Addition of the p110α inhibitor BYL719 overcomes targeted therapy resistance in cells from Her2-positive-PTEN-loss breast cancer

Abstract Breast cancer is one of the leading causes of death for women worldwide. Among various subtypes of breast can- cer, human epidermal growth factor receptor 2 (HER2)-positive and phosphatase and tensin homolog (PTEN) loss breast cancer is a cause of great concern in terms of its resistance to HER2- targeted therapies and its poor prognosis. Phosphatidylinositol 3-kinase (PI3K)/AKT hyperphosphorylation is considered one of key mechanisms leading to this resistance, thus combination therapy of PI3K inhibitors and HER2 antibodies is promising for overcoming this problem, and more specific regimens should be designed in this age of precision medicine. In this study, we established an HER2-positive and PTEN loss cell line and confirmed it by western blot analysis. This cell line and its orthotopic xenograft models were exposed to p110α-specific inhibitor BYL719, p110β-specific inhibitor AZD6482, or pan- PI3K inhibitor BKM120, respectively, and the results showed sensitivity to both BYL719 and BKM120 but not AZD6482, which indicated a p110α-reliance for HER2-positive-PTEN- loss breast cancer. Then, the addition of BYL719 to HER2 antibody greatly reduced tumor growth both in vitro and in vivo, accompanied by inhibited PI3K effector phosphoryla- tion. Therefore, our findings suggest that the combination of p110α-selective inhibitor BYL719 with HER2 antibody could be a potential strategy for more personalized treatment of HER2-posistive-PTEN-loss breast cancer; and in addition, the optimal schedule of this combination therapy needs to be fur- ther explored.

Keywords : HER2 . PTEN . Breast cancer . p110α . p110β

Introduction

Breast cancer is one of the most frequently diagnosed cancers and remains the second leading cause of cancer death in women worldwide. Among women, breast cancer is not only the most commonly diagnosed cancer at ages 30 to 59 years but also is the leading cause of cancer death in women younger than 45 years [1, 2]. Breast can- cer is a complex and heterogeneous disease, which can be classified through histopathological features, responses to therapy, or molecular profiling [3–5]. One of the common subgroups is the human epidermal growth factor receptor 2 (HER2, also referred to as ERBB2 or Neu)-positive subtype, which occurs in 15–20 % of breast cancer cases and is often associated with poor prognosis [6, 7].

Multiple HER2-targeted therapies are available for patients with metastatic HER2-positive (HER2+) cancer, including the HER2-specific monoclonal antibodies trastuzumab and the epidermal growth factor receptor (EGFR)/HER2 small-molecule inhibitor lapatinib. Although remarkable clinical efficacies in HER2+ breast cancer are observed in these therapies, resistances com- monly occur [8–10].

Several potential mechanisms of HER2-targeted therapy resistance have been identified, and hyperactivation of phos- phatidylinositol 3-kinase (PI3K)/AKT signaling downstream HER2 is considered one of the key mechanisms [11, 12].

PI3Ks are subdivided into three subclasses and the class IA PI3Ks have been demonstrated to be involved in human can- cer [13, 14]. Class IA PI3Ks are heterodimeric proteins com- posed of a catalytic p110 subunit and a regulatory p85 subunit. There are three class IA p110 isoforms in mammalian cells, of which p110δ is mostly restricted to the immune system, whereas p110α and p110β are ubiquitously expressed [15]. Recent studies have demonstrated that p110α and p110β maintain distinct functions in signaling and cellular transfor- mation [14, 16, 17]. Active PI3Ks catalyze the phosphoryla- tion of phosphatidylinositol 4,5-bisphosphate (PIP2) to form the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3), which activates AKT and other effectors. Lipid phos- phatase and tumor suppressor phosphatase and tensin homo- log (PTEN), which converts PIP3 back into PIP2, antagonize this process. Loss of PTEN, which is relevant in almost 40 % in HER2-overexpressing breast cancers, has been reported to confer trastuzumab/lapatinib resistance through enhanced PI3K/AKT signaling [18, 19].

Clinical and preclinical trials of PI3K inhibitors are ongoing and show modestly promising results. The earli- est trials have largely featured so-called pan-PI3K inhibi- tors that block the action of all receptor-coupled class IA PI3K isoforms, which may result in excess toxicities [20]. Indeed, for the development of more personalized thera- pies, isoform-selective inhibitors are now emerging, and much work is ongoing to determine the isoform depen- dence of different cancers [21–23]. For example, the p110δ isoform inhibitor GS1101 has been proven ex- tremely effective in certain B cell neoplasias; p110α- selective inhibitors have shown promise in early phase trials for patients with breast cancer bearing PIK3CA mu- tations [24, 25]. In addition, p110α is proven to be critical for activated receptor tyrosine kinases or oncogenes such as vascular endothelial growth factor receptor (VEGFR), EGFR, platelet-derived growth factor receptor (PDGFR), and HER2, whereas p110β seems to be essential in many tumors deficient of PTEN [16, 17, 26]. Thus, for HER2+ PTEN-loss breast cancer, in order to obtain an optimized therapeutic effect, it is critical to assess the efficacy of p110α/β isoform-selective inhibitors in combination with HER2-targeted therapy separately.

In the present study, we established cell lines isolated from spontaneous mammary tumors that arose in HER2 activation and PTEN loss mouse models, and tested the efficacy of p110α selective inhibitor (BYL719), p110β selective inhibitor (AZD6482), and pan-PI3K inhibitor (BKM120), both in vitro and in vivo. Our results demon- strated a significant anti-tumor effect in BYL719 and BKM120 but not in AZD6482. Furthermore, combination therapy of BYL719 and lapatinib indicated an obvious synergistic anti-tumor effect compared to lapatinib alone both in vitro and in vivo. These suggest that combining

HER2 inhibitors with p110α-selective inhibitors would be an effective and practical personalized therapy for HER2+ and PTEN-loss breast cancer.

Materials and methods

Cell lines and orthotopic xenograft models

MMTV-Neu-IRES-Cre (NIC) mice [27] and PTENL/L mice [28] (Charle River, USA) were backcrossed to the FVB/N background generating MMTV-NIC-PTENL/L (NIC-PTENL/L) mice. Primary NIC-PTENL/L tumor cells were generated from a mammary tumor of an NIC- PTENL/L genetically engineered FVB/N female mouse. To establish this cell line, fresh mammary tumors from NIC-PTENL/L mice were excised and then digested in 9- ml DMEM/F12 basic medium ( Gibco) and 1-ml collagen ase/h yaluronidase m ix ture (StemCell Technologies, Canada) at 37 °C for 9–15 h. After washing and removing red blood cells, cells were resuspended to form single-cell suspensions and filtered through a 40-μm cell strainer (BD Biosciences) to remove clumps. Cells were cultured in complete medium (complete EpiCult-B heparin + rhEGF, bFGF, Heparin + 10 %FBS, all bought from Gibco). Log-phase cells were harvested with tryp- sin– ethylenediaminetetraacetic acid ( EDTA) and suspended in PBS for bilateral mammary fat pad (the forth pair) injection of FVB/N female nude mice at 8 weeks of age. Each subgroup contained five individuals. Each mammary fat pad was injected with 5 × 106 cells. FVB/N mice were obtained from Charles River, USA. Animals were under treatment until the tumor volume reached 100 mm3. The animals’ weights, the longest di- ameter (LD), and the shortest diameter (SD) of the tumors were evaluated each time. The tumor volume was deter- mined by the formula V = LD × SD2/2.
All animals were housed and treated in accordance with protocols approved by the Institutional Animal Care and Use Committee at Dana-Farber Cancer Institute and Harvard Medical School.

Inhibitor studies in vitro

Primary NIC-PTENL/L tumor cells were treated with p110α-specific inhibitor BYL719 (Novartis), p110β- specific inhibitor AZD6482 (MedChemexpress), and pan-PI3K inhibitor BKM120 (Novartis) individually. The detailed concentrations for each drug were demonstrated in figures. Furthermore, cells were exposed to BYL719 with or without lapatinib (Chemexpress)/trastuzumab (Roche) as indicated in the text.

Inhibitor administration in vivo

Animals were under treatment until the tumor volume reached 100 mm3. Forty mice were randomized divided into two parts, the first part was under the treatment of BYL719, AZD6482, BKM120 or PBS respectively, and each group contained five individuals; the second part was under the treatment of lapatinib with BYL719 or PBS (lapatinib were administrated 30 min earlier than BYL719), each group contained five indi- viduals. BYL719 (45 mg/kg) in 10 % 2-hyroxypropyl-β- cyclodextrin (Sigma) was administered daily by intraperitone- al (IP) injection as described [29]. AZD6482 (20 mg/kg) in 7.5 % NMP and 40 % PEG400 (polyethylene glycol 300; Fluka Analytical) were dosed twice a day by IP injection as described [30]. BKM120 was reconstituted 1:9 in NMP (1- methyl-2 pyrrolidone, Sigma) and PEG300 (polyethylene glycol 300; Fluka Analytical), and mice were given this compound formulation at 45 mg/kg daily by oral gavage [31]. Lapatinib (100 mg/kg) was administered daily by oral gavage in 0.5 % hydroxypropyl methylcellulose, 0.1 % Tween80 [32].

Anchorage-independent growth assay

First, 5 × 104 cells were plated into individual 60-mm dishes with a 0.6 % agarose bottom layer and a 0.3 % agarose top layer. Colonies were photographed and counted using a Nikon microscope after 3 weeks of incubation [33]. Each experiment was performed in triplicate measurements.

Crystal violet assay

The effects of trastuzumab with or without BYL719 on the proliferation of NIC-PTENL/L tumor cells were also explored by crystal violet assay. Briefly, cells were seeded in 96-well plates and grown to 75 % confluency. After incubation for 72 h with the drugs mentioned above, cells were incubated with crystal violet in a 7 % (v/v) solution of ethanol/PBS at 37 °C for 10 min and washed twice with PBS [34]. Images of the cells were captured on a Microtek ScanMaker 8700 (Microtek International, Inc.). Each experiment was per- formed in triplicate measurements.

Cell viability assay

Cell viability was assessed using CellTiter 96® AQueous Cell Proliferation Assay (Promega). Equal numbers of cells were plated in 96-well plates and treated with serial dilutions of lapatinib or BYL719 for 72 h. The exact con- centrations of Lapatinib are as follows: 0, 0.01, 0.1, 0.5, 1, 5, and 10 μM; the concentrations of BYL719 are 0, 1, and 2 μM. Each experiment was performed in triplicate measurements.

Western blotting

Cells were lysed in 1 % NP-40 buffer supplemented with protease and phosphatase inhibitors (Roche). Equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes for western blot. The primary antibodies used included p-AKT473 (1:200 dilution, #4060); p-AKT308 (1:200 dilution,#2965); p-PRAS40 (1:1000 dilution, #2997); p-S6RP (1:1000 dilution, #4858); p-4E-BP-1 (1:1000 dilution, #9456); p-HER2 (1:500 dilution, #2243); p-EGFR (1:500 dilution, #8543); PTEN (1:1000 dilution, #9583); Total AKT (1:1000 dilution, #4685), p-ERK (1:1000 dilution, #4370); NeuT (1:500 dilution, #2165) (all from Cell Signaling Technology, CST); and vinculin (1:2000 dilution, #V9264, Sigma). Secondary IRDye 800 goat anti-mouse (1:1000 dilution, #926-32210, LI-COR) and IRDye 680 goat anti-rabbit (1:800 dilution, #926-32221, LI-COR) IgG fluorophore conjugated antibodies were used to visualize the indicated proteins on an Odyssey scanner. Quantification of band intensities was performed using Odyssey 3.0 software.

Statistical analyses

Results are presented as mean ± SD. GraphPad Prism 5 soft- ware (GraphPad Software Inc., San Diego, CA, USA) was used for the two-way ANOVA and Student’s t test where appropriate. Differences were considered statistically signifi- cant at p < 0.01.

Results

Acquisition and characterization of NIC-PTENL/L cell line

In order to rapidly and stably mimic HER2+ PTEN-loss breast cancer in our study, we attempted to establish cell line and orthotopic xenograft models from spontaneous HER2-positive and PTEN-deficient (MMTV-NIC- PTENL/L) mammary tumors. General procedures were de- scribed concretely in the “Materials and methods” section (Fig. 1). As shown in Fig. 2, the critical molecular pheno- type of the cell line was confirmed. Compared with wild- type cells, hyperactivations of p-Akt 473 and 308 were observed in PTEN loss cells.

Exploration of NIC-PTENL/L cells’ reliance upon p110α and p110β

After generation of the NIC-PTENL/L cell line, we investi- gated the reliance of p110α or p110β isoform in the breast cancer cell line harboring HER2+ and PTEN-loss. We assessed cell proliferation and HER2/PI3K signaling in NIC-PTENL/L cell line exposed to BYL719, AZD6482, or BKM120 individually. As indicated in our results, BYL719 and BKM120 not only inhibited cell proliferation but also suppressed the phosphorylation of PI3K pathway such as AKT, PRAS40, S6RP, and 4E-BP-1, while AZD6482 was not as effective as the other two drugs in either aspect (Fig. 3). This result suggested that it was p110α not p110β that played an important role in both NIC-PTENL/L cell proliferation and PI3K signaling. Then, we built orthotopic xenograft models from NIC-PTENL/L cells to  further confirm this conclusion in vivo. Animals were un- der the treatment of BYL719, AZD6482, or BKM120 as described in the “Materials and methods” section and the outcome was in harmony with our previous results (Fig. 4). As demonstrated in Fig. 4, AZD6482 inhibited neither tu- mor growth nor phosphorylation of PI3K pathway includ- ing AKT, PRAS40, S6RP, and 4E-BP-1; by contrast, both BYL719 and BKM120 showed significant effects on the suppression of tumor growth and PI3K signaling. Taken together, we gained the conclusion that it was p110α and not p110β that played an important role in both NIC- PTENL/L tumor growth and PI3K signaling.

Fig. 4 Effects of p110α or p110β inhibition on signaling and growth of orthotopic tumor transplants derived from NIC-PTENL/L cells. a Growth curves of NIC-PTENL/L cell xenografts treated with BYL719, AZD6482, or BKM120. *P < 0.01, two-way analysis of variance. b Western blot analysis of PI3K signaling in NIC-PTENL/L cell xenograft tissues treated with BYL719, AZD6482, or BKM120 (K976, K977, K978, et al. were
serial numbers of the nude mice under treatment, each group contained five individuals)
Combined inhibition of p110α and HER2 on NIC-PTENL/L cells both in vitro and in vivo.

To test whether p110α inhibition could overcome lapatinib or trastuzumab resistance in HER2+ and PTEN-loss cells in vitro, NIC-PTENL/L cells were exposed to BYL719 of different concentrations with or without lapatinib/ trastuzumab. Cell growth and PI3K signaling were assessed. Lapatinib or trastuzumab alone failed to signifi- cantly inhibit tumor growth, while the addition of BYL719 provided obvious improvement over lapatinib or trastuzumab monotherapy (Fig. 5a, b). In addition, dual HER2 and p110α inhibition significantly decreased PI3K pathway phosphorylation (Fig. 5c). Further, we utilized orthotopic xenograft models from NIC-PTENL/L cells and showed that the addition of p110α inhibitor to lapatinib indeed delayed tumor development (Fig. 6). Our results suggested that combined HER2 and p110α inhibition should be a durable treatment for patients with HER2+ and PTEN-loss breast cancer.

Discussion

HER2-targeted therapy resistance is a clinically devastat- ing problem for HER2-positive breast cancer, especially when concomitant with PTEN loss. In the present study, we established NIC-PTENL/L cell line and built their orthotopic xenograft models as in vitro and in vivo models, closely mimicking the trastuzumab/lapatinib- resistant human breast cancer with the aim of obtaining better strategies to overcome this resistance in the clinic. Our results showed that the HER2 antibody and p110α inhibitor BYL719 combination treatment suppressed PI3K phosphorylation and led to a much better anti-tumor effect.

Previous studies have proved hyperphosphorylation of PI3K signaling to be one of the major mechanisms leading to drug resistance of different cancers, thus PI3K is predicted as a promising target to combat these resistances [11, 12, 35–37]. Several novel inhibitors have been developed and assessed in preclinical trials, including pan-PI3K inhibitors and PI3K isoform-selective inhibitors. Samuel W. Brady et al. and others proved that pan-PI3K inhibitors or p110α selective inhibitors usually worked better in tumors with ac- quired activation of receptor tyrosine kinases such as VEGF receptor, EGF receptor, PDGF receptor, or HER2; Shidong Jia et al. and some other researchers proved that ablation of p110β but not of p110α impeded tumorigenesis of prostate tumors with PTEN loss; while Britta Weigelt et al. demon- strated PTEN-mutant endometrioid endometrial cancer (EEC) cell lines were sensitive to p110α selective and pan-PI3K inhibitors but resistant to the p110β inhibitors [16, 17, 26, 38–42]. Considering the complexity of the matter, isoform dependence is urging to be explored in a given subtype of cancer for more precise targeted therapies. Our data suggested that HER2-positive-PTEN- loss breast cancer depended on p110α for tumor develop- ment and targeting p110α was necessary to overcome their resistance to HER2-targeted therapies.

Of note, most existing clinical trials concerned pan- PI3K inhibitors alone or in combination with other thera- pies, and proved those inhibitors such as BKM120, BEZ235, or XL765 to be effective in improving anti- tumor activities in several kinds of cancers [43–47]. Besides, although demonstrated to be safe for most pa- tients, adverse events like asthenia, hyperglycemia, hyper- lipidemia rash, liver toxicity, and others have been report- ed for those inhibitors due to the inhibition of all class IA PI3K isoforms. Thus, with the ongoing work to determine the isoform dependence of different cancers, new PI3K isoform-selective inhibitors are emerging in the clinic as a better choice than pan-PI3K inhibitors. PI3K alpha iso- form inhibitor BYL719 is suggested to be well-tolerated, and showed signs of clinical activity with manageable side effects and a predictable PK profile [48, 49]. PI3K beta isoform inhibitor AZD6482 and delta isoform inhib- itor CAL-101 also have been examined in clinical trials for safety, pharmacokinetic, and pharmacodynamic stud- ies [50, 51]. More clinical trials are examining these in- hibitors to explore outcomes and the safety of combina- tion therapies with different drugs.

With the development of biological techniques, targeted therapies are more accurately selected for pop- ulations on the basis of the molecular features of their tumors, putting forward the concept of precision medi- cine. Along with the recent emphasis on precision med- icine especially in oncology, increasing attention has been drawn to new biomarkers that may help account for more effective regimens. PTEN usually works as a tumor suppressor and loss of PTEN could promote the growth of breast cancer, which should be considered during therapy determination. Combining our reports to- gether with those many previous researches, we provide a possible regimen for breast cancer with consideration of HER2 and PTEN: HER2-targeted therapy is good for tumors which are both HER2- and PTEN-positive, p110β selective inhibitor should be a choice for tumors with HER2-negative but PTEN-deficient, while combi- nation therapy of HER2 antibody and p110α selective inhibitor is suitable for HER2-positive-PTEN-loss tumors.

In summary, our study implicated that it was p110α rather than p110β that played an important role in the resistance of HER2+ PTEN-loss breast cancer to HER2- directed therapies, and combining BYL719 with lapatinib/trastuzumab showed significant synergistic ef- fect. In addition, the combination of p110α selective inhibitors could be a potential strategy for personalized treatment of HER2 antibody resistant breast cancer, par- ticularly mediated by PTEN loss. Further preclinical and clinical researches are expected to find the optimal schedules.