Aiyappa, Radhika
(2020)
Targeting DNA repair and hypoxia to improve radioresponse in breast cancer.
PhD thesis, University of Nottingham.
Abstract
Radiotherapy is an essential treatment modality in the clinical management of breast cancers, with an established role to reduce the risk of local recurrences. However, the repair of radiotherapy-induced DNA damage by cancer cells and the presence of tumour hypoxia reduces the efficacy of radiotherapy as a treatment modality. The present study aimed to assess how agents targeting DNA repair and hypoxia can modulate radioresponse in breast cancer. The aim of the current thesis was to investigate the single agent and radiosensitising efficacy of the FEN-1 inhibitor PTPD, and novel hypoxia-activated prodrug (HAP) KP167, in luminal and triple negative breast cancer models.
The efficacy of PTPD and KP167 were evaluated in two luminal (MCF-7 and T47D) and three triple negative (MDAMB-231, MDAMB-468 and BRCA-1 deficient MDAMB-436) breast cancer cell lines. The well-established DNA repair inhibitors such as the ATM inhibitor, KU-55933 and PARP-1/2 inhibitor, olaparib were used as controls against the FEN-1 inhibitor, PTPD in 2D normoxia cultures. The hypoxia activated prodrug, AQ4N was used as a comparator against its novel analogue KP167 in 3D spheroids, 2D normoxia and 2D hypoxia (1% O2) cultures. The single agent and radiosensitising efficacy of PTPD and KP167 were assessed by clonogenic survival assays. The expression of cytochrome P450 enzymes, CYP2S1 and CYP2W1, involved in the bio-reductive activation of the HAPs were assessed in breast cancer cells by Western blot and in a large cohort of breast cancer patients (n=1426) by immunohistochemistry.
In vitro results show that PTPD was cytotoxic only in the BRCA-1 deficient MDAMB-436 cells and resulted in a 60% reduction in clonogenic survival, compared to 20-40% and 40-80% reduction across all breast cancer cells with KU-55933 and olaparib. KU-55933 and olaparib induced significant radiosensitisation across all phenotypes of breast cancer cells with sensitiser enhancement ratio’s (SER0.01) ranging between 1.61-3.42 and 1.60-2.35 respectively. PTPD was effective in radiosensitising only the triple negative cell lines, especially the BRCA-1 deficient MDAMB-436 with SER0.01 of 1.50 compared to MDAMB-231 (SER0.01 1.31) and MDAMB-468 (SER0.01 1.13) cells. PTPD did not induce any radiosensitisation in the luminal MCF-7 or T47D cells (SER0.01 1.00). The radiosensitisation observed in MDAMB-436 cells may be due, in part, to increased DNA double strand breaks, as assessed by the γH2AX assay, due to PTPD inhibition of single strand repair, along with its inherent double strand break repair deficiency. Such data indicate that PTPD is effective, particularly in the context of BRCA-1 deficiency. PTPD was able to induce some levels of radiosensitisation in the TNBC cell lines, this was however, lower compared to the control DNA repair inhibitors.
In vitro results suggest that AQ4N and KP167 may undergo hypoxia-specific activation as shown by increases in single agent cytotoxicity in 3D spheroid and 2D hypoxia models of breast cancer compared to response obtained in 2D normoxia cultures. KP167 was 2-100 fold more effective in reducing survival with IC50 values of (0.14-12.8) µM compared to AQ4N IC50 of (0.2–22.8) µM in breast cancer 3D cultures. The luminal MCF-7 and T47D cells were more sensitive to single agent AQ4N and KP167 across all 3 culture conditions and this may be due to the higher expression of CYP2W1 (assessed by Western blot), essential for both estrogen metabolism and HAP activation.
The radiosensitivity induced by HAPs were greater in 3D spheroid and 2D hypoxia models of breast cancer compared to normoxia cultures. Compared to AQ4N SER0.01 of 1.30-3.33, a 100-fold increase in radiosensitisation was seen in KP167 treated cells with SER0.01 of 1.46-4.55 in 3D spheroid cultures. Greater levels of radiosensitisation with KP167 was also seen in 2D normoxia (SER0.01 of 1.21-1.93) and 2D hypoxia (SER0.01 of 1.56-2.37) cultures compared to AQ4N SER0.01 of 1.11-1.64 and 1.13-1.94 respectively. Such data indicate that KP167 is a potent radiosensitiser across both luminal and triple negative breast cancer cells and may hold promising potential in the treatment of hypoxic breast cancers.
Immunohistochemical studies show that high cytoplasmic CYP2S1 and nuclear CYP2W1 were able to identify a sub-group of ER-positive, HER-2 negative patients, above the age of 50 that may potentially benefit from HAP treatment. Low cytoplasmic CYP2S1 (P=0.034) and nuclear CYP2W1 (P=0.012) were significantly associated with adverse breast cancer specific survival in the total patient cohort, which retained its significance in multivariate analysis (P=0.002 and P=0.007 respectively). Low cytoplasmic CYP2S1 and nuclear CYP2W1 were also associated with adverse survival in ER-positive (P=0.031) and ER-negative patients (P=0.020) respectively. The association of low CYP2S1 and CYP2W1 with adverse survival may suggest involvement of these enzymes in the metabolism of anti-cancer agents such as tamoxifen and taxanes used in breast cancer treatment. Such data establishes the prognostic significance of CYP2S1 and CYP2W1 in breast cancer.
Collectively, although further confirmation is needed, KP167 appears to be a promising agent, either single or combined with radiation, to target hypoxic breast cancer cells and improve patient outcomes. CYP2S1 and CYP2W1 may be of prognostic significance in breast cancer.
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