FCN3 functions as a tumor suppressor of lung adenocarcinoma through induction of endoplasmic reticulum stress

Abstract

In this study, we report a novel function of FCN3 (Ficolin 3), a secreted lectin capable of activating the complement pathway, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of FCN3 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of FCN3 was shown to be significantly correlated with increased mortality among LUAD patients. Interestingly, while ectopic expression of FCN3 led to cell cycle arrest and apoptosis in A549 and H23 cells derived from LUAD, the secreted form of the protein had no effect on the cells. Rather, we found evidence indicating that activation of the unfolded protein response from endoplasmic reticulum (ER) stress is induced by ectopic expression of FCN3. Consistently, inhibition of ER stress response led to enhanced survival of the LUAD cells. Of note, the fibrinogen domain, which is not secreted, turned out to be both necessary and sufficient for induction of apoptosis when localized to ER, consistent with our proposed mechanism. Collectively, our data indicate that FCN3 is a tumor suppressor gene functioning through induction of ER stress.

Fig. 1. FCN3 is down-regulated in LUAD.

A Box-plots of FCN3 RNA expression in normal-tumor matched 102 LUAD patients in this study (left panel) and of normal-tumor matched 58 patients from TCGA data (right panel). The bold line in the middle of each box represents the median level. B Conventional RT-PCR confirmation using normal (N) and tumor (T) tissue samples from 5 representative LUAD patients. ACTB was used as an endogenous reference gene. Note that mRNA expression of FCN3 is markedly down-regulated in tumor tissues. NTC stands for no template control. C Immunoblot analysis of FCN3 expression in normal (N) and tumor (T) tissue samples of 4 representative patients. ɑ–Tubulin was used as the loading control. FCN3 down-regulation is confirmed. D Kaplan–Meier survival curves of 58 TCGA LUAD patients divided into two groups based on FCN3 expression. Long-term survival was seen to be significantly correlated with high expression of FCN3.

Fig. 2. Cell growth is inhibited by FCN3.

A A549 and H23 cells were transduced with control empty virus (EV), FCN3-expressing virus, or CDKN2A-expressing virus and allowed to grow for 8 and 12 days, respectively. Resulting colonies were stained with Coomassie blue, and the number was determined. Graphs to the right show result from three independent experiments as mean ± SEM. Note the strong decrease in the number of colonies in FCN3-expressing cells. B Photographs of mice with xenograft tumors (top) and of excised tumors (bottom). The mice were sacrificed 35 days after cell injection. Note that tumors from the FCN3-expressing A549-injected group show decreased tumor development compared to mice in the control group injected with control EV-transduced A549 cells. C Growth curves of xenograft tumor. Tumor volume was measured every 5 days. N = 10 for both groups. D Body weight of BALB/c nude mice was measured every 5 days. E The weight of all tumors isolated from EV group and FCN3 group. The lines in the middle represent the median levels. (**) and (***) represent P-values of <0.01 and <0.001 from t tests, respectively.

Fig. 3. Ectopic expression of FCN3 induces cell cycle arrests and apoptosis.

A A549 and H23 cells were infected with control virus (EV) or FCN3-expressing virus, and cell cycle progression was analyzed by flow cytometry 72 h after infection. Representative histograms are shown, and proportions of cell cycle distribution were presented as bar graphs. G1 arrest was observed in A549 cells, while G2/M arrest occurred in H23 cells. Data are mean ± SEM of three independent experiments. B Immunoblots of various cell cycle markers in A549 and H23 cells 72 h after viral transduction. ɑ–Tubulin was used as the loading control. C A549 and H23 cells were transduced with control virus (EV) or FCN3-expressing virus, and apoptotic cells were analyzed with flow cytometry 96 h after infection. Proportions of apoptotic cells are presented as bar graph. The percentage of apoptotic cells is increased with FCN3 over-expressed cells compared to that in control. Data summarized in the graph are mean ± SEM of three independent experiments. D Immunoblots showing PARP in A549 and H23 cells after 72 h of viral infection. Cleaved PARP is increased in FCN3-expressing cells. (*) and (**) represent P-values of <0.05 and <0.01 from t tests, respectively.

Fig. 4. Tumor suppressor activity of FCN3 involves an intracellular mechanism.

A Immunoblot showing the secreted FCN3 in cultured media of FCN3-virus-transduced A549 cells but not in culture media of control virus (EV)-transduced cells. Indicated amounts of recombinant FCN3 protein are also shown. B Culture media from control virus- or FCN3-virus-transduced cells were applied to A549 cells, and apoptosis was analyzed by flow cytometry after 96 h. Data summarized in the graph are mean ± SEM of three independent experiments. Note no significant difference was seen. C Cell cycle analyses of A549 cells by flow cytometry 72 h after application of indicated doses recombinant FCN3. No alteration in cell cycle progression was observed. Data are mean ± SEM of three independent experiments. D Apoptosis was examined 96 h after application of indicated doses recombinant FCN3. No alteration in proportions of apoptotic cells was noticed. Data are mean ± SEM of three independent experiments. E A549 cells with or without viral transduction were mixed and co-cultured for 24 h and 72 h. Proportions of GFP-negative and -positive cell populations were analyzed by flow cytometry. After 72 h, GFP-positive proportion was decreased only in the case of FCN3-virus-transduced cells. Data are mean ± SEM of three independent experiments, and (**) represents P-value of <0.01 from t test.

Fig. 5. Transcriptomic analyses indicate FCN3 induces ER stress.

A Expression heatmap view using 1,055 differentially expressed genes (DEGs) from duplicate samples of control empty virus (EV) and FCN3-virus-transduced cells. Genes with indicated gene symbols were used in validation experiment. B Gene set analysis on 595 up-regulated genes (top) and 460 down-regulated genes (bottom) for KEGG pathways and Gene Ontology (GO) biological processes. Top 5 scoring terms are shown in the plots. The full list is provided in Supplementary Table 3. Black dashed vertical lines represent the FDR threshold of 0.05. C Quantitative real time RT-PCR was performed to confirm RNA-Seq results. Five up-regulated and five down-regulated genes were examined after transduction with control or FCN3-expressing virus. D Immunoblots of ER stress markers HSPA5 and DDIT3 in A549 cells with or without 4-phenylbutyric acid (4-PBA) treatment. A549 cells were mock treated, EV transduced or FCN3-virus-transduced and cultured for 48 h prior to sampling. HSPA5 and DDIT3 were induced in FCN3-virus-transduced cells, and 4-PBA treatment effectively down-regulated these ER stress markers. ɑ–Tubulin was used as the loading control. E Apoptosis of A549 cells were examined by flow cytometry 96 h after viral transduction with or without 4-PBA. The percentage of apoptotic cells decreased with the alleviation of ER stress by 4-PBA treatment. Graph to the bottom summarizes the results. Data are mean ± SEM of three independent experiments, and (**) represents P-value of <0.01 from t test. F Subcellular localization of FCN3 in A549 cells. Nuclear GFP expression indicates the FCN3-virus-transduced cell. V5 staining shows localization of ectopic FCN3 in ER.

Fig. 6. The FBG domain localized to ER is necessary and sufficient for tumor suppressor activity of FCN3.

A Schematic illustration of various FCN3 derivatives: wild type FCN3, FBG domain, SP (signal peptide) + FBG domain, and FCN3 1-90. B Immunoblots showing ectopic expression of various FCN3 derivatives in A549 cells. Antibody against V5 epitope was used. ɑ–Tubulin was used as the loading control. C Expression and subcellular localization of FCN3 derivatives in A549 cells. Nuclear GFP expression indicates the virus-transduced cell. V5 staining shows localization of various FCN3 derivatives. Note no V5 staining in control virus (EV)-transduced cells and nuclear localization of FBG. D Colony formation assay after transducing with control virus or viruses expressing the indicated FCN3 derivatives in A549 cells. Note SP + FBG has growth inhibition effect. Numbers of colonies were counted and presented in bar graphs. Data are mean ± SEM of three independent experiments. E Apoptosis of A549 cells was evaluated with flow cytometry after transducing with control virus or viruses expressing the indicated FCN3 derivatives. (*) and (**) represent P-values of <0.05 and <0.01 from t tests, respectively.