Introduction
Triple negative breast cancer (TNBC), which comprises approximately 15% of all breast carcinomas [
1], is defined as breast carcinoma that does not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor type 2 (HER2). These tumors are characterized by occurrence in younger women, aggressive behaviors with a high recurrence rate, metastasis potential and poor prognosis [
1‐
3]. Because of a lack of targeted therapies (such as hormone therapy or anti-HER2 therapy) for TNBC, chemotherapy is currently the main treatment. There is, therefore, an urgent and unmet need to develop targeted therapy for TNBC. Discovering the critical molecular mechanisms of TNBC and developing new compounds targeting these mechanisms may advance the development of TNBC treatments.
Bortezomib is the first proteasome inhibitor to be approved for treatment for multiple myeloma and mantle cell lymphoma [
4,
5]. Bortezomib has been shown to block proteasome degradation of IκB, an inhibitor of nuclear factor-κB (NF-κB), and demonstrated remarkable anti-tumor activity against these hematological malignancies. The transcription factor NF-κB is believed to play a vital role in the action of bortezomib as it is involved in cancer cell proliferation, apoptosis, invasion, metastasis, tumorigenesis and angiogenesis [
4‐
6]. In addition, bortezomib affects several other cellular pathways, such as tumor suppressor protein p53, cell cycle regulators p21, p27, proapoptotic (Noxa, bax, and so on) and antiapoptotic (mcl-1, bcl-2, and so on) bcl-2 family proteins that lead to apoptosis [
7]. Preclinical studies have demonstrated an
in vitro antitumor effect of bortezomib in breast cancer models [
8‐
10]. In the clinical arena bortezomib as a single agent showed limited clinical efficacy (objective response) in two single institutional phase II clinical trials for patients with previously treated metastatic breast cancers (MBC) [
11,
12]. In contrast, combinational trials of bortezomib with other therapeutics for MBC seem promising: a phase II study combining bortezomib with pegylated liposomal doxorubicin demonstrated a response rate of 8% in patients with MBC [
13]; another phase I/II study showed that a combination of bortezomib and capecitabine is well tolerated and has moderate antitumor activity (15% overall response rate) in heavily pretreated MBC patients [
14]; and another phase I/II study combining bortezomib with docetaxel showed a more promising response rate of 38% at the maximum tolerated dose for anthracycline-pretreated advanced/metastatic breast cancer [
15]. Bortezomib is currently being tested in combination with fulvestrant, a novel estrogen antagonist, in a randomized phase II study for patients with ER positive MBC (NCT01142401). Although the reason why the single bortezomib regimen is not significantly active in clinical trials might be explained by the possibility of the activation of multiple drug resistance pathways in heavily pretreated populations, particularly those previously exposed to anthracycline [
16], alternative mechanisms might also confer sensitivity to bortezomib in patients with breast cancers. Interestingly, in the phase II study by Yang
et al. [
12], the inhibition of proteasome activity was measured in bortezomib-treated patients and did not translate into a meaningful therapeutic benefit in these patients, implying that bortezomib's mechanism of action may not necessarily depend on its proteasome inhibitory effect [
12]. Therefore, the exact anti-tumor mechanisms of bortezomib in breast cancers, and to our interest TNBC, warrant further elucidation.
In this regard, our previous study showed that downregulation of phospho-Akt (p-Akt) plays a key role in determining the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib-induced apoptosis [
17]. Importantly, we found that the differential cytotoxic effects of bortezomib on HCC are independent of its proteasome inhibition [
17]. Akt is a well-known key player in cancer cell survival and apoptosis regulation. It is noteworthy that activated p-Akt signaling has been shown to be higher in TNBC tumor samples than in other breast tumor types [
18]. Negative regulation of Akt signaling can be achieved by phosphatases, such as phosphatase and tensin homologue deleted on chromosome ten (PTEN), and protein phosphatase 2A (PP2A). PTEN dephosphorylates phosphatidylinositol 3, 4, 5-triphosphate (PIP3) at the 3-position to counteract phosphatidylinositol-3-kinase (PI3K), thereby inhibiting Akt signaling. In contrast, PP2A is a complex serine/threonine protein phosphatase that can directly dephosphorylate oncogenic kinases such as p-Akt and p-ERK [
19], by which PP2A can function as a tumor suppressor through regulating apoptosis, cell cycle, cell survival and proliferation [
20]. Our recent data indicated that the bortezomib enhances PP2A activity thereby downregulating p-Akt and inducing apoptosis in HCC cells [
21]. We also found that bortezomib can act synergistically with sorafenib to induce apoptosis in HCC cells through this PP2A-dependent p-Akt inactivation [
22]]. In addition, several cellular upstream inhibitors of PP2A such as SET [
23], and cancerous inhibitor of protein phosphatase 2A (CIP2A) [
24] have been identified. SET is a nucleus/cytoplasm-localized phosphoprotein that has been shown to be predominantly a myeloid leukemia-associated protein [
25]. In contrast, CIP2A has emerged as a novel oncoprotein and a growing number of reports have shown its overexpression in many human malignancies, including breast cancers [
21,
24,
26‐
31]. CIP2A has been shown to promote anchorage-independent cell growth and
in vivo tumor formation by inhibiting PP2A activity toward c-Myc [
32]. Importantly, Come
et al. [
33] found that CIP2A is associated with clinical aggressiveness in human breast cancer and promotes the malignant growth of breast cancer cells, suggesting CIP2A as a new target for breast cancer therapy.
In this study, we revealed that CIP2A, a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of bortezomib. Bortezomib induced significant apoptosis in TNBC cell lines but not in hormone receptor positive or HER2-overexpressing cells. Our data indicate that bortezomib's downregulation of CIP2A and p-Akt correlated with its drug sensitivity. Through ectopic overexpression and silencing CIP2A, we confirmed that CIP2A is the predominant mediator of bortezomib-induced apoptosis in TNBC cells. This CIP2A-dependent p-Akt inhibitory mechanism that mediates the efficacy of bortezomib was confirmed in vivo in a nude mouse model. Furthermore, CIP2A expression can be demonstrated in tumor samples from TNBC patients. Our results suggest that CIP2A may be a novel therapeutic target for treatment of TNBC.
Materials and methods
Reagents and antibodies
Bortezomib (Velcade®) was kindly provided by Millennium Pharmaceuticals (Cambridge, MA, USA)For in vitro studies, bortezomib at various concentrations was dissolved in dimethyl sulfoxide (DMSO) and then added to cells in (D)MEM medium (Invitrogen, Carlsbad, CA, USA). The final DMSO concentration was 0.1% after addition to the medium. Antibodies for immunoblotting such as anti-I-κB and CIP2A were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). Other antibodies such as anti-caspase-3, Akt, and P-Akt (Ser473) were from Cell Signaling (Danvers, MA, USA).
Cell culture and western blot analysis
The HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and MCF-7 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). All breast cancer cells were maintained in (D)MEM medium supplemented with 10% FBS, 0.1 mM nonessential amino acids (NEAA), 2 mM L-glutamine, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, and 25 μg/mL amphotericin B in a 37°C humidified incubator and an atmosphere of 5% CO
2 in air. Lysates of breast cancer cells treated with drugs at the indicated concentrations for various periods of time were prepared for immunoblotting of caspase-3, P-Akt, Akt, CIP2A, and so on. Western blot analysis was performed as previously reported [
17].
Apoptosis analysis
Drug-induced apoptotic cell death was assessed using the following methods: (a) Western blot analysis of caspase activation or poly ADP-ribose polymerase (PARP) cleavage (in caspase deficient MCF-7 cells), and (b) measurement of apoptotic cells by flow cytometry (sub-G1 analysis).
Proteasome inhibitory activity
A 20S Proteasome Activity Assay kit (Chemicon International, Temecula, CA, USA) was used to determine the proteasome inhibition in drug-treated cells. All procedures were conducted according to the manufacturer's instructions [
17]. In brief, cells were treated with or without bortezomib for the indicated length of time. Then, cells were lysed and total protein was quantified. Equal amounts of total protein of each sample were used for incubation with the proteasome substrate (fluorophore-labeled substrate). Proteasome activity measurement was based on detection of the fluorophore after cleavage from the labeled substrate by a fluorometer with a 380/460 nm filter set.
Gene knockdown using siRNA
Smartpool siRNA reagents, including control (D-001810-01), and CIP2A were all purchased from Dharmacon (Chicago, IL, USA). The procedure has been described previously [
17]. Briefly, cells were transfected with siRNA (final concentration, 100 nM) in six-well plates using the Dharma-FECT1 transfection reagent (Dharmacon) according to the manufacturer's instructions. After 72 hours, the medium was replaced and the breast cancer cells were incubated with bortezomib, harvested, and separated for western blot analysis and for apoptosis analysis by flow cytometry.
Generation of MDA-MB-231 and MDA-MB-468 cells with constitutive active CIP2A
Cells were transfected with active CIP2A construct by procedures as previously described [
26]. Briefly, following transfection, cells were incubated in the presence of G418 (1.40 mg/mL). After eight weeks of selection, surviving colonies, that is, those arising from stably transfected cells, were selected and individually amplified. CIP2A cDNA (KIAA1524) was purchased from Origene (RC219918; Rockville, MD, USA) and constructed into pCMV6 vector.
Xenograft tumor growth
NCr athymic nude mice (five to seven weeks of age) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in groups and maintained in a specific pathogen free (SPF)-environment. All experimental procedures using these mice were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital. Each mouse was inoculated s.c. in the dorsal flank with 2 to 4 × 106 breast cancer cells suspended in 0.1 to 0.2 mL serum-free medium containing 50% Matrigel (BD Biosciences, Bedford, MA, USA) under isoflurane anesthesia. Tumors were measured using calipers and their volumes calculated using a standard formula: width2 × length × 0.52. When tumors reached 100 mm3, mice were administered an i.p. injection of bortezomib (0.5 mg/kg body weight) twice weekly for three to four weeks. Controls received vehicle.
Reverse transcription-PCR
Total RNA was extracted from cultured cells using TRIzol® Reagent (Invitrogen, San Diego, CA, USA) and RT-PCR was performed according to the manufacturer's instructions (MBI Fermentas, Vilnius, Lithuania). RT-PCR analyses were performed as previously described, using specific primers for human CIP2A (forward, 5'-TGGCAAGATTGACCTGGGATTTGGA-3'; reverse, 5'-AGGAGTAATCAAACGTGGGTCCTGA-3'; 172 bps); the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene (forward, 5'-CGACCACTTTGTCAAGCTCA-3'; reverse, 5'-AGGGGTCTACAT GGCAACTG-3'; 228 bps) was chosen as an internal control.
Real-time quantitative PCR was performed in a LightCycler 480II instrument (Roche Diagnostics, Indianapolis, IN, USA) using a LightCycler 480 SYBR Green I Master kit (Roche). Primers are the same as above described.
Immunohistochemical staining
Paraffin-embedded tissue microarray of tumor samples from TNBC patients' sections (4-μm) on poly-1-lysine-coated slides were first de-waxed in xylene and re-hydrated through graded alcohols, followed by a rinse using 10 mM Tris-HCl (pH 7.4) and 150 mM sodium chloride, then treated with 3% hydrogen peroxide for five minutes. Slides were incubated with a 1:100 dilution of rabbit polyclonal anti-p90 autoantigen (CIP2A) antibody (ab84547) (Abcam, Cambridge, UK) for one hour at room temperature, then thoroughly washed three times with PBS. Bound antibodies were detected using the LSAB+ kit (Dako, Glostrup, Denmark). The slides were then counterstained with hematoxylin stain solution. Paraffin-embedded sections of human colon carcinoma and ovarian carcinoma of cytoplasmic immunoreactivity were used as positive controls. Negative controls had the primary antibody omitted and replaced by PBS. CIP2A immunoreactivity was scored as negative, weak, moderate, and strong expression, respectively.
This study was approved by the ethics committee of the Institutional Review Board of Taipei Veterans General Hospital. All informed consents from sample donors were in accordance with the Declaration of Helsinki and were obtained at time of their donation.
Statistical analysis
Data are expressed as mean ± standard deviation (SD) or standard error (SE). Statistical comparisons were based on nonparametric tests and statistical significance was defined at P < 0.05. All statistical analyses were performed using SPSS for Windows software, version 12.0 (SPSS, Inc., Chicago, IL, USA).
Discussion
This study reveals a novel mechanism by which bortezomib induces apoptosis in triple negative breast cancer cells (that is, CIP2A-dependent p-Akt downregulation). This finding has several potentially important implications: First, we identified CIP2A as a molecular determinant of cell sensitivity to bortezomib-induced apoptosis and demonstrated that bortezomib-induced apoptosis does not necessarily depend on its proteasome inhibition. We showed that bortezomib exerts similar proteasome inhibitory effects in sensitive and resistant cells as demonstrated by proteasome activity analysis and by efficient I-kB (a proteasome substrate) accumulation (Figure
1B and
1C). In contrast, bortezomib induced differential apoptotic effects in these breast cancer cells, which correlated with CIP2A downregulation (Figure
2 and
3). This
in vitro finding was supported by previous
in vivo evidence showing that bortezomib-induced proteasome inhibition did not correlate well with clinical therapeutic benefit in patients with breast cancers in a phase II study [
12]. Indeed, despite the excellent anti-cancer activity of bortezomib in multiple myeloma and mantle cell lymphoma via its proteasome inhibition, cumulative clinical data have shown that bortezomib is less efficient or shows transient anti-cancer activity in solid tumors and other hematological malignancies [
11,
12,
37‐
39]. Our data may, further, partly explain why bortezomib showed limited anti-tumor activity in breast cancer patients in the phase II trials [
11,
12]. In addition to the several known mechanisms of bortezomib resistance in cancers [
4,
40], the CIP2A-PP2A-p-Akt pathway may contribute to bortezomib resistance. Future studies correlating response to bortezomib with downregulation and/or pre-treatment expression levels of CIP2A in breast cancer patients may help to establish a clinical role for CIP2A as a predictive factor in breast cancer.
Second, our data strengthen the case for the use of CIP2A as an anti-cancer target. Accumulating evidence from our studies and the studies of others suggests that targeting CIP2A may be an ideal approach [
26,
32,
33,
41‐
43]. CIP2A expression is very low in most human tissues and, importantly, undetectable in normal mammary glands [
32,
33], thereby creating a potential therapeutic window for CIP2A targeting agents. Come
et al. [
33] demonstrated that depletion of CIP2A by siRNA inhibits tumor growth of MDA-MB-231 xenograft tumors. Our
in vivo data also showed bortezomib downregulated CIP2A in HCC-1937 xenograft tumors and inhibited their tumor growth (Figure
5A to
5C). Moreover, it has been shown that the traditional chemotherapeutic agent doxorubicin downregulates CIP2A expression and that increased CIP2A expression confers doxorubicin resistance in breast cancer cells [
44]. More recently, a natural Chinese medicinal herbal extract of
Rabdosia coetsa, rabdocoetsin B, was also shown to inhibit proliferation and induce apoptosis in a variety of lung cancer cells via CIP2A-dependent p-Akt downregulation [
43]. Taken together, these structurally unrelated agents show a common target in various cancer cells suggesting that CIP2A is a novel anti-cancer target.
Our data showed that 50/57 (87.7%) tumor samples from TNBC patients demonstrated variable CIP2A expressions. As stated earlier, CIP2A expression has been shown to correlate with disease aggressiveness [
33] in breast cancer. Higher CIP2A expression has been shown as a prognostic factor predicting survival in gastric cancer [
29], non-small cell lung cancer [
27,
45], renal cell carcinoma [
46], serous ovarian cancer [
47] and early stage tongue cancer [
47]. Very recently, an IHC-based study [
48] demonstrated that the CIP2A signature clustered with basal-type and HER2-positive breast cancer signatures and suggested that CIP2A is linked to these two subtypes of breast cancer. It would also be interesting to further investigate the prognostic role of CIP2A among various subtypes of breast cancer, in addition to TNBC subtypes, by large immunohistochemistry-based studies.
Despite the current results, the detailed mechanism by which bortezomib inhibits CIP2A remains unknown and further mechanistic studies are needed. Our data showed that bortezomib did not affect the half-life of CIP2A protein degradation after translation was stopped by cyclohexamide and that bortezomib suppressed CIP2A transcription (Figure
5), suggesting that the effect of bortezomib on CIP2A occurs pre-translation and is possibly irrelevant to its proteasome inhibition. The possible mechanisms through which bortezomib may affect the transcription of CIP2A include direct or indirect promoter regulation of CIP2A mRNA, epigenetic regulation of the
CIP2A gene by DNA methylation or micro-RNA machinery, or affecting other uncovered molecules that regulate CIP2A expression.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CYL and KFC were responsible for coordination and manuscript editing as well as acting as corresponding authors. CYL, KFC and CWS participated in the research design. LMT, KCC, CYL and PYC conducted experiments. LMT, CYL and CWS performed data analysis. LMT, CYL and KCC wrote or contributed to the writing of the manuscript. All authors have read and approved the final manuscript.