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, 7 (26), 40329-40347

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

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

Chen-Hung Ting et al. Oncotarget.

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.

Keywords: FOSB; TP4; calcium; cationic antimicrobial peptide; triple-negative breast cancer.

Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1
Figure 1. TP4 selectively kills breast cancer cells through inducing necrosis
Cell viability in A. MB231, B. MB453, C. MCF7, D. M10, and E. HDF was determined by MTS assay following treatment with varying doses of TP4 (2.5-20 μg mL−1) at the indicated time-points (3-24h). Sextuplicate wells were analyzed for each assay. Results represent the mean±SD (N = 3, statistical analyses are shown in Supplementary Table S1). F. Detection of DNA fragmentation in TP4-treated MB231 cells by 2% agarose gel electrophoresis. Lane P: Positive control (Actinomycin D-treated HL60 cell lysate); lane N: Negative control (MB231 cell lysate); lane T: TP4-treated MB231 cell lysate; Lane M: DNA molecular weight marker. G. LDH levels in MB231 cells were determined following treatment with varying doses of TP4 (2.5-20 μg mL−1) at 3h. Sextuplicate wells were analyzed for each assay. Results represent the mean±SEM (N = 3, One way ANOVA: ***, P < 0.001 versus control, ns: not significant).
Figure 2
Figure 2. Induction of FOSB by TP4 in breast cancer cells
A. Gene ontology (GO) analyses of the microarray study classified dysregulated genes into nine defined categories (P < 0.01). The graph shows the number of genes of each category that were found to be differentially expressed in MB231 cells following TP4 treatment, as compared to untreated controls. Annotation terms were determined using David 6.7 software. B. Heat maps depicting the changes of expression of genes in MB231 or HDF cells following TP4 treatment (scale bar indicates log2-fold changes). AP-1 transcription factor members are shown in red. C1, C2 and T1, T2 indicate the mock and TP4-treated samples collected from two independent assays, respectively. C. TP4- (14 μg mL−1) or mock-treated cells were stained with FOSB antibody (red) and βactin (green). Hoechst 33342 dye was used for nuclear staining (blue). Bar: 50 μm. D. Total lysates from MB231, Hs578T, or HDF cells with (+) or without (−) TP4 treatment were analyzed by Western blot using antibodies against GAPDH and FOS/JUN family proteins. E, F. Quantitative analysis of the blots shown in (D) using GAPDH as a control for normalization. Results represent the mean±SEM (N = 3, Student's t-test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. versus control, ns: not significant). G. Total lysates from control cells (lane 1), and cells treated with TP4 (lane 2), doxorubicin (lane 3), epirubicin (lane 4), docetaxel (lane 5), or paclitaxel (lane 6) were analyzed by Western blot using antibodies against GAPDH and FOSB. The relative amounts of FOSB plus FOSΔB in each lane are expressed as relative densitometric units (RDUs), calculated by dividing the FOSB plus FOSΔB signal by the GAPDH signal. H. MTS assay was used to measure cell viability in cells treated with PD98059 and TP4. Sextuplicate wells were analyzed for each assay. Results represent the mean±SEM (N = 3). Statistical comparisons of the differences between groups treated with or without PD98059 were performed using Student's t-test. ns: not significant; *, P < 0.05; ***, P < 0.001.
Figure 3
Figure 3. TP4 triggers TNBC cell death through FOSB induction
A. Normal adjacent tissue (NAT, N = 26) and different grades of TNBC samples (N = 6, 19, 10 for grade I, II, III samples, respectively) were stained with FOSB (red) antibody and Hoechst 33342 (blue). Bar: 200 μm. Bottom graph, quantitation of the FOSB fluorescent signal indicated that FOSB level is associated with TNBC pathological grade. AU: arbitrary unit. B. Total lysates from mock (M) and TP4-treated groups were examined by Western blot. Bottom graph, quantitative analysis of total FOSB (FOSB plus FOSΔB) induction, normalized to GAPDH. C. Phase contrast and fluorescent images of MB231 cells transfected with FOSB or FOSDB vector. Bar: 50 μm. Cell viability was determined by ATP assay. At least fourteen replicate wells were analyzed for each dose. D. Total lysates from mock and TP4-treated (14 μg mL−1, 6h) MB231 cells transduced with control or FOSB shRNA lentivirus were analyzed by Western blot. E. Induction of FOSB levels, as normalized to GAPDH. F. The effect of TP4 treatment on the viability of the indicated cells, as determined by MTS assay. Sextuplicate wells were analyzed for each assay. G. Total lysates from MB231 cells (mock (−) or TP4-treated (+)) were analyzed by Western blot. H, I. Quantitative analyses of FRA1 (H) and CDH1 (I) levels, normalized to GAPDH. J. DNA-protein complexes were immunoprecipitated from mock (M-) or TP4-treated (T-) MB231 nuclear extracts (NEs) using the indicated antibodies. Forty picomoles of wild-type (WT) or mutated (MT) AP-1-binding oligonucleotides were used in the competition assay. K-562 cell NEs stimulated with TPA were used as a positive control. K, L. cJUN was immunoprecipitated from mock (C) or TP4-treated (T) NE with anti-cJUN antibody. Total lysates from mock or TP4-treated groups were used as positive controls. Immunoprecipitation with nonspecific IgG was performed as a negative control. Coimmunoprecipitation of FRA1 (K) and FOSB (L) with cJUN were examined by Western blot. Results represent mean±SEM (N = 3) by Student's t-test (A, D, E, H-J), one-way ANOVA (B), or two-way ANOVA (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns: not significant.
Figure 4
Figure 4. TP4 is bound to the TNBC cell membrane and intracellular organelles
A–D. Cellular localization of biotinylated-TP4 in MB231 (A-C) and HDF cells (D). Cells were stained with biotin (green), Golgi marker (giantin; red) (A,D), ER marker (calreticulin; red) (B), and mitochondrial marker (mitotracker; red) (C) antibodies. The plasma membrane was labeled with Alexa Flour-647-conjugated WGA (purple). Hoechst 33342 was used for nuclei staining (blue). Boxed regions are shown magnified in the panels to the right of the merged images. Yellow and white arrows indicate co-localization of biotinylated-TP4 with plasma membrane and Golgi or mitochondria, respectively. Bar: 50 μm. E, F. Quantitation of the fluorescent signals, indicating that mitochondrial membrane potential was significantly decreased in TP4-treated MB231 cells (E). Statistical comparisons between mock and TP4-treated cells were performed using Student's t-test. ns: not significant; *, P < 0.05; ***, P < 0.001. G. Quantitation of the mitochondria fluorescent signals in Controlsh– and FOSBsh–MB231 cells treated with TP4 (14 μg mL−1 for 0.5−6.0 h), indicating that mitochondrion intensity could be partially restored in FOSB-knockdown MB231 cells by TP4 treatment. Statistical comparisons between mock and TP4 treatment groups were performed using Student's t-test (N = 50 in each group). ns: not significant; *, P = 0.0221; **, P < 0.01; ***, P < 0.001. AU: arbitrary unit. H. Quantitation of the fluorescent intensity in vehicle control (V), eGFP-transfected, and FOSB-transfected MB231 cells, indicating that mitochondrion fluorescent intensity was decreased in FOSB-transfected MB231 cells. Statistical comparisons between mock and TP4-treated groups were performed using Student's t-test. ns: not significant; ***, P < 0.001.
Figure 5
Figure 5. FOSB induction in TNBC cells requires calcium signaling
A. Ca2+ levels were measured by the addition of fluorescent Ca2+ indicator (Fluo-4) after treatment with the indicated doses of TP4 for 5-30 min. Octuplicate wells were analyzed for each assay. Results represent the mean±SEM (N = 3, Student's t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001). B. Mitochondrial Ca2+ levels were measured kinetically using a fluorescent Ca2+ indicator (Rhod-2 AM) after treatment with the indicated doses of TP4 every 30 sec for 30 min. Results represent the mean±SEM (N = 3, one-way ANOVA: ***, P < 0.001). C, F. Total lysates from control (lane 1), BAPTA/AM (calcium chelator)-treated or AIP2 (CaMKII inhibitor)-treated cells (lane 2), TP4-treated cells (lane 3), and combination-treated cells (lane 4) were analyzed by Western blot, using antibodies against GAPDH and FOSB. D, G. Quantitative analyses of the blots shown in (C, F); levels of FOSB plus FOSΔB were normalized to GAPDH. Results represent the mean±SEM (N = 3, Student's t-test: *, P < 0.05; **, P < 0.01). E, H. Cell viability was measured in cells treated with Ca2+ chelator or CaMKII inhibitor and TP4. Sextuplicate wells were analyzed for each assay. Results represent the mean±SEM. Statistical comparisons of the differences between groups treated with or without inhibitors were performed using Student's t-test. ***, P < 0.001. I. Proposed mechanism-of-action of TP4 against TNBC. TP4 binds the cell membrane and selectively binds the mitochondria. This in turn results in Ca2+ release and induction of FOSB expression. FOSB/c-JUN becomes the predominant AP-1 complex that triggers downstream cell death.
Figure 6
Figure 6. TP4 inhibits TNBC xenograft growth in nude mice
A. Xenograft growth in nude mice (N = 5). B. Quantitation of tumor size at the indicated days after the commencement of TP4 treatment. Statistical comparisons between KY and TP4-treated groups were performed by two-way ANOVA with post hoc analysis (Bonferroni test). ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. C. Xenograft tumor weight (left) and mouse body weight (right) were determined when the mice were sacrificed (Student's t-test, **, P < 0.01; ns, not significant). D. H&E staining of xenograft tumors. White dotted lines mark necrotic regions. E. Immunohistochemical staining of Ki-67-positive (left) and FOSB-positive (right) cells in xenograft tumors. Bar: 200 μm.
Figure 7
Figure 7. TP4 treatment prolongs survival in TNBC xenograft zebrafish
A. Survival analysis of M10 and TNBC cells with or without TP4 treatment. Statistical comparisons were performed by Log-rank test. *, P < 0.05; ***, P < 0.001. B. Quantitation of the eGFP fluorescent signals in M10 and TNBC xenografts with or without TP4 treatment (3 μg mL−1 for 5d). Statistical comparisons between mock and TP4-treated cells were performed using Student's t-test (N = 11). ns: not significant; ***, P < 0.001. AU: arbitrary unit. C. Transgenic zebrafish (fli:eGFP) with mOrange2-expressing TNBC xenografts (red) underwent mock or TP4 treatment (3 μg mL−1 for 5d) and were then stained with FOSB antibody (white). Each panel is a merged image of photographs taken of the posterior and anterior parts. Boxed regions are shown magnified in the lower-right corner of the figures. Blue arrows indicate disseminated tumor foci. Yellow arrows indicate TNBC xenografts with positive FOSB signals. Bar: 200 μm. D, E. Quantitation of the primary tumor area (D) and disseminated tumor foci (E) in TNBC xenograft zebrafish. Results represent mean±SEM, and were analyzed by Student's t-test (N = 4 in each group). ns: not significant; *, P = 0.0221; **, P < 0.01; ***, P < 0.001. F. Time-lapse study of transgenic zebrafish (fli:eGFP) with mOrange2-expressing TNBC xenografts (red) during a single treatment with TP4 (3 μg mL−1). Time series images were taken every 1 h, including z-stacks. Selected planes within 48hrs are shown. Arrows in xenograft zebrafish indicate blood vessel invasion of TNBC cells. Boxed regions are shown magnified in the images above the figures. G. Schematic drawing of TNBC xenograft migration through the common cardinal vein (CCV) in zebrafish. H. Survival analysis of TNBC xenografts with or without TP4 (3 μg mL−1, single treatment) at 72-120 hpf. Statistical comparisons of survival curves between groups were performed by Log-rank test. *, P < 0.05. I, J. Quantitation analysis of xenograft tumor growth, based on normalized tumor area (I) and fluorescence intensity (J), in zebrafish with or without TP4 treatment. Results represent mean±SD, and were analyzed by two-way ANOVA; ***, P < 0.001.

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