Research On TG2 As A Prognostic Marker And Its Role And Mechanism In Promoting Cancer Cell Proliferation In Breast Cancer

Background and Objective:Breast cancer(BC)occupies the first rank in the morbidity of Chinese female malignancies.In China,BC possesses the characteristics of young onset,easy recurrence and high risk of metastasis,which seriously threatens the health of women.Although in recent 20 years,with the development of BC molecular type,surgery,chemoradiotherapy,targeted and endocrine therapy,immunotherapy and other technologies,the overall survival among patients with BC has already gained great improvement.However,the mechanism of BC occurrence and development remains unclear.And new biomarkers need to be developed urgently in clinical work to meet the needs of diagnosis,evaluation of efficacy and prognosis.The main signaling pathways involved in the promotion of BC are PI3K/AKT/mTOR and RAS/RAF/MEK,which are involved in the regulation of cell proliferation,invasion,and other biological functions.In addition,the mitogenactivated protein kinase(MEK)and extracellular signal-related protein kinase(E RK)pathway can regulate lactate dehydrogenase(LDH)and promote glycolysis to reprogram cancer cell metabolism.Tissue transglutaminase(TG)can bind to and hydrolyze guanosine triphosphate(GTP),and catalyze the enzymatic transamidation reaction that crosslinks primary amines with glutamine residues.Among TGs,TG2 is the most widely distributed and extensively studied.Research has found that TG2 expression is increased in various cancers and is associated with drug resistance,distant metastasis,and poor prognosis.However,there are few reports on the correlation between TG2 and glycolysis in BC.Therefore,we aim to investigate the mechanism by which TG2 may influence BC proliferation through the regulation of glycolysis.Methods:(1)Bioinformatics analysis was conducted to investigate the expression profile of TG2 in BC and its potential involvement in biological functions and signaling pathways.Initially,the expression of TG2 in various types of BC cell lines was analyzed using the CCLE website.Subsequently,the expression of TG2 in BC and normal tissues,as well as its expression in the context of positive or negative lymph node metastasis,was examined through the TIMER and bcgenex websites.Furthermore,the relationship between TG2 expression and overall survival in BC patients,as well as the association between TG2 expression levels and overall survival in patients with different molecular subtypes of BC,was analyzed.Additionally,potential biological processes and related genes involved with TG2 were explored using the online bioinformatics website LinkedOmics.Finally,gene set enrichment analysis(GSEA)was performed to identify potential signaling pathways in which TG2 might participate.(2)We enrolled the BC tissue samples of 210 female patients with BC who had received modified radical mastectomy or breast-conserving surgery from January 2012 to December 2015 at a tertiary hospital in Jilin Province.Furthermore,30 para-tumor tissues were enrolled as controls.The study was approved by the Ethics Committee of the hospital above and conducted with written informed consent from all patients.To identify the level of TG2 in BC tissues,immunohistochemical(IHC)staining was performed.TG2 mRNA expression in BC samples,paired samples and BC cell lines were detected by RT-qPCR.Chi-square test,Kaplan-Meier and Cox regression were used to explore the relationships between TG2 expression,clinicopathologic parameters and overall survival.(3)RT-qPCR was used to detect TG2 mRNA expression levels in human mammary epithelial line MCF-10A,human BC cell lines SK-BR-3,BT-474,T47D,ZR-75 and MCF-7,and idenify cell lines with high-or low expression of TG2.(4)Lenti-virus was used to overexpress TG2(TG2-OE)in BT-474 cells.The CCK8 assay was employed to assess the impact of TG2 overexpression on cell proliferation.Additionally,Transwell assays were performed to evaluate the effects on migration and invasion.Biochemical kits were utilized to measure the levels of glucose and lactic acid in the culture supernatants.Western Blot was conducted to detect the protein expression levels of TG2,p-MEK,MEK,p-ERK,ERK,N-cadherin,Snail,Slug,Vimentin,HKⅡ,PFKP,PKM2,LDHA,and LDHB.RT-qPCR was used to assess the mRNA expression levels of LDHA and LDHB.Furthermore,we treated the cells with the MEK inhibitor U0126(50 μM)and again employed the CCK-8 assay to evaluate its impact on cell proliferation.Glucose and lactate levels in the culture supernatants were also reassessed.Western Blot was repeated to detect changes in the protein expression levels of TG2,pMEK,MEK,p-ERK,ERK,LDHA,and LDHB.Additionally,RT-qPCR was performed to reassess the mRNA expression levels of LDHA and LDHB.(5)Lentivirus was utilized to knockdown(KD)TG2 expression in SK-BR-3 cells.CCK-8 assays were performed to assess the impact of TG2 knockdown on cell proliferation.Additionally,Transwell assays were conducted to evaluate the effects on cell migration and invasion.Biochemical kits were used to measure the levels of glucose and lactate in the culture supernatant.Western blot was conducted to detect the protein expression levels of TG2,p-MEK,MEK,p-ERK,ERK,N-cadherin,Snail,Vimentin,Slug,HK Ⅱ,PFKP,PKM2,LDHA,and LDHB.RT-qPCR was employed to measure the mRNA expression levels of LDHA and LDHB.Furthermore,the MEK inhibitor U0126(50 μM)was added to the system,and CCK-8 assays were again performed to investigate its impact on cell proliferation.Finally,the levels of glucose and lactate in the culture supernatant were re-assessed.(6)To investigate the influence of TG2 on proliferation in BC and glycolysisrelated proteins in vivo,six female Balb/c nude mice aged 4-5 weeks were randomly assigned to two groups.SK-BR-3 cells(scramble control,SC or knockdown,KD)were injected into the fourth mammary fat pads of the mice to develop tumor-bearing nude mice model.Calipers was employed to measure tumor size every 3 days for 30 consecutive days.At the end of the experimental period,after euthanasia,the mice xenograft tumors resection and tumor weight evaluating were conducted.Protein levels of TG2,p-MEK,MEK,p-ERK,ERK,LDHA and LDHB of cancer tissue were detected by Western Blot.All procedures of animal experimentation were approved by the Laboratory Animal Ethics Committee.Results:(1)Bioinformatics analyses were conducted using public databases.The results revealed that TG2 expression was relatively low in BT-474 cells obtained from primary tumor sites but significantly upregulated in SK-BR-3 cells derived from metastatic foci.Analysis of tissue samples from BC patients indicated that TG2 expression was elevated in ER-negative,PR-negative,and HER-2-positive subgroups,with higher expression levels observed in patients with positive lymph node involvement compared to those with negative lymph nodes.Subgroup analysis across various molecular subtypes revealed a shortened overall survival in patients with high TG2 expression within the Luminal B and HER-2-positive subtypes.KEGG analysis suggested that TG2 might positively correlate with biological functions such as DNA replication and glycolysis while negatively correlating with extracellular protein transport.Further analysis of TG2-related genes revealed significant positive correlations with genes like LDHB and negative correlations with genes like MLPH.Gene Set Enrichment Analysis(GSEA)identified potential signaling pathways regulated by TG2,including epithelialmesenchymal transition,K-RAS,and hypoxia-related pathways.(2)In this study,we collected 30 adjacent non-cancerous samples and 210 BC samples.IHC staining revealed negative or weak expression of TG2 in the adjacent non-cancerous samples,whereas positive staining was observed in 183 out of 210(87.14%)BC samples.Additionally,RT-qPCR confirmed higher levels of TG2 mRNA expression in 30 BC samples compared to their paired adjacent non-cancerous samples.To analyze the relationship between TG2 expression and clinicopathological parameters in BC samples,patients were categorized into low(negative to weak staining)and high(moderate to strong staining)expression groups based on staining intensity.Kaplan-Meier analysis demonstrated significantly poorer overall survival(OS)in patients with high TG2 expression.Cox regression analysis further indicated that high TG2 expression was an independent risk factor for OS in BC patients.(3)Compared with normal breast epithelium MCF-10A cells,the TG2 mRNA level was lower in BT-474(P<0.01),while higher in T47D,ZR-75,MCF-7,and SKBR-3 cells(P<0.01).TG2 mRNA level in SK-BR-3 was the highest among all cell lines above.(4)The influence of TG2-OE on the cell proliferation of BC cells.① Lenti-virus was used to increases TG2 expression in BT-474 cells.After overexpression,TG2 mRNA levels in BT-474 cells(OE)increased 2.13 times(P<0.01),which indicated TG2-OE cell line was successfully generated.Compared with the control group(EV),TG2-OE cell proliferation by CCK-8 was increased(OD 450nm at 72 h,EV vs OE:1.67±0.12 vs 2.34±0.13,P<0.05).②In order to further evaluate the influence of TG2 on epithelial-mesenchymal transition(EMT)in BC,the EMT-related proteins in BT-474 cells with TG2-OE were detected.The results showed that TG2 overexpression increased the ability of migration and invasion of BT-474 cells,meanwhile,resulted in higher expression of mesenchymal related proteins(N-cadherin,Snail,Vimentin and Slug)(P<0.05),while E-cadherin,a epithelial related protein,was decreased(P<0.05).③To further investigate the influence of TG2 on the glycolysis of BC cells,the glucose consumption and lactate product in BT-74(OE)cell line were detected.TG2OE resulted in increased glucose consumption and lactate production(P<0.05);TG2OE increased the protein expression of HKII,PFKP and PKM2(P<0.01).④ Compared with EV group,higher phosphorylation of MEK(P<0.05)and ERK(P<0.05)were observed in TG2-OE cells.Increased protein(P<0.05)and mRNA levels of LDHA and LDHB(P<0.01)were also observed in OE group.These findings demonstrated that TG2 can regulate glycolysis of BC cells through the MEK/ERK/LDH axis.⑤ To further confirm the mechanism of MEK/ERK/LDH pathway in TG2 promoting BC cell proliferation,the MEK inhibitor U0126 was used to perform rescue experiments.The results revealed that U0126 significantly inhibited the proliferation of TG2-OE cells(OD 450nm of TG2-OE at 72 h,DMSO vs U0126:2.74±0.22 vs 1.93±0.16,P<0.01);and reduced glucose consumption and lactate production(P<0.01).Furthermore,the activation of p-MEK,p-ERK,LDHA and LDHB by TG2-OE was rescued by U0126.The mRNA levels of LDHA(64%decreased,P<0.01)and LDHB(68%decreased,P<0.01)was detected by RT-qPCR.(5)The influence of TG2-KD on the proliferation of BC cells.① Lentivirus was used to knockdown TG2(Knockdown,KD)in SK-BR-3 cells.TG2-KD cell line was successfully generated in SK-BR-3(mRNA level decreased by 73%in TG2-KD,P<0.01).Compared with the control group(SC),proliferation of TG2-KD cell line by CCK-8 was inhibited(OD 450nm at 72 h,SC vs KD:2.87±0.07 vs 2.13±0.09,P<0.05).②In order to evaluate the influence of TG2 on epithelial-mesenchymal transition(EMT)in BC,SK-BR-3 cells with TG2-KD were detected.TG2 knockdown reduced the ability of migration and invasion.Meanwhile,compared to the SC group,TG2 knockdown resulted in lower expression of mesenchymal related protein,such as Ncadherin,Snail,Vimentin and Slug(P<0.05),while E-cadherin,a epithelial related protein,was higher(P<0.05).③To further investigate the influence of TG2 on the glycolysis of BC cells,detecting the glucose consumption and lactate product in SK-BR-3(KD)cell line was conducted.TG2-KD resulted in decreased glucose consumption and lactate production(P<0.05);TG2-KD decreased the protein expression of HKII,PFKP and PKM2(P<0.01).④Compared with SC group,lower phosphorylation of MEK(P<0.05)and ERK(P<0.05)were observed in TG2-KD cells.Decreased protein and mRNA levels of LDHA and LDHB(P<0.05)were also observed in KD group(P<0.05).These findings demonstrated that TG2 can regulate glycolysis of BC cells through the MEK/ERK/LDH axis.⑤ To further confirm the mechanism of MEK/ERK/LDH pathway in TG2 promoting BC cell proliferation,the MEK inhibitor U0126 was used to perform rescue experiments.In SK-BR-3 cell line,the proliferation was decreased(OD 450 nm,TG2SC at 72 h,DMSO vs U0126:2.84±0.15 vs 2.16±0.19,P<0.01);and glucose consumption and lactate production were decreased(P<0.01).⑥ Compared with the SC group,tumor weight(1.21 ±0.08 g vs 0.42±0.07 g)and tumor volume(125.40±8.26 mm3 vs 53.47±5.10 mm3)were significantly lower in the KD group of nude mice(P<0.01),and the phosphorylation of MEK and ERK proteins in the KD group of nude mice(P<0.01)was lower,and LDHA and LDHB protein levels were also significantly lower(P<0.01).Conclusion:(1)TG2 is overexpressed in breast cancer tissues and serves as an independent risk factor for overall survival in patients with breast cancer.(2)TG2 promotes epithelial-mesenchymal transition of breast cancer cell.(3)TG2 activates MEK/ERK/LDH pathway and enhances proliferation in breast cancer cell lines.In summary,TG2 overexpresses in breast cancer and acts as an independe nt risk prognostic factor.TG2 promotes cancer cells proliferation by activating the MEK/ERK/LDH pathway.