Development of the Anti-cancer Therapeutic Modalities Overcoming Radio-resistance in Metastatic Brain Tumors

Abstract

Brain metastases are the most common form of adult central nervous system (CNS) tumors, outnumbering primary brain tumors by 10:1. Brain metastases occur late in the progression of multiple types of cancer and are associated with poor patient survival. These metastases most commonly arise from cancers of the lung, breast, and skin (melanoma). Standard treatment options include symptomatic therapy with corticosteroids and whole-brain radiotherapy (WBRT), and more aggressive approaches such as surgery or radiosurgery are used in a subset of patients. The most successful therapeutic tool in brain metastases has been radiation therapy. However, resistance to radiation is a major cause of recurrence or treatment failure. Imperative goals for future research include optimizing the efficacy of radiation therapy against metastatic tumor cells compared with normal brain cells, and preventing the cognitive losses that a proportion of patients suffer. In this study, we seek to find functional radio-sensitization targets to translate to the clinic. Recently, signal pathways about DNA damage checkpoints after irradiation and correlation of c-Met and radio-resistance have been noticed. However, it is insufficient of clues to translate to the clinic in metastatic brain cancers. Therefore, we investigated whether Chk1, one of the components of DNA damage checkpoint signaling and c-Met, a hepatocyte growth factor receptor, could be targeted for radiosensition in lung cancer brain metastases and breast cancer brain metastases, respectively. We proved that there is a correlation between Chk1 activation and prognosis of brain metastases from patients with metastatic brain tumor and Chk1 is highly activated in respond to radiation in radio-resistant lung cancer cells. To prove a role for Chk1 as a target for radio-sensitization, we investigated radio-sensitizing effects of the Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastases. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation, stabilized Cdc25A and suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastases, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA showed an enhancement of sensitivity to radiation in PC14PE6 cells. Also, life span was significantly increased by depletion of Chk1 in combination with radiation treatment in a radio-resistant lung cancer brain metastatic xenograft model. The results of this study strongly support our hypothesis that Chk1 is a good target for radio-sensitivity enhancement. To find out the correlation between c-Met and radio-resistance, we investigated c-Met protein intensity and c-Met mRNA level after radiation in different breast cancer cell lines. In MDA-MB-435 and MDA-MB-231 cell lines, c-Met protein intensities and c-Met mRNA levels were increased after radiation. Also, c-Met was elevated in the radio-resistant clone, which was acquired from xenograft models of breast cancer brain metastases. A combination of c-Met silencing, using shRNA and radiation, showed regression of tumor growth in the breast cancer orthotopic xenograft model and prolonged median survival and decreased tumor mass volume in the brain metastatic xenograft model. Also, a combination of c-Met silencing and radiation significantly induced apoptosis in the breast cancer brain metastatic model. The results of this study support our hypothesis that c-Met is a good target for radiosensitivity enhancement. Taken together, we suggest that Chk1 and c-Met can be functional targets in lung and breast cancer brain metastases for developing radio-sensitizers, which can be translated to the clinic.