The concept of ‘oncogene addiction,’ whereby cancer cells become dependent on a specific oncogenic signaling pathway for survival 1, is highlighted by the recent success of molecularly targeted agents 2. However, the initial enthusiasm over marked clinical responses has now been tempered by the bedside observation that these responses are not durable because tumors acquire drug resistance 2–4. The identification of resistance mechanisms is essential to developing a strategy to enhance molecularly targeted therapy and prolong the efficacy of these agents.
Mutations and amplification of receptor tyrosine kinases (RTKs) were nominated as driver events in lung adenocarcinoma and gastric cancer 5,6. In RTK mutated or amplified cancers, RTK inhibition markedly reduces cell viability and invariably leads to downregulation of the PI3K (phosphatidylinositol 3-kinase)/AKT (anaplastic lymphoma kinase) and the MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase) pathways 3,7. Therapies targeting RTKs are part of the arsenal of agents that are used to treat lung, gastric, colorectal, and other cancers. Specific drugs used include small-molecule tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib [epidermal growth factor receptor (EGFR) TKIs], crizotinib (ALK TKI), etc. Despite the initial widely observed clinical efficacy of these TKIs, resistance invariably develops, typically within 1–2 years 3,4. Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged 3,4,8, which include mutation of RTK to a drug-resistant state; activation of a bypass pathway; and histologic transformation to an epithelial–mesenchymal transition 3. However, a large fraction of clinical cases including both de novo and acquired resistance cases are still driven by unknown mechanisms 9,10. Novel de novo and acquired resistance mechanisms continue to be identified.
The Bcl-2 protein family determines the commitment of cells to apoptosis, an ancient cell suicide program that is important to cancer development and drug response 11. A common deletion polymorphism of a Bcl-2 family member Bim mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in chronic myeloid leukemia and non-small-cell lung cancer 12. Genetic or pharmacological inhibition of Bcl-2 increased the sensitivity of lung cancer cells to EGFR inhibitors 13,14.
Here, we examine the role of Bcl-2 and Bcl-xL in the mechanisms underlying acquired resistance to small-molecule TKIs in lung and gastric cancers cells driven by the targeted RTKs. We modeled acquired resistance to various TKIs by exposing RTK-driven cells to increasing concentrations of TKIs. Our results suggest a novel common mechanism of resistance to TKIs, potentially leading to new co-treatment strategies.
Materials and methods
Antibodies and reagents
The following antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA): anti-MET (Cat#8198), anti-phospho-MET (Cat#3077), anti-EGFR (Cat#4267), anti-phospho-EGFR (Cat#3777), anti-HER2 (Cat#4290), anti-phospho-HER2 (Cat#2247), anti-ALK (Cat#3633), anti-phospho-ALK (Cat#12127), anti-AKT (pan, Cat#4691), anti-phospho-AKT (S473, Cat#4058), anti-phospho-S6 (Cat#2215), anti-ERK (Cat#9102), anti-phospho-ERK (Cat#9101), anti-Bcl-2 (Cat#4223), anti-Bcl-xL (Cat#2764), anti-Bim (Cat#2933), and anti-cleaved PARP (Cat#5625). Anti-β-actin (Cat#A3854) and anti-β-tubulin (Cat#T4026) antibodies were purchased from Sigma. The anti-Mcl-1 antibody (Cat#sc-12756) was from Santa Cruz Biotechnology (Dallas, Texas, USA). HRP-linked enhanced chemiluminescence (ECL) mouse (Cat#NA931V) and rabbit IgG (Cat#NAV934V) were purchased from GE Healthcare Life Sciences (Chicago, Illinois, USA).
The small-molecule inhibitors TAE684 (ALK TKI), Lapatinib (Dual EHER2/EGFR TKI), and ABT-263 (Bcl-2 and Bcl-xL inhibitors) were obtained from Selleck Biochemicals (Houston, Texas, USA). BEZ235 (Dual PI3K/mTOR inhibitor), GSK1120212 (MEK inhibitor), PHA665752 (MET TKI), Erlotinib (EGFR TKI), and Gefitinib (EGFR TKI) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). AZD6094 (MET TKI) was from Chemietek (Indianapolis, Indiana, USA).
Cell culture and transfection
HCC827, PC9, HCC4006, NCI-N87, and MKN45 cells were from the American Type Culture Collection. H3122 cells were purchased from the Tumor/Cell Line Repository at NCI (Bethesda, Maryland, USA). EBC-1 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. All cell lines were authenticated by providers utilizing short tandem repeat profiling. Cells were used over a course of no more than 3 months after resuscitation of frozen aliquots. Cells were grown in RPMI supplemented with 2 mmol/l glutamax [Life Technologies (Carlsbad, California, USA)] and 10% fetal bovine serum [BioAbChem (Ladson, South Carolina, USA)] at 37°C under 5% CO2.
Bcl-2 siRNAs (Cell Signaling, Danvers, Massachusetts, USA) were transfected into cells with Lipofectamine 3000 reagents [Thermo Fisher Scientific (Waltham, Massachusetts, USA)] according to the manufacturer’s instructions.
Establishment of RTK TKIs-resistant cells
Cells were exposed to increasing concentrations of TKI every 3 weeks starting from 50 nmol/l until a concentration of 5 μmol/l was reached at the end of a 5-month period. RTK TKI-resistant cells were expanded successfully in 10% FBS culture medium containing 1 μmol/l of TKI.
Cells were harvested in lysis buffer consisting of 50 mmol/l Tris pH 7.4, 150 mmol/l NaCl, 1% NP-40, and 5 mmol/l EDTA. Following 30 min of incubation in lysis buffer at 4°C, lysates were cleared by centrifugation at 16 000g for 10 min at 4°C, and then protein concentrations were determined using the DC Protein Assay [BioRad (Hercules, California, USA)]. Peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse [1 : 10 000, NA934 and NA931, respectively; GE Healthcare) antibodies were incubated for 1 h at room temperature. The ECL prime kit (GE Healthcare) was used to detect chemiluminescence.
Cell viability assays
Cells were seeded overnight at a density of 3000 cells/well in 96-well plates in RPMI 1640 containing 10% FBS and then treated with the relevant agents for 3 days. Viable cell numbers were determined using the CellTiterGLO assay kit according to the manufacturer’s protocols (Promega (Madison, Wisconsin, USA)]. Each assay consisted of three replicate wells and was repeated at least three times. Data were expressed as the percentage of surviving cells compared with control. This was calculated from the absorbance corrected for background.
NU/NU nude male mice of 4–6 weeks old were obtained from Charles River Laboratories (Wilmington, Massachusetts, USA). All studies were carried out in compliance with institutional guidelines under an IACUC-approved protocol at Medical University of South Carolina (Charleston, South Carolina, USA). HCC827-GR xenograft tumors were established in nude mice by subcutaneously injecting 5×106 cells suspended in PBS into the right flank. Mice with well-established tumors were selected and randomized 10 days after implantation (size>150 mm3). Mice were treated with vehicle or ABT-263 (25 mg/kg/day) or the combination of BEZ235 (25 mg/kg/day) and GSK1120212 (25 mg/kg/day), or the combination of three chemicals by oral gavage once daily for 6 days a week. All drugs were dissolved in 0.5% HPMC/0.1% Tween 80. Tumor dimensions were measured with a caliper and tumor volumes were calculated as: [tumor volume (mm3)=(length×width2)/2].
The results of quantitative studies are reported as mean±SD or mean±SEM. The SEM/SD was calculated on the basis of the number of independent experiments. Differences were analyzed using Student’s t-test. P values of less than 0.05 were considered significant.
Bcl-2 and Bcl-xL are upregulated upon treatment with TKIs in RTK-dependent lung and gastric cancer cell lines
The growth and survival of a number of lung and gastric cancer cell lines have been found to be highly dependent on RTK signaling because of gene mutations or amplifications. These cell lines include lung cancer cell lines HCC827 (egfr deletion) 15, PC9 (egfr deletion) 16, HCC4006 (egfr mutation) 17, EBC-1 (met amplification) 18, H3122 (eml4–alk fusion) 19, and gastric cancer cell lines MKN45 (met amplification) 7, NCI-N87 (her2 amplification) 20, and KATOIII (fgfr2 amplification) 21. We have previously shown that treatment of EBC-1 or MKN45 cells with the small-molecule MET TKIs induces the expression of Bcl-2 22. To determine whether this is a generalizable phenomenon, we treated HCC827, H3122, and NCI-N87 cells with the EGFR TKI gefitinib, the ALK TKI TAE684, and the dual HER2/EGFR TKI lapatinib, respectively. All inhibitors strongly blocked the RTK signaling that they specifically target shown by strongly reduced phosphorylation of EGFR, ALK, or HER2 as well as reduced phosphorylation of the downstream AKT (Fig. 1a and b). The effectiveness of these inhibitors in inducing apoptosis was confirmed by the induction of cleaved PARP, a marker for apoptosis (Fig. 1a and b). In agreement, downregulation of antiapoptotic Mcl-1 and upregulation of proapoptotic Bim were observed (Fig. 1). However, we found that the expression of antiapoptotic Bcl-2 and Bcl-xL was upregulated in all of these cells (Fig. 1). In addition, EGFR inhibition by gefitinib in PC9 cells or a second EGFR inhibitor erlotinib in HCC4006 cells led to increased protein levels of Bcl-2 and Bcl-xL (Fig. 1a) as well.
The combination of TKI and Bcl-2, Bcl-xL inhibitor is superior to TKI alone
Given the well-established antiapoptotic roles of Bcl-2 and Bcl-xL, we speculated that inhibition of Bcl-2 and Bcl-xL would strengthen the efficacy of TKIs as they caused a feedback upregulation of Bcl-2 and Bcl-xL (Fig. 1). Indeed, ABT-263, a small-molecule Bcl-2 Bcl-xL inhibitor, was only minimally active, whereas the combination of ABT-263 and an EGFR TKI erlotinib significantly decreased cell viability (Fig. 2a) and increased PARP cleavage (Fig. 2a) induced by erlotinib. To confirm that this was because of the inhibition of Bcl-2, we applied the Bcl-2 siRNAs and obtained similar results (Supplementary Fig. S1A, Supplemental digital content 1, http://links.lww.com/ACD/A215). This effect was also observed in the ALK-driven lung cancer cell line H3122 treated with the ALK TKI TAE684 and ABT-263 (Fig. 2a) or the Bcl-2 siRNAs (Supplementary Fig. S1B, Supplemental digital content 1, http://links.lww.com/ACD/A215), and the HER2-dependent gastric cancer cell line NCI-N87 treated with the HER2 TKI laptinib and ABT-263 (Fig. 2a).
ABT-263 blocks the formation of resistant colonies induced by TKIs
The long-term treatment of HCC827 with the small-molecule EGFR TKI erlotinib led to the killing of the majority of these tumor cells but also to the outgrowth of resistant colonies (Fig. 2b). Although ABT-263 had little effect on the growth of these tumor cells, the addition of this inhibitor to erlotinib suppressed the outgrowth of resistant colonies induced by this EGFR inhibitor. Similar results were obtained in H3122 cells treated with TAE684 and NCI-N87 cells treated with lapatinib (Fig. 2b), suggesting the possibility that upregulation of Bcl-2 and Bcl-xL contribute toward the emergence of resistance to TKIs.
Resistant cells express higher levels of Bcl-2 and/or Bcl-xL protein
To examine whether Bcl-2 and Bcl-xL levels were also increased in TKI-resistant clones, RTK-dependent cancer cells were grown in increasing concentrations of corresponding TKIs similar to previously described methods 23,24. Cells were then expanded in these TKIs (1 µmol/l) and designated as follows: HCC827-GR (gefitinib resistant), HCC827-ER (erlotinib resistant), PC9-GR (gefitinib resistant), PC9-ER (erlotinib resistant), H3122-TR (TAE684 resistant), NCI-N87-LR (lapatinib resistant), EBC-1-PR (PHA665752 resistant), EBC-1-AR (AZD6094 resistant), MKN45-PR (PHA665752 resistant), and MKN45-AR (AZD6094 resistant). These resistant cells indeed contain elevated levels of Bcl-2 or Bcl-xL (Fig. 3a), but not Mcl-1 (Fig. 3b) as shown by immunoblot assays.
The combination of inhibitors of PI3K/AKT, MEK/ERK, and Bcl-2/Bcl-xL overcomes resistance to TKIs
In RTK-dependent cancers, inhibition of the corresponding RTK leads to the suppression of key downstream signaling pathways, such as the PI3K/AKT and the MEK/ERK pathways, resulting in cell growth arrest and death 25,26. Indeed, the combination of PI3K/AKT and MEK/ERK inhibitors acted similar to TKIs in inducing apoptosis in parental cells as indicated by PARP cleavage (Fig. 4). We and others have shown that, in resistant cells, the PI3K/AKT and the MEK/ERK pathways are no longer regulated by RTK activity 15,22,27. However, these pathways can still be blocked by their own inhibitors in resistant cells (Fig. 4). Nevertheless, the blockade of these key pathways in resistant cells was not enough to induce sufficient apoptosis (Fig. 4a) and reduce cell viability (Fig. 4b) as observed in parental cells, suggesting that the function of apoptosis machinery is compromised. We reasoned that increased levels of Bcl-2 or Bcl-xL in resistant cells play an important role. Indeed, when we treated resistant cells with the combination of ABT-263, BEZ235, and GSK1120212, it caused significant apoptosis and marked cell death (Fig. 5a).
In addition, we treated the xenograft tumors derived from HCC827 cells with acquired resistance (HCC827-GR) with the BEZ235/GSK1120212/ABT-263 combination and observed marked tumor growth inhibition compared with the control group (Fig. 5b). The treatment also induced cleaved PARP, suggesting that apoptosis occurred in these tumors (Fig. 5b). This combination appeared to be well tolerated as there was no significant body weight loss or signs of toxicity in the treatment group throughout the experiment (Cen B, unpublished data).
All patients who initially benefit from RTK-targeted therapies eventually develop resistance within 1 or 2 years. An increasing understanding of the number and complexity of resistance mechanisms highlights the Herculean challenge of killing tumors that are resistant to TKIs. Our growing knowledge of resistant pathways provides an opportunity to develop new mechanism-based inhibitors and combination therapies to prevent or overcome therapeutic resistance in tumors. In this study, we present a previously unobserved resistance mechanism mediated by Bcl-2 and Bcl-xL that is shared by multiple RTKs. Acute exposure of RTK-dependent lung and gastric cancer cells to their corresponding TKIs leads to increased expression of Bcl-2 and Bcl-xL (Fig. 1). The upregulation of these two molecules is maintained in cells that have acquired resistance through long-term TKI exposure (Fig. 3). It would be important to determine whether in specimens of patients who received TKI treatment there is increased expression of Bcl-2 and/or Bcl-xL. Here, we propose two potential uses for Bcl-2 and Bcl-xL inhibitors. First, they may be combined with a TKI to enhance the efficacy of the TKI (Fig. 2a) and to prevent resistance as this combination is significantly better at delaying the onset of resistance than a TKI alone (Fig. 2b). A second potential clinical use for Bcl-2/Bcl-xL inhibitors is to combine with PI3K/AKT and MEK/ERK inhibitors to overcome resistance to TKIs (Fig. 5). To date, in completed clinical trials, the activity of single-agent Bcl-2/Bcl-xL inhibitors is limited in solid tumors, suggesting the need to develop novel strategies to utilize these drugs.
Mechanisms of acquired resistance in RTK-driven cancer are broadly divided into two categories. The first involves development of additional genetic alterations in the primary RTK genes, which facilitates continued downstream signaling 25. This commonly arises through secondary mutations in the kinase target or through gene amplification of the kinase itself. Alternatively, resistance can develop independent of genetic changes in the target. This occurs through activation of downstream signaling pathways, changes in tumor histology, or alterations in drug metabolism 25,28. Studies indicate that multiple resistance mechanisms may operate within an individual tumor to promote acquired resistance 3,4. One potential solution to overcome multiple mechanisms of resistance is to target downstream pathways that mediate the balance between survival and apoptosis. A therapeutic strategy that directly activates the machinery that is necessary for apoptosis may circumvent multiple mechanisms of acquired resistance to RTK-targeted therapies and may therefore be a broader approach than aiming to inhibit one resistant mechanism at a time. Given the prominent antiapoptotic roles of Bcl-2 and Bcl-xL and their upregulation in response to TKI treatment, targeting these molecules in the context of RTK-targeted therapies may have the potential to overcome or prevent acquired resistance resulting from various mechanisms.
It was shown previously that induction of Bim is essential for apoptosis triggered by EGFR TKIs 29. Our data show that Bim upregulation is present in all RTK-dependent lung and gastric cancer cell lines upon treatment with corresponding TKIs (Fig. 130), which underscores the importance of RTK signaling for survival. However, in response to TKI treatment, cells trigger a feedback mechanism by increasing the expression of Bcl-2 and Bcl-xL. Augmented transcription of Bim can account for at least part of the increase in Bim protein after treatment with EGFR TKIs in EGFR-dependent lung cancer cells and the ERK pathway is responsible for Bim increase 29. In agreement, our data show that the MEK inhibitor GSK1120212, but not the PI3K/AKT inhibitor BEZ235, induced Bim expression in RTK-dependent cells, although the combination of these two inhibitors seems to show further induction of Bim (Fig. 4). This combination not only failed to induce PARP cleavage but also showed reduced Bim induction in resistant cells (Fig. 4), likely resulting from Bcl-2 and/or Bcl-xL upregulation. Indeed, the addition of ABT-263 effectively led to significantly better induction of apoptosis (Fig. 5). Studies are underway to determine the molecular mechanisms by which TKIs upregulate Bcl-2 and Bcl-xL expression. Our previous work suggests that Pim-1 kinase may be involved in the regulation 30. This Yin (Bim upregulation) and Yang (Bcl-2 and Bcl-xL upregulation) relationship highlights the opportunity and challenge that we are facing in treating these cancers. Although the combination of EGFR TKI and a Bcl-2 inhibitor has been explored before preclinically 29 and in clinical trials 31, we, for the first time, provide a biological basis for targeting Bcl-2 and Bcl-xL and show that this strategy may also work for other TKIs.
To understand the mechanism underlying the acquisition of TKI resistance, we adopted a classical method, which is the establishment and characterization of drug-resistant cancer cells selected from drug-sensitive cell lines in vitro following exposure to stepwise increasing doses of the drug 16. This method has successfully led to the identification of many resistance mechanisms to TKIs that are also observed in the clinics 15,32. Our data support the further clinical evaluation of Bcl-2/Bcl-xL inhibitors in combination with TKIs. This combination strategy may be extended to other targeted agents that induce apoptosis. Clinical trials are underway to investigate the potential of ABT-263 in combination with the MEK inhibitor tramatinib in KRAS mutant tumors or with the BRAF inhibitor dabrafenib in BRAF mutant melanoma 33.
The authors thank Dr. Robert Gemmill (Medical University of South Carolina) for providing PC9-GR cells and Dr. Michael B. Lilly (Medical University of South Carolina) for sharing equipment.
This work is supported by the South Carolina Clinical & Translational Research (SCTR) Institute, with an academic home at the Medical University of South Carolina CTSA, NIH/NCATS grant number UL1TR001450 (to B.C.), and the Natural Science Foundation of Guangxi, China (2015GXNSFEA139003, to J.J.).
J.J. contributed to the study concept and design, and data analysis and interpretation, and provided materials. X.Y. and B.C. conducted all the experiments and contributed toward data analysis and interpretation. B.C. wrote the manuscript and contributed toward the study concept and design, and study coordination. All authors reviewed and approved the manuscript.
Conflicts of interest
There are no conflicts of interest.
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Bcl-2; Bcl-xL; gastric cancer; lung cancer; molecularly targeted therapy; receptor tyrosine kinase; tyrosine kinase inhibitor
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