BLCA patient tumors from the TCGA cohort have mutations in DDR genes
We used GDC Data Portal, COSMIC, and cBioPortal to analyze mutation rates in DNA repair genes [28] in BLCA patient tumors from the TCGA. The data revealed that ATM, ERCC2, BRCA2, ATR, and TP53 mutations are highly prevalent in BLCA tissues from the TCGA cohort (Table 1). Mutations in genes involved in indirect DNA stability were also associated with high death rates from BLCA in this cohort (Table 1). These data confirmed previous findings which showed that ~34% of BLCA harbor mutations in DDR genes [24, 34, 35]. The results provided the rationale for our study to test the relative efficacy of commercially available PARPi against BLCA cells.
PARPi suppress cell survival of BLCA cells
We determined the IC50 of cisplatin as well as the PARPi (olaparib, niraparib, rucaparib, veliparib, and talazoparib) in UM-UC-3 cells by treating with 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 μM concentrations for 72 h. The AAT Bioquest (https://www.aatbio.com/tools/ic50-calculator) online tool was used to calculate IC50 values. The IC50 curves and the calculated IC50 values for each of these agents are summarized in Fig. 1A-F. IC50 values (μM) were as follows: Niraparib (8.6093); olaparib (8.2312); rucaparib (15.5063); talazoparib (1.0989); veliparib (39.4209); and cisplatin (3.163).
Next, to determine working concentrations for each of the PARPi, we treated UM-UC-3 and T-24 as well as SV-HUC-1 cells with 5, 10, or 20 μM of olaparib, niraparib, veliparib, or rucaparib or 0.5, 1, or 2 μM of talazoparib for 3 days. As demonstrated in Fig. 2A, olaparib, niraparib, talazoparib, and rucaparib significantly blocked the survival of UM-UC-3, T-24, and SV-HUC-1 cells. Veliparib did not achieve >30% inhibition of BLCA cell survival even at very high concentrations. Our findings indicate that talazoparib and niraparib achieved >50% reduction in survival of BLCA cells at low concentrations.
PARPi reduce proliferation of BLCA cells
We treated BLCA cell lines UM-UC-3 and T-24 and the normal urothelial cells SV-HUC-1 with sub-IC50 concentrations of PARPi for 5 days. As shown in Fig. 2C, the percentages of proliferation in niraparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 77.75 ± 8.63 (p = 0.034), 61.43 ± 13.24 (p = 0.011), and 47.71 ± 9.58 (p = 0.0012), respectively, compared with their respective DMSO-treated controls. The percentages of proliferation in olaparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 84.67 ± 3.10 (p = 0.041), 70.11 ± 6.36 (p = 0.035), and 76.39 ± 1.84 (p = 0.022), respectively, compared with their respective DMSO-treated controls. The percentages of proliferation in rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 90.84 ± 3.41 (p = 0.07), 79.78 ± 0.85 (p = 0.039), and 83.03 ± 4.51 (p = 0.046), respectively. The percentages of proliferation in talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 94.22 ± 5.18 (p = 0.43), 75.54 ± 0.66 (p = 0.036), and 81.61 ± 2.42 (p = 0.047), respectively, compared with their respective DMSO-treated controls. These results indicated that PARPi suppressed the proliferation of UM-UC-3 and T-24 more significantly compared with that of SV-HUC-1 cells, suggesting that PARPi can be used as potential therapeutic agents against BLCA.
PARPi inhibit the clonogenic ability of BLCA cells
We treated BLCA cell lines UM-UC-3 and T-24 as well as the normal urothelial cells SV-HUC-1 with sub-IC50 concentrations of PARPi for 72 h and performed clonogenic assays as described earlier [31]. The percentages of colonies formed in niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 72.30 ± 2.66 (p = 0.036), 56.44 ± 5.62 (p = 0.022), and 74.43 ± 8.30 (p = 0.013), respectively, compared with their DMSO-treated controls. The percentages of colonies formed in olaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 48.84 ± 4.36 (p = 0.0021), 76.99 ± 2.12 (p = 0.037), and 85.52 ± 5.75 (p = 0.047), respectively, compared with their DMSO-treated controls. The percentages of colonies formed in rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 70.76 ± 2.66 (p = 0.033), 87.42 ± 7.53 (p = 0.048), and 94.73 ± 4.4 (p = 0.71), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 45.76 ± 9.81 (p = 0.0019), 56.44 ± 2.81 (p = 0.0031), and 68.23 ± 0.97 (p = 0.002), respectively, compared with their respective DMSO-treated controls. The results showed that PARPi inhibited the clonogenic ability of BLCA cells and normal urothelial cells significantly (Fig. 3A, B), indicating that PARPi may suppress the ability of BLCA cells to recover from treatment and form colonies.
PARPi synergize with cisplatin treatment in vitro
Cisplatin is the mainstay of BLCA therapy. However, cisplatin treatment produces life-threatening toxicities in ~50% of BLCA patients. Strategies to overcome these drawbacks are needed urgently. Towards this end, we sought to determine whether co-treatment with PARPi can be used to reduce the effective dosage of cisplatin against BLCA cells. Hence, we treated BLCA cells UM-UC-3 and T-24 as well as the normal urothelial cells SV-HUC-1 with sub-IC50 concentrations of PARPi in combination with sub-IC50 concentrations of cisplatin and measured cell survival, proliferation, and clonogenic ability of the treated cells compared with vehicle-treated cells. The percentages of cells surviving in cisplatin-treated SV-HUC-1, UM-UC-3, and T-24 groups were 94.46 ± 8.5 (p = 0.38), 96.09 ± 4.8 (p = 0.42), and 98.85 ± 1.66 (p = 0.31), respectively, compared with their DMSO-treated controls. The percentages of cells surviving in niraparib-treated vs. CP+Niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 18.44 ± 7.56 vs. 11.85 ± 1.74 (p = 2.663e-8), 55.34 ± 3.10 vs. 22.19 ± 7.56 (p = 1.57e-6), and 24.33 ± 3.53 vs. 23.44 ± 8.49 (p = 0.00059), respectively, compared with their respective DMSO-treated controls. The percentages of cells surviving in olaparib-treated vs. CP+Olaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 56.14 ± 6.08 vs. 40.03 ± 3.54 (p = 3.69e-4), 36.75 ± 6.22 vs. 20.84 ± 1.08 (p = 1.22e-5), and 52.83 ± 1.86 vs. 31.69 ± 4.21 (p = 3.19e-3), respectively, compared with their respective DMSO-treated controls. The percentages of cells surviving in rucaparib-treated vs. CP+Rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 42.53 ± 7.03 vs. 31.04 ± 2.57 (p = 4.17e-4), 62.60 ± 4.29 vs. 34.29 ± 1.32 (p = 3.81e-4), and 54.20 ± 2.93 vs. 31.05 ± 0.86 (p = 6.43e-4), respectively, compared with their respective DMSO-treated controls. The percentages of cells surviving in talazoparib-treated vs. CP+Talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 35.35 ± 4.38 vs. 22.22 ± 5.20 (p = 4.21e-6), 47.01 ± 0.69 vs. 27.95 ± 4.16 (p = 0.0014), and 36.63 ± 1.41 vs. 30.48 ± 2.67 (p = 2.19e-3), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in niraparib-treated vs. CP+Niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 77.75 ± 8.63 vs. 70.74 ± 0.32 (p = 0.049), 61.43 ± 13. 24 vs. 37.49 ± 1.30 (p = 0.031), and 47.71 ± 9.58 vs. 33.97 ± 3.06 (p = 0.015), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in olaparib-treated vs. CP+Olaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 84.67 ± 3.10 vs. 74.14 ± 1.89 (p = 0.06), 70.11 ± 6.36 vs. 36.39 ± 3.83 (p = 0.037), and 76.39 ± 1.84 vs. 45.69 ± 3.24 (p = 0.022), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in rucaparib-treated vs. CP+Rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 90.84 ± 3.41 vs. 75.45 ± 11.12 (p = 0.072), 79.78 ± 0.85 vs. 41.35 ± 3.77 (p = 0.043), and 83.03 ± 4.51 vs. 42.32 ± 9.77 (p = 0.044), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in talazoparib-treated vs. CP+Talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 94.22 ± 5.18 vs. 76.56 ± 14.34 (p = 0.069), 75.54 ± 0.66 vs. 44.24 ± 8.88 (p = 0.016), and 81.61 ± 2.42 vs. 43.22 ± 1.6 (p = 0.0449), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in niraparib-treated vs. CP+Niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 72.30 ± 2.66 vs. 57.30 ± 1.33 (p = 0.044), 56.44 ± 5.62 vs. 45.09 ± 1.59 (p = 0.021), and 74.43 ± 8.3 vs. 34.21 ± 5.23 (p = 0.031), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in olaparib-treated vs. CP+Olaparib-treated SV-HUC-1, UM-UC3, and T-24 groups were 48.84 ± 4.36 vs. 28.07 ± 2.90 (p = 0.002), 76.99 ± 2.12 vs. 62.88 ± 5.62 (p = 0.023), and 85.52 ± 5.75 vs. 63.90 ± 5.86 (p = 0.049), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in rucaparib-treated vs. CP+Rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 70.76 ± 2.66 vs. 70 ± 5.45 (p = 0.073), 87.42 ± 7.53 vs. 70.55 ± 6.46 (p = 0.066), and 94.73 ± 4.40 vs. 77.25 ± 3.94 (p = 0.048), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in talazoparib-treated vs. CP+Talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 45.76 ± 9.81 vs. 28.84 ± 5.02 (p = 2.47e-5), 56.44 ± 2.81 vs. 37.42 ± 4.61 (p = 1.22e-3), and 68.23 ± 0.97 vs. 43.98 ± 6.50 (p = 0.0031), respectively, compared with their respective DMSO-treated controls. The combinations of PARPi with sub-IC50 concentrations of cisplatin inhibited cell survival (Fig. 2B), proliferation (Fig. 2C), and clonogenic ability (Fig. 3A, B) of BLCA cells significantly, compared with the effects observed as single agents. The combination treatments also inhibited the survival, proliferation, and clonogenicity of normal urothelial cells. These results implied that PARPi may synergize with cisplatin in inhibiting the growth and survival of BLCA cells and effectively reduce the amount of cisplatin needed for anti-cancer effects.
PARPi induce apoptosis in BLCA cells in vitro
We treated BLCA cell lines UM-UC-3 and T-24 as well as the normal urothelial cells SV-HUC-1 with sub-IC50 concentrations of PARPi either alone or in combination with sub-IC50 concentration of cisplatin for 72 h and subjected the resulting cell lysates to Western blotting with the apoptosis markers cleaved caspases 3 and 9, or cleaved PARP. As shown in Fig. 4, cells treated with the combination of cisplatin and PARPi showed higher levels of caspase cleavage as well as that of PARP, indicating that PARPi not only suppress growth and proliferation of BLCA cells but also induce apoptosis.
In vivo synergism between PARPi and cisplatin
To establish that PARPi can synergize with cisplatin treatment in vivo, we generated xenografts of UM-UC-3 cells in male SCID mice followed by treatment with PARPi (olaparib, niraparib, talazoparib, or rucaparib), cisplatin, or cisplatin+different PARPi [6]. Toxicity was monitored by examining weight loss and serum ALP or AST activity. The xenografts were treated for 3 weeks and tumor tissues were collected. Immunohistochemistry was used to examine FFPE tumor sections for ki-67 and caspases 3, 7, and 9 to measure proliferation and apoptosis in the tumor tissues, respectively. At the end of the experiment, the vehicle control tumors measured 2287.021 ± 150.51 mm3. Niraparib (alone 1086.25 ± 76.2 mm3, p = 0.013 vs. CP+Niraparib 1037.48 ± 77.9 mm3, p = 0.034) and rucaparib (alone 1233.75 ± 61.65 mm3, p = 0.046 vs. CP+Rucaparib 1023.54 ± 64.21 mm3, p = 0.037) reduced tumor growth to a similar extent singly as well as in combination with cisplatin (Fig. 5A and C). On the other hand, olaparib showed strongly additive effects in reducing tumor growth when used in combination with cisplatin, while not being very effective as a single agent (olaparib alone 1865.63 ± 93.25 mm3, p = 0.07 vs. CP+Olaparib 641.28 ± 32.05 mm3, p = 0.002) (Fig. 5B). Talazoparib not only reduced tumor growth as a single agent but inhibited tumor growth virtually completely when used in combination with cisplatin (talazoparib alone 824.57 ± 41.2 mm3, p = 0.0061 vs. CP+Talazoparib 333.95 ± 16.65 mm3, p = 0.0018) (Fig. 5D). The P values shown are in comparison with the vehicle-treated tumors. There were no significant differences in average mouse weights between different treatment groups (Fig. 5E and Suppl. Fig. 1). ALP and AST activities in the sera, denoting potential hepatic injury, were not significantly different between the different treatment groups (Suppl. Fig. 2). Immunohistochemistry analyses demonstrated that tumor proliferation was suppressed in the PARPi and cisplatin combination treatments, while induction of apoptosis is evidenced by higher levels of cleaved caspases in the xenografts treated with the PARPi and cisplatin combinations (Fig. 6A and B).