BRUTON’S TYROSINE KINASE INHIBITORS PREVENT THERAPEUTIC ESCAPE IN BREAST CANCER CELLS

Abstract

We have reported that a novel isoform of BTK (BTK-C) expressed in breast cancer protects these cells from apoptosis. In this study, we show that recently developed inhibitors of BTK, such as ibrutinib (PCI-32765), AVL-292 and CGI-1746, reduce breast cancer cell survival and prevent drug resistant clones from arising. Ibrutinib treatment impacts HER2+ breast cancer cell viability at lower concentrations than the established breast cancer therapeutic lapatinib. In addition to inhibiting BTK, ibrutinib, but not AVL-292 and CGI-1746, efficiently blocks the activation of EGFR, HER2, ErbB3, and ErbB4. Consequently, the activation of AKT and ERK signaling pathways are also blocked leading to a G1/S cell cycle delay and increased apoptosis. Importantly, inhibition of BTK prevents activation of the AKT signaling pathway by NRG or EGF that has been shown to promote growth factor-driven lapatinib resistance in HER2+ breast cancer cells. HER2+ breast cancer cell proliferation is blocked by ibrutinib even in the presence of these factors. AVL-292, which has no effect on EGFR family activation, prevents NRG- and EGF-dependent growth factor-driven resistance to lapatinib in HER2+ breast cancer cells. In vivo, ibrutinib inhibits HER2+ xenograft tumor growth. Consistent with this, immunofluorescence analysis of xenograft tumors shows that ibrutinib reduces the phosphorylation of HER2, BTK, Akt and Erk and histone H3 and increases cleaved caspase-3 signals. Since BTK-C and HER2 are often co-expressed in human breast cancers, these observations indicate that BTK-C is a potential therapeutic target and that ibrutinib could be an effective drug especially for HER2+ breast cancer.

Introduction

Bruton’s tyrosine kinase (BTK) belongs to the TEC family of cytoplasmic tyrosine kinases(1). BTK was identified in 1993 as a novel non-receptor protein tyrosine kinase that is mutated in X-linked agammaglobulinaemia (XLA)(2, 3). It is predominantly expressed in hematopoietic cells including erythroid progenitors and myeloid cells(4) and is established as a critical regulator of B-cell receptor (BCR) signaling, development, differentiation and survival(5–7). Due to its role in BCR signaling-induced proliferation, BTK has emerged as a novel target for the treatment of rheumatoid arthritis and other immune diseases. Recent studies have focused on the essential role of BTK in many B cell leukemias and lymphomas(8, 9) which provides a rationale for targeting the kinase in these malignancies. BTK inhibitors including ibrutinib(10), AVL-292(11) and CGI-1746(12) developed as immunosuppressants have been used in clinical trials for blood malignancies(13). Recently, ibrutinib (Imbruvica) gained FDA approval for the treatment of mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström’s macroglobulinemia(14). An alternate isoform of BTK, BTK-C, was identified as a novel survival factor for breast cancer cells in a large-scale loss-of-function analysis of human tyrosine kinases using an RNA interference library(15). This study showed that, although BTK-C is expressed at relatively low levels in several human breast cancer cell lines and tumor tissues, it provides an essential function protecting breast cancer cells from apoptosis.

It has long been appreciated that HER2 is overexpressed or amplified in tumors of about 20% of patients with early stage breast cancer and confers an increased disease recurrence and a worse prognosis(16). HER2-directed therapies including trastuzumab, pertuzumab, ado-trastuzumab and lapatinib have been used in clinic and has significantly improved the outlook for patients with HER2-positive breast cancer(17). However, a significant proportion of these patients still relapses and succumbs to their disease(18). Therefore, new classes of drugs are needed, especially for HER2-positive advanced-stage breast cancer and those that have developed resistance to current therapies.

Here we describe the effects of treating breast cancer cells with recently developed BTK inhibitors. We find that among the new inhibitors, ibrutinib is particularly effective at inhibiting breast cancer cell growth in vitro and in vivo. Ibrutinib inhibits BTK and all members of the ERBB family of receptor tyrosine kinases making it especially effective at reducing HER2-positive breast cancer cell growth and survival. Ibrutinib induces a G1-S arrest and apoptosis in these cells. We also show that ibrutinib’s effects on HER2-positive breast cancer cells are not mitigated by NRG1 or EGF stimulation, as occurs with lapatinib. As the expression of BTK-C and HER2 are positively correlated in surgical specimens of human breast cancer tissues, these results indicate that ibrutinib is a potential therapy for this solid tumor type.

Materials and Methods

Cell culture and chemicals

Breast cancer and MCF-10A cell lines were obtained from the American Type Culture Collection in 2011. All cell lines were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 units/μl of penicillin-streptomycin (Cellgro), except for MCF-10A cells cultured as indicated in (15). All cell lines were authenticated in March 2016 by the SUNY-Albany Center for Functional Genomics Molecular Core Facility using a short tandem repeat method (Promega GenePrint 10 system). BTK inhibitors were supplied as follows. Ibrutinib was purchased from ChemieTek, AVL-292 was purchased from MedKoo Biosciences, CGI-1764 was purchased from Axon Medchem. Recombinant human –β1 and Matrigel were purchased from R&D. Src inhibitor Saracatinib was purchased from Selleckchem. LY294002 was purchased from Cell Signaling.

Cell viability assays

For live cell counts, cells were grown on 96-well plates, fixed with 4% formaldehyde and counterstained with Hoechst 33342. Images of cells were acquired using an In Cell Analyzer 1000 (GE Healthcare) high-content imaging system. At least 30 fields were imaged per well. Statistics were performed using the In Cell Investigator 3.4 image analysis software (GE Healthcare).

3D cell culture was performed as previously described (19). BT474 cells were propagated in DMEM with 10% FBS. Single cells in medium containing 5% Matrigel were seeded at a density of 5 × 10⁴ cells/cm² on a Matrigel-coated well. The top medium with 5% Matrigel and ibrutinib or lapatinib was changed every 3 days. 1μM ethidium bromide was used to stain cell death during 3d culture.

Cells were cultured in 6-well plates for indicated number of days. Cells were fixed with 3.7% paraformaldehyde for 10 min and washed with PBS. After washing, cells were stained with 0.05% Crystal Violet for 30 min and washed with tap water. 0.5ml methanol was added to the dry plates to solubilize the dye. Crystal Violet staining was measured by reading OD 540 of each sample. Tumorspheres were counted under low magnification

Cell cycle analysis

Cells were cultured in six-well plates and treated with vehicle, lapatinib or ibrutinib at the concentration as indicated for 16 hours. After trypsinization, cells were collected and washed with cold PBS. Cells were fixed with 70% ethanol, washed and stained with PI/Triton X-100 staining solution (0.1% triton X-100, 2μg/ml PI and 0.2mg/ml DNAse-free RNAse) for 30 min. Samples were analyzed by flow cytometry.

Immunofluorescence

Human breast cancer tissue sections (BR10010b, US Biomax, Inc. MD) were baked for 1h at 62°C, subjected to serial alcohol treatments for rehydration and microwaved in 0.01M sodium citrate for 20min for antigen retrieval. The sections were serum blocked for 30min, incubated overnight at 4°C with primary antibodies in phosphate-buffered saline and subsequently with Cy5-labeled secondary antibodies for 60min, nuclei were stained with Hoechst 33342. The stained sections were mounted with anti-fade solution for microscopy. The two by two tables for human data was analyzed by Fisher’s Exact Test. Significance was determined by the alpha level of 0.05.

Immunoblotting

Immunoblotting was performed essentially as described previously. Equal amounts of proteins were used. Antibodies used were anti-EGFR (1:1000,), anti-HER2 (1:1000,), anti-ERBB3, ERBB4 antibody, anti-BTK, anti-AKT, ERK, anti-PLCγ1, PLCγ2 and anti-PARP (1:1000, cell signaling), anti-Flag antibody (1:1000, Sigma), anti-rabbit IgG-HRP and mouse IgG-HRP (1:5000, Jackson ImmunoResearch)(20).

Apoptosis assay

Apoptotic cells were identified using an Alexa Fluor 488 annexin V apoptosis Kit (Invitrogen). Cells were treated with lapatinib or ibrutinib for 24 hours. Cells were trypsinized and washed with cold PBS and resuspended in 1×annexin-binding buffer to 1×10⁶cells/ml. 5μl Alexa Fluor 488 annexin V and 1μl 100μg/ml PI was added to each 100μl of cell suspension. Cells were incubated for 15 min at room temperature. After the incubation, 400μl 1×annexin-binding buffer was added, mixed gently and the sample kept on ice. Samples were analyzed on a BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA). The data were analyzed using the FlowJo software package (Treestar Inc., Ashland, OR).

Animal experiments

NOD/SCID mice were purchased from Jackson Lab (The Jackson Laboratory, Bar Harbor, ME, USA). All mouse procedures were approved by the Animal Care and Use Committees of SUNY Albany and performed in accordance with institutional policies. For xenograft tumor studies, 1×10⁶ SKBR3 cells were suspended in 50 μl Matrigel (BD Biosciences) diluted 1:2 with DMEM and injected into mammary fat pad. Treatment began when tumors were palpable. Ibrutinib was given p.o. 6mg/kg/day or 12mg.kg.day in a vehicle of 1% DMSO/30% polyethylene glycol/1% Tween 80. Lapatinib was given p.o. 75mg/kg/day or 37.5mg/kg/day. The tumor volume in mm³ is calculated by the formula: volume = (width)² × length/2 every 7 days.

Results

Activity of BTK inhibitors in human breast cancer cells

In a functional genomic screen, a novel BTK isoform has been identified as a gene whose expression protects breast cancer cells from apoptosis (15). In addition to genetic evidence, the early BTK inhibitor LFM-A13 was shown to increase apoptosis levels in breast cancer cells. The recently developed BTK inhibitors, including ibrutinib, AVL-292 and CGI-1746, are more potent, more specific and more useful clinically compared to LFM-A13(12, 21, 22). In 2013, ibrutinib was approved by the FDA for treatment of B cell malignancies(23, 24). As a first step in exploring the potential clinical utility of the recently developed BTK inhibitors, we performed cell growth assays to determine the effect of these inhibitors on breast cancer cells. We find that ibrutinib results in decreased cell number in breast cancer cells MCF7, SKBR3 and MDA-MB-231, but not in MCF-10A cells (Fig. 1A). These results are consistent with our previous findings(15). Surprisingly, we observe that HER2-positive breast SKBR3 cancer cells are more sensitive to ibrutinib (1μM) than non-tumorigenic, luminal or triple negative cell lines (Fig. 1A). Ibrutinib reduces cell numbers by more than 80% at 3 days. To extend our findings to another HER2-positive breast cancer cell, we tested the effect of ibrutinib on BT474 cell survival. The result shows that ibrutinib decreases HER2-positive breast cancer cell growth by 20% ~30% at a concentration of 10 nanomolar(Fig. 1B). The IC50 for ibrutinib’s effect on HER2-positive breast cancer cells measured at 3 days of culture is 30 nM. We also explored whether HER2-positive breast cancer cells are sensitive to other BTK inhibitors. AVL-292 and CGI-1746 inhibit BTK-C kinase activity to the same degree as ibrutinib (Supplementary data, Figure 1). They are, however, less effective anti-proliferative agents than ibrutinib, achieving 30~40% decreases in cell numbers in HER2-positve breast cancer cell lines at concentrations two orders of magnitude higher than the IC50 of ibrutinib (Fig. 1C). These results suggest that ibrutinib not only inhibits BTK-C activity, but also affects other targets specifically required for HER2-positive breast cancer cell survival.

Figure 1. Growth inhibitory effects of BTK inhibitors on breast cancer cell lines.

A, Cell counts of MCF-10A, MCF-7, SKBR3 and MDA-MB-231 cells treated with vehicle, 1μM and 10μM of ibrutinib (Ibr) for 3 days. B, Cell counts of SKBR3 and BT474 cells treated with vehicle and different concentrations of ibrutinib, as indicated for 3 days. C, Cell counts of BT474 cells treated with vehicle and ibrutinb, AVL-292 and CGI-1746, at indicated concentrations, for 3 days. Results are presented as percentage of control (vehicle). Error bars indicate the s.d. from three individual experiments, *p<0.05, **p<0.01 compared with control.

Ibrutinib effects in HER2-positive breast cancer cells

Ibrutinib is a covalent inhibitor of BTK(10) that irreversibly binds to a cysteine residue (Cys-481) near the ATP binding pocket of BTK. Sequence alignments show that only 10 kinases in the human genome have a cysteine residue at an analogous position. They include Blk, Btk, Bmx, EGFR, HER2, ErbB4, Itk, Jak3, Tec and Txk(25). Given the well-characterized role of the ERBB family in breast cancer we suspected that, in addition to BTK, Ibrutinib may exert its potent anti-proliferative affect through these targets. To compare the inhibitory effect of ibrutinib with lapatinib on HER2-positive breast cancer cells, we first tested their activity in monolayer culture. Lapatinib, a selective, reversible inhibitor of both EGFR and HER2 is currently used to treat HER2-positive breast cancer patients. Treatment with ibrutinib at different concentrations as indicated for 3 days reduces BT474 cell number. 0.01μM of ibrutinib reduces cell populations to 30% of control (p<0.01). However, lapatinib, at the same concentration (0.01μM) fails to reduce HER2-positive breast cancer cell numbers significantly (Fig. 2A). These results are generally consistent with recently published data also showing ibrutinib inhibits cell viability of HER2-positve breast cancer cells(26). A number of studies have shown that cells’ microenvironment can impact drug response(27, 28). In order to test whether HER2-positive breast cancer cells are still sensitive to ibrutinib in 3D matrigel culture condition, treatment of BT474 cells with 0.1 μM or 0.5μm of ibrutinib for 9 days significantly reduces cell number when compared with the control (Fig. 2B). Thus, we find that ibrutinib reduces HER2-positive breast cancer cell number in both monolayer and 3D culture, and that ibrutinib has a more potent effect on HER2-positive breast cancer cells than lapatinib.

Figure 2. Effect of lapatinib and ibrutinib on cell growth and signal transduction in BT474 cells.

A, Cell counts of BT474 cells treated with vehicle, lapatinib (Lap) and ibrutinib (Ibr) for 3 days. Results are presented as percentage of control (vehicle). Error bars indicate the s.d. from three individual experiments, *p<0.05, **p<0.01 compared with control. B, BT474 cells were treated with laptanib and ibrutinib, as indicated, for 9 days on Matrigel. C, Alignment of EGFR family members with C481 region of BTK. D, effects of BTK inhibitors on EGFR family activation. BT474 cells were treated with different concentrations of BTK inhibitors ibrutinib and AVL-292 or lapatinib for 2h. Whole cell lysates were prepared for immunoblotting using antibodies against p-EGFR, p-HER2, p-ERBB3, p-ERBB4, p-Akt, p-ERK proteins. Immunoblotted GAPDH levels provide loading controls.

In HER2 overexpressing breast cancer cells, HER2 dimerizes with its partner EGFR or Her3. HER2/Her3 heterodimers directly phosphorylate the p85 regulatory subunit of PI3K activating the PI3K/Akt pathway(29). In parallel, HER2/EGFR heterodimers also activate the MAPK pathway in most cases(30). To examine the effect of ibrutinib on levels of activated Akt or Erk, we treated BT474 cells with 0.05μM or 0.1μM of ibrutinib and 0.1μM of AVL-292 for 2 hours. We find that ibrutinib inhibits the phosphorylation of EGFR, HER2, Her3 and ErbB4, which results in blocking downstream signaling that requires Akt or Erk activation. Compared with ibrutinib, AVL-292 does not block EGFR family signaling pathway activation, even though AVL-292 also covalently binds Cys481 on BTK. Lapatinib, a dual kinase inhibitor of EGFR and HER2, inhibits both AKT and ERK phosphorylation (Fig. 2D)(31). These results indicate that in breast cancer cells, ibrutinib, in addition to inhibiting BTK irreversibly (Supplementary data, Figure 2)., also serves as a pan-EGFR family inhibitor blocking the activation of each kinase. That ibrutinib is able to block two distinct types of kinases both of which have been established as important to the growth and survival likely explains its greater impact on HER2-positive breast cancer cells compared to other breast cancer cells (Fig. 1).

Ibrutinib effects on proliferation and survival of HER2-positive breast cancer cells

In HER2-positive breast cancer cells, the activation of HER2 stimulates both the MAPK and Akt signaling pathways, which results in cell proliferation due to increased G1-S phase transition and cell cycle progression (31). Treatment of HER2 overexpressing BT474 human breast cancer cells for 24 hours with ibrutinib or lapatinib leads to an appreciable G1-S arrest. A significant 50% decrease in the number of cells in the S phase of the cell cycle is observed at a concentration of 0.03μM for ibrutinib when compared with controls (Fig. 3A). Similar results are seen for lapatinib(31). This cell cycle delay is correlated with an increase in p27, an inhibitor of cell cycle progression, and a decrease in cyclinD1 (Fig. 3B).

Figure 3. Effect of ibrutinib on cell cycle and apoptosis.

A, effect of ibrutinib and lapatinib on cell cycle progression. BT474 cells were treated with the indicated concentration of ibrutinib and lapatinib for 16h. Cells were stained with propidium iodide and analyzed by flow cytometry. B, BT474 cells were treated with ibrutinib or lapatinib for 16h. Cell lysates were analyzed by immunoblotting using anti-cyclin D1, anti-p27 antibodies. C, BT474 cells were treated with ibrutinib or lapatinib for 16h. Apoptotic cells were identified by Alexa Fluor 488 Annexin V kit. *p<0.05, **p<0.01 compare with control. D, immunoblots showing apoptosis in BT474 cells after indicated concentration of ibrutinib treatment for 16 h. Immunoblotted GAPDH levels provide loading controls.

An increase in the number of apoptotic cells following treatment with 0.05 or 0.1μM ibrutinib or lapatinib for 24 hours is observed. Our results are consistent with the results of earlier studies, which have shown the effect of 0.1 or 0.5μM lapatinib on cell survival (31). Compared with lapatinib, ibrutinib induces 1.5 fold more apoptosis in HER2-positive breast cancer cells (Fig. 3C). The effect of ibrutinib on cell survival regulatory proteins was also examined. Ibrutinib blocks PLCγ1, PLCγ2, Akt and ERK phosphorylation, and increases cleaved PARP (Fig. 3D). Taken together, these results suggest that ibrutinib decreases cell numbers by inducing both a G1-S delay and apoptosis in HER2-positive breast cancer cells.

BTK inhibition blocks the reactivation of AKT and ERK pathways induced by NRG1 or EGF in lapatinib treated HER2-positive breast cancer cells

Cancer cells typically express multiple receptor tyrosine kinases (RTKs) which control cell survival signals(32). RTK ligands are produced via autocrine tumor-cell production, paracrine tumor stroma production and systemic production(33). An increase in RTK ligands has been shown to result in the resistance of cancer cells to RTK inhibitors which ultimately produces therapeutic escape (34). In HER2-positive breast cancer cells, NRG1 is the most broadly active ligand, followed by EGF(35). We hypothesized that the greater efficacy of ibrutinib may impact the occurrence of growth factor dependent lapatinib resistance. We first tested the effect of exposing HER2-positive cells to 50ng/ml of NRG1 with different concentrations of lapatinib or ibrutinib for 3 days. The results again show that either lapatinib or ibrutinib potently suppresses cell growth. However, NRG1 application is able to rescue lapatinib-induced growth inhibition in HER2-positive breast cancer cells allowing resistant cells to emerge from the treatment (Fig. 4A). In contrast, HER2-positive breast cancer cells cannot be rescued from ibrutinib-induced growth inhibition by NRG1. Similar results are observed using 50ng/ml of EGF.

Figure 4. Inhibition of pro-survival pathway re-activation by ibrutinib.

A, Live cell count assay showing the NRG effects on drug-treated BT474 cells(72h), complete rescue, lapatinib (Lap) with NRG treatment; no rescue lapatinib, ibrutinib(Ibr) and ibrutinib with NRG treatment. B, crystal violet cell staining of BT474 or SKBR3 cells treated with lapatinib (1uM) with or without NRG1 (50ng/ml), ibrutinib (1uM) with or without NRG(50ng/ml). C, Immunoblots showing effects of NRG (50ng/ml) on p-AKT and a-ERK after cells treated with lapatinib or ibrutinib (2h) in BT474 and SKBR3 cells. Immunoblotted GAPDH levels provide loading controls. D, Live cell count assay showing the NRG effects on drug-treated BT474 cells(72h), complete rescue, Lapatinib with NRG1 treatment; no rescue, lapatinib alone or lapatinib and AVL with NRG1 treatment.

The ability of NRG1 or EGF signaling to rescue HER2-positive breast cancer cells from growth inhibition by HER2 kinase inhibitor lapatinib is consistent with other findings(34). However, the findings that neither NRG1 nor EGF was able to rescue HER2-positive breast cancer cells from the growth inhibition by BTK inhibitor ibrutinib were unexpected. To further confirm these findings, additional long-term co-treatment experiments were performed. Nine days lapatinib/NRG1 co-treatment yields cells that exhibit lapatinib resistance in both BT474 and SKBR3 cell populations. In contrast, 9-days ibrutinib/NRG1 co-treatment does not yield ibrutinib resistant cells in HER2-positive breast cancer cell lines (Fig. 4B). Similar results are observed in experiments using EGF where cells are co-treated with EGF/lapatinib or EGF/ibrutinib (Supplementary data, Figure 3).

To investigate the signaling pathway which mediates the NRG1- or EGF- induced rescue of HER2-positive breast cancer cells, we examined the activation of two downstream survival signaling pathways commonly engaged by RTKs: the PI3K-AKT and MAPK pathway(35). In the presence of lapatinib or ibrutinib alone, the activation of HER2, AKT and ERK are blocked. When cells are co-treated with NRG1/lapatinib, NRG1 induces phosphorylation of AKT while HER2 remains inhibited. Surprisingly, NRG1 fails to re-activate AKT under conditions of NRG1/ibrutinib co-treatment (Fig. 4C). These results suggest that the NRG1 rescuing effect is blocked by ibrutinib and that ibrutinib may target a second activated kinase. The results also suggest that lapatinib cannot block this second kinase.

Since ibrutinib was originally designed to block BTK activation(25) and since BTK has been shown to activate AKT in B cells(36), the possibility existed that BTK-C provided a pro-survival signal in the genesis of lapatinib resistance. That we identified BTK-C as a critical cell survival gene in a large scale RNAi screen in these cells was consistent with this notion(15). For these reasons, we hypothesized that BTK-C mediated the re-activation of AKT induced by NRG1. However, since ibrutinib is as potent as an inhibitor of EGFR family activation as lapatinib in HER2-positive breast cancer cells, we needed to dissociate the effects of EGFR inhibition from the effects of BTK inhibition. To test whether BTK-C is a secondary kinase that mediates NRG1 rescue in HER2-positive breast cancer cells, we treated BT474 and SKBR3 cells with lapatinib, lapatinib/NRG1 or lapatinib/NRG1 plus AVL-292, a BTK inhibitor that does not inhibit the EGFR family (Fig. 2). We find that NRG1 rescue is blocked by simultaneously targeting BTK and the EGFR family when cells treated with lapatinib and AVL-292 (Fig. 4D). These results provide evidence that BTK-C signaling is involved in the appearance of ligand-dependent lapatinib resistance in treated HER2-positive breast cancer cell populations.

BTK-C signaling in HER2-positive breast cancer cells

The BTK signaling pathway has been extensively studied in hematopoietic cells. Upon antigen binding to the BCR, PI3K is activated. PI3K activity recruits BTK to the cell membrane through a PIP3-PH domain interaction, which allows SYK and LYN to fully activate BTK(37–39). In our previous studies, we showed that a novel isoform of BTK (BTK-C) is expressed in human breast cancer cell lines and tissues. To explore the signaling activation of BTK-C in breast cancer cells, we assessed two potential upstream regulatory molecules of BTK-C: PI3K and Src(40). First, we treated the SKBR3-BTK-C cells for 24 hours with established concentrations of the PI3K inhibitor LY294002 (5 or 10 μM) or the Src inhibitor saracatinib (5 or 10 μM). The phosphorylation of BTK-C is appreciably decreased by saracatinib at 10μM (41). The phosphorylation of AKT, as a downstream target of BTK-C, also decreases. In contrast, 10μM LY294002 does not suppress BTK-C activation (Fig. 5A). Since the possibility exists that this lower concentration of LY294002 may not block BTK-C activation, we increased the concentration of LY294002 to 50μM and repeated the test. The results show that LY294002 at 50μM completely blocks AKT activation, but not BTK-C activation (Fig. 5B). Collectively, these results suggest that Src, or a closely related kinase or kinases, is a significant player in the upstream signaling pathway of BTK-C activation in HER2-positive breast cancer cells. The Src/FAK signaling pathway is involved in the lapatinib-induced kinome reprogramming (42) that contributes to drug resistance in HER2-positive breast cancer. Inhibition of Src/FAK signaling enhances lapatinib growth inhibition in these cells. Consistent with the notion that Saracatinib inhibits BTK-C activation we find that blocks the NRG1-mediated rescue of lapatinib sensitivity in HER2-positive breast cancer cells (Fig. 5C). These findings suggest that BTK-C is a downstream target of Src or a closely related kinase or kinases and that this signaling contributes to NRG1-mediated drug resistance in HER2-positive breast cancer cells.

Figure 5. BTK-C activation by Src in breast cancer cells.

A, SKBR3-BTKC cells were treated with ibrutinib, LY294002 and saracatinib at indicated concentrations for 24h. Cell lysates were probed for p-BTK, p-AKT and p-ERK. B, SKBR3-btkc cells were treated with ibrutinib and different concentration of LY294002 for 24h. Cell extraction were tested for phosphorylation of the indicated protein. Anti-ERK as a loading control. C, crystal violet cell staining of BT474 cells treated with lapatinib (1uM) with or without NRG1 (50ng/ml), and lapatinib with NRG(50ng/ml) and saracatinib (5uM). Immunoblotted GAPDH levels provide loading controls.

Effects of ibrutinib treatment on HER2-positive breast tumor xenografts growth in vivo

Results from molecular experiments carried out in vitro pointed to the possibility that ibrutinib treatment might be useful in inhibiting HER2-positive tumor progression. To test this possibility, we assessed the effect of ibrutinib on xenografts of SKBR3 in NOD/SCID mice. Ibrutinib treatment inhibits tumor growth when administered to animals between 6mg/kg/day and 12mg/kg/day. At day 28, tumor volumes in mice that received 12mg/kg are 45% smaller than the volumes in mice that receive a vehicle control (p<0.01)(Fig 6A). To determine whether inhibition of tumor growth was correlated with inhibition of the target molecules, HER2 and BTK phosphorylation were examined in tumor tissues by immunofluorescence staining as well as the phosphorylation of downstream targets AKT and ERK. Phosphorylation of HER2, BTK, AKT and ERK are inhibited by ibrutinib treatment (Fig 6B). In addition, changes in proliferation and apoptosis markers in tumor tissues reflect the effect that ibrutinib has in xenografts of SKBR3. The ability of ibrutinib to target both HER2 and BTK-C in these xenografts confirms the in vitro findings and provides a strong rationale for the use of ibrutinib in HER2-positive breast cancer chemotherapy.

Figure 6.

Ibrutinib inhibits Her2+ breast cancer cells growth in vivo. A, Images of tumors formed in animals after mammary fat pad injection of SKBR3 cancer cells. Animals were treated with vehicle, lapatinib (37.5mg/kg or 75mg/kg) or ibrutinib (6mg/kg or 12mg/kg) for 4 weeks. B, Tumor growth curves obtained following fat pad injection of SKBR3 cells. The data represent the mean ± s.e. (n=5, *P<0.01). (c) Histological analysis of tumors from SKBR3 cells treated with ibrutinib (12mg/kg) or vehicle. Shown are pHH3, caspase 3, pHER2, pBTK, pAkt and pErk staining.

The expression of BTK-C in HER2-positive breast cancer tissue

The above results suggest that simultaneously targeting HER2 and BTK represents an improved approach to the treatment of HER2-positive breast cancer. We identified the BTK-C isoform as a gene whose function is critical for breast cancer cell survival even though its expression levels are quite low. For this gene product to be a useful target, it is necessary to determine the frequency of its expression in HER2+ breast cancer. Although currently routine for many genes, establishing BTK-C expression patterns in tumors by querying available databases is not possible. The BTK-C isoform has only recently been described and little is understood regarding its expression. This is due in part to the fact that Affymetrix probes for this region have only been included in exon microarrays very recently. Additionally, the BTK-C isoform encodes the entire B-cell version sequence (BTK-A) and is annotated as a 5′ UTR splice variant of BTK-A(15). To provide further evidence about the expression of BTK-C in breast cancer tissues, especially HER2-positive breast cancer tissues, we examined the co-expression of BTK-C and HER2 in surgical specimens of human breast cancer tumor tissues by immunofluorescence. Consistent with our previous studies(15), we find that 30% of breast cancer tumor tissues stain positively for BTK (Fig 7). Among 23 HER2-positive human breast cancer tissues, 43% are positive for BTK. There is a statistically significant association of expression of BTK with HER2 expression (p<0.01) (Fig. 7B). These findings suggest that the expression of BTK-C and HER2 are positively correlated in human breast cancer tissues. This increases the likelihood that ibrutinib has breast cancer therapeutic utility as the opportunity to target both kinases simultaneously should occur in a significant percentage of HER2-positive breast cancers.

Figure 7. Positive correlation between BTK and HER2 expression in surgical specimens of human breast cancer tissue.

A, Her2 positive and negative surgical specimens of human breast tumors (BR10010b) were subject to stain BTK and HER2. The data represent 50 of breast tumor specimens. Case 1 and case 2 represent positive staining BTK-C, case1 and case 3 represent positive staining HER2. B, Summary of the positive correlation. The correlation between the overexpression of BTK-C and HER2 was analyzed by Fisher’s exact test.

Discussion

Our previous findings showed that a novel isoform of BTK (BTK-C) is frequently expressed in human breast cancer cells and tissues and that this isoform plays a crucial role in cell survival. Consistent with these findings, we report that several recently developed BTK inhibitors reduce breast cancer cell number in a variety of cell types including MCF7, MDA-MB-231 and SKBR3 cells. Importantly, we found that HER2-positive breast cancer cells are highly sensitive to ibrutinib. Ibrutinib, with an IC50 for these cells of 0.03 μM, is much better at decreasing cell numbers than the other BTK inhibitors, AVL-292 and CGI-1746, and compares favorably with lapatinib. In vitro, enzymatic activity assays show that IC50 of lapatinib for EGFR and HER2 are10.8 and 9.2nM respectively. Our results indicate that the sensitivity of HER2-positive breast cancer cells to ibrutinib is due to the drug’s ability to simultaneously suppress the activation of both BTK-C as well as the EGFR family. Consistent with previous studies, ibrutinib treatment results in induction of a pronounced G1/S delay and increased apoptosis in HER2-positive breast cancer cells(31). We also find that BTK-C plays an essential role in the ability of HER2-positive breast cancer cells to develop resistance to lapatinib under conditions when the growth factors NRG1 or EGF are present. NRG1 or EGF are unable to re-activate PI3K/Akt or MAPK signaling pathways and allow cells to escape the inhibitory effects of lapatinib when BTK-C is blocked by AVL-292, which is incapable of inhibiting the EGFR family. Ibrutinib inhibits the activity of the EGFR family and inhibits the BTK-dependent reactivation of the PI3K/Akt or MAPK signaling pathways. It is, therefore, a strong candidate to replace lapatinib as a combination therapy with trastuzumab for HER2-positive breast cancer patients who might otherwise develop resistance to lapatinib. As we find that the expression of BTK-C and HER2 are positively correlated in human breast cancer tissue with 40% of HER2-positive breast cancer tissues also being BTK-C positive, the potential utility of the drug is significant.

In accord with the in vitro studies, our results show that ibrutinib treatment at 12mg/kg/day causes a significant 45% inhibition of HER2-positive tumor growth in vivo. Animals tolerate ibrutinib at this dose, although ibrutinib–related toxicity was observed. Consistent with ibrutinib’s antitumor effects, staining of the proliferation marker, phosphorylated histone H3, decreased in treated xenografts tumor tissues compared with untreated control tumor tissues. Moreover, we observed focal apoptotic lesions caused by ibrutinib treatment, as evidenced by caspase 3 staining in xenografts tumor tissues. In these experiments, lapatinib treatment at 75mg/kg/day was less potent in SKBR3 tumor xenograft models than ibrutinib treatment at 12mg/kg/day. This is likely due to the ability of ibrutinib to target irreversibly both BTK and EGFR family members and block re-activation of PI3K/AKT or MAPK caused by growth factors such as NRG1 in vivo. Taken together, our findings show that ibrutinib actively blocks BTK and EGFR family activation efficiently inhibiting HER2-positive breast cancer growth in vivo. As a result, this study supports the use of ibrutinib as a HER2-positive breast cancer treatment and indicates that targeting BTK-C with recently developed BTK inhibitors such as ibrutinib or AVL-292 may avert the development of drug resistance in breast cancer patients.

The advent of HER2-directed therapy has significantly improved the prognosis of patients with metastatic and early HER2-positive breast cancer. Currently, there are two classes of HER2-targeting drugs used in the clinic. Trastuzumab, Trastuzumab-DM1 and Pertuzumab are antibody based drugs, whereas lapatinib is a small molecule that reversibly inhibits both EGFR and HER2, blocking the PI3K/Akt and MAPK pathways. Lapatinib is the only small compound used to treat HER2-positive breast cancer that has metastasized to the brain due to its ability to cross the blood-brain barrier. Despite this recent progress, acquired resistance to HER2-directed therapy still results in relapse and progression of HER2-positive disease. The primary mechanism of resistance to lapatinib stems from increased levels of EGFR or HER3 ligands such as NRG1 or EGF in the tumor microenvironment which re-activate the PI3K/Akt and MAPK signaling pathways that are blocked by the drug (34). The activation of the signaling pathway downstream of EGFR family members other than HER2 is dependent on heterodimerization of the EGFR family member triggered by NRG1 or EGF binding to the extracellular ligand-binding domain. Increased expression of HER2 at the cell membrane leads to constitutive signaling of downstream pathways: PI3K/Akt and the Ras/Raf/MEK/MAPK, which are involved in regulating cell growth, survival, differentiation, migration and metastasis. Reactivation of these pathways allows cells to escape from the anti-proliferative and anti-apoptotic effects of the HER2 inhibitors.

Ibrutinib was the first reported covalent inhibitor of BTK(25). As an orally bioavailable, selective and irreversible inhibitor, ibrutinib has been undergoing multiple clinical trials targeting treatment of various B-cell malignancies and has shown promising clinical efficacy(10). Based on sequence alignments, 10 kinases in the human genome have an orthologous cysteine residue(25). The EGFR family includes EGFR/Her1, HER2, Her3 and Her4 all of which are involved in several aspects of tumorigenesis. These kinases include EGFR, HER2 and Her4 covalently bind to the thiol group of Cys481 in the ATP pocket region of BTK (position Cys515 of BTK-C). In vitro, enzymatic activity assays show that and have IC50 of ibrutinib for each enzyme in the nm range BTK, EGFR, HER2 and Her4 are 0.5, 5.6, 9.4 and 0.6nM respectively. Addition, ibrutinib also inhibits ITK, an essential enzyme in Th2 T cells, which shifts the balance between Th1 and Th2 T cells and potentially enhances antitumor immune responses (43). Given the relevance of this spectrum of targets, it is therefore not surprising that ibrutinib is effective in decreasing the proliferation and increasing apoptosis in HER2-positive breast cancer cells.

In summary, this work demonstrates that BTK-C is a novel therapeutic target for breast cancer, and that current recently developed BTK inhibitors may be useful in the treatment of HER2-positive breast cancers. Based on its efficacy in breast cancer cells, its ability to reduce xenograft growth and its effects on EGFR family members, tests of ibrutinib used in a neoadjuvant or adjuvant setting with trastuzumab or other chemotherapeutic compounds for HER2-positive breast cancer is warranted. Moreover, since BTK inhibition abolishes the ability of either NRG1 or EGF to re-activate Akt or Erk in HER2-positive breast cancer cells, drugs such as AVL-292 might also be useful in suppressing therapeutic escape in these cancers.