Alteration of the Nucleotide Excision Repair (NER) Pathway in Soft Tissue Sarcoma

Abstract

Clinical responses to anticancer therapies in advanced soft tissue sarcoma (STS) are unluckily restricted to a small subgroup of patients. Much of the inter-individual variability in treatment efficacy is as result of polymorphisms in genes encoding proteins involved in drug pharmacokinetics and pharmacodynamics. The nucleotide excision repair (NER) system is the main defense mechanism for repairing DNA damage caused by carcinogens and chemotherapy drugs. Single nucleotide polymorphisms (SNPs) of NER pathway key genes, altering mRNA expression or protein activity, can be significantly associated with response to chemotherapy, toxicities, tumor relapse or risk of developing cancer. In the present study, in a cohort of STS patients, we performed DNA extraction and genotyping by SNP assay, RNA extraction and quantitative real-time reverse transcription PCR (qPCR), a molecular dynamics simulation in order to characterize the NER pathway in STS. We observed a severe deregulation of the NER pathway and we describe for the first time the effect of SNP rs1047768 in the ERCC5 structure, suggesting a role in modulating single-stranded DNA (ssDNA) binding. Our results evidenced, for the first time, the correlation between a specific genotype profile of ERCC genes and proficiency of the NER pathway in STS.

Keywords: ERCC; SNP; nucleotide excision repair (NER); pharmacogenomic; soft tissue sarcoma.

Figure 1

Nucleotide excision repair pathway (NER). GGR, global genomic NER; TCR, transcription coupled NER.

Figure 2

Presence/absence of the ERCC variants in the STS cohort. STS, soft tissue sarcoma.

Figure 3

(A) Linkage disequilibrium (LD) block and haplotype frequencies for rs2296147 and rs1047768 in ERCC5 gene. (B) LD block and haplotype frequencies for r1318, rs799793 and rs11615 in ERCC1 and ERCC2 genes.

Figure 4

Expression plots showing trends of individual NER genes in STS samples. (A) ERCC1; (B) ERCC2; (C) ERCC5. SD, standard deviation; STS, soft tissue sarcoma.

Figure 5

Correlation analysis between relative gene expression and genotype in 5 different SNPs (A) ERCC1_rs11615, (B) ERCC2_rs13181, (C) ERCC2_rs1799793, (D) ERCC5_rs2296147, (E) ERCC_rs1047768. The relative gene expression is presented as log2 Fold Change. In every plot, the samples were separated according to the presence (left side) or absence (right side) of at least one wild-type allele. The significance value at the top of every plot was obtained via Welch’s t-test. FC, fold change.

Figure 6

(A) Relative expression clusters of ERCC1, ERCC2 and ERCC5 per patient. The rows represent each sample and its respective ID, sex, histotype and grade. The columns represent the relative expression of ERCC1, ERCC2 and ERCC5 as log Fold Change values. (B) Correlation matrix of ERCC1, ERCC2 and ERCC5 relative gene expression with Pearson’s correlation similarity. M, male; F, female.

Figure 7

(A) Model of the ERCC2 electrostatic interaction network between the negative loop Pro311-Pro320 and positive charges; mutant Asp312Asn (position in magenta) destabilise the interaction and loop. (B) Loss of C-terminal α-helix and β-sheet (transparent segment in magenta) following mutation Lys751*.

Figure 8

Protein local flexibility is represented with the root mean square fluctuation profile (RMSF, nm) for wild type (solid line) and Asp312Asn mutant (dashed line) as calculated from the molecular dynamics simulations. The secondary structure is reported at the bottom (helix for α-helices, line for loops). Loop segment Pro310-Pro320 (black thick line) shows flexibility lower than helices thanks to the extended network of electrostatic interactions. Point mutation Asp312Asn (indicated by arrow) causes electrostatic network disruption and segment flexibility to increase up to 4-fold. The RMSF of the remaining structure is not affected, suggesting only local conformational change occurs without hampering the overall folding.

Figure 9

Model of activated ERCC5 using the DNA in the T4 RNase H crystal structure (PDB 2IHN). Close-up views of the gateway and the canyon from the model shown in panel B. Residues mutated in silico are colored in magenta. The undamaged ssDNA fits in the crevice formed by the hydrophobic wedge (green), the β-pin motif (cyan) and α-helix 9 (light blue). The crevice accommodates the phosphate backbone and is stabilised by charge interactions with bottom Lys828, His80 and Lys972; the MD simulation also showed wedge flexible loop to participate in binding by His46 driven closure onto the phosphate backbone. The His46Gln mutant largely lost such interaction.