4B). Similarly, inhibiting PDH in the PGK1-depleted T47D, MCF7, MDAMB453 and HCC70 cells sensitized them to the erastin-induced cell death (Fig. 4C). Notably, 30-hour PDH inhibition produced a cell death level nearly comparable to 72-hour ferroptosis induction in the PGK1-depleted cells (Fig. 4C), suggesting PDH upregulation as a possible resistance mechanism. These results imply that PGK1 depletion can sensitize certain subtypes of breast cancer to ferroptosis induction by disrupting metabolic balance, and this sensitization correlates with PDH downregulation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Targeting PGK1-mediated ferroptosis enhances anti-tumor immunity.\u003c/h2\u003e\n \u003cp\u003eTo assess the importance of the PGK1-regulated ferroptosis \u003cem\u003ein viv\u003c/em\u003eo, we inoculated the murine TNBC 4T1 cells with or without depletion of PGK1, followed by treatment with either vehicle or imidazole ketone erastin (IKE), a ferroptosis inducer (Fig. 5A). These experiments demonstrated that IKE significantly slowed tumor growth, with a more pronounced reduction in tumor volume observed in mice bearing PGK1-depleted tumors in those with tumors expressing PGK1 (Fig. 5C\u0026ndash;5E). Furthermore, IKE treatment completely abrogated lung metastasis in mice bearing the PGK1-depleted 4T1 tumors, whereas mice with control tumors exhibited metastatic progression (Fig. 6A, 6B). Also, we observed a progressive decrease of the spleen size in the mice of the treatment groups, with the following extent: control 4T1-inoculated (vehicle-treated)\u0026thinsp;\u0026gt;\u0026thinsp;control 4T1-inoculated (IKE-treated)\u0026thinsp;\u0026gt;\u0026thinsp;PGK1-depleted 4T1-inoculated (vehicle-treated)\u0026thinsp;\u0026gt;\u0026thinsp;PGK1-depleted 4T1-inoculated (IKE-treated) (Fig. 6C, 6D). Western blot analysis of the T cells isolated from those spleens showed a corresponding increase in the expression of Granzyme B, a key cytolytic enzyme, and CD25, a T cell activation marker, following the same pattern as above (Fig. 6E). These observations imply that PGK1 depletion in combination with IKE treatment can enhance T cell activation. To further evaluate T cell functionality, we co-cultured CD8\u0026thinsp;+\u0026thinsp;T cells from the spleens of the mice with 4T1 tumor cells, followed by cytotoxicity assay. Figure 6F shows that CD8\u0026thinsp;+\u0026thinsp;T cells from the mice bearing PGK1-depleted 4T1 cells and receiving IKE treatment had a significantly enhanced tumor cell killing ability than CD8\u0026thinsp;+\u0026thinsp;T cells from the mice bearing control 4T1cells (Fig. 6F), indicating that targeting PGK1 in combination with ferroptosis induction can strengthen the cytotoxic T lymphocyte-mediated antitumor immunity. Additionally, immunohistochemical analysis of tumor sections showed a progressive reduction in GPX4 expression across the treatment groups: GPX4 expression was high in the control 4T1-inoculated (vehicle-treated) tumors, moderately reduced in the control 4T1-inoculated (IKE-treated) tumors, further decreased in the PGK1-depleted 4T1-inoculated (vehicle-treated) tumors, and completely absent in the PGK1-depleted 4T1-inoculated (IKE-treated) tumors (Fig. 6G).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePGK1 was selected for this study due to its elevated expression in aggressive breast cancer subtypes and its association with poor prognosis, as revealed in TCGA and GEO datasets. While prior studies have implicated PGK1 in promoting breast cancer progression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the role of PGK1 in modulating ferroptosis, an important form of cell death, has not been well elucidated. Our findings demonstrate that PGK1 inhibition not only destabilizes the ferroptosis regulator GPX4 but also reprograms pyruvate metabolism by modulating PDH levels, ultimately influencing the cellular susceptibility to ferroptosis under normoxic and hypoxic conditions. These insights uncover previously uncharacterized function of PGK1 in regulating ferroptosis resistance, offering a novel metabolic target for therapeutic intervention in breast cancer.\u003c/p\u003e\u003cp\u003eWe show that PGK1 is highly expressed in advanced stages of breast cancers, especially in HER2\u0026thinsp;+\u0026thinsp;and TNBC, and that PGK1 expression appears to be causally associated with tumor sensitivity to ferroptosis induction. We further show that modulation of ferroptosis by PGK1 is associated with metabolic reprogramming, and that ferroptosis-resistant breast cancers have high expression of PGK1, which has a critical role in controlling GPX4, thereby contributing to cellular sensitivity to ferroptosis induction. Importantly, our study indicates that the effect of tumoral PGK1 on ferroptosis induction may vary among the subtype of breast cancers, with TNBCs showing greater vulnerability to ferroptosis following PGK1 depletion as compared to the luminal subtypes. While multiple studies have implicated PGK1 overexpression in oncogenic activities such as tumor progression and therapy resistance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], others have argued for its anti-tumorigenic as was the case in Lewis lung cancer[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, how PGK1 metabolically reprograms tumor cells to survive under ferroptotic conditions remains to be fully elucidated.\u003c/p\u003e\u003cp\u003eThere are studies showing the metabolic underpinnings of ferroptosis including fatty acid metabolism, iron handling, mevalonate pathway, and thiol metabolism on lipid peroxidation, a catalyst for ferroptosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Here, we demonstrate that PGK1 has a role in regulating the levels of ferroptosis markers ACSL4, FSP1, Ferritin, and GPX4, the evidence supporting the ferroptosis-modulating function of PGK1. Contradictory to a previous study showing that pyruvate dehydrogenase enhances the autooxidation of dihydrolipoamide, promoting ferroptosis in human fibrosarcoma cells[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we show that PGK1 modulates ferroptosis susceptibility via its downstream target PDH. This discrepancy might be due to cancer-specific roles of PDH in ferroptosis response. Notwithstanding, we demonstrate that PGK1 promotes ferroptosis resistance in the TNBC MDAMB231 cells by regulating PDH, which prevents an inefficient compensatory glycolytic shift under ferroptotic conditions. This stabilization reduces metabolic stress, extracellular acidification, and lipid peroxidation, ultimately inhibiting ferroptosis. In addition, PGK1 depletion disrupts metabolic balance, with the glycolytic shift, contributing to ferroptosis susceptibility in certain breast cancer subtypes. Of note, we observed that TNBC MDAMB231 cells were more sensitive to ferroptosis under normoxia compared to hypoxic conditions, a phenomenon that is linked to the differential expression of PDH under these circumstances. Elevated PDH expression under normoxic conditions enhance oxidative phosphorylation, resulting in elevated lipid peroxidation and mitochondrial ROS, hallmarks known to promote ferroptosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In contrast, during hypoxic conditions or after chemical inhibition of ATP synthase, PDH is significantly attenuated, reducing mitochondrial activity and a shift towards the glycolytic phenotype. Hypoxia-induced metabolic reprogramming may limit ferroptosis susceptibility by maintaining redox balance and attenuating oxidative stress. Therefore, the oxygen-mediated regulation of PDH seems to modulate the metabolic response and ferroptosis sensitivity in MDAMB231, highlighting a mechanistic basis for their differential response to ferroptosis induction under varying oxygen tensions.\u003c/p\u003e\u003cp\u003eIt is likely that PGK1 and PDH are differentially regulated by oxygen availability. Under normoxic conditions, PGK1 facilitates pyruvate production and glycolytic flux, maintaining PDH depression and mitochondrial metabolism. However, hypoxia already limits oxidative phosphorylation, and combined with PGK1 depletion further restricts glycolytic output and pyruvate supply. This metabolic stress possibly induces PDH suppression through feedback mechanisms, likely through upregulated phosphorylation by hypoxia-induced pyruvate dehydrogenase kinases (PDKs) that ultimately inactivate PDH [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, this two-way impairment of mitochondrial metabolism underlies the observed PDH downregulation and may explain reduced sensitivity to ferroptosis under hypoxia. To summarize our findings in the context of metabolic regulation and ferroptosis, we propose a schematic model (Supp Fig.\u0026nbsp;2A) that illustrates how PGK1 and PDH influence ferroptosis sensitivity under normoxic and hypoxic conditions. This diagram integrates our experimental data with established metabolic pathways to provide a unified mechanistic framework. New insights have revealed that ferroptosis resistance not only directly contributes to malignant phenotypes of breast cancer but may also modulate the immunosuppressive features of the TME through its interaction with immune cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, we show the immunomodulatory potential of targeting PGK1 in combination with ferroptosis induction. Although we observed that IKE treatment shrinks tumor in immune-competent BALB/c mice, the effect of PGK1 depletion during IKE treatment is striking, which shows the abrogation of both micro and macroscopic tumor metastasis to the lungs. Also, we observed a reduction in the spleen size and a corresponding increase in T cell activation markers such as Granzyme B and CD25, suggesting that this combined treatment not only promotes ferroptosis but also improves anti-tumor immunity. It remains to be determined whether the reduced spleen size indicates decreased tumor-induced immunosuppression or a reduction in tumor-associated immune cell populations. However, CD8⁺ T cells extracted from IKE-treated mice bearing the PGK1-depleted tumors exhibited enhanced cytotoxicity when cocultured with 4T1 murine TNBCs (Fig.\u0026nbsp;6F), suggesting an augmented T cell-mediated anti-tumor response. Beyond its role in glycolysis and immune modulation, PGK1 is also linked with cellular stress pathways through the AMPK\u0026ndash;mTOR axis. This axis integrates energy sensing (via AMPK) and anabolic signaling (via mTOR), both of which are essential for regulating autophagy and tumor cell survival. Therapeutic combinations such as metformin (an AMPK activator) and rapamycin (an mTOR inhibitor) have demonstrated efficacy in TNBC models [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Given the link between ferroptosis and metabolic stress, co-targeting PGK1 with modulators of the AMPK\u0026ndash;mTOR pathway may intensify oxidative damage and enhance ferroptotic cell death. Thus, these strategies might help overcome therapy resistance and improve treatment outcomes in aggressive breast cancers.\u003c/p\u003e\u003cp\u003eRecent efforts have led to the development of promising small-molecule PGK1 inhibitors. Notably, CBR-470-1 was reported to inhibit PGK1 enzymatic activity, attenuating glycolytic flux and shrinking tumor growth in preclinical models [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In a similar trend, Ilicicolin H, a fungal metabolite and a dual inhibitor of PGK1 and mitochondrial complex III, disrupted tumor energy metabolism and exhibited anticancer effects[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The proof-of-concept experiments using these agents suggest that PGK1 is a druggable target, and their emerging profiles support the feasibility of combining PGK1 inhibition with ferroptosis inducers to enhance metabolic vulnerability in breast cancers. Further investigation of these compounds in the context of ferroptosis sensitivity may broaden therapeutic strategies, especially in metabolically active and therapy-resistant subtypes of malignancies such as TNBC.\u003c/p\u003e\u003cp\u003eOur Study identifies PGK1 as a mediator of ferroptosis resistance and provides a rationale for targeting PGK1 in conjunction with ferroptosis induction as a potential therapeutic strategy for aggressive breast cancers such as TNBC. By simultaneously inhibiting tumor growth and enhancing anti-tumor immune responses, this approach might offer a multifaceted attack on cancer cells for improving the effectiveness of cancer immunotherapy such as immune checkpoint inhibitors.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"630\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003e2-DG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003e2-Deoxy-D-glucose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eACSL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eAcyl-CoA synthetase long-chain family member 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eAMPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eAMP-activated protein kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eAnalysis of Variance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eATP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eAdenosine triphosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eBCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eBicinchoninic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eCD25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eCluster of Differentiation 25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eCD71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eCluster of Differentiation 71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003e4\u0026acirc;\u0026euro;\u0026sup2;,6-diamidino-2-phenylindole\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eDMEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eDulbecco\u0026acirc;\u0026euro;\u0026trade;s Modified Eagle Medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eECAR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eExtracellular Acidification Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eFBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eFetal Bovine Serum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eFFPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eFormalin-Fixed Paraffin-Embedded\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eGEO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eGene Expression Omnibus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eGPX4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eGlutathione Peroxidase 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eHRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eHorseradish Peroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eIC50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eHalf maximal inhibitory concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eIKE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eImidazole Ketone Erastin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eKM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eKaplan-Meier\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eLDHA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eLactate Dehydrogenase A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eOverall Survival\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003ePhosphate Buffered Saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePBST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003ePBS with Tween 20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003ePyruvate Dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePGK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003ePhosphoglycerate Kinase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003ePost-Progression Survival\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePVDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003ePolyvinylidene Fluoride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eRFS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eRelapse-Free Survival\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eRibonucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eRPMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eRoswell Park Memorial Institute medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eStandard Deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eSDS-PAGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eSodium Dodecyl Sulfate\u0026acirc;\u0026ldquo;Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eTCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eTricarboxylic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eTCGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eThe Cancer Genome Atlas\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eTME\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eTumor Microenvironment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eTNBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eTriple-Negative Breast Cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003ecDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eComplementary DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003emTOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eMechanistic Target of Rapamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eqRT-PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eQuantitative Real-Time Polymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003eshRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eShort Hairpin RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 210px;\"\u003e\n \u003cp\u003esiRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 420px;\"\u003e\n \u003cp\u003eSmall Interfering RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Availability of Data and Materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are publicly available in Gene Expression Omnibus (GEO) at\u0026nbsp;GSE148297.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u0026quot; in this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical Trial Number\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u0026quot; in this section.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis investigation was self-funded by the Yang Lab.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u003cem\u003eAuthor Contribution\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFO performed some bioinformatic investigation into PGK1 expression across a. cancer subtypes, and b. tumor staging. She also analyzed the raw data from the PDH depletion experiments using statistical tools. TO conducted a bioinformatic analysis of the prognostic significance of PGK1 expression in relation to post-progression survival, relapse-free survival, and overall survival in patients with luminal and HER2-positive breast cancer. OO conducted bioinformatic investigation into the differences between sensitive and ferroptosis-resistant cell lines using GEO datasets. He also investigated the Protein and gene expression of PGK1 across various tissues and their corresponding tumors. AA brought in his methods for evaluating different protein expressions of PGK1 downstream targets. He also reviewed the draft of this manuscript. CN and AS worked in the curation of Data as well as their validation for this research work. XR and J Y supervised and sourced for funds and resources to carry out this research work. At the time of this work, Oluwafunminiyi Obaleye, Amos Akinyemi, Chrispus Ngule, Andrew Shinkle, Xingcong Ren, and Jin-Ming Yang were affiliated with the Department of Toxicology and Cancer Biology, University of Kentucky, USA. Folake Oyelami was affiliated with the Department of Microbiology, University of Ilorin, Nigeria. Tijesunimi Oyetunde was an independent bioinformatics collaborator based in Manitoba, Canada.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026ldquo;Breast Cancer Statistics | How Common Is Breast Cancer? | American Cancer Society.\u0026rdquo; Accessed: Dec. 10, 2024. [Online]. Available: https://www.cancer.org/cancer/types/breast-cancer/about/how-common-is-breast-cancer.html\u003c/li\u003e\n\u003cli\u003eR. L. Siegel, A. N. Giaquinto, and A. Jemal, \u0026ldquo;Cancer statistics, 2024,\u0026rdquo; CA Cancer J Clin, vol. 74, no. 1, pp. 12\u0026ndash;49, Jan. 2024, doi: 10.3322/CAAC.21820.\u003c/li\u003e\n\u003cli\u003eB. Faubert, A. Solmonson, and R. J. DeBerardinis, \u0026ldquo;Metabolic reprogramming and cancer progression,\u0026rdquo; Science, vol. 368, no. 6487, Apr. 2020, doi: 10.1126/SCIENCE.AAW5473.\u003c/li\u003e\n\u003cli\u003eC. McCann and E. M. Kerr, \u0026ldquo;Metabolic Reprogramming: A Friend or Foe to Cancer Therapy?,\u0026rdquo; Cancers (Basel), vol. 13, no. 13, p. 3351, Jul. 2021, doi: 10.3390/CANCERS13133351.\u003c/li\u003e\n\u003cli\u003eC. Schiliro and B. L. Firestein, \u0026ldquo;Mechanisms of Metabolic Reprogramming in Cancer Cells Supporting Enhanced Growth and Proliferation,\u0026rdquo; Cells, vol. 10, no. 5, May 2021, doi: 10.3390/CELLS10051056.\u003c/li\u003e\n\u003cli\u003eG. Lei, L. Zhuang, and B. Gan, \u0026ldquo;Targeting ferroptosis as a vulnerability in cancer,\u0026rdquo; Nat Rev Cancer, vol. 22, no. 7, pp. 381\u0026ndash;396, Mar. 2022, doi: 10.1038/s41568-022-00459-0.\u003c/li\u003e\n\u003cli\u003eZ. Chen, W. Wang, S. R. Abdul Razak, T. Han, N. H. Ahmad, and X. Li, \u0026ldquo;Ferroptosis as a potential target for cancer therapy,\u0026rdquo; Cell Death Dis, vol. 14, no. 7, Jul. 2023, doi: 10.1038/S41419-023-05930-W.\u003c/li\u003e\n\u003cli\u003eC. Zhang, X. Liu, S. Jin, Y. Chen, and R. Guo, \u0026ldquo;Ferroptosis in cancer therapy: a novel approach to reversing drug resistance,\u0026rdquo; Mol Cancer, vol. 21, no. 1, Dec. 2022, doi: 10.1186/S12943-022-01530-Y.\u003c/li\u003e\n\u003cli\u003eQ. Fu and Z. Yu, \u0026ldquo;Phosphoglycerate kinase 1 (PGK1) in cancer: A promising target for diagnosis and therapy,\u0026rdquo; Life Sci, vol. 256, Sep. 2020, doi: 10.1016/J.LFS.2020.117863.\u003c/li\u003e\n\u003cli\u003eH. Liu, X. Wang, P. Shen, Y. Ni, and X. Han, \u0026ldquo;The basic functions of phosphoglycerate kinase 1 and its roles in cancer and other diseases,\u0026rdquo; Eur J Pharmacol, vol. 920, p. 174835, 2022, doi: 10.1016/j.ejphar.2022.174835.\u003c/li\u003e\n\u003cli\u003eK. Zhang, L. Sun, and Y. Kang, \u0026ldquo;Regulation of phosphoglycerate kinase 1 and its critical role in cancer,\u0026rdquo; Cell Commun Signal, vol. 21, no. 1, p. 240, Dec. 2023, doi: 10.1186/S12964-023-01256-4.\u003c/li\u003e\n\u003cli\u003e\u0026ldquo;Kaplan-Meier plotter [Breast cancer].\u0026rdquo; Accessed: Jun. 22, 2025. [Online]. Available: https://kmplot.com/analysis/index.php?p=service\u003c/li\u003e\n\u003cli\u003eJ. Liu et al., \u0026ldquo;An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics,\u0026rdquo; Cell, vol. 173, no. 2, pp. 400-416.e11, Apr. 2018, doi: 10.1016/j.cell.2018.02.052.\u003c/li\u003e\n\u003cli\u003eA. Ghoochani et al., \u0026ldquo;Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer,\u0026rdquo; Cancer Res, vol. 81, no. 6, p. 1583, Mar. 2021, doi: 10.1158/0008-5472.CAN-20-3477.\u003c/li\u003e\n\u003cli\u003eM. D. Iglesia et al., \u0026ldquo;Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages,\u0026rdquo; Nat Cancer, vol. 5, no. 11, pp. 1713\u0026ndash;1736, Oct. 2024, doi: 10.1038/s43018-024-00773-6.\u003c/li\u003e\n\u003cli\u003eF. Yang et al., \u0026ldquo;Ferroptosis heterogeneity in triple-negative breast cancer reveals an innovative immunotherapy combination strategy,\u0026rdquo; Cell Metab, vol. 35, no. 1, pp. 84-100.e8, Jan. 2023, doi: 10.1016/j.cmet.2022.09.021.\u003c/li\u003e\n\u003cli\u003eX. Gao et al., \u0026ldquo;Acetylation of PGK1 at lysine 323 promotes glycolysis, cell proliferation, and metastasis in luminal A breast cancer cells,\u0026rdquo; BMC Cancer, vol. 24, no. 1, Dec. 2024, doi: 10.1186/S12885-024-12792-8.\u003c/li\u003e\n\u003cli\u003eZ. Guo et al., \u0026ldquo;Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle,\u0026rdquo; Nat Commun, vol. 15, no. 1, Dec. 2024, doi: 10.1038/S41467-024-51232-W.\u003c/li\u003e\n\u003cli\u003eX. Li et al., \u0026ldquo;Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis,\u0026rdquo; Mol Cell, vol. 61, no. 5, pp. 705\u0026ndash;719, Mar. 2016, doi: 10.1016/J.MOLCEL.2016.02.009.\u003c/li\u003e\n\u003cli\u003eY. Zhang et al., \u0026ldquo;Macrophage-Associated PGK1 Phosphorylation Promotes Aerobic Glycolysis and Tumorigenesis,\u0026rdquo; Mol Cell, vol. 71, no. 2, pp. 201-215.e7, Jul. 2018, doi: 10.1016/J.MOLCEL.2018.06.023.\u003c/li\u003e\n\u003cli\u003eS. J. Tang et al., \u0026ldquo;Phosphoglycerate kinase 1-overexpressing lung cancer cells reduce cyclooxygenase 2 expression and promote anti-tumor immunity in vivo,\u0026rdquo; Int J Cancer, vol. 123, no. 12, pp. 2840\u0026ndash;2848, Dec. 2008, doi: 10.1002/IJC.23888.\u003c/li\u003e\n\u003cli\u003eJ. Zheng and M. Conrad, \u0026ldquo;The Metabolic Underpinnings of Ferroptosis,\u0026rdquo; Cell Metab, vol. 32, no. 6, pp. 920\u0026ndash;937, Dec. 2020, doi: 10.1016/J.CMET.2020.10.011.\u003c/li\u003e\n\u003cli\u003eJ. W. Kim, J. Y. Lee, M. Oh, and E. W. Lee, \u0026ldquo;An integrated view of lipid metabolism in ferroptosis revisited via lipidomic analysis,\u0026rdquo; Exp Mol Med, vol. 55, no. 8, pp. 1620\u0026ndash;1631, Aug. 2023, doi: 10.1038/s12276-023-01077-y.\u003c/li\u003e\n\u003cli\u003eY. Zhang et al., \u0026ldquo;The molecular mechanisms of ferroptosis and its role in cardiovascular disease,\u0026rdquo; Biomed Pharmacother, vol. 145, p. 112423, Jan. 2022, doi: 10.1016/J.BIOPHA.2021.112423.\u003c/li\u003e\n\u003cli\u003eJ. I. J. Leu, M. E. Murphy, and D. L. George, \u0026ldquo;Functional interplay among thiol-based redox signaling, metabolism, and ferroptosis unveiled by a genetic variant of TP53,\u0026rdquo; Proc Natl Acad Sci U S A, vol. 117, no. 43, pp. 26804\u0026ndash;26811, Oct. 2020, doi: 10.1073/PNAS.2009943117/-/DCSUPPLEMENTAL.\u003c/li\u003e\n\u003cli\u003eA. M. Vučković et al., \u0026ldquo;Aerobic pyruvate metabolism sensitizes cells to ferroptosis primed by GSH depletion,\u0026rdquo; Free Radic Biol Med, vol. 167, pp. 45\u0026ndash;53, May 2021, doi: 10.1016/J.FREERADBIOMED.2021.02.045.\u003c/li\u003e\n\u003cli\u003eS. Chen, Q. Li, H. Shi, F. Li, Y. Duan, and Q. Guo, \u0026ldquo;New insights into the role of mitochondrial dynamics in oxidative stress-induced diseases,\u0026rdquo; Biomed Pharmacother, vol. 178, p. 117084, Sep. 2024, doi: 10.1016/J.BIOPHA.2024.117084.\u003c/li\u003e\n\u003cli\u003eL. J. Su et al., \u0026ldquo;Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis,\u0026rdquo; Oxid Med Cell Longev, vol. 2019, p. 5080843, 2019, doi: 10.1155/2019/5080843.\u003c/li\u003e\n\u003cli\u003eD. Nolfi-Donegan, A. Braganza, and S. Shiva, \u0026ldquo;Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement,\u0026rdquo; Redox Biol, vol. 37, p. 101674, Oct. 2020, doi: 10.1016/J.REDOX.2020.101674.\u003c/li\u003e\n\u003cli\u003eJ. W. Kim, I. Tchernyshyov, G. L. Semenza, and C. V. Dang, \u0026ldquo;HIF-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia,\u0026rdquo; Cell Metab, vol. 3, no. 3, pp. 177\u0026ndash;185, Mar. 2006, doi: 10.1016/j.cmet.2006.02.002.\u003c/li\u003e\n\u003cli\u003eK. Cui, K. Wang, and Z. Huang, \u0026ldquo;Ferroptosis and the tumor microenvironment,\u0026rdquo; J Exp Clin Cancer Res, vol. 43, no. 1, p. 315, Nov. 2024, doi: 10.1186/S13046-024-03235-0.\u003c/li\u003e\n\u003cli\u003eQ. Wen, J. Liu, R. Kang, B. Zhou, and D. Tang, \u0026ldquo;The release and activity of HMGB1 in ferroptosis,\u0026rdquo; Biochem Biophys Res Commun, vol. 510, no. 2, pp. 278\u0026ndash;283, Jan. 2019, doi: 10.1016/J.BBRC.2019.01.090.\u003c/li\u003e\n\u003cli\u003eP. Chen et al., \u0026ldquo;ACSL4 promotes ferroptosis and M1 macrophage polarization to regulate the tumorigenesis of nasopharyngeal carcinoma,\u0026rdquo; Int Immunopharmacol, vol. 122, p. 110629, Sep. 2023, doi: 10.1016/J.INTIMP.2023.110629.\u003c/li\u003e\n\u003cli\u003eA. Qiu et al., \u0026ldquo;Phosphoglycerate Kinase 1: An Effective Therapeutic Target in Cancer,\u0026rdquo; Front Biosci (Landmark Ed), vol. 29, no. 3, 2024, doi: 10.31083/J.FBL2903092.\u003c/li\u003e\n\u003cli\u003eS. Mukhopadhyay, A. Chatterjee, D. Kogan, D. Patel, and D. A. Foster, \u0026ldquo;5-aminoimidazole-4-carboxamide-1-\u0026beta;-4-ribofuranoside (AICAR) enhances the efficacy of rapamycin in human cancer cells,\u0026rdquo; Cell Cycle, vol. 14, no. 20, pp. 3331\u0026ndash;3339, 2015, doi: 10.1080/15384101.2015.1087623.\u003c/li\u003e\n\u003cli\u003eX. Qian et al., \u0026ldquo;Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy,\u0026rdquo; Mol Cell, vol. 65, no. 5, pp. 917-931.e6, Mar. 2017, doi: 10.1016/j.molcel.2017.01.027.\u003c/li\u003e\n\u003cli\u003eJ. Zheng et al., \u0026ldquo;PGK1 inhibitor CBR-470-1 protects neuronal cells from MPP+,\u0026rdquo; Aging (Albany NY), vol. 12, no. 13, pp. 13388\u0026ndash;13399, 2020, doi: 10.18632/AGING.103443.\u003c/li\u003e\n\u003cli\u003eM. Li, A. Zhang, X. Qi, R. Yu, and J. Li, \u0026ldquo;A novel inhibitor of PGK1 suppresses the aerobic glycolysis and proliferation of hepatocellular carcinoma,\u0026rdquo; Biomed Pharmacother, vol. 158, Feb. 2023, doi: 10.1016/j.biopha.2022.114115.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable I: IC50 values of Breast cancer cell lines to erastin treatment after 72hrs\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell line\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCategory\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC50 (\u003c/strong\u003e\u003cstrong\u003e\u0026mu;\u003c/strong\u003e\u003cstrong\u003eM\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;SEM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMDAMB231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eBT549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMDAMB468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMDAMB453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHCC1187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e41.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHCC70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026gt;60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eT47D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eLuminal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026gt;60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMCF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eLuminal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026gt;60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eTable I: Viability measurement of human triple-negative breast cancers MDAMB231, BT549, MDAMB468, MDAMB453, HCC1187, and luminal cancers T47D and MCF7 cell lines treated with increasing doses of erastin for 72 hours. Data are represented as the mean\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026plusmn;\u003c/em\u003e\u003cem\u003e\u0026nbsp;SD (n=6).\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTable II: IC50 values of Breast cancer cell lines to NG52 treatment after 72hrs\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell line\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCategory\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC50 (\u003c/strong\u003e\u003cstrong\u003e\u0026mu;\u003c/strong\u003e\u003cstrong\u003eM\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;SEM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMDAMB231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eBT549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e40.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMDAMB468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e35.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMDAMB453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHCC1187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHCC70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTriple-Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e40.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eT47D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eLuminal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e42.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMCF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eLuminal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable II: Viability measurement of human triple-negative breast cancers MDAMB231, BT549, MDAMB468, MDAMB453, HCC1187, and luminal cancers T47D and MCF7 cell lines treated with increasing doses of PGK1 inhibitor NG52 for 72hours. Data are represented as the mean \u0026plusmn; SD (n=6)\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PGK1, ferroptosis, GPX4, breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-7323200/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7323200/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTherapeutic resistance and recurrence are among the major contributors to poor outcomes for patients with breast cancer. Induction of ferroptosis, a form of cellular death characterized by toxic lipid peroxide overload, has emerged as a promising therapeutic strategy against breast cancers including triple-negative breast cancer(TNBC). Nevertheless, certain types of cancer are impervious to induction of ferroptosis and the underlying mechanisms remain incompletely clear. In this study, we show that phosphoglycerate kinase 1 (PGK1), an important enzyme in glycolysis, is highly expressed in breast tumors, and the elevated levels of PKG expression correlate with advanced tumor stages, poor prognosis and ferroptosis insensitivity, particularly in TNBCs. Using genetic or pharmacological inhibition, we demonstrate that knockdown or inhibition of PGK1 enhances ferroptosis sensitivity in both TNBC and luminal breast cancer cell lines. We further demonstrate that depletion of PGK1 destabilizes glutathione peroxidase 4 (GPX4), an anti-ferroptotic defense peroxidase, thereby disturbing cellular redox homeostasis and promoting lipid peroxidation. Moreover, targeting PGK1 disrupts glycolytic metabolism and sensitizes breast cancer cells to ferroptosis induction in tumor cells subjected to glucose deprivation or treated with glycolytic inhibitors. In orthotopic TNBC models, loss of tumoral PGK1 augments the action of the ferroptosis inducer, imidazole ketone erastin (IKE), in inhibiting tumor growth and metastasis, and enhances CD8\u0026thinsp;+\u0026thinsp;T cell-mediated anti-tumor immunity. These results indicate that PGK1 has a critical role in modulating breast cancer invulnerability to induction of ferroptosis, implying that this kinase may be exploited as a therapeutic target to sensitize breast cancers, especially, TNBC, to ferroptosis inducers.\u003c/p\u003e","manuscriptTitle":"Expression of PGK1 in Breast Cancers Alters Their Sensitivity to Ferroptosis Induction via Metabolic Reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 09:30:07","doi":"10.21203/rs.3.rs-7323200/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-03T12:37:41+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T11:49:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-21T06:01:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2025-08-17T03:55:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"080717ac-3924-4e00-af87-4b319d75c675","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T11:04:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 09:30:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7323200","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7323200","identity":"rs-7323200","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}