PRKACA Mediates Resistance to HER2-Targeted Therapy in Breast Cancer Cells and Restores Anti-Apoptotic Signaling

Oncogene (2015) 34, 2061–2071 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling SE Moody1,2,3, AC Schinzel1, S Singh1, F Izzo1, MR Strickland1, L Luo1,2, SR Thomas3, JS Boehm3, SY Kim4, ZC Wang5,6 and WC Hahn1,2,3 Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL- XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease. Oncogene (2015) 34, 2061–2071; doi:10.1038/onc.2014.153; published online 9 June 2014 INTRODUCTION of these molecular mechanisms contributes to resistance in The receptor tyrosine kinase HER2 is amplified and/or over- HER2-positive human breast cancers is largely unknown. expressed in 20–30% of all breast cancers,1,2 leading to Although strategies to target the MAPK and PI3K pathways in constitutive proliferative and survival signaling via the down- resistant cancers are being pursued, these mechanisms likely fail stream Ras/extracellular-signal-regulated kinase (ERK) and PI3K to account for the development of resistant disease in all patients. (phosphoinositide 3-kinase)/Akt pathways. Amplification or over- Hence we conducted an unbiased screen to determine whether expression of HER2 is associated with poor prognosis, and pathways other than those directly downstream of canonical HER2 perturbation of HER2 signaling with trastuzumab or lapatinib signaling might also confer resistance. Here we describe a systematic interrogation of mechanisms of resistance to suppres- has led to clinical benefit in HER2-positive breast cancer – sion of HER2, to identify the major mechanisms of resistance to patients.3 7 However, many patients with early-stage disease HER2-directed therapy. experience tumor recurrence despite adjuvant treatment with trastuzumab, and patients with metastatic disease inevitably develop resistance to anti-HER2 therapies. For trastuzumab, a monoclonal antibody that binds to the RESULTS extracellular domain of HER2, a number of potential mechanisms fi We conducted two kinome open reading frame (ORF) screens in of resistance have been identi ed. These include cleavage of the parallel to identify genes that confer resistance to the lapatinib- extracellular domain of HER2, as well as heterodimerization with like dual EGFR/HER2 inhibitor AEE788 and to suppression of HER2 HER3 or IGF1R; both of these mechanisms result in continued with a short hairpin RNA (shRNA). We reasoned that the ‘off-target’ downstream MAPK (mitogen-activated protein kinase) and PI3K 8–10 effects of a small molecule inhibitor and a shRNA should be signaling. Resistance also has been shown to occur in the different, such that the intersection of hits from both screens setting of PTEN loss, activating PIK3CA mutations, CCNE amplifica- would help to identify biological pathways that can confer 11–17 tion or c-SRC activation. Resistance to lapatinib, which inhibits resistance to anti-HER2 therapy. We tested six independent anti- intracellular tyrosine kinase activity, can be induced in HER2- HER2 shRNAs in BT474 cells and found that there was a strong amplified breast cancer cells by activating PIK3CA mutations, loss correlation between the degree of HER2 protein suppression and of PTEN18 or by activation of mammalian target of rapamycin loss of viability/proliferation. We chose the most effective shRNA, complex 1 (mTORC1).19 As clinically resistant breast cancer sh4355, for the screen (Supplementary Figure S1A). We titrated samples have not been extensively molecularly characterized, the AEE788 dose in BT474 cells, and selected 0.85 μM for the due in part to limited sample availability, the extent to which each screen because it reduced cell viability to ~ 40% that of the 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 2Department of Medicine, Bringham and Women's Hospital and Harvard Medical School, Boston, MA, USA; 3Broad Institute of MIT and Harvard, Cambridge, MA, USA; 4Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA; 5Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA and 6Department of Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. Correspondence: Dr WC Hahn, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Dana 1538, Boston, MA 02115, USA. E-mail: [email protected] Received 24 February 2014; revised 12 April 2014; accepted 18 April 2014; published online 9 June 2014 PRKACA-induced resistance in breast cancer SE Moody et al 2062 control, allowing a sufficient window for rescue to be detected Three genes that have not been previously described as (Supplementary Figure S1B). downstream targets of HER2 signaling scored more than 2 s.d. We then used the Broad Institute/Center for Cancer Systems above the median in both screens: PRKACA, PIM1, and PIM2. In Biology (CCSB) V5 epitope-tagged kinase ORF collection to validation studies we found that, of these three molecules, identify genes that mediate resistance to these manipulations20 PRKACA expression rescued BT474 cells most strongly from (Supplementary Figure S2). Of the 597 ORFs, 14 scored more than lapatinib, although PIM1 and PIM2 were expressed at much lower two standard deviations (s.d.) above the median of all ORFs in the levels in these experiments (Supplementary Figure S3). AEE788 screen, and 20 did so in the shRNA screen (Table 1 and PRKACA is the alpha catalytic subunit of cyclic AMP-activated Figure 1a). Seven genes scored in both screens, including the protein Kinase A (PKA), whose activity is inhibited by PKA activated forms of HRAS, KRAS and MAPK/ERK kinase (MEK), which regulatory subunits. The second messenger cyclic AMP activates were screened as positive controls because they are known to PKA by causing the release of PRKACA or PRKACB from the signal downstream of HER2. AKT1, which signals downstream of regulatory subunits. Myriad effects of PKA activation have been HER2 to promote survival, scored strongly in both screens. In described, including promotion of survival signaling. In addition, addition, MAP2K6, CRKL and AKT3, which are known to signal Vegran et al.22 demonstrated that PRKACA was one of the through the Ras–ERK pathway or the PI3K–AKT pathway, scored 16 upregulated genes within a transcriptional signature more than two s.d. above the median in the shHER2 screen and that distinguishes breast cancers that failed to achieve a more than 1.5 s.d. above the median in the AEE788 screen. pathologic complete response (pCR) after trastuzumab plus These observations confirm prior work implicating MAPK and docetaxel neoadjuvant chemotherapy from those that did PI3K signaling as major mechanisms of resistance to HER2 achieve a pCR. inhibition.9–12,21 We validated our findings by performing dose titration curves for lapatinib in the setting of ectopic PRKACA expression in three HER2-amplified breast cancer cell lines. PRKACA expression Table 1. ORF screen identifies mediators of resistance to anti-HER2 increased the viability of BT474, SKBr3 and ZR-75-30 cells treatment propagated in the presence of lapatinib (Figure 1b). PRKACA expression also increased the viability of trastuzumab-treated AEE788 (>Median+2 s.d.) shHER2 (>Median+2 s.d.) HER2-amplified cells (Supplementary Figure S4). By counting viable cells, we found that lapatinib treatment of control cells Gene Relative viability Gene Relative viability expressing LACZ resulted in cell death, whereas overexpression of PRKACA in BT474 cells prevented cell death but failed to restore HRASV12 1.033 FGR 0.889 proliferation (Figure 1c). Based on these observations, we HRASV12 0.870 STYK1 0.391 LIMK2 0.774 MEKDD 0.390 hypothesized that PRKACA expression interferes with lapatinib- RP6-213H19.1 0.690 MAP2K6 0.351 induced apoptosis. We found that, indeed, lapatinib treatment PRKACA 0.624 PIM2 0.343 induced caspase 3/7 cleavage, and this event was significantly BLK 0.591 PIM1 0.324 reduced by PRKACA overexpression (Figure 1d, LACZ vs PRKACA, PIM1 0.585 DDR1 0.315 P = 0.0003 for ZR-75-30, P = 0.0002 for BT474). These findings ERBB3 0.579 KRASV12 0.314 indicate that PRKACA rescues HER2-amplified cells from lapatinib PIM2 0.553 PRKACA 0.306 treatment at least in part through the restoration of anti-apoptotic KRASV12 0.550 CRKL 0.304 survival signaling. AKT1 0.543 LYN 0.291 We then investigated mechanism(s) by which PRKACA con- MEKDD 0.538 JAK2 0.290 HIPK1 0.529 ABL2 0.290 ferred resistance to trastuzumab and lapatinib treatment. Since TNK1 0.523 AKT3 0.286 HER2 is thought to signal primarily through the Ras/Raf/MAPK and AKT1 0.285 PI3K/AKT pathways, we first examined the activation of these ABL1 0.283 pathways in the presence of AEE788 or lapatinib in BT474 cells MAP3K15 0.282 transduced with a control ORF, LACZ.

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