Mitochondrial Lactate Uptake And Oxidation Complex Contributes To Stemness And Metastatic Potentials Of Detached Breast Cancer

【Background】Breast cancer is the most common cancer in women worldwide and ranks first among the causes of cancer deaths in developing countries.Metastasis is one of the main causes of poor prognosis for patients with breast cancer,and ninety percent of deaths from breast cancer are related to lung metastasis,so how to effectively inhibit lung metastasis of breast cancer is the key to improving the survival of breast cancer patients.Recent studies have shown that a small number of tumor cells can form clinically visible metastatic foci in secondary organs,that is,metastasis-initiating cells（MICs）,which can drive tumor formation,have the stemness potentials of self-renewal and multidirectional differentiation,and are the root cause of tumor metastasis and recurrence.How to target and eliminate MICs is currently a hot and difficult issue in the study of tumor metastasis mechanisms and anti-metastasis treatment.MICs may originate from cancer stem cells（CSCs）in the primary tumor,or from tumor cells that acquire the potential to initiate metastasis during the metastatic process.Our previous studies have found that detachment stress increases the number of original CSCs and improves their stemness potentials,and induces transformation of non-CSCs into CSCs,that is,reprogramming into CSCs.The latter can further acquire the potential of initiating metastasis and promote the process of tumor metastasis.Therefore,in-depth analysis of the common mechanism of maintaining and inducing the stemness and metastatic potentials of MICs under detachment environment can provide important theoretical basis for the targeted eradication of MICs.Under different microenvironmental stress conditions,cells can adjust and reshape nutrient acquisition and metabolic pathways to meet the needs of survival and growth,and thus play an important role in the process of tumor metastasis.For example,during the dissemination and metastasis of detached tumor cells in the blood circulation,cell polarity is lost and glucose transport is defective.At this time,lactate,a circulating glycolytic metabolite,can be utilized by lung cancer cells and serve as fuel for Oxidative phosphorylation（OXPHOS）to promote the process of tumor metastasis.This suggests that breast cancer cells may reshape their metabolic substrates and pathways under detachment stress,but the energy substrates that maintain the stemness and metastatic potentials of MICs and the specific metabolic pathways remain unclear.This study aims to deeply explore the mechanism of the origin of MICs from the perspective of cellular metabolic remodeling,and provide a new target for the development of more effective targeted strategies against MICs,so it has important theoretical and clinical significance.【Objectives】1.To explore the metabolic characteristics of detached breast cancer cells and identify the main energy source of detached breast cancer cells.2.To investigate the effects of energy substrate of detached breast cancer cells on the stemness and metastatic potentials of MICs and figure out the specific mechanism.3.To study the molecular mechanism involved in energy substrate uptake and oxidation in detached breast cancer cells and its targeted intervention strategies.【Methods】1.Bioinformatics was used to analyze the significantly different pathways between primary and metastatic tumors of breast cancer,and to identify the key metabolic factors that may affect breast cancer metastasis.A cellular detachment model was established and metabolomics analysis was used to screen the potential energy sources of detached breast cancer cells.Lactate uptake and utilization of detached breast cancer cells were verified through intracellular and extracellular lactate concentration measurement and ¹³C-lactate metabolic flux analysis.Human stem cell transcription factor PCR microarray was used to screen the significantly upregulated stem cell transcription factor in detached and lactate-treated breast cancer cells.In vitro,the effects of lactate on the maintenance and acquisition of stemness potentials in detached breast cancer cells or its ALDH^-subpopulations were observed through flow cytometry,tumorsphere formation and immunoblotting.In vivo,the effects of lactate on tumor growth and metastasis in mice was observed using in vivo imaging,hematoxylin-eosin staining,immunofluorescence staining,and circulating tumor cells（CTCs）isolation.2.Analyzing the metabolic direction of lactate in ¹³C-lactate metabolic flux assay after its uptake by detached breast cancer cells.The effects of lactate on the tricarboxylic acid cycle（TCA cycle）and OXPHOS metabolism of detached breast cancer cells were studied through seahorse mitochondrial stress testing,ATP production and mitochondrial complex activity testing.Metabolite concentration measurement,immunofluorescence,dot blot and chromatin immunoprecipitation were used to investigate the specific molecular mechanism of how lactate promotes the stemness and metastatic potentials of detached breast cancer cells through TCA cycle.3.²H-lactate metabolic flux technology was used to identify the subcellular localization of lactate uptake and oxidation in detached breast cancer.Proteomics was employed to analyze and screen potential key molecules that may regulate lactate uptake and oxidation in breast cancer cells.Immunofluorescence,co-immunoprecipitation,transmission immunoelectron microscopy,protease K protection,and mitochondrial subfraction isolation assays were been conducted to confirm the existence of a lactate uptake and oxidation complex on the mitochondrial inner membrane of detached breast cancer cells.4.We constructed breast cancer cells with CD147 knocked out or exclusively expressed in mitochondria.Utilizing these cell lines,techniques such as ²H-lactate metabolic flux,flow cytometry,tumorsphere formation,western blotting,hematoxylin-eosin staining,immunofluorescence staining,and CTCs isolation were employed to observe the effects of mitochondrial CD147 on the uptake and oxidation of lactate,as well as lactate-induced stemness and metastatic potentials in detached breast cancer cells.We constructed a eukaryotic expression vector of a single chain variable fragment targeting mitochondrial CD147（mito-CD147 ScFv）and observed the blocking effects of mito-CD147 ScFv on lactate-induced stemness and metastatic potentials in detached breast cancer in vitro and in vivo.【Results】1.Detached breast cancer cells took up and oxidized lactate,using lactate as their main energy source;lactate promoted stemness and metastatic potentials in detached breast cancer cells:Compared to primary tumors,small molecule metabolic pathways were significantly enriched in breast cancer metastases,suggesting that changes in energy metabolism may play an important role in breast cancer metastasis.Detached breast cancer cells showed decreased uptake of glucose,glutamine,and fatty acids,but a significant increase in the uptake of lactate.The key proteins involved in lactate uptake and oxidation,MCT1 and LDHB,were significantly upregulated,and the uptake and oxidation of lactate provided carbon sources for the TCA cycle of detached breast cancer cells.In vitro,lactate increased the proportion of ALDH⁺or CD44^highCD24^-/lowsubpopulation,enhanced the expression of the stemness transcription factor SOX2,and promoted the tumorsphere formation in detached breast cancer cells,indicating that lactate boosted the stemness potentials of these cells.In vivo,lactate elevated the number of CTCs and lung nodules in breast cancer-bearing mice,suggesting that lactate enhanced the metastatic potentials of detached breast cancer cells.In addition,lactate could induce ALDH^-non-CSCs reprograms to CSCs in breast cancer by acquiring stemness potentials.2.Lactate induced stemness potentials in detached breast cancer cells by promoting TCA cycle and OXPHOS:Lactate induced stemness potentials in detached breast cancer cells by promoting TCA cycle and OXPHOS,independent of histone acetylase P300 or the lactate receptor GPR81.In terms of molecular mechanism,lactate promoted stemness potentials in detached breast cancer cells throughα-KG-DNMT3B-mediated SOX2 demethylation.3.The mitochondrial lactate uptake and oxidation complex promoted lactate-induced stemness and metastatic potentials in detached breast cancer cells:Detached breast cancer cells directly took up lactate into the mitochondria,where it was then oxidized to pyruvate.There was a CD147/MCT1/LDHB lactate uptake and oxidation complex on the inner membrane of mitochondria in detached breast cancer cells,and mitochondrial CD147 played a key role in this complex.Mitochondrial CD147/MCT1/LDHB complex can promote lactate uptake and oxidation,thus promoting lactate-induced stemness and metastatic potentials in detached breast cancer.4.Mitochondrial CD147 promoted lactate-induced stemness and metastatic potentials in detached breast cancer;mito-CD147 ScFv inhibited lactate-induced stemness and metastatic potentials in detached breast cancer:Mitochondrial CD147can promote lactate uptake and oxidation,thereby promoting stemness and metastatic potentials of detached breast cancer cells by enhancing OXPHOS andα-KG-DNMT3B-mediated SOX2 demethylation.Mito-CD147 ScFv inhibited lactate uptake and oxidation by disrupting the formation of mitochondrial CD147/MCT1/LDHB complex,thereby reducing lactate-induced stemness and metastatic potentials of detached breast cancer.【Conclusions】Our study firstly proposed that mitochondrial CD147-mediated lactate uptake and oxidation may be one of the important mechanisms for the reprogramming of detached breast cancer cells.Furthermore,we clarified the specific molecular mechanisms of reprogramming in detached breast cancer cells as follows:mitochondrial CD147 promoted lactate uptake and oxidation by forming a mitochondrial lactate uptake and oxidation complex with MCT1/LDHB.This process mediated the OXPHOS metabolic pathway,leading to the reprogramming of detached breast cancer cells.Additionally,we developed a specific ScFv targeting mitochondrial CD147 for the first time.By disrupting the formation of the mitochondrial CD147/MCT1/LDHB lactate uptake and oxidation complex,this ScFv reduced lactate-induced stemness and metastatic potentials in detached breast cancer cells,providing new insights and strategies for the treatment of breast cancer metastasis.