Down-regulated Beclin 1 expression is closely linked to lung carcinogenesis by reversing aggressive phenotypes

Background Beclin1 is involved in numerous biological processes, including autophagy, stress, senescence, and death. This article aims to clarify the roles of Beclin1 expression in lung carcinogenesis and subsequent progression. The Beclin 1 expression was examined in lung cancer and compared it with clinicopathological parameters and survival data of the cancers using a large number of lung cancer samples. Additionally, we observed the effects of forced Beclin 1 overexpression and its silencing on the proliferation, glucose metabolism, apoptosis, autophagy, invasion, migration, lamellipodia formation, and chemoresistance of lung cancer cells and analyzed the relevant mechanisms. Finally, in vivo effects of Beclin 1 overexpression on tumor growth was determined in nude mice.


Introduction
Beclin1, a central role in autophagy, is involved in numerous biological processes, including autophagy, stress, senescence, and death. Beclin 1 dysfunction participates in many diseases, such as cancer and 1 × 10 6 cells were trypsinized, collected, washed by PBS twice, and xed in cold 10ml 75% ethanol for more than 2 h. And then, cells were washed by PBS twice and incubated with RNase at 37°C for 1 h. The tube with cells was added by PI to 50µg/ml and incubated at 4°C in the dark for 30 min. Finally, ow cytometry was employed to examine the PI signal.

Apoptosis assay by ow cytometry
Flow cytometry was performed with PI and FITC-labeled annexin V (BD Pharmingen, USA) to detect phosphatidylserine externalization (on the surface of cell membrane) as an endpoint indicator of early apoptosis as the protocol recommends. The 1 × 10 6 cells were collected, washed with cooled PBS at 4°C and centrifuged at 500×g for 3 min twice. FITC-labeled Annexin V (5 µl) and PI (5 µl) were added to 490 µl of the 1 × 10 6 cell suspension and mixed gently. After incubation at room temperature for 15 min in the dark, the cells were analyzed by ow cytometry (Epics XL, Beckman-Coulter, Miami, FL). β-galactosidase staining β-galactosidase staining kit (Beyotime, Shanghai, China) was employed to indicate the senescence. The protocol was executed as recommended by the kit instruction.
Cell migration and invasion assays.
For migration assay, 5 × 10 4 cells were resuspended in serum-free RPMI 1640, and seeded in the controlmembrane insert on the top portion of the chamber (BD Bioscience). The lower compartment of the chamber contained 10% FBS as a chemo-attractant. After incubated for 24 h, cells on the membrane were scrubbed, washed with PBS and xed in 100% methanol and stained with Giemsa dye. For invasive assay, the procedures were the same as above excluding the matrigel-coated insert (BD Bioscience).

Immuno uorescence
Cells were grown on glass coverslips and treated as described in the gure legends. Cells were washed twice with PBS, xed with PBS containing 4% formaldehyde for 10 minutes at room temperature, and permeabilized with 0.2% Triton X-100 in PBS for 10 minutes at room temperature. After washing with PBS, cells were incubated overnight at 4 ℃ with the mouse antibody against Beclin 1 (Sigma, 1:50). They were then washed with PBS and incubated with Alexa Fluor 594 IgG (Invitrogen, 1:2000). Alexa Fluor® 568 phalloidin (Invitrogen) was employed to observe the lamellipodia.Nuclei were stained with 1 µg/ml DAPI (Sigma) for 30 minutes at 37 ℃. Finally, coverslips were mounted with SlowFade® Gold antifade reagent (invitrogen) and observed under laser confocal scanning microscope (Leica, Germany).

Subjects
Lung cancer (n = 655) were collected from surgical resection in our hospital. The patients with lung cancer were 439 men and 216 women (26 ~ 88 years, mean = 65.1 years). Among them, 271 cases have tumors accompanied with lymph node metastasis. Fresh samples of lung cancer (n = 76) and normal tissues (n = 22) were also collected in our hospital. None of the patients underwent chemotherapy, radiotherapy or adjuvant treatment before surgery. They all provided consent for use of tumor tissue for clinical research and our University Ethical Committee approved the research protocol. We followed up the patients by consulting their case documents and through telephone.

Xenograft models
Mice were housed in plastic cages with paper chips for bedding, three mice per house, in speci c pathogen-free conditions in a temperature-controlled animal room with a 12-h light/dark illumination cycle. All had access to standard rodent food and water ad libitum. All experiments involving mice were conducted using protocols approved by the Committee on Animal Experimentation of our hospital. Locally bred female Balb/c nude (nu/nu) mice were used for implantation at the age of 6-8 weeks. They were maintained under speci c pathogen-free conditions as described above. Subcutaneous xenografts were established by injection of 1× 10 6 cells per mouse to axilla (n = 20 mice /group). From tumor diameter reached 8mm, we began to intraperitoneally inject 20 mg/kg cisplatin into mice from 8th, 10th, and 12th day of cell injection. After anesthetization, the mice were photographed, and sacri ced. For each tumor, measurements were made using calipers, and tumor volumes were measured or calculated as follows: width 2 ×length×0.52. The part of tumors was subsequently xed in 4% paraformaldehyde for 24 h, and then embedded in para n for following experiments.

Pathology
All tissues of human and animal were xed in 10% neutral formalin, embedded in para n and cut into pieces at 4 µm. These sections were stained by hematoxylin-and-eosin (HE) to con rm their histological characteristics. The staging for human lung cancer was evaluated according to the Union Internationale Contre le Cancer (UICC) system (Sobin,2009). Histological architecture and grading of lung cancer were expressed in terms of WHO's classi cation. Furthermore, tumor size, pleural, lymphatic and venous invasion were determined (Travis et al., 2000).

Western blot
Denatured protein was separated on an SDS-polyacrylamide gel (10% acrylamide) and transferred to PVDF membrane (Amersham, Germany), which was then blocked overnight in 5% non-fat milk in TBST (10mmol/L Tris-HCl, 150mmol/L NaCl, 0.1% Tween 20). For immunoblotting, the membrane was incubated for 120 min with primary antibody (Table 3). Then, it was rinsed by TBST and incubated with anti-rabbit, anti-mouse IgG conjugated to horseradish peroxidase (DAKO, 1:1000) for 60 min. Bands were visualized with X lm (Fuji, Japan) by ECL-Plus detection reagents (Santa Cruz, USA). Either β-tubulin or GAPDH was employed as an internal control. Tissue microarray and immunohistochemistry Representative areas of solid tumors were identi ed in HE stained sections of the selected tumor cases and a two mm-in-diameter tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Japan). Consecutive sections were depara nized with xylene, dehydrated with alcohol, and subjected to antigen retrieval by irradiating in target retrieval solution (TRS, DAKO, USA), and subsequent immunostaining as described previously (Zhao Y et al., 2014).
Terminal digoxigenin-labeled dUTP nick-end labeling (TUNEL) Cell apoptosis was assessed using TUENL, a method that is based on the speci c binding O-TdT to the 3-OH ends of DNA, ensuring the synthesis of a polydeoxynucleotide polymer. For this purpose, ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore) was employed according to the recommendation. Omission of the working strength TdT enzyme was considered as a negative control.

Meta-analysis
We performed a publication search using PubMed, Web of Science, BIOSIS and SciFinder updated on Dec, 2019. The following search terms were used: (Beclin 1 OR Becn1) AND (lung OR pulmonary) AND (cancer OR carcinoma OR adenocarcinoma).The articles to observe the alteration in Beclin 1 expression in lung cancer by immunohistochemistry were included in the present study. The following information were included in each study: name of rst author, year of publication, country, ethnicity, cancer types, source of control, antibody company, numbers of cases and controls, expression alteration, correlation with aggressive features, and follow-up times. Regarding survival analysis, we used Engauge Digitizer software to extract data from Kaplan-Meier curves and calculated the Hazard ratios (HR) and their corresponding 95% con dence intervals (CI). Any disagreement was resolved through discussion until the two reviewers reached a consensus. Two reviewers independently assessed the quality of the included studies according to Newcastle Ottawa Scale (NOS) (http://www.ohri.ca/programs /clinical_epidemiology/oxford.htm).

Bioinformaticsanalysis
The prognostic signi cance of Becn1 mRNA was analyzed using Kaplan-Meier plotter (http://kmplot.com). The relationship between Becn1 mRNA and overall or progression-free or postprogression survival rate was analyzed, even strati ed by clinicopathological parameters.

Statistical analysis
Statistical evaluation was performed using Spearman correlation test to analyze the rank data, and Mann-Whitney U to differentiate the means of different groups. Kaplan-Meier survival plots were generated and comparisons were made with the log-rank statistic. SPSS 17.0 software was employed to analyze all data.

Results
The in vitro and in vivo effects of Beclin 1 expression on the phenotypes of lung cancer cells To clarify the roles of Beclin 1, we successfully transfected the Becn1-expressing plasmid into KJ and SQ-5 cells, evidenced by RT-PCR, Western blot and immuno uorescence (Fig. 1A). Compared with the control and mock, Beclin 1 overexpression decreased cell viability, glycolysis and mitochondrial respiration, induced apoptosis, S or G 2 arrest (Figs. 1B-1E, p < 0.05). According to Oil red O staining, GFP-LC-3B transfection and β-galactosidase staining, a higher level of fat accumulation, autophagy or senescence was observed inBecn1 transfectants than the control and mock ( Fig. 1F-1H). Based on wound healing, transwell chamber assay and F-actin uorescence staining, cell migration, invasion and lamellipodia formation were weakened in Becn1 transfectants (Figs. 1I-1K). After exposed to cisplatin, SQ-5 and KJ transfectants showed higher viability and lower apoptosis than the control, evidenced by MTT assay and FAS assay respectively (Fig. 1L). At the protein level, Beclin 1 overexpression decreased the expression of Bcl-2, β-catenin and HSP90, whereas increased the expression of LC-3B, ADFP, cytochrome c, p-p38, and p-NF-кB in transfectants. (Fig. 1M).
We subcutaneously transplanted SQ-5 cancer cells and itsBecn1 transfectants into immune-de cient mice and found that the tumor volume and weight of Becn1transfectants were smaller than the control by rule measurement and calculation ( Fig. 2A,p < 0.05). Interestingly, Becn1 transfectants showed a lower inhibition rate of tumor growth than the control after the exposure to cistplatin (Figs. 2A and 2B). Becn1 transfectants showed higher Beclin 1 expression, lower proliferation, higher apoptosis and autophagy than the control, evidenced by Beclin 1, Ki-67 and LC-3B immunostaining, and TUNEL respectively (Fig. 2C). Becn1 knockdown had the converse results (Fig. 3C, p < 0.05).
Compared with normal tissue, lung cancer showed a low expression of Becn1 mRNA by real-time RT-PCR (Fig. 4A, p < 0.05). Becn1 mRNA expression was negatively related to tumor size, and higher in female and younger patients with lung cancer than the counterpart (Fig. 4A, p < 0.05). A higher level of Becn1 mRNA was detectable in adenocarcinoma (Ad) than squamous cell carcinoma (Sq, p < 0.05). According to the Kaplan-Meier plotter, we found that a higher Becn1 mRNA expression was positively correlated with overall, and post-progression survival rates of all cancer patients (Fig. 4B, p < 0.05). As shown in Table 1, the female, male, Ad, Stage-I and -II, T-4, margin-negative, no-chemotherapy, radiotherapy, no-radiotherapy, and smoking cancer patients with high Becn1 mRNA expression showed a long overall survival time than those with its low expression (p < 0.05). There appeared a positive relationship between Becn1 mRNA expression and the progression-free survival rate of the Ad and staging-I cancer patients (p < 0.05). The post-progression survival rates of the male, T-1, margin-negative, no-chemotherapy, no-radiotherapy, and smoking patient was higher in the group of high Becn1mRNA expression than its low expression (p < 0.05).   (Fig. 6M, p > 0.05).  Reportedly, low Beclin 1 expression was signi cantly associated with shorter survival as an independent factor of lung cancer (Wang et al., 2015). Katagiri et al. (2015)found that negative expression of Beclin 1 was associated with a shorter progression-free survival than positive Beclin 1 expression in ovarian clear cell carcinoma who received cytoreductive surgery and then a standard platinum-based chemotherapy regimen.Becn1 mRNA was found to be negatively correlated with overall survival in breast cancer patients receiving tamoxifen treatment, and it was the same for Beclin 1 expression in ER-positive breast cancer patients (Tan Set al., 2016).According to bioinformatics analysis, we found that Becn1 mRNA expression was positively correlated with overall or post-progression survival rates of all cancer patients, even strati ed by sex, histological subtyping, chemotherapy, radiotherapy or smoking. The no correlation between Beclin 1 immunopositivity and survival rate might be due to a short follow-up time and too many cases for early lung cancer for survival analysis. These ndings suggested that higher Becn1 mRNA was an indicator for the favorable prognosis of lung cancer patients.
In summary, our study indicated that down-regulated Becn1 expression might have impact on the carcinogenesis and histogenesis of lung cancer, and should be considered as a good biomarker for