Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia

Abstract

Background: Poly-(ADP-ribose)-polymerase1 (PARP1) is involved in repair of DNA single strand breaks. PARP1-inhibitors (PARP1-i) cause an accumulation of DNA double strand breaks, which are generally repaired by homologous recombination (HR). Therefore, cancer cells harboring HR deficiencies are exceptionally sensitive to PARP1-i. For patients with HR-proficient tumors, HR can be temporarily inhibited by hyperthermia, thereby inducing synthetic lethal conditions in every tumor type. Since cisplatin is successfully used combined with hyperthermia (thermochemotherapy), we investigated the effectiveness of combining PARP1-i with thermochemotherapy.

Results: The in vitro data demonstrate a decreased in cell survival after addition of PARP1-i to thermochemotherapy, which can be explained by increased DNA damage induction and less DSB repair. These in vitro findings are in line with in vivo model, in which a decreased tumor growth is observed upon addition of PARP1-i.

Materials and methods: Survival of three HR-proficient cell lines after cisplatin, hyperthermia and/or PARP1-i was studied. Cell cycle analyses, quantification of γ-H2AX foci and apoptotic assays were performed to understand these survival data. The effects of treatments were further evaluated by monitoring tumor responses in an in vivo rat model.

Conclusions: Our results in HR-proficient cell lines suggest that PARP1-i combined with thermochemotherapy can be a promising clinical approach for all tumors independent of HR status.

Keywords: PARP1-inhibitor; cDDP; hyperthermia; synthetic lethality.

Figure 1. The effects of PARP1-i, HT, cDDP and combined treatments on BRCA2, Rad51 and cell survival are shown

(A) Western blots demonstrating R1, SiHa and HeLa cells are BRCA2 proficient. After HT, BRCA2 is downregulated. (B) γ-H2AX and Rad51 co-localization, to investigate activity of homologous recombination. (C) Clonogenic assays were performed to study the effect of the different treatment combinations, 10–12 days after treatments. The addition of PARP1-i to cDDP-based thermochemotherapy resulted in a significantly lower cell survival compared to cDDP-based thermochemotherapy alone. R1: p = 0.0008, SiHa: p = 0.034, HeLa: p = 0.021. The bar graph shows the mean of at least five independent experiments. From left to right: R1, SiHa, Hela cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. DSBs were analysed using the γ-H2AX assay

(A) The induction of DSBs in R1 and SiHa was significantly higher after addition of PARP1-i to cDDP-based thermochermotherapy. In HeLa cells this was not found to be significant, although a trend is seen. R1: p = 0.048, SiHa: p = 0.035, HeLa: p = 0.068 From left to right: R1, SiHa, Hela cells. (B) One representative cell is depicted for each condition. Bars represent the mean of three independent experiments with the standard error of the mean (SEM). *p < 0.05.

Figure 3. Cell cycle distribution using BrdU incorporation

While the monotherapies hardly ever changed the cell cycle analyses, any treatment containing cDDP resulted in a larger proportion of cells in S-phase. The differences found after adding PARP1-i to thermochemotherapy were not significant. From left to right: R1, SiHa, Hela cells.

Figure 4. Apoptotic levels measured using the Nicoletti assay

An ~1.5-fold induction in apoptosis is found after treatment with PARP1-i, cDDP and HT compare to cDDP with HT in R1 and SiHa cells (R1: p = 0.0007, SiHa: p = 0.0054). A smaller, though significant effect was found in HeLa cells after addition of a PARP1-i (HeLa: p = 0.026). From left to right: R1, SiHa, Hela cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. In vivo experiments performed on rats

(A) Relative tumour volume demonstrated that PARP1-i enhanced the effectiveness of cDDP-based thermochemotherapy and reduced the in vivo tumour outgrowth. Arrows present the time of treatment. (B) Time needed to achieve ten times the start volume (T10 × SV). Animals treated with the triple modality showed a delay in reaching T10 × SV compared to thermochemotherapy or any single treatment. All animal experiments included at least five rats per group.