DNA Damage Response during Replication Correlates with CIN70 Score and Determines Survival in HNSCC Patients

Abstract

Aneuploidy is a consequence of chromosomal instability (CIN) that affects prognosis. Gene expression levels associated with aneuploidy provide insight into the molecular mechanisms underlying CIN. Based on the gene signature whose expression was consistent with functional aneuploidy, the CIN70 score was established. We observed an association of CIN70 score and survival in 519 HNSCC patients in the TCGA dataset; the 15% patients with the lowest CIN70 score showed better survival (p = 0.11), but association was statistically non-significant. This correlated with the expression of 39 proteins of the major repair complexes. A positive association with survival was observed for MSH2, XRCC1, MRE11A, BRCA1, BRCA2, LIG1, DNA2, POLD1, MCM2, RAD54B, claspin, a negative for ERCC1, all related with replication. We hypothesized that expression of these factors leads to protection of replication through efficient repair and determines survival and resistance to therapy. Protein expression differences in HNSCC cell lines did not correlate with cellular sensitivity after treatment. Rather, it was observed that the stability of the DNA replication fork determined resistance, which was dependent on the ATR/CHK1-mediated S-phase signaling cascade. This suggests that it is not the expression of individual DNA repair proteins that causes therapy resistance, but rather a balanced expression and coordinated activation of corresponding signaling cascades.

Keywords: CIN70 score; Chk1 inhibition; HNSCC; chromosomal instability (CIN); radiosensitization; radiotherapy; replication stress.

Figure 1

Functional aneuploidy indicates worse survival in HNSCC patients and is associated with high mRNA expression of DNA repair proteins. (A) Probability of survival after treatment of tumors with a low compared to a high CIN70 score; (B–H) mRNA expression in tumors with low and high CIN70 scores involved in the ATR/ATM signaling cascade, mismatch repair, single-strand break repair, homologous recombination, classical non-homologous end-joining, ICL repair and DNA replication fork stability (h) was analyzed. Asterisks (*) represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Student´s t-test). Blue bars represent variation of housekeeping genes.

Figure 2

mRNA expression of HR proteins correlates with CIN70 score (A) The mRNA expression for the proteins RAD54B, BRCA1, MRE11, and ERCC1 was plotted against the corresponding CIN70 score of 519 HNSCC patients and calculated with linear regression. (B) Relative HR capacity in HNSCC cell lines (data taken from Wurster et al., 2016). (C) Protein expression of BRCA1, ATR, MRE11A, CHK1, and RAD51 of 7 unirradiated HNSCC cell lines. (D) Cellular survival after irradiation, mitomycin C and topotecan determined by the colony formation assay.

Figure 3

Survival probability is determined by mRNA expression of the DNA repair proteins MSH2, XRCC1, ERCC1, BRCA1, BRCA2, RAD54B, LIG1, CLSPN, MRE11A, DNA2, POLD1 and MCM2 in 519 HNSCC patients of the TCGA dataset using Kaplan-Meier survival curves up to 60 months after therapy. The 15% of patients with the lowest and/or highest protein expression were compared with the remaining patients. Differences between both groups were determined by the log-rank test.

Figure 4

Resistance is mediated by DNA replication fork stability and active DNA damage response of S phase. (A,B) Treatment scheme and examples of replication tracts and replication tract lengths before (CldU) (C) and after (IdU) (D) irradiation. Correlation of replication tract length and cellular radiosensitivity (E). (F) Qualitative and quantitative activation of CHK1/pCHK1 after UV irradiation. Replication tract length before (left) and after UV irradiation (right) (G). p-values represent errors of the mean. Asterisks (*) represent significant differences (**** p < 0.0001; Student´s t-test).

Figure 5

Inhibition of S phase checkpoint control radiosensitizes exclusively resistant HNSCC cell lines. (A) Concentration-dependent inhibition of CHK1 by siRNA treatment in FaDu and HSC4 cells. (B,C) Effect of siRNA or CHK1 inhibitor MK8776 treatment in combination with irradiation on replication tract length compared to irradiation alone. (D) Cellular radiosensitization only in resistant cell lines. Error bars represent the error of means of at least three independent experiments. (Student´s t-test).