Paola Maycotte

Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It 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 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. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.

Autophagy and cancer therapy

Although autophagy has been shown to have a clear role as a tumor suppressor mechanism, its role in cancer treatment is still controversial. Because autophagy is a survival pathway activated during nutrient deprivation and other stresses, it is reasonable to think that autophagy can function as a tumor cell survival mechanism activated after cancer treatment. Such a mechanism could be widely important because most cancer treatments induce autophagy in tumor cells. Indeed, many papers have presented data suggesting that tumor cell autophagy induced by anti-cancer treatment inhibits tumor cell killing. However, it has also been proposed that autophagy is a cell death mechanism that could function as a backup when apoptosis is disabled. The fact that there are active clinical trials in patients both using autophagy inhibitors or inducers together with other cancer treatments underscores the importance of understanding and distinguishing between these opposing ideas. Here we discuss some of the recent work studying the role of autophagy with different cancer therapies.

Autophagy Inhibition for Chemosensitization and Radiosensitization in Cancer: Do the Preclinical Data Support This Therapeutic Strategy?

Recognition of the cytoprotective functions of autophagy that occur in tumor cells exposed to various forms of chemotherapy or radiation has generated intense interest in the possibility that pharmacological interference with autophagy could provide a clinical strategy for overcoming therapeutic resistance. Multiple clinical trials are currently in progress to evaluate the antimalarial agent chloroquine (generally in its clinical formulation as hydroxychloroquine) and its impact on various forms of cancer therapy. In this commentary/review, we focus on the relatively limited number of studies in the literature where chloroquine has been tested in combination with chemotherapy or radiation in experimental tumor-bearing animal models. We also present recent data from our own laboratories, in cell culture experiments as well as in vivo studies, which demonstrate that neither chloroquine nor silencing of an autophagy regulatory gene was effective in conferring radiation sensitivity in an experimental model of breast cancer. The capacity for sensitization by chloroquine appears to be quite wide-ranging, with dramatic effects for some drugs/tumor models and modest or minimal effects in others. One possible caveat is that, with only a few exceptions, experiments have generally been performed in xenograft models, thereby eliminating the involvement of the immune system, which might ultimately be proven to play a central role in determining the effectiveness of autophagy inhibition in chemosensitization or radiosensitization. Nevertheless, a careful review of the current literature suggests that caution is likely to be warranted in translating preclinical findings relating to autophagy inhibition as an adjunctive therapeutic strategy.

STAT3-mediated autophagy dependence identifies subtypes of breast cancer where autophagy inhibition can be efficacious

Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases, including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition, especially in combination with other therapy, could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for survival even in nutrient rich conditions without any additional stress, whereas others need autophagy only when stressed. Survival under unstressed conditions is due to cell type-specific autophagy regulation of STAT3 activity and this phenotype is enriched in triple-negative cell lines. This autophagy-dependency affects response to therapy because autophagy inhibition reduced tumor growth in vivo in autophagy-dependent but not in autophagy-independent breast tumors, whereas combination treatment with autophagy inhibitors and other agent was preferentially synergistic in autophagy-dependent cells. These results imply that autophagy-dependence represents a tumor cell-specific characteristic where autophagy inhibition will be more effective. Moreover, our results suggest that autophagy inhibition might be a potential therapeutic strategy for triple-negative breast cancers, which currently lack an effective targeted treatment.

Regulation of autophagy and chloroquine sensitivity by oncogenic RAS in vitro is context-dependent

Chloroquine (CQ) is an antimalarial drug and late-stage inhibitor of autophagy currently FDA-approved for use in the treatment of rheumatoid arthritis and other autoimmune diseases. Based primarily on its ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as primary or adjuvant therapy in multiple clinical trials for cancer treatment. Oncogenic RAS has previously been shown to regulate autophagic flux, and cancers with high incidence of RAS mutations, such as pancreatic cancer, have been described in the literature as being particularly susceptible to CQ treatment, leading to the hypothesis that oncogenic RAS makes cancer cells dependent on autophagy. This autophagy “addiction” suggests that the mutation status of RAS in tumors could identify patients who would be more likely to benefit from CQ therapy. Here we show that RAS mutation status itself is unlikely to be beneficial in such a patient selection because oncogenic RAS does not always promote autophagy addiction. Moreover, oncogenic RAS can have opposite effects on both autophagic flux and CQ sensitivity in different cells. Finally, for any given cell type, the positive or negative effect of oncogenic RAS on autophagy does not necessarily predict whether RAS will promote or inhibit CQ-mediated toxicity. Thus, although our results confirm that different tumor cell lines display marked differences in how they respond to autophagy inhibition, these differences can occur irrespective of RAS mutation status and, in different contexts, can either promote or reduce chloroquine sensitivity of tumor cells.

Autophagy Supports Breast Cancer Stem Cell Maintenance by Regulating IL6 Secretion

Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem cell (CSC) or tumor-initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44+/CD24low/− population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival, and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer. Implications: Modulation of autophagy in breast cancer has different and even opposite effects, indicating the need for a selection strategy when trying to manipulate autophagy in the context of cancer therapy. Mol Cancer Res; 13(4); 651–8. ©2015 AACR.

Autophagy Controls the Kinetics and Extent of Mitochondrial Apoptosis by Regulating PUMA Levels

Macroautophagy is thought to protect against apoptosis; however, underlying mechanisms are poorly understood. We examined how autophagy affects canonical death receptor-induced mitochondrial outer membrane permeabilization (MOMP) and apoptosis. MOMP occurs at variable times in a population of cells, and this is delayed by autophagy. Additionally, autophagy leads to inefficient MOMP, after which some cells die through a slower process than typical apoptosis and, surprisingly, can recover and divide afterward. These effects are associated with p62/SQSTM1-dependent selective autophagy causing PUMA levels to be kept low through an indirect mechanism whereby autophagy affects constitutive levels of PUMA mRNA. PUMA depletion is sufficient to prevent the sensitization to apoptosis that occurs when autophagy is blocked. Autophagy can therefore control apoptosis via a key regulator that makes MOMP faster and more efficient, thus ensuring rapid completion of apoptosis. This identifies a molecular mechanism whereby cell-fate decisions can be determined by autophagy.

Targeting autophagy in breast cancer.

Macroautophagy (referred to as autophagy here) is an intracellular degradation pathway enhanced in response to a variety of stresses and in response to nutrient deprivation. This process provides the cell with nutrients and energy by degrading aggregated and damaged proteins as well as compromised organelles. Since autophagy has been linked to diverse diseases including cancer, it has recently become a very interesting target in breast cancer treatment. Indeed, current clinical trials are trying to use chloroquine or hydroxychloroquine, alone or in combination with other drugs to inhibit autophagy during breast cancer therapy since chemotherapy and radiation, regimens that are used to treat breast cancer, are known to induce autophagy in cancer cells. Importantly, in breast cancer, autophagy has been involved in the development of resistance to chemotherapy and to anti-estrogens. Moreover, a close relationship has recently been described between autophagy and the HER2 receptor. Here, we discuss some of the recent findings relating autophagy and cancer with a particular focus on breast cancer therapy.

Autophagy inhibition delays early but not late stage metastatic disease

The autophagy pathway has been recognized as a mechanism of survival and therapy resistance in cancer, yet the extent of autophagy’s function in metastatic progression is still unclear. Therefore, we used murine models of metastatic cancer to investigate the effect of autophagy modulation on metastasis development. Pharmacologic and genetic autophagy inhibition were able to impede cell proliferation in culture, but did not impact the development of experimentally induced 4T1 and B16-F10 metastases. Similarly, autophagy inhibition by adjuvant chloroquine (CQ) treatment did not delay metastasis in an orthotopic 4T1, tumor-resection model. However, neoadjuvant CQ treatment or genetic autophagy inhibition resulted in delayed metastasis development, whereas stimulation of autophagy by trehalose hastened development. Cisplatin was also administered either as a single agent or in combination with CQ. The combination of cisplatin and CQ was antagonistic. The effects of autophagy modulation on metastasis did not appear to be due to alterations in the intrinsic metastatic capability of the cells, as modulating autophagy had no impact on migration, invasion, or anchorage-independent growth in vitro. To explore the possibility of autophagy’s influence on the metastatic microenvironment, bone marrow–derived cells (BMDCs), which mediate the establishment of the premetastatic niche, were measured in the lung and in circulation. Trehalose-treated mice had significantly more BMDCs than either vehicle- or CQ-treated mice. Autophagy inhibition may be most useful as a treatment to impede early metastatic development. However, modulating autophagy may also alter the efficacy of platinum-based therapies, requiring caution when considering combination therapies.

Abstract 4121: Autophagy influences the development of the pre-metastatic niche

Background: Autophagy is a lysosomal degradation process that is characterized by cellular self-consumption. Autophagy allows for the recycling and removal of damaged cytoplasmic material. Many types of cancer have elevated autophagic activity in order to sustain a high rate of metabolism and circumvent stress-induced cell death. However, little is known of autophagy9s role in metastasis. Therefore, we tested the effect of autophagy modulation in different mouse models and cell based assays that reflect different points along the metastatic cascade. Methods: Pharmacologic autophagy inhibition in the murine mammary carcinoma cell line 4T1 was achieved with 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1). Knockdown of autophagy critical genes Becn1 or Atg7 by lentiviral delivery of shRNA was also used as a means of inhibition. Proliferation, invasion, migration, and anchorage independent growth of cells were assessed in the presence or absence of active autophagy. For an experimental model of metastasis, Balb/c mice were challenged with 4T1 cells expressing luciferase (4T1-luc) injected via the tail vein. Mice were pre-treated with CQ (60 mg/kg, qd, i.p.) and monitored until the development of luciferase positive metastases. Mice were also challenged with 4T1 Becn1 knockdowns or cells pre-treated with CQ for 2h to assess effect of cellular autophagy impairment. An orthotopic model was also utilized by implanting 4T1-luc cells into the mammary fat pad, with tumors resected at 100-200 mm 3 and mice followed until metastasis development. CQ was given either adjuvantly, 24h after surgery, or neoadjuvantly, 24h after implantation. Autophagy stimulation, 2% Trehalose in drinking water, was also given as a neoadjuvant therapy. Bone marrow derived cells (BMDCs), mediators of pre-metastatic niche formation, were measured by flow cytometry in the lungs and blood of treated mice prior to the arrival of tumor cells. Results: Autophagy inhibition was able to reduce cell proliferation, but did not significantly delay metastasis in the experimentally induced model. Similarly, CQ was not effective as an adjuvant therapy. Yet, as a neoadjuvant therapy both CQ and Becn1 knockdown were able to significantly delay metastases. Autophagy stimulation actually sped up metastasis development. Autophagy modulation had no effect on invasion, migration, or anchorage independent growth. Conversely, altering autophagy did impact the number of BMDCs present in the lung and blood. Trehalose treated mice had more than either vehicle or CQ. In the blood, CQ treated mice had even less BMDCs present than vehicle treated mice. Conclusions: Autophagy does not seem to be required for metastatic colonization and survival. Nor does it seem to affect the metastatic capability of cells. Rather, autophagy may be influencing the pre-metastatic microenvironment by impacting BMDCs. Thus autophagy appears to be most essential before the arrival of tumor cells to the site of metastasis. Citation Format: Rebecca A. Barnard, Daniel Regan, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Autophagy influences the development of the pre-metastatic niche. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2015-4121