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Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that harbors therapy-resistant cancer stem cells (CSCs). Due to the limited effectiveness of conventional chemotherapies and radiation treatments against CSCs, there is a critical need for the development of innovative therapeutic approaches. Our previous research revealed the significant expression of embryonic stemness genes, NANOG and OCT4, in CSCs, suggesting their role in enhancing cancer-specific stemness and drug resistance. In our current study, we employed RNA interference (RNAi) to suppress the expression of these genes and observed an increased susceptibility of CSCs to the anticancer drug, temozolomide (TMZ). Suppression of NANOG expression induced cell cycle arrest in CSCs, specifically in the G0 phase, and it concomitantly decreased the expression of PDK1. Since PDK1 activates the PI3K/AKT pathway to promote cell proliferation and survival, our findings suggest that NANOG contributes to chemotherapy resistance in CSCs through PI3K/AKT pathway activation. Therefore, the combination of TMZ treatment with RNAi targeting NANOG holds promise as a therapeutic strategy for GBM.

Cell identity is the control of gene transcription by Transcription Factors (TF) and chromatin. The extent to which human cell identity can be recapitulated outside of the cell using TFs and nucleosomes in unknown. I investigate if hESC-like nucleosome positioning, MNase-sensitivity and OCT4 and SOX2 (OS) binding can be recapitulated in vitro. Nucleosomes reconstitute well into cell-like positions upon human DNA sequences in vitro as determined by MNase-seq. These in vitro nucleosomes display fragility, but these nucleosomes are not fragile in hESCs. A sub-population of 110bp nucleosomes are reconstituted, which demonstrate similar MNase-resistance as the 150bp population of nucleosomes. The addition of OS to the reconstituted nucleosomes reveals SOX2-lead binding to MNase-sensitive 110bp nucleosomes, recapitulating OS binding to MNase-sensitive nucleosomes in hESCs. However, binding locations and hESC motifs are not recapitulated in vitro. These results contribute to the understanding of human nucleosome properties, and pioneer factor targeting of chromatin.

Dichlorodiphenyltrichloroethane (DDT) is the most widespread persistent pollutant with endocrine-disrupting properties. DDT has been shown to disrupt secretory and morphogenetic processes in the adrenal cortex. The present investigation aimed to evaluate transcriptional regulation of postnatal growth of the adrenal medulla and formation of the pools necessary for self-renewal of medullary cells in rats that developed under low-dose exposure to DDT. The study was performed using male Wistar rats exposed to low doses of o,p’-DDT during prenatal and postnatal development. Light microscopy and histomorphometry revealed diminished medulla growth in the DDT-exposed rats. Evaluation of Ki-67 expression in chromaffin cells found later activation of proliferation indicative of retarded growth of the adrenal medulla. All DDT-exposed rats exhibited a gradual decrease in tyrosine hydroxylase production by adrenal chromaffin cells. Immunohistochemical evaluation of nuclear β-catenin, transcription factor Oct4, and ligand of sonic hedgehog revealed increased expression of all factors after termination of growth in the control rats. The DDT-exposed rats demonstrated diminished increases in Oct4 and sonic hedgehog expression and lower levels of canonical Wnt signaling activation. Thus, developmental exposure to the endocrine disruptor o,p’-DDT alters the transcriptional regulation of morphogenetic processes in the adrenal medulla and evokes a slowdown in its growth and in the formation of a reserve pool of cells capable of dedifferentiation and proliferation that maintain cellular homeostasis in adult adrenals.

Background The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. Results We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. Conclusions Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.

Pluripotent stem cells could self-renew and differentiate into various cells. In particular, porcine pluripotent stem cells are useful for preclinical therapy, transgenic animals, and agricultural usage. These stem cells have naïve and primed pluripotent states. Naïve pluripotent stem cells represented by mouse embryonic stem cells form chimeras after blastocyst injection. Primed pluripotent stem cells represented by mouse epiblast stem cells and human embryonic stem cells. They could not produce chimeras after blastocyst injection. Populations of embryonic stem cells are not homogenous; therefore, reporter systems are used to clarify the status of stem cells and to isolate the cells. For this reason, studies of the OCT4 reporter system have been conducted for decades. This review will discuss the naïve and primed pluripotent states and recent progress in the development of porcine OCT4 reporter systems.

OCT4 is a key mediator of induced pluripotent stem cell (iPSC) reprogramming, but the mechanistic insights into the role of exogenous OCT4 and timelines that initiate pluripotency remain to be resolved. Here, using measles reprogramming vectors, we present microRNA (miRNA) targeting of exogenous OCT4 to shut down its expression during the mesenchymal to the epithelial transition phase of reprogramming. We showed that exogenous OCT4 is required only for the initiation of reprogramming and is dispensable for the maturation stage. However, the continuous expression of SOX2, KLF4, and c-MYC is necessary for the maturation stage of the iPSC. Additionally, we demonstrate a novel application of miRNA targeting in a viral vector to contextually control the vector/transgene, ultimately leading to an improved reprogramming efficiency. This novel approach could be applied to other systems for improving the efficiency of vector-induced processes., Graphical abstract, Devaux and colleagues develop a novel miRNA-targeted measles vector that contextually suppress exogenous OCT4 during mesenchymal to epithelial transition (MET) and improve human reprogramming efficiency and kinetics. They show that while SOX2, KLF4, and cMYC are required for both the initiation and maturation stage of reprogramming, OCT4 is essential only for the initiation stage.

The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

Background A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter. Results We identify Platr10 as an Oct4 - Sox2 binding lncRNA that is activated in somatic cell reprogramming. Platr10 is essential for the maintenance of pluripotency, and lack of this lncRNA causes stem cells to exit from pluripotency. In fibroblasts, ectopically expressed Platr10 functions in trans to activate core stem cell factor genes and enhance pluripotent reprogramming. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we show that Platr10 interacts with multiple pluripotency-associated genes, including Oct4, Sox2, Klf4, and c-Myc, which have been extensively used to reprogram somatic cells. Mechanistically, we demonstrate that Platr10 helps orchestrate intrachromosomal promoter-enhancer looping and recruits TET1, the enzyme that actively induces DNA demethylation for the initiation of pluripotency. We further show that Platr10 contains an Oct4 binding element that interacts with the Oct4 promoter and a TET1-binding element that recruits TET1. Mutation of either of these two elements abolishes Platr10 activity. Conclusion These data suggest that Platr10 functions as a novel chromatin RNA molecule to control pluripotency in trans by modulating chromatin architecture and regulating DNA methylation in the core stem cell factor network.

Background Aberrant expression of stem cell markers has been observed in several types of neoplasms. This trait attributes to the acquired stem-like property of tumor cells and can impact patient prognosis. The objective of this study was to comparatively analyze the expression and significance of SOX2 and OCT4 in various types of odontogenic cysts and tumors. Methods Fifty-five cases of odontogenic cysts and tumors, including 15 ameloblastomas (AM), 5 adenomatoid odontogenic tumors (AOT), 5 ameloblastic fibromas (AF), 5 calcifying odontogenic cysts (COC), 10 dentigerous cysts (DC) and 15 odontogenic keratocysts (OKC) were investigated for the expression of SOX2 and OCT4 immunohistochemically. Results Most OKCs (86.7 %) and all AFs expressed SOX2 in more than 50 % of epithelial cells. Its immunoreactivity was moderate-to-strong in all epithelial cell types in both lesions. In contrast, SOX2 expression was undetectable in AOTs and limited to the ameloblast-like cells in a minority of AM and COC cases. Most DCs showed positive staining in less than 25 % of cystic epithelium. Significantly greater SOX2 expression was noted in OKC compared with DC or AM, and in AF compared with COC or AOT. OCT4 rarely expressed in odontogenic lesions with the immunoreactivity being mild and present exclusively in OKCs. Conclusions SOX2 is differentially expressed in odontogenic cysts and tumors. This could be related to their diverse cells of origin or stages of histogenesis. The overexpression of SOX2 and OCT4 in OKC indicates the acquired stem-like property. Future studies should investigate whether the overexpression of OCT4 and SOX2 contributes to the aggressive behaviors of the tumors.

Transcription factors Sox2 and Oct4 are essential in maintaining the pluripotency of embryonic stem cells and conferring stemness in cancer stem-like (CSL) cells. SORE6, an in-vitro reporter system, was designed to quantify the transcription activity of Sox2/Oct4 and identify CSL cells in non-hematologic cancers. Using SORE6, we identified and enriched CSL cells in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). Two ALK + ALCL cell lines, SupM2 and UCONN-L2, contained approximately 20% of SORE6+ cells, which were purified based on their expression of green fluorescent protein. We then performed functional studies using single-cell clones derived from SORE6− and SORE6+ cells. Compared to SORE6− cells, SORE6+ cells were significantly more chemoresistant and clonogenic in colony-formation assays. Sox2/Oct4 are directly involved in conferring these CSL properties, since the shRNA knockdown of Sox2 in SORE6+ significantly lowered their chemoresistance, while enforced expression of Sox2/Oct4 in SORE6− cells produced opposite effects. Using Western blots, we found that the expression and subcellular localization of Sox2/Oct4 were similar between SORE6− and SORE6+ cells. However, in SORE6+ but not SORE6− cells, Sox2 and Oct4 abundantly bound to a probe containing the SORE6 consensus sequence. c-Myc, previously shown to regulate cancer stemness in ALK + ALCL, regulated the SORE6 activity. In conclusion, SORE6 is useful in identifying/enriching CSL cells in ALK + ALCL.

Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability.Chow diet-fed atherosclerosis-prone ApoeWe show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs.Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.

Summary Polycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages., Graphical Abstract, Highlights • RBBP4 deficiency in ESCs leads to spontaneous differentiation into mesendodermal lineages • Rbbp4 binding sites in ESCs substantially overlap with PRC2 binding • Rbbp4 is absolutely essential for PRC2 chromatin occupancy • Rbbp4 is necessary for sustaining the expression levels of Oct4 and Sox2, Polycomb group (PcG) proteins play a critical role in silencing developmental genes, yet the underlying mechanisms remain elusive. Yikai et al. report that PcG protein Rbbp4 represses developmental gene expression by recruiting PRC2 to the genes' promoter regions, thereby maintaining stem cell self-renewal and identity.

Transcription-induced chimeric RNAs are an emerging area of research into molecular signatures for disease biomarker and therapeutic target development. Despite their importance, little is known for chimeric RNAs-relevant roles and the underlying mechanisms for cancer pathogenesis and progression. Here we describe a unique ASTN2-PAPPA(antisense) chimeric RNA (A-P-as chiRNA) that could be the first reported chimeric RNA derived from the splicing of exons and intron antisense of two neighboring genes, respectively. Aberrant A-P-as chiRNA level in ESCC tissues was associated with tumor progression and patients' outcome. In vitro and in vivo studies demonstrated that A-P-as chiRNA aggravated ESCC metastasis and enhanced stemness through modulating OCT4. Mechanistic studies demonstrated that ERK5-mediated non-canonical PAF1 activity was required for A-P-as chiRNA-induced cancer malignancy. The study defined an undocumented function of chimeric RNAs in aggravating cancer stemness and metastasis.

The overexpression of stem cell-related genes such as octamer-binding transcription factor 4 (OCT4) and (sex determining region Y)-box 2 (SOX2) has been indicated to play several critical roles in stem cell self-renewal; moreover, the elevation of the self-renewal of cancer cells with stem cell-like properties has been suggested. The clustered and regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) protein fused to transactivation domains can be used to activate gene expression in human cells. CRISPR-mediated activation (CRISPRa) systems represent an effective genome editing tool for highly specific gene activation in which a nuclease-deficient Cas9 (dCas9) is utilized to target a transcriptional activator to the gene’s regulatory element, such as a promoter and enhancer. The main drawback of typical delivery methods for CRISPR/Cas9 components is their low transfection efficiency or toxic effects on cells; thus, we generated superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) to improve the delivery of CRISPR/Cas9 constructs into human foreskin fibroblast cells. The delivery system with magnetic PEI-coated nanoparticles complex was applied to constitute plasmid DNA lipoplexes. CRISPRa systems were used to overexpress the endogenous OCT4 and SOX2 in fibroblast cells. The quantitative polymerase chain reaction (QPCR) assessment exhibited a three-times higher expression of OCT4 and SOX2 transfected by CRISPRa using MNPs. Moreover, no additional cytotoxicity was observed with the application of magnetic nanoparticles (MNPs) compared to lipofectamine. Our results demonstrate that MNPs enable the effective delivery of the CRISPR/Cas9 construct into human foreskin fibroblasts with low cell toxicity and a consequential overexpression of endogenous OCT4 and SOX2.

In contrast to the case in mammals, the fish optic nerve can spontaneously regenerate and visual function can be fully restored 3–4 months after optic nerve injury (ONI). However, the regenerative mechanism behind this has remained unknown. This long process is reminiscent of the normal development of the visual system from immature neural cells to mature neurons. Here, we focused on the expression of three Yamanaka factors (Oct4, Sox2, and Klf4: OSK), which are well-known inducers of induced pluripotent stem (iPS) cells in the zebrafish retina after ONI. mRNA expression of OSK was rapidly induced in the retinal ganglion cells (RGCs) 1–3 h after ONI. Heat shock factor 1 (HSF1) mRNA was most rapidly induced in the RGCs at 0.5 h. The activation of OSK mRNA was completely suppressed by the intraocular injection of HSF1 morpholino prior to ONI. Furthermore, the chromatin immunoprecipitation assay showed the enrichment of OSK genomic DNA bound to HSF1. The present study clearly showed that the rapid activation of Yamanaka factors in the zebrafish retina was regulated by HSF1, and this sequential activation of HSF1 and OSK might provide a key to unlocking the regenerative mechanism of injured RGCs in fish.

Objective Retinal degenerative diseases remain the dominant causes of blindness worldwide, and cell replacement is viewed as a promising therapeutic direction. However, the resources of seed cells are hard to obtain. To further explore this therapeutic approach, human embryonic stem extracellular vesicles (hESEVs) were extracted from human embryonic stem cells (hESCs) to inspect its effect and the possible mechanism on retinal Müller cells and retinal function. Methods hESEVs were extracted by multi-step differential centrifugation, whose morphologies and specific biomarkers (TSG101, CD9, CD63, and CD81) were observed and measured. After hESEVs were injected into the vitreous cavity of RCS rats, the retinal tissues and retinal functions of rats were assessed. The alteration of Müller cells and retinal progenitor cells was also recorded. Microvesicles (MVs) or exosomes (EXOs) were extracted from hESCs transfected with sh-HSP90 or pcDNA3.1-HSP9, and then incubated with Müller cells to measure the uptake of EVs, MVs, or EXOs in Müller cells by immunofluorescence. The retrodifferentiation of Müller cells was determined by measuring Vimentin and CHX10. qRT-PCR and western blot were used to detect HSP90 expression in MVs and evaluate Oct4 level in Müller cells, and Co-IP to inspect the interaction of HSP90 and Oct4. Results RCS rats at the postnatal 30 days had increased retinal progenitor cells which were dedifferentiated from Müller cells. hESEVs were successfully extracted from hESCs, evidenced by morphology observation and positive expressions of specific biomarkers (TSG101, CD9, CD63, and CD81). hESEVs promoted Müller cells dedifferentiated and retrodifferentiated into retinal progenitor cells evidenced by the existence of a large amount of CHX10-positive cells in the retinal inner layer of RCS rats in response to hESEV injection. The promotive role of hESEVs was exerted by MVs demonstrated by elevated fluorescence intensity of CHX10 and suppressed Vimentin fluorescence intensity in MVs rather than in EXOs. HSP90 in MVs inhibited the retrodifferentiation of Müller cells and suppressed the expression level of Oct4 in Müller cells. Co-IP revealed that HSP90 can target Oct4 in Müller cells. Conclusion hESEVs could promote the retrodifferentiation of Müller cells into retinal progenitor cells by regulating the expression of Oct4 in Müller cells by HSP90 mediation in MVs.

Objective The aim of this project is to identify the differences in expression levels of stem cell related genes (SCRGs) in normal colon tissue, histopathologically staged colon polyps and colon cancer, and to explain the role of SCRGs in the formation of CC and for contributing the practical usage of SCRGs in the diagnosis and treatment of CC. Methods Normal colon tissue, hyperplastic polyps, histopathologically (HGD and LGD) staged tubular, tubulovillous and villous polyps and colon cancer paraffin tissue (FFPE) samples were used. Transcription factor genes (OCT4, KLF4, SOX2, MYC, NANOG, and REX1) and cell surface markers (CD133, LGR5), which are associated with embryonic stem cells, induced pluripotent stem cells, and cancer stem cells, have been selected for measuring expression levels from the selected tissues. After isolation of total RNA from FFPE tissues, SCRGs expressions were measured by RT-qPCR method. Results SCRGs expression differences were detected in normal-adenoma-cancer progression. A significant increase was observed in the expression of LGR5 (p: 0.01) and PROM1 (p: 0.005) genes in villous HGD polyps, LGR5 (p: 0.003) gene in G1, and LGR5 (p: 0.0002) and MYC (p: 0.002) genes in G2 stage tumor tissues. When compared with each other, a significant increase in SCRGs expression is noticeable in the formation from adenoma to cancer tissues regarding malign phenotype. Conclusion This study shows that the increase of SCRGs expressions occurs with high-grade dysplasia (HGD), villous features, and the malignant phenotype. Increased expression levels of LGR5, PROM1, KLF4, SOX2, and MYC in HGD and cancerous tissues support the malignant phenotype and the existence of cancer stem cells and demonstrate that they can be used to assess diagnosis and prognosis. Identification of tissue-specific SCRGs expressions will help design new therapies to control the development and progression of colonic neoplasia.

The Role and Specific Mechanism of OCT4 in Cancer Stem CellsRecently, evidences show that cancer stem cells (CSCs) are a type of cancer cell group with self-renewal and play a huge role in tumor recurrence, metastasis, and drug resistance. Finding new treatment directions and targets for cancer prognosis and reducing mortality has become a top priority. OCT4, as a transcription factor, participates in maintaining the stem characteristics of CSCs, but the mechanism of OCT4 is often overlooked. In this review, we try to illustrate the mechanism by which OCT4 plays a role in CSCs from the perspective of genetic modification of OCT4, non-coding RNA, complexes and signaling pathways associated with OCT4. Our ultimate goal is to provide new targets for cancer treatment to prolong the survival of cancer patients.

Liver cancer stem cells (LCSCs) are a small subset of oncogenic cells with a self-renewal ability and drug resistance, and they promote the recurrence and metastasis of hepatocellular carcinoma (HCC). However, the mechanisms regulating LCSCs have not been fully explored. By enriching LCSCs from spheroid cultures and performing transcriptomic analysis, we determined that alanine–glyoxylate aminotransferase (AGXT), which participates in the metabolism of serine and glycine, was significantly upregulated in spheroid cultures, and its function in LCSCs remains unknown. Through the exogenous overexpression or short hairpin RNA knockdown of AGXT in HCC cells, we observed that changes in the AGXT level did not affect the spheroid ability and population of LCSCs. The knockdown of AGXT in LCSCs reduced the number of spheroids and the population of LCSCs; this implies that AGXT is required for the maintenance of cancer stemness rather than as a driver of LCSCs. Mechanistically, AGXT may sustain the self-renewal potential of LCSCs by upregulating the expression of SRY-box transcription factor 2 (SOX2) and octamer-binding transcription factor 4 (OCT4), two well-known master regulators of cancer stemness. Taken together, our study demonstrates the role of AGXT in supporting LCSCs; thus, AGXT merits further exploration.

Ruiyun Zhang,1,* Jun Xia,2,* Yiqiu Wang,1,* Ming Cao,1 Di Jin,1 Wei Xue,1 Yiran Huang,1 Haige Chen1 1Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 2Department of Pathology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ming Cao; Haige ChenDepartment of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaEmail chowming2007@gmail.com; rjbladder@163.comObjective: Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) features display enhanced malignant and metastatic potential. This study aimed to introduce a new methodology developed in order to investigate the co-expression of a stemness (OCT4) and EMT markers on single circulating tumor cells (CTCs) of patients with localized urinary bladder cancer and their potential prognostic prediction value.Methods and Materials: Between April 2015 and July 2015, blood samples of 51 consecutive patients diagnosed with high risk bladder cancer (cT1-3N0M0) were prospectively investigated for CTCs. Peripheral blood (5 mL) was drawn before primary transurethral resection. Detection of CTCs was performed using the CanPatrolTM system. Nucleic acid probes were used to identify CTCs, and expression levels of epithelial and mesenchymal genes in CTCs were examined by situ hybridization assay.Results: All patients received radical cystectomy with pelvic lymph nodes dissection. CTCs were detected in 44 of 51 (86.3%) patients, respectively. The overall mean number of CTCs was 6.1 (range: 0∼ 29; median: 4). A total of 311 CTCs were detected in PB. High OCT4 expression (OCT4high) was detected more frequently in Epi−Mes+ cells (p=0.001). Patients with pathological confirmed muscle-invasive bladder cancer (MIBC) had higher Epi−Mes+ CTCs positive rates (p=0.001) and OCT4high CTCs positive rates (p=0.019) than pathological confirmed non muscle-invasive bladder cancer (NMIBC). Regarding co-expression of these markers, Epi−Mes+/OCT4high CTCs were more frequently evident in the MIBC setting (30.4% vs 3.6% of patients, p = 0.016).Conclusion: A differential expression pattern for these markers was observed both in NMIBC and MIBC disease. A subgroup of CTCs showed a CTCs expressing high OCT4, along with Mes were more frequently detected in patients with MIBC, suggesting that these cells may prevail during tumor muscle invasion and disease progression.Keywords: circulating tumor cells, epithelial-to-mesenchymal transition, OCT4, muscle-invasive bladder cancer

Several studies have confirmed the involvement of cancer stem cells (CSC) in tumour progression, metastasis, drug resistance and cancer relapse. SATB2 (special AT‐rich binding protein‐2) acts as a transcriptional co‐factor and modulates chromatin architecture to regulate gene expression. The purpose of this review was to discuss the pathophysiological roles of SATB2 and assess whether it could be used as a therapeutic target for cancer. SATB2 modulated the expression of those genes which regulated pluripotency and self‐renewal. Overexpression of SATB2 gene in normal epithelial cells was shown to induce transformation, as a result transformed cells gained CSC’s characteristics by expressing stem cell markers and pluripotency maintaining factors, suggesting its role as an oncogene. In addition, SATB2 induced epithelial‐mesenchymal transition (EMT) and metastasis. Interestingly, the expression of SATB2 was positively correlated with the activation of β‐catenin/TCF‐LEF pathway. Furthermore, SATB2 silencing inhibited EMT and their positive regulators, and tumour growth, and suppressed the expression of stem cell markers, pluripotency maintaining factors, cell cycle and cell survival genes, and TCF/LEF targets. Based on the cancer genome atlas (TCGA) expression data and published papers, SATB2 alone or in combination with other proteins could be used a diagnostic biomarker for cancer. Although there is no pharmacological inhibitor of SATB2, studies using genetic approaches suggest that SATB2 could be a potential target for cancer treatment and prevention.

Embryonic stem cells provide their major contribution to embryogenesis through formation of germ layers as they have pluripotency potential and capacity for self-renewal. Retention of pluripotency of these stem cells depends on expression/level of transcription factors, i.e., SOX2, OCT4 and NANOG. During organogenesis, the altered expression of the molecules also influences these stem cells to lose their pluripotency and turn toward the lineage selection. As the differentiation progresses, the maintenance of the somatic cells including the oral squamous cells also depends on differential expression of the transcription factors to some extent. Recently, many experimental and observational studies documented the significant contribution in carcinogenesis of various human cancers. In this review, we have attempted to summarize the evidences indicating about the putative role of these master pluripotency regulators in various phases of oral carcinogenesis i.e. initiation , progression and prognosis of oral squamous cell carcinoma.

Aims The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells. Methods and Results Normal cervices and squamous intraepithelial lesions (SIL) were studied with immunohistochemistry, methylation of SOX17, human papilloma virus (HPV) genotyping, and in situ hybridization. In the TZ squamous metaplasia originating from these reserve cells can still show SOX17 expression, while also remnants of SOX17‐positive immature metaplasia can be recognized in the normal squamous epithelium. SOX17 expression is gradually lost during maturation, resulting in the exclusive expression of SOX2 in the majority of (SIL). This loss of SOX17 expression is independent of methylation of the CpG island in its promotor region. HPV can be detected in SOX17‐positive immature metaplastic regions in the immediate vicinity of SOX2‐positive SIL, suggesting that switches in SOX17 and 2 expression can occur upon HPV infection. Conclusions This switch in expression, and the strong association between the distribution of reserve cells and squamous areas within the columnar epithelium in the TZ, suggests that reserve cell proliferations, next to basal cells in the squamous epithelium, are potential targets for the formation of squamous lesions upon viral infection., Switches of SOX17 and SOX2 expression in the development of squamous metaplasia and squamous intraepithelial lesions of the uterine cervix are associated with viral infection.

Background: Current study investigates the role of Oct4, Nanog and CD24 in locally-advanced oral squamous cell carcinoma (OSCC), to evaluate whether the expression of these markers can predict efficacy of neoadjuvant-chemo-radiotherapy and survival of patients. Methods: Biomarker expression was evaluated in 50 homogenously treated patients of locally-advanced OSCC. Results: Clinical response was complete in 30% (n=15), partial response in 46% (n=23), no response in 24% (n=12). Pathologically, 74% patents (n=37) were responders and 26% were non-responders (n=13). Biomarker-overexpression was seen in 46% cases for Oct4, 54% cases for Nanog and 58% cases for CD24. Oct4, Nanog and CD24 expression showed significant correlation with clinical and pathological response (p

The present study is aimed to validate expression of transcriptional factors mediating postnatal development of adrenal zona fasciculata in rats exposed to low doses of endocrine disrupter dichlorodiphenyltrichloroethane prenatally and postnatally. Histological and immunohistochemical examination of the adrenals was performed. Impaired blood circulation, dystrophy and cell death were found in zona fasciculata of pubertal rats after developmental exposure to low doses of dichlorodiphenyltrichloroethane. Reparation of zona fasciculata was associated with increased number of Sonic hedgehog- and Oct4-expressing adrenal cortical cells but not in areas of regeneration. These data suggest that cell death may promote upregulation of factors inducing and maintaining pluripotent state in fasciculata cells for restoration of tissue homeostasis. Termination of growth of the adrenals after puberty was associated with upregulation of antiproliferative factor Hhex and decrease of cell proliferation. Dichlorodiphenyltrichloroethane exposure disrupted transcriptional control of cell proliferation by downregulation of Hhex expression in fasciculata cells. Decrease of proliferation in the exposed rats was mediated by inhibition of Sonic hedgehog and Oct4 expression and suppression of canonical Wnt signaling. The present study elucidated an alternative mechanism of proliferation control activated by endocrine disrupter dichlorodiphenyltrichloroethane through transition of fasciculata cells from pluripotent state and higher proliferative potential to differentiation. Activation of the alternative mechanism of growth control may probably affect maintenance of tissue homeostasis of zona fasciculata in postnatal development.

Background Octamer-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. Methods RT-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. Results The 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. Conclusions Co-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.

Background/purpose: Oct4, a key transcription factor, could reprogram human somatic fibroblasts into embryonic stem cell-like pluripotent cells. The exact mechanism of cyclosporine A (CsA)-induced gingival overgrowth is still unclear. The aim of this study was to investigate the effects of CsA on the expression of Oct4 in cultured human gingival fibroblasts (HGFs) in vitro. Materials and methods: The effects of CsA on HGFs were used to elucidate whether Oct4 expression could be induced by CsA by using quantitative real-time reverse transcription-polymerase chain reaction and western blot. Cell growth in CsA-treated HGFs with Oct4 lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay. Results: CsA was found to upregulate Oct4 transcript in a dose-dependent manner (p

Temperature is an important microenvironmental factor that functions epigenetically in normal embryonic development. However, the effect of hyperthermia in the stem cells is not fully understood. Oct4 is a tightly regulated master regulator of pluripotency maintenance in stem cells and during early embryonic development. We report here that Oct4 protein level was significantly reduced under hyperthermia in mouse blastocysts and embryonic stem cells. The reduction in Oct4 in the mouse embryonic stem cells under hyperthermia was mediated by a ubiquitin-proteasome pathway that was dependent on the activity of death-associated protein kinase 1 (Dapk1) to phosphorylate its substrate, Pin1. Our results imply that the depletion of Oct4 via brief hyperthermia, such as a high fever, during early pregnancy might severely impair the growth of the mammalian embryo or even cause its death.

Knockdown of THG-1 in TE13 esophageal squamous cell carcinoma (ESCC) cells is known to suppress tumorsphere growth. THG-1 was identified as an NRBP1 binding protein, and NRBP1 was reported to downregulate an stemness-related transcriptional factor SALL4, so we decided to examine the possibility that tumorigenic function of THG-1 is achieved by the competition to the tumor-suppressive function of NRBP1. SALL4 was decreased in THG-1 deficient TE13 cells with reduced tumorsphere formation, while exogenous SALL4 expression in THG-1 deficient TE13 cells recovered expression of stemness genes (NANOG and OCT4) and partially, but significantly, recovered tumorsphere formation ability. Additionally, we found that NRBP1 induced ubiquitination of SALL4, and THG-1 interrupted the ubiquitination of SALL4 by antagonizing NRBP1 binding to SALL4. These results suggest that THG-1 promotes tumorsphere growth of ESCC cells by the stabilization of SALL4 protein and induction of the target stemness genes through competitive binding to NRBP1. (C) 2019 The Authors. Published by Elsevier Inc.

Objective: Cancer stem cell is one of the important causes of tumorigenesis as well as a drug target in the treatment of malignant tumor. However, at present, there is no immune vaccine targeting these cells. Octamer-binding transcription factor 4 (OCT4), a marker of embryonic stem cells and germ cells, often highly expresses in the early stages of tumorigenesis and is therefore a good candidate for cancer vaccine development. Methods: To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked three different OCT4 epitope antigens to a carrier protein, keyhole limpet hemocyanin (KLH), combined with Toll-like receptor 9 agonist (TLR9). Results: Immunization with OCT4-3 + TLR9 produced the strongest immune response in mice. In prevention assays, significant tumor growth inhibition was achieved in BABL/c mice treated with OCT4-3 + TLR9 (P < 0.01). Importantly, the results showed that cytotoxic T lymphocyte activity and the inhibition of tumor growth were enhanced in mice immunized with OCT4-3 combined with TLR9. Meanwhile, multiple cytokines [such as interferon (IFN)-γ (P < 0.05), interleukin (IL)-12 (P < 0.05), IL-2 (P < 0.01), and IL-6 (P < 0.05)] promoting cellular immune responses were shown to be greatly enhanced in mice immunized with OCT4-3 + TLR9. Moreover, we considered safety considerations in terms of the composition of the vaccines to help facilitate the development of effective next-generation vaccines. Conclusions: Collectively, these experiments demonstrated that combination therapy with TLR9 agonist induced a tumor-specific adaptive immune response, leading to the suppression of primary tumor growth in testis embryonic carcinoma.

Background Recent advancements in cancer biology field suggest that glucose metabolism is a potential target for cancer treatment. However, little if anything is known about the metabolic profile of cancer stem cells (CSCs) and the related underlying mechanisms. Methods The metabolic phenotype in lung CSC was first investigated. The role of collagen XVII, a putative stem cell or CSC candidate marker, in regulating metabolic reprogramming in lung CSC was subsequently studied. Through screening the genes involved in glycolysis, we identified the downstream targets of collagen XVII that were involved in metabolic reprogramming of lung CSCs. Collagen XVII and its downstream targets were then used to predict the prognosis of lung cancer patients. Results We showed that an aberrant upregulation of glycolysis and oxidative phosphorylation in lung CSCs is associated with the maintenance of CSC-like features, since blocking glycolysis and oxidative phosphorylation reduces sphere formation, chemoresistance, and tumorigenicity. We also showed that the Oct4-hexokinase 2 (HK2) pathway activated by collagen XVII-laminin-332 through FAK-PI3K/AKT-GSB3β/β-catenin activation induced the upregulation of glycolysis and maintenance of CSC-like features. Finally, we showed that collagen XVII, Oct4, and HK2 could be valuable markers to predict the prognosis of lung cancer patients. Conculsions These data suggest the Oct4-HK2 pathway regulated by collagen XVII plays an important role in metabolic reprogramming and maintenance of CSC-like features in lung CSCs, which may aid in the development of new strategies in cancer treatment.

Deciphering the dynamic changes of core factors at different reprogramming stages plays an important role in elucidating the reprogramming mechanism of induced pluripotent stem cells (iPSCs) and improving their induction efficiency. The use of transcription factors (TFs) in combination with histone modification is vital to understand the multiple regulatory of pioneer factor. However, existing studies are not enough to consider the classification of stage-specific gene clusters from the perspective of multi-omic in the process of cell reprogramming. In this study, three stage-specific gene clusters of reprogramming initiation, maturation and stabilization phase were identified by using differential expression analysis. Considering the effects of regional binding preference, we further constructed a quantitative model on different genome regions (promoter, enhancer and enhancer subdivision region) by integrating the DNA binding profiles of Oct4 and three histone modifications (HMs). For promoter and enhancer regions, the receiver operating characteristic curve (Roc curve) of support vector machine (SVM) model was above 0.75 and predictive with the accuracy (Acc) about 66~69%. But on enhancer subdivision region, the convolutional neural network (CNN) model we constructed showed more faithful predictive performance than the model on promoter and enhancer, which Roc curve area can reach 0.87. Taken together, our studies quantitatively reveal the cooperative effects of TFs and HMs on reprogramming stage-specific gene clusters, hoping to provide new sights in mining the key regulators of reprogramming.

The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2.

Due to their therapeutic potential, mesenchymal stem cells are the subject of intensive research on the use of their potential in the treatment of, among others, neurodegenerative diseases or immunological diseases. They are among the newest in the field of medicine. The presented study aimed to evaluate the expression of eight genes from the IAP family and the gene regulating IAP—XAF1—in stem cells derived from human milk, using the qPCR method. The relationships between the expression of genes under study and clinical data, such as maternal age, maternal BMI, week of pregnancy in which the delivery took place, bodyweight of the newborn, the number of pregnancies and deliveries, and the time elapsed since delivery, were also analyzed. The research was carried out on samples of human milk collected from 42 patients hospitalized in The Clinic of Obstetrics and Perinatology of the Independent Public Clinical Hospital No. 4, in Lublin. The conducted research confirmed the expression of the following genes in the tested material: NAIP, BIRC2, BIRC3, BIRC5, BIRC6, BIRC8, XIAP, XAF1, OCT4 and SOX2. Moreover, several dependencies of the expression of individual genes on the maternal BMI (BIRC5, XAF1 and NAIP), the time since childbirth (BIRC5, BIRC6, XAF1 and NAIP), the number of pregnancies and deliveries (BIRC2, BIRC5, BIRC6 and XAF1), the manner of delivery (XAF1 and OCT4), preterm labor (BIRC6 and NAIP) were demonstrated. Additionally, we found positive relationships between gene expression of BIRC7, BIRC8 and XAF1 and the main factors of pluripotency: SOX2 and OCT4. This work is the first to investigate the expression of genes from the IAPs family in mother’s milk stem cells.

Background The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear. Methods HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo. Results We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells. Conclusions The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b–mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.

Background:In the previous theories of cancer, they considered that cancer was a homogeneous which mean that the tumor had only tumor cells and for this reason the treatment for any tumor directed to kill these tumor cells. But, with rising of the metastatic cases of cancer patients, another theory have been raised, that the cancer is a heterogeneous disease which composed of tumor cells that previously the chemotherapy and other cancer therapies directed toward them, in addition there is another group of cells, called cancer stem cells (CSCs), these are more aggressive than the tumor cells that can force the poor microenvironment of the cancer tissue and survive and also they are undifferentiated cells so can undergo mitosis to produce more tumor cells and another group of cancer stem cells in contrast to the tumor cells, which considered a post mitotic and not divided. Objective:Demonstrate some of cancer stem cell markers that considered an important indicators of early cancer development and lately to detect cases of metastasis. Conclusion:The theory of the presence of cancer stem cells is more acceptable and applicable and so the cancer therapy must be directed to these groups of cancer stem cells.

The biological properties of mesenchymal stem cells (MSCs) from synovial fluid from horse limb joints were analysed. The samples were characterized by their peculiarity of adhering to the plastic of the culture flasks. The electromagnetic field technique (MACS) and the surface biomarker STRO-1 were used to optimize their isolation by enriching mesenchymal precursor cells (MSC-LS). To determine the expression of multipotency genes, total RNA was extracted and M-MLV reverse transcriptase was used for the cDNA, which served as a template for qPCR (quantitative PCR). qPCR was performed using the BioRad system with Sybr Green quantification. The multipotency genes quantified were OCT4 and NANOG. The cells selected using MACS with the surface biomarker STRO-1 (CMM-LS STRO-1) proliferate more rapidly than the CMM-LS obtained without purification. In addition, they showed a greater predisposition to differentiate into chondrocytes and osteocytes compared to non-purified LS-MSCs, and a lower predisposition to differentiate into adipocytes. Likewise, the expression of OCT-4 was significantly higher in the CMM-LS STRO-1 compared to the non-purified CMM-LS. NANOG expression was slightly higher in STRO-1 CMM-LS, but without significant differences. The results suggest that the co-expression of OCT-4 and Nanog could increase the physiological functions of MSC-LS STRO-1 related to better proliferation, self-renewal, and the predisposition to differentiate in osteochondral lineages; however, the functional role of these pluripotency markers in adult stem cells needs to be clarified., Se analizaron las propiedades biológicas de las células madre mesenquimales (CMM) provenientes del líquido sinovial de las articulaciones de las extremidades de caballo. Las muestras fueron caracterizadas por su peculiaridad de adherirse al plástico de los frascos de cultivos. Se utilizó la técnica de campos electromagnéticos (MACS) y el biomarcador de superficie STRO-1 con la finalidad de optimizar su aislamiento mediante el enriquecimiento de células precursoras mesenquimales (CMM-LS). Para determinar la expresión de los genes multipotencia, se extrajo el ARN total y se usó la transcriptasa reversa M-MLV para el ADNc, el que sirvió como molde para la qPCR (PCR cuantitativa). El qPCR se realizó utilizando el sistema BioRad con cuantificación mediante Sybr Green. Los genes de multipotencia cuantificados fueron OCT4 y NANOG. Las células seleccionadas utilizando MACS con el biomarcador de superficie STRO-1 (CMM-LS STRO-1), proliferan más rápidamente que las CMM-LS obtenidas sin purificar. Además, manifestaron una mayor predisposición a diferenciarse en condrocitos y osteocitos en comparación a las CMM-LS sin purificar, y una menor predisposición a diferenciarse en adipocitos. Así mismo, la expresión de OCT-4 fue significativamente superior en la CMM-LS STRO-1 en comparación con las CMM-LS sin purificar. La expresión de NANOG fue ligeramente superior en las CMM-LS STRO-1, pero sin diferencia significativa. Los resultados hacen suponer que la coexpresión de OCT-4 y Nanog podrían incrementar las funciones fisiológicas de las CMM-LS STRO-1 relacionadas con una mejor proliferación, autorrenovación y la predisposición a diferenciarse en los linajes osteocondrales; sin embargo, se necesita aclarar el papel funcional de estos marcadores de pluripotencia en células madre adultas.

Includes bibliographical references. Number of sources in the bibliography: 198. Thesis (Ph. D.) -- University of Cyprus, Faculty of Pure and Applied Sciences, Department of Biological Sciences, 2021. The University of Cyprus Library holds the printed form of the thesis. Ο μεταγραφικός παράγοντας Oct4 διαδραματίζει σαφείς ρόλους στην εμβρυ?κή ανάπτυξη και σχετίζεται με την πολυδυναμία των κυττάρων. Πρόσφατα, οι λειτουργίες του έχουν ταυτιστεί και με την καρκινογένεση. Παρόλα αυτά, η μεταγεννητική λειτουργεία του Oct4 δεν έχει καθοριστεί με σαφήνεια. Έχουμε επιβεβαιώσει πως το Oct4 υπερ-εκφράζεται σε καρκίνους του τράχηλου της μήτρας όπου το επιθήλιο είναι θετικό στη μόλυνση με τον ιό των κονδυλωμάτων HPV. Η υπερ-έκφραση του Oct4, καθώς και οι φαινότυποι που σχετίζονται με τον πολλαπλασιασμό τoν κυττάρων στον καρκίνο του τράχηλου της μήτρας όπου εκφράζεται ο HPV, συνδέετε εν μέρη με την αλληλεπίδραση του Oct4 και της ογκο-πρωτε?νης Ε7 του HPV. Για να διερευνήσουμε την μοριακή λειτουργεία του Oct4 και να κατανοήσουμε τους διαφορετικούς φαινοτύπους σε καρκίνους της μήτρας που είναι αρνητικοί (HPV (-)) ή θετικοί στον ιό (HPV (+)), θελήσαμε να διερευνήσουμε την αλληλεπίδραση του Oct4 με τις πρωτεΐνες του ξενιστή, χρησιμοποιώντας δύο μεθόδους: φασματομετρία μάζας και βιοπληροφορική. Και οι δύο αυτές προσεγγίσεις ταυτοποίησαν πρωτεΐνες που αλληλεπιδρούν με το Oct4 στον καρκίνο του τράχηλου της μήτρας, οι οποίες ανήκουν στο σύμπλεγμα πρωτεϊνών NuRD (NuRD complex). Επαληθεύσαμε αυτές τις αλληλεπιδράσεις χρησιμοποιώντας την τεχνική της ανοσοκαθίζησης. Συγκεκριμένα, βρήκαμε ότι διαφορετικά μέλη του συμπλέγματος NuRD αλληλεπιδρούν με το Oct4 στην παρουσία της Ε7. Στα καρκινικά κύτταρα που δεν εκφράζεται ο ιός HPV (HPV (-)), το Oct4 αλληλεπιδρά με το σύμπλεγμα Mbd2-NuRD, ενώ στην παρουσία της Ε7 το Oct4 αλληλεπιδρά με το σύμπλεγμα Mbd3-NuRD. Για να διερευνήσουμε περεταίρω την βιολογική λειτουργεία του πρωτεϊνικού συμπλέγματος Oct4 -Mbd2-NuRD στα καρκινικά κύτταρα αρνητικά για τον ιό, (HPV (-)), χρησιμοποιήσαμε ένα αναστολέα που στοχεύει την λειτουργία του Mbd2. Η αναστολή του Mbd2 τροποποίησε την μεταγραφική λειτουργία του Oct4 καθώς και το φαινότυπο που σχετίζεται με τον πολλαπλασιασμό των καρκινικών κυττάρων όταν η Ε7 δεν εκφράζεται. Η αλληλεπίδραση του Oct4 με τις διαφορετικές παραλλαγές του συμπλέγματος NuRD, μπορεί να εξηγήσει τις διαφορετικές λειτουργίες του Oct4 στα καρκινικά κύτταρα. The stem cell-related transcription factor, Octamer binding transcription factor-4 (Oct4), has well-defined roles in embryonic stem cells (ESCs) for maintaining self-renewal and pluripotency and has recently been implicated in carcinogenesis. However, the postnatal function of Oct4 is poorly understood. We and others have demonstrated that HPV-associated cervical cancers over-express Oct4. The upregulation of Oct4, as well as its proliferation-associated phenotypes in HPV (+) cervical cancers, are in part linked to an interaction between Oct4 and the E7 oncoprotein of HPV. To explore the molecular function of Oct4 and understand the differential Oct4-mediated phenotypes in HPV (+) and HPV (-) cervical cancers, we aimed to investigate the Oct4 interactome using parallel proteomics and bioinformatics approaches. Mass spectrometry and bioinformatics data have identified several members of the NuRD (Nucleosome Remodelling and Deacetylase) complex as relevant interactors of Oct4 in cervical cancer cells. We have validated these protein-protein interactions using co-immunoprecipitation approaches. Notably, using co-immunoprecipitation studies, we have found that different members of the NuRD complex interact with Oct4 in the presence and absence of E7. In HPV (-) C33A cells, Oct4 interacts with the Mbd2-NuRD variant whereas in the context of E7 expression, Oct4 co-immunoprecipitated with components of the Mbd3-NuRD variant. To further investigate the biological role of Mbd2-NuRD variant in cervical cancer cells we used a pharmacological inhibitor targeting the function of Mbd2. The inhibition of Mbd2 modified the Oct4-controlled transcriptional output and the Oct4-regulated proliferation phenotype only in cervical cancer cells where E7 was not expressed. The binding of Oct4 to distinct variants of the NuRD complex may explain the diverse functions of Oct4 in different cell contexts.

Background Asf1 is a well-conserved histone chaperone that regulates multiple cellular processes in different species. Two paralogous genes, Asf1a and Asf1b exist in mammals, but their role during fertilization and early embryogenesis remains to be investigated further. Methods We analyzed the dynamics of histone chaperone Asf1a and Asf1b in oocytes and pre-implantation embryos in mice by immunofluorescence and real-time quantitative PCR, and further investigated the role of Asf1a and Asf1b during fertilization and pre-implantation development by specific Morpholino oligos-mediated knock down approach. Results Immunofluorescence with specific antibodies revealed that both Asf1a and Asf1b were deposited in the nuclei of fully grown oocytes, accumulated abundantly in zygote and 2-cell embryonic nuclei, but turned low at 4-cell stage embryos. In contrast to the weak but definite nuclear deposition of Asf1a, Asf1b disappeared from embryonic nuclei at morula and blastocyst stages. The knockdown of Asf1a and Asf1b by specific Morpholino oligos revealed that Asf1a but not Asf1b was required for the histone H3.3 assembly in paternal pronucleus. However, knockdown of either Asf1a or Asf1b expression decreased developmental potential of pre-implantation embryos. Furthermore, while Asf1a KD severely reduced H3K56 acetylation level and the expression of Oct4 in blastocyst stage embryos, Asf1b KD almost eliminated nuclear accumulation of proliferating cell marker-PCNA in morula stage embryos. These results suggested that histone chaperone Asf1a and Asf1b play distinct roles during fertilization and pre-implantation development in mice. Conclusions Our data suggested that both Asf1a and Asf1b are required for pre-implantation embryonic development. Asf1a regulates H3K56ac levels and Oct4 expression, while Asf1b safeguards pre-implantation embryo development by regulating cell proliferation. We also showed that Asf1a, but not Asf1b, was necessary for the assembly of histone H3.3 in paternal pronuclei after fertilization.

Cancer stem cells (CSCs) are distinct cancer populations with tumorigenic and self-renewal abilities. CSCs are drivers of cancer initiation, progression, therapeutic failure, and disease recurrence. Thereby, novel compounds targeting CSCs offer a promising way to control cancer. In this study, the hydroquinone 5-O-cinnamoyl ester of renieramycin M (CIN-RM) was demonstrated to suppress lung cancer CSCs. CIN-RM was toxic to lung cancer cells with a half-maximal inhibitory concentration of around 15 µM. CIN-RM suppressed CSCs by inhibiting colony and tumor spheroid formation. In addition, the CSC population was isolated and treated and the CSCs were dispatched in response to CIN-RM within 24 h. CIN-RM was shown to abolish cellular c-Myc, a central survival and stem cell regulatory protein, with the depletion of CSC markers and stem cell transcription factors ALDH1A1, Oct4, Nanog, and Sox2. For up-stream regulation, we found that CIN-RM significantly inhibited Akt and consequently decreased the pluripotent transcription factors. CIN-RM also inhibited mTOR, while slightly decreasing p-GSK3β (Ser9) but rarely affected the protein kinase C (PKC) signal. Inhibiting Akt/mTOR induced ubiquitination of c-Myc and promoted degradation. The mechanism of how Akt regulates the stability of c-Myc was validated with the Akt inhibitor wortmannin. The computational analysis further confirmed the strong interaction between CIN-RM and the Akt protein with a binding affinity of −10.9 kcal/mol at its critical active site. Taken together, we utilized molecular experiments, the CSC phenotype, and molecular docking methods to reveal the novel suppressing the activity of this compound on CSCs to benefit CSC-targeted therapy for lung cancer treatment.

The embryonic stem cell marker Oct4 is expressed in several human cancers and is positively correlated with a poor outcome in cancer patients. However, its physiological role in cancer progression remains poorly understood. Tumor cells block apoptosis to escape cell death so that they can proliferate indefinitely, leading to ineffective therapy for cancer patients. In this study, we investigated whether Oct4 regulates the apoptosis pathway and contributes to poor prognosis in patients with lung adenocarcinoma. Our results revealed that Oct4 expression is correlated with Stat1 expression in lung adenocarcinoma patients and Oct4 is directly bound to the Stat1 promoter to transactivate Stat1 in lung adenocarcinoma cells. Expression of the Stat1 downstream gene Mcl-1 increased in Oct4-overexpressing cancer cells, while Stat1 knockdown in Oct4-overexpressing cancer cells sensitized them to cisplatin-induced apoptosis. Furthermore, Oct4 promoted Stat1 expression and tumor growth, whereas silencing of Stat1 reduced Oct4-induced tumor growth in human lung tumor xenograft models. Taken together, we demonstrate that Oct4 is a pro-survival factor by inducing Stat1 expression and that the Oct4/Stat1/Mcl-1 axis may be a potential therapeutic target for lung adenocarcinoma.

Objectives: To study the correlation between the putative cancer stem cell (CSC) markers aldehyde dehydrogenase 1 (ALDH1), cluster of differentiation 44 (CD44), sex-determining region Y-box 2 (SOX2), and octamer-binding protein 4 (OCT4) and human papilloma virus (HPV) infection using p16, the surrogate marker of HPV in oral epithelial dysplasia (OED) and normal mucosa. Methods: Five sections each from 40 histopathologically diagnosed cases of different grades of OED and 10 cases of normal oral mucosa without dysplasia were immunohistochemically stained with p16, ALDH1, CD44, SOX2, and OCT4, respectively. Results: Expression of ALDH1 and SOX2 was significantly increased in OED cases, whereas CD44 and OCT4 expression was increased in normal mucosa. P16-positive OED cases showed upregulation of ALDH1 and OCT4 expression as compared to p16-negative cases, while CD44 and SOX2 expression was downregulated in p16-positive OED cases; however, the results were not statistically significant. Conclusion: The present study indicated a suggestive link between p16 and cancer stem cell marker expression in HPV-associated OED, and that p16 has a significant role in CSC progression in OED. This is the first study to evaluate the expression of putative CSC markers in HPV-associated OED. However, low study numbers are a potential limiting factor in this study.

Cancer stem cells (CSCs) are distinct cancer populations with tumorigenic and self-renewal abilities. CSCs are drivers of cancer initiation, progression, therapeutic failure, and disease recurrence. Thereby, novel compounds targeting CSCs offer a promising way to control cancer. In this study, the hydroquinone 5-O-cinnamoyl ester of renieramycin M (CIN-RM) was demonstrated to suppress lung cancer CSCs. CIN-RM was toxic to lung cancer cells with a half-maximal inhibitory concentration of around 15 µM. CIN-RM suppressed CSCs by inhibiting colony and tumor spheroid formation. In addition, the CSC population was isolated and treated and the CSCs were dispatched in response to CIN-RM within 24 h. CIN-RM was shown to abolish cellular c-Myc, a central survival and stem cell regulatory protein, with the depletion of CSC markers and stem cell transcription factors ALDH1A1, Oct4, Nanog, and Sox2. For up-stream regulation, we found that CIN-RM significantly inhibited Akt and consequently decreased the pluripotent transcription factors. CIN-RM also inhibited mTOR, while slightly decreasing p-GSK3β (Ser9) but rarely affected the protein kinase C (PKC) signal. Inhibiting Akt/mTOR induced ubiquitination of c-Myc and promoted degradation. The mechanism of how Akt regulates the stability of c-Myc was validated with the Akt inhibitor wortmannin. The computational analysis further confirmed the strong interaction between CIN-RM and the Akt protein with a binding affinity of −10.9 kcal/mol at its critical active site. Taken together, we utilized molecular experiments, the CSC phenotype, and molecular docking methods to reveal the novel suppressing the activity of this compound on CSCs to benefit CSC-targeted therapy for lung cancer treatment.

Background The administration of mesenchymal stem cells (MSCs) remains the most promising approach for cardiac repair after myocardial infarct (MI). However, their poor survival and potential in the ischemic environment limit their therapeutic efficacy for heart repair after MI. The purpose of this study was to investigate the influence of FoxC1-induced vascular niche on the activation of octamer-binding protein 4 (Oct4) and the fate of MSCs under hypoxic/ischemic conditions. Methods Vascular microenvironment/niche was induced by efficient delivery of FoxC1 transfection into hypoxic endothelial cells (ECs) or infarcted hearts. MSCs were cultured or injected into this niche by utilizing an in vitro coculture model and a rat MI model. Survival and neovascularization of MSCs regulated by Oct4 were explored using gene transfer and functional studies. Results Here, using gene expression heatmap, we demonstrated that cardiac ECs rapidly upregulated FoxC1 after acute ischemic cardiac injury, contributing to an intrinsic angiogenesis. In vitro, FoxC1 accelerated tube-like structure formation and increased survival of ECs, resulting in inducing a vascular microenvironment. Overexpression of FoxC1 in ECs promoted survival and neovascularization of MSCs under hypoxic coculture. Overexpression of Oct4, a FoxC1 target gene, in MSCs enhanced their mesenchymal-to-endothelial transition (MEndoT) while knockdown of Oct4 by siRNA altering vascularization. In a rat MI model, overexpression of FoxC1 in ischemic hearts increased post-infarct vascular density and improved cardiac function. The transplantation of adOct4-pretreated MSCs into these ischemic niches augments MEndoT, enhanced vascularity, and further improved cardiac function. Consistently, these cardioprotective effects of FoxC1 was abrogated when Oct4 was depleted in the MSCs and was mimicked by overexpression of Oct4. Conclusions Together, these studies demonstrate that the FoxC1/Oct4 axis is an essential aspect for survival and neovascularization of MSCs in the ischemic conditions and represents a potential therapeutic target for enhancing cardiac repair.

Octamer-binding transcription factor 4 (OCT4) and cancerous inhibitor of protein phosphatase 2A (CIP2A) are upregulated in testicular cancer and cell lines. However, its contribution to orchitis (testicular inflammation) is unclear and was thus, investigated herein. Cell-based experiments on a lipopolysaccharide (LPS)-induced orchitis mouse model revealed robust inflammation, apoptotic cell death, and redox disorder in the Leydig (interstitial), Sertoli (supporting), and, germ cells. Meanwhile, real-time quantitative PCR revealed low OCT4 and CIP2A levels in testicular tissue and LPS-stimulated cells. A gain-of-function study showed that OCT4 overexpression not only increased CIP2A expression but also repressed LPS-induced inflammation, apoptosis, and redox disorder in the aforementioned cells. Furthermore, the re-inhibition of CIP2A expression by TD-19 in OCT4-overexpressing cells counteracted the effects of OCT4 overexpression on inflammation, apoptosis, and redox equilibrium. In addition, our results indicated that the Keap1-Nrf2-HO-1 signaling pathway was mediated by OCT4 and CIP2A. These findings provide insights into the potential mechanism underlying OCT4- and CIP2A-mediated testicular inflammation.

Primitive extraembryonale Endoderm (pXEN) Stam-Zelllinien der Ratte repraesentieren wahrscheinlich die festgelegten Vorläufer des extraembryonalen. Die im mesenchymalen Zustand gehaltenen pXEN-Zellen können in vitro weiter zu parietalen und viszeralen Endoderm-ähnlichen Zellen differenzieren. pXEN-Zellen zusätzlich halten moderate Konzentrationen des ICM-Markers Oct4 aufrecht. Die Bedeutung von Oct4 in pXEN-Zellen ist jedoch unbekannt. Bei höheren Zelldichten, beobachteten wir eine erhöhte Oct4-Expression und gleichzeitig eine Tendenz zu Epithelialisierung (MET) und viszeral endodermaler (VE) Differenzierung. Um zu klären, ob die Oct4-Expression kausal beteiligt ist, modulierten wir die Oct4-Konzentration. Transienter Knockdown von Oct4 reduzierte tendenziell die Expression von MET / VE-assoziierten Genen; umgekehrt förderte die Doxycycline-induzierte Expression eines menschlichen Oct4-Transgens die MET / VE-Differenzierung und verhinderte die Bildung charakteristischer Gang-Strukturen. Im letzteren Fall ging dem MET eine anfängliche Zell-Verlängerung und eine erhöhte Zellmotilität voraus. Da ein GSK3-Inhibitor und Activin A auch den MET / VE-Phänotyp stimulierten, fragten wir uns, ob Oct4 über die Wnt/β-Catenin oder TGFβ Signalwege wirkt. Die verschiedene Schritte der Wnt/β-Catenin Signalgebung hemmen, blockierten die hOct4-induzierte MET- und VE-Expression nicht. Im Gegensatz dazu verhinderte Repsox, ein Inhibitor von Alk5 (TGFBR1), das hOct4-induzierte MET und die Expression von MET- und VE-Genen und stimulierte eher die Expression von parietalen Endoderm (PE) Genen. Zusammengefasst zeigen diese Daten eine Rolle für Oct4 bei der MET / VE-Differenzierung auf, wahrscheinlich durch Stimulation eines TGFβ Signalweges. Weiterführende Experimente sind erforderlich um zu bestimmen, wie die zwei Prozesse der MET- und VE-Differenzierung innerhalb der extraembryonalen Endoderm-Linie unterschieden und in Beziehung gesetzt werden. Rat primitive extraembryonic endoderm (pXEN) cell lines appear to represent the committed precursors of the extraembryonic endoderm. The pXEN cells maintained in the mesenchymal state can further differentiate to the parietal endoderm and visceral endoderm like-cells in vitro. In addition, pXEN cells maintain moderate levels of the ICM marker Oct4, a transcription factor that plays important roles in pluripotency, plasticity, and differentiation. However, the significance of Oct4 in pXEN cell lineage specification is unknown. We observed that rat pXEN cells show increased Oct4 expression at higher densities, a condition that also promotes their epithelialization (MET) and visceral endodermal (VE) differentiation. In order to elucidate whether the Oct4 expression is causally involved, we modulated the Oct4 levels. Transient knockdown of Oct4 tended to reduce the expression of MET/VE-associated genes; conversely, the doxycycline-induced expression of a human Oct4 transgene promoted MET/VE differentiation and prevented the formation of characteristic duct structures. In the latter case, the MET was preceded by an initial elongation and increased cell motility. Since GSK3 inhibitor and Activin A also stimulated the MET/VE phenotype, we then asked whether Oct4 acts through the Wnt/β-catenin or TGFβ pathways. Wnt inhibitors did not block the hOct4-induced MET and VE expression. By contrast, Repsox, an inhibitor of Alk5 (TGFBR1), prevented the hOct4-induced MET and the expression of MET and VE genes and rather stimulated the expression of parietal endoderm (PE) genes. Taken together, these data indicate a role for Oct4 in MET/VE differentiation via stimulation of TGFβ signaling. Further work is needed to determine how the two MET and VE differentiation processes are distinguished and related within the extraembryonic endoderm lineage.

Background Currently, no clinically useful biomarkers for radioresistance are available in head and neck squamous cell carcinoma (HNSCC). This study assesses the usefulness of Cell Line Microarray (CMA) method to enhance immunohistochemical screening of potential immunohistochemical biomarkers for radioresistance in HNSCC cell lines. Methods Twenty-nine HNSCC cell lines were cultured, cell pellets formalin-fixed, paraffin-embedded, and arrayed. Radioresistance features of the cell lines were combined to immunohistochemical stains for p53, NDFIP1, EGFR, stem cell marker Oct4, and PP2A inhibitor CIP2A. Results Expression of p53, EGFR or CIP2A did not indicate intrinsic radioresistance in vitro. Stem cell marker Oct4 nuclear positivity and NDFIP1 nuclear positivity was correlated with increased intrinsic radioresistance. Conclusion The usefulness of CMA in analysis of HNSCC cell lines and discovery of biomarkers is demonstrated. CMA is very well adapted to both testing of antibodies in a large panel of cell lines as well as correlating staining results with other cell line characteristics. In addition, CMA-based antibody screening proved an efficient and relatively simple method to identify potential radioresistance biomarkers in HNSCC.

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3′ processing endoribonuclease tRNase ZL, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells., Highlights • TRUE gene silencing is a gene suppression technology with tRNase ZL and sgRNA. • We designed and synthesized an sgRNA targeting the OCT4 mRNA. • The sgRNA induced amnion cells to take a morphology resembling initialized cells. • The sgRNA upregulated OCT4 and alkaline phosphatase expression in HL60 cells.

This study investigates the relevance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis of stromal/tumoral CD4+, CD8+ and FOXP3+ TILs is performed in 125 OSCC patients. Potential relationships with the expression of tumoral PD-L1 and cancer stem cell (CSC) markers (NANOG, SOX2, OCT4, Nestin and Podoplanin (PDPN)) are assessed. CD4+ and CD8+ TILs are significantly associated with smoking and alcohol habits. CD4+ and CD8+ TILs show an inverse relationship with NANOG and SOX2 expression, and FOXP3+ TILs is significantly correlated with Nestin and PDPN expression. High infiltration of CD4+ and CD8+ TILs and a high tumoral CD8+/FOXP3+ ratio are significantly associated with tumors harboring positive PD-L1 expression. Infiltration of stromal/tumoral FOXP3+ TILs and a low stromal CD8+/FOXP3+ ratio are significantly associated with better disease-specific survival. Multivariate analysis reveals that the stromal CD8+/FOXP3+ TILs ratio is a significant independent prognostic factor. Regarding OSCC patient survival, the CD8+/FOXP3+ TILs ratio is an independent prognostic factor. TILs may act as biomarkers and potential therapeutic targets for OSCC.