Autophagic cell death is characterized by the presence of double or multiple-membrane enclosed vesicles and is involved in bulk degradation through an autophagosomic-lysosomal pathway (Rohatgi et al., 2015). Different from the other findings (Yu et al., 2013; Zhang et al., 2014), we demonstrated down-regulated Beclin 1 expression in lung cancer according to RT-PCR, western blot, and meta-analysis, suggesting that Beclin 1 loss was positively linked to lung carcinogenesis as described previously (Kim et al., 2011; Okura et al., 2011; Weh et al., 2016). As for our knowledge, the histogenesis of lung cancer included squamous dysplasia-carcinoma, glandular cell-adenocarcinoma, Type II alveolar or Clara cells-bronchoalveolar adenocarcinoma, and neuroendocrine cells-large cell carcinoma (Travis et al., 2000). Therefore, the paradoxical data might be attributed to the various cell types of lung normal and cancer tissue, more blood cells, complicated histogenesis of lung cancer, and Beclin 1 expression in stromal cells. A body of evidence showed that Beclin 1 hypoexpression was inversely correlated with the tumor size, tumor stage, lymph node metastasis, histological grade, lymphatic invasion, distant metastasis, depth of invasion of meningiomas, cholangiocellular, lung, cervical, ovarian, esophageal, pancreatic, colorectal or gastric cancer in agreement with meta-analysis(Cheng et al., 2012; Dong et al., 2011; Kim et al., 2011; Lin et al., 2013; Wang et al., 2015; Weh et al., 2016; Yu et al., 2013; Kuo et al.,2019 ). Song et al. (2020) found that Beclin-1 expression was positively correlated with tumoral FOXP3 overexpression and with a greater number of infiltrated Tregs in gastric adenocarcinoma. Here, we found that Becn1 mRNA expression level was negatively correlated with tumor size of lung cancer, and higher in Ad than Sq, indicating that Becn1 mRNA expression might be involved in tumor growth and histogenesis of lung cancer.
Reportedly, Beclin 1 overexpression inhibited proliferation, migration and invasion, and lamellipodia formation and tumor growth, induced cell cycle arrest, autophagy, and apoptosis of colorectal cancer cells (Zhang et al.,). Becn1 knockdown increased cell growth of ovarian clear cell carcinoma cells (Katagiri et al., 2015), but suppressed cell growth, invasion and metastasis of osteosarcoma cells with downregulated expression of PI3Kp85α, p-AKT, and MMP-9 (Zhang et al., 2015). Cheng et al.(2019) found that BECN1 promotes the migration of NSCLC cells via interaction with Vimentin, followed by its K48-linked ubiquitination via ubiquitin-specific peptidase 14 (USP14). Hasan et al. (2020) found that gedunin suppressed interaction between Hsp90:Beclin-1:Bcl-2, finally to inhibit autophagy (Beclin-1, Atg5-12 complex, and LC3) and antiapoptotic protein Bcl-2, which may result in ER stress-induced apoptosis of A549 cells. Hu et al. (2020) observed Becn1 knockdown markedly promoted motility and invasion of CRC cell with STAT3 phosphorylation via regulation of the interaction between STAT and JAK2. In the present study, we found that Beclin 1 overexpression suppressed proliferation, glycolysis, mitochondrial respiration, migration, invasion, lamellipodia formation and tumor growth of lung cancer cells, while versa for Becn1 knockdown, indicating that Beclin 1 might be employed as a potential target of gene therapy for lung cancer. Pirtoli et al. (Pirtoli et al., 2009) also reported that Beclin 1 overexpression was positively correlated with apoptosis, and negatively with cell proliferation in high-grade glioma.
In apoptosis, Bcl-2 can interact with Bax on the mitochondrial membrane to suppress the release of cytochrome c, which can initiate mitochondrial apoptosis (Cosentino et al., 2014). Sun et al. (2019) found that Beclin-1 expression in hepatocellular carcinoma was negatively correlated with the expression of PCNA, NET-1, and anti-apoptotic protein Bcl-2, but positively correlated with pro-apoptotic protein Bax expression. Therefore, the apoptotic induction of Beclin 1 might result from Bcl-2 hypoexpression and cytochrome c hyperexpression of mitochondrial pathway. GSK-3 phosphorylated β-catenin for its entrance into the nucleus, where the interaction of β-catenin with TCF family transcription factors regulates gene expression of c-myc, VEGF, survivin and Cyclin D1 (Hendersonet al., 2011).Hsp90 is a chaperone protein that assists other proteins to fold properly, stabilizes proteins against heat stress, and aids in oncoprotein degradation, including EGFR, Akt, v-Src, and Bcr/Abl (Esfahani et al., 2016). We speculated that Beclin 1 down-regulated the expression of β-catenin and Hsp90, finally to suppress proliferation, invasion and metastasis of lung cancer cells. LC3B is an ubiquitin-like protein involved in the formation of the autophagosome and mediates membrane expansion in order to selectively engulf cargo and deliver it to the lysosome for degradation (Amaya et al., 2015).Adipophilin (ADFP) is a ubiquitous component of lipid droplets and its overexpression is a useful marker for pathologies characterized by increased lipid droplet accumulation (Morrissey et al., 2015). Beclin 1 overexpression induced fat accumulation and autophagy, possibly by up-regulating the expression ADFP and LC-3B respectively.
Tan et al. (2016) demonstrated that miR-409-3p sensitized colon cancer cells to oxaliplatin by inhibiting Beclin-1-mediated autophagy. In clinical practice, Beclin 1-negative ovarian clear cell carcinomas were no more resistant to primary adjuvant chemotherapy than Beclin 1-positive ones (Katagiri et al., 2015). Gu et al. (2016) found that a tamoxifen-resistant and ER-positive breast cancer cell displayed Beclin 1 and HER2 overexpression, and Becn1 silencing decreased HER2 level and significantly promoted its tamoxifen sensitivity. In neuroblastoma, autophagy was induced by chemotherapy and associated with chemoresistance (Belounis et al., 2016), and autophagy protected human lung cancer cells from gemcitabine-induced apoptosis (Wu et al., 2016). In contrast, Zhang et al. (2015) found that Becn1 knockdown suppressed chemotherapy-induced cytotoxicity in osteosarcoma cells. In the present study, we found that Beclin 1 overexpression in vivo and vitro mediated chemoresistance of lung cancer cells to cisplatin, which was positively linked with apoptotic resistance, and p-NF-Кb overexpression. Therefore, we should carefully utilize Becn1 as a gene therapy target only if its chemoresistance would be ameliorated and avoided in clinical practice.
Reportedly, low Beclin 1 expression was significantly 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 stratified 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 findings 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 unfavorable prognosis. Beclin 1 overexpression might reverse the aggressive phenotypes and might be employed as a molecule target for gene therapy only if its chemoresistance would be ameliorated and avoided in the future.