Chloroquine (CQ) inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of some tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. It also has been shown that CQ sensitizes cancer cells with an autophagy-indipendent pathway.
Literature articles
Since autophagy is thought to act as a cell-survival pathway in cancer, CQ has been used in combination with diverse chemotherapeutic drugs and radiation and has been shown to enhance tumor cell killing. Increased signaling through the PI3K / mTOR pathway induces cellular transformation, as well as tumor progression and metastasis; in addition to its role in cell growth, the PI3K/mTOR pathway is one of the main regulators of macroautophagy. PI3K pathway inhibition decrease tumor growth in vitro but apoptosis was only induced after the addition of lysosomotropic agents. These effects were attributed to the inhibitory effect of CQ in autophagy induced PI3K pathway inhibition.
Although autophagy has become a well-accepted survival pathway, it has also been suggested that when cellular damage is extensive, autophagy could be cytotoxic. Both mechanisms could be important during cancer treatment since many chemo-therapeutic drugs as well as radiation therapy induce autophagy in a variety of cancer cell lines. CQ-mediated chemosensitization to therapy may be acting through mechanisms other than by inhibition of autophagy; however the role of autophagy during cancer treatment is controversial. Although it is widely accepted that many anticancer agents induce autophagy in cancer cells, it is not yet clear if this autophagy represents a survival mechanism activated in response to stress induced by the treatment, if it is a cell death pathway activated when apoptosis is disabled, if it has no effect on tumor cell viability at all, or if all three effects arise in different contexts. It will be necessary to better understand under which circumstances autophagy is or is not chemo-protective and to develop more selective autophagy inhibitors if we are to maximize the benefits of autophagy manipulation during cancer therapy.
Autophagy is recognized as a crucial cell survival pathway that enables tumor cells to overcome stressors in the tumor microenvironment as well as injuries caused by treatments such as endocrine therapy, chemotherapy, and radiation therapy. Because the abrogation of autophagy via knockdown of autophagy-related molecules potentiates the resensitization of therapy-resistant cancer cells to conventional cancer therapies, there has been great interest in developing clinically relevant autophagy inhibitors. CQ-based, ‘‘customized’’ combinatorial approaches could prove to be more effective and autophagy-specific when combined with molecularly targeted drugs in biomarker-identified patient subpopulations whose carcinomas are addicted to specific, autophagy-related mechanisms of malignant transformation.
Some evidences show that protective autophagy regulates primary resistance to trastuzumab and that adding CQ to trastuzumab-based regimens may significantly improve outcomes among women with autophagy-addicted, HER2-positive breast cancer. These tumor cells display increased cellular levels of the LC3-II protein (the mammalian homolog of Atg8) - a finding that correlates with increased numbers of autophagosomes - and decreased levels of the autophagy receptor p62 / SQSTM1, a protein selectively degraded by autophagy. CQ impedes autophagic resolution of the accumulation of autophagolysosomes formed in the presence of trastuzumab, cells commit to die by apoptosis. Importantly, the simultaneous combination of trastuzumab and CQ increased expression of Bax while reducing that of Bcl-2, which resulted in the synergistic increase in Bax/Bcl-2 ratio (a pro-apoptotic pathway).
It’s important to say that the supra-additive interaction between CQ and trastuzumab may involve other molecular mechanisms. For the first time, these findings confirm this scenario in vivo, as co-treatment with CQ and trastuzumab was dramatically more effective in reducing the tumor growth of trastuzumab-refractory xenograft tissues implanted in mice than treatment with each agent alone.
This study illustrates a novel approach to improve the efficacy of breast cancer radiation therapy by blocking endosomal pathways, which enhances radiation-induced cell death within the field and drives antitumor immunity to assist therapeutic cure. Recently CQ was shown to enhance death preferentially in cells overexpressing the oncogene Myc. The fact that low-dose CQ is chemopreventative in vivo suggests that immune-mediated tumor rejection may also be enhanced. Although this hypothesis has received little exploration in the context of immunotherapy for breast cancer, there is some evidence that CQ stimulates immunogenic tumor cell death in response to some chemotherapeutic agents. The main observations of this study are that CQ given after radiation increases the rapid death of MCaK breast cancer cells in vitro and that such cells are more immunogenic and can enhance radiation-induced tumor regression in vivo. CQ treatment of irradiated MCaK cells led to rapid apoptotic cell death that was more immunogenic, and this was probably the main reason for the efficacy of CQ in vivo versus in vitro when combined with radiation; the in vivo effects seemed more dramatic in terms of tumor cure, which was evident in immune-competent but not immune-incompetent mice. This strongly suggests that the drug has potent immune-modulating effects. We conclude that CQ has potential to add survival benefits for some cancer patients undergoing radiation therapy, although its effectiveness may vary depending upon the mode of cell death induced by irradiation. However, in the best situation, promotion of an immunogenic form of cell death and better antigen cross-presentation together with increased intrinsic radiosensitivity could result in superior local tumor control by radiation therapy and drive immune elimination of micrometastases.
This is a commentary/review, where authors focus on the relatively limited number of studies in the literature where CQ has been tested in combination with chemotherapy or radiation in experimental tumor-bearing animal models. They saw that studies in xenograft models alone may be insufficient for accurately determining the capacity of autophagy inhibition to sensitize tumors to chemotherapy or radiation, as a recent study has shown that therapeutic success may be highly dependent on the immune system; indeed, autophagy was determined to be essential for the release of immunogenic ATP from dying cells. Taken together with the studies in animal models discussed in this review, it can be predicted that autophagy inhibition is unlikely to be uniformly effective in increasing radiation or chemotherapeutic sensitivity across the tumor spectrum. However, this is not unusual or unexpected given that the sensitivities of malignancies of different origins to chemotherapeutic approaches can differ by orders of magnitude. It is possible, but as yet untested, that the strength of autophagy induction in each system might determine its susceptibility to autophagy inhibition and sensitization. It follows that it will be critical to identify biomarkers that will distinguish between malignancies that are likely to benefit from autophagy inhibition as a strategy for chemosensitization or radiation sensitization and those tumors which are unlikely to benefit from this therapeutic approach.
Conclusions
After some researches, it’s clear that this topic is still in an early development. The relationship between autophagy and cell fate is complex, as autophagy can be a survival or a death pathway depending on the context. It’s also important to say that CQ has different mechanism other than blocking autophagy (e.g. the immune activation against tumor cells), and it has not the same effect in all type of tumors. However, in considering the variability and uncertainty in the current literature relating to autophagy inhibition as a therapeutic strategy caution appears to be warranted in extrapolating to the clinic from the limited preclinical data in animal models of cancer that have combined CQ with chemotherapy or radiotherapy. Indeed, there are some clue that CQ could be used against cancer, but it’s too early for a clinical application.
Matteo Barba
Alessandro Di Stefano
