Combination treatment with ABT-737 and chloroquine in preclinical models of small cell lung cancer
© Zinn et al; licensee BioMed Central Ltd. 2013
Received: 27 August 2012
Accepted: 27 February 2013
Published: 2 March 2013
New therapies are urgently needed for patients with small cell lung cancer (SCLC). Chemotherapy and targeted therapies, including the Bcl-2 inhibitor ABT-737, may induce tumor cell autophagy. Autophagy can promote survival of cancer cells under stress and comprise a pathway of escape from cytotoxic therapies.
We explored the combination of ABT-737 and chloroquine, an inhibitor of autophagy, in preclinical models of SCLC. These included cell culture analyses of viability and of autophagic and apoptotic pathway induction, as well as in vivo analyses of efficacy in multiple xenograft models.
Combination treatment of SCLC lines with ABT-737 and chloroquine decreased viability and increased caspase-3 activation over treatment with either single agent. ABT-737 induced several hallmarks of autophagy. However, knockdown of beclin-1, a key regulator of entry into autophagy, diminished the efficacy of ABT-737, suggesting either that the effects of chloroquine were nonspecific or that induction but not completion of autophagy is necessary for the combined effect of ABT-737 and chloroquine. ABT-737 and chloroquine in SCLC cell lines downregulated Mcl-1 and upregulated NOXA, both of which may promote apoptosis. Treatment of tumor-bearing mice demonstrated that chloroquine could enhance ABT-737-mediated tumor growth inhibition against NCI-H209 xenografts, but did not alter ABT-737 response in three primary patient-derived xenograft models.
These data suggest that although ABT-737 can induce autophagy in SCLC, autophagic inhibition by choroquine does not markedly alter in vivo response to ABT-737 in relevant preclinical models, arguing against this as a treatment strategy for SCLC.
KeywordsAutophagy Apoptosis ABT-737 Chloroquine Primary xenograft
Autophagy is an evolutionarily conserved, reversible process in which the cell degrades its own cytoplasmic components utilizing lysosomal pathways. Typically, autophagy is activated under conditions of cell stress, including nutrient deprivation, environmental stressors, accumulation of misfolded proteins, and growth factor signal interruption. Cytoplasmic components are sequestered into double-membraned vesicles, or autophagosomes, which then fuse with lysosomes and the contained material is degraded to amino acids, nucleotides, sugars, and fatty acids that are released into the cytoplasm for biosynthesis and metabolism [1, 2].
Recent evidence suggests that autophagy is frequently activated in tumor cells treated with chemotherapy and radiation [3–7]. While persistent activation of autophagy may lead to tumor cell death, accumulating evidence suggests that it can serve as a protective pathway for cells to escape apoptotic cell death, allowing survival through periods of stress, growth factor withdrawal, or nutrient deprivation [8, 9]. Several studies have shown that autophagy delayed apoptotic death in cancer cells undergoing chemotherapeutic treatment. Treatment of these cells with inhibitors of autophagy, such as chloroquine, or knockdown of essential autophagy genes (beclin-1, ATG genes) resulted in enhanced therapy-induced apoptosis [10–14]. Chloroquine acts as a lysosomal pH inhibitor, therefore preventing the fusion of autophagosomes with lysosomes and inhibiting late stages of autophagy [15, 16]. These findings have led to the initiation of multiple clinical trials combining autophagy inhibitors and chemotherapeutic agents for diverse cancer types .
Small cell lung cancer (SCLC) comprises approximately 15% of all lung cancers and is a remarkably aggressive disease . SCLC is characterized by rapid proliferation and early dissemination to metastatic sites. While standard of care platinum-based chemotherapy induces responses in up to 80% of newly diagnosed SCLC cases, these responses are short-lived: the median survival in patients with advanced SCLC is approximately 9 months from the time of diagnosis . New therapies are critically needed for this disease.
The anti-apoptotic protein Bcl-2 is overexpressed in the majority of SCLC cases [20, 21]. Suppression of Bcl-2 can trigger apoptosis in SCLC, and enhance the anti-cancer efficacy of cytotoxic therapies . ABT-737, a Bcl-2 homology domain 3 mimetic (BH3), which inhibits the activity of Bcl-2 and Bcl-XL, has been shown to have potent activity against many SCLC cell lines . However, the efficacy of ABT-737 is variable across SCLC cell lines, and this agent has only modest single agent activity against most primary xenograft models of SCLC . Consistent with the observed level of activity in primary xenografts, while clinical trials of ABT-263 (an orally bioavailable derivative of ABT-737 for human use) have shown pharmacodynamic evidence of on-target activity, the overall response rate in SCLC patients has been disappointing [25, 26].
We hypothesized that SCLC resistance to ABT-737 may be in part due to cancer cell induction of autophagy. Bcl-2 has been shown to directly bind and inhibit Beclin-1 (BECN1), a critical mediator of initiation of autophagy . Treatment of HeLa and MCF7 cells with ABT-737 or gossypol, another purported BH3 mimetic, has been shown to inhibit the interaction of Beclin-1 and Bcl-2/Bcl-XL and stimulate autophagy [28–30]. We sought to define whether the treatment of SCLC cell lines with ABT-737 induces autophagy and whether this induction is a mechanism of therapeutic resistance. We also assessed the in vivo efficacy of the combination of ABT-737 and chloroquine using patient-derived xenograft (PDX) models of SCLC.
We have previously reported on a series of PDX tumors, generated by direct transfer of human tumor into immunosuppressed mice, which we have used as a preclinical platform for testing novel therapeutic strategies, including ABT-737 [24, 31]. For correlative in vitro analyses, we have also generated derivative cell lines from two of these primary xenografts, LX22 and LX33. Cell lines derived from LX22 and LX33 both demonstrated greater loss of viability, as determined by MTS, in response to the combination of ABT-737 and chloroquine than to either agent alone (p < 0.003 for both, see Figure 1B).
These results suggested that inhibition of autophagy could enhance the efficacy of ABT-737. Alternatively, chloroquine could be having a cytotoxic effect unrelated to inhibition of autophagy in these cells. We interrogated this intersection between apoptotic and autophagic pathways using an alternative pharmacologic inhibitor of autophagy, 3-methyladenine (3MA). Treatment with 3MA, with or without ABT-737, in H209 and H345 cells resulted in similar results to those obtained with chloroquine: clear evidence of enhanced cytotoxic response in cells treated with the combination relative to either agent alone (p < 0.0015 for both, see Figure 1C).
Similar suppressive effects on apoptotic induction were seen in H209 and H345. Taken together with data demonstrating that ABT-737 can activate autophagy (as determined by LC3-II levels and autophagosome formation), and that two inhibitors of autophagy (chloroquine and 3MA) can augment ABT-737 efficacy, the entirely unexpected results obtained using BECN1 knockdown suggest that the complex of Bcl-2 and Beclin-1 may have functional significance in the regulation of apoptosis by Bcl-2, independent of the suppressive effect on autophagy.
In this study we sought to assess whether the efficacy of the targeted Bcl-2 inhibitor, ABT-737, against SCLC could be improved by concurrent inhibition of autophagy with the clinically available agent chloroquine. In vitro analyses of the combination of ABT-737 and chloroquine in several SCLC cell lines did show a decrease in viability and a concomitant increase in caspase-3 activation. In addition, we found evidence that ABT-737 induced autophagy in several cell lines by the conversion of LC3-I to LC3-II seen on western blots and increased autophagosome formation as documented by TEM.
However, a targeted genetic approach to blocking entry into autophagy based on shRNA knockdown of BECN1 yielded the opposite result: a consistent decrease in apoptotic response to ABT-737, with or without chloroquine. There are several possible explanations for these seemingly contradictory results. First, the effects seen with chloroquine may not have been specific to the autophagy pathway. Indeed, we were able to demonstrate that the combination of ABT-737 and chloroquine can lead to marked inhibition of Mcl-1 and upregulation of NOXA, both of which would serve to facilitate apoptotic induction, independent of an effect on autophagy. It is known that chloroquine can also act on several other pathways including those involved in immune modulation and DNA damage .
Second, while chloroquine inhibits the late stages of autophagy, i.e. the fusion of autophagosomes with lysosomes leading to degradation of vesicular contents, Beclin-1 is required for an initial commitment toward autophagy. It is possible that successful initiation of autophagy without the ability to complete the process leads to intracellular accumulation of toxic materials, leading to enhanced apoptotic cell death, while blockade of entry into this pathway does not. Some reports do suggest that specific stage of autophagy inhibition can result in different therapeutic outcomes [5, 36]. Our data with 3MA would argue against this explanation to some extent: 3MA inhibits initiation of autophagy at a stage closely related to that of Beclin-1, but unlike Beclin-1 suppression, augmented the apoptotic response to ABT-737 in multiple lines. 3MA is a class I and class III PI3K inhibitor , and could, like chloroquine, have other relevant effects leading to enhanced programmed cell death.
Third, and perhaps most intriguingly, Beclin-1 and Bcl-2 are known to directly interact, and shRNA suppression of BECN1 may affect Bcl-2 function or localization independent of any effect on induction of autophagy. While previous data suggest that Beclin-1 does not affect the anti-apoptotic activity or localization of Bcl-2, this finding may be context and cell type-specific; these studies were performed in HeLa cervical carcinoma cells and mouse embryonic fibroblasts . One possible model to explain our results would be if Beclin-1 sequesters some fraction of the cellular Bcl-2 away from the mitochondrial membrane, where Bcl-2 primarily acts to suppress outer mitochondrial membrane disruption. Targeted loss of Beclin-1 could lead to a preferential localization of Bcl-2 to mitochondria, decreasing ABT-737 efficacy by functional upregulation of the target. The novel observations in this study suggest several testable hypotheses to be addressed in future studies, which may provide additional insights into the interface between regulators of apoptosis and autophagy in SCLC.
Several ongoing clinical trials seek to evaluate the efficacy of autophagy inhibitors in combination with cytotoxic therapies in cancer types including SCLC. The data presented here have important implications for studies in SCLC. Different strategies for modulating autophagy can have markedly discordant effects on the efficacy of cytotoxic therapies. It will be important to study the modulation of autophagy closely in relevant preclinical models to anticipate possible combinatorial effects.
In work presented here, we analyzed for the first time the effect of combining ABT-737 and chloroquine in SCLC models, including assessment of activity against PDX tumors, models that may better reflect the biology of disease than do cell line-based xenografts [31, 38]. Our in vivo xenograft data, particularly in the three primary xenograft models, suggest that despite some intriguing molecular interactions, the combination of ABT-737 and chloroquine is unlikely to have significant anti-cancer activity against SCLC, relative to ABT-737 alone.
All cell lines were obtained from American Type Culture Collection and grown in the recommended media. Primary xenograft cell lines (LX) were derived from patient tumors as previously described .
MTS cell proliferation assays
Cells were plated in quadruplicate and treated with ABT-737 and chloroquine for 72 hours at indicated doses. MTS cell proliferation assays were performed following manufacturer instructions using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega).
Caspase-Glo 3/7 assay. Assay was performed following manufacturer instructions (Promega). Briefly, 20,000 viable cells from each sample were transferred to 96-well white-walled plates in 75–100 μl total volume of culture medium. An equal volume of Caspase-Glo 3/7 Reagent was added to each well. Assays were performed in quadruplicate. Plates were covered and incubated at room temperature for 1–2 hours before measuring luminescence on a plate reading luminometer. The luminescence reading is directly proportional to caspase-3 activity.
Protein was isolated from cell pellets using radioimmunoprecipitation assay buffer (Sigma) and run on 10 or 12% Bis-Tris gels with MOPS or MES running buffer (Invitrogen). Membranes were probed with antibodies for LC3, Beclin-1, GAPDH, β-Actin (Santa Cruz), Mcl-1 (BD Pharmigen; #559027), Bax (Santa Cruz; #sc-6236), Bcl-XL (Cell Signaling; #2764), Bcl-2 (Santa Cruz; #sc-492), NOXA (Imgenex; #IMG-349A), PUMA (Cell Signaling; #4976), and cleaved PARP (Cell Signaling; #5625) following manufacturer instructions.
Samples were fixed in 2.5% glutaraldehyde, 3 mM CaCl2, 1% sucrose, in 0.1 M sodium cacodylate buffer, pH 7.2 for one hour at room temperature. After buffer rinse, samples were postfixed in 1% osmium tetroxide in buffer (1 hr) on ice in the dark. Following a DH2O rinse, plates were stained with 2% aqueous uranyl acetate (0.22 μm filtered, 1 hr, dark), dehydrated in a graded series of ethanol and embedded in Eponate 12 (Ted Pella) resin. Samples were polymerized at 60°C overnight. Thin sections, 60 to 90 nm, were cut with a diamond knife on the Reichert-Jung Ultracut E ultramicrotome and picked up with naked 200 mesh copper grids. Grids were stained with 2% uranyl acetate in 50% methanol and observed with a Philips CM 120 at 80 kV. Images were captured with an Advanced Microscopy Techniques transmission electron microscopy (1 K × 1 K) camera.
Lentiviral shRNA and cDNA overexpression experiments
Lentiviral particles were generated using a three-plasmid system and infected as per the RNAi Consortium Library Production and Performance Protocols, Broad Institute . shRNA constructs were obtained from the Broad RNAi Consortium and screened for knockdown. The BECN1 shRNA clone ID TRCN0000033550 was used these experiments. pLKO.1-shRNA scramble vector was obtained from Dr. David M. Sabatini through Addgene (Addgene plasmid 1864) as previously described .
In vivo studies
Patient-derived xenograft tumor models LX22, LX33, and LX44 were generated as previously described [24, 31]. NCI-H209 cells (1 × 107), or SCLC patient derived tumor cells LX22 (1.5 × 106), LX33 (1.6 × 106), and LX44 (3.5 × 106), were suspended in a 1:1 ratio of PBS and matrigel (BD Biosciences). Cell suspensions were injected subcutaneously on right hind flanks of female nu/nu 6 – 8 week old mice (Charles River). Drug treatments began when tumor sizes reached 200 mm3. Mice were treated daily with 100 mg/kg ABT-737 (IP), 60 mg/kg chloroquine (IP), combination or vehicle treated for 21 days. Tumors were measured with a manual caliper every 3 days and volumes were calculated using the formula: tumor weight (mg) = [length (mm) x width2 (mm2)]/2. In order to test whether treatment inhibited tumor growth, we used a mixed effects linear model comparing tumor growth between ABT-737 and ABT-737 and CQ treatment groups, modeling the log of tumor size over time and including an interaction term. All in vivo experiments were conducted with protocol approval by the Johns Hopkins Animal Care and Use Committee.
The student’s t-test was used to compare combination treatment effects vs. single agent treatment effects in cell lines. For in vivo analyses of whether treatment inhibited tumor growth, we used a mixed effects linear model comparing tumor growth between treatment groups, modeling the log of tumor size over time and including an interaction term. A p value <0.05 was considered significant.
Small cell lung cancer
Transmission electron microscopy
The authors would like to thank members of the Rudin and Hann laboratories for discussion and advice regarding this work. In addition, we would like to thank Barbara Smith for her technical assistance with electron microscopy. Grant support was provided by the Flight Attendant Medical Research Institute, the Burroughs Wellcome Fund, the Anti-Cancer Drug Development Training Grant (NIH T32 CA009243), and the Cancer Center Support Grant (NCI P30 CA006973).
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