Inhibition of autophagy with chloroquine is effective in melanoma

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Inhibition of autophagy with chloroquine is effective in melanoma Michael E. Egger, MD,a,b Justin S. Huang, BS,b,c Wenyuan Yin, BS,b,c Kelly M. McMasters, MD, PhD,a,b and Lacey R. McNally, PhDb,c,* a

Hiram C. Polk Jr MD Department of Surgery, University of Louisville, Louisville, Kentucky James Graham Brown Cancer Center, Louisville, Kentucky c Department of Medicine, University of Louisville, Louisville, Kentucky b

article info

abstract

Article history:

Background: Cancer cells adapt to the stress resulting from accelerated cell growth and

Received 5 January 2013

a lack of nutrients by activation of the autophagy pathway. Two proteins that allow cell

Received in revised form

growth in the face of metabolic stress and hypoxia are hypoxia-inducible factor-1a (HIF-1a)

4 April 2013

and heat shock protein 90 (Hsp 90). We hypothesize that chloroquine (CQ), an antimalarial

Accepted 24 April 2013

drug that inhibits autophagosome function, in combination with either echinomycin,

Available online 17 May 2013

a HIF-1a inhibitor, or 17-dimethylaminoethylamino-17-dimethoxygeldanamycin (17DMAG), an Hsp 90 inhibitor, will result in cytotoxicity in melanoma.

Keywords:

Materials and methods: Multiple human melanoma cell lines (BRAF wild-type and mutant)

Melanoma

were tested in vitro with CQ in combination with echinomycin or 17-DMAG. These treatments

Chloroquine

were performed in hypoxic (5% O2) and normoxic (18% O2) conditions. Mechanism of action

Echinomycin

was determined through Western blot of autophagy-associated proteins HIF-1a and Hsp 90.

Hypoxia-inducible factor-1

Results: Chloroquine, echinomycin, and 17-DMAG each induced cytotoxicity in multiple

Heat shock protein 90

human melanoma cell lines, in both normoxia and hypoxia. Chloroquine combined with

Autophagy

echinomycin achieved synergistic cytotoxicity under hypoxic conditions in multiple

Autophagy inhibition

melanoma cell lines (BRAF wild-type and mutant). Western blot analysis indicated that

Autophagosome

echinomycin reduced HIF-1a levels, both alone and in combination with CQ. Changes in LC3

Cancer hypoxia

flux indicated inhibition of autophagy at the level of the autophagosome by CQ therapy. Conclusions: Targeting autophagy with the antimalarial drug CQ may be an effective cancer therapy in melanoma. Sensitivity to chloroquine is independent of BRAF mutational status. Combining CQ with the HIF-1a inhibitor echinomycin improves cytotoxicity in hypoxic conditions. ª 2013 Published by Elsevier Inc.

1.

Introduction

Outcomes for patients with cutaneous melanoma are quite good if the diagnosis is made early and aggressive surgical

resection is performed prior to metastatic spread; however standard systemic therapies for metastatic disease have dismal complete response rates of approximately 5% [1]. Newer agents targeting T-cell regulation (ipilimumab) or the

* Corresponding author. Department of Medicine, University of Louisville, 505 S. Hancock Street, CTR Rm 307, Louisville, KY 40202. Tel.: þ1 502 852 2288; fax: þ1 502 852 2123. E-mail address: [email protected] (L.R. McNally). 0022-4804/$ e see front matter ª 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2013.04.055

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V600E BRAF mutation (vemurafenib) have shown promising improvements in response rates, but most patients will progress or develop resistance [2e4]. The broad applicability of vemurafenib is limited, as only 50% of melanoma patients carry the V600E BRAF mutation [5,6]. Novel therapies are needed with improved durable response rates and wider target populations to treat metastatic melanoma. A potential target for cancer therapy is autophagy inhibition. Autophagy is a normal cellular response to stress in which organelles, cytoplasm, proteins, and metabolic byproducts are degraded; this process is crucial to the survival and growth of apoptosis-deficient cancer cells [7]. Autophagic processes protect the cellular genome and preserve limited metabolic resources in cancer cells in which dysregulated growth processes produce metabolic stress [8,9]. Inhibition of autophagy may disrupt this compensatory process, resulting in the accumulation of metabolic stress products and induction of cell death [7,10,11]. In this study, we evaluate chloroquine, 17dimethylaminoethylamino-17dimethoxygeldanamycin (17DMAG), and echinomycin to target cell stress response pathways. The antimalarial drug chloroquine (CQ) disrupts autophagy by inhibiting the acidification of the lysosomes that fuse with the autophagosomes, thereby preventing the degradation of metabolic stress products and inducing apoptosis [12e15]. Chloroquine-mediated inhibition of autophagy has been demonstrated in melanoma [16e18]. Heat shock protein 90 (Hsp 90) is an important molecular chaperone involved in protein folding and a regulator protein involved in the cellular response to metabolic stress that may be a useful target in cancer cells [19,20]. 17-DMAG, an analogue of geldanamycin, is an inhibitor of Hsp 90. Hypoxiainducible factor-1 (HIF-1) is a regulatory transcription factor involved in the hypoxic stress response and a potential anticancer target [21]. Echinomycin is an inhibitor of HIF-1a DNA binding activity [22]. Using a combination approach targeting both autophagy and the hypoxic stress response, we hypothesize that CQ is an effective antimelanoma therapy in vitro, particularly in hypoxic conditions found in melanoma. Combining CQ with either 17-DMAG or echinomycin may enhance the cytotoxicity of CQ therapy in normoxic and hypoxic conditions.

type (personal communication with D.S. Tyler regarding DM6 cell line) [23].

2.2.

Materials and methods

2.1.

Cell lines and culture conditions

Human melanoma cells lines SK-MEL-2 and A375 were obtained from the American Type Culture Collection (Rockville, MD). Human melanoma cell line DM6 was kindly provided by D.S. Tyler (Duke University, Durham, NC). SK-MEL-2 and A375 were cultured in Dulbecco’s modified Eagle medium, and DM6 cells were cultured in Iscove’s modified Dulbecco medium. All media were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% L-glutamine. Cell culture reagents were purchased from Gibco (Life Technologies, Grand Island, NY). Cells were cultured in 37 C, 5% CO2 incubators. Both DM6 and A375 melanoma cell lines have the V600E BRAF mutation, whereas SK-MEL-2 is BRAF wild-

Reagents

Chloroquine(Thermo Fisher, Waltham, MA), 17-DMAG, and echinomycin were purchased from Acros (Thermo Fisher Scientific, NJ), Tocris Bioscience (Bristol, United Kingdom), and Cayman Chemical (Ann Arbor, MI), respectively. Chloroquine stock solution of 10 mM was prepared with phosphatebuffered saline. Echinomycin and 17-DMAG stock solutions were prepared in dimethyl sulfoxide (10 mM and 1 mM, respectively). Reagents were stored at 20 C (echinomycin) or 80 C (chloroquine and 17-DMAG) until use.

2.3.

Cytotoxicity determination

Human melanoma cell lines were plated in 96-well black plates at a concentration of 2  103 cells/well. Twenty-four hours following plating and incubation under normoxic conditions, cells were treated with increasing concentrations of CQ , 17-DMAG, and echinomycin in quadruplicate, then incubated under normoxic or hypoxic conditions. Normoxic conditions were held in standard laboratory incubators at 37 C, 5% CO2, and 18% (ambient) O2 levels. Hypoxic conditions were maintained in an Oasis hypoxic incubator (Caron, Marietta, OH) at 37 C, 5% CO2, and 5% O2. Control cells were left untreated. Cell viability was determined at 24 and 48 h following treatment using the ATPlite assay system and read on a TopCount microplate counter (PerkinElmer, Waltham, MA). Percent cell viability was determined by dividing the treated cells’ luminescence counts by the corresponding control cells. Combination therapies with CQ and 17-DMAG and CQ and echinomycin were evaluated in a similar manner. Combination therapy results were analyzed with Calcusyn version 2.1 (Biosoft, Cambridge, United Kingdom) and synergistic effects were determined by calculating the combination indices by the method of Chou-Talalay (combination index < 1.0 indicates synergism) [24]. The nonparametric Kruskal-Wallis test was used to compare differences in viability across treatments. Statistical analysis was performed using SAS 9.3 (SAS, Cary, NC).

2.4.

2.

275

Protein analysis

Human melanoma cells A375 and DM6 were plated in 6-well plates and allowed to attach for 24 h in normoxic conditions. Cells were then left untreated (control), or treated with CQ alone (50 mM), echinomycin alone (50 pM), or CQ and echinomycin together. Treated cells were then incubated under either normoxic (18% O2) or hypoxic (5% O2) conditions as described above. Whole-cell protein lysates were collected 6 and 12 h following treatment using sodium orthovanadate (Sigma-Aldrich, St. Louis, MO) immediately upon removal from hypoxic or normoxic incubators. NuPAGE 4e12% Bis Tris gels were used for protein electrophoresis (Life Technologies, Grand Island, NY). Transfer to nitrocellulose membranes was performed using the iBLOT gel transfer device from Invitrogen (Life Technologies). Odyssey blocking buffer was used for Western blotting (LI-COR, Lincoln, NE). Primary antibodies for Western blotting were as follows: HIF-1a, LC3, and Atg 7

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Fig. 1 e Chloroquine, 17-DMAG, and echinomycin achieve cytotoxicity in multiple human melanoma cell lines. SK-MEL-2, A375, and DM6 human melanoma cell lines were plated at 2 3 103 cells/well in quadruplicate and treated at the indicated concentration with chloroquine (A), 17-DMAG (B), and echinomycin (C). Plated cells were then incubated with either ambient 18% O2 levels (normoxia) or 5% O2 (hypoxia). Cell viability was measured as a percentage of untreated cell viability as determined by ATPlite assay 48 h following treatment. Error bars represent 1 standard deviation.

(Novus Biologicals, Littleton, CO); Hsp 60 and Hsp 90 (Abcam, Cambridge, MA); Atg 5 (Epitomics, Burlingame, CA). Secondary antibodies were rabbit and mouse IRDye antibodies (LI-COR). Primary antibodies were incubated at 4 C overnight using the following concentrations: LC3 (1:500), actin (1:1000), Hsp 60 (1:1000), Hsp 90 (1:1000), Atg 5 (1:5000), Atg 7 (1:5000), HIF-1a (1:2000). Corresponding secondary antibodies were incubated for 1 h at 25 C (1:3000). Blots were developed and analyzed using the Odyssey Infrared Imaging System (LI-COR).

3.

Results

3.1.

Single-agent cytotoxicity

Cytotoxicity in multiple human melanoma cell lines was achieved with CQ in both normoxic and hypoxic environments (P  0.0015 for differences across treatment doses for each cell line by Kruskal-Wallis test) (Fig. 1A). Treatment using micromolar concentrations of 17-DMAG resulted in reduced cell viability when used alone in multiple melanoma cell lines (P  0.0011 for differences across treatment doses for each cell line by Kruskal-Wallis test) (Fig. 1B). Echinomycin was a potent cytotoxic agent in both normoxic and hypoxic environments (P  0.0125 for differences across treatment doses for each cell line by Kruskal-Wallis test) (Fig. 1C). Greater than 80% cytotoxicity was achieved after 48 h with picomolar concentrations of the drug in both BRAF wild-type and mutant human melanoma cell lines. In summary, CQ , 17-DMAG, and echinomycin

were cytotoxic to melanoma in vitro under both normoxic and hypoxic conditions, independent of BRAF mutation status.

3.2. Combination therapy of 17-DMAG or echinomycin with chloroquine Although combining CQ with 17-DMAG did not result in synergistic cytotoxicity in multiple human melanoma cell lines, CQ combined with echinomycin achieved synergistic cytotoxicity in hypoxic environments (Fig. 2). In both normoxic and hypoxic conditions, treatment with CQ , 17-DMAG, echinomycin, or combinations of CQ þ 17-DMAG/echinomycin resulted in significant decreases in cell viability (P < 0.05 by Kruskal-Wallis test). We next evaluated the synergistic effect of CQ combined with 17-DMAG or echinomycin in normoxic and hypoxic conditions. Although combination treatment of CQ and 17-DMAG did not result in synergy under normoxic conditions for any cell line (P > 0.05), CQ combined with 17-DMAG resulted in synergistic cytotoxicity in DM6 cells under hypoxia (combination index ¼ 0.052, P < 0.05 for index < 1.0). For the combination of CQ and echinomycin, synergistic cytotoxicity was seen in SK-MEL-2 (combination indices ¼ 0.816 and 0.326 for normoxia and hypoxia, respectively, P < 0.05 for index < 1.0) and DM6 (combination indices ¼ 0.899 and 0.523 for normoxia and hypoxia, respectively, P < 0.05 for index < 1.0) but not in the A375 cell line (combination indices ¼ 0.738 and 0.406 for normoxia and hypoxia, respectively, P > 0.05 for index < 1.0). In conclusion, combining CQ with echinomycin resulted in synergistic cytotoxicity in human melanoma cells

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Fig. 2 e Combining 17-DMAG with CQ does not significantly increase cytotoxicity in either normoxic or hypoxic conditions, whereas echinomycin and CQ together increase cytotoxicity in both normoxic and hypoxic conditions. Human melanoma cells SK-MEL-2, A375, and DM6 were plated at 2 3 103 cells/well in quadruplicate. Cells were treated with chloroquine (50 mM) and 17-DMAG (0.5 mM) separately or together in combination (A); likewise, cells were treated with chloroquine (25 mM) and echinomycin (25 pM) separately or in combination (B). Cell viability was compared with untreated (control) cell viability by ATPlite assay after incubating for 24 h in either normoxic (18% ambient O2) or hypoxic (5% O2) conditions. Error bars represent 1 standard error of the mean. Synergistic effect of combination therapy noted by * (combination index < 1.0, P < 0.05).

in vitro in both normoxic and hypoxic conditions; CQ combined with 17-DMAG was less effective.

3.3. Morphologic changes after chloroquine and echinomycin treatment We continued to study the effects of combining CQ with echinomycin because of the significant cytotoxicity found with echinomycin alone and in combination with CQ compared with 17-DMAG. Following 12 h of treatment with echinomycin, accumulation of the autophagosomes in the cytoplasm of treated cells was observed due to the deacidification of the lysosomes and the subsequent failure of the autophagosomes to undergo fusion with the lysosome and subsequent degradation of the contents of the autophagosome (Fig. 3). Echinomycin treatment alone does not produce the same macrovesicles in the cellular cytoplasm, but the large autophagosomes are again observed in combination therapy of CQ and echinomycin. The accumulation of large cytosolic autophagosomes after treatment with CQ and echinomycin indicate interruption of autophagy at the level of the autophagosome-autophagolysosome fusion process.

3.4. Protein changes following chloroquine and echinomycin treatment Protein analysis demonstrates that in multiple human melanoma cell lines, CQ treatment causes an increase in the LC3-II fraction relative to the LC3-I fraction (Fig. 4). Echinomycin

alone does not alter the LC3-I and LC3-II fractions. The autophagy regulator proteins Atg 7 and Atg 5 are not altered by CQ or echinomycin treatment, nor are Hsp 90 and Hsp 60 levels. As expected, HIF-1a levels are increased in the hypoxic control cells compared with the normoxic controls. HIF-1a levels are increased after chloroquine treatment alone in normoxic and hypoxic conditions. Echinomycin reduces HIF-1a levels compared with controls. Protein changes in LC3-I and LC3-II are consistent with the interruption of autophagy at the level of the autophagosome-autophagolysosome fusion, whereas the reduction of HIF-1a levels after treatment with echinomycin indicates a possible mechanism for the synergistic effect of CQ and echinomycin.

4.

Discussion

The most important finding in this study is that CQ is an effective melanoma therapy in multiple human melanoma cell lines in vitro in both normoxic and hypoxic environments. Echinomycin, a HIF-1a inhibitor, is a potent cytotoxic agent against multiple human melanoma cell lines. Synergistic cytotoxicity was achieved by combining CQ with picomolar concentrations of echinomycin. The mechanism of CQinduced toxicity is in part due to the interruption of autophagy at the level of the autophagosome. Echinomycin improves CQ-induced cytotoxicity in part by reducing levels of HIF-1a. Chloroquine is a well-characterized drug that has been used successfully for prophylaxis and treatment of malaria

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Fig. 3 e Morphologic changes, including the development of macroautophagic vesicles, following treatment with chloroquine and echinomycin. A375, SK-MEL-2, and DM6 human melanoma cell lines 12 h following treatment with chloroquine (50 mM) or echinomycin (50 pM) after incubation under normoxic conditions at 5% CO2 and 37 C. Magnification 3400, bar represents 50 mm, arrows indicate macroautophagic vesicles.

[25]. Chloroquine increases the pH of cellular lysosomes by disrupting the proton pump mechanism, raising the lysosomal pH by approximately 0.5e1.5 [12]. CQ has been shown to be an effective autophagy inhibitor that causes cancer cell death in a variety of cancers, including lymphoma, lung cancer, glioma, fibrosarcoma, breast cancer, retina cells, and colon cancer [13e15,18,26e30]. In melanoma, limited studies of CQ have shown promising results. Intraperitoneal injections of CQ prolonged survival in mice with subcutaneous B16 melanoma cell tumors [16]. More recent research has focused on autophagy inhibition by CQ in melanoma cells in the context of nutrient or hypoxic stress. The inhibition of hypoxia-induced autophagy by hydroxychloroquine promoted tumor regression in an in vivo subcutaneous B16 melanoma mouse model [31]. Chloroquine treatment combined with calorie restriction inhibited melanoma tumor growth in a subcutaneous mouse model of B16 melanoma cells [18]. Similarly, deprivation of leucine in combination with CQ decreased the growth of human melanoma tumors in a mouse xenograft model [17]. Our work offers a novel evaluation of CQ therapy in melanoma along two lines of reasoning: we evaluated CQ therapy in combination with novel agents that target the metabolic and hypoxic stress responses and we evaluated CQ therapy under hypoxic conditions. Combination therapy of CQ with alternative agents is feasible and has been demonstrated with several agents and therapies. CQ has been combined with all-trans retinoic acid and Akt inhibitors in breast cancer cells and high-dose interleukin 2 in pancreatic cancer cells [30,32,33]. In

melanoma cells, CQ enhances sensitivity to cucurbitacin B when used in combination [34]. Two novel targets of the metabolic stress response, Hsp 90 and HIF-1a, were chosen in this study for further evaluation of combination therapy with CQ. The Hsp 90 inhibitor 17-DMAG was evaluated in this study because it has shown promise as an anticancer therapy in limited preclinical data. Hsp 90 shuttles a wide variety of proteins through the many environmental stresses that must be addressed by the rapidly dividing cancer cell, including nutrient and proteotoxic stress, hypoxia, and genetic instability [35,36]. 17-DMAG is currently in phase I and II clinical trials in advanced solid tumor malignancies [37,38]. The HIF-1a inhibitor echinomycin was chosen in this study because of its potent effect on HIF-1a levels and the promising concept of targeting the hypoxic stress response in addition to autophagy. Echinomycin has been used to sensitize multiple myeloma cells to melphalan [39]. HIF-1 activity is increased in human malignant melanoma cells [40]. Thus, by combining CQ with agents that targeted the metabolic and hypoxic stress responses, we hypothesized that the effect of CQ would be enhanced. Our results demonstrated that chloroquine, 17-DMAG, and echinomycin are cytotoxic to melanoma in vitro. CQ and 17-DMAG were cytotoxic at micromolar concentration levels, whereas echinomycin was much more potent, with greater than 80% cytotoxicity with picomolar concentrations after 48 h of treatment. Combining CQ and 17-DMAG did not significantly improve cytotoxicity and was not synergistic,

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Fig. 4 e LC3-I and LC3-II fractions are altered by CQ and echinomycin treatment, both in normoxic conditions (N) and in 5% O2 hypoxic conditions (H), without changes in upstream autophagy regulators Atg5 or Atg7. HIF-1a levels are increased in hypoxia and with CQ treatment. Echinomycin decreases HIF-1a levels, both alone and in combination with CQ. HSP 90 and HSP 60 levels are not appreciably altered. Whole-cell protein lysates were collected 12 h after no treatment (control), CQ (50 mM), echinomycin (50 pM), or both in A375 and DM6 human melanoma cell lines. Normoxic conditions were at ambient (18%) O2 levels, 5% CO2, and 37 C. Hypoxic conditions were maintained at 5% O2 level, 5% CO2, and 37 C. Proteins were resolved on standard electrophoresis gels and transferred to nitrocellulose membranes, and standard blotting techniques were performed.

with the exception of one cell line (DM6) in hypoxia. These findings suggest that the autophagy and Hsp 90 pathways may not be related very closely in human melanoma. We saw improved additive and, in some cell lines, synergistic cytotoxicity with CQ and echinomycin combination therapy in both normoxia and hypoxia. The protein studies suggest a possible mechanism for this interaction. As expected, HIF1a levels were increased in hypoxic controls compared with normoxic controls. An unexpected finding was the CQinduced increase in HIF-1a levels compared with untreated controls in both normoxic and hypoxic conditions. This may be a compensatory mechanism by the cell in response to autophagy inhibition, or it may be due to the collection of hypoxic and metabolic stress-related proteins as a result of autophagy inhibition. This increase in HIF-1a may be a compensatory mechanism that limits the cytotoxicity of CQ when used as a single therapy. Echinomycin, as expected, decreased HIF-1a levels, both compared with controls and compared with the CQ-treated cells when echinomycin was combined with CQ. Thus, echinomycin may reduce the cell’s compensatory increase in HIF-1a as a result of increased metabolic stress following autophagy inhibition by CQ , thus improving CQ cytotoxicity. Protein analysis also demonstrated that CQ inhibits autophagy at the level of the autophagosome and autophagolysosome, leaving upstream autophagy regulators Atg 5 and Atg 7 relatively unchanged. LC3 proteins are associated with autophagosome membranes; LC3-I is cytosolic and LC3II is membrane bound [41]. Thus, LC3-II levels correlate with the levels of autophagosomes prior to fusion with lysosomes to form the autophagolysosomes in which protein

degradation takes place [41,42]. Thus, increased levels of LC3-II in the context of CQ treatment suggests that there is accumulation of autophagosomes near the terminal end of the autophagosome process due to the deacidification of lysosomes and the failure to progress through the autophagolysosome fusion process and subsequent degradation. Morphologic changes in the cellular cytoplasm support this mechanism, as large cytoplasmic vesicles accumulate after CQ treatment. These in vitro experiments are promising first steps in the characterization of both CQ and echinomycin as potential therapies for metastatic melanoma. Both CQ and echinomycin are effective against both BRAF wild-type and mutant cell lines. These findings must be replicated in animal studies. An important consideration for the future application of this work is the importance of having metastatic melanoma models in which to test these therapies. The critical weakness of the melanoma cells to autophagy inhibition is based on the reliance of aggressive, actively growing cells on increased autophagy to support unregulated cell growth. Higher levels of autophagy have been correlated with invasive and metastatic melanoma in human tissue samples [43]. Hypoxic conditions were used in the in vitro studies to more closely mimic the tumor microenvironment, enhancing the translational relevance of our findings to in vivo work. These cells lines grew readily in hypoxia. In fact, cell growth was statistically significantly increased in hypoxic controls by 25%e50% compared with normoxic controls after 24 h (data not shown). Aggressive metastatic melanoma subtypes thus may be more susceptible to CQ and echinomycin therapies that target the hypoxic stress response and autophagy.

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In conclusion, CQ and echinomycin are effective melanoma therapies in multiple human melanoma cell lines, regardless of BRAF mutation status. These agents are effective in aggressive melanoma cells in both normoxic and hypoxic conditions. Modulations of the hypoxic stress response in combination with the inhibition of autophagy are the likely mechanisms.

[18]

[19] [20]

Acknowledgment

[21]

This project was also supported by intramural funding from the University of Louisville.

[22]

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