sign in sign up
<<
Home , ABL (gene), Lapatinib

ANTICANCER RESEARCH 31: 789-796 (2011)

Inhibition of c-ABL Sensitizes Breast Cells to the Dual ErbB Receptor Tyrosine Inhibitor Lapatinib (GW572016)

YUAN-HUNG LO, PO-CHUN HO, HUAJUN ZHAO and SHAO-CHUN WANG*

Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521, U.S.A.

Abstract. Background: The dual kinase inhibitor lapatinib Activation of the ErbB receptors triggers downstream signaling (Tykerb) has been applied for advanced . However, pathways manifested by PI3K-AKT, MAPK, and mTOR the effectiveness in the clinic has been elusive and the activation (3). Pre-clinical studies demonstrate that treatment development of novel approaches to enhance the responsiveness with these drugs inhibits phosphorylation of the ErbB receptors, is needed. In this study, we test whether the non-receptor down-regulates downstream signaling, and subsequently c-Abl regulates the responsiveness of breast suppresses cancer cell growth and induces cell death. However, cancer cells to lapatinib and, if so, whether the combination in several clinical trials where the ErbB inhibitors were treatment with lapatinib plus the c-ABL kinase inhibitor administered as a monotherapy, responses have been seen in (STI571; Gleevec) can sensitize breast cancer cells to only a small minority of breast (4). Lapatinib treatment the treatment. Materials and Methods: The endogenous c-ABL as a single-agent frontline therapy resulted in only modest kinase was silenced by RNA interference or inhibited by clinical benefit in ErbB2-positive advanced breast cancer (5). imatinib to test whether the co-treatment improves the The non- c-ABL plays a fundamental responsiveness of the lapatinib-resistant breast cancer cell lines role in the regulation of important cell functions, including cell MDA-MB-468 and T47D, by measuring cell growth and cell- migration, responses to oxidative stress and DNA damage, progression. Conclusion: The responsiveness to lapatinib proliferation, and survival (6-11). Expression of the c-ABL can be improved by targeting the function of c-ABL, suggesting proto- is a frequent event in breast cancer (12). c-ABL that combination treatment of lapatinib plus imatinib can lead has been shown to positively regulate the function of EGFR by to significant gains in therapeutic benefit. protecting the activated EGFR receptor from phosphatase inhibition or by preventing the membrane EGFR from Intensive research over the past decades has led to the internalization (13, 14). In breast cancer, the c-ABL kinase identification of numerous that play pivotal roles in the functions to promote cell growth and invasion. Accordingly, development of breast cancer. Among the most studied include blocking c-ABL activity with RNA interference or the clinically the receptor tyrosine (RTKs) of the ErbB family; in approved inhibitor imatinib inhibits cell proliferation and particular, the epidermal receptor (EGFR) and anchorage-independent growth and sensitizes cell to ErbB2 (also known as HER2/neu), against which multiple caused by chemotherapeutic agents and nutrient deprivation (15- targeted therapeutic approaches have been developed. These 17). Taken together, these studies have identified c-ABL as a include small molecules that inhibit the tyrosine kinase activity promoting factor in breast cancer downstream of the ErbB of these RTKs, such as , , , and the RTKs, and warrant further investigation of whether c-ABL dual kinase inhibitor lapatinib that interacts with and inhibits inhibition can sensitize breast cancer cells to lapatinib in resistant several ErbB family members and is therefore expected to be breast cancer cell lines with distinct cellular contexts. superior to other member-specific anti-ErbB inhibitors (1, 2). Materials and Methods

Cell culture, antibodies, and chemicals. T47D and MDA-MB-468 cell Correspondence to: Shao-Chun Wang, Department of Cancer and Cell lines were purchased from the American Type Culture Collection. Biology, 3125 Eden Ave., Cincinnati, OH 45267-0521, U.S.A. Tel: +1 T47D/shAbl and T47D/shCtrl cells were generated as described 5135584259, Fax: +1 5135582445, e-mail: [email protected] previously (12). All cells were grown in DMEM/F12 (1:1) with 10% fetal bovine serum. The following antibodies were purchased: α- Key Words: Breast cancer, lapatinib, imatinib, EGFR, ErbB2, c-ABL, tubulin (Sigma, St. Louis, MO, USA); c-ABL (Calbiochem, San RTK. Diego, CA, USA); phosphotyrosine, p27 (Millipore/Upstate, Billerica,

0250-7005/2011 $2.00+.40 789 ANTICANCER RESEARCH 31: 789-796 (2011)

Figure 1. Knockdown of c-Abl sensitizes the cells to lapatinib. The endogenous c-ABL in MDA-MB-468 (a) and T47D (b) cells was silenced with stable expression of an shRNA targeting c-ABL (shABL) or a control scramble shRNA (shCtrl). The cells were treated with different doses of imatinib as indicated for 72 h and cell growth was evaluated by MTT assay. *p<0.05; **p<0.01; ***p<0.001. Insets show the level of c-ABL in the shCtrl and shABL cells.

MA, USA); EGFR, CrkL, phospho-CrkL, AKT, phospho-S473-AKT, Immunoprecipitation. Cultured cells were washed twice with ice- phospho-T308-AKT, cyclin D1 (Cell Signaling, Danvers, MA, USA); cold PBS. The cells were then lysed in NETN buffer (150 mM PCNA, cyclin E (Santa Cruz, Santa Cruz, CA, USA). Lapatinib and NaCl, 1 mM EDTA pH8.0, 20 mM Tris pH 8.0, 0.5% NP-40, imatinib were purchased from LC Laboratories (Woburn, MA, USA). 25 mM NaF, 2 mM Na3VO4, 20 μl/ml aprotinin, 0.1 M PMSF) on ice for 30 min. For immunoprecipitations, the lysate was incubated Western blotting. Cell lysates of the treated cells were isolated by with primary antibody at 4˚C overnight. The immunocomplexes incubation with NETN buffer (150 mM NaCl, 1 mM EDTA pH 8.0, were then incubated with 50 μl G agarose (Roche, 20 mM Tris pH 8.0, 0.5% NP-40, phosphatase inhibitors 25 mM Indianapolis, IN, USA) at 4˚C for 2 h. The beads were washed four NaF and 2 mM Na3VO4, and the protease inhibitors 20 μl/ml times with ice-cold NETN buffer and the protein complexes were aprotinin and 0.1 M PMSF). Cell lysates were separated on then eluted by boiling the beads in 40 μl of 2× loading buffer. acrylamide gels, transferred to a PVDF membrane (Bio-Rad; Hercules, CA, USA), and probed with the indicated antibodies. Cell-cycle analysis by flow cytometry. Cells were harvested by Bands were visualized by a chemiluminescence-based detection trypsin, washed with PBS, and then fixed in 70% of ethanol. The method (Fisher/Pierce, Rockford, IL, USA) that used a horseradish fixed cells were stained with 25 μg/ml propidium iodide (Sigma) in peroxidase-conjugated secondary antibody. the presence of 1 μg/ml RNase (Sigma), then subjected to

790 Lo et al: Combination Treatment with Lapatinib plus Imatinib

Figure 2. Combination treatment with lapatinib and imatinib inhibited cell growth. MDA-MB-468 (a) and T47D (b) cells were inoculated in 96- well plates and treated with lapatinib alone, imatinib alone, or their combination for 72 h at the indicated doses. Cell growth was evaluated by MTT assay. **p<0.01, ***p<0.001. fluorescence-activated cell sorting (FACS) analysis. The data were 468 cells) or only a modest effect (in T47D cells) on cell collected using a FACSCalibur flow cytometer and analyzed by the growth (Figure 2). The activity of c-ABL was assessed by software ModFit (Verity, Topsham, ME, USA). The cell-cycle the phosphorylation of CrkL. CrkL encodes an adapter distribution was evaluated by counting >10,000 cells per sample. protein functioning in signal transduction and is best known Statistical analysis: Data were expressed as means±SD (n=3). Statistical differences between two groups were determined by the as a substrate of the BCR-ABL kinase in chronic Student’s t-test. P<0.05 was considered significantly different. myelogenous . It has been shown that down- regulation of CrkL significantly decreased cell proliferation, Results cell-cycle progression, and cell survival, while overexpression of CrkL led to enhanced growth factor- The dual ErbB inhibitor lapatinib is not a potent growth independent cell growth (19, 20). Although treatment with inhibitor in T47D and MDA-MB-468 cells, in comparison imatinib inhibited the phosphorylation of CrkL, the treatment with other sensitive breast cancer cell lines (18). These two had only limited effects on the growth of MDA-MB-468 or cell lines are intrinsically resistant to lapatinib and differ T47D cells. This is in a drastic contrast to the combined significantly with regard to their molecular makeup. For treatment of lapatinib plus imatinib, which induced example, T47D expresses a high level of ErbB2 and is significant inhibition of cell growth in both cell lines (Figure negative for EGFR expression, while MDA-MB-468 2). These results indicate that functional gains of multiple expresses a very high level of EGFR and is negative for pathways downstream of the ErbB and c-ABL kinases ErbB-2 expression. Both cell lines are positive for c-ABL convey the growth advantage of these breast cancer cells, expression (12). To determine whether c-ABL is involved in offering a therapeutic opportunity of combination treatment the resistance to lapatinib in these cells, the endogenous by targeting both pathways. c-ABL was silenced by shRNA introduced by lentiviral To further characterize the mechanisms of growth infection in MDA-MB-468 and T47D cells (Figure 1). The inhibition mediated by the combination treatment, flow results showed that down-regulation of c-ABL resulted in cytometric analysis was performed on cells that received increased sensitivity to different doses of lapatinib, as mock treatment, lapatinib alone, imatinib alone, or lapatinib demonstrated by the magnitude of cell-growth inhibition. plus imatinib. The combination conferred a prominent effect These results prompted us to test whether combination on cell-cycle progression in that MDA-MB-468 cells treatment of lapatinib plus imatinib improves the exhibited a reduced proportion of cells in G2/M, and a responsiveness in these cells. Treatment with lapatinib concomitant increase in G1 cells (Figure 3a); while T47D inhibited the activity of endogenous ErbB proteins, cells were blocked at S-phase progression which was indicating that the drug was able to target the designated accompanied by an increase in G1 cells (Figure 3b). At the pathway in each cell line as expected. However, treatment molecular level, lapatinib treatment inhibited the with lapatinib alone had no significant effect (in MDA-MB- phosphorylation of EGFR and ErbB-2 in MDA-MB-468

791 ANTICANCER RESEARCH 31: 789-796 (2011)

Figure 3. Combination effect of lapatinib and imatinib on cell-cycle progression. MDA-MB-468 (a) and T47D (b) cells treated with lapatinib (Lap), imatinib (STI), or both in combination for 48 h were fixed and processed for FACS analysis. The relative proportions of G1, S, G2/M cells are summarized in the table.

(Figure 4) and T47D cells (Figure 5), respectively, without imatinib-treated cells, which provided a potential explanation affecting the protein levels of these RTKs. However, as shown for the observed AKT up-regulation by imatinib. Taken in Figure 2, the down-regulation of EGFR and ErbB2 by together, these results demonstrate that both ABL and ErbB lapatinib alone did not result in significant cell-growth tyrosine kinases are involved in the growth advantage gained inhibition. Lapatinib inhibited AKT activity in MDA-MB-468 in these cancer cells, and that the simultaneous inhibition of cells, which was demonstrated by the decreased these pathways confers significant growth inhibition. phosphorylation of the kinase at Ser473 and Thr308. In addition, phosphorylation of CrkL was inhibited upon Discussion imatinib treatment. In addition, combined treatment with lapatinib and imatinib caused a pronounced decrease in Although imatinib has been widely applied to gastrointestinal activated AKT kinase and further increased the cyclin- tumors and hematopoietic malignancies (23), its application dependent kinase inhibitor p27. in breast cancer has not been fully explored. To the best of As in MDA-MB-468 cells, co-treatment with lapatinib and our knowledge, the current study is the first exploring the imatinib also resulted in significant cell-growth suppression in efficacy of treatment combining an ErbB inhibitor with T47D cells. As expected, tyrosine phosphorylation of ErbB2 imatinib in breast cancer cells. We have shown that lapatinib and CrkL was inhibited by lapatinib and imatinib, respectively, had a modest growth-inhibiting effect on the ErbB2- in this cell line. The combination further reduced the expressing T47D cells, while having no effect on the growth expression of S-phase cyclins, in particular cyclin E, in of MDA-MB-468 cells, which express high levels of EGFR. agreement with the S-phase inhibition effect revealed by flow This observation is consistent with the previous report by cytometry analysis. Intriguingly, treatment with imatinib Konecny et al. (18). Interestingly, it has been shown that the induced AKT phosphorylation in both MDA-MB-468 and therapeutic benefit of lapatinib is associated with the T47D cells, which was curbed by the addition of lapatinib. To expression of ErbB2, but not with the status of EGFR in explore this unexpected finding further, we examined the effect breast tumors (21, 22), leading to the suggestion that of imatinib on the activation of the p70 S6K protein and found lapatinib targets ErbB2, but not EGFR, in breast cancer (22). that imatinib treatment downregulated the kinase activity of The current study demonstrates that cooperation between S6K, as shown by the reduced phospho-S6K level in the lapatinib and imatinib can overcome the insensitivity to

792 Lo et al: Combination Treatment with Lapatinib plus Imatinib

Figure 4. Combination treatment of lapatinib and imatinib resulted in enhanced inhibition of growth-promoting signals in MDA-MB-468 cells. Cells were treated with lapatinib alone, imatinib alone, or both drugs in combination for 24 h. Cellular protein extracts were then subjected to immunoprecipitation followed by Western analysis (a), or Western analysis to examine the levels of the indicated proteins (b).

lapatinib in the EGFR-overexpressing MDA-MB-468 cells. which activation of S6K results in inhibition of the - It should be noted that a sensitization effect was not observed related substrate and subsequently inhibits AKT activity to in combinations of imatinib and the COX-2 inhibitor NS398 prevent over-activation of the PI3K-AKT pathway (25). Our (data not shown), suggesting a pathway-dependent specificity data suggest that imatinib treatment can lead to AKT for the synergism. activation through the down-regulation of S6K activity, which T47D and MDA-MB-468 differ with respect to their was suppressed by lapatinib co-treatment. This observation molecular makeup, but their growth was similarly inhibited warrants further investigation to determine whether co- in response to the combination treatment. This suggests that activation of AKT is a potential mechanism accounting for combination treatment with these two drugs inhibits the the lack of therapeutic efficacy of imatinib as a single-agent distinct growth-promoting pathways in these two cancer cells in advanced breast cancer (26), and whether combined in a complementary fashion. For example, in MDA-MB-468 targeting of the ErbB kinases can achieve significant clinical cells, co-treatment with lapatinib and imatinib resulted in an benefit. Our results also highlight the potential of combining enhanced decrease in phospho-AKT, while the level of the lapatinib and imatinib, or other regimens designed with a cell-cycle inhibitor p27 increased and cells accumulated at the similar concept in mind, to target advanced breast cancer. G1 phase of the cell cycle. In T47D cells, combined treatment Acknowledgements resulted in enhanced inhibition of cyclin D1 and cyclin E and a decrease in the numbers of S-phase cells. The Authors thank Maryellen Daston and Glenn Doerman for Our data also show that imatinib treatment resulted in assistance in editing the manuscript and preparing the graphics. This increased AKT activation. It is noteworthy that the activity of work was supported in part by the following grants: Susan G. AKT is under the control of a negative-feedback loop in Komen Research Award KG080540 (to S.-C. W.), the Department

793 ANTICANCER RESEARCH 31: 789-796 (2011)

Figure 5. Western blotting of T47D cells in response to the individual and combination treatment with lapatinib and imatinib. Cells were treated with lapatinib alone, imatinib alone, or a combination of both for 24 h. Cellular protein extracts were then subjected to immunoprecipitation followed by Western analysis (a), or Western analysis to examine the levels of the indicated proteins (b).

of Defense Prostate Cancer Research Award PC073951 (to S.-C. 6 Kharbanda S, Yuan ZM, Weichselbaum R and Kufe D: W.), the Marlene Harris-Ride Cincinnati Pilot Grant (to S.-C. W.), Determination of cell fate by c-Abl activation in the response to and the PHS Grant P30 DK078392. DNA damage. Oncogene 17(25): 3309-3318, 1998. 7 Lin J, Sun T, Ji L, Deng W, Roth J, Minna J et al: Oncogenic References activation of c-Abl in non-small cell lung cancer cells lacking FUS1 expression: inhibition of c-Abl by the tumor suppressor 1 Yarden Y and Sliwkowski MX: Untangling the ErbB signalling gene product Fus1. Oncogene 26(49): 6989-6996, 2007. network. Nat Rev Mol Cell Biol 2(2): 127-137, 2001. 8 Pendergast AM: The Abl family kinases: mechanisms of 2 Rusnak DW, Alligood KJ, Mullin RJ, Spehar GM, Arenas-Elliott regulation and signaling. Adv Cancer Res 85: 51-100, 2002. C, Martin AM et al: Assessment of 9 Plattner R and Pendergast AM: Activation and signaling of the receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression Abl tyrosine kinase: bidirectional link with phosphoinositide levels and response to lapatinib (Tykerb, GW572016) in an signaling. Cell Cycle 2(4): 273-274, 2003. expanded panel of human normal and tumour cell lines. Cell 10 Shaul Y: c-Abl: activation and nuclear targets. Cell Death Differ Prolif 40(4): 580-594, 2007. 7(1): 10-16, 2000. 3 Alvarez RH, Valero V and Hortobagyi GN: Emerging targeted 11 Wang JY: Regulation of cell death by the Abl tyrosine kinase. therapies for breast cancer. J Clinical Oncol 28(20): 3366-3379, Oncogene 19(49): 5643-5650, 2000. 2010. 12 Zhao H, Ou-Yang F, Chen IF, Hou MF, Yuan SS, Chang HL et 4 Mendelsohn J and Baselga J: Status of epidermal growth factor al: Enhanced resistance to tamoxifen by the c-ABL proto- receptor antagonists in the biology and treatment of cancer. oncogene in breast cancer. Neoplasia 12(3): 214-223, 2010. J Clin Oncol 21(14): 2787-2799, 2003. 13 Birge RB, Fajardo JE, Mayer BJ and Hanafusa H: Tyrosine- 5 Gomez HL, Doval DC, Chavez MA, Ang PC, Aziz Z, Nag S et phosphorylated epidermal and cellular al: Efficacy and safety of lapatinib as first-line therapy for p130 provide high affinity binding substrates to analyze Crk- ErbB2-amplified locally advanced or . phosphotyrosine-dependent interactions in vitro. J Biol Chem J Clin Oncol 26(18): 2999-3005, 2008. 267(15): 10588-10595, 1992.

794 Lo et al: Combination Treatment with Lapatinib plus Imatinib

14 Tanos B and Pendergast AM: Abl tyrosine kinase regulates 22 Amir E, Ocana A, Seruga B, Freedman O and Clemons M: endocytosis of the epidermal growth factor receptor. J Biol Lapatinib and HER2 status: Results of a meta-analysis of Chem 281(43): 32714-32723, 2006. randomized phase III trials in metastatic breast cancer. Cancer 15 Sims JT, Ganguly S, Fiore LS, Holler CJ, Park E-S and Plattner Treat Rev 36(5): 410-415, 2010. R: STI571 sensitizes breast cancer cells to 5-fluorouracil, 23 Capdeville R, Buchdunger E and Zimmermann J: Matter a. cisplatin and camptothecin in a cell type-specific manner. Glivec (STI571, imatinib), a rationally developed, targeted Biochem Pharmacol 78(3): 249-260, 2009. anticancer drug. Nat Rev Drug Discov 1(7): 493-502, 2002. 16 Srinivasan D and Plattner R: Activation of Abl tyrosine kinases 24 Dann SG, Selvaraj A and Thomas G: mTOR Complex1-S6K1 promotes invasion of aggressive breast cancer cells. Cancer Res signaling: at the crossroads of obesity, diabetes and cancer. 66(11): 5648-5655, 2006. Trends Mol Med 13(6): 252-259, 2007. 17 Srinivasan D, Sims JT and Plattner R: Aggressive breast cancer 25 Inoki K, Corradetti MN and Guan K-L: Dysregulation of the cells are dependent on activated Abl kinases for proliferation, TSC-mTOR pathway in human disease. Nat Genetics 37(1): 19- anchorage-independent growth and survival. Oncogene 27(8): 24, 2005. 1095-1105, 2008. 26 Cristofanilli M, Morandi P, Krishnamurthy S, Reuben JM, Lee 18 Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, BN, Francis D et al: Imatinib mesylate (Gleevec) in advanced Rahmeh M et al: Activity of the dual kinase inhibitor lapatinib breast cancer-expressing C-Kit or PDGFR-beta: clinical activity (GW572016) against HER-2-overexpressing and trastuzumab- and biological correlations. Ann Oncol 19(10): 1713-1719, 2008. treated breast cancer cells. Cancer Res 66(3): 1630-1639, 2006. 27 Yamasaki F, Zhang D, Bartholomeusz C, Sudo T, Hortobagyi 19 Kim YH, Kwei KA, Girard L, Salari K, Kao J, Pacyna- GN, Kurisu K et al: Sensitivity of breast cancer cells to erlotinib Gengelbach M et al: Genomic and functional analysis identifies depends on cyclin-dependent kinase 2 activity. Mol Cancer Ther CRKL as an oncogene amplified in lung cancer. Oncogene 6(8): 2168-2177, 2007. 29(10): 1421-1430, 2010. 20 Feller SM: CrK family adaptors-signalling complex formation and biological roles. Oncogene 20(44 REV. ISS. 5): 6348-6371, 2001. 21 Finn RS, Press MF, Dering J, Arbushites M, Koehler M, Oliva C et al: Estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor expression and benefit from lapatinib in a randomized trial of paclitaxel with lapatinib or placebo as first- Received January 12, 2011 line treatment in HER2-negative or unknown metastatic breast Revised February 28, 2011 cancer. J Clin Oncol 27(24): 3908-3915, 2009. Accepted February 28, 2011

795