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.

Fig. 1

EY-L demonstrates antiproliferative effect and radiosensitization in RCC cells in vitro. MTS assay in 786-O (A) and Renca (B) cells treated with increasing concentrations of E-L, Y-L, or EY-L for 72 h (n = 4 wells per treatment condition). Clonogenic assay in 786-O (C) and Renca (D) cells for determining radiosensitization in vitro. Representative images of the colonies were included. Colonies greater than 50 cells were counted under a microscope and surviving fractions were plotted for 786-O (E) and Renca (F) cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Western blot analysis of various DNA damage repair proteins from lysates of 786-O (G) and Renca (H) cells treated with sub-IC50 concentrations of E-L, Y-L, and EY-L for 48 h followed by exposure to 2 Gy radiation and incubation of 1 h and 6 h. A ‘no radiation’ control was included for each of the treatment groups

Fig. 2

EY-L demonstrates a remarkable antitumor effect in a subcutaneous syngeneic mouse model of RCC. (A) Growth curves for subcutaneous Renca tumors treated with E-L, Y-L, and EY-L (n = 5 mice per group). (B) Representative images of H&E and Ki67 stained tumor sections from the above experiment. Bar length = 200 μm. Quantitation of percentage of Ki67-positive nuclei (C), Ki67-positive nuclei count (D), and total nuclei count (E) in tumor sections (n = 30, 10 visual fields 0.25 mm² each from 3 different tumor sections per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

EY-L demonstrates a remarkable antitumor effect in an orthotopic syngeneic mouse model of RCC. (A) Bioluminescence images for orthtotopic Renca tumors treated with E-L, Y-L, and EY-L (n = 4 for the control group, n = 5 for EY-L group). 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. (C) Median overall survival from the above experiment. * p < 0.05, ** p < 0.01

Fig. 4

EY-L augments radiation therapy in an RCC xenograft model. (A) Timeline of the experiment. (B) Growth curves of subcutaneous 786-O tumors treated with EY-L, Radiation, and their combination (n = 5). Untreated control and R(Early) groups were also included for comparison. (C) A similar experiment was performed but was stopped 2 days after the final dose of radiation to harvest the tumors for immunohistochemistry. Here, only the R group was included to keep the washout period the same between treatments. The tumor growth curves were steeper here due to inoculation of a higher number of cells. (D) Representative images of H&E and Ki67 stained tumor sections from the above experiment. Bar length = 200 μm. Quantitation of percentage of Ki67-positive nuclei (E), Ki67-positive nuclei count (F), and total nuclei count (G) in tumor sections (n = 30, 10 visual fields 0.25 mm² each from 3 different tumor sections per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 5

EY-L augments radiation therapy in a murine syngeneic RCC model. (A) Timeline of the experiment. (B) Growth curves of subcutaneous Renca tumors treated with EY-L, and its combination with radiation. Untreated control and R(Early) groups were also included for comparison. No statistics was performed due to different endpoints. (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. Quantitation of percentage of Ki67-positive nuclei (E), Ki67-positive nuclei count (F), total nuclei count (G), CD45 + cells (H), CD3 + cells (I), and CD8 + cell (J) in tumor sections (n = 30, 10 visual fields 0.25 mm² each from 3 different tumor sections per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 6

EY-L induces mitotic catastrophe in RCC tumors which is further enhanced by radiation therapy. Representative images of H&E-stained tumor sections from experiments shown in Fig. 5 (A) and Fig. 2 (C) showing the presence of large multinucleated cells (indicated by white arrows) as evidence of mitotic catastrophe. (B, D) Quantification of mitotic catastrophe in these tumor sections (n = 30, 10 visual fields 0.25 mm² each from 3 different tumor sections per group). * p < 0.05, **** p < 0.0001