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, 11 (1)

FOSB⁻PCDHB13 Axis Disrupts the Microtubule Network in Non-Small Cell Lung Cancer

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FOSB⁻PCDHB13 Axis Disrupts the Microtubule Network in Non-Small Cell Lung Cancer

Chen-Hung Ting et al. Cancers (Basel).

Abstract

Non-small cell lung cancer (NSCLC) is among the leading causes of human mortality. One reason for high rates of NSCLC mortality is that drug resistance is a major problem for both conventional chemotherapies and less-toxic targeted therapies. Thus, novel mechanistic insights into disease pathogenesis may benefit the development of urgently needed therapies. Here we show that FBJ murine osteosarcoma viral oncogene homolog B (FOSB) was induced by an antimicrobial peptide, tilapia piscidin-4 (TP4), through the dysregulation of mitochondrial Ca2+ homeostasis in NSCLC cells. Transcriptomic, chromatin immunoprecipitation quantitative PCR, and immunocytochemical studies reveal that protocadherin-β13 (PCDHB13) as a target of FOSB that was functionally associated with microtubule. Overexpression of either PCDHB13 or FOSB attenuated NSCLC growth and survival in vitro and in vivo. Importantly, downregulation of both FOSB and PCDHB13 was observed in NSCLC patients and was negatively correlated with pathological grade. These findings introduce the FOSB⁻PCDHB13 axis as a novel tumor suppressive pathway in NSCLC.

Keywords: FOSB; antimicrobial peptide; cytoskeleton; mitochondrial stress; non-small cell lung cancer (NSCLC); protocadherin.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Mitochondrial stress-mediated FOSB activation in non-small cell lung cancer (NSCLC) cells. (A) Total lysates from control and NSCLC cells without (Co) or with TP4 treatment (T, 6.71 μM) were analyzed by Western blot using antibodies against GAPDH and FOSB. Quantitative analysis of FOSB (normalized to GAPDH) is shown below the blot. (B) Cell viability was determined by the ATP assay for NSCLC cells transfected with FOSB or FOSΔB plasmid. Eight replicate wells were analyzed for each dose. (C) Total lysates from A549 cells transfected with control (Neg-si) or FOSB small interfering RNAs (siRNAs) were analyzed by Western blot using antibodies against GAPDH and FOSB. (D) Quantitative analyses of FOSB levels (C), normalized to GAPDH. (E) Viability of A549 cells transfected with FOSB siRNAs were determined by ATP assay. Eight replicate wells were analyzed for each condition. (F) Intracellular localization of biotinylated-TP4 in A549 cells. Cells were stained with biotin, prohibitin (upper), giantin (middle), and calreticulin (lower) antibodies. Hoechst33342 was used to stain nuclei. Boxed regions are magnified in the panels to the right of the merged images. Yellow arrows indicate colocalization of biotinylated-TP4 with mitochondria. Bar: 40 μm. (G) Mitochondrial fractions from A549 cells without (Co) or with TP4 treatment (T, 5.03 μM) were analyzed by Western blot using antibodies against organelle markers and TP4. (H) Mitochondria in A549 cells were stained by MitoTracker Red CMXRos dye. Fluorescence intensity of the mitochondria in each cell was quantified after 5.03 and 6.71 μM TP4 treatment for 3 h. Bar: 20 μm. (I) Mitochondrial Ca2+ levels were measured kinetically (every 30 s for 30 min) using Rhod-2 AM dye after treatment with the indicated doses of TP4. (J) Ca2+ levels were measured by the addition of Fluo-4 dye after treatment with the indicated doses of TP4 for 15–60 min. Eight wells were analyzed for each treatment in an independent repeat. (K) Total lysates from A594 cells without (Co) or with 6.71 μM TP4 treatment (T) for 45 min to 6 h were analyzed by Western blot using antibody against GAPDH, FOSB, and ERK. The relative amounts of FOSB + FOSΔB, ERK1/2, and p-ERK1/2 in each lane are expressed as RDU and normalized to GAPDH signal. Experiments were independently repeated with comparable results. (L) Total lysates from control, BAPTA/AM-treated, TP4-treated cells, and combination-treated cells were analyzed by Western blot using antibodies against GAPDH and FOSB. Quantitative analyses of the blots shown in left; levels of FOSB + FOSΔB were normalized to GAPDH. (N) Cell viability was measured in cells treated with BATPA/AM and TP4. Eight wells were analyzed for each independent replicate. Quantitative results are presented as the mean ± SD. (n = 3, two tailed t-test in (A,D,E,H,J,L,M), one-way ANOVA followed by Bonferroni’s test in (B): * p < 0.05; ** p < 0.01; *** p < 0.001). Co in (A,G,K): control group.
Figure 2
Figure 2
Loss of cytoskeletal integrity upon FOSB induction. (A,B) Gene ontology (GO) analyses of dysregulated genes revealed three distinct functional categories (A). Twelve out of twenty-two annotation terms were assigned to the cellular component ontology, including genes that are involved in the regulation of cytoskeleton and membrane (B). (C,D) Total lysates from A549 cells transfected with EGFP or FOSB-tGFP plasmid were analyzed by Western blot using antibodies against GFP, GAPDH, FOSB, EMT markers, PCDHB13, and Stathmin. (E,F) Total lysates from A549 cells without (Co.) or with TP4 (T) were analyzed by Western blot using antibody against GAPDH, EMT markers, PCDHB13, and Stathmin. (G,H) Total lysates from A549 cells transfected with control (Neg-si) or FOSB siRNAs (FOSB-si-1+2) with or without TP4 treatment were analyzed by Western blot using antibodies against GAPDH, EMT markers, and PCDHB13. Quantitative measurements of protein levels were normalized to GAPDH in (D,F,H). Quantitative results represent the mean ± SD (n = 3, two-tailed t-test in (D,G,H): * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 3
Figure 3
PCDHB13 is a downstream target of FOSB. (A) Total lysate from A549 cells transfected with control (Neg.) or FOSB siRNAs were analyzed by Western blot using antibodies against GAPDH and PCDHB13. (B) Quantitative analyses of PCDHB13 levels in (A) normalized to GAPDH. (CG) Quantitative PCR following ChIP assay to confirm putative FOSB target sites in the PCDHB13 promoter. Two primer sets were used for qPCR studies (C). RNA Pol II antibody and a qPCR primer-pair flanking the GAPDH promoter served as positive controls, while non-specific rabbit IgG antibody served as a negative control. The ChIP-qPCR signals are normalized to Rabbit IgG signals, showing the fold enrichment relative to the background (DG). Results represent mean ± SD. PCR amplicons were electrophoresed on a 1.5% agarose gel to confirm specificity of amplification. (H) Transmitted light and fluorescent images of A549 cells transfected with reporter or cotransfected with reporter and FOSB. Bar: 100 μm. (I) Total lysates from A549 cells transfected with reporter or cotransfected with reporter and FOSB were analyzed by Western blot using antibodies against tGFP, GAPDH, and FOSB. (J) Quantitative measurements of protein levels were normalized to GAPDH (I). Quantitative results represent the mean ± SD (n = 3, two tailed t-test: * p < 0.05).
Figure 4
Figure 4
PCDHB13 disrupts microtubule dynamics in NSCLC cells. (A) Cellular localization of PCDHB13 in A549 cells. Cells were stained with PCDHB13 (green) and α-Tubulin (red). Hoechst33342 was used to stain nuclei (blue). Boxed regions are magnified in the panels to the right. Red arrows indicate the filamentary structure of PCDHB13. White circles indicate the cellular aggregates formed by PCDHB13 and α-Tubulin. Bar: 20 μm. (B) PCDHB13 and α-Tubulin are shown in red and green, respectively. Cytoskeletal projections of each protein were simulated by Imaris software. Yellow color indicates colocalization of PCDHB13 and α-Tubulin. Bar: 8 μm. (C) A549 cell lysates without (lane 1 and 3) or with tGFP-tagged PCDHB13 overexpression (lane 2 and 4) were harvested for co-immunoprecipitation (Co-IP) assay using the anti-tGFP conjugated magnetic beads. IP eluates were analyzed by Western blot for PCDHB13 and α-Tubulin. (D,E) GFP (D) or PCDHB13-tGFP vector (E) transfected A549 and H1975 cells pretreated with 20 μM nocodazole were stained for PCDHB13 (red) and α-Tubulin (white) at 0, 10, and 30 min after nocodazole washout. Higher magnifications of the areas in the white boxes marked by * or # are shown in the corresponding panel. Hoechst33342 was stained for nuclei (blue). Green arrows indicate MTOC with defective microtubule outgrowth. The spatial correlation of PCDHB13 with microtubules in the indicated region (pink lines mark two opposite ends, indicated by pink arrows) is shown by a line-series analysis. The red and white lines in the right panels represent the PCDHB13 and α-Tubulin fluorescence intensities, respectively. au: arbitrary units. Bar: 20 μm. (F) Quantification of MTOC formation in cells transfected by GFP or PCDHB13 after nocodazole washout. g Illustration of the phenomena shown in (D,E).
Figure 5
Figure 5
The extracellular domain of PCDHB13 regulates microtubule polymerization. (A) Schematic showing the functional domains of PCDHB13. Recombinant proteins with EC1–EC3, EC1–EC6, and CR of PCDHB13 were used for the in vitro tubulin polymerization assay. (BD) Dynamic assessment of the effects of PCDHB13 domains on microtubule polymerization. Pure tubulins were assembled in the presence of indicated amounts of CR domain (B) or EC domains (C,D). Tris-HCl-10% DMSO, paclitaxel, and nocodazole served as vehicle, positive control, and negative control, respectively. (E) Fluorescent tubulins were used to probe the formation of microtubules in the presence of PCDHB13 EC domains. Boxed regions are magnified in the panels to the left. Bar: 20 μm. Three wells were analyzed for each independent replicate. Quantitative results are presented as the mean ± SD (n = 3).
Figure 6
Figure 6
Elevated expression of PCDHB13 inhibits cell invasion and triggers cell death. (A,C) The underside of Matrigel-coated polycarbonate membranes used for cell invasion assays on EGFP-, PCDHB13-, and FOSB-transfected cells (A) or cells incubated with 1.68 and 3.35 μM TP4 (C). Cells were stained by Hoechst33342 shown in blue (A) or white (C). Bar: 200 μm. (B,D) Quantification of the cells that migrated across the membrane. Data were calculated by normalizing GFP/tGFP-positive to the total cell counts (n = 463 in EGFP transfected groups, n = 300 in PCDHB13-tGFP transfected groups, and n = 297 in FOSB-tGFP transfected group) in (B) or by counting Hoechst-dye stained cells in (D). (E) Schematic of the A549 cell xenotransplantation procedure. Cells were used for RO injection at 24 h post-transfection. Cell-transplanted zebrafish were cultured for 4 d. (F) Transmitted light and fluorescent images of A549 cells without or with transfection. Bar: 100 μm. (G) Quantification of fluorescent signal in dissected organs (n = 5 in the nontransfected/NT group and n = 9 in the transfected groups). au: arbitrary unit. (H) Tissue extracts from tGFP-, PCDHB13-, and FOSB-transfected A549-transplanted zebrafish were analyzed by Western blot using antibodies against N-Cadherin, α/β-Tubulin, and tGFP. P: Lysates from tGFP/PCDHB13/FOSB-transfected A549 cells. N: Lysate from nontransfected A549 cell. (I) Quantification of the EGFP/tGFP levels from Western blots (n = 9). (J) Cell viability was determined by the ATP assay for A549 and NCI-H1975 cells after transfection with PCDHB13. Ten replicate wells were analyzed for each dose (n = 3). (K) LDH release in A549 and NCI-H1975 cultures were determined 24 h after FOSB and PCDHB13 transfection. Triton-X was used as a positive control. Each independent replicate was measured at least in triplicate (n = 3). (L) Total lysates from A549 cells transfected with control (Neg.) or PCDHB13 siRNAs were analyzed by Western blot using antibodies against GAPDH and PCDHB13. Bottom, PCDHB13 levels were quantified and normalized to GAPDH. (M) Cell viability was determined by the ATP assay for A549 cells transfected with siRNAs targeting PCDHB13. Eight replicate wells were analyzed for each condition (n = 3). Quantitative results represent the mean ± SD (One-way ANOVA test in (K); two tailed t-test in (M): ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 7
Figure 7
Downregulation of FOSB and PCDHB13 in lung cancer patients. (A) Matched NAT (n = 9) and lung cancer samples (n = 31, 42, 21, and 1 for grade I, II, III, and IV samples, respectively) were stained with DAB signal enhancement for FOSB and PCDHB13. Scale bar: 50 μm. (B,C) Quantification of DAB staining intensity for FOSB (B) and PCDHB13 (C) in alive, dead, and matched NAT samples. (D) Correlation between FOSB and PCDHB13 levels in the alive group (p < 0.001, Spearman’s r = 0.6070). (E,F) DAB staining intensity of FOSB (E) and PCDHB13 (F) were normalized to the respective values from matched NAT and were classified according to the disease stage. (G) Survival analysis of lung cancer patients according to relative FOSB expression (upper panel) and PCDHB13 expression (lower panel) level. Statistical comparisons of survival curves between groups were performed by Log-rank test. ** p < 0.001 for FOSB expression and *p < 0.05 for PCDHB13 expression. (H,I) Tissue extracts from twelve NSCLC patients were analyzed by Western blot using antibodies against FOSB, PCDHB13, and GAPDH. P: Lysates from EGFP/PCDHB13/FOSB-transfected A549 cells. N: normal adjacent tissue; T: tumor. (J) Quantitation of the FOSB and PCDHB13 levels shown in (H) and (I) (n = 11). Quantitative results represent the mean ± SD (two-tailed t-test: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

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