Role of OCT4 in cancer stem-like cells and chemotherapy resistance

Abstract

Cancer stem-like cells (CSCs) contribute to the tumorigenicity, progression, and chemoresistance of cancers. It is not known whether CSCs arise from normal stem cells or if they arise from differentiated cancer cells by acquiring self-renewal features. These CSCs share stem cell markers that normal stem cells express. There is a rising interest in octamer-binding transcription factor 4 (OCT4), one of the stem cell factors that are essential in embryogenesis and pluripotency. OCT4 is also overexpressed in CSCs of various cancers. Although the majority of the studies in CSCs reported a positive association between the expression of OCT4 and chemoresistance and an inverse correlation between OCT4 and clinical prognosis, there are studies rebuking these findings, possibly due to the sparsity of stem cells within tumors and the heterogeneity of tumors. In addition, post-translational modification of OCT4 affects its activity and warrants further investigation for its association with chemoresistance and prognosis.

Introduction

The two distinctive features of normal stem cells are self-renewal and pluripotency, differentiation into cells with physiological functions [1, 2]. The evidence of cancer stem-like cells (also called as tumor-initiating cells or side population, CSCs) arose in hematologic malignancies. For example, in chronic myelogenous leukemia (CML), the DNA at the site of chromosomal translocation t(9;22)(q34;q11) showed that the site of breakage and rejoining of the translocated fragments is identical in all the leukemic cells in any given patient, but that the site differs from one patient to another [3]. This implies that CML arises from a single aberrant cell. Later studies discovered CSCs in solid tumors. In human teratocarcinomas, an experiment of nature in which differentiated tissues such as muscle and bone can appear in the tumor mass presents evidence for the existence of CSCs in solid tumors [4, 5]. In breast cancer studies, researchers found a tumorigenic stem-cell population with unique stem cell surface marker profile [6]. Also, CD133 positive stem cells with self-renewal and pluripotency have been isolated from human central nervous system tumors [7]. However, it is not known whether CSCs originate from normal stem cells, whether cancer cells acquire self-renewal, or whether it is a multi-step process of normal stem cells accumulating mutations followed by the acquisition of self-renewal capacity.

Although CSCs share markers with normal stem cells and give rise to differentiated cancer cells, the differentiated cancer cells have limited physiological functions when compared to normal stem cells. Takahashi et al. showed that octamer-binding transcription factor 4 (OCT4, also known as OCT3 or OCT¾), a homeodomain transcription factor of the POU family, is required to induce pluripotency in both human and mouse somatic cells along with c-MYC, KFL4, and SOX2 [8]. In recent years OCT4 has emerged as a master regulator of the induction and maintenance of cellular pluripotency [9]. Although studies have shown that pluripotency can also be induced involving different transcription factors or small molecules, transcription factors have been applicable in inducing pluripotency in a greater variety of cell types [8, 10]. Of these transcription factors, OCT4 is well-studied as an indispensable transcription factor that controls self-renewal and pluripotency in embryonic stem cells [11] as well as in CSCs, although no set of surface or non-surface markers thus far have been defined to be generalized in all types of CSCs. Other studies also showed that higher expression of OCT4 occurs in embryonic stem cells and CSCs [10–17], but the functional role of OCT4 in cancers other than CSCs is yet to be defined.

In addition to their tumorigenicity and generation of cellular progeny, CSCs are involved in fundamental processes of cell proliferation and metastatic dissemination [18]. Upon achieving complete remission of tumors after chemotherapy or radiation, CSCs in quiescence survive and give rise to relapse tumors that are more invasive and chemoresistant. The oncogenic role of CSCs has been reported in most types of human cancers, including breast, brain, liver, lung, gastric, colon, prostate, pancreatic, and head and neck cancers, as well as multiple myeloma, leukemia, melanoma, etc. [19]. OCT4 has been studied as one of the drivers of stemness in CSCs, and the current review provides current knowledge on the potential role of OCT4 in chemoresistance and clinical prognosis of cancer patients.

Molecular features of OCT4

OCT4 is encoded by the POU domain (Pit1, Oct1/Oct2, and Unc86), class 5 transcription factor 1 (POU5F1) gene, which is located on chromosome 6p21 and 17B1 in human and mouse genome, respectively [20–22]. The POU5F1 gene is approximately 6-kb in length and includes five exons (from E1 to E5) and four introns. There have been six POU5F1 pseudogenes identified on human chromosomes 1q22 (POU5F1P4), 3 (POU5F1P6), 8q24 (POU5F1P1 and P2), 10q21 (POU5F1P5) and 12p13 (POU5F1P3) by using a bioinformatics approach to analyze the genomic nucleotide sequences [23]. Therefore, RT-PCR analysis showed that POU5F1P1 and POU5F1P5 pseudogenes were transcribed in various cancers but not in fibroblasts, normal tissues, and embryonic carcinoma cells examined. The RNA transcription of POU5F1P1 and POU5F1P5 in different types of human cancers may play a role in the regulation of POU5F1 gene activity thus might be pertinent to carcinogenesis. Interestingly, POU5F1 remains capable of generating many RNA transcripts, including OCT4A, OCT4B variant 2, OCT4B variant 3, OCT4B variant 5, OCT4B1, OCT4B2 and OCT4B3, driven either by choosing different promoters or alternative splicing in their 5’-untranslated regions (UTRs) [24]. In addition, OCT4B was identified to act as a competing endogenous RNA (ceRNA) to modulate OCT4A expression in tumor cells [25].

POU5F1 is primarily expressed in pluripotent cells, including the inner cell mass of the mammalian blastocyst (early embryo), embryonic stem cells (ESCs), embryonal carcinoma cells, embryonic germ cells, and CSCs [26–29]. The OCT4 protein acts as a master integrator not only playing a role in development but also in pluripotency and signal-induced differentiation of ESCs where its inactivation results in apoptosis and loss of pluripotency [6–8]. The OCT4 protein comprises three distinct domains, a critical POU domain with bipartite DNA-binding structure flanked by an NH₂-terminal domain and a COOH-terminal transactivation domain [30]. The POU domain, which consists of a POU specific domain (POU_S) and a POU homeodomain (POU_HD) fused by a flexible α-helix linker [31], is responsible for specific binding to the consensus DNA sequence (octamer motif 5’-ATTTGCAT-3’) within the promoter or enhancer regions of its downstream target genes. While the POU domain residues confer OCT4 uniqueness for inducing pluripotency in ESCs it also plays a reprogramming role in both human and mouse fibroblasts into induced pluripotent stem (iPS) cells [10, 32–34]. A recent report indicates that OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells [35]. Moreover, OCT4 along with other three stemness-related transcription factors, SOX2, KLF4, and c-MYC, can form a set of reprogramming factors named OSKM Yamanaka factors or Y4 [18–20]. Notably, MYC can be omitted from reprogramming cocktails because iPS cells can also be generated in the absence of c-MYC from mouse and human fibroblasts [36, 37]. Unlike the other three Yamanaka factors, OCT4 is essential, indispensable, and non-replaceable by its family members for cell reprogramming [36, 37].

OCT4 transcriptional regulation has been extensively studied, but its post-translational regulation, including phosphorylation, ubiquitylation, sumoylation, glycosylation, and acetylation is not fully understood [29–33]. Of the post-translational modifications of OCT4, phosphorylation, in particular, can control its protein stability and activity [38, 39]. An in vitro PTM/mass spectrometry approach in a cell-free system has identified 15 OCT4 phosphorylation sites (13 in serine and 2 in threonine but no in tyrosine residue) that were commonly present in cell-free systems (293FT, NCCIT, and U87) or in a particular cellular context [40]. OCT4^T235 was reported to be phosphorylated by AKT/protein kinase B [41], OCT4^S236 (equivalent to mouse OCT4^S229) was phosphorylated by protein kinase A (PKA) [39], and a total of 5 sites (S111, S236, S289, S355, and T118) were phosphorylated by serine/threonine kinase ERK-1/2 or p38/MAPK. While several large-scale studies with phosphoproteomic approach have identified protein phosphorylation events in pluripotent stem cells, 14 phosphorylation sites on OCT4 were further confirmed (3 known and 11 new) [42–44]. Functional analyses of two highly conserved residues, T235 and S236, conducted by mutating these two sites to mimic constitutive phosphorylation (T235E and S236E) reduced transcriptional activation from an OCT4 responsive reporter and decreased reprogramming efficiency from somatic cells into iPS cells [42], suggesting that phosphorylation within the POU_HD region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Subsequent studies also showed that 3 out of 14 phosphorylation sites on OCT4, including S111, T118, and S355, were validated to be phosphorylated experimentally by ERK-2 [42]. ERK-1/2 interact with and phosphorylate OCT4^S111 to regulate OCT4 protein subcellular distribution and degradation in ESCs [45]. Furthermore, the phosphorylation regulation of OCT4 by Aurora kinase B/protein phosphatase 1 has been demonstrated important for re-setting OCT4 to pluripotency and cell cycle genes in determining the identity of ESCs [46]. Although OCT4 protein phosphorylation has been well studied in iPS cells and ECSs, limited data are available on OCT4 phosphorylation in CSCs. Such phosphoproteomic studies will enable the comparison of protein network contributing to OCT4 phosphorylation among human ESCs, iPS cells and CSCs.

OCT4 and chemotherapy resistance

One of the self-protective mechanisms of normal stem cells is quiescence, spending most of their time in G0 phase [47, 48]. CSCs that share many properties of the normal stem cells might also possess relative quiescence, leading to chemoresistance of CSCs [49]. This suggests that new modalities are needed in developing chemotherapeutic agents to target the CSC population. Another potential issue of targeting cancer cells is the heterogeneity [50]. Cancer cells are able to change their phenotype between less and more differentiated states, especially when epithelialmesenchymal transition or mesenchymal-epithelial transition occurs [51].

Chemotherapy or radiation resulted in the enrichment of CSCs in various cancers [52–55], and the CSCs, showed the changes in phenotypes as well as the increased expression of stem cell markers, including OCT4, CD133, SOX2, NANOG, and ALDH [56–59]. In glioblastoma-derived circulating tumor cells, the expression of SOX2, OCT4, and NANOG was increased, and the cells were resistant to gamma radiation and temozolomide than matched tumor cells [60]. In a study of glioblastoma cells, more cells became CD133 positive after gamma radiation [61]. In lung cancer, cells with high OCT4 expression were resistant to conventional therapy, including cisplatin, etoposide, doxorubicin, paclitaxel, and gamma radiation [62], or to targeted therapy, gefitinib [63]. Chemotherapy-induced OCT4 expression in association with drug resistance and tumor recurrence was also observed in bladder cancer [64]. Mesothelioma cells with high expression of OCT4 and SOX2 were resistant to cisplatin [65]. In breast cancer patients as both whole group and in the ER+ group but not in hormone (−) group, OCT4 expression was associated with tamoxifen resistance as well as poor clinical outcome [66]. In canine osteosarcoma cells treated with doxorubicin and cisplatin, the enrichment of CSCs with SOX2, OCT4, and CD133 was observed [67]. While many studies related CD133+ in direct association with increased stem cell marker [62, 67, 68], retinoblastoma cells with low CD133 expression showed higher stem cell genes, although the study showed that higher stem cell gene expression was associated with carboplatin [69].

Several groups exogenously expressed OCT4 to determine its effect on the resistance to chemotherapeutic drugs. PLC/PRF/5 hepatoma cells with transient OCT4 overexpression showed increased resistance to cisplatin and doxorubicin via the activation of OCT4-AKT-ATP-binding cassette G2 pathway [70], but the chemoresistance was not observed by OCT4 overexpression in a normal hepatic cell line [70]. Another study by Yin et al. demonstrated that MHCC97-L, another HCC cell line, with OCT4 and NANOG overexpression are resistant to cisplatin [71]. Although the studies provided convincing data, whether the OCT4 overexpression effect on chemoresistance applies to other types of cancer cells, but not in normal cells, requires further investigation. Melanoma cells with exogenous expression of OCT4 possessed a higher proliferate rate and were more resistant to hypoxia and cisplatin relative to control cells [72]. Also, Li et al. transfected OCT4 isoforms in cervical cancer cells and found that the cells became resistant to cisplatin [73].

In lieu of exogenous expression of OCT4, some studies knocked-down OCT4 to evaluate its effect on drug resistance. Chen et al. and Cortes-Dericks et al. demonstrated that OCT4 knock-down increases sensitivity to cisplatin and irradiation in lung cancer cells [62, 74]. Ikushima et al., in their glioma-initiating cells, showed that SOX2 and OCT4 are critical in retaining tumorigenicity, and that knock-down of OCT4 increased the sensitivity of glioma cells to temozolomide [75]. Similarly, OCT4 was increased in oral squamous cancer cells treated with cisplatin. In a mouse model of ovarian cancer, Samardzija et al. showed that OCT4 mediates metastasis and disease-free survival by knocking down OCT4 [76]. Another ovarian cancer study demonstrated that the activation of IGF-1R-Akt signaling is associated with high OCT4 and platinum-paclitaxel resistance [77]. In more recent studies, knock-down of OCT4 sensitized cells to cisplatin [78] and reduced cell proliferation and potentiated apoptosis by gefitinib, an EGFR inhibitor, in non-small cell lung cancer [79]. In reviewing OCT4’s role in chemoresistance, cisplatin was almost exclusively used in the majority of the in vitro studies. The changes in OCT4 expression with additional agents, the effect of OCT4 expression in chemoresistance measured in vivo, and potentially its effect in clinical samples obtained post-treatment need to be evaluated to generalize the information. In the context of OCT4 knock-down resulting in reduced cell proliferation of CSCs, normal stem cells are generally quiescent, spending most of their time in the G0/G1 phase of cell cycle. Therefore, if increased cell proliferation in OCT4 expressing CSCs needs additional confirmation to determine whether it could serve as a distinction between CSCs and normal stem cells.

In contrast to what has been reported thus far, OCT4 expression and chemoresistance were inversely associated in testicular germ cell tumor (TGCT), in which the exposure to cisplatin decreased OCT4 expression and increased drug resistance. Although the biology behind low OCT4 being related to chemoresistance in TGCT cells is now clear [80–82], one of the studies suggested increased sumoylation of OCT4 after cisplatin treatment as one of the mechanisms [81]. TGCT being presented as a mixture of differentiated and undifferentiated cells is one of the challenges in drawing conclusions on the role of OCT4 in chemoresistance for the specific cancer. Gidekel et al. analyzed different subtypes of TGCT and showed that the levels of OCT4 increased with the malignancy of these tumors [83].

Activators and suppressors of OCT4

As OCT4 plays an important role in inducing CSCs to cause chemoresistance, understanding the activation mechanisms of OCT4 may provide insights on how to target CSCs to enhance drug activity. One of the well-reported upstream activators of OCT4 is the WNT pathway. In embryonic stem cell studies, β-catenin/TCF transcriptional activation has been identified as OCT4 upstream activator [84]. A study previously referred as OCT4 associated chemoresistance also has shown that WNT activation induces stemness, including OCT4 increase in circulating tumor cells of glioblastoma [60]. Several groups demonstrated that WNT-Notch crosstalk is important in development as well as other diseases, including cancer [85–87]. Therefore, it is anticipated that the Notch pathway is often activated in cancers with high OCT4 expression [88]. Beta-catenin in the WNT pathway is another molecule that increases OCT4 by binding to the promoter region of the OCT4 encoding gene, POU5F1, and ATG7 has been reported as the upstream of β-catenin activation [89]. Then, OCT4 can also drive β-catenin/WNT activation via mir-1246 in HCC cells [90], indicating that OCT4 and the WNT pathway may result in a vicious cycle of activation to induce cancer stemness.

It is well-documented that hypoxia plays a role in the maintenance of CSCs via hypoxiainducible factors (HIF1-α or HIF2-α). Of the HIFs, HIF2-α activates NF-κb or other inflammatory mediators to induce stemness [88, 91–93]. In testicular germ cell tumor, overexpression of SENP1 reduced the sumoylated OCT4 level induced by SUMO1gg, active form of SUMO1, thereby regulating OCT4 levels and enhancing chemosensitivity [94]. In 11 different cell lines of various cancers, HIF induces human embryonic stem cell markers, including SOX2, OCT4, NANOG, KLF4, and c-MYC [95]. Keith and Simon’s review extensively lists the studies of HIFs activating specific signaling pathways, such as Notch and increasing the expression of transcription factors such as Oct4 that control stem cell self-renewal and multipotency [96].

The phosphoinositide 3-kinase (PI3K) pathway has been identified as one of the upstream activator of OCT4 in both embryonic stem cells as well as CSCs. Insulin-like growth factor-1R (IGF-1R) activates Akt signaling, which resulted in high OCT4 expression [77]. One of the downstream transcription factors of PI3K, FOXC1, induced stemness in non-small cell lung cancer by promoting β-catenin and the formation of tumor in xenografts [91]. In rhodamine 123-low-retaining melanoma cells with high OCT4 expression, both HIF1-α and PI3K/Akt signaling pathway genes were highly expressed [97]. Hu et al. showed that transcription factors, transcription activator-like effector protein (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas9) system activated OCT4 gene in both human and mouse somatic cells [98]. In non-small cell lung cancer, VEGFC/VEGFR3 increased TGF-β1 induced epithelial-mesenchymal transition, a process in which OCT4 is also increased [99].

Several upstream mediators have been reported to negatively regulate OCT4 expression. MyD88 has been recently reported to downregulate OCT4, NANOG, and SOX2 during retinoic acid-induced differentiation [100]. The carboxy terminus of HSP-70 interacting protein (CHIP) has been shown to be downregulated in breast cancer, and exogenously expressed CHIP decreased OCT4 [101]. Figure 1 summarizes the upstream activators and suppressors of OCT4 that have been discovered in CSCs.

Figure 1.

Activators and suppressors of OCT4 in cancer stem cells. Five representative pathways of OCT4 activation or suppression are illustrated. (1) WNT/NOTCH pathway: ATG7 activates β-catenin via an unknown mechanism in the WNT pathway, and then β-catenin binds to TCF and transcriptionally activate OCT4. (2) PI3K pathway: FOXC1 in the PI3K pathway activates β-catenin which increases gene transcription of POU5F1 and OCT4 protein in turn. (3) HIF: HIF2-α increases OCT4 expression via NF-κB activation. (4) Hypoxia: Hypoxia suppresses post-transcriptional OCT4 protein activity by inhibiting SENP1 which removes sumoylation (Su) from OCT4. Post-translational sumoylation of OCT4 reduces OCT4 protein activity. (5) Other pathways: VEGFC/VEGFR3 activates OCT4 via TGF-β1 and TALE/Cas9. ATG7: Autophagy-related protein 7, TCF: transcription factor 3, FOXC1: forkhead box protein C1, HIF2-α: hypoxia-inducible factor-alpha, NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells, SENP1: Sentrin-specific protease 1, VEGFC/VEGFR3: vascular endothelial growth factor C/ vascular endothelial growth factor receptor 3, TGF-β 1: transforming growth factor beta 1, Su: sumoylation, TALE/Cas9: transcription activator-like effector protein (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas9) system.

Drugs to overcome OCT4-induced resistance

As reviewed above, the drugs that are associated with increased OCT4 are noted in two categories, conventional chemotherapy and tyrosine kinase inhibitors. Conventional chemotherapy includes many different drugs with various mechanisms, but the studies on chemoresistance in association with OCT4 expression have been limited to only a few drugs, such as cisplatin, doxorubicin, paclitaxel, and tamoxifen. Tyrosine kinase inhibitors are another category of drugs with chemoresistance issues. Thus, it is important to understand the role of OCT4 in inducing drug resistance. Several studies have attempted to target upstream activators of OCT4. One study utilized Dickkopf-1 (DKK-1), a WNT pathway inhibitor, and L685458, an inhibitor of the Notch pathway, and showed that the combination reduced the stem phenotype of breast cancer cells in vitro and in vivo [88]. Similarly, a γ-secretase inhibitor along with sorafenib has been shown to reduce stemness assessed by OCT4 expression in vivo of hepatocellular carcinoma [102]. The decrease in OCT4, as well as other stem cell markers, by SAHA resulted in overcoming resistance in imatinib-resistant chronic myelogenous leukemia [103]. Retinoic acid, a vitamin A derivative, is used in the treatment of acute promyelocytic leukemia and high-risk neuroblastoma patients as well as in various cancer cells for its differentiation effect [100, 104–107]. All-trans retinoic acid is also shown to inhibit stemness in radioiodine-refractory thyroid tumor cells [108].

OCT4 in cancer prognosis

In various preclinical/clinical studies, OCT4 expression was shown to be associated with chemoresistance in the majority of cancers with TGCT as an exception. Thus, it is worth asking if OCT4 expression can serve as a prognostic factor in cancers. Numerous studies have analyzed the expression of OCT4 mRNA, protein, or both in relation to clinical outcome in cancers with the epithelial origin, non-epithelial origin or mixed. High OCT4 expression of either mRNA or protein was associated with poor clinical outcome in bladder cancer, ovarian cancer, prostate cancer, rectal cancer, glioma, melanoma, medulloblastoma, acute myeloid leukemia, hepatocellular carcinoma, and esophageal squamous cell carcinoma [1, 2, 21, 23–29, 72, 109–121]. Thus far, there has not been any data to dispute these findings.

In gastric cancer, an inverse correlation between the expression of OCT4 and differentiation of the tumor was observed [122]. A recent study reported that higher OCT4 expression was associated with shorter survival [123]. In a study evaluating a larger set of clinical samples, patients with positive expression of OCT4 had longer overall survival, and low OCT4 had more tumor invasion and metastasis [124]. Pancreatic cancer also showed conflicting results. A study by Wen et al. reported that precancerous lesions have higher OCT4 expression than pancreatic cancer tissues [125]. More recent studies, however, showed that high OCT4 expression was associated with worse overall survival of pancreatic cancer patients [126–128]. Oral squamous carcinoma is one of the cancers with bad clinical prognosis [129]. In evaluating the association of OCT4 expression and clinical prognosis, five of the six studies showed a positive correlation between high OCT4 and lower overall survival of oral squamous cell carcinoma [130–134]. In laboratory studies, exogenous expression of OCT4 increased tumorigenicity, and stable silencing of OCT4 resulted in tumor volume reduction [135]. However, one study compared OCT4 expression in tumor-adjacent tissues with tumor tissues and showed the tumor expression of OCT4 was lower relative to that of adjacent-tumor tissues [136].

The number of clinical studies on the relationship between OCT4 expression and outcome in breast cancer is limited. Liu et al. reported a correlation between OCT4 expression, vasculogenic mimicry formation and poor clinical prognosis in human breast cancer [137]. Preclinical studies on the correlation between the survival of breast cancer cells and the expression of OCT4 showed conflicting results. Kim et al., in their mouse model of breast cancer, showed that OCT4 expressing cells were more tumorigenic than OCT4-non-expressing cells [138]. Similarly, OCT4 knock-down resulted in more apoptosis in stem cell-like breast cancer cells in vitro and in vivo in another study by Hu et al. [139] while in another study of a breast cancer cell line, OCT4 knock-down induced EMT, cell migration, and invasion [140].

Conclusions

The abundance in data supporting the importance of CSCs in association with chemoresistance led to a rising interest in OCT4. The majority of the studies have shown positive correlations between OCT4 expression and chemoresistance. However, these findings were observed in a limited number of chemotherapy drugs, and there is enough evidence disputing these results. The conflicting reports are partly because the mechanisms of OCT4 in inducing chemoresistance are not very well delineated. Thus, studies on the downstream activation of OCT4 in conjunction with drug resistance will provide insights into a better understanding of the roles of OCT4 in chemoresistance. Another limitation of the currently available data is that these studies used mRNA and/or protein expression of OCT4 with limited information on post-translational modification of OCT4 which is required to maintain its stability and transcriptional activity. Therefore, studies correlating the expression of POU5F1 mRNA and OCT4 protein with the levels of post-transcriptional modifications of the protein may enable the use of POU5F1 mRNA and OCT4 protein as prediction markers in chemoresistance and prognosis of cancer.

Highlights.

• Overview of biological regulation of octamer-binding transcription factor 4 (OCT4), one of the stem cell factors that are essential in embryogenesis and pluripotency as well as the roles of OCT4 in pluripotency with OCT4 in cancer stem-like cells.

• Up-to-date information on the impact of OCT4 expression in chemoresistance occurring in various cancers.

• Collective data of OCT4 expression in association with clinical prognosis of various cancers and insights into the directions of future studies.