Targeting FOSB with a Cationic Antimicrobial Peptide, TP4, for Treatment of Triple-Negative Breast Cancer

www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 26 Research Paper Targeting FOSB with a cationic antimicrobial peptide, TP4, for treatment of triple-negative breast cancer Chen-Hung Ting1, Yi-Chun Chen1, Chang-Jer Wu2, Jyh-Yih Chen1 1Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Jiaushi, Ilan 262, Taiwan 2Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwan Correspondence to: Jyh-Yih Chen, email: [email protected] Keywords: TP4, cationic antimicrobial peptide, triple-negative breast cancer, calcium, FOSB Received: February 12, 2016 Accepted: May 02, 2016 Published: May 26, 2016 ABSTRACT Triple-negative breast cancer (TNBC) currently lacks a suitable therapeutic candidate and is thus difficult to treat. Here, we report that a cationic antimicrobial peptide (CAP), tilapia piscidin 4 (TP4), which was derived from Nile tilapia (Oreochromis niloticus), is selectively toxic to TNBC. TP4 acts by inducing an AP-1 protein called FOSB, the expression of which is negatively associated with the pathological grade of TNBC. We show that TP4 is bound to the mitochondria where it disrupts calcium homeostasis and activates FOSB. FOSB overexpression results in TNBC cell death, whereas inhibition of calcium signaling eliminates FOSB induction and blocks TP4-induced TNBC cell death. Both TP4 and anthracyclines strongly induced FOSB, particularly in TNBC, indicating that FOSB may be suitable as a biomarker of drug responses. This study thus provides a novel therapeutic approach toward TNBC through FOSB induction. INTRODUCTION normally zwitterionic [13, 14]. This characteristic allows some selective cytotoxic agents, such as cationic Breast cancer (BC) is the most common malignancy antimicrobial peptides (CAPs), to attack cancers through that causes death in women. Global gene-expression profile electrostatic interactions [15–17]. Cationic antimicrobial studies have classified breast cancers into different subtypes peptides (CAPs) are evolutionarily conserved components [1–3]. The subtypes which lack expression of estrogen of the innate immune system, integral for activity against receptors (ER), progesterone receptors (PR), and human pathogens [18, 19]. The defensive capabilities of CAPs epidermal growth factor receptor 2 (HER2) are clustered arise from their structures, which allow them to penetrate as triple negative BC (TNBC:ER-/PR-/HER2-). Hormone anionic bacterial membrane [16, 20] or cancer cells with or targeted therapies are not usually effective against negatively-charged outer membrane [21–23]. Treatment TNBC, with the exception of anthracycline or taxane-based of cancer cells with large amounts of CAPs leads to conventional chemotherapy [4, 5]. However, TNBC patients, transient membrane lysis [14, 24–29]. However, low even after treatment with chemotherapy, often present with concentrations of CAPs can trigger apoptosis [30, 31] distant metastases and have poor prognosis. The main cause and/or necrosis of cancer cells [24, 32–34]. Mechanisms of chemotherapeutic agent failure is the development of of cancer killing by CAP involve changes in calcium multidrug-resistant (MDR) cancer cells under standard homeostasis and induction of activator protein-1 (AP-1) regimens [6], and chemotherapy also causes adverse side- [14, 23, 31, 35]. Calcium signaling appears to be activated effects [7]. Use of non-cross-resistant drugs [8–11] or early on in response to CAP-induced stress, and enhances biological agents [12] in combination with chemotherapeutic downstream AP-1 signaling [23]. AP-1 members form drugs is a possible option for TNBC patients with metastases. a dimer with proteins of the JUN proto-oncogene However, even though such options improve the outcome, (c-JUN) family (c-JUN, JUNB, JUND) or FBJ murine the prognosis of metastatic TNBC patients remains poor. osteosarcoma viral oncogene homolog (FOS) family Metastatic cancer cells that respond poorly (c-FOS, FOSB, FRA1/2). The dimer composition of to treatment usually possess negatively-charged AP-1 activates downstream gene expression in response phosphatidylserine (PS) or anionic structures on their to cellular stimuli or in different cellular contexts, as outer membrane, in contrast to healthy cells that are well as controlling cell fate decisions [36]. In TNBC, www.impactjournals.com/oncotarget 40329 Oncotarget FRA1 controls tumor cell growth and metastasis through Gene ontology (GO) analysis revealed that TP4 treatment repression of CDH1 in poorly differentiated cells [37, 38] caused dramatic changes in the gene expression profiles which lack FOSB expression [39, 40]. However, little is of TNBC cells (Figure 2A and 2B), but minor changes known about the role of FOSB in TNBC. in HDF cells (Figure 2B). Of note, FOS members TNBC cells with negatively-charged PS may be good (FOSB, c-FOS) and ATF3 were significantly induced in candidate for CAP-based therapy. In the present study, TNBC cells (Figure 2B). Immunocytochemical studies we investigate the therapeutic potential of a CAP, tilapia and Western blotting confirmed that FOS members, piscidin 4 (TP4), derived from Nile Tilapia (Oreochromis particularly FOSB and FOSΔB (a truncated splice variant niloticus) [41] for treatment of TNBC. Transcriptome of FOSB), were induced in tested BC cell-lines (Figures analyses of TNBC cells and normal fibroblasts were 2C-2E and Supplementary Figure S2A-S2D). Unlike carried out to identify potential molecular targets of TP4. JUNB and JUND, cJUN was not significantly influenced The mechanism of action was investigated using in vitro in TNBC cells (Figure 2D and 2E). Neither FOS nor JUN BC cell models. The therapeutic efficacy of TP4 was family members were significantly activated in control further evaluated through intratumoral injection of null HDF cells (Figure 2D and 2F). To explore the therapeutic mice bearing BC xenotransplants. In addition, zebrafish role of FOSB, we investigated whether FOSB induction embryonic models have been shown to have metastatic could be observed in TNBC cells during treatment with potential in BC xenografts, and the optical transparency of anthracycline or taxane-based chemotherapeutic agents. this model organism enables the study of tumor growth and Interestingly, anthracyclines (doxorubicin and epirubicin) metastasis [42]. Here, we employed zebrafish as a second (Figure 2G, lanes 3 and 4) induced strong FOSB model for testing the therapeutic potential of TP4 in vivo. expression, comparable to that induced by TP4 treatment of TNBC cells (Figure 2G, lane 2). Taxane-based agents RESULTS (docetaxel and paclitaxel), however, induced FOSB in MDA-MB453 (denoted as MB453) and MCF7 cells, but TP4 induces selective necrosis of TNBC cells not in MB231 cells (Figure 2G, lanes 5 and 6). These findings suggest that TP4 and anthracyclines act through Different molecular subtypes of BC cell-lines a similar therapeutic pathway in TNBC cells. In addition, (MDA-MB231, MDA-MB453, and MCF7) were Kyoto Encyclopedia of Genes and Genomes (KEGG) subjected to the MTS assay to investigate whether TP4 analysis of the microarray data revealed a significant can selectively kill BC cells in vitro. It was observed that effect of TP4 treatment on MAPK signaling (Table 1); treatment with 15 μg mL-1, 5.03 μM of TP4 is sufficient to this signaling pathway is known to increase AP-1 activity kill over 50% BC cells at 6h, while the same dose had only [43], and we further examined the molecules involved minor effects on the viability of control normal human by Western blotting. We observed that active forms of mammary epithelial cells (M10) or dermal fibroblasts both JNK and p38 were significantly decreased by TP4 (HDFs) (Figure 1A–1E, statistical analyses are shown in treatment in TNBC cells, but not in control HDF cells Supplementary Table S1). Genomic DNA samples from (Supplementary Figure S3A-C). Activation of ERK TP4-treated MDA-MB231 cells (denoted as MB231) proteins had no significant effect (Supplementary Figure were taken at different time-points and subjected to a S3A and S3B), but inhibition of ERK activity by PD98059 DNA laddering assay; no obvious DNA fragmentation disrupted TP4-induced TNBC cell death, as shown by was observed after TP4 treatment, indicating that TP4 MTS assay (Figure 2H); these findings suggest that ERK does not induce apoptosis in TNBC cells (Figure 1F). signaling is required for TP4-activated cell death. In addition, TUNEL staining of TP4-treated MB231 or HDF cells revealed very limited DNA fragmentation TP4 induces FOSB to trigger TNBC cell death (Supplementary Figure S1A and S1B) and no obvious caspase3 activation was observed in TP4-treated TNBC Strong induction of FOSB by TP4 in TNBC cells cells (Supplementary Figure S1C and S1D). On the other suggested possible involvement of FOSB in TP4-activated hand, a necrotic marker, Lactate dehydrogenase (LDH), TNBC cell death. A previous study indicated that FOSB is was significantly increased at 3h post-TP4 treatment in highly expressed in normal ductal mammary epithelium, TNBC cells (Figure 1G). Taken together, these findings but not in poorly differentiated ductal carcinoma [40]. indicate that TP4 induces necrotic death in TNBC cells. To address whether FOSB expression is associated with TNBC progression, we analyzed FOSB expression in FOS family members were induced by TP4 various grades of tumor samples from TNBC patients by in TNBC cells immunohistochemical analysis. Expression of FOSB in breast normal adjacent tissue (NAT, N = 26)

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