LDH-A Promotes Malignant Progression Via Activation Of Epithelial-to-mesenchymal Transition And Conferring Stemness In Muscle-invasive Bladder Cancer

Bladder cancer is the seventh most common cancer worldwide,and the first leading cause of death among genitourinary malignancies in China.Muscle-invasive bladder cancer(MIBC)is amongst the most aggressive and deadliest cancers necessitating more radical and aggressive therapies.However,MIBC progresses rapidly to become metastatic and generates the bulk of patient mortality.Cancer stem cells(CSCs),as the driver of tumor growth,invasion,metastasis,therapy resistance and recurrence,constitute a sub-population of malignant cells within a tumor mass and posses the ability to self-renew giving rise to heterogeneous tumor cell populations.A second process and gene program critical for cancer progression is epithelial-to-mesenchymal transition(EMT),i.e.,concomitant loss of epithelial marker E-cadherin and gain of mesenchymal markers including N-cadherin,Fironectin,and Vimentin.EMT-inducing transcription factors,such as Snai1/2,Zeb1/2,or Twist1/2,have been found to activate EMT programs,resulting in enhanced migratory and invasive potentials of epithelial tumors.Furthermore,induction of EMT potentiates self-renewal and the acquisition of CSC properties.Considering the vital role of EMT and CSC in tumor aggressiveness,it is deserving of more in-depth investigation to explore genetic and epigenetic alterations involved in regulation of EMT and CSC in order to attack their specific vulnerabilities and deliver durably effective and precise cancer treatments.Cancer-associated EMT is a complicated and comprehensive reprogramming involved in metabolism and epigenetics,through which differentiated epithelial cancer cells reverse to an undifferentiated state,not only expressing stem cell markers,but also acquiring stem cell-like functions.Epigenetic regulatory mechanisms,especially histone deacetylases(HDACs)are involved in regulating both EMT and CSC.Reprogramming of energy metabolism is the seventh hallmarker of cancer.The best characterized metabolic phenotype observed in tumor cells is the Warburg effect,which is a shift of ATP generation from oxidative phosphorylation to glycolysis,where glucose is converted to lactate at high rates even in oxygen-rich conditions.LDH-A,as the enzyme responsible for transforming pyruvate into lactate,plays an important role in the Warburg effect.Recent evidence has confirmed that epigenetic modifications require the substrates or cofactors that are intermediate metabolites of cells and capable of diffusing through nuclear pores,such as acetyl-Co A,NAD+,SAM,á-KG,and FAD,providing a direct link between metabolic output and epigenetics[15].Cancer cells effectively maintain low levels of pyruvate to prevent inhibition of HDAC1/HDAC3 and thereby to evade cell death.Thus,it is tempting to deduce that LDH-A,responsible for transforming pyruvate into lactate,can regulate EMT and CSC via activation of HDACs.In this study,we first examined the expression of LDH-A in MIBC specimens and correlated its expression with clinicopathological parameters and overall survival(OS)and elucidated the close relationship between LDH-A expression and CSC/EMT markers,and therefore provides a potential therapeutic target for MIBC.Part Ⅰ The relationship of LDH-A expression with ITGC prognosisObjectives: To investigate the expression of LDH-A in MIBC cell lines and tissues,the relationship of LDH-A expression with clinicopathological parameters,Oct4,E-cadherin,overall survival,disease-specific survival.Methods: Westernblot was applied to detect LDH-A expression in the human bladder cancer SW780,BIU87,5637,T24,UMUC-3 cell lines and the human immortalized bladder epithelial cell line SV-HUC-1.A statistical analysis of 136 MIBC specimens and the corresponding non-cancerous tissues was performed on the basis of immunohistochemistry results.All patients had follow-up information for 5 years.The follow-up deadline was March 2015.Survival analysis of overall survival and disease-specific survival was assessed.Results:1.Westernblot analysis showed that the invasive cell lines T24 and UMUC-3 exhibited high levels of LDH-A expression compared to SV-HUC-1 cells,as was high-risk superficial cell line 5637;A comparative analysis of immunohistochemistry(IHC)results of 136 MIBC specimens indicated that LDH-A was differentially upregulated in MIBC tissues compared to adjacent non-cancerous tissues(P<0.001).2.The increased LDH-A expression was significantly related to invasion depth(P=0.001),regional lymph node metastases(P =0.009),.positive Oct4(P=0.016)and negative E-cadherin(P=0.031).We did not find any significant correlations between LDH-A expression and other clinicopathologic variables,including patient age,sex,tumor size and differentiation3.Univariate analysis revealed that patients harbouring tumors with high LDH-A expression showed significantly worse overall survival and disease-specific survival(P<0.001,Log-rank test).4.The multivariate Cox regression model indicated that high expression of LDH-A is an independent prognostic marker for overall survival(P=0.003)and disease-specific survival(P=0.007).Conclusions: Upregulation of LDH-A is frequently detected in MIBC.MIBC patients harbouring tumors with high LDH-A expression showed significantly worse overall survival and disease-specific survival.The increased LDH-A expression was significantly related to positive Oct4 and negative E-cadherin,suggesting LDH-A promoted poorer prognosis by activating EMT and upregulating stemness gene Oct4.Knockout and knockin experiment was performed to testify the deduction.Part Ⅱ LDH-A promotes malignant progression via activation of epithelial-to-mesenchymal transition and conferring stemness in muscle-invasive bladder cancerObjectives: Three si RNAs targeting LDH-A messenger RNA(m RNA)and a scrambled si RNA used as a negative control(NC)were designed,and the si RNA with the best inhibition of LDH-A expression in UMUC-3 was selected for subsequent experiments according to the real-time RT-PCR and western blot results.Plasmid transfection was performed to knock in LDH-A expression in UMUC-3.We detected the m RNA expression of stemness genes including Yamanaka factors and EMT-related genes after LDH-A knock in and knock down;the corresponding protein expression of genes presenting remarkable change at m RNA lever was confirmed further by westernblot.Methods: 1.The real-time RT-PCR and westernblot were applied to detect LDH-A expression after plasmid transfection and select the si RNA with the best inhibition of LDH-A expression in UMUC-3 from three si RNAs.2.The m RNA expression of stemness genes including Yamanaka factors and EMT-related genes was detected by real-time RT-PCR 72 hours after transiently infected by the selected si RNA and plasmid.3.Westernblot was applied to detect the protein expression of genes presenting remarkable change at m RNA level.3.Cell proliferation was carried out by CCK-8 cell proliferation assay,cell apoptosis was detected by flow cytometry,and migration assay and matrigel invasion assay were performed to detected the migratory and invasive ability,respectively.Results: 1.According to the real-time RT-PCR and westernblot results,plasmid transfection significantly upregulated LDH-A expression and the si RNA3(knock down,KD)showed the best inhibition of LDH-A expression in UMUC-3 cells 2.Real-time RT-PCR analysis revealed that 72 h after LDH-Asi RNA infection in UMUC-3 cells,the expression of stemness genes(Oct4,Sox2,Naong,c-myc and Bami1)and mesenchymal markers(Snail,N-cadherin,Fibronectin and Vimentin)as well as HDAC2 was remarkably reduced(P<0.001),while the expression of epithelial marker E-cadherin was significantly upregulated(P<0.001),and HDAC1 remained unchanged(P>0.05).The downregulation of Snail,Fibronectin and Oct4,and upregulation of E-cadherin at the protein level was confirmed by westernblot.3.Real-time RT-PCR analysis revealed that 72 h after LDH-A overexpression in UMUC-3 cells,the expression of stemness genes(Oct4,Sox2,Naong,c-myc and Bami1)and mesenchymal markers(Snail,N-cadherin,Fibronectin and Vimentin)as well as HDAC1(P<0.001)and HDAC2(P<0.01)was remarkably reduced,while the expression of epithelial marker E-cadherin was significantly downregulated(P<0.001).The upregulation of Snail,Fibronectin and Oct4,and downregulation of E-cadherin at the protein level was confirmed by westernblot.4.LDH-A knockdown significantly decreased cell proliferation,migratory and invasive ability,while increased apoptosis ratio;LDH-A knockin significantly increased cellproliferation,migratory and invasive ability,while decreased apoptosis ratio.Conclusions: LDH-A promotes malignant progression via activation of EMT/CSC,and therefore offers a promising therapeutic strategy for IMBC.