| BackgroundThe morbidity and mortality of colorectal cancer(CRC)have been increasing in recent years,which seriously threatens the health and life of the people.Although a comprehensive CRC treatment system including surgery,chemotherapy,radiotherapy,targeted therapy,and immunotherapy has been formed,the prognosis of CRC is still poor due to its high metastasis and recurrence rate.For progressive and metastatic CRC patients,chemotherapy is used as the primary treatment,among which 5-fluorouracil(5-FU)and its derivatives are the first-line drugs and the most critical components for CRC single-agent or combination chemotherapy.Although 5-FU and its derivatives play an important role in the treatment of CRC,it is common for patients to develop chemoresistance in the process of receiving 5-FU treatment,which is also one of the important reasons for treatment failure in CRC patients.Therefore,it is necessary to search for molecular markers for the prediction and dynamic monitoring of 5-FU chemoresistance in CRC,explore the mechanisms of the occurrence and development of 5-FU chemoresistance in CRC,and find a universally applicable method for reversing 5-FU chemoresistance.These issues are important basic and clinical translation problems in the current CRC research field.5-FU is an antimetabolite chemotherapeutic agents,which directly participates in the nucleotide metabolism of tumor cells,thereby extensively affecting the metabolism of carbohydrates,lipids,amino acids and other substances.The adaptive metabolic shift of tumor cell is known as metabolic reprogramming,which could ensure the supply of energy and provide raw materials for growth and proliferation of tumors cells.Metabolic reprogramming is a key factor in tumor development and chemoresistance.Targeting metabolic reprogramming may be a potential strategy to directly or indirectly increase chemosensitivity.The metabolic reprogramming that occurs in the 5-FU resistant CRC has not been thoroughly studied.Therefore,this study intends to explore the glycolipid metabolic reprogramming events in the 5-FU resistant CRC.We also aimed to reveal its potential molecular mechanism,and find a effective strategy for reversing 5-FU resistance in CRC.Glucose metabolic reprogramming could affect the energy supply mode of tumor cells and determine its malignant biological behaviors.If the glucose metabolic reprogramming events of 5-FU resistant CRC can be identified,it may offer a new insight into the molecular mechanism of 5-FU resistance and find a possible strategy to reverse 5-FU resistance.A correlation has also been reported between enhanced glycolysis and chemoresistance.Based on these,We hypothesized that HIF-1α-mediated glucose metabolic reprogramming may lead to the 5-FU resistance of CRC.Overexpressed CD 147 has been observed in chemoresistant tumors of ovarian cancer,renal cancer,and Kaposi’s sarcoma.Presently,no relevant research has been reported the changes and roles of CD147 in 5-FU resistant CRC.CD147 is treated as a regulator of glycolysis due to it is an important membrane protein involved in lactate transport.Thus,we speculated that CD 147 may affect the 5-FU sensitivity of CRC by regulating glycolysis.Tumor cells are highly dependent on glucose to maintain energy metabolism and produce malignant phenotypes such as proliferation and invasion.Additionally,there is accumulating evidence that the energy supply of tumor cells also depends on remodeled lipid metabolism.Lipid metabolic reprogramming events of tumor cells usually manifest as decreased utilization of exogenous fatty acids,increased fatty acid de novo synthesis pathway,and enhanced fatty acid oxidation(FAO)capacity.However,there is no research to deeply explore the lipid metabolic reprogramming events in 5-FU resistant CRC.Therefore,this study intends to take this as a breakthrough point to explore the lipid metabolic reprogramming events in 5-FU resistant CRC,and look for possible strategy to reverse the 5-FU resistance.Studies have reported that CD 147 is involved in the de novo fatty acid synthesis pathway and FAO of tumor cells.Considering the close relationships between CD 147 and chemoresistance,we hypothesized that CD147-mediated lipid metabolic reprogramming may lead to CRC cells becoming resistant to 5-FU.ObjectivePresent study intends to explore the glycolipid metabolic reprogramming events of 5-FU resistant CRC,and to determine whether HIF-1α and CD 147 are involved in regulating the glycolipid metabolic reprogramming and mediating the acquisition of 5-FU resistance in CRC.To evaluate whether HIF-1α and CD 147 could be used as biomarkers for the prediction and dynamic monitoring of 5-FU resistance in CRC patients,and to clarify whether HIF-1α and CD 147 have potential value as therapeutic targets for 5-FU resistant CRC.In addition,in-depth exploration of the molecular regulatory mechanisms of HIF-1α and CD 147 in 5-FU resistant CRC is needed.MethodsFirst,we attempted to establish research models of 5-FU resistant CRC.We treated CRC cells with a gradually increasing concentration of 5-FU and generated stable acquired 5-FU resistance cell models successfully.To evaluate the 5-FU resistance of cell models,the ability of proliferation,cell cycle,and apoptosis of 5-FU resistant cell models were detected after 5-FU treatment.To carry out in vivo experiments,BALB/c nude mice were subcutaneously injected with cell suspensions to establish cell-derived xenograft(CDX)models,and NOD/SCID mice were subcutaneously implanted with human tumor tissue(derived from a TNM stage Ⅲ,male,rectal cancer patient who received neoadjuvant chemotherapy containing 5-FU before surgery)to construct patient derived xenograft(PDX)models.In addition,tumor specimens and clinicopathological data of 42 CRC patients were collected,and all CRC patients were TNM stage Ⅲ or stage Ⅳ.According to whether patients received and responded to preoperative chemotherapy,they were divided into 3 groups,non-chemotherapy group,response group,and non-response group.We carried out a series of experiments to explore the glucose metabolic reprogramming events of 5-FU resistant CRC.The assessments of the mitochondrial number,morphology and function were made by flow cytometry,transmission electron microscopy,and dynamic monitoring of cellular oxygen consumption rate(OCR).The changes in mitochondrial respiratory chain were detected by western blot(WB)and real-time quantitative polymerase chain reaction(RT-qPCR).By detecting glucose uptake,intracellular lactate content and cellular lactate release of 5-FU resistant CRC cells,as well as the glucose uptake capacity and lactate content of CDX models,the metabolic characteristics of glucose and lactate in 5-FU resistant CRC cells were evaluated.Additionally,to evaluate the fluxes of glycolysis,pentose phosphate pathway and tricarboxylic acid cycle in 5-FU resistant CRC cells,ultra-high pressure liquid chromatography-coupled tandem mass spectrometry(UHPLC-MS/MS),WB,RT-qPCR,extracellular acidification rate(ECAR)dynamic monitoring were performed.Finally,the expressions of glucose metabolism-related enzymes and membrane transporters were detected in CDX models and tumor specimens from CRC patients.To have a clear understanding of the lipid metabolic reprogramming events in 5-FU resistant CRC cells,we carried out the following experiments.The content of triglyceride and total cholesterol in 5-FU resistant CRC cells was detected.The abilities to form lipid droplets and process exogenous fatty acids of 5-FU resistant CRC cells were detected by Oil Red O staining after culturing in high-fat medium.In addition,the ability to FAO of 5-FU resistant CRC cells was measured by FAOBlue probe.Finally,the expressions of FAO-related enzymes were detected in CDX models and tumor specimens from CRC patients.Next,the expressions and distributions of HIF-1α and CD147 in 5-FU resistant CRC cells were detected by WB,RT-qPCR,immunocytofluorescence and other methods.To clarify whether HIF-1α and CD 147 are involved in regulating the glycolipid metabolic reprogramming and mediating the acquisition of 5-FU resistance in CRC cells,we inhibited HIF1A and CD147 gene expressions by small interfering RNA(siRNA)or lentiviral loaded short hairpin RNA(shRNA),and 5-FU chemosensitivity and glycolipid metabolic characteristics were detected in HIF1A or CD147-inhibited 5-FU resistant CRC cells.Tumor specimens,circulating tumor cells(CTC)and clinicopathological data of CRC patients,and CRC patient dataset from the GEO database were detected and analyzed to evaluate whether HIF-1α and CD 147 could be used as biomarkers for the prediction and dynamic monitoring of 5-FU resistance in CRC patients.Furthermore,to determine whether HIF-1α and CD147 have potential value as therapeutic targets for 5-FU resistant CRC patients,tumor proliferation and 5-FU chemosensitivity were assessed in HIF-1α or CD147-inhibited CDX models and PDX models.Finally,in-depth exploration of the molecular regulatory mechanisms of HIF-1α and CD 147 in 5-FU resistant CRC was performed.To verify whether reactive oxygen species(ROS)could regulate HIF-1α expression through the PI3K/Akt signaling pathway,tert-butylhydroperoxide(t-BHP)and N-acetylcysteine was used to establish ROS overload and scavenging cell models,respectively.In ROS-changed cell models,the activity of PI3K/Akt signaling pathway,HIF-1α expression,5-FU chemosensitivity and glucose metabolic phenotypes were evaluated by WB and RT-qPCR.In order to clarify whether the Wnt/β-Catenin signaling pathway is involved in the regulation of HIF-1α expression in 5-FU resistant CRC,β-Catenin inhibitor or siRNA was used to inhibit CTNNB1 gene expression,and WB,RT-qPCR,immunofluorescence,protein half-life detection,co-immunoprecipitation(co-IP)and other methods were carried out to prove there is an interaction between β-Catenin and HIF-la.To verify whether CD147 could upregulate HIF-1α and enhance glycolysis by activated PI3K/Akt/mTOR signaling pathway,shRNA-targeted CD147 gene combined agonists or inhibitors of mTOR were added to 5-FU resistant CRC cells,and HIF-1αexpression and glycolytic activity were detected.In order to investigate whether CD 147 could downregulate the expression of PPARα and inhibit the FAO of 5-FU resistant CRC through the activated MAPK signaling pathway,shRNA-targeted CD147 gene combined agonists or inhibitors of ERK MAPK were added to 5-FU resistant CRC cells,and PPARa expression and FAO were performed.ResultsThe resistance index(RI)of 5-FU resistant CRC cells was detected by CCK-8 cytotoxicity test,and the RI was more than 10.The ability of proliferation,cell cycle,and apoptosis of 5-FU resistant CRC cells after 5-FU treatment have not changed noticeably.These results indicated that 5-FU resistant CRC cell models were successfully established.The mitochondria of 5-FU resistant CRC cells were detected,and it was found that the mitochondrial content was decreased,the shape was damaged,the OCR was significantly decreased,and the protein expression of mitochondrial respiratory chain components was decreased,indicating that the mitochondrial structure and function of 5-FU resistant CRC cells were damaged.The utilization of glucose and lactate in 5-FU resistant CRC cells was evaluated,and it was found that glucose uptake,glucose dependence and lactate release were all increased.Consistently,both enhanced glucose uptake and increased lactate content of the tumor were observed in CDX models.By detecting the contents of intermediates and the expressions of metabolic enzymes in central carbon metabolism(tricarboxylic acid cycle,glycolysis and pentose phosphate pathway),it was found that the weakened TCA cycle,and the enhancement of glycolysis and pentose phosphate pathway in 5-FU resistant CRC cells.The lipid metabolism characteristics of 5-FU resistant CRC cells were evaluated,and increased triglyceride and total cholesterol content,enhanced exogenous fatty acid uptake,increased lipid droplet formation,weakened exogenous fatty acid consumption,and decreased FAO were observed in 5-FU resistant CRC cells.It was observed that HIF-1α had increased expression at both mRNA and protein levels,increased protein stability,enhanced nuclear localization,and HIF-1α was more sensitive to hypoxia.It also found the increased expression of CD 147 at both mRNA and protein levels,and membrane expression of CD147 was increased.5-FU resistance and glycolipid metabolic reprogramming events in 5-FU resistant CRC cells were partially reversed after inhibiting HIF1A or CD147 gene expression by shRNA.These observations suggested that HIF-la and CD 147 are involved in the regulation of 5-FU chemosensitivity and glycolipid metabolic reprogramming in 5-FU resistant CRC cells.By analyzing tissue and blood samples of CRC patients,and clinicopathological data,we found that 5-FU resistance was associated with increased expression of HIF-1α or CD147 in tumor cells,especially the HIF-1α-positive CTCs were detected.In addition,we found significant correlations between HIF-1α or CD147 expression and disease-free survival in patients previously treated with 5-fluorouracil analog,while the analysis of GEO database revealed that the expression of HIF-1α in primary tumors before chemotherapy don’t have a predictive value for chemotherapy efficacy.Finally,it was found that inhibiting gene expression by shRNA or injecting small molecule inhibitors to inhibit the expression of HIF1A or CD 147 could inhibit tumor proliferation and partially restore the 5-FU chemosensitivity in CDX models and PDX models.These evidence supported that HIF-1α and CD147 are targets for possible therapeutic intervention in 5-FU resistant CRC patients.Finally,the molecular regulation mechanisms of the expression of HIF-la and CD147 in 5-FU resistant CRC cells were studied.Overloaded ROS upregulated HIF-la by activating the PI3K/Akt signaling pathway,resulting in increased glycolysis and 5-FU resistance;In addition,β-Catenin bound to HIF-1α and formed the β-Catenin/HIF-1α transcriptional complex,which promoted HIF-1α migration into the nucleus and increased HIF-1α stability.CD147 enhanced HIF-1α-induced glycolysis by activating the PI3K/AKT/mTOR signaling pathway and suppressed PPARα-mediated FAO by activating the MAPK signaling pathway.ConclusionProlonged exposure of CRC cells to 5-FU resulted in the irreversible damage to mitochondrial structure and function,and dysfunction of mitochondria is one of the most important driving factors of glucose metabolic reprogramming in 5-FU resistant CRC.The main consequence of the destruction of mitochondria is impaired aerobic oxidation in 5-FU resistant CRC cells.But with an decrease of energy supply,glycolysis and pentose phosphate pathway are in a state of compensatory activation.Metabolic reprogramming provides sufficient energy supply with additional biosynthetic raw materials to CRC cells,and endows CRC cells with a new phenotype,5-FU resistance.According to the metabolic reprogramming characteristics of 5-FU resistant CRC cells,HIF-la was selected as the candidate target for monitoring,assessing and therapy in 5-FU resistant CRC patients.HIF-la may serve as a potential biomarker for assessment and prognosis in CRC patients with 5-fluorouracil analog treatment,and both gene knockdown and pharmacological inhibition of HIF-1α could improve 5-FU resistance in vivo.Furthermore,our results suggested that ROS-induced HIF-1α high expression in 5-FU-R CRC cells is mediated via PI3K/Akt pathway activation,and β-catenin plays important roles in stabilization and nuclear translocation of HIF-1α in 5-FU-R CRC cells.We identified oxygen-independent regulations of HIF-1α in 5-FU resistant CRC cells,which may lead to identifying more therapeutic targets.We demonstrated CD147 is involved in the glycolipid metabolic reprogramming in 5-FU resistant CRC cells.CD147 enhanced HIF-1α-induced glycolysis by activating the PI3K/AKT/mTOR signaling pathway and suppressed PPARα-mediated FAO by activating the MAPK signaling pathway.In addition,our findings showed that overexpressed CD 147 confers 5-FU resistance to CRC by reprogramming glycolipid metabolism.We demonstrated that CD147 is a potential 5-FU resistance biomarker for CRC patients and a valuable therapeutic target for 5-FU-resistant CRC. |
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