The Clinical Influence of Autophagy-Associated Proteins on Human Lung Cancer

Abstract

Exploitation of autophagy might potentially improve therapeutic strategy. Here, we analyzed the protein expression of autophagy-associated genes including LC3A, LC3B, Beclin-1, p62, and Atg5 in 88-131 primary lung tumors by immunohistochemistry (IHC) on tissue-microarrays (TMAs). Additionally, the DNA methylation pattern of LC3A was investigated by bisulfite sequencing (BS) and methylation-specific-PCR (MSP). It turned out that the higher expression of LC3A protein was associated with adenocarcinoma compared to squamous cell carcinoma of lung (p = 0.008), positive staining of LC3B was significantly related to tumor grade (p = 0.006), and the protein expression of Beclin-1 was significantly correlated to pN stage (p = 0.041). The expression of p62 and Atg5 was however not significantly associated with any clinicopathological parameters. Downregulation of LC3A was related to DNA methylation in lung cancer cell lines, while in primary lung tumor samples, protein expression of LC3A was not significantly correlated with DNA methylation, and the methylation status of LC3A was not related to clinicopathological features. Taken together, our results suggest that autophagy-associated proteins such as LC3A, LC3B, and Beclin-1 might be potential biomarkers for subclassification, differentiation, and local metastasis in primary lung tumor, and epigenetic mechanism is partially responsible for gene silencing of LC3A in lung cancer cell lines.

Figure 1

(a) LC3A mRNA expression analysis in lung cancer cell lines and human bronchial epithelial cells (HBEC). LC3A mRNA expression was analyzed by real-time RT-PCR, showing that LC3A expression was increased in lung cancer cell lines H2170 and H1650 and downregulated in H226, COLO677, H322, and H1975 compared to HBEC. Relative LC3A mRNA expression was normalized to GAPDH mRNA expression. (b) Demethylation tests in lung cancer cell lines. Real-time RT-PCR showed that after treatment with 5 μM of 5-aza-2′-deoxycytidine (5-Aza) for 96 h, LC3A mRNA expression was heterogeneously upregulated in the 4 cell lines. Student's t-test was applied to compare the gene expression levels. ^∗∗ p < 0.01; ^∗∗∗ p < 0.001.

Figure 2

(a) Methylation status of LC3A in 5 lung cancer cell lines and HBEC. Bisulfite sequencing (BS) showed a heterogeneous methylation pattern in the promoter region and exon 4 of the LC3A gene in lung cancer cell lines. White: unmethylated; grey: partially methylated or only one allele is methylated; black: totally methylated or two alleles are methylated. 1–6: 6 CpG sites in promoter region; 1–8: 8 CpG sites in exon 4. (b) Representative results from methylation-specific PCR (MSP) showing that LC3A was unmethylated in cases 1, 4, and 5 while partially methylated in cases 3, 6, 7, and 8. Case 2 was excluded for the statistical analysis (see Supplementary Table 3), since no PCR products were observed most probably due to degraded genomic DNA. U: unmethylated; M: methylated.

Figure 3

Representative images for the expression of autophagy-associated proteins in primary lung cancer. (a) LC3A protein expression in lung adenocarcinoma (ADC). (b) LC3A protein expression in lung squamous cell carcinoma (SCC). (c) LC3B protein expression in lung ADC. (d) LC3B protein expression in lung SCC. (e) Beclin-1 protein expression in lung ADC. (f) Beclin-1 expression in lung SCC.