The Role And Molecular Mechanism Of Glycolysis In Inflammasome Activation-induced Progression Of Colorectal Cancer

Colorectal cancer(CRC)is the 3rd most commonly diagnosed cancer in the world.Although early diagnosis of CRC has markedly improved CRC patients survival in recent years,the 5-year survival rate is still very low in advanced CRC patients with distant metastasis.Therefore,clarifying the mechanism of CRC metastasis and finding potential molecular targets are essential to improve treatment and prognosis for CRC patients.Traditionally,inflammation is a protective response to remove internal and extermal harmful stimuli and restore tissue homeostasis.However,many studies indicated that carcinogenesis and tumor progression could be promoted by inflammation.Nowadays,it is widely accepted that inflammation is highly associated with CRC.Inflammasomes have a critical role in inflammation.The inflammasome,which consists of the PRRs(pattern recognition receptors),the ASC(apoptosis-associated speck-like protein containing a CARD)adaptor and an inactive zymogenic pro-caspase-1(cysteinyl aspartate specific proteinase-1,Caspasel),is an important cytosolic multiprotein complex that functions during inflammatory responses.The molecular targets of PRRs usually include PAMPs(pathogen associated molecular patterms)and DAMPs(danger associated molecular patterns),which are from microbial components and damaged host cells,respectively.Formation of inflammasomes in response to microbes or danger signals leads to cleavage of pro-caspase-1 into active caspase-1 enzyme,which further cleaves proinflammatory cytokines,IL-1? and IL-18,into their active forms,and these cytokines play key roles in inflammation.Although inflammasome activation in immune cells affects carcinogenesis and cancer progression,the inflammasome in tumor cells is poorly understood.Therefore,our research were dedicated to explore the functions of inflmmasome in tumor cells.LPS(lipopolysaccharide),endotoxin derived from the cell wall of Gram-negative bacteria,is one of the most common ligands for PRRs.Generally,LPS have been recognized as a potent inducer of inflammasome activation in immune cells such as macrophage and DC.However,the effect of LPS induced inflammasome activation in tumor cells is still not well-understood.In order to study the degree of inflammasome activation in CRC cells,we detected the protein expression of inflammasome activation markers P20 and IL-1?-P17 in 8 CRC cell lines.The results showed that inflammasome acitvation is lower in RKO cells than in other cell lines.RKO cell lines were selected as a cell model.Different concentrations(0,1,2,4,8?g/ml)of LPS were used to detect the activation of inflammasome at different time(0,4,8,12,24,48h).Among the concentrations and times we used,1?g/ml LPS treatment 24hrs had the most potent effect on inflammasome activation,so we used the above treatment condition in the subsequent experiments.Moreover,eompared to untreated group,LPS treatment potentiated RKO cells migration and invasive capacity,and similar results were observed in DLD1 cells.To determine whether LPS-mduced cell movements are regulated by the inflammasome-dependent pathways,CRC cells were incubated with specific inhibitor for caspasel(Ac-YVAD-CHO)before stimulation with LPS.We found that Ac-YVAD-CHO treatment could inhibit LPS-mduced inflammasome activation and cell mobility.These results revealed that LPS promotes CRC cell movements through inducing inflammasome activation.NF-?B 1s a nuclear transcription factor with multiple members,which can be involved in a variety of biological processes,including cell proliferation and differentiation,immunity and inflammatory response.The NF-?B protein family consists of five members:p50(NF-?B1),p52(NF-?B2),P65(RelA),c-Rel(Rel)and RelB,of which p65(RelA)is the most widely studied member of the NF-?B family(p65NF-?B).Growing evidence indicates that NF-?B has been implicated in the tumor progression proccsse.In addition,NF-?B plays a central role in tumor metastasis by affecting inflammasome activation in immune cells.Thus,we next examined the function of NF-?B in LPS-induced CRC inflammasome activation.The results showed that knockdown of p65NF-?B not only reduced P20 and IL-1?-P17 expression,but also impaired LPS-mduced upregulation of caspasel enzyme activity in CRC cells,indicating that LPS treatment induced inflammsone activation in a p65NF-?B-dependent manner in CRC cells.We then asked whether LPS promote CRC cells invasion and migration via p65NF-?B.As expected,the results showed that LPS-induced invasion and migration was significantly inhibited by cells transfected with p65NF-?B-siRNA,suggesting that LPS enhanced CRC cell movements through p65NF-?B.Recently,a positive correlation between EMT transcription factors and NF-?B activation has been described in several human cancers.Increasing evidence suggests that transcription factor NF-?B can regulate the expression of Snail,and then promote the progression of tumor.Therefore,we then asked whether LPS affect the expression of Snail and what role does p65NF-?B play in this process?We found that LPS could no longer upregulate Snail expression after knockdown of p65NF-?B.These data strongly suggested that LPS upregulates Snail expression through p65NF-?B.To further uncover the mechanism involved in LPS-mediated enhancement of invasion and migration of CRC,we focused on the expression of Snail,which is thought to be an oncogenic transcription factors of tumor progression.However,after knockdown of Snail,LPS treatment no longer affected tumor movements,indicating that Snail abundance was essential to LPS induced migration and invasion.To further examine the effect of Snail on the motility of CRC cells,we transfected Snail-knockdown vector or control vector into RKO and DLDI cells respectively.We found that loss of Snail significantly decreased invasion and migration of LPS-treated CRC cells.The results showed that the expression of Snail serves as a critical factor to LPS-induced cell mobility.It has been reported that tumor cells have different metabolic patterns from normal cells.This metabolic pattern is called "metabolic reprogramming".In 1930,German biochemist Otto Warburg found that cancer cells glycolysis level is higher than normal cells(Warburg Effect).Emerging studies indicate that the upregulation of glycolysis is an important feature of malignant phenotype of invasive cancer.We then asked whether LPS promote CRC cell mobility via upregulation of glycolysis.Firstly,in order to test whether glucose abundance is required for CRC cells invasion and migration,we compared cell motility in a glucose-starved and glucose-exsited condition under LPS treated or untreated condition.The results implying that LPS could promote CRC cells mobility only in a glucose-nourished environment,but in a glucose-starved condition,LPS do not exert its functions.We next sought to identify whether LPS have effect on CRC cells glucose metabolism.We performed glucose and lactate concentration assays to determine the effect of LPS on glycolysis.Examining metabolic change induced by LPS treatment,we found that LPS could promote glucose consumption and lactate production.Therefore,our observations suggest a possibility that metabolic reprogramming towards glycolysis could promote CRC cells invasion and migration under LPS treatment.Recent studies have shown that Snail is critically involved in glucose metabolism.While LPS is critical for glycolysis as well as for cancer progression,the underlying mechanism of Snail in metabolic reprogramming under LPS stimulationm is not well-understood.Therefore,we investigated the role of Snail in LPS-induced glucose metabolism change.The results showed that LPS upregulated glucose consumption and lactate producrtion in CRC cells,whereas knockdown of Snail significantly decreased the upregulation of glycolysis level induced by LPS.On the basis of the strong effect of LPS on glycolysis,we hypothesized that LPS might upregulate glycolysis through upregulation of glycolysis rate-limiting enzyme.There are three key rate-limiting enzymes in glycolysis including hexokinase(Hexokinase,HK),6-phosphofructokinase 1(PFK-1)and pyruvate kinase(pyruvate kinase.HK includes four types including HK1,HK2,HK3 and HK4.HK4 only catalyze glucose and mainly expressed in liver,thus,we did not detect HK4 protein expression.Studies have shown that PFKP(an isoform of PFK-1)and PKM2(an isoform of PK)mainly expressed in cancer.Therefore,we detected the effects of LPS treatment on the expression of HK1,HK2,HK3,PFKP and PKM2 in colorectal cancer cells.Western Blot analysis showed that LPS markedly increased HK3 expression,but had no significant effect on protein expressions of HK1,HK2,PFKP,and PKM2.p65NF-kB and Snail are important transcription factors in cells,which can play role in transcriptional regulation of a variety of genes.The results of Real-Time PCR in colorectal cancer tissue samples showed that there was a significant positive correlation between p65NF-kB and HK3(r? 0.237,P? 0.035),Snail and HK3(r? 0.327,P?0.003)at the expression level of mRNA.We next investigated whether LPS upregulated HK3 expression through transcription factor p65NF-?B or Snail.Western Blot assay showed that LPS failed to upregulate HK3 expression in cells depleted of either p65NF-?B or Snail,indicating that both proteins are required for LPS-upregulated HK3 expression.Since p65NF-?B and Snail were demonstrated to upregulate the HK3 protein expression in CRC cells under LPS stimulation,we flurther determine if transcriptional factor p65NF-?B and Snail regulate HK3 promoter activity.We directly transfected luciferase reporter plasmid containing HK3 promoter region to CRC cells and exalined the activity of HK3 promoter.The results showed that LPS failed to upregulate HK3 promoter activity in cells depleted of either p65NF-?B or Snail,indicating that these two proteins are required to LPS-activated HK3 promoter.Based on the above experimental results,we speculate that there might be protein interaction between p65NF-kB and Snail to jointly regulate the transcription of HK3,which is subsequently confirmed by co-immunoprecipitation(co-IP)and cellular immunofluorescence(IF)techniques.The results of co-IP showed that LPS could promote the formation of p65NF-?B/Snail protein complex.Furthermore,the location of LPS-induced NF-?B/snail protein complex was confirmed by IF assay,which indicated that LPS could promote the formation of p65NF-kB/Snail protein complex in the nucleus,to jointly regulate the transcription of HK3.Together,we draw the following conclusions:1.LPS could promote the activation of inflammasome in colorectal cancer cells and promote cell migration and invasion.2.LPS could promote glucose consumption and lactate production in colorectal cancer cells.3.LPS upregulates the glycolysis level of colorectal cancer cells through NF-?B/Snail-HK3 signal pathway,and then promotes cell migration and invasion.