Introduction
Triple-negative breast cancer (TNBC), defined by the lack of expression of estrogen, progesterone, and epidermal growth factor receptor/human epidermal growth factor receptor 2 (ERBB2/HER2) receptors [
1], represents 15% to 20% of all breast cancer cases [
2] and occurs in young premenopausal women with a higher frequency [
3]. TNBC is commonly associated with basal-like phenotype and characterized by high histological grade, preference for brain or lung metastasis and aggressive behavior with shorter time to recurrence and death [
1,
3]. Patients with this subtype usually have a worse clinical outcome [
2]. In addition to an intricate relationship with basal-like breast carcinomas, TNBC is gaining attention due to its lack of effective tailored therapies. Chemotherapy is the systemic therapy currently available for TNBC, but no standard regimen is recommended. Some TNBC tumors are sensitive to paclitaxel-containing and doxorubicin-containing chemotherapies [
4]. However, TNBC patients became rapidly chemoresistant and frequently relapsed, and showed a worse prognosis [
5,
6]. New therapeutic strategies are urgently needed.
NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a family of transcription factors involved in the regulation of immune responses and inflammation, and plays a major role in tumorigenesis of many cancer types [
7,
8]. It is restricted to the cytoplasm by binding with inhibitory IκB proteins. In response to stimulations, IκB kinase (IKK) complex is activated to phosphorylate IκB proteins. The phosphorylated IκB proteins are then ubiquitinated and degraded by 26S proteasome [
9], leading to NF-κB nuclear translocation. NF-κB controls the expressions of several pro-tumorigenic genes which are associated with angiogenesis, apoptosis, invasion, migration, and cell survival [
10,
11]. Aberrant activation of NF-κB also enhances resistance to chemotherapy in cancer cells [
12]. Inactivation of NF-κB through blocking IκB degradation by bortezomib, a proteasome inhibitor, has shown clinical benefits for the treatment of hematological malignancies [
13]. Although NF-κB activation and overexpression of its target genes have been observed in TNBC tumors [
14,
15], bortezomib showed limited clinical benefits in phase II trials [
16]. These disappointing results suggest that the survival of TNBC may only be partially addicted to NF-κB. Therefore, new strategies making TNBC more addicted to NF-κB activity may be able to improve the therapeutic efficacy of proteasomal inhibitors.
Lapatinib (GW572016, Tykerb), a dual EGFR and HER2 tyrosine kinase inhibitor (TKI), has been approved for trastuzumab-resistant HER2-positive advanced breast cancer patients, [
17]. However, acquired resistance still occurred within six to twelve months after initial treatment [
18]. The elevation of NF-κB activity was found in lapatinib-treated HER2-positive breast cancer cells [
19,
20], and targeting RelA (p65) protein expression enhanced the lapatinib-induced apoptosis [
20]. Lapatinib has recently been found to up-regulate the gene expression of proapoptotic TRAIL death receptors DR4 and DR5 [
21]. Our recent study also showed that lapatinib can induce the NF-κB-targeted gene COX-2 in a HER2/EGFR-independent manner [
22]. These observations raise the possibility that lapatinib may increase NF-κB activity independently of targeting EGFR and HER2.
In this study, we demonstrated that lapatinib, but not specific EGFR inhibitors gefitinib and erlotinib, can induce the phosphorylation and nuclear translocation of p65 and the subsequent expression of NF-κB target genes in both HER2-positive and TNBC cells. We further revealed the involvement of Src family kinase (SFK)-dependent p65 and IκBα phosphorylations in this event. Although lapatinib or bortezomib alone did not elicit the expected clinical benefits for TNBC, co-treatment can enhance the anti-tumor activity of bortezomib by increasing the oncogenic addiction of these cancer cells to NF-κB. These findings not only decipher the molecular mechanisms of lapatinib-induced NF-κB activation, but also suggest remarkable therapeutic benefits with combination of bortezomib and lapatinib in TNBC patients.
Methods
Cell lines and reagents
All cancer cell lines were purchased from the American Type Culture Collection (ATCC – Manassas, VA, USA) and maintained in a humidified 5% CO2 incubator at 37°C. SkBr3, BT474, MDA-MB-231, MDA-MB-468 and HBL100 were cultured in (Dulbecco’s) modified Eagle’s medium ((D)MEM)/F12. HS-578 T was maintained in Roswell Park Memorial Institute medium (RPMI). All the media were supplemented with 10% FBS, 100 unit/ml penicillin and 100 mg/ml streptomycin. To establish lapatinib-, erlotinib-, and gefitinib-selected cells, cancer cells were treated with increasing concentrations of lapatinib, erlotinib, or gefitinib up to 1 μM. p65 shRNA clones were purchased from the National RNAi Core Facility at Academia Sinica (Taipei, Taiwan).
Protein extraction and immunoblot
For total cell lysates, cells were washed with ice-cold PBS one time and lysed in RIPA buffer (20 mM Tris–HCl, pH7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM ethylene glycol tetraacetic acid (EGTA)). For subcellular fractionation, the methods were done as previously described. Protease inhibitors and phosphatase inhibitors cocktails were added in the RIPA buffer. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane and blotted with indicated antibodies.
Immunofluorescence staining
Cells were grown on gelatin cover slips and fixed at day 2 with 4% paraformaldehyde in PBS for 15 minutes. For immunofluorescence staining, cells were next treated with 0.5% Triton X-100 in PBS for 15 minutes and blocked with 10% BSA in PBS for 1 hour followed by incubation with anti-p65 antibody at 4°C overnight. After incubation with horseradish peroxidase (HRP)-labeled secondary antibody, cells were further stained with the nucleic acid stain, diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA), and mounted with ProLong Gold antifade mounting reagent (Invitrogen).
Microarray analysis and ingenuity pathway analysis
Total RNA was extracted by Trizol® Reagent (Invitrogen) according to the instruction manual. RNA was quantified at OD260 nm by using a ND-1000 spectrophotometer (Nanodrop Technology, Wilmington, Delaware USA) and qualitated by using a Bioanalyzer 2100 (Agilent Technology, Santa Clara, California USA) with RNA 6000 nano labchip kit (Agilent Technologies). Total RNA (0.5 mg) was amplified by a Quick-Amp Labeling kit (Agilent Technologies) and labeled with Cy3 or Cy5 (CyDye, PerkinElmer, Waltham, Massachusetts USA) during the
in vitro transcription process. CyDye-labled cRNA (0.825 mg) was fragmented to an average size of about 50 to 100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes. Correspondingly fragmented labeled cRNA was then pooled and hybridized to Agilent Human Whole Genome Oligo 4 × 44 K Microarray (Agilent Technologies) at 60°C for 17 hours. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images were analyzed by Feature extraction 9.5.3 software (Agilent Technologies), an image analysis and normalization software was used to quantify signal and background intensity for each feature, and the data substantially normalized using the rank-consistency-filtering LOWESS method. The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE51889 [
23].
NF-κB-targeted gene expressions were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base (IPA Ingenuity Systems [
24]). The network of these NF-κB-targeted genes was then algorithmically generated based on their connectivity and the molecular relationships between these genes/gene products were presented graphically. The NF-κB-targeted genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base. The intensity of the node color indicates the degree of positive (red) or negative (green) correlation. Nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed with various labels that describe the nature of the relationship between the nodes.
RNA extraction and quantitative reverse transcription PCR
Total RNA was extracted by Trizol™ reagent (Roche, Basel, Switzerland) and converted into cDNA with M-MLV reverse transcriptase (Invitrogen). Synthesized cDNA was used as the template for SYBR qPCR, and changes in gene expression level were normalized to GAPDH in respective cDNA samples. Primers used were as described below: 5′-CGGACTGCCCTTCACCTCGC-3′ (IκBα, forward), 5′-GTATCCGGGTGCTTGGGCGG-3′ (IκBα, reverse), 5′-CCCCCGACTGGACGAGAGGG-3′ (ICAM1, forward), 5′-TGAGTGCTCCTGGCCCGACA-3′ (ICAM1, reverse), 5′-CTTCAGGCAGGCCGCGTCAG-3′ (IL-1β, forward), 5′-TGCTGTGAGTCCCGGAGCGT-3′ (IL-1β, reverse), 5′-TGGCACCTGACCGGAGCATGTA-3′ (TRAF1, forward), 5′-CAACAGGTGGCCTCTGGGCTGT -3′ (TRAF1, reverse), and 5′GCTTAAACAGGAGCATCCTGA-3′ (COX-2, forward), and 5′-GGGTAATTCCATGTTCCAGC-3′ (COX-2, reverse).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay
In vitro cell viability was measured using an MTT colorimetric assay. Cells were trypsinized to seed at a density of 5 × 103 to 1 × 104 cells/well in 96-well plates. After various treatments and culturing periods, the culture medium was removed and 100 μL of serum-free medium with 5 mg/mL MTT solution (Sigma, St. Louis, MO, USA), 25 μL/well, was added and the cells incubated for three hours. Finally, 100 μL of dimethyl sulfoxide (DMSO) was added to lyse the cells and the O.D.550 wavelength was detected by ELISA reader.
Clonogenic assay
For clonogenic assay, cells were plated at 5 × 103 to 1 × 104 cells/well in 6-well plates and pretreated with 1 μM lapatinib or gefitinib for three days followed by addition of MG132 or bortezomib. Colonies were fixed and stained with 30% ethanol containing 1% crystal violet.
Reporter gene assay
Cells were seeded at 2 × 105 cells/well in 12-well plates and transfected with the indicated plasmids in each experiment, including IL-1 β promoter-luciferase plasmids containing NF-κB binding sites. Twenty-four hours after transfection, the luciferase activities in cell lysates were determined by the Luciferase Assay System (Promega, Madison, WI, USA) and normalized to β-galactosiadase activities.
Tumor xenograft mouse model
All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of China Medical University and Hospital (No. 100-61-N). Female severe combined immunodeficient (SCID) mice at 4 to 6 weeks of age were used in the orthotopic tumor-xenograft model. MDA-MB-231 cells (6 × 106 cells/mouse; re-suspended in a 1:1 mixture of PBS and growth factor–reduced Matrigel (BD Biosciences, San Jose, CA, USA) in a total volume of 50 μL) were injected into the mammary fat pads of SCID mice, and the tumor size was measured regularly. Once the tumor size reached 100 mm3, mice were treated orally with saline, lapatinib (20 mg/kg), bortezomib (0.02 mg/kg), or a combination of lapatinib (20 mg/kg) and bortezomib (0.02 mg/kg) every day. One month later, all mice were sacrificed, and tumors were resected and their size was measured. The tumor growth rates were analyzed by measuring the tumor length (L) and width (W) with calipers and by calculating the volume with the formula LW2/2.
Immunohistochemical Staining
Five-micron thick paraffin-embedded tissue sections were deparaffinized and rehydrated. The tissue sections were incubated for two hours with mouse monoclonal anti-human p65 and anti-Bax antibodies (100 dilution, Santa Cruz, Dallas, Texas USA). After washing to remove unbound primary antibody, sections were treated with a dextran polymer backbone conjugated to secondary antibodies and labeled with HRP according to the manufacturer’s instructions (DAKO Envision system for mouse and rabbit primary antibodies, DAKO Corporation, Carpinteria, CA, USA) for 30 minutes. Tissue sections were incubated in the chromogenic peroxidase substrate, diaminobenzidine, for five minutes. The specificity of labeling by this procedure was verified by negative control reactions using buffer to replace the primary antibody and isotype-specific immunoglobulin G (IgG).
Statistical analysis
All results are presented as the mean ± SD. A two-tailed Student’s t-test was used to calculate the statistical significance between the groups. The tumor volume was analyzed by a two-sided t-test.
Discussion
Unlike the luminal or HER2-enriched subtypes, characterized by the expression of hormone receptors or HER2, respectively, TNBC cells are almost insensitive to the established endocrine treatments and HER2-targeted agents due to the lack of known oncogenic drivers. Although synthetic lethal targeting of BRCA-deficient cells with PARP inhibitors has been proposed as a promising therapeutic strategy for TNBC in preclinical studies [
31], the low mutation rate (near 20%) of BRCA1/2 [
32] suggests that PARP inhibitors may only benefit a small number of such patients. New therapeutic strategies are therefore urgently needed for such patients.
Recent results from gene expression profiling analysis revealed that the NF-κB pathway may represent a key regulator of TNBC [
33]. Various small molecules inhibiting NF-κB, including aspirin [
34], genistein [
35] and synthesized phospho-ibuprofen [
36], have shown significant anti-tumor activity in preclinical studies for treating TNBC tumors. These studies revealed NF-κB as a potential therapeutic target for TNBC patients. Bortezomib (Velcade), a potent inhibitor of the 26S proteasome, has been approved for melanoma and hematopoietic malignancies. Preclinical studies have further demonstrated that bortezomib also showed remarkable anti-tumor activity against TNBC
in vitro[
37]. However, bortezomib alone [
16] or in combination with aromatase inhibitor or tamoxifen [
38] failed to show a significant clinical benefit in patients with metastatic breast cancer. The molecular heterogeneity and complexity of addicted status in TNBCs might explain these disappointing results [
39]. Therefore, strategies to trap cancer cells to the NF-κB signal pathway by an artificial addiction shift may be able to potentiate the anti-tumor activity of proteasome inhibitors [
40]. In this study, we reported that the off-target activity of lapatinib in augmenting NF-κB activity may enhance the therapeutic benefits of bortezomib for TNBC patients.
Induction of NF-κB activity was observed in lapatinib-treated HER2-positive breast cancer cells,and, thus, has been considered as a potential target for circumventing lapatinib resistance [
19,
20]. In addition to confirming the involvement of NF-κB activation in contributing to lapatinib resistance in HER2-positive breast cancer cells (Figure
5A), our data also showed that treatment with lapatinib elevates NF-κB activity in TNBC cell lines (Figure
1), uncovering the off-target effect of lapatinib on NF-κB activation. Lapatinib was tested as monotherapy or in combination with other systemic therapies in phase II trials for TNBC or HER2-negative breast cancers [
41,
42]. Inhibition of EGFR by lapatinib was considered as a promising therapeutic strategy for TNBC patients [
43] since EGFR is overexpressed in 80% of TNBC [
44]. Unfortunately, results from most of these studies showed limited clinical benefits for these patients. Nevertheless, the increased NF-κB activity renders lapatinib-treated TNBCs more vulnerable to NF-κB inhibition by p65 shRNA or proteasome inhibitors (Figure
5). These results strongly suggest that lapatinib may augment the oncogenic addiction of cancer cells to NF-κB, which may become the Achilles’ heel in TNBCs. The switch of survival pathway to NF-κB was also observed in various types of solid tumors during the acquisition of resistance to camptothecin and rendered these camptothecin-resistant cells more sensitive to the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) [
45]. Our results further demonstrated that co-treatment with lapatinib can sensitize TNBC cells to proteasome inhibitors both
in vitro and
in vivo (Figure
6), suggesting that the artificial trap of cancer cells to NF-κB signaling by lapatinib may be a potential strategy to increase the anti-tumor activity of bortezomib for TNBC patients.
Several lines of evidence from this study and the literature indicate that the induction of NF-κB by lapatinib is independent of EGFR and HER2 inhibition. First, regardless of the HER2 status, lapatinib-induced NF-κB activation was found in both HER2-positive and TNBC cells in this study (Figure
1A and B, respectively). Second, although lapatinib also possesses inhibitory activity against EGFR, our data showed that treatments with specific EGFR inhibitors, including erlotinib and gefitinib, suppress rather than induce p65 phosphorylations in both HER2-positive BT474 and triple-negative MDA-MB-231 breast cancer cells (Figure
1E and F). Also, only lapatinib but not gefitinib can synergize the anti-tumor activity of MG-132 in TNBC cells (Figure
6B). Third, inhibition of either HER2 [
46] or EGFR [
47] by its specific siRNA has been reported to decrease but not increase NF-κB activity. Similar to this EGFR/HER2-independent role in NF-κB activation, lapatinib has also been reported to up-regulate the expression of pro-apoptotic TRAIL death receptors DR4 and DR5 through an off-target mechanism in colon cancer cells and, thus, sensitizes these cancer cells to TRAIL-induced apoptosis [
21]. The induction of DR5 by lapatinib was evident only with high drug concentrations (>5 μM) [
21]. However, our data showed that 1 μM of lapatinib is sufficient for NF-κB activation. Therefore, lapatinib-enhanced NF-κB activity is unlikely through induction of DR5 expression although TRAIL has been known to initiate signaling to NF-κB activation [
45].
The lapatinib-augmented NF-κB activity, derived from the EGFR/HER2-independent off target effect, involves both classic and SFK-mediated NF-κB activation pathways. Although our data revealed that reduction of the IκBα protein level was not accompanied with lapatinib-induced NF-κB activation, IκBα Ser32/36 phosphorylations and a higher turnover rate of IκBα were still found in lapatinib-treated cells, indicating that IκBα degradation remains necessary for liberating NF-κB. However, the
de novo synthesis of IκBα, which is mediated by lapatinib-activated NF-κB, accounts for the unchanged IκBα protein level but did not feedback bind to and inhibit NF-κB. Up-regulation of SFK activity has been found in the acquired lapatinib-resistant cells with HER2 overexpression [
48]. Our data further showed that lapatinib also activates SFK in both HER2-positive and TNBC cells to mediate IκBα Tyr42 phosphorylation, which prevents the feedback inhibition of NF-κB from IκBα protein binding [
28,
29]. These events lead to the constitutive activation of NF-κB in lapatinib-treated cells. Interestingly, bortezomib not only inhibits p65 phosphorylation but also reduces SFK tyrosine phosphorylation (data not shown). It suggests that the anti-tumor activity of bortezomib with co-treatment of lapatinib in our study may be partly attributed to inhibition of SFK activity by bortezomib. However, the potential possibility and molecular mechanisms underlying bortezomib-mediated SFK inhibition await further investigations.
Acknowledgements
This work was supported by grants from E-Da Hospital (EDAHT100024, EDAHT100026), the National Science Council of Taiwan (NSC 102-2320-B-039-054-MY3, NSC 102-2320-B-039-052, NSC 101-2911-I-002-303, and NSC 101-2320-B-039-049 to W.C.H), China Medical University and Hospital (CMU101-S-28, DMR-102-113), and the National Health Research Institutes of Taiwan (NHRI-EX-100-9812BC to W.C.H).
Competing interests
The authors declare they have no competing interests.
Authors’ contributions
YJC carried out the western blot analysis and drafted the manuscript. MHY participated in the design and coordination of the study and drafted the manuscript. MCY carried out the IPA analysis and immunoprecipitation assays, and was involved in drafting the manuscript. YLW, WSC and JYC participated in the animal studies and helped to revise the manuscript. CYS, YLY, CHC, CYT, PHC and TCH performed the immunoblot, immunohistochemical staining and reporter assays, and helped to revise the manuscript. SHL carried out the statistical analysis and drafted the manuscript. WCH conceived of the study, participated in the design of the study and contributed to the manuscript. All authors read and approved the final manuscript.