Oct4 Transcription Research Articles

5-Desmethylsinensetin isolated from Artemisia princeps suppresses the stemness of breast cancer cells via Stat3/IL-6 and Stat3/YAP1 signaling

AimsTo study 5-desmethylsinensetin exhibiting potential anticancer activity against breast cancer stem cells and the related molecular mechanism. Main methodsIn this study, isolation of a cancer stem cell (CSC) inhibitor of Artemisia princeps was performed using a silica gel column, a Sephadex gel column, and high-performance liquid chromatography. A single compound was purified via activity-based isolation using mammosphere formation assays. An MTS was used to examine the proliferation of breast cancer cells, and flow cytometry was used to analyze apoptosis and cancer stem cell markers. Western blotting was used to detect the signaling pathway. ResultsThe isolated compound was identified as 5-desmethylsinensetin using nuclear magnetic resonance and mass spectrometry. 5-Desmethylsinensetin suppresses the proliferation and mammosphere formation of breast cancer cells, reduces the subpopulations of CD44+/CD24- and ALDH1+ cancer cells, and reduces the transcription of the stemness markers Oct4, c-Myc, Nanog and CD44 in Breast CSCs. 5-Desmethylsinensetin inhibits the total and nuclear expression of Stat3 and p-Stat3, as well as the translocation of YAP1. Additionally, 5-desmethylsinensetin reduces the mRNA and protein levels of IL-6. ConclusionOur results show that 5-desmethylsinensetin exhibits potential anticancer activity against breast cancer stem cells via Stat3-IL-6 and Stat3-YAP1 signaling.

Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells.

Embryonic stem cell (ESC) self-renewal and cell fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during early stem cell differentiation. Notably, many RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly targeted by this factor. Using cross-linking and immunoprecipitation (CLIP-seq), we find that the pluripotency-associated STAT3 and OCT4 transcription factors interact with RNA in hESCs and confirm the binding of STAT3 to the conserved NORAD long-noncoding RNA. Our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated.

Development, characterization, and hematopoietic differentiation of Griscelli syndrome type 2 induced pluripotent stem cells

BackgroundGriscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2.MethodsHere, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media.ResultsAll GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage.ConclusionsUsing the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.

Identification of Suitable Housekeeping Genes for Quantitative Gene Expression Analysis During Retinoic Acid-induced Differentiation of Embryonal Carcinoma NCCIT Cells

Real-time quantitative PCR (qRT-PCR) is often used as an effective experimental method for analyzing gene expression. In this method, normalization of target gene expression levels must be performed using housekeeping genes (HKGs). HKGs are used to compensate for difference between samples due to diverse quality and quality of RNAs and different reverse transcription yield. For an ideal reference gene, constant expression levels across different samples of one experiment is necessary. In the current study, expression stability of four candidate references genes including Beta actin (ACTB), glyceraldeyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase (HPRT1) and Beta-2-Microglobulin (β2M) following retinoic acid (RA) treatment in embryonal carcinoma NCCIT cells were evaluated.NCCIT cells were exposed to RA (10 µM) for 14 days to induce differentiation. RT-qPCR for candidate references genes was performed and normalization between untreated and RA-treated cells was performed using identical sample input amounts. Expression of OCT4, SOX2, NANOG during RA-induced differentiation was assessed by quantitative real-time PCR. RT-qPCR results indicated significant difference in expression level of GAPDH between untreated (Ct mean: 19.36667± 0.28) and RA-treated (Ct mean: 28.94± 0.18) NCCIT cells. However, transcriptional level of ACTB, HPRT and β2M remained unchanged after RA treatment. qRT-PCR analysis using ACTB, HPRT and β2M showed treatment of NCCIT cells with RA lead to significant down regulation of OCT4 (79%), NANOG (71%) and SOX2 (96%) transcript. ACTB, HPRT and β2M were recognized as valid reference genes for analysis of gene expression during RA-induced differentiation of NCCIT cells, while GAPDH was not suitable.

ESE3/EHF, a promising target of rosiglitazone, suppresses pancreatic cancer stemness by downregulating CXCR4

Background and aimsThe crosstalk between cancer stem cells (CSCs) and their niche is required for the maintenance of stem cell-like phenotypes of CSCs. Here, we identified E26 transformation-specific homologous factor...

Embryonic Stem Cell-like Population in Hypertrophic Port-wine Stain

Objective: To identify and characterize an embryonic stem cell (ESC)-like population within hypertrophic port-wine stain (HPWS). Methods: HPWS tissue samples from 15 patients underwent immunohistochemical staining for induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC. Immunofluorescence staining was performed on 2 of these tissue samples to investigate colocalization of these markers. In situ hybridization and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were performed on 6 of the HPWS samples to investigate transcript expression of these iPSC markers. Western blotting and RT-qPCR were performed on 3 HPWS-derived primary cell lines, to determine protein and transcript expression of these markers, respectively. Results: Immunohistochemical staining demonstrated expression of OCT4, SOX2, KLF4, and c-MYC but not NANOG on the endothelium and media of lesional vessels and on cells within the stroma in all 15 HPWS tissue samples. Immunofluorescence staining showed the presence of an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like subpopulation within the endothelium and media of the lesional vessels, and cells within the stroma of HPWS. In situ hybridization detected OCT4, SOX2, KLF4, and c-MYC transcripts in all 6 HPWS tissue samples. RT-qPCR demonstrated transcripts of all 5 iPSC markers in the HPWS tissue samples and in the HPWS-derived primary cell lines, which expressed OCT4, SOX2, KLF4, and c-MYC but not NANOG proteins by Western blotting. Conclusion: This study demonstrated an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like population within the endothelium and media of the lesional vessels and the cells within the stroma of HPWS.

An Overview on the Complexity of OCT4: at the Level of DNA, RNA and Protein

OCT4 plays critical roles in self-renewal and pluripotency maintenance of embryonic stem cells, and is considered as one of the main stemness markers. It also has pivotal roles in early stages of embryonic development. Most studies on OCT4 have focused on the expression and function of OCT4A, which is the biggest isoform of OCT4 known so far. Recently, many studies have shown that OCT4 has various transcript variants, protein isoforms, as well as pseudogenes. Distinguishing the expression and function of these variants and isoforms is a big challenge in expression profiling studies of OCT4. Understanding how OCT4 is functioning in different contexts, depends on knowing of where and when each of OCT4 transcripts, isoforms and pseudogenes are expressed. Here, we review OCT4 known transcripts, isoforms and pseudogenes, as well as its interactions with other proteins, and emphasize the importance of discriminating each of them in order to understand the exact function of OCT4 in stem cells, normal development and development of diseases.

Maternal and zygotic activin signaling promotes adequate pattern and differentiation of mesoderm through regulation of pluripotency genes during zebrafish development.

To investigate the role of maternal Activin-like factors in the preservation of stemness and mesendoderm induction, their effects were promoted and inhibited using synthetic human Activin A or SB-505124 treatments, respectively, before the maternal to zygotic transition (MZT). To study the role of zygotic Activin-like factors, SB-505124 treatment was also used after the MZT. Promoting the signaling intensity of maternal Activin-like factors led to premature differentiation, loss of stemness, and no mesendoderm malformation, while its alleviation delayed the differentiation and caused various malformations. Inhibition of the zygotic Activin-like factors was associated with suppressing the ndr1, ndr2, oct4 (pou5f3), mycb and notail transcription as well as differentiation retardation at the oblong stage, and a broad spectrum of anomalies in a dose-dependent manner. Together, promoting the signal intensity of maternal Activin-like factors drove development along with mesendodermal differentiation, while suppression of the maternal or zygotic ones maintained the pluripotent state and delayed differentiation.

Ubiquitin specific protease 7 maintains pluripotency of mouse embryonic stem cells through stabilization of β-catenin.

Embryonic stem cells (ESCs), which are derived from the undifferentiated inner cell mass of the embryo, can differentiate every cell type of the body regarding their pluripotency. Therefore, human or mouse ESCs can be used as an unlimited cell source for numerous researches or therapeutical approaches. However, pluripotency maintenance of ESCs during in vitro culture is challenging because of their endless differentiation capacity. In the current study, the effect of USP7 on pluripotency maintenance of mouse ESCs (mESCs) has been investigated with the help of cell viability assay, morphological analysis, alkaline phosphatase (ALP) staining, qPCR analysis, and Western Blotting. 600 nM P5091 application, which showed no significant toxicity in mESCs, increased the total ubiquitinated protein amount as a proof of the accomplishment of proper USP7 inhibition. Morphological analysis and ALP activity evaluation indicated that dual inhibition of GSK3 and MEK together with leukemia inhibitory factor (LIF) treatment protects the pluripotency in presence of active USP7 enzyme. Yet, inactivation of USP7 reduced the ALP activity and altered the cell morphology in each treatment group. This morphological change and decreased ALP activity refer to differentiated mESCs. These findings were supported by gene expression and protein analysis. Gene expressions and protein amounts of pluripotency related Oct4, Nanog, c-Myc, Sox2 and Klf4 transcription factors decreased significantly after USP7 inhibition. Together with this observation, a remarkable reduction in β-Catenin expression was also noticed. It was also observed that USP7 inactivation shortens the half-live of β-Catenin and GSK3β proteins. This study demonstrates that USP7 activation is crucial for proper pluripotency maintenance, which is provided through β-Catenin stabilization.

Generation of neural progenitor cells from porcine-induced pluripotent stem cells.

In this study, porcine embryonic fibroblasts (pEFs) were reprogrammed into porcine-induced pluripotent stem cells (piPSCs) using either human or mouse specific sequences for the OCT4, SOX2, c-Myc, and KLF4 transcription factors. In total, three pEFs lines were reprogrammed, cultured for at least 15 passages, and characterized regarding their pluripotency status (alkaline phosphatase expression, embryoid body formation, expression of exogenous and endogenous genes, and immunofluorescence). Two piPSC lines were further differentiated, using chemical inhibitors, into putative neural progenitor-like (NPC-like) cells with subsequent analyses of their morphology and expression of neural markers such as NESTIN and GFAP as well as immunofluorescent labeling of NESTIN, β-TUBULIN III, and VIMENTIN. NPC-like cells were positive for all the neural markers tested. These results evidence of the generation of porcine NPC-like cells after in vitro induction with chemical inhibitors, representing an adequate model for future regenerative and translational medicine research.

Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs.

Simple SummaryA non-integrating and self-replicating Venezuelan equine encephalitis RNA replicon system can potentially make a great contribution to the generation of clinically applicable canine induced pluripotent stem cells. Our study shows a new method to utilize the synthetic RNA-based approach for canine somatic cell reprogramming regarding transfection and reprogramming efficiency.Canine induced pluripotent stem cells (ciPSCs) can provide great potential for regenerative veterinary medicine. Several reports have described the generation of canine somatic cell-derived iPSCs; however, none have described the canine somatic cell reprogramming using a non-integrating and self-replicating RNA transfection method. The purpose of this study was to investigate the optimal strategy using this approach and characterize the transition stage of ciPSCs. In this study, fibroblasts obtained from a 13-year-old dog were reprogrammed using a non-integrating Venezuelan equine encephalitis (VEE) RNA virus replicon, which has four reprogramming factors (collectively referred to as T7-VEE-OKS-iG and comprised of hOct4, hKlf4, hSox2, and hGlis1) and co-transfected with the T7-VEE-OKS-iG RNA and B18R mRNA for 4 h. One day after the final transfection, the cells were selected with puromycin (0.5 µg/mL) until day 10. After about 25 days, putative ciPSC colonies were identified showing TRA-1-60 expression and alkaline phosphatase activity. To determine the optimal culture conditions, the basic fibroblast growth factor in the culture medium was replaced with a modified medium supplemented with murine leukemia inhibitory factor (mLIF) and two kinase inhibitors (2i), PD0325901(MEK1/2 inhibitor) and CHIR99021 (GSK3β inhibitor). The derived colonies showed resemblance to naïve iPSCs in their morphology (dome-shaped) and are dependent on mLIF and 2i condition to maintain an undifferentiated phenotype. The expression of endogenous pluripotency markers such as Oct4, Nanog, and Rex1 transcripts were confirmed, suggesting that induced ciPSCs were in the late intermediate stage of reprogramming. In conclusion, the non-integrating and self-replicating VEE RNA replicon system can potentially make a great contribution to the generation of clinically applicable ciPSCs, and the findings of this study suggest a new method to utilize the VEE RNA approach for canine somatic cell reprogramming.

Improvement of in vitro proliferation of cockerel spermatogonial stem cells using different combinations of growth factors

ABSTRACT 1. This study examined whether in vitro proliferation and maintenance of cockerel spermatogonial stem cells (SSCs) could be improved by adding different combinations of growth factors (GFs), including glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) or leukaemia inhibitory factor (LIF) into the culture medium. 2. The SSCs were isolated from the testes of immature cockerels. For short-term cultures, a medium supplemented with different combinations of GFs for 7 d in 5 replicates was used. The groups were classified as follows: without GF (control group); with GDNF (G group); with GDNF and bFGF (GF group); and with GDNF, bFGF and LIF (GFL group). The number of colonies and cells per colony, as well as the transcript abundance of STRA8 and OCT4 genes, was determined 7 d after the initial culturing. Immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, besides periodic acid-Schiff (PAS) staining, was carried out. 3. The number of colonies and cells per colony increased in the G, GF and GFL groups, compared to the control group (P < 0.01); however, the highest proliferation and colony formation were observed in the GFL group. The positive immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, as well as PAS staining, confirmed the self-renewal and colonisation of cockerel SSCs. The proliferation results were supported by the increased STRA8 and OCT4 transcript abundance in the treated groups (G, GF and GLF), compared to the control group. The SSC proliferation was associated with the higher transcript abundance of STAR8 and OCT4 genes in the GFL group, compared to the G and GF groups (P < 0.01). 4. The results showed that proliferation and colony-forming capacity of cockerel SSCs were positively improved by GDNF, bFGF and LIF. However, the most significant effect was observed when the medium was supplemented with LIF in combination with GDNF and bFGF.

A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

Deregulation of Stemness-Related Genes in Endometriotic Mesenchymal Stem Cells: Further Evidence for Self-Renewal/Differentiation Imbalance

Background:Any irregularities in self-renewal/differentiation balance in endometriotic MSCs can change their fate and function, resulting in endometriosis development. This study aimed to evaluate the expression of OCT4 transcripts (OCT4A, OCT4B, and OCT4B1), SOX2, and NANOG in endometriotic MSCs to show their aberrant expression and to support self-renewal/differentiation imbalance in these cells. Methods:MSCs were isolated from three endometriotic and three normal endometrium samples and characterized and analyzed for the expressions of OCT4A, OCT4B, OCT4B1, SOX2, and NANOG using the qRT-PCR. Results:The expressions of OCT4 transcripts and NANOG increased significantly in endometriotic MSCs, whereas SOX2 expression did not show any significant difference. Conclusion:Our findings provide further evidence for confirming the self-renewal/ differentiation imbalance in endometriotic MSCs, as the main underlying cause of endometriosis development. This study also paves the way for further research on endometriosis treatment by focusing on endometriotic stem cells.

Regulation of β-catenin by IGFBP2 and its cytoplasmic actions in glioma

IGFBP2 is one of the highly expressed genes in glioblastoma (GBM). It has both IGF dependent and independent activities. IGF independent actions are mediated by the activation of integrin signalling through its RGD motif present at C-terminal domain. One of the actions of IGFBP2 is to regulate β-catenin by the inactivation of GSK3β, which preferentially accumulates in the cytoplasm. The mechanism of nuclear β-catenin regulation by IGFBP2 and role of cytoplasmic β-catenin is not clear. We aimed to understand the mechanism in GBM cell lines. The gene expression studies were performed by RT-PCR, western blot analysis; the knockdown of genes was performed by shRNA transfection; RNAIP and luciferase reporter assays were utilized to study the cytoplasmic regulation of genes by β-catenin; neurosphere assays were performed to study the stemness of cells. IGFBP2 overexpression or treatment in GBM cells regulates β-catenin, TRIM33 (E3 ubiquitin ligase) and Oct4 genes. TRIM33 was induced by IGFBP2. β-catenin was found to accumulate predominantly in the cytoplasm and nuclear β-catenin was depleted by IGFBP2 induced TRIM33. IGFBP2 regulated cytoplasmic β-catenin binds to 3' UTR of Oct4 RNA. IGFBP2 was also able to induce stemness of glioma cells. IGFBP2 induces TRIM33 which regulates the nuclear β-catenin protein. In addition, IGFBP2 stabilizes the cytoplasmic β-catenin which is involved in the regulation of Oct4 transcript and consequently induction of stemness of glioma cells.

Model-based analysis of chromatin interactions from dCas9-Based CAPTURE-3C-seq.

Deciphering long-range chromatin interactions is critical for understanding temporal and tissue-specific gene expression regulated by cis- and trans-acting factors. By combining the chromosome conformation capture (3C) and biotinylated dCas9 system, we previously established a method CAPTURE-3C-seq to unbiasedly identify high-resolution and locus-specific long-range DNA interactions. Here we present the statistical model and a flexible pipeline, C3S, for analysing CAPTURE-3C-seq or similar experimental data from raw sequencing reads to significantly interacting chromatin loci. C3S provides all steps for data processing, quality control and result illustration. It can automatically define the bin size based on the binding peak of the dCas9-targeted regions. Furthermore, it supports the analysis of intra- and inter-chromosomal interactions for different mammalian cell types. We successfully applied C3S across multiple datasets in human K562 cells and mouse embryonic stem cells (mESC) for detecting known and new chromatin interactions at multiple scales. Integrative and topological analysis of the interacted loci at the human β-globin gene cluster provides new insights into mechanisms in developmental gene regulation and network structure in local chromosomal architecture. Furthermore, computational results in mESCs reveal a role for chromatin interacting loops between enhancers and promoters in regulating alternative transcripts of the pluripotency gene OCT4.

Differential impacts of carp and salmon pituitary extracts on induced oogenesis, egg quality, molecular ontogeny and embryonic developmental competence in European eel.

Low egg quality and embryonic survival are critical challenges in aquaculture, where assisted reproduction procedures and other factors may impact egg quality. This includes European eel (Anguilla anguilla), where pituitary extract from carp (CPE) or salmon (SPE) is applied to override a dopaminergic inhibition of the neuroendocrine system, preventing gonadotropin secretion and gonadal development. The present study used either CPE or SPE to induce vitellogenesis in female European eel and compared impacts on egg quality and offspring developmental competence with emphasis on the maternal-to-zygotic transition (MZT). Females treated with SPE produced significantly higher proportions of floating eggs with fewer cleavage abnormalities and higher embryonic survival. These findings related successful embryogenesis to higher abundance of mRNA transcripts of genes involved in cell adhesion, activation of MZT, and immune response (dcbld1, epcam, oct4, igm) throughout embryonic development. The abundance of mRNA transcripts of cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 was relatively stable during the first eight hours, followed by a drop during MZT and low levels thereafter, indicating transfer and subsequent clearance of maternal mRNA. mRNA abundance of zar1, epcam, and dicer1 was associated with cleavage abnormalities, while mRNA abundance of zar1, sox2, foxr1, cldnd, phb2, neurod4, and neurog1 (before MZT) was associated with subsequent embryonic survival. In a second pattern, low initial mRNA abundance with an increase during MZT and higher levels persisting thereafter indicating the activation of zygotic transcription. mRNA abundance of ccna1, npm2, oct4, neurod4, and neurog1 during later embryonic development was associated with hatch success. A deviating pattern was observed for dcbld1, which mRNA levels followed the maternal-effect gene pattern but only for embryos from SPE treated females. Together, the differences in offspring production and performance reported in this study show that PE composition impacts egg quality and embryogenesis and in particular, the transition from initial maternal transcripts to zygotic transcription.

Prognostic value of OCT4A and SPP1C transcript variant co-expression in early-stage lung adenocarcinoma

BackgroundOctamer-binding transcription factor 4A (OCT4A) is essential for cell pluripotency and reprogramming both in humans and mice. To date, however, the function of human OCT4 in somatic and/or tumour tissues is largely unknown.MethodsRT-PCR was used to identify full-length splice forms of OCT4 transcripts in normal and cancer cells. A FLAG-tagged OCT4 genomic transgene was used to identify OCT4-positive cancer cells. A potential role for OCT4 in somatic cancer cells was examined by cell ablation of OCT4-positive cells using promoter-driven diphtheria toxin A. OCT4 and secreted phosphoprotein 1 (SPP1) transcripts in early-stage lung adenocarcinoma tumours were analysed and compared with pathohistological features.ResultsThe results show that, unlike in murine cells, OCT4A and OCT4B variants are transcribed in both human cancer cells and in adult tissues such as lung, kidney, uterus, breast, and eye. We found that OCT4A and SPP1C are co-expressed in highly aggressive human breast, endometrial, and lung adenocarcinoma cell lines, but not in mesothelial tumour cell lines. Ablation of OCT4-positive cells in lung adenocarcinoma cells significantly decreased cell migration and SPP1C mRNA levels. The OCT4A/SPP1C axis was found in primary, early-stage, lung adenocarcinoma tumours.ConclusionsCo-expression of OCT4 and SPP1 may correlate with cancer aggressiveness, and the OCT4A/SPP1C axis may help identify early-stage high-risk patients with lung adenocarcinoma. Contrary to the case in mice, our data strongly suggest a critical role for OCT4A and SPP1C in the development and progression of human epithelial cancers.

Human papillomavirus E7 binds Oct4 and regulates its activity in HPV-associated cervical cancers.

Octamer binding transcription factor-4 (Oct4), is highly expressed in stem cells and has indispensable roles in pluripotency and cellular reprogramming. In contrast to other factors used for cellular reprogramming, a role for Oct4 outside embryonic stem cells has been elusive and highly controversial. Emerging evidence implicates Oct4 in the carcinogenic process, but the mechanism through which Oct4 may be functioning in cancers is not fully appreciated. Here, we provide evidence that Oct4 is expressed in human cervical cancer and this expression correlates with the presence of the human papillomavirus (HPV) oncogenes E6 and E7. Surprisingly, the viral oncogenes can complement exogenously provided Oct4 in reprogramming assays, providing functional validation for their ability to activate Oct4 transcription in Mouse Embryonic Fibroblasts (MEFs). To interrogate potential roles of Oct4 in cervical cancers we knocked-down Oct4 in HPV(+) (HeLa & CaSki) and HPV(-) (C33A) cervical cancer cell lines and found that Oct4 knockdown attenuated clonogenesis, only in the HPV(+) cells. More unexpectedly, cell proliferation and migration, were differentially affected in HPV(+) and HPV(-) cell lines. We provide evidence that Oct4 interacts with HPV E7 specifically at the CR3 region of the E7 protein and that introduction of the HPV oncogenes in C33A cells and human immortalised keratinocytes generates Oct4-associated transcriptional and phenotypic patterns, which mimic those seen in HPV(+) cells. We propose that a physical interaction of Oct4 with E7 regulates its activity in HPV(+) cervical cancers in a manner not seen in other cancer types.

Nonenzymatic function of Aldolase A downregulates miR-145 to promote the Oct4/DUSP4/TRAF4 axis and the acquisition of lung cancer stemness

Drug resistance remains a serious issue of clinical importance and is a consequence of cancer stemness. In this study, we showed that the level of Aldolase A (ALDOA) expression is significantly associated with the IC50 value of chemotherapy drugs in lung cancer. Our data revealed that ALDOA overexpression resulted in a significant increase of lung tumor spheres. The use of ingenuity pathway analysis (IPA) resulted in the identification of POU5F1 (Oct4) as the leading transcription factor of ALDOA. We observed high expression of ALDOA, Oct4 and stemness markers in collected spheroid cells. DUSP4 and TRAF4 were confirmed as major downstream targets of the ALDOA-Oct4 axis. Knockdown of these molecules significantly decreased the stemness ability of cells. In addition, we investigated whether miR-145 targets the 3′-UTR of Oct4 and is regulated by ALDOA due to the involvement of ALDOA in glycolysis and metabolic reprogramming. Furthermore, we constructed several mutant forms of ALDOA that disrupted its enzymatic activity and showed that they still induced significant in vitro sphere formation and in vivo tumorigenicity. These results demonstrated that ALDOA-mediated spheroid formation is independent of its enzymatic activity. In the clinical component, we also showed that the combination of ALDOA and TRAF4 or DUSP4 is positively correlated with poor overall survival in a xenograft model and cancer patients through immunohistochemical analyses. The results of our study revealed novel functional roles of ALDOA in inducing cancer stemness via the inhibition of miR-145 expression and the activation of Oct4 transcription. These findings offer new therapeutic strategies for modulation of lung cancer stemness to enhance chemotherapeutic responses in lung cancer patients.