Abstract
OCT4A is well established as a master transcription factor for pluripotent stem cell (PSC) self-renewal and a pioneer factor for initiating somatic cell reprogramming, yet its presence and functionality in somatic cancer cells remain controversial and obscure. By combining the CRISPR-Cas9-based gene editing with highly specific PCR assays, highly sensitive immunoassays, and mass spectrometry, we provide unequivocal evidence here that full-length authentic OCT4A transcripts and proteins were both present in somatic cancer cells, and OCT4A proteins were heterogeneously expressed in the whole cell population and when expressed, they are predominantly localized in cell nucleus. Despite their extremely low abundance (approximately three orders of magnitude lower than in PSCs), OCT4A proteins bound to the promoter/enhancer regions of the AP-1 transcription factor subunit c-FOS gene and critically regulated its transcription. Knocking out OCT4A in somatic cancer cells led to dramatic reduction of the c-FOS protein level, aberrant AP-1 signaling, dampened self-renewal capacity, deficient cell migration that were associated with cell growth retardation in vitro and in vivo, and their enhanced sensitivity to anticancer drugs. Taken together, we resolve the long-standing controversy and uncertainty in the field, and reveal a fundamental role of OCT4A protein in regulating FOS/AP-1 signaling-centered genes that mediate the adhesion, migration, and propagation of somatic cancer cells.
Highlights
POU5F1 gene belongs to the class 5 POU (Pit-Oct-Unc) family of homeodomain transcription factors (TFs) whose transcript can generate three main isoforms by alternative splicing, namely OCT4A, OCT4B, and OCT4B11
The full-length OCT4A-1184 band variably appeared in somatic cancer cells but missed in all non-tumor samples including 293T cells (Fig. 1b, middle panel), LO2 cells, and normal human liver tissues with the exception of HUVEC cells (Supplementary Figure 1C) whose identity was subsequently confirmed by DNA sequencing (Supplementary Figure 1D, E)
The RNA-Seq data revealed that the transcript levels of other known OCT4 isoforms and major POU5F1 pseudogenes were similar to that of OCT4A in HeLa cells and the OCT4A mRNA level difference between HeLa and NCCIT was in line with that of qRT-PCR (Fig. 1e)
Summary
POU5F1 gene belongs to the class 5 POU (Pit-Oct-Unc) family of homeodomain transcription factors (TFs) whose transcript can generate three main isoforms by alternative splicing, namely OCT4A (often referred to as OCT4), OCT4B, and OCT4B11. Main caveats exist in those studies that include: the possible presence of other OCT4 isoforms and multiple POU5F1 pseudogenes that cannot be effectively distinguished by most PCR primers[20,21,22]; commercially available OCT4 antibodies cannot ensure their specific detection of OCT4A protein only[7,22,23]. Considerable efforts have been made by shRNA/siRNA approach in order to verify or validate the presence and functionality of OCT4A in somatic cancer cells[24,25]. ShRNA/siRNA approach can only provide incomplete gene silencing, leaving residual OCT4 mRNAs and proteins that may still function; it has relatively high off-target effects that cannot eliminate possible indirect contributions from reducing POU5F1 pseudogenes
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