Profiling of Pluripotency Factors in Single Cells and Early Embryos.

Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.

ERK signalling eliminates Nanog and maintains Oct4 to drive the formative pluripotency transition.

Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states.

Translational control by maternal Nanog promotes oogenesis and early embryonic development.

Many maternal mRNAs are translationally repressed during oocyte development and spatio-temporally activated during early embryogenesis, which is crucial for oocyte and early embryo development. By analyzing maternal mutants of nanog (Mnanog) in zebrafish, we demonstrated that Nanog tightly controls translation of maternal mRNA during oogenesis via transcriptional repression of eukaryotic translation elongation factor 1 alpha 1, like 2 (eef1a1l2). Loss of maternal Nanog led to defects of egg maturation, increased endoplasmic reticulum stress, and an activated unfold protein response, which was caused by elevated translational activity. We further demonstrated that Nanog, as a transcriptional repressor, represses the transcription of eefl1a1l2 by directly binding to the eef1a1l2 promoter in oocytes. More importantly, depletion of eef1a1l2 in nanog mutant females effectively rescued the elevated translational activity in oocytes, oogenesis defects and embryonic defects of Mnanog embryos. Thus, our study demonstrates that maternal Nanog regulates oogenesis and early embryogenesis through translational control of maternal mRNA via a mechanism whereby Nanog acts as a transcriptional repressor to suppress transcription of eef1a1l2.

H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate.

Mouse embryonic stem cells have an inherent propensity to explore gene regulatory states associated with either self-renewal or differentiation. This property depends on ERK, which downregulates pluripotency genes such as Nanog. Here, we aimed at identifying repressive histone modifications that would mark Nanog for inactivation in response to ERK activity. We found that the transcription factor ZFP57, which binds methylated DNA to nucleate heterochromatin, is recruited upstream of Nanog, within a region enriched for histone H3 lysine 9 tri-methylation (H3K9me3). Whereas before differentiation H3K9me3 at Nanog depends on ERK, in somatic cells it becomes independent of ERK. Moreover, the loss of H3K9me3 at Nanog, induced by deleting the region or by knocking out DNA methyltransferases or Zfp57, is associated with reduced heterogeneity of NANOG, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. Hence, a network axis centred on DNA methylation, ZFP57 and H3K9me3 links Nanog regulation to ERK activity for the timely establishment of new cell identities. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.

Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells.

Autophagy is required for benign hepatic tumors to progress into malignant hepatocellular carcinoma. However, the mechanism is unclear. Here, we report that mitophagy, the selective removal of mitochondria by autophagy, positively regulates hepatic cancer stem cells (CSCs) by suppressing the tumor suppressor p53. When mitophagy is enhanced, p53 co-localizes with mitochondria and is removed by a mitophagy-dependent manner. However, when mitophagy is inhibited, p53 is phosphorylated at serine-392 by PINK1, a kinase associated with mitophagy, on mitochondria and translocated into the nucleus, where it binds to the NANOG promoter to prevent OCT4 and SOX2 transcription factors from activating the expression of NANOG, a transcription factor critical for maintaining the stemness and the self-renewal ability of CSCs, resulting in the reduction of hepatic CSC populations. These results demonstrate that mitophagy controls the activities of p53 to maintain hepatic CSCs and provide an explanation as to why autophagy is required to promote hepatocarcinogenesis.

KLF15 suppresses stemness of pancreatic cancer by decreasing USP21-mediated Nanog stability.

The existence of cancer stem cells (CSCs) in pancreatic ductal adenocarcinoma (PDAC) is considered to be the key factor for metastasis and chemoresistance. Thus, novel therapeutic strategies for eradicating CSCs are urgently needed. Here we aimed to explore the role of KLF15 in stemness and the feasibility of using KLF15 to inhibit CSCs and improve chemotherapy sensitivity in PDAC. In this study, we report that KLF15 is negatively associated with poor survival and advanced pathological staging of PDAC. Moreover, tumorous KLF15 suppresses the stemness of PDAC by promoting the degradation of Nanog, and KLF15 directly interacts with Nanog, inhibiting interaction between Nanog with USP21. We also demonstrate that the KLF15/Nanog complex inhibit the stemness in vivo and in PDX cells. Tazemetostat suppresses stemness and sensitizes PDAC cells to gemcitabine by promoting KLF15 expression in PDAC. In summary, the findings of our study confirm the value of KLF15 level in diagnosis and prognosis of PDAC, it is the first time to explore the inhibition role of KLF15 in stemness of PDAC and the regulation mechanism of Nanog, contributing to provide a new therapeutic strategy that using Tazemetostat sensitizes PDAC cells to gemcitabine by promoting KLF15 expression for PDAC.

Microgravity as a Tool to Investigate Cancer Induction in Pleura Mesothelial Cells.

The present work shows that the exposure of mesothelial cells to simulated microgravity changes their cytoskeleton and adhesion proteins, leading to a cell switch from normal towards tumoral cells. Immunohistochemical and molecular data were obtained from both MeT-5A exposed to simulated microgravity and BR95 mesothelioma cell lines. Simulated microgravity was found to affect the expression of actin, vinculin, and connexin-43, altering their quantitative and spatial distribution pattern inside the cell. The analysis of the tumoral markers p27, CD44, Fibulin-3, and NANOG and the expression of genes related to cancer transformation such as NANOG, CDH-1, and Zeb-1 showed that the simulated microgravity environment led to expression patterns in MeT-5A cells similar to those observed in BR95 cells. The alteration in both quantitative expression and structural organization of the cytoskeleton and adhesion/communication proteins can thus be considered a pivotal mechanism involved in the cellular shift towards tumoral progression.

ALKBH5 regulates somatic cell reprogramming in a phase-specific manner.

Establishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors [octamer binding transcription factor 4 (OCT4; also known as POU5F1), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4) and cellular myelocytomatosis (c-MYC)] provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA-specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, whereas overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5, mediated through its catalytic activity, in regulating somatic cell reprogramming at the post-transcriptional level. This article has an associated First Person interview with the first author of the paper.

Research progress of nanog gene in fish.

Nanog is a crucial regulatory factor in maintaining the self-renewal and pluripotency of embryonic stem cells. It is involved in various biological processes, such as early embryonic development, cell reprogramming, cell cycle regulation, the proliferation and migration of primordial germ cells. While research on this gene has primarily focused on mammals, there has been a growing interest in studying nanog in fish. However, there is a notable lack of comprehensive reviews regarding this gene in fish, which is essential for guiding future research. This review aims to provide a thorough summary of the gene's structure, expression patterns, functions and regulatory mechanisms in fish. The findings suggest that nanog probably has both conserved and divergent functions in regulating cell pluripotency, early embryonic development, and germ cell development in teleosts compared to other species, including mammals. These insights lay the foundation for future research and applications of the nanog gene, providing a new perspective for understanding the evolution and conserved charactristics of teleost nanog.

Integrated analysis of the role of PR/SET domain 14 in gastric cancer.

BACKGROUND: Gastric cancer is one of the most common tumors worldwide, and most patients are deprived of treatment options when diagnosed at advanced stages. PRDM14 has carcinogenic potential in breast and non-small cell lung cancer. however, its role in gastric cancer has not been elucidated. METHODS: We aimed to elucidate the expression of PRDM14 using pan-cancer analysis. We monitored the expression of PRDM14 in cells and patients using quantitative polymerase chain reaction, western blotting, and immunohistochemistry. We observed that cell phenotypes and regulatory genes were influenced by PRDM14 by silencing PRDM14. We evaluated and validated the value of the PRDM14-derived prognostic model. Finally, we predicted the relationship between PRDM14 and small-molecule drug responses using the Connectivity Map and The Genomics of Drug Sensitivity in Cancer databases. RESULTS: PRDM14 was significantly overexpressed in gastric cancer, which identified in cell lines and patients' tissues. Silencing the expression of PRDM14 resulted in apoptosis promotion, cell cycle arrest, and inhibition of the growth and migration of GC cells. Functional analysis revealed that PRDM14 acts in epigenetic regulation and modulates multiple DNA methyltransferases or transcription factors. The PRDM14-derived differentially expressed gene prognostic model was validated to reliably predict the patient prognosis. Nomograms (age, sex, and PRDM14-risk score) were used to quantify the probability of survival. PRDM14 was positively correlated with sensitivity to small-molecule drugs such as TPCA-1, PF-56,227, mirin, and linsitinib. CONCLUSIONS: Collectively, our findings suggest that PRDM14 is a positive regulator of gastric cancer progression. Therefore, it may be a potential therapeutic target for gastric cancer.

A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics.

The self-renewal and differentiation potential of embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. Expression levels of the core pluripotency factor Nanog are tightly regulated by a negative feedback autorepression loop. However, it remains unclear how ESCs perceive NANOG levels and execute autorepression. Here, we show that a dose-dependent induction of Fgfbp1 and Fgfr2 by NANOG activates autocrine-mediated ERK signaling in Nanog-high cells to trigger autorepression. pERK recruits NONO to the Nanog locus to repress transcription by preventing POL2 loading. This Nanog autorepression process establishes a self-perpetuating reciprocal NANOG-pERK regulatory circuit. We further demonstrate that this reciprocal regulatory circuit induces pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells. Collectively our data suggest that NANOG induces Fgfr2 and Fgfbp1 to activate ERK signaling in Nanog-high cells to establish a NANOG-pERK reciprocal regulatory circuit. This circuit regulates ERK signaling dynamics and Nanog autoregulation in pluripotent cells.

Targeting Nanog expression increased Cisplatin chemosensitivity and inhibited cell migration in Gastric cancer cells.

Regardless of significant advances in cancer treatment, gastric cancer (GC) incidence rate is increasing worldwide. As one of the main transcription factors participating in stemness, Nanog plays a pivotal role in various aspects of tumorigenesis, metastasis, and chemosensitivity. Given that, the current research intended to evaluate the potential effects of Nanog suppression on the GC cell Cisplatin chemosensitivity and in vitro tumorigenesis. First, bioinformatics analysis was performed to evaluate the effect of Nanog expression on GC patients' survival. The MKN-45 human GC cells were transfected with specific siRNA targeting Nanog and/or treated with Cisplatin. Then, to study cellular viability and apoptosis, MTT assay and Annexin V/PI staining were done, respectively. Also, the scratch assay was performed to investigate cell migration, and MKN-45 cell stemness was followed using colony formation assay. Western blotting and qRT-PCR were used for gene expression analysis. The findings demonstrated that Nanog overexpression was significantly correlated with poor survival of GC patients, and siRNA-mediated Nanog silencing strongly increased MKN-45 cell sensitivity to Cisplatin through apoptosis induction. Also, Nanog suppression combined with Cisplatin resulted in the upregulation of the Caspase-3 and Bax/Bcl-2 ratio at mRNA levels and increased Caspase-3 activation. Moreover, reduced expression of Nanog, separately or combined with Cisplatin, inhibited MKN-45 cell migration by downregulating MMP2 mRNA and protein expression levels. The results also evidenced CD44 and SOX-2 downregulation aligned with a decreased rate of MKN-45 cell colony formation ability through treatments. Besides, Nanog downregulation significantly decreased MDR-1 mRNA expression. Taken together, the results of this study indicated that Nanog could be suggested as a promising target combined with Cisplatin-based GC therapies for reducing drug side effects and improving patients' outcomes.

Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

INTRODUCTION: Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. METHODS: Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. RESULTS: Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. CONCLUSIONS: Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

CRET-based immunoassay on magnetic beads for selective and sensitive detection of Nanog antigen as a key cancer stem cell marker.

A method is presented for chemiluminescence resonance energy transfer (CRET) using APTES-Fe3O4 as a highly efficient energy acceptor with strong magnetic effectiveness over extended distances, while an Au@BSA-luminol composite acts as the donor. In order to boost the chemiluminescence reactions, CuO nanoparticles were successfully employed. The distance between the donor and acceptor is a crucial factor in the occurrence of the CRET phenomenon. A sensitive and high-throughput sandwich chemiluminescence immunosensor has been developed accordingly with a linear range of 1.0 × 10-7 g/L to 6.0 × 10-5 g/L and a limit of detection of 0.8 × 10-7 g/L. The CRET-based sandwich immunosensor has the potential to be implemented to early cancer diagnosis because of its high sensitivity in detecting Nanog, fast analysis (30 min), and simplicity. Furthermore, this approach has the potential to be adapted for the recognition of other antigen-antibody immune complexes by utilizing the corresponding antigens and their selective antibodies.

Unraveling the Significance of Nanog in the Generation of Embryonic Stem-like Cells from Spermatogonia Stem Cells: A Combined In Silico Analysis and In Vitro Experimental Approach.

Embryonic stem-like cells (ES-like cells) are promising for medical research and clinical applications. Traditional methods involve "Yamanaka" transcription (OSKM) to derive these cells from somatic cells in vitro. Recently, a novel approach has emerged, obtaining ES-like cells from spermatogonia stem cells (SSCs) in a time-related process without adding artificial additives to cell cultures, like transcription factors or small molecules such as pten or p53 inhibitors. This study aims to investigate the role of the Nanog in the conversion of SSCs to pluripotent stem cells through both in silico analysis and in vitro experiments. We used bioinformatic methods and microarray data to find significant genes connected to this derivation path, to construct PPI networks, using enrichment analysis, and to construct miRNA-lncRNA networks, as well as in vitro experiments, immunostaining, and Fluidigm qPCR analysis to connect the dots of Nanog significance. We concluded that Nanog is one of the most crucial differentially expressed genes during SSC conversion, collaborating with critical regulators such as Sox2, Dazl, Pou5f1, Dnmt3, and Cdh1. This intricate protein network positions Nanog as a pivotal factor in pathway enrichment for generating ES-like cells, including Wnt signaling, focal adhesion, and PI3K-Akt-mTOR signaling. Nanog expression is presumed to play a vital role in deriving ES-like cells from SSCs in vitro. Finding its pivotal role in this path illuminates future research and clinical applications.

Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Characterizing CD133 and NANOG Expression in Melanoma: Associations with Histological and Epidemiological Parameters.

Background/Objectives: Melanoma is an aggressive skin malignancy, and the majority of deaths associated with melanoma result from malignant skin lesions. Our study aims to evaluate the expression of the markers CD133 and NANOG, associated with tumor stem cells, and to analyze their link with epidemiological and histological parameters, thus contributing to early diagnosis and the development of targeted therapies. Methods: We performed a retrospective study in the Mureș Clinical County Hospital, Romania, which included 66 cases of melanoma: 50 primary cutaneous melanomas, 10 metastases, and 6 local recurrences. CD133 and NANOG marker expression was assessed by immunohistochemistry and quantified using the H score. Statistical analyses were applied to determine the correlations between marker expression and clinicopathological parameters. Results: CD133 expression was identified in six cases (12%) of primary melanoma, with a mean H-Score of 29, and was associated with an increased Breslow index and a higher number of mitoses. NANOG expression was positive in 30 cases (60%) of primary melanoma, with a median H-Score of 15 and with increased expression observed in cases with pagetoid migration and lesions in situ. In metastases, eight cases (80%) were positive for NANOG and four (40%) for CD133. Local recurrences showed positive expression for NANOG in four cases (66%). Conclusions: The expression of CD133 and NANOG markers highlights the role of tumor stem cells in melanoma progression. Early identification of these markers could improve diagnosis and treatment, including the application of targeted therapies.

Reciprocal interplays between MicroRNAs and pluripotency transcription factors in dictating stemness features in human cancers.

The interplay between microRNAs (miRNAs) and pluripotency transcription factors (TFs) orchestrates the acquisition of cancer stem cell (CSC) features during the course of malignant transformation, rendering them essential cancer cell dependencies and therapeutic vulnerabilities. In this review, we discuss emerging themes in tumor heterogeneity, including the clonal evolution and the CSC models and their implications in resistance to cancer therapies, and then provide thorough coverage on the roles played by key TFs in maintaining normal and malignant stem cell pluripotency and plasticity. In addition, we discuss the reciprocal interactions between miRNAs and MYC, OCT4, NANOG, SOX2, and KLF4 pluripotency TFs and their contributions to tumorigenesis. We provide our view on the potential to interfere with key miRNA-TF networks through the use of RNA-based therapeutics as single agents or in combination with other therapeutic strategies, to abrogate the CSC state and render tumor cells more responsive to standard and targeted therapies.

Sumoylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells.

AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.

SOX9 and HMGB3 co-operatively transactivate NANOG and promote prostate cancer progression.

BACKGROUND: The homeodomain-containing transcription factor NANOG is overexpressed in prostate adenocarcinoma (PCa) and predicts poor prognosis. The SOX family transcription factor SOX9, as well as the transcription co-activator HMGB3 of the HMGB family, are also overexpressed and may play pivotal roles in PCa. However, it is unknown whether SOX9 and HMGB3 interact with each other, or if they regulate NANOG gene transcription. METHODS: We identified potential SOX9 responsive elements in NANOG promoter, and investigated if SOX9 regulated NANOG transcription in co-operation with HMGB3 by experimental analysis of potential SOX9 binding sites in NANOG promoter, reporter gene transcription assays with or without interference or artificial overexpression of SOX9 and/or HMGB3, and protein-binding assays of SOX9-HMGB3 interaction. Clinicopathologic and prognostic significance of SOX9-HMGB3 overexpression in PCa was analyzed. RESULTS: SOX9 activated NANOG gene transcription by preferentially binding to a highly conserved consensus cis-regulatory element (-573 to -568) in NANOG promoter, and promoted the expression of NANOG downstream oncogenic genes. Importantly, HMGB3 functioned as a partner of SOX9 to co-operatively enhance transactivation of NANOG by interacting with SOX9, predominantly via the HMG Box A domain of HMGB3. Overexpression of SOX9 and/or HMGB3 enhanced PCa cell survival and cell migration and were significantly associated with PCa progression. Notably, Cox proportional regression analysis showed that co-overexpression of both SOX9 and HMGB3 was an independent unfavorable prognosticator for both CRPC-free survival (relative risk [RR] = 3.779，95% confidence interval [CI]: 1.159-12.322, p = 0.028) and overall survival (RR = 3.615，95% CI: 1.101-11.876, p = 0.034). CONCLUSIONS: These findings showed a novel SOX9/HMGB3/NANOG regulatory mechanism, deregulation of which played important roles in PCa progression.