The Autophagy Inhibitor Chloroquine, Alone or in Combination with mTOR Inhibitors, Displays Anti-Tumor Effects in In Vitro and In Vivo Lung Carcinoid Models

Simple Summary Neuroendocrine neoplasms of the lung (lung carcinoids) are often diagnosed when they are not surgically curable, and treatment options are limited. One of the approved options for treating inoperable tumors is everolimus, an mTOR inhibitor. Activation of mTOR inhibits autophagy, which is a cell survival mechanism; everolimus may paradoxically encourage cancer cell survival via stimulation of autophagy. Chloroquine, a known antimalarial compound, inhibits autophagy. Our research is focused on the hypothesis that autophagy plays a key role in the development of tumor resistance to everolimus, and that chloroquine addition to an mTOR inhibitor increases their inhibitory effect on tumor growth. In this study, we examined the effects of chloroquine alone or in combination with mTOR inhibitors on lung neuroendocrine tumor models (cell lines and mice). We have shown that chloroquine alone suppresses tumor cells' viability and proliferation and increases their cytotoxicity and apoptosis; these effects are augmented when chloroquine is added to mTOR inhibitors. Apparently, chloroquine suppresses tumor cell growth in lung neuroendocrine neoplasms models, potentiating the effects of the mTOR inhibitors, and implying that more research is warranted to unravel its possible role in the clinical setting, in patients with advanced lung neuroendocrine neoplasms. (1) Background: Neuroendocrine neoplasms of the lung (LNENs, lung carcinoids) are often diagnosed at an advanced stage when they are not surgically curable, and treatment options are limited. One of the approved options for treating inoperable tumors is everolimus-an mTOR inhibitor (mTORi). Activation of mTOR, among many other effects, inhibits autophagy, which is a cell survival mechanism in general, and in tumor cells in particular. Everolimus may paradoxically encourage cancer cell survival. In practice, the drug inhibits tumor development. Chloroquine (CQ) is a known antimalarial compound that inhibits autophagy. Our research is focused on the hypothesis that autophagy plays a key role in the development of tumor resistance to mTORi, and that the addition of autophagy inhibitors to mTORi exerts a synergistic effect on suppressing tumor cell proliferation. We have recently demonstrated that the combination of CQ with different mTORi increases their potency compared with mTORi alone in both in vitro and in vivo models of pancreatic NENs. In this study, we examined the effects of CQ and mTORi on in vitro and in vivo LNEN models. Aims: Testing the effects of CQ together with mTORi on cell proliferation, apoptosis, and autophagy in in vitro and in vivo LNEN models. (2) Methods: The NCI-H727 LNEN cells were treated with CQ +/- mTORi. Cells' viability and proliferation were measured using XTT and Ki-67 FACS staining. The effects of the treatments on the mTOR pathway and autophagy were examined using Western blotting. Cytotoxicity was measured using a cytotoxicity kit; apoptosis was measured by PI FACS staining and Western blotting. We further established an LNEN subcutaneous murine xenograft model and evaluated the effects of the drugs on tumor growth. (3) Results: CQ alone suppressed LNEN cells' viability and proliferation and increased their cytotoxicity and apoptosis; these effects were augmented when CQ was added to an mTORi. We also showed the possible mechanisms for these results: on the one hand we could see a decrease in P62 levels and the absence of LC3-II (both inversely related to autophagy) following treatment with the mTORi, and on the other hand we could demonstrate an increase in their levels when CQ was added. The effect was less apparent in the murine xenograft model. (4) Conclusions: By inhibiting autophagy and inducing apoptosis, CQ suppresses tumor cell growth in LNENs. CQ potentiates mTORi effects, implying that further studies are needed in order to elucidate its possible role in tumor inhibition in patients with LNENs.

Automated summary

In advanced stage lung neuroendocrine neoplasms, treatment options are limited. The mTOR inhibitor everolimus can paradoxically encourage cancer cell survival. Chloroquine, an autophagy inhibitor, suppresses tumor cell growth and enhances the effects of mTOR inhibitors. More research is needed to explore the potential role of chloroquine in treating patients with advanced lung neuroendocrine neoplasms.