Overexpression Of Oct4 Research Articles

Investigation of specific binding of designed oligodeoxynucleotide decoys to transcription factors in HT29 cell line undergoing epithelial-mesenchymal transition (EMT).

Expression of master transcriptional regulators of stem cells (Oct4 and Sox2) is associated with mediating tumor proliferation and tumor differentiation. The main goal of this study is the investigation of specific binding of designed Oct4-Sox2 transcription factors decoy oligodeoxynucleotides (ODNs) sequence to their nucleus-extracted proteins in HT29-ShE cells containing enriched cancer stem-like cells (SCLCs). First, gene expression of Oct4, Sox2, and E-cadherin revealed the overexpression of Oct4 and Sox2 and downregulation of E-cadherin in HT29-ShE cells compared with HT29 wild-type and HT29-ShC cells. Next, Oct4-Sox2 complex decoy ODNs were designed according to their elements in the promoter region of Sox2 gene. Then, the interactions of Oct4 and Sox2 proteins to designed ODNs were evaluated in silico. Finally, DNA-protein interactions of decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Analysis of gel shift retardation assay admitted the specific binding of designed ODNs sequence to the nuclear extracted Oct4 and Sox2 proteins. The results will be a promising approach to target cancer stem cells for potential use in differentiation therapy before chemotherapy and radiotherapy of cancers.

BDNF and NT3 Reprogram Human Ectomesenchymal Dental Pulp Stem Cells to Neurogenic and Gliogenic Neural Crest Progenitors Cultured in Serum-Free Medium.

Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising types of cells to regenerate nerve tissues. Standard DMEM+10% fetal bovine serum (FBS) culture medium allows a fast expansion of hDPSC as a surface-adherent cell monolayer. However, the use of FBS also compromises the clinical use of these protocols, and its longterm presence favors hDPSCs differentiation toward mesenchymal cell-derived lineages, at the expense of a reduced capability to generate neural cells. The objective of this work was to characterize the role of neurotrophin signaling on hDPSCs using a serum-free culture protocol, and to assess the neurogenic and gliogenic capacity of hDPSCs for future nerve tissue bioengineering and regeneration. We compared the different expression of neurotrophin receptors by RT-PCR, Q-PCR, and IF of hDPSCs cultured with different growth media in the presence or absence of serum. Moreover, we assessed the response of hDPSCs to stimulation of neurotransmitter receptors by live cell calcium imaging under these different media. Finally, we compared the osteogenic potential of hDPSCs by Alizarin red staining, and the differentiation to gliogenic/neurogenic fates by immunostaining for Schwann lineage and neuronal lineage markers. We tested a commercial serum-free medium designed for the growth of mesenchymal stem cells: StemPro MSCTM (STP). hDPSCs cultured in STP generated small non-adherent floating dentospheres that showed very low proliferation rates, in contrast to standard FBS-containing medium. We found that hDPSCs grown in STP conditions overexpressed neurotrophin receptor genes NTRK2 (TrkB) and NTRK3 (TrkC). Interestingly, the stimulation of these receptors by adding their respective ligands BDNF and NT-3 to STP medium enhanced the neural crest (NC) progenitor features of cultured hDPSCs. We observed a 10 to 100-fold increase of migratory NC cell markers HNK1 and P75NTR, and a significant overexpression of pluripotency core factors SOX2, OCT4 and NANOG. Moreover, hDPSCs cultured in BDNF/NT-3 supplemented STP showed a largely increased potential to differentiate towards neuronal and Schwann glial lineage cells, assessed by positive immunostaining for DCX, NeuN and S100ß, p75NTR markers, respectively. Our results demonstrate that the use of BDNF and NT-3 combined with STP induced the partial reprogramming of ectomesenchymal hDPSCs to generate early NC progenitor cells, which are far more competent for neuronal and glial differentiation than hDPSCs grown in the presence of FBS.

ERα is required for suppressing OCT4-induced proliferation of breast cancer cells via DNMT1/ISL1/ERK axis.

ObjectivePOU5F1 (OCT4) is implicated in cancer stem cell self‐renewal. Currently, some studies have shown that OCT4 has a dual function in suppressing or promoting cancer progression. However, the precise molecular mechanism of OCT4 in breast cancer progression remains unclear.Materials and MethodsRT‐PCR and Western blot were utilized to investigate OCT4 expression in breast cancer tissues and cells. Cell proliferation assays and mouse models were applied to determine the effects of OCT4 on breast cancer cell proliferation. DNMT1 inhibitors, ChIP, CoIP, IHC and ERα inhibitors were used to explore the molecular mechanism of OCT4 in breast cancer.ResultsOCT4 was down‐regulated in breast cancer tissues, and the overexpression of OCT4 promoted MDA‐MB‐231 cell proliferation and inhibited the proliferation of MCF‐7 cells in vitro and in vivo, respectively. Two DNMT1 inhibitors (5‐aza‐dC and zebularine) suppressed OCT4‐induced MDA‐MB‐231 cell proliferation through Ras/Raf1/ERK inactivation by targeting ISL1, which is the downstream of DNMT1. In contrast, OCT4 interacted with ERα, decreased DNMT1 expression and inactivated the Ras/Raf1/ERK signalling pathway in MCF‐7 cells. Moreover, ERα inhibitor (AZD9496) reversed the suppression of OCT4‐induced proliferation in MCF‐7 cells via the activation of ERK signalling pathway.ConclusionsOCT4 is dependent on ERα to suppress the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis.

The Prognostic and Clinicopathologic Characteristics of OCT4 and Lung Cancer: A Meta-Analysis.

The relationship between OCT4 and clinicopathological features in lung cancer is shown to be controversial in recent publications. Therefore, we conducted this meta-analysis to quantitatively investigate the prognostic and clinicopathological characteristics of OCT4 in lung cancer. A comprehensive literature search of the PubMed, EMBASE, Cochrane Library, WOS, CNKI and Wanfang databases was performed to identify studies. Correlations between OCT4 expression and survival outcomes or clinicopathological features were analyzed using meta-analysis methods. Twenty-one studies with 2523 patients were included. High OCT4 expression showed a poorer overall survival (OS) (univariate: HR= 2.00, 95% CI = (1.68, 2.39), p<0.0001; multivariate: HR= 2.43, 95% CI = (1.67, 3.55), p<0.0001) and median overall survival (MSR = 0.51, 95% CI = (0.44, 0.58), p < 0.0001), disease-free survival (DFS) (HR= 2.18, 95% CI = (1.30, 3.67), p = 0.003) and poorer disease-specific survival (DSS) (HR= 2.23, 95% CI = (1.21, 4.11), p = 0.010). Furthermore, high OCT4 expression was found to be related with lower 5 year disease-specific survival rate (OR= 0.24, 95% CI = (0.14, 0.41), p<0.0001) and 10 year overall survival rate (OR= 0.22, 95% CI = (0.12, 0.40), p=0.0001). Additionally, OCT4-high expression was also strongly associated with higher clinical TNM stage, lymph node metastasis, tumor distant metastasis, higher histopathologic grade, but not related with gender, smoking status, tumor size and histologic type of lung cancer. OCT4 over-expression in lung cancer was strongly related to poorer clinicopathological features and worse survival outcomes, which suggests that OCT4 could be a valuable prognostic marker in lung cancer.

Clinicopathological and Prognostic Significance of Octamer-Binding Transcription Factor 4 in Patients with Gastric Cancer: A Systematic Review and Meta-Analysis

The role of octamer-binding transcription factor 4 (Oct4) in gastric cancer (GC) progression is still under debate and reported results are inconsistent. Therefore, we conducted a meta-analysis to evaluate the clinicopathological and prognostic significance of Oct4 expression in patients with GC. Relevant articles were retrieved from a diverse number of databases, and meta-analysis was completed using STATA software 12.0. Total of 21studies were included in this analysis (3209 samples). Expression of Oct4 was associated with incidence, tumor size, lymph node metastasis, histological differentiation, pTNM stage, tumor depth of infiltration, vascular invasion and distal metastasis. Additionally, Oct4 expression was correlated with poor overall survival rate. The Oct4 overexpression suggested aggressive biological behaviors and imply that Oct4 may be a useful prognostic biomarker in gastric cancers.

OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases

BackgroundSelf-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved.MethodsA lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCsOCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected.ResultsThe overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCsOCT4 and hHFMSCsEGFP, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCsOCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCsOCT4, and triggered cell senescence.ConclusionsOCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Attempting to Convert Primed Porcine Embryonic Stem Cells into a Naive State Through the Overexpression of Reprogramming Factors.

Establishing pig embryonic stem cells (pESCs) remains a challenge due to differences in the genetic backgrounds of mouse, human, and pig. Therefore, pig-specific pluripotency markers and cellular signaling must be identified. In this study, doxycycline (DOX)-inducible vectors carrying Oct4, sex-determining region Y-box 2 (Sox2), Nanog, Kruppel-like family 4 (Klf4), or Myc, which are known reprogramming factors, were transduced into pESCs. And pluripotency genes were analyzed in one or two reprogramming factor-expressed pESCs. When cultured without DOX, pESCs were stably maintained in basic fibroblast growth factor-supplemented media. However, when treated with DOX, the cells lost their alkaline phosphatase (AP) activity and differentiated within 2 weeks. Subsequently, we investigated the expression of genes related to pluripotency in DOX-treated pESCs using quantitative reverse transcription-polymerase chain reaction (PCR). Expression levels of Oct4, E-cadherin, and Fut4 were significantly increased by Oct4 overexpression, and Oct4 and Fut4 were upregulated in the Sox2-infected group. When a combination of two reprogramming factors, including Oct4 or Sox2, was introduced, weak AP activity remained. In addition, several of the two reprogramming factor transduction groups could be maintained after subculturing with transgene activation. Although long-term culture failed, pESCs transduced with Oct4 and Nanog, Oct4 and Klf4, or Sox2 and Nanog combinations could be subcultured even under transgene activation conditions. Analysis of the cause of long-term culture failure by quantitative PCR confirmed that the expression of intermediate reprogramming markers was not maintained. Given these results, additional methods are needed to support the completion of each reprogramming phase to succeed in the conversion of the pluripotent state of pESCs. This study improves our understanding of pluripotent networks and can be used to aid in the establishment of bona fide pig pluripotent stem cells.

Reprogramming of Extraembryonic Trophoblast Stem Cells into Embryonic Pluripotent State by Fusion with Embryonic Stem Cells.

Pluripotential reprogramming has been examined using various technologies, including nuclear transfer, cell fusion, and direct reprogramming. Many studies have used differentiated cells for reprogramming experiments, and nearly all type of somatic cells can acquire pluripotency. However, within the embryo, other cells types are present in addition to somatic cells. The blastocyst stage embryo consists of two main types of cells, inner cell mass and trophectoderm (TE). TE cells are the first differentiated form of the totipotent zygote and differ from epiblast cells. Thus, we examined whether extraembryonic cells can be reprogrammed using a cell-cell fusion method. Trophoblast stem cells (TSCs), which can be obtained from the TE, are known to acquire pluripotency by transcription factor Oct4 overexpression or somatic cell nuclear transfer. In this study, we demonstrated that TSCs can acquire pluripotent properties by cell fusion with embryonic stem cells (ESCs). TSC-ESC hybrids reactivated Oct4-GFP and displayed self-renewal properties. They expressed the pluripotency markers Oct4 and Nanog, whereas the expression of Cdx2 and Tead4, trophoblast lineage markers, was diminished. Moreover, these cells developed into three germ layers similarly to other pluripotent stem cells. RNA-seq analysis showed that global gene expression patterns of TSC-ESC hybrids are more similar to ESCs than TSCs. Thus, we demonstrated that TSCs successfully complete reprogramming and acquire pluripotency by cell fusion-induced reprogramming.

VPA selectively regulates pluripotency gene expression on donor cell and improve SCNT embryo development.

SCNT technology has been successfully used to clone a variety of mammals, but the cloning efficiency is very low. This low efficiency is likely due to the incomplete reprogramming of SCNT embryos. Histone modification and DNA methylation may participate in these events. Thus, it would be interesting to attempt to improve the efficiency of SCNT by using a HDACi VPA. In order to guarantee the effect of VPA and reduce its cytotoxicity, a comprehensive analysis of the cell proliferation and histone modification was performed. The results showed that 0.5 and 1mM VPA treatment for 24h were the optimal condition. According to the results, H3K4me3 was increased in 0.5 and 1mM VPA groups, whereas H3K9me2 was significantly decreased. These are the signals of gene-activation. In addition, VPA treatment led to the overexpression of Oct4 and Nanog. These indicated that VPA-treated cells had similar patterns of histone to zygotic embryos, and may be more favorable for reprograming. A total of 833 cloned embryos were produced from the experimental replicates of VPA-treated donor cells. In 1mM treatment group, the blastocyst rates were significantly increased compared with control. At the same time, our findings demonstrated the interrelation between DNA methylation and histone modifications.

MicroRNA-299-3p regulates proliferation, migration and invasion of human ovarian cancer cells by modulating the expression of OCT4

Ovarian cancer is among the most prevalent and lethal types of cancers in women. Several factors such as late diagnosis, unavailability of the reliable biomarkers, frequent relapses and dearth of efficient therapeutic targets form bottleneck in the treatment of ovarian cancers. In this study we investigated the potential of less studied miR-299-3p as the therapeutic target for the treatment of ovarian cancer. The results of the present investigation revealed that miR-299 is significantly upregulated in the ovarian cancers and suppression of its expression inhibits the proliferation by induction of apoptosis as well suppresses migration and invasion of the SKOV3 cancers cells. Further, OCT-4 was found to be putative target of miR-99-3p in ovarian cancer and inhibition of OCT-4 had similar effects as that of miR-299 inhibition on cell migration and invasion. Intriguingly, even overexpression of miR-299-3p could not rescue the effects of OCT-4 suppression on SKOV3 cell proliferation, migration and invasion. On contrary, overexpression of OCT-4 in SKOV3 cells transfected with miR-299-3p transfected could nullify the effects of miR-200-3p on proliferation, migration and invasion of the SKOV3 cells. Taken together, miR-299-3p regulated cell proliferation and metastasis by modulating the expression of OCT-4 and as such may prove to be an important therapeutic target.

Control of PD-L1 expression by miR-140/142/340/383 and oncogenic activation of the OCT4\u2013miR-18a pathway in cervical cancer

PD-L1, a key inhibitory immune receptor, has crucial functions in cancer immune evasion, but whether PD-L1 promotes the malignant properties of cervical cancer (CC) cells and the mechanism by which PD-L1 is regulated in CC remains unclear. We report that PD-L1 is overexpressed in CC, and shRNA-mediated PD-L1 depletion suppresses the proliferation, invasion, and tumorigenesis of CC cells. Loss of miR-140/142/340/383 contributes to PD-L1 upregulation. miR-18a enhances PD-L1 levels by targeting PTEN, WNK2 (ERK1/2 pathway inhibitor), and SOX6 (Wnt/β-catenin pathway inhibitor and p53 pathway activator) to activate the PI3K/AKT, MEK/ERK, and Wnt/β-catenin pathways and inhibit the p53 pathway, and miR-18a also directly suppresses the expression of the tumor suppressors BTG3 and RBSP3 (CTDSPL). miR-18a overexpression in CC cells is triggered by OCT4 overexpression. Our data implicate PD-L1 as a novel oncoprotein and indicate that miR-140/142/340/383 and miR-18a are key upstream regulators of PD-L1 and potential targets for CC treatment.

Suppression of dsRNA response genes and innate immunity following Oct4, Stella, and Nanos2 overexpression in mouse embryonic fibroblasts

The self-renewal capacity of germline derived stem cells (GSCs) makes them an ideal source for research and use in clinics. Despite the presence of active gene network similarities between embryonic stem cells (ESCs) and GSCs, there are unanswered questions regarding the roles of evolutionary conserved genes in GSCs. To determine the reprogramming potential of germ cell- specific genes, we designed a polycistronic gene cassette expressing Stella, Oct4 and Nanos2 in a lentiviral-based vector. Deep transcriptome analysis showed the activation of a set of pluripotency and germ-cell-specific markers and the downregulation of innate immune system. The global shut down of antiviral genes included MHC class I, interferon response genes and dsRNA 2′-5′-oligoadenylate synthetase are critical pathways that has been affected . Individual expression of each factor highlighted suppressive effect of Nanos2 on genes such as Isg15 and Oasl2. Collectively, to our knowledge this is the first report showing that Nanos2 could be considered as an immunosuppressive factor. Furthermore, our results demonstrate suppression of endogenous retrotransposons that harbor immune response but further analysis require to uncover the correlation between transposon suppression and immune response in germ cell development.

Post-translational modification of OCT4 in breast cancer tumorigenesis

Recurrence and drug resistance of breast cancer are still the main reasons for breast cancer-associated deaths. Cancer stem cell (CSC) model has been proposed as a hypothesis for the lethality of breast cancer. Molecular mechanisms underlying CSC maintenance are still unclear. In this study, we generated mammospheres derived from breast cancer MDA-MB231 cells and MCF7 cells to enrich CSCs and performed DNA microarray analysis. We found that the expression of carboxy terminus of HSP70-interacting protein (CHIP) E3 ubiquitin ligase was significantly downregulated in breast CSCs. CHIP depletion increased mammosphere formation, whereas CHIP overexpression reversed this effect. We identified interactomes by mass spectrometry and detected CHIP directly interacted with OCT4, a stemness factor. CHIP overexpression decreased OCT4 stability through proteasomal degradation. CHIP induced OCT4 ubiquitination, whereas H260Q, a catalytic CHIP mutant, did not. Interestingly, we determined that OCT4 was ubiquitinated at lysine 284, and CHIP overexpression did not degrade K284R mutant OCT4. CHIP overexpression decreased the proliferation and side population of breast cancer cells, but these were not occurred in K284R mutant OCT4 overexpressed cells. Only 1000 cells showing CHIP depletion or OCT4 overexpression sufficiently generated breast tumors and lung metastases in xenografted mice. Ubiquitination-defective mutant of OCT4(K284R) overexpressed cells drastically generated tumor burdens in mice. Patients with breast cancer who showed low CHIP expression had poor survival probability. Taken together, we suggest that CHIP-induced OCT4 ubiquitination is important in breast CSCs. Regulation of CHIP expression and OCT4 protein stability is a considerable approach for breast cancer therapy.

Reciprocity of Action of Increasing Oct4 and Repressing p53 in Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiac Myocytes.

p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53+/+), p53 heterozygous mutant (p53+/-), and p53 homozygous mutant (p53-/-) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53+/- and p53-/- MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53+/- and p53-/- MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53+/+ and p53+/- MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53+/- MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.

MicroRNA‑32 silences WWP2 expression to maintain the pluripotency of human amniotic epithelial stem cells and β islet‑like cell differentiation.

Human amniotic epithelial stem cells (HuAECs) exhibit pluripotent characteristics, which are similar to those of embryonic stem cells, and can differentiate into various adult tissues and cells through directed induction. However, in culture, HuAECs tend to lose their pluripotency, and their directed differentiation capability declines with increasing passage number. The stem cell pluripotency factor octamer-binding protein 4 (Oct4) is an important transcription factor that promotes stem cell self-proliferation and maintains their pluripotency. Previous studies have demonstrated that WW domain containing E3 ubiquitin protein ligase 2 (WWP2) negatively regulates Oct4 expression and stem cell pluripotency. Therefore, the present study aimed to investigate the regulation of WWP2 by microRNAs (miRs), and to evaluate the expression of the downstream factor Oct4 and the maintenance of HuAEC pluripotency. Bioinformatics analysis identified a complementary binding site for miR-32 in the 3′untranslated region of the WWP2 gene, thus suggesting that it may be a target gene of miR-32. Post-infection of HuAECs with a vector overexpressing miR-32, the endogenous expression of WWP2 was significantly decreased, whereas Oct4 expression was significantly increased. Furthermore, miR-32-infected cells differentiated into β islet-like cells by directed induction. The results indicated that after induction, HuAECs overexpressing miR-32 also overexpressed the biomarkers of β islet-like cells. In addition, the ability to secrete insulin was markedly enhanced in response to glucose stimulation, in cells overexpressing miR-32. In conclusion, the present study suggested that miR-32 may effectively inhibit WWP2 expression in HuAECs and promote Oct4 overexpression to maintain their pluripotency.

The expression of the embryonic gene Cripto-1 is regulated by OCT4 in human embryonal carcinoma NCCIT cells.

Cripto-1 and OCT4, expressed in stem cells and cancers, play important roles in tumorigenesis. Here, we demonstrate that Cripto-1 expression is regulated by OCT4 in human embryonic carcinoma NCCIT cells. The endogenous expression of Cripto-1 and OCT4 is significantly reduced during differentiation. Cripto-1 expression is increased by OCT4 overexpression, but decreased by shRNA-mediated OCT4 knockdown. OCT4 overexpression significantly activates Cripto-1 transcriptional activity. A 5'-upstream minimal promoter sequence in the gene-encoding Cripto-1 is significantly activated by OCT4 overexpression. Mutation of the putative OCT4-binding site abolishes OCT4-mediated activation of the Cripto-1 promoter. The OCT4 transactivation domains mediate transcriptional activity of the Cripto-1 minimal promoter through direct interaction. Taken together, OCT4 plays an important role as a transcriptional activator of Cripto-1 expression in NCCIT cells.

Overexpression of OCT4 induced by modulation of histone marks plays crucial role in breast cancer progression

OCT4 is known as the gatekeeper of pluripotent embryonic state as it is responsible for maintenance of pluripotency via self-renewal of embryonic stem cells and acquisition of induced pluripotency via somatic cell reprogramming. OCT4 is responsible for oncogenic transformation by disrupting pre-scheduled differentiation programs and in general, favoring evolution of cancer cells into a more aggressive cancer stem cell phenotype. In this study, we have investigated in details, the epigenetic regulatory mechanisms responsible for over-expression and subsequent aberrant function of OCT4 in breast cancer. Expression of OCT4 in breast cancer tissues and cell lines was determined by qRT-PCR, Western blot, immunohistochemistry and immunocytochemistry followed by investigation of pro-tumorigenic properties such as cell proliferation, migration and apoptosis by gene knockdown and treatment with epigenetic modulators. Ectopic treatment of epigenetic modulators was done followed by MS-PCR and chromatin immunoprecipitation. OCT4 is over-expressed in both of the breast cancer cell lines; MCF7 and MDA-MB-231 and its inhibition resulted in drastic decrease in rate of cell proliferation, metastatic ability and induced apoptosis. After treatment with epigenetic modulators, general increase in expression of OCT4 was observed at both gene and protein levels; however, changes of promoter DNA methylation was not found to be significant during OCT4 over-expression. Active histone marks especially H3K4me3 and H3K9acS10p were enriched in the promoter region with very low levels of repressive marks H3K9me3 and H3K27me3 indicating that active histone modifications are the deciding factor in inducing over-expression of OCT4 during breast carcinogenesis. These findings could provide the basis on which epigenetic therapy, targeting reversible epigenetic modifications and their perpetuating enzymes, can be designed for effective treatment of aggressive breast cancer.

OCT4 supports extended LIF-independent self-renewal and maintenance of transcriptional and epigenetic networks in embryonic stem cells

Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.

Abstract B19: The role of ABCB1 genotypes and targeting cancer stem cells in pancreatic cancer: Effects of metformin and dietary agents

Abstract Pancreatic cancer carries a poor prognosis and survival rate. Developing effective agents or repurposing agents with low toxicity, such as metformin, an insulin-lowering drug, has recently been investigated for pancreatic cancer. Metformin has long been associated with decreased cancer risk, particularly in diabetic patients. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately leads to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3, 15 and 17. Treatment of cells with the dietary agent, indole-3-carbinol alone at 100 or 200 µM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. Expression levels of drug transporters in pancreatic tissue can also affect potential treatment of pancreatic cancer. High level of expression of ABCB1 causes multidrug-resistance. Metformin modulated the ATP-binding cassette gene, ABCB1, expression in pancreatic cancer cell lines with different ABCB1 genotypes. Metformin (100 µM or 200 µM) alone (p=0.0212 or p=0.0161) or in combination with gemcitabine (15nM) (p=0.0248 or p=0.0174) significantly decreased ABCB1 expression in MIAPaCa2 pancreatic cells after 24 hr or 72 hr treatments. This correlated to increased cell death. Metformin in combination with indole-3-carbinol also significantly (p=0.0317) decreased ABCB1 expression. However, a sex or genotype difference was noted when two female pancreatic cell lines, SU86.86 and AsPC1, were used. Metformin did not down-regulate ABCB1 expression and was chemoresistance in these two cell lines. AsPC1 and SU86.86 both carry the 2677GG-3435CC ABCB1 haplotype compared to MiaPaca-2, which carries the 2677TT-3134TT ABCB1 haplotype. The 2677TT and 3435TT genotypes/haplotypes are known to be associated with lower risk of developing pancreatic cancer. These data demonstrated that these genotypes are also more sensitive to metformin treatment compared to the 2677GG or 3435CC genotypes. Further research is needed to ascertain whether sex or genotype is contributing to different efficacy of metformin in pancreatic cancer cells with different genotypes. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells. Furthermore, downregulation of ABCB1 expression varied with treatment with specificity to certain genotypes. Citation Format: Beverly D. Lyn-Cook, Beverly Word, George Hammons. The role of ABCB1 genotypes and targeting cancer stem cells in pancreatic cancer: Effects of metformin and dietary agents [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B19.

Induction of reprogramming of human amniotic epithelial cells into iPS cells by overexpression of Yap, Oct4, and Sox2 through the activation of the Hippo-Yap pathway

The present study has reported a novel method for producing induced pluripotent stem (iPS) cells. Primary human amniotic epithelial cells (HuAECs) were isolated from the amniotic membranes of pregnant women who received Cesarean sections. These cells were infected with retroviruses carrying octamer-binding transcription factor 4 (Oct4), (sex determining region Y)-box 2 (Sox2) and Yes-associated protein (Yap) (OSY). Following in vitro culture for ~14 days, epithelial-like HuAECs exhibited several iPS clone-like cell colonies (OSY-iPS). These cell clones presented positive alkaline phosphatase features and expressed high levels of embryonic stem cell-like markers (Nanog homeobox, Sox2, Oct4, reduced expression protein 1, and SSES3/4). Additionally, epigenetic analysis results indicated that the methylation of CpG islands on endogenous Oct4 and Sox2 promoters was reduced in OSY-iPS cells. Furthermore, the majority of the histone H3 at lysine 9 sites that interacted with the Oct4 and Sox2 promoters were acetylated, suggesting that the transcription activities of the above two transcription factors significantly increased. In vivo and in vitro induced differentiation experiments demonstrated that OSY-iPS could develop into embryoid bodies in vitro, and express numerous cellular markers in the three germ layers. Furthermore, OSY-iPS could form teratomas in immunodeficient mice. The pathological detection results suggest that these teratomas contain numerous types of cells from the three germ layers. However, the results from the quantitative polymerase chain reaction and western blot analyses suggest that the Hippo-Yap signaling pathway was significantly activated in OSY-iPS cells. In conclusion, a novel method for iPS induction was established in the present study. HuAECs were successfully induced to reprogram iPS cells through the introduction of OSY to activate the Hippo-Yap signaling pathway.