Targeting mTOR and Survivin Concurrently Potentiates Radiation Therapy in Renal Cell Carcinoma by Suppressing DNA Damage Repair and Amplifying Mitotic Catastrophe

Abstract

Background: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation.

Experimental design: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization.

Results: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy.

Conclusion: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Keywords: Mitotic catastrophe; Radiation Therapy; Renal Cancer; Survivin; mTOR.

Figure 1. EY-L demonstrates a remarkable antiproliferative effect in RCC in vitro and in vivo.

MTS assay in 786-O (A) and Renca (B) cells treated with increasing concentrations of E-L, Y-L, or EY-L for 72 hours (n=4 wells per treatment condition). Interestingly, E-L did not show a noticeable effect in vitro whereas Y-L and EY-L show almost similar curves, which suggests that YM-155 is solely responsible for the antiproliferative effect of EY-L in RCC, at least in vitro. Here, 1% liposome is equivalent to ~4.1 μM (in E-L) or ~4.04 μM (in EY-L) everolimus, and ~6.7 μM (in Y-L) or ~6.51 μM (in EY-L) YM155. (C) Approximately 1 × 10⁶ Renca cells were injected subcutaneously in 6–8 weeks-old female Balb/c mice. When the average size of tumors reached 100 mm³, twice a week i.v. treatment started with E-L (1.94 mg/kg E), Y-L (1.44 mg/kg Y), or EY-L (1.94 mg/kg E, 1.44 mg/kg Y) (n=5 mice per group). The treatment was continued for 4 weeks, and tumor growth was monitored weekly. Both E-L and EY-L showed excellent inhibition of tumor growth, but EY-L was the most effective. Surprisingly, Y-L did not show any noticeable tumor growth inhibition. * denotes p<0.05. (D) Representative images of H&E and Ki67 stained tumor sections from the above experiment. EY-L showed noticeable reductions in Ki67 staining. Bar length = 200 μm. (E) Quantitation of Ki67-positive nuclei in tumor sections. EY-L showed significant reductions in Ki67-positive nuclei compared to all other groups. * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. EY-L demonstrates a remarkable antitumor effect in an orthotopic syngeneic mouse model of RCC.

(A) Approximately 1 × 10⁵ luciferase-transfected Renca cells were injected orthotopically into the left kidneys of 6–8 weeks-old female Balb/c mice. After 14 days, twice a week i.v. treatment started with EY-L (1.94 mg/kg E, 1.44 mg/kg Y) (n=4 for the control group, n=5 for EY-L group). The treatment was continued for 4 weeks, and tumor growth was monitored weekly by bioluminescence imaging. EY-L showed excellent inhibition of tumor growth as measured by reductions in the bioluminescence signals. The control mice reached the endpoint due to aggressive tumor growth after 2 weeks of starting treatment. (B) Tumor growth curves plotted as a fold change in RLU from initial measurements showed strong inhibition of tumor growth due to EY-L treatment. * and ** denote p<0.05 and p<0.01, respectively. (C) EY-L improved median overall survival compared to the control group (43 vs 15) as well. ** denotes p<0.01.

Figure 3. EY-L sensitizes RCC cells toward radiation in vitro by inhibiting cell cycle checkpoints and DNA damage repair.

A) 786-O and Renca cells were treated for sub-IC50 concentrations of E-L, Y-L, and EY-L (0.01% liposomes for 786-O, 0.125% liposomes for Renca) for 48 hours followed by exposure to 2 Gy radiation. A ‘no radiation’ control was included for each of the treatment groups. Cells were then harvested and re-seeded in 12 well plates at a concentration of 100 cells/well. Colonies were allowed to grow for 14 days followed by fixation with 4% paraformaldehyde and staining with 0.2% crystal violet solution. Representative images of the colonies were included. (B-C) Colonies greater than 50 cells were counted under a microscope and surviving fractions were determined. EY-L treatment led to the highest reduction in the surviving fraction in both cell lines. Bliss synergy scores were 0.81 and 0.50 for 786-O (B) and Renca (C) cells, respectively, signifying a moderate to strong synergistic effect of EY-L. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (D) 786-O and (E) Renca cells were treated for sub-IC50 concentrations of E-L, Y-L, and EY-L (0.01% liposomes for 786-O, 0.125% liposomes for Renca) for 48 hours followed by exposure to 2 Gy radiation. A ‘no radiation’ control was included for each of the treatment groups. Cells were then harvested and lysed, and Western Blot analysis was employed to determine alterations in expressions of various DNA damage repair proteins were analyzed. EY-L showed strong inhibition of ATR, Chk1, ATM, Chk2, and PARP1.

Figure 4. EY-L augments radiation therapy in an RCC xenograft model.

(A) Approximately 5 × 10⁶ 786-O cells were injected subcutaneously in the right flanks of 6–8 weeks-old female SCID mice. After 30 days, twice a week i.v. treatment started with EY-L (1.94 mg/kg E, 1.44 mg/kg Y) (n=4 for the control group, n=5 for EY-L group) and continued for 3 weeks. Two doses of focused 10 Gy radiation each were administered to the tumors on days 12 and 19 for mice belonging to the radiation-only (R) and combination group (EY-L + R). Additionally, a separate group of mice (R-early) received two doses of focused 10 Gy radiation on days 5 and 12. Treatment was stopped after 3 weeks, but weekly tumor growth monitoring was continued for an additional 3 weeks of washout period. The EY-L + R treatment group showed the highest inhibition of tumor growth. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (C) Representative images of H&E and Ki67 stained tumor sections from the above experiment. All the treatment groups showed noticeable reductions in Ki67 staining from the control group. Bar length = 200 μm. (D) Quantitation of Ki67-positive nuclei in tumor sections. All the treatment groups showed significant reductions in Ki67-positive nuclei compared to the control groups but were not significantly different among themselves. This might be due to the 3-week-long washout period. * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. EY-L augments radiation therapy in a murine syngeneic RCC model.

(A) Approximately 1 × 10⁶ Renca cells were injected subcutaneously in the right flanks of 6–8 weeks-old female Balb/c mice. After 14 days, twice a week EY-L (1.94 mg/kg E, 1.44 mg/kg Y, i.v.) treatment started (n=5 mice per group) and continued for 3 weeks. Two doses of focused 10 Gy radiation each were administered to the tumors on days 12 and 19 for mice belonging to the combination group (EY-L + R). The R (Early) group received two doses of focused 10 Gy radiation on days 5 and 12. Here we included only the R (Early) group based on the results obtained from the 786-O experiment for a stringent comparison. Treatment was stopped after 3 weeks, but twice-a-week tumor growth monitoring was continued throughout the washout period until the endpoint. The EY-L + R treatment group showed the highest inhibition of tumor growth. (C) A similar experiment was performed but was stopped 2 days after the final dose of radiation to harvest the tumors for immunohistochemistry. Here, the R (Early) group was replaced with the regular R group to keep the washout period the same between treatments. (D) Representative images of H&E, Ki67, CD45, CD3, and CD8 stained tumor sections from the above experiment. Bar length = 200 μm. (E) Quantitation of Ki67-positive nuclei in tumor sections (n=30, 10 visual fields each from 3 sections per group). The EY-L + R treatment group showed a significant reduction in Ki67-positive nuclei compared to all the other groups. * p<0.05, **** p<0.0001. (F-H) Quantitation of CD45+ (F), CD3+ (G), and CD8+ (H) cells in tumor sections (n=30, 10 visual fields each from 3 sections per group). No significant changes in CD45+ cells were observed, but the EY-L + R group showed the highest CD3+ and CD8+ staining suggesting greater infiltration of CD3+ and CD8+ T cells in the tumor. * p<0.05, ** p<0.01.

Figure 6. EY-L induces mitotic catastrophe in RCC tumors which is further enhanced by radiation therapy.

(A). Representative confocal images of fresh frozen tumor sections from the above experiment stained for DAPI (top panel) and pericentrin (middle panel). The bottom panel shows the merged images. Magnification = 40X. (B-C) Plots showing the pericentrin count per visual field (B) or pericentrin/nuclei ratio (C). Individually, the EY-L and R groups showed higher values than the control, but EY-L + R demonstrated the highest pericentrin count or pericentrin/nuclei ratio. A higher pericentrin/nuclei ratio indicates a higher incidence of mitotic catastrophe. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.