Bruton's Tyrosine Kinase Inhibitors Prevent Therapeutic Escape in Breast Cancer Cells

Bruton's tyrosine kinase (BTK) belongs to the TEC family of cytoplasmic tyrosine kinases (1). BTK was identified in 1993 as a novel nonreceptor protein tyrosine kinase that is mutated in X-linked agammaglobulinaemia (XLA; refs. 2, 3). It is predominantly expressed in hematopoietic cells including erythroid progenitors and myeloid cells (4) and is established as a critical regulator of the B-cell receptor (BCR) signaling, development, differentiation, and survival (5–7). Because of 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 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 have significantly improved the outlook for patients with HER2⁺ 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⁺ 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⁺ 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⁺ breast cancer cells are not mitigated by NRG1 or EGF stimulation, as occurs with lapatinib. As the expression of BTK-C and HER2 is positively correlated in surgical specimens of human breast cancer tissues, these results indicate that ibrutinib is a potential therapy for this solid tumor type.

Breast cancer and MCF-10A cell lines were obtained from the ATCC in 2011. All cell lines were cultured in DMEM (Hyclone) supplemented with 10% FBS (Hyclone) and 100 U/μL of penicillin–streptomycin (CellGro), except for MCF-10A cells cultured as indicated in ref. (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 Systems. Src inhibitor saracatinib was purchased from Selleckchem. LY294002 was purchased from Cell Signaling Technology.

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).

Three-dimensional (3D) cell culture was performed as described previously (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. 1 μmol/L 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 minutes and washed with PBS. After washing, cells were stained with 0.05% crystal violet for 30 minutes and washed with tap water. Methanol (0.5 mL) was added to the dry plates to solubilize the dye. Crystal violet staining was measured by reading OD 540 nm of each sample. Tumorspheres were counted under low magnification.

Cells were cultured in 6-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, 2 μg/mL PI, and 0.2 mg/mL DNAse-free RNAse) for 30 minutes. Samples were analyzed by flow cytometry.

Human breast cancer tissue sections (BR10010b, US Biomax, Inc.) were baked for 1 hour at 62°C, subjected to serial alcohol treatments for rehydration and microwaved in 0.01 mol/L sodium citrate for 20 minutes for antigen retrieval. The sections were serum blocked for 30 minutes, incubated overnight at 4°C with primary antibodies in PBS and subsequently with Cy5-labeled secondary antibodies for 60 minutes, and 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 were analyzed by Fisher exact test. Significance was determined by the alpha level of 0.05.

Immunoblotting was performed essentially as described previously. Equal amounts of proteins were used. Antibodies used were anti-EGFR (1:1,000), anti-HER2 (1:1,000), anti-ERBB3, ERBB4 antibody, anti-BTK, anti-AKT, ERK, anti-PLCγ1, PLCγ2 and anti-PARP (1:1,000, Cell Signaling Technology), anti-Flag antibody (1:1,000, Sigma), anti-rabbit IgG-HRP, and mouse IgG-HRP (1:5,000, Jackson ImmunoResearch; ref. 20).

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 × 10⁶ cells/mL. Alexa Fluor 488 Annexin V (5 μL) and 100 μg/mL PI (1 μL) was added to each 100 μL of cell suspension. Cells were incubated for 15 minutes at room temperature. After the incubation, 400 μL 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). The data were analyzed using the FlowJo software package (Treestar Inc.).

NOD/SCID mice were purchased from The Jackson Laboratory. All mouse procedures were approved by the Animal Care and Use Committees of The State University of New York at Albany (SUNY Albany, Albany, NY) 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 6 mg/kg/day or 12 mg/kg/day orally in a vehicle of 1% DMSO/30% polyethylene glycol/1% Tween 80. Lapatinib was given 75 mg/kg/day or 37.5 mg/kg/day orally. The tumor volume in mm³ was calculated by the formula: volume = (width)² × length/2 every 7 days.

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 with 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 observed that HER2⁺ breast SKBR3 cancer cells are more sensitive to ibrutinib (1 μmol/L) than nontumorigenic, 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⁺ breast cancer cell, we tested the effect of ibrutinib on BT474 cell survival. The result shows that ibrutinib decreases HER2⁺ breast cancer cell growth by 20%–30% at a concentration of 10 nmol/L (Fig. 1B). The IC₅₀ for ibrutinib's effect on HER2⁺ breast cancer cells measured at 3 days of culture is 30 nmol/L. 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 Fig. S1). They are, however, less effective antiproliferative agents than ibrutinib, achieving 30%–40% decreases in cell numbers in HER2⁺ 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⁺ breast cancer cell survival.

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 antiproliferative affect through these targets. To compare the inhibitory effect of ibrutinib with lapatinib on HER2⁺ 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⁺ breast cancer patients. Treatment with ibrutinib at different concentrations as indicated for 3 days reduces BT474 cell number. Ibrutinib (0.01 μmol/L) reduces cell populations to 30% of control (P < 0.01). However, lapatinib, at the same concentration (0.01 μmol/L) fails to reduce HER2⁺ breast cancer cell numbers significantly (Fig. 2A). These results are generally consistent with recently published data also showing ibrutinib inhibits cell viability of HER2⁺ breast cancer cells (26). A number of studies have shown that cells' microenvironment can impact drug response (27, 28). To test whether HER2⁺ breast cancer cells are still sensitive to ibrutinib in 3D Matrigel culture condition, treatment of BT474 cells with 0.1 μmol/L or 0.5 μmol/L of ibrutinib for 9 days significantly reduces cell number when compared with the control (Fig. 2B). Thus, we find that ibrutinib reduces HER2⁺ breast cancer cell number in both monolayer and 3D culture, and that ibrutinib has a more potent effect on HER2⁺ breast cancer cells than lapatinib.

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 0.05 μmol/L or 0.1 μmol/L of ibrutinib and 0.1 μmol/L 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; ref. 31). These results indicate that in breast cancer cells, ibrutinib, in addition to inhibiting BTK irreversibly (Supplementary Fig. S2), 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⁺ breast cancer cells compared with other breast cancer cells (Fig. 1).

In HER2⁺ 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 0.03 μmol/L 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 cyclin D1 (Fig. 3B).

An increase in the number of apoptotic cells following treatment with 0.05 or 0.1 μmol/L 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 0.5 μmol/L lapatinib on cell survival (31). Compared with lapatinib, ibrutinib induces 1.5 fold more apoptosis in HER2⁺ 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⁺ breast cancer cells.

Cancer cells typically express multiple receptor tyrosine kinases (RTK) 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⁺ 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⁺ cells to 50 ng/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⁺ breast cancer cells allowing resistant cells to emerge from the treatment (Fig. 4A). In contrast, HER2⁺ breast cancer cells cannot be rescued from ibrutinib-induced growth inhibition by NRG1. Similar results are observed using 50 ng/mL of EGF.

The ability of NRG1 or EGF signaling to rescue HER2⁺ 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⁺ breast cancer cells from the growth inhibition by BTK inhibitor ibrutinib were unexpected. To further confirm these findings, additional long-term cotreatment experiments were performed. Nine-day lapatinib/NRG1 cotreatment yields cells that exhibit lapatinib resistance in both BT474 and SKBR3 cell populations. In contrast, 9-day ibrutinib/NRG1 cotreatment does not yield ibrutinib-resistant cells in HER2⁺ breast cancer cell lines (Fig. 4B). Similar results are observed in experiments using EGF where cells are cotreated with EGF/lapatinib or EGF/ibrutinib (Supplementary Fig. S3).

To investigate the signaling pathway which mediates the NRG1- or EGF-induced rescue of HER2⁺ 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, and the activation of HER2, AKT, and ERK are blocked. When cells are cotreated with NRG1/lapatinib, NRG1 induces phosphorylation of AKT while HER2 remains inhibited. Surprisingly, NRG1 fails to reactivate AKT under conditions of NRG1/ibrutinib cotreatment (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.

As ibrutinib was originally designed to block BTK activation (25) and as BTK has been shown to activate AKT in B cells (36), the possibility existed that BTK-C provided a prosurvival 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 reactivation of AKT induced by NRG1. However, as ibrutinib is as potent as an inhibitor of EGFR family activation as lapatinib in HER2⁺ 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⁺ 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⁺ breast cancer cell populations.

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 μmol/L) or the Src inhibitor saracatinib (5 or 10 μmol/L). The phosphorylation of BTK-C is appreciably decreased by saracatinib at 10 μmol/L (41). The phosphorylation of AKT, as a downstream target of BTK-C, also decreases. In contrast, 10 μmol/L LY294002 does not suppress BTK-C activation (Fig. 5A). As the possibility exists that this lower concentration of LY294002 may not block BTK-C activation, we increased the concentration of LY294002 to 50 μmol/L and repeated the test. The results show that LY294002 at 50 μmol/L 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⁺ breast cancer cells. The Src/FAK signaling pathway is involved in the lapatinib-induced kinome reprogramming (42) that contributes to drug resistance in HER2⁺ 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⁺ 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⁺ breast cancer cells.

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 6 mg/kg/day and 12 mg/kg/day. At day 28, tumor volumes in mice that received 12 mg/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⁺ breast cancer chemotherapy.

The above results suggest that simultaneously targeting HER2 and BTK represents an improved approach to the treatment of HER2⁺ 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. In addition, 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⁺ breast cancer tissues, we examined the coexpression 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⁺ 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⁺ breast cancers.

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 μmol/L, 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 9.2 nmol/L respectively. Our results indicate that the sensitivity of HER2⁺ 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⁺ breast cancer cells (31). We also find that BTK-C plays an essential role in the ability of HER2⁺ 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 reactivate 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⁺ 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 12 mg/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 75 mg/kg/day was less potent in SKBR3 tumor xenograft models than ibrutinib treatment at 12 mg/kg/day. This is likely due to the ability of ibrutinib to target irreversibly both BTK and EGFR family members and block reactivation 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⁺ breast cancer growth in vivo. As a result, this study supports the use of ibrutinib as a HER2⁺ 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⁺ 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⁺ 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⁺ 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 reactivate 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 antiproliferative and antiapoptotic 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). On the basis of 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 nanomolar range BTK, EGFR, HER2 and Her4 are 0.5, 5.6, 9.4, and 0.6 nmol/L respectively. In 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⁺ 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⁺ breast cancers. On the basis of 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⁺ breast cancer is warranted. Moreover, as BTK inhibition abolishes the ability of either NRG1 or EGF to reactivate Akt or Erk in HER2⁺ breast cancer cells, drugs such as AVL-292 might also be useful in suppressing therapeutic escape in these cancers.

X. Wang, L. Kokabee, and D.S. Conklin are currently inventors on the patents but do not have ownership interest at this juncture. No potential conflicts of interest were disclosed by the other authors.

Some of the data in this study have been used to support one or more patent applications.

Conception and design: X. Wang, D.S. Conklin

Development of methodology: X. Wang, J. Wong, L. Kokabee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Wang, J. Wong, L. Kokabee, D.S. Conklin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Wang, L. Kokabee, Y. Sun, D.S. Conklin

Writing, review, and/or revision of the manuscript: X. Wang, J. Wong, F. Khan, Y. Sun, D.S. Conklin

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Wang, J. Wong, L. Kokabee, F. Khan, Y. Sun, D.S. Conklin

Study supervision: X. Wang, D.S. Conklin

Other (conception and design of IHC experiment):C.J. Sevinsky

The authors thank members of the Conklin laboratory and the University at Albany Laboratory Animal Care Facility for help with experimentation and critical reading of the manuscript.

This work was supported by NCI R01CA136658 and a Technology Accelerator Fund Award from The Research Foundation for the State University of New York (to D.S. Conklin).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.