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Molecular targeting for tumor radiosensitization: implications of apoptosis and autophagy signaling in combined anticancer therapy

par Moretti, Luigi
Président du jury Bergmann, Pierre
Promoteur Van Laethem, Jean-Luc
Co-Promoteur Van Houtte, Paul
Publication Non publié, 2015-11-19

Thèse de doctorat

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Résumé :	The central hypothesis supporting the present work is that the effectiveness of radiation therapy for cancer is often limited due to defects in key apoptosis regulators, such as Bcl-2 family members, that contribute to cancer ability to evade apoptosis. One way to bypass this resistance to radiotherapy is to target cell death pathways, aiming to sensitize tumours to radiation and enhance the therapeutic ratio in cancer. To test this central hypothesis, we took a dual approach: one targeted apoptosis and the other targeted autophagy.
	First, we focused on the apoptotic signaling. The Bcl-2 family comprises antiapoptotic members, such as Bcl-2, Mcl-1, and Bcl-XL, and proapoptotic members, such as Bax, Bak, and Bid. The Bcl-2 family controls the integrity of the outer mitochondrial membrane and is critical in determining the susceptibility of cells to apoptosis induced by the intrinsic pathway. The balance between cell survival and cell death is modulated by the ratios and interactions of antiapoptotic and proapoptotic Bcl-2 family proteins. Overexpression of Bcl-2 or Bcl-XL is observed in several cancers, including lung, colorectal, prostate, and breast cancers, and has been shown to confer resistance to various anticancer agents, including radiotherapy. In cancer cells, alterations in the amounts of these antiapoptotic Bcl-2 proteins promote cell survival, among others by contributing to their evasion from treatment-induced apoptosis. We made the observation that lung cancer cells have different radiosensitivity. On the basis of their relative response to radiotherapy, we stratified lung cancer cells into two groups (higher or lower sensitivity), and selected a representative cell line of each group for more in-depth study: A549 (resistant) and HCC2429 (sensitive). We found that the expression levels of Bcl-XL expression, which is antiapoptotic, was dramatically higher in A549, whereas almost not detected in HCC2429. We then hypothesized that AT-101, a pan-Bcl-2 inhibitor, had the potential to radiosensitize lung cancer by restoring radiation-induced apoptosis. When administered alone, AT-101 resulted in increased apoptosis in a concentration-dependent manner in both groups, with enhanced activity in HCC2429 even at lower concentration. Furthermore, AT-101 promoted radiosensitivity of A549 and HCC2429 cells (p < 0.005). A549 cells required increased AT-101 dose to achieve the same level of cytotoxicity than HCC2429 cells. These investigations suggest that the Bcl-2 family members may serve as effective therapeutic targets in lung cancer. However, the potential of AT-101 as an agent that enhances the therapeutic ratio of radiotherapy varies depending on the lung cancer clone.
	Next, we turned to a different approach, focusing on the inhibition of apoptosis instead of its promotion. This work hypothesis was based on previous observations looking at the role of radiation-induced apoptosis by knockdown of Bak and Bax. The radiosensitivity of breast and lung cancer in vitro was increased through autophagy, an alternate type of programmed cell death. Consistently, radiation-induced apoptosis accounts for a minor portion of cell death in irradiated solid tumors. The hypothesis of our work was that apoptosis inhibition would increase radiation-induced autophagy and tumor sensitivity to radiation. To block apoptosis, we used Z-VAD, a broad-spectrum caspase inhibitor, and examined its in vitro and in vivo effects on breast and lung cancer models. Z-VAD markedly radiosensitized breast and lung cancer cells in vitro, with a radiation dose enhancement ratio of 1.31 (P < 0.003). The enhanced tumor cytotoxicity was associated with induction of autophagy. In both breast and lung cancer mice xenograft models, the administration of Z-VAD concurrent with radiation produced a significant tumor growth delay compared with radiation alone and was well tolerated. Interestingly, Z-VAD also had a dramatic antiangiogenic effect when combined with radiation both in vitro and in vivo. Thus, Z-VAD represents an attractive anticancer therapeutic strategy. We further explored the potential of apoptosis inhibition as a way to sensitize cancer to radiation using a more selective chemical, M867, which is a reversible caspase-3 inhibitor. In an in vivo mouse hind limb lung cancer model, the administration of M867 with ionizing radiation was well tolerated, and produced a significant tumor growth delay compared with radiation alone. A dramatic decrease in tumor vasculature and tumor cell proliferation was observed with M867 despite the reduced levels of apoptosis. The radiosensitizing effect of M867 through the inhibition of caspases was validated using a caspase-3/-7 double-knockout (DKO) mouse embryonic fibroblasts (MEF) cell model. Consistent with our previous results, autophagy contributed to the mechanism of increased cell death, following inhibition of apoptosis. Finally, we investigated the mechanism by which radiation triggers autophagy in caspase-3/7-deficient cells, and found the involvement of endoplasmic reticulum (ER) stress. The ER activates a survival pathway, the unfolded protein response, which involves ER-localized transmembrane proteins such as protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1, and activating transcription factor-6. In this study, we found that PERK is essential for radiation-induced autophagy and radiosensitivity in caspase-3/7 double-knockout cells. Irradiation of these cells increased expression of phosphorylated-eIF2a. Similar results were seen after administration of tunicamycin (TM), a well-known ER stress inducer. We found that the administration of TM with radiation in MCF-7 breast cancer cells, which are lacking functional caspase-3 and are relatively resistant to many anticancer agents, enhances radiation sensitivity. Our findings revealed ER stress as a novel potential mechanism of radiation-induced autophagy in caspase-3/7-deficient cells and as a potential strategy to maximize efficiency of radiation therapy in breast cancer. Our data suggested that caspase-3 has a critical role in modulating the PERK/eIF2a pathway after radiation.
	Many cancers exhibit multiple deregulations in cell death pathways, allowing for the subsequent promotion of tumor cell survival, and contributing to a relatively low response rate to therapies based on the use of pro-apoptotic strategies. As we have showed, there is a potential for novel anticancer strategies to overcome resistant cancer cells with defective apoptosis machinery in order to improve overall therapeutic outcomes. Such novel approach is to drive cancer cells towards autophagy, as demonstrated by our experiments that studied the effect of radiation on the induction of autophagy in caspase-deficient models.