Study On Biomarkers For Locally Advanced Rectal Cancer And 5-fluorouracil Based Neoadjuvant Chemoradiotherapy

Background: Rectal cancer is one of the most common cancers in the world.Due to late detection or delayed diagnosis,patients are often diagnosed with locally advanced rectal cancer(LARC).The clinical lymph node stage is of importance for diagnosis of LARC but few biomarkers for lymph node status are currently available.Neoadjuvant chemoradiotherapy(n CRT)prior to surgery is standard treatment for LARC patients,while parts of them show poor response accompanying with therapy adverse effects.Predictive biomarkers for n CRT response could facilitate the guidance on treatment decisions but are still insufficient until now,which limited the clinical applications of n CRT in LARC patients.Besides,mechanism research would be useful for exploring new therapeutic targets in LARC.Purposes: Our study aims to explore markers for lymph node staging of rectal cancer and prediction of therapeutic response to 5-fluorouracil based neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer,and to reveal the roles and underlying mechanism of candidate markers in n CRT.Methods: Plasma samples from patients with pathologically confirmed rectal adenocarcinoma were prospectively collected before treatment,and all patients were divided into lymph node positive group(LN_P)and lymph node negative group(LN_N).According to admission time,patients were divided into discovery cohort and validation cohort.Samples of the discovery cohort were analyzed using label-free proteomic approach by mass spectrometry.Differentially expressed proteins(DEPs)between LN_N and LN_P group screened out from discovery cohort were then validated through parallel reaction monitoring(PRM)approach in validation cohort.Diagnostic performance was assessed by receiver operating characteristic(ROC)analysis.Thirty-seven formalin-fixed paraffin-embedded tumor biopsies were retrospectively obtained from LARC patients before 5-flurouracil based n CRT.Proteomics analysis were conducted to identify the DEPs between total responders(TR)and poor responders(PR).The DEPs were validated via ROC plotter and their predictive performance were evaluated by ROC analysis.Functional enrichment analyses were performed to further explore the potential mechanisms underlying n CRT response.Five types of human colorectal cancer(CRC)cell lines(HCT116,Lo Vo,SW480,HT29,and HR8348)in vitro and two tumor xenografts(HCT116 and HT29)in nude mice in vivo were established to evaluate their sensitivity to chemoradiotherapy(CRT),and were then divided into sensitive group and resistant group.Proteomic analysis were performed using CRC cells,cell supernatant,and tumor xenograft tissues to figure out differentially expressed proteins between sensitive and resistant group.The intersection of DEPs among cells,cell supernatant,tumor xenograft,as well as tumor biopsies were figured out,which was HSPA4.HSPA4 was overexpressed or knocked down in Lo Vo and HT29 through Lentivirus infection and si RNA transfection,respectively.CCK8(cell counting kit-8),Western Blot,and flow cytometry method were used to evaluate the effect of HSPA4 on cell survival rate after chemoradiotherapy,cell cycle distribution,and cell apoptosis rate.Enrichment analyses were also conducted to reveal the biological relevance of HSPA4 to n CRT response in CRC cell lines.Results: A total of 350 proteins were identified through proteomic analysis in plasma samples of the discovery cohort,of which 51 DEPs were screened out between LN_N and LN_P group.After PRM approach,IGF2 、 CBPB2 、 FA11 、 A1AG2 、 MBL2 、 HELZ2 were independently validated by the validation cohort.IGF2 and HELZ2 showed stable diagnostic performance by ROC curve analysis both in the discovery cohort and the validation cohort.IGF2 reached an area under cure(AUC)of 0.857(95% CI: 0.673~0.960,p = 0.0001.sensitivity = 75.57%,specificity = 100.00%)in the discovery cohort and 0.853(95% CI:0.702~0.945,p < 0.0001.sensitivity = 95.00%,specificity = 73.68%)in the validation cohort.HELZ2 achieved an AUC of 0.775(95% CI: 0.557~0.897,p = 0.0073.sensitivity =71.43%,specificity = 78.57%)in the discovery cohort and 0.808(95% CI: 0.652~0.915,p <0.0001.sensitivity = 95.00%,specificity = 70.00%)in the validation cohort.Among 3998 total proteins identified in biopsies of LARC patients,91 DEPs between TR and PR were screened out.HSPA4,NIPSNAP1,SPTB with AUC around 0.8 in the internal discovery cohort were independently validated by the external m RNA datasets(AUC around0.7),and their protein levels were linearly correlated with the graded responses to n CRT in the total internal cohort.The combination of HSPA4 and SPTB could distinctly discriminate TR and PR group(AUC = 0.980,p < 0.0001).Moreover,multiple combinations of the three proteins realized better specificity and/or sensitivity,and achieved favorable predictive value when moderate responders were introduced into ROC analysis.Pathways including DNA damage repair,cell cycle and epithelial mesenchymal transition(EMT)were involved in n CRT response according to enrichment analysis results.In vivo results showed that after CRT,xenograft tumor size of HCT116 and HT29 were significantly distinct(p < 0.05).The mean size of HT29 xenografts constantly grew while the mean size of HCT116 xenografts showed a decrease after therapy.Thus,HCT116 xenografts were defined as sensitive group and HT29 xenografts as resistant group.In vitro,survival rates after CRT of HT29 and HR8348 were significant higher than HCT116,SW480,and Lo Vo(p < 0.05),so the former two cell lines were defined as resistant group and the latter three cell lines were defined as sensitive group.The intersection of DEPs among cells,cell supernatant,tumor xenografts,as well as tumor biopsies of LARC patients results were figured out,which was HSPA4.HSPA4 knockdown increased the survival rate of Lo Vo after CRT.While overexpression of HSPA4 significantly increased the percent of G2/M cells accompanied with a decrease of survival rate after CRT in both Lo Vo and HT29,indicating that higher level of HSPA4 confered more sensitivity to CRT upon the tumor cells.And this sensitization were negated by knockdown of HSPA4.But cell apoptosis rate after chemoradiotherapy was decreased in HSPA4 overexpressed group of Lo Vo cells compared with negative control group.Furthermore,enrichment analysis found that biological functions in cells with HSPA4 overexpression were related to regulation of cell cycle,oxidative phosphorylation,and EMT.And in rectal cancer,m RNA expression of HSPA4 were positively or negatively correlated to genes of pathways closely related to tumor resistance,suggesting its limited value in sensitization of colorectal cancer to chemoradiotherapy.Conclusions: HELZ2 and IGF2 showed a stable diagnostic performance in discrimination between lymph node positive and negative rectal cancer,with AUCs around 0.8.Proteomic analysis of plasma could provide valuable information for accurate lymph node staging,thus benefiting the diagnosis of LARC.Tumor tissues-derived HSPA4,SPTB and NIPSNAP1,as well as their optional combinations might be potential predictive markers for n CRT response in LARC patients.HSPA4,as a predictive marker,potentially plays a role in n CRT response through regulation of cell cycle,in which oxidative phosphorylation,EMT,etc are also involved.Nevertheless,in rectal cancer,HSPA4 may play negative or positive roles in cancer related pathways,such as apoptosis,cell cycle,and DNA damage repair pathways,resulting in its limited values in chemoratiotherapy sensitization of colorectal cancer.