OCT4 is an embryonic stemness factor that plays key roles to maintain the self-renewal and pluripotency of embryonic stem cells, and is also associated with tumorigenesis. Our study showed that in lung cancer, lung adenocarcinoma (LAC) and squamous-cell carcinoma (LSCC) express distinct isoforms of OCT4. An unspliced OCT4B RNA was found in LSCC (but not LAC), which played an important role in cell survival. Knockdown the unspliced OCT4B with shRNAs targeting intron regions led to LSCC cell death, but showed no effect in LAC cells. An open-reading-frame flanking exon 2ab and Intron 2 on the unspliced OCT4B RNA was identified, which encodes a functional protein (indicated as “ORF1”) with a molecular weight ~14 kD. Customized antibody confirmed the expression of endogenous ORF1 protein in LSCC cell lines and patient samples.
Bioinformatic analysis showed that ORF1 could be involved in cancer stemness regulation and EMT and invasion phenotypes. We also found that ORF1 can positively regulate SOX2, a key stemness factor and amplified lineage-survival oncogene in LSCC. Specifically, ORF1 does not regulate SOX2 promoter activity, but likely modulates SOX2 mRNA stability. Overexpression of SOX2 through an exogenous promoter cannot recover the cell death mediated by knockdown of ORF1, emphasizing the key role of ORF1 in LSCC survival.
In summary, ORF1 is a novel protein derived from unspliced OCT4B RNA, which plays a key role in LSCC cell survival. The mechanisms that promote the generation of ORF1 in LSCC, and its clinical significance will be further elucidated.