As a member of the inhibitors of apoptosis protein (IAPs) family, BIRC5 is preferentially expressed in human cancer cells and has multiple functions, including the inhibition of cell apoptosis , control of the cell cycle [15, 16], promotion of tumor angiogenesis [17, 18], resistance to chemotherapy or radiotherapy , acceleration of metastasis and recurrence [20, 21], and regulation of cancer cell autophagy , all of which favour cancer cell survival and tumor maintenance. Therefore, multiple strategies have been employed to target BIRC5 for cancer therapy by silencing BIRC5 expression with small interfering RNA  or antisense oligonucleotides , inhibiting the BIRC5 promoter activity with small-molecule antagonists , and interfering BIRC5 function with dominant-negative mutant forms of the protein . Some of these strategies are being applied in clinical trials at various phases, and the initial results are promising when combined with other treatments, such as chemotherapy or radiotherapy [16, 27]. Although certain strategies for cancer therapy targeting BIRC5 have shown a varied extent of antitumor efficacy, the potential benefit of single anti-BIRC5 treatment in different types of cancers is uncertain. Although the down-regulation of BIRC5 expression by anti-BIRC5 agents can inhibit the growth of cancer, tumors consistently obtain growth capabilities in later stages, demonstrating that this treatment approach remains poorly characterized and requires further study.
BIRC5 expression is precisely regulated at transcriptional and post-translational levels. The signal transducer and activator of transcription 3 (Stat-3), β-catenin-activated T-cell factor (TCF) transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α) and Sp1 transcription factor promote BIRC5 expression by increasing BIRC5 promoter activity [28–31]. Sp1-mediated BIRC5 expression can be suppressed by p53 . The stability of BIRC5 protein represents another potential method of regulating BIRC5 function. BIRC5 protein is phosphorylated at Thr34 by cdc2 kinase, which prevents BIRC5 proteosome-mediated clearance or degradation . Recently, OCT4 was reported to have a regulatory effect on BIRC5 expression .
OCT4 belongs to the family of POU-domain transcription factors, which are involved in the regulation of cell growth and differentiation in a variety of tissues [11, 34]. Many studies have shown that OCT4 expression is restricted to germline and pregastrulation embryos and also to embryonal carcinomas and testicular germ cell tumors , but not expressed in mature somatic cells. Further evidence has shown that some cancer cells, such as breast, bladder, prostate, liver, head and neck squamous cell cancer and non-small cell lung cancers, are positive for OCT4 expression [7–9, 35–39]. Therefore, OCT4 acts as a multifunctional factor not only in stem cells but also in many cancers, and the expression of OCT4 causes more malignant histological phenotypes, including rapid progression, great metastasis, and short cancer-related survival. However, one study unexpectedly found that adult human peripheral blood mononuclear cells, which are genetically stable and mainly terminally differentiated cells with a limited lifespan, express OCT4; this finding challenges the paradigm of OCT4 as a marker of pure stem cells and provides novel insight into the role of OCT4 in fully differentiated cells . OCT4 functions by directly or indirectly activating a series of downstream target genes. By characterizing the genes in OCT4-mediated regulatory networks, it has been found that many candidate target genes that are directly regulated by OCT4 have an OCT4-binding octamer motif . However, a large number of target genes, such as BIRC5 , have no OCT4 motifs and might be indirectly regulated by OCT4. Therefore, transcriptional regulation of target genes by OCT4 is very complicated, and it is necessary to understand the key gene network that maintains cell pluripotency in embryo development and governs cell differentiation and proliferation in cancer progression.
To clarify the relationship between OCT4 and BIRC5 in HCC, we first analyzed the OCT4 and BIRC5 expression levels in HCC cell lines, including Hep3B, HepG2, PLC/PRF5, SMMC-7721, BEL-7402 and BEL-7404. All cell lines were positive for BIRC5 expression, although only the Hep3B, HepG2 and PLC/PRF5 cells were positive for OCT4 expression; SMMC-7721 cells were weakly positive for OCT4 expression. OCT4 and BIRC5 expression was also investigated by immunohistochemistry in 49 pairs of cancer and liver tissues taken from HCC patients. They were overexpressed in HCC compared with the corresponding liver tissues (Additional file 2: Table S1). BIRC5 immunostaining was mainly localized in cancer cell cytoplasm and nuclei, and OCT4 expression was localized in cancer cell nuclei (Additional file 3: Figure S1).
We found that the expression levels of OCT4 in HCC cell lines were consistent with the percentages of CD133-positive cells, suggesting that OCT4 expression might be related to CD133 expression. By manipulating the expression of OCT4 and BIRC5, we found that BIRC5 expression silencing did not influence OCT4 expression in Hep3B and BEL-7404 cells. However, down-regulation of OCT4 expression inhibited BIRC5 expression, even in the OCT4-negative BEL-7404 cells, and increasing OCT4 expression by infection with adenovirus carrying the OCT4 gene in BEL-7404 cells up-regulated BIRC5 expression. In exploring the superior-subordinate relationship between BIRC5 and OCT4, we found that the relative activity of the BIRC5 promoter in HCC cells was controlled by OCT4. These results seemingly demonstrated that BIRC5 is a downstream target gene of OCT4.
Functional binding sites for the transcription factors SP1, KLF5, HIF-1α, Rb/E2F, TCF4 and Egr1 have been found in the BIRC5 gene promoter, suggesting that these factors regulate BIRC5 gene expression . However, a binding site for OCT4 is not found in the BIRC5 promoter region, suggesting that OCT4 may indirectly regulate BIRC5 expression [13, 40]. In addition to the Rb suppressor and E2F activators (i.e., E2F1, E2F2 and E2F3) that bind directly to the BIRC5 promoter and regulate BIRC5 transcription , the regulatory proteins CDK4, SKP2, Rad51, BRCA2, E2F-DP1, CCND1, Stat3, Rb and p21 can activate the SP1 promoter , which then indirectly leads to an increase in BIRC5 expression. These factors are all involved in cell cycle regulation. In addition, OCT4 modulates the cell cycle by up-regulating CDKN1B, CDKN1C, CDK6 and MAPK4 . Coincidentally, by screening the binding sites in the promoter regions of these cell cycle regulators, we found an octamer motif for OCT4 at −252 to −245 in the CCND1 proximal promoter. Further studies have confirmed that CCND1 expression and promoter activity is strictly correlated with OCT4 expression levels in OCT4-positive Hep3B cells. When the OCT4 motif in the CCND1 promoter was mutated or modified with a PORE motif that could bind two OCT4 molecules, the promoter activity was suppressed or enhanced, respectively. We also observed high CCND1 promoter activity in OCT4-negative BEL-7404 cells. These results suggested that the OCT4 motif might participate in the regulation of CCND1 promoter activity, and that there are other factors that regulate CCND1 promoter activity in HCC cells.
In the in vitro experiments, silencing of BIRC5 expression effectively induced apoptosis and cell cycle arrest in HCC cells, thereby inhibiting cancer cell proliferation and decreasing cancer cell viability. Co-suppression of OCT4 and BIRC5 further enhanced the inhibitory effect on cancer cell proliferation. In the in vivo experiments, BIRC5-shRNA expression inhibited the growth of HCC xenograft tumors by inducing cell apoptosis, although tumor growth was restored in the late stage after the adenovirus injections ceased. The Dual-shRNA that targeted both OCT4 and BIRC5 inhibited tumor growth with great efficiency for a long period of time. These results showed that OCT4 and BIRC5 collusively educe cell proliferation. Clinical follow-up information also demonstrated that the HCC patients who showed co-expression of OCT4 and BIRC5 in cancer tissues had poorer disease-free survival (DFS) and overall survival (OS) than patients who were negative for both OCT4 and BIRC5 (Additional file 4: Figure S2).