Objective:The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner.Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poo...Objective:The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner.Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer.Nevertheless,the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined.Methods:The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases.The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry.CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells.Cell-counting kit-8,colony formation,cell cycle,and EdU incorporation assays,as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation.Quantitative RT-PCR,western blotting,immunofluorescence staining,and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation.Results:High SHP2 expression is correlated with poor prognosis in patients with breast cancer.SHP2 is required for the proliferation of breast cancer cellsin vitro and tumor growthin vivo through regulation of Cyclin D1 abundance,thereby accelerating cell cycle progression.Notably,SHP2 modulates the ubiquitin–proteasome-dependent degradation of Cyclin D1viathe PI3K/AKT/GSK3βsignaling pathway.SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3β.GSK3βthen mediates phosphorylation of Cyclin D1 at threonine 286,thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin–proteasome system.Conclusions:Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation.SHP2 may therefore potentially serve as a therapeutic target for breast cancer.展开更多
Objective:Pyruvate kinases M(PKM),including the PKM1 and PKM2 isoforms,are critical factors in glucose metabolism.PKM2promotes aerobic glycolysis,a phenomenon known as"the Warburg effect".The purpose of this...Objective:Pyruvate kinases M(PKM),including the PKM1 and PKM2 isoforms,are critical factors in glucose metabolism.PKM2promotes aerobic glycolysis,a phenomenon known as"the Warburg effect".The purpose of this study was to identify the roles of PKM2 in regulating cellular metabolism.Methods:The CRISPR/Cas9 system was used to generate the PKM-knockout cell model to evaluate the role of PKM in cellular metabolism.Lactate levels were measured by the Vitros LAC slide method on an autoanalyzer and glucose levels were measured by the autoanalyzer AU5800.The metabolism of ^(13)C_6-glucose or ^(13)C_5-glutamine was evaluated by liquid chromatography/mass spectrometry analyses.The effects of PKM on tumor growth were detected in vivo in a tumor-bearing mouse model.Results:We found that both PKM1 and PKM2 enabled aerobic glycolysis,but PKM2 converted glucose to lactate much more efficiently than PKM1.As a result,PKM2 reduced glucose levels reserved for intracellular utilization,particularly for the production of citrate,and thus increased theα-ketoglutarate/citrate ratio to promote the generation of glutamine-derived acetylcoenzyme A through the reductive pathway.Furthermore,reductive glutamine metabolism facilitated cell proliferation under hypoxia conditions,which supports in vivo tumor growth.In addition,PKM-deletion induced a reverse Warburg effect in tumorassociated stromal cells.Conclusions:PKM2 plays a critical role in promoting reductive glutamine metabolism and maintaining proton homeostasis.This study is helpful to increase the understanding of the physiological role of PKM2 in cancer cells.展开更多
Glucose is one of major nutrients and its catabolism provides energy and/or building bricks for cell proliferation. Glucose deficiency results in cell death. However, the underlying mechanism still remains elusive. By...Glucose is one of major nutrients and its catabolism provides energy and/or building bricks for cell proliferation. Glucose deficiency results in cell death. However, the underlying mechanism still remains elusive. By using our recently developed method to monitor real-time cellular apoptosis and necrosis, we show that glucose deprivation can directly elicit necrosis, which is promoted by mito- chondrial impairment, depending on mitochondrial adenosine triphosphate （ATP） generation instead of ATP depletion. We demon- strate that glucose metabolism is the major source to produce protons. Glucose deficiency leads to lack of proton provision while mitochondda[ electron transfer chain continues consuming protons to generate energy, which provokes a compensatory lysosomal proton efflux and resultant increased lysosomal pH. This [ysosomal aikaUnization can trigger apoptosis or necrosis depending on the extent of alkalinization. Taken together, our results build up a metabolic connection between glycolysis, mitochondrion, and iysosome, and reveal an essential role of glucose metabolism in maintaining proton homeostasis to support cell survival.展开更多
Background:Tertiary lymphoid structures(TLS)are major components in the immune microenvironment,correlating with a favorable prognosis in colorectal cancer.However,the methods used to define and characterize TLS were ...Background:Tertiary lymphoid structures(TLS)are major components in the immune microenvironment,correlating with a favorable prognosis in colorectal cancer.However,the methods used to define and characterize TLS were not united,hindering its clinical application.This study aims to seek a more stable method to characterize TLS and clarify their prognostic value in larger multicenter cohorts.Methods:A total of 1609 patients from four hospitals and The Cancer Genome Atlas database were analyzed.We quantified the number and maximum length of TLS along the invasive margin of tumor using hematoxylin and eosin-stained whole-slide images(WSIs).Additionally,the length of the invasive margin was determined to calculate the TLs density.The prognostic value of TLS for overall survival was evaluated.In addition,we examined the association between TLS density and immune cell infltration using immunohistochemistry-stained WSIs.The performance for predicting overall survival was measured using hazard ratios(HR)with 95%confidence intervals(CI).Results:Among the three TLS quantification methods,TLS density has the strongest discriminative performance.Survival analysis indicated that higher TLS density correlated with better overall survival[HR for high vs.low 0.57(95%CI 0.42-0.78)in the primary cohort;0.49(0.35-0.69)in the validation cohort;0.35(0.18-0.67)in TCGA cohort].A high TLS density was associated with a high level of CD3+Tcell infiltration.Conclusions:Based on this comparative multicenter analysis,TLs density was identified as a simple,robust,and effective immune prognostic index for colorectal cancer.展开更多
基金This work was supported by grants from the National Natural S&ence Foundation of China(grant Nos.81372844,81472474,81772804 and 81903092)Tianjin Municipal Science and Technology Commission(grant No.16JCYBJC25400)+1 种基金Changjiang Researchers and Innovative Research Team(grant No.IRT_14R40)Postgraduate Innovation Fund of"13th Five-Year Comprehensive Investment,"Tianjin Medical University(grant No.YJSCX201716).
文摘Objective:The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner.Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer.Nevertheless,the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined.Methods:The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases.The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry.CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells.Cell-counting kit-8,colony formation,cell cycle,and EdU incorporation assays,as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation.Quantitative RT-PCR,western blotting,immunofluorescence staining,and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation.Results:High SHP2 expression is correlated with poor prognosis in patients with breast cancer.SHP2 is required for the proliferation of breast cancer cellsin vitro and tumor growthin vivo through regulation of Cyclin D1 abundance,thereby accelerating cell cycle progression.Notably,SHP2 modulates the ubiquitin–proteasome-dependent degradation of Cyclin D1viathe PI3K/AKT/GSK3βsignaling pathway.SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3β.GSK3βthen mediates phosphorylation of Cyclin D1 at threonine 286,thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin–proteasome system.Conclusions:Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation.SHP2 may therefore potentially serve as a therapeutic target for breast cancer.
基金supported by the funds from National Natural Science Foundation of China(Grant No.81672762,81622037 and 81602446)
文摘Objective:Pyruvate kinases M(PKM),including the PKM1 and PKM2 isoforms,are critical factors in glucose metabolism.PKM2promotes aerobic glycolysis,a phenomenon known as"the Warburg effect".The purpose of this study was to identify the roles of PKM2 in regulating cellular metabolism.Methods:The CRISPR/Cas9 system was used to generate the PKM-knockout cell model to evaluate the role of PKM in cellular metabolism.Lactate levels were measured by the Vitros LAC slide method on an autoanalyzer and glucose levels were measured by the autoanalyzer AU5800.The metabolism of ^(13)C_6-glucose or ^(13)C_5-glutamine was evaluated by liquid chromatography/mass spectrometry analyses.The effects of PKM on tumor growth were detected in vivo in a tumor-bearing mouse model.Results:We found that both PKM1 and PKM2 enabled aerobic glycolysis,but PKM2 converted glucose to lactate much more efficiently than PKM1.As a result,PKM2 reduced glucose levels reserved for intracellular utilization,particularly for the production of citrate,and thus increased theα-ketoglutarate/citrate ratio to promote the generation of glutamine-derived acetylcoenzyme A through the reductive pathway.Furthermore,reductive glutamine metabolism facilitated cell proliferation under hypoxia conditions,which supports in vivo tumor growth.In addition,PKM-deletion induced a reverse Warburg effect in tumorassociated stromal cells.Conclusions:PKM2 plays a critical role in promoting reductive glutamine metabolism and maintaining proton homeostasis.This study is helpful to increase the understanding of the physiological role of PKM2 in cancer cells.
文摘Glucose is one of major nutrients and its catabolism provides energy and/or building bricks for cell proliferation. Glucose deficiency results in cell death. However, the underlying mechanism still remains elusive. By using our recently developed method to monitor real-time cellular apoptosis and necrosis, we show that glucose deprivation can directly elicit necrosis, which is promoted by mito- chondrial impairment, depending on mitochondrial adenosine triphosphate （ATP） generation instead of ATP depletion. We demon- strate that glucose metabolism is the major source to produce protons. Glucose deficiency leads to lack of proton provision while mitochondda[ electron transfer chain continues consuming protons to generate energy, which provokes a compensatory lysosomal proton efflux and resultant increased lysosomal pH. This [ysosomal aikaUnization can trigger apoptosis or necrosis depending on the extent of alkalinization. Taken together, our results build up a metabolic connection between glycolysis, mitochondrion, and iysosome, and reveal an essential role of glucose metabolism in maintaining proton homeostasis to support cell survival.
基金supported by the National Science Fund for Distinguished Young Scholars of China(Grant No.81925023)Joint Funds of the National Natural Science Foundation of China(Grant No.U23A20478)the National Science Foundation for Young Scientists of China(Grant No.82202267,82101996).
文摘Background:Tertiary lymphoid structures(TLS)are major components in the immune microenvironment,correlating with a favorable prognosis in colorectal cancer.However,the methods used to define and characterize TLS were not united,hindering its clinical application.This study aims to seek a more stable method to characterize TLS and clarify their prognostic value in larger multicenter cohorts.Methods:A total of 1609 patients from four hospitals and The Cancer Genome Atlas database were analyzed.We quantified the number and maximum length of TLS along the invasive margin of tumor using hematoxylin and eosin-stained whole-slide images(WSIs).Additionally,the length of the invasive margin was determined to calculate the TLs density.The prognostic value of TLS for overall survival was evaluated.In addition,we examined the association between TLS density and immune cell infltration using immunohistochemistry-stained WSIs.The performance for predicting overall survival was measured using hazard ratios(HR)with 95%confidence intervals(CI).Results:Among the three TLS quantification methods,TLS density has the strongest discriminative performance.Survival analysis indicated that higher TLS density correlated with better overall survival[HR for high vs.low 0.57(95%CI 0.42-0.78)in the primary cohort;0.49(0.35-0.69)in the validation cohort;0.35(0.18-0.67)in TCGA cohort].A high TLS density was associated with a high level of CD3+Tcell infiltration.Conclusions:Based on this comparative multicenter analysis,TLs density was identified as a simple,robust,and effective immune prognostic index for colorectal cancer.
