Genetic polymorphisms in genes regulating cell death and prognosis of patients with rectal cancer receiving postoperative chemoradiotherapy

Abstract

Objective: The identification of biomarkers for predicting chemoradiotherapy efficacy is essential to optimize personalized treatment. This study determined the effects of genetic variations in genes involved in apoptosis, pyroptosis, and ferroptosis on the prognosis of patients with locally advanced rectal cancer receiving postoperative chemoradiotherapy (CRT).

Methods: The Sequenom MassARRAY was used to detect 217 genetic variations in 40 genes from 300 patients with rectal cancer who received postoperative CRT. The associations between genetic variations and overall survival (OS) were evaluated using hazard ratios (HRs) and 95% confidence intervals (CIs) computed using a Cox proportional regression model. Functional experiments were performed to determine the functions of the arachidonate 5-lipoxygenase (ALOX5) gene and the ALOX5 rs702365 variant.

Results: We detected 16 genetic polymorphisms in CASP3, CASP7, TRAILR2, GSDME, CASP4, HO-1, ALOX5, GPX4, and NRF2 that were significantly associated with OS in the additive model (P < 0.05). There was a substantial cumulative effect of three genetic polymorphisms (CASP4 rs571407, ALOX5 rs2242332, and HO-1 rs17883419) on OS. Genetic variations in the CASP4 and ALOX5 gene haplotypes were associated with a higher OS. We demonstrated, for the first time, that rs702365 [G] > [C] represses ALOX5 transcription and corollary experiments suggested that ALOX5 may promote colon cancer cell growth by mediating an inflammatory response.

Conclusions: Polymorphisms in genes regulating cell death may play essential roles in the prognosis of patients with rectal cancer who are treated with postoperative CRT and may serve as potential genetic biomarkers for individualized treatment.

Keywords: ALOX5; Rectal neoplasms; genetic variation; overall survival; regulated cell death.

Figure 1

The Kaplan-Meier survival curves of rectal cancer patients treated with postoperative chemoradiotherapy by genotypes or different number of risk genotypes. (A) rs571407 (log-rank test, P = 0.002); (B) rs612987 (log-rank test, P = 0.025); (C) rs623114 (log-rank test, P = 0.015); (D) rs543923 (log-rank test, P = 0.045); (E) rs702365 (log-rank test, P = 0.014); (F) rs2242332 (log-rank test, P = 0.005); (G) rs4948673 (log-rank test, P = 0.009); (H) rs17883419 (log-rank test, P = 0.001); (I) rs2071749 (log-rank test, P = 0.010); (J) Four risk groups (log-rank test, P < 0.001) and (K) Three risk groups (log-rank test, P < 0.001) of joint effect of unfavorable genotypes (CASP4 rs571407 TT, ALOX5 rs2242332 CC, HO-1 rs17883419 CT + TT). (L) Four risk groups (log-rank test, P < 0.001) of the unfavorable genotype joint effects (CASP4 rs571407 TT, ALOX5 rs2242332 CC, HO-1 rs17883419 CT + TT, hsa-miR-4274 rs202195689 CCCCA-del + del-del, and PMS1 rs5743030 GG).

Figure 1

The Kaplan-Meier survival curves of rectal cancer patients treated with postoperative chemoradiotherapy by genotypes or different number of risk genotypes. (A) rs571407 (log-rank test, P = 0.002); (B) rs612987 (log-rank test, P = 0.025); (C) rs623114 (log-rank test, P = 0.015); (D) rs543923 (log-rank test, P = 0.045); (E) rs702365 (log-rank test, P = 0.014); (F) rs2242332 (log-rank test, P = 0.005); (G) rs4948673 (log-rank test, P = 0.009); (H) rs17883419 (log-rank test, P = 0.001); (I) rs2071749 (log-rank test, P = 0.010); (J) Four risk groups (log-rank test, P < 0.001) and (K) Three risk groups (log-rank test, P < 0.001) of joint effect of unfavorable genotypes (CASP4 rs571407 TT, ALOX5 rs2242332 CC, HO-1 rs17883419 CT + TT). (L) Four risk groups (log-rank test, P < 0.001) of the unfavorable genotype joint effects (CASP4 rs571407 TT, ALOX5 rs2242332 CC, HO-1 rs17883419 CT + TT, hsa-miR-4274 rs202195689 CCCCA-del + del-del, and PMS1 rs5743030 GG).

Figure 2

rs702365 variant at ALOX5 intron region functions in an allele-specific manner. (A) eQTL analysis of ALOX5 rs702365 (left), rs2242332 (middle), and rs4948673 (right) variants and associations with mRNA expression from GTEx in normal colon tissue. (B) Functional annotation of three SNPs in RegulomeDB (up) and GWAVA database (down). (C) Relative luciferase activity of the reporter gene with reconstructed plasmid containing rs702365[C]- or [G]-allele in constructs containing SV40 in HCT8 (left) and HCT116 (right) cells. (D) Relative luciferase activity of the reporter gene with reconstructed plasmids containing rs702365[C]- or [G]-allele in constructs containing ALOX5 promoter in HCT8 (left) and HCT116 (right) cells. Relative luciferase activities are shown as fold changes relative to luciferase expression in cells transfected with empty vector. All constructs were co-transfected with pRL-TK to standardize transfection efficiency. Data represent the mean ± SEM from three independent experiments, each with three replicates. P-values were obtained using Student’s t-test. (E, F) EMSAs with biotin-labeled probes containing rs702365-[C] or -[G] allele in HCT8 (E) and HCT116 (F) cells. Arrow, allele-specific bands that interact with nuclear extracts. 10× and 100× indicate 10-fold and 100-fold excess amounts of an unlabeled probe compared with the amount of the labeled probe, respectively. “−” and “+” represent not added and added, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

rs702365 variant at ALOX5 intron region functions in an allele-specific manner. (A) eQTL analysis of ALOX5 rs702365 (left), rs2242332 (middle), and rs4948673 (right) variants and associations with mRNA expression from GTEx in normal colon tissue. (B) Functional annotation of three SNPs in RegulomeDB (up) and GWAVA database (down). (C) Relative luciferase activity of the reporter gene with reconstructed plasmid containing rs702365[C]- or [G]-allele in constructs containing SV40 in HCT8 (left) and HCT116 (right) cells. (D) Relative luciferase activity of the reporter gene with reconstructed plasmids containing rs702365[C]- or [G]-allele in constructs containing ALOX5 promoter in HCT8 (left) and HCT116 (right) cells. Relative luciferase activities are shown as fold changes relative to luciferase expression in cells transfected with empty vector. All constructs were co-transfected with pRL-TK to standardize transfection efficiency. Data represent the mean ± SEM from three independent experiments, each with three replicates. P-values were obtained using Student’s t-test. (E, F) EMSAs with biotin-labeled probes containing rs702365-[C] or -[G] allele in HCT8 (E) and HCT116 (F) cells. Arrow, allele-specific bands that interact with nuclear extracts. 10× and 100× indicate 10-fold and 100-fold excess amounts of an unlabeled probe compared with the amount of the labeled probe, respectively. “−” and “+” represent not added and added, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

ALOX5 may promote proliferation of colon cancer cells by inducing an inflammatory response. (A) The effects of ALOX5 knockdown on the proliferation of HCT8 (left) and HCT116 (right) cells. (B) The expression of ALOX5 in HCT8 (left) and HCT116 (right) cells detected by RT-qPCR after transfection with siControl or siALOX5. GAPDH was used as the reference gene. (C) Enrichment analysis results of ALOX5 co-expression genes in Metascape. (D) The expression of inflammation-related protein in HCT8 (up) and HCT116 (down) cells transfected with siControl or siALOX5. (E) Schematic of the mechanism by which ALOX5 rs702365 [G] > [C] substitution functions as a tumor suppressor in CRC. ALOX5 rs702365 [G] > [C] change directly decreased ALOX5 expression. The down-expression of ALOX5 led to decreased expression of p-p65 and iNOS, which contributed to the inhibition of proliferation of colon cancer cells. Data represent the mean ± SEM based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

ALOX5 may promote proliferation of colon cancer cells by inducing an inflammatory response. (A) The effects of ALOX5 knockdown on the proliferation of HCT8 (left) and HCT116 (right) cells. (B) The expression of ALOX5 in HCT8 (left) and HCT116 (right) cells detected by RT-qPCR after transfection with siControl or siALOX5. GAPDH was used as the reference gene. (C) Enrichment analysis results of ALOX5 co-expression genes in Metascape. (D) The expression of inflammation-related protein in HCT8 (up) and HCT116 (down) cells transfected with siControl or siALOX5. (E) Schematic of the mechanism by which ALOX5 rs702365 [G] > [C] substitution functions as a tumor suppressor in CRC. ALOX5 rs702365 [G] > [C] change directly decreased ALOX5 expression. The down-expression of ALOX5 led to decreased expression of p-p65 and iNOS, which contributed to the inhibition of proliferation of colon cancer cells. Data represent the mean ± SEM based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.