Overexpression Of SOX2 Research Articles

Keratinocyte-Specific SOX2 Overexpression Suppressed Pressure Ulcer Formation after Cutaneous Ischemia–Reperfusion Injury via Enhancement of Amphiregulin Production

Ischemia-reperfusion (I/R) injury is a key player in the pathogeneses of pressure ulcer formation. Our previous work demonstrated that inducing the transcription factor SOX2 promotes cutaneous wound healing through EGFR signaling pathway enhancement. However, its protective effect on cutaneous I/R injury was not well-characterized. We aimed to assess the role of SOX2 in cutaneous I/R injury and the tissue-protective effect of SOX2 induction in keratinocytes (KCs) in cutaneous I/R injury. SOX2 was transiently expressed in KCs after cutaneous I/R injury. Ulcer formation was significantly suppressed in KC-specific SOX2-overexpressing mice. SOX2 in skin KCs significantly suppressed the infiltrating inflammatory cells, apoptotic cells, vascular damage, and hypoxic areas in cutaneous I/R injury. Oxidative stress-induced mRNA levels of inflammatory cytokine expression were suppressed, and antioxidant stress factors and amphiregulin were elevated by SOX2 induction in skin KCs. Recombinant amphiregulin administration suppressed pressure ulcer development after cutaneous I/R injury in mice and suppressed oxidative stress-induced ROS production and apoptosis invitro. These findings support that SOX2 in KCs might regulate cutaneous I/R injury through amphiregulin production, resulting in oxidative stress suppression. Recombinant amphiregulin can be a potential therapeutic agent for cutaneous I/R injury.

Expression of circ-PHC3 enhances ovarian cancer progression via regulation of the miR-497-5p/SOX9 pathway

BackgroundAccumulating studies have reported indispensable functions of circular RNAs (circRNA) in tumor progression through regulation of gene expression. However, circRNA expression profiles and functions in human ovarian carcinoma (OC) are yet to be fully established.MethodsIn this research, deep sequencing of circRNAs from OC samples and paired adjacent normal tissues was performed to establish expression profiles and circ-PHC3 levels between the groups further compared using RT-qPCR. The effects of ectopic overexpression of miR-497-5p and SOX9 and siRNA-mediated knockdown of circ-PHC3 and an miR-497-5p inhibitor were explored to clarify the regulatory mechanisms underlying circ-PHC3 activity in OC proliferation and metastasis. Information from public databases and the luciferase reporter assay were further utilized to examine the potential correlations among circ-PHC3, miR-497-5p and SOX9.ResultsOur results showed significant upregulation of circ-PHC3 in both OC cell lines and tissues. In the luciferase reporter assay, downregulation of circ-PHC3 led to suppression of metastasis and proliferation, potentially through targeted effects on the miR-497-5p/SOX9 axis in OC. SOX9 overexpression or miR-497-5p suppression rescued OC cell proliferation and invasion following silencing of circ-PHC3. Moreover, SOX9 inhibition induced restoration of OC cell invasion and proliferation under conditions of overexpression of miR-497-5p. Thus, circ-PHC3 appears to exert effects on cancer stem cell differentiation through regulation of the miR-497-5p/SOX9 axis.ConclusionTaken together, our findings suggest that circ-PHC3 enhances OC progression through functioning as an miR-497-5p sponge to promote SOX9 expression, supporting its potential as a promising candidate target for OC therapy.

Profibrotic role of the SOX9–MMP10–ECM biosynthesis axis in the tracheal fibrosis after injury and repair

Fibroblast activation and extracellular matrix (ECM) deposition play an important role in the tracheal abnormal repair process and fibrosis. As a transcription factor, SOX9 is involved in fibroblast activation and ECM deposition. However, the mechanism of how SOX9 regulates fibrosis after tracheal injury remains unclear. We investigated the role of SOX9 in TGF-β1-induced fibroblast activation and ECM deposition in rat tracheal fibroblast (RTF) cells. SOX9 overexpression adenovirus (Ad-SOX9) and siRNA were transfected into RTF cells. We found that SOX9 expression was up-regulated in RTF cells treated with TGF-β1. SOX9 overexpression activated fibroblasts and promoted ECM deposition. Silencing SOX9 inhibited cell proliferation, migration, and ECM deposition, induced G2 arrest, and increased apoptosis in RTF cells. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) assays identified MMP10, a matrix metalloproteinase involved in ECM deposition, as a direct target of SOX9, which promotes ECM degradation by increasing MMP10 expression through the Wnt/β-catenin signaling pathway. Furthermore, in vivo, SOX9 knockdown ameliorated granulation proliferation and tracheal fibrosis, as manifested by reduced tracheal stenosis. In conclusion, our findings indicate that SOX9 can drive fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/β-catenin signaling pathway. The SOX9–MMP10–ECM biosynthesis axis plays an important role in tracheal injury and repair. Targeting SOX9 and its downstream target MMP10 may represent a promising therapeutic approach for tracheal fibrosis.

Control of SOX2 protein stability and tumorigenic activity by E3 ligase CHIP in esophageal cancer cells.

SOX2 is highly expressed and controls tumor initiation and cancer stem cell function in various squamous cell carcinomas including esophageal squamous cancer. However, the molecular mechanism leading to SOX2 overexpression in cancer is incompletely understood. Here, we identified CHIP, a chaperone-associated ubiquitin E3 ligase, as a novel negative regulator of SOX2 protein stability and tumorigenic activity in esophageal squamous carcinoma cells. We showed that CHIP interacted with SOX2 primarily via chaperone HSP70, together they catalyzed SOX2 ubiquitination and degradation via proteasome. In contrast, HSP90 promoted SOX2 stability and inhibition of HSP90 activity induced SOX2 ubiquitination and degradation. Notably, unlike the case in normal esophageal tissues where CHIP was detected in both the cytoplasm and nucleus, CHIP in clinical esophageal tumor specimens was predominantly localized in the cytoplasm. Consistent with this observation, we observed increased expression of exportin-1/CRM-1 in clinical esophageal tumor specimens. We further demonstrated that CHIP catalyzed SOX2 ubiquitination and degradation primarily in the nuclear compartment. Taken together, our study has identified CHIP as a key suppressor of SOX2 protein stability and tumorigenic activity and revealed CHIP nuclear exclusion as a potential mechanism for aberrant SOX2 overexpression in esophageal cancer. Our study also suggests HSP90 inhibitors as potential therapeutic agents for SOX2-positive cancers.

Molecular Characterization of Esophageal Squamous Cell Carcinoma Using Quantitative Proteomics

Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC.

GSK3β-driven SOX2 overexpression is a targetable vulnerability in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest forms of human malignancy that currently lacks approved targeted therapeutics. Accumulating evidence suggests that SOX2 overexpression is a key driving factor for ESCC and various squamous cell carcinoma. Here, through screening a small-molecule kinase inhibitor library, we identified GSK3β as a kinase that is critically required for robust SOX2 expression in ESCC cells. GSK3β did not promote SOX2 transcriptionally but was required for SOX2 protein stability. We demonstrated that GSK3β interacts with and phosphorylates SOX2 at residue S251, which blocks SOX2 from ubiquitination and proteasome-dependent degradation instigated by ubiquitin E3 ligase CUL4ADET1-COP1. Pharmacological inhibition or knockdown of GSK3β by RNA interference selectively impaired SOX2-positive ESCC cell proliferation, cancer stemness, and tumor growth in mouse xenograft model, suggesting that GSK3β promotes ESCC tumorigenesis primarily by driving SOX2 overexpression. GSK3β was found to be frequently overexpressed in clinical esophageal tumors, and there was a positive correlation between GSK3β and SOX2 protein levels. Notably, we found that SOX2 enhanced GSK3β expression transcriptionally, suggesting the existence of a vicious cycle that drives a coordinated GSK3β and SOX2 overexpression in ESCC cells. Finally, we demonstrated in tumor xenograft model that GSK3β inhibitor AR-A014418 was effective in suppressing SOX2-positive ESCC tumor progression and inhibited tumor progression cooperatively with chemotherapeutic agent carboplatin. In conclusion, we uncovered a novel role for GSK3β in driving SOX2 overexpression and tumorigenesis and provided evidence that targeting GSK3β may hold promise for the treatment of recalcitrant ESCCs.

SOX2 downregulation of PML increases HCMV gene expression and growth of glioma cells.

The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment.

Abstract 27: Characterizing 3q26 copy number gain in lung squamous cell carcinoma through a novel genetically engineered mouse model

Abstract Lung squamous cell carcinoma (LUSC) is a major histological subtype of lung cancer accounting for ~30% of all lung cancer diagnoses. LUSC patients suffer from poor therapeutic response, high relapse rate, and poor prognosis. A major reason for this bleak outcome is the lack of well characterized oncogenic drivers of LUSC. This has stalled the generation of relevant genetic pre-clinical mouse models amenable to studying LUSC tumorigenesis in vivo and developing targeted therapeutics to treat LUSC patients. Our group has identified concomitant 3q26 copy number gain (CNG) and TP53 loss as defining genetic alterations present in ~90% of LUSC tumors. Furthermore, we have identified three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a potential cell of origin for LUSC) is both necessary and sufficient to transform these cells into orthotopic LUSC tumors in syngeneic mice. These findings indicate that 3q26 amplification and TP53 loss play a critical role in the initiation and progression of LUSC. Based on these data, we have developed the first autochthonous genetically engineered mouse model (GEMM) to study the role of 3q26 CNG in LUSC tumorigenesis in vivo. Our GEMM allows inducible overexpression of Prkci, Ect2, and Sox2 and knockout of Trp53 (PES/Trp53−/−) in lung tissue. Tumorigenesis will be initiated in PES/Trp53−/− mice using an established protocol to target transgene recombination to LBSCs in vivo. These mice will be assessed for tumor latency, survival, tumor burden, and metastatic potential. In related studies, we used bioinformatics analyses on primary LUSC tumors from the Cancer Genome Atlas’ database to identify 13 genes in addition to PRKCI, ECT2 and SOX2, whose expression is driven by 3q26 amplification. These genes were functionally characterized by assessing the effect of shRNA-mediated gene silencing on soft-agar growth of human H520 LUSC cells, a cell line that harbors 3q26 amplification. Our results revealed that knockdown of each of these genes independently resulted in a significant decrease in soft agar colony formation. We further assessed these genes through RT-qPCR mRNA expression profiling comparing PES/Trp53−/- transformed LBSCs to non-transformed LBSCs. These genes were significantly upregulated in PES/Trp53−/- transformed LBSCs, suggesting that they play important roles in LBSC transformation. Future work will further assess the molecular mechanisms by which these additional 3q26 genes contribute to LUSC cell transformation and assess the role of alternate cells of origin (Club and AT2) in 3q26-driven LUSC tumor formation using our PES/Trp53−/− GEMM. Citation Format: Kayleah M. Meneses, Capella Weems, Yi Liu, Verline Justilien, Nicole R. Murray, Alan P. Fields. Characterizing 3q26 copy number gain in lung squamous cell carcinoma through a novel genetically engineered mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 27.

SOX17 is a Critical Factor in Maintaining Endothelial Function in Pulmonary Hypertension by an Exosome-Mediated Autocrine Manner.

Endothelial dysfunction is considered a predominant driver for pulmonary vascular remodeling in pulmonary hypertension (PH). SOX17, a key regulator of vascular homoeostasis, has been found to harbor mutations in PH patients, which are associated with PH susceptibility. Here, this study explores whether SOX17 mediates the autocrine activity of pulmonary artery ECs to maintain endothelial function and vascular homeostasis in PH and its underlying mechanism. It is found that SOX17 expression is downregulated in the endothelium of remodeled pulmonary arteries in IPH patients and SU5416/hypoxia (Su/hypo)-induced PH mice as well as dysfunctional HPAECs. Endothelial knockdown of SOX17 accelerates the progression of Su/hypo-induced PH in mice. SOX17 overexpression in the pulmonary endothelium of mice attenuates Su/hypo-induced PH. SOX17-associated exosomes block the proliferation, apoptosis, and inflammation of HPAECs, preventing pulmonary arterial remodeling and Su/hypo-induced PH. Mechanistic analyses demonstrates that overexpressing SOX17 promotes the exosome-mediated release of miR-224-5p and miR-361-3p, which are internalized by injured HPAECs in an autocrine manner, ultimately repressing the upregulation of NR4A3 and PCSK9 genes and improving endothelial function. These results suggest that SOX17 is a key gene in maintaining endothelial function and vascular homeostasis in PH through regulating exosomal miRNAs in an autocrine manner.

Delivery of dCas9 Activator System Using Magnetic Nanoparticles Technology as a Vector Delivery Method for Human Skin Fibroblast

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.

Dexmedetomidine ameliorates ischemia-induced nerve injury by up-regulating Sox11 expression.

Dexmedetomidine (Dex) is associated with several biological processes. Ischemic stroke has the characteristics of high morbidity and mortality. Herein, we aimed to explore whether Dex ameliorates ischemia-induced injury and determine its mechanism. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to measure gene and protein expression. Cellular viability and proliferation were assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, respectively. Cell apoptosis was detected by flow cytometry. An oxygen-glucose deprivation/reoxygenation model of SK-N-SH and SH-SY5Y cells was constructed. A middle cerebral artery occlusion (MCAO) model was also built to assess Dex function in vivo. Neuronal function was assessed using the Bederson Behavior Score and Longa Behavior Score. We found that Dex positively and dose-dependently regulated Sox11 expression and prevented damage caused by oxygen-glucose deprivation/reoxygenation (OGD/R), enhancing cell viability and proliferation and reducing apoptosis in SK-N-SH and SH-SY5Y cells. The overexpression of Sox11 antagonized OGD/R-induced SK-N-SH and SH-SY5Y cell apoptosis and promoted cell growth in vitro. Furthermore, cell proliferation was decreased and cell apoptosis was increased after Sox11 knockdown in Dex-treated SK-N-SH and SH-SY5Y cells. We demonstrated that Dex prevented OGD/R-induced cell injury by up-regulating Sox11. Furthermore, we also confirmed that Dex protected rat from ischemia-induced injury in the MCAO model. The role of Dex in cell viability and survival was verified in this study. Moreover, Dex protected neurons from MCAO-induced injury by up-regulating the expression of Sox11. Our research proposes a potential drug to improve the functional recovery of stroke patients in the clinic.

Hyperglycemia-induced endothelial exosomes trigger trophoblast dysregulation and abnormal placentation through PUM2-mediated repression of SOX2.

Hyperglycemia is closely related to adverse pregnancy outcomes including pre-eclampsia (PE), a life-threatening complication with a substantial morbidity and mortality. However, the pathogenesis of abnormal placentation in gestational diabetes mellitus (GDM)-associated PE remains elusive. Here we isolated exosomes from the human umbilical vein endothelial cells (HUVECs) treated with normal level of glucose (NG) and high levels of glucose (HG). The exosomes were added to HTR-8a/SVneo cells, a trophoblast cell line. High-throughput RNA-sequencing was performed to analyzed the changed RNAs in the exosomes and exosome-treated HTR-8a/SVneo cells. HTR-8a/SVneo cell phenotypes were evaluated from the aspects of cell proliferation, cell invasion and DNA damage. After treatment with HG, the changed RNAs in exosomes was enriched in RNA stabilization and oxidative stress. The altered RNAs in the HTR-8a/SVneo cells treated with exosomes from HG-induced HUVECs were enriched in pathways related to cell adhesion, migration, DNA damage response and angiogenesis. The HG-induced exosomes impaired the proliferation and invasion of HTR-8a cells and caused the DNA damage. HG up-regulated PUM2 in the exosomes and exosome-treated HTR-8a/SVneo cells. PUM2 interacted with SOX2 mRNA, resulting in the mRNA degradation. Overexpression of SOX2 prevented the damage to HTR-8a/SVneo cells caused by the exosomes from HG-induced HUVECs. We demonstrate that high glucose-induced endothelial exosomes mediate abnormal phenotypes of trophoblasts through PUM2-mediated repression of SOX2. Our results reveal a novel regulatory mechanism of hyperglycemia in development of abnormal placentation and provide potential targets for preventing adverse pregnancy outcomes.

SOX2 Promotes Radioresistance in Non-small Cell Lung Cancer by Regulating Tumor Cells Dedifferentiation.

Background: Radiation therapy plays an important role in the treatment of patients with non-small cell lung cancer (NSCLC). However, the radiocurability is greatly limited because of radioresistance which leads to treatment failure, tumor recurrence, and metastasis. Cancer stem cell (CSC) has been identified as the main factor that contributes to radiation resistance. SOX2, one of the transcription factors specifically expressed in CSC, is involved in tumorigenesis, progression, and maintenance of cell stemness. But the association between SOX2 and NSCLC radioresistance is not clear now. Methods: We constructed the radiotherapy-resistant cell line of NSCLC by multiple radiotherapy treatments. Colony formation assay, western blot, and immunofluorescence were performed to detect the radiosensitivity of cells. Western blot, qRT-PCR, and sphere formation assay were used to detect CSC characteristics of cells. Wound healing assay and Transwell assay were used to determine cell migration motility. The SOX2-upregulated model and SOX2-downregulated model was constructed by lentivirus transduction. Finally, the expression and clinical relevance of SOX2 in NSCLC were investigated by bioinformatics analysis based on TCGA and GEO datasets. Results: The expression of SOX2 was increased in radioresistant cells and a trend of dedifferentiation were observed. The results of wound healing assay and Transwell assay showed that SOX2 overexpression significantly promote the migration and invasion of NSCLC cells. Mechanistically, overexpression of SOX2 enhanced radioresistance and DNA damage repair capability of parental cells, while down-regulation of SOX2 led to decreased radioresistance and DNA repair ability in radioresistant cells, all of which were related to cells dedifferentiation regulated by SOX2. In addition, bioinformatics analysis show that high expression of SOX2 was strongly associated with the progression and poor prognosis of patients with NSCLC. Conclusions: Our study revealed that SOX2 regulates radiotherapy resistance in NSCLC via promoting cell dedifferentiation. Therefore, SOX2 may be a promising therapeutic target for overcoming radioresistance in NSCLC, providing a new perspective to improve the curative effect.

SOX2-high cancer cells exhibit an aggressive phenotype, with increases in stemness, proliferation and invasion, as well as higher metabolic activity and ATP production.

Cancer stem cells (CSCs) are responsible for cancer recurrence, treatment failure and metastatic dissemination. As such, the elimination of CSCs represents one of the most important approaches for the future of cancer treatment. Among other properties, CSCs show the activation of particular cell signalling pathways and the over-expression of certain transcription factors, such as SOX2. Herein, we describe a new model system to isolate stem-like cancer cells, based on the functional transcriptional activity of SOX2. Briefly, we employed a SOX2-enhancer-GFP-reporter system to isolate cancer cells with high SOX2 transcriptional activity by FACS sorting. The over-expression of SOX2 in this sub-population was validated by Western blot analysis and flow cytometry. SOX2-high cancer cells showed CSCs features, such as greater mammosphere forming ability, validating that this sub-population was enriched in CSCs. To further explore the model, we analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, corroborating that SOX2-high cells were more metabolically active, proliferative, migratory, invasive, and drug-resistant. SOX2-high MDA-MB-231 cells also showed a loss of E-cadherin expression, and increased Vimentin expression, consistent with an epithelial-mesenchymal transition (EMT). Therefore, endogenous SOX2 transcriptional activity and protein levels are mechanistically linked to aggressive phenotypic behaviours and energy production in CSCs.

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

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. 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. 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). These findings showed a novel SOX9/HMGB3/NANOG regulatory mechanism, deregulation of which played important roles in PCa progression.

A pro B cell population forms the apex of the leukemic hierarchy in Hoxa9/Meis1-dependent AML

Relapse is a major challenge to therapeutic success in acute myeloid leukemia (AML) and can be partly associated with heterogeneous leukemic stem cell (LSC) properties. In the murine Hoxa9/Meis1-dependent (H9M) AML model, LSC potential lies in three defined immunophenotypes, including Lin−cKit+ progenitor cells (Lin−), Gr1+CD11b+cKit+ myeloid cells, and lymphoid cells (Lym+). Previous reports demonstrated their interconversion and distinct drug sensitivities. In contrast, we here show that H9M AML is hierarchically organized. We, therefore, tracked the developmental potential of LSC phenotypes. This unexpectedly revealed a substantial fraction of Lin− LSCs that failed to regenerate Lym+ LSCs, and that harbored reduced leukemogenic potential. However, Lin− LSCs capable of producing Lym+ LSCs as well as Lym+ LSCs triggered rapid disease development suggestive of their high relapse-driving potential. Transcriptional analyses revealed that B lymphoid master regulators, including Sox4 and Bach2, correlated with Lym+ LSC development and presumably aggressive disease. Lentiviral overexpression of Sox4 and Bach2 induced dedifferentiation of H9M cells towards a lineage-negative state in vitro as the first step of lineage conversion. This work suggests that the potency to initiate a partial B lymphoid primed transcriptional program as present in infant AML correlates with aggressive disease and governs the H9M LSC hierarchy.

Abstract B023: Sox9 drives an aberrant transcriptional program impeding intestinal differentiation in colorectal cancer initiation

Abstract Despite the implementation of screening and preventative strategies, colorectal cancer (CRC) remains the second most deadly cancer worldwide and is responsible for an alarming trend of increasing prevalence among younger patients. Inappropriate WNT activation is the initiating step in sporadic CRC development, typically through deleterious mutations in the pathway negative regulator Adenomatous polyposis coli (APC). WNT pathway hinders whereas transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling supports differentiation of progenitors into mature enterocytes, establishing a crypt-villus gradient. Genomic alterations that dysregulate intestinal stem cell differentiation are central to CRC development. Impairment of differentiation and inappropriate Sox9 overexpression are conserved events in CRC initiation based on the evaluation of two genetically engineered mouse models, two carcinogen-induced mouse models, and a patient with familial adenomatous polyposis (FAP), a hereditary polyposis syndrome in which a mutant copy of APC is inherited. Single cell RNA-sequencing (scRNA-seq) of adenomas from an ApcKO mouse model and FAP sample implicates an aberrant stem cell-like program (herein referred to as AbSC) as a key aspect of CRC initiation. The AbSc program is characterized by selective intestinal stem cell activity, indiscriminate attenuation of differentiated lineages, and aberrant activation of genes associated with fetal intestinal development. Histopathology and scRNA-seq of adenomas and derivative organoids from a patient with FAP demonstrated a partial block in differentiation and confirmed reactivation of genes reserved for intestinal development. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) of APCKO epithelial cells revealed increased chromatin accessibility at fetal intestinal genes. Genetic inactivation of Sox9 prevented adenoma formation in ApcKO mice and induced differentiation of ApcKO organoids, obstructing the emergence of the ApcKO transcriptional state, including reactivation of a fetal-like intestinal program, restored multi-lineage differentiation, and markedly reduced the gained chromatin accessibility at developmental genes in neoplasia. These studies indicate that an aberrant transcriptional state hindering intestinal differentiation mediated by Sox9 is an early conserved event in CRC and carry important implications for developing therapeutics directed at inducing intestinal differentiation. Citation Format: Pratyusha Bala, Jonathan P. Rennhack, Clare Morris, Daulet Aitymbayev, Sydney M. Moyer, Gina Nicole Duronio, Paul Doan, William C. Hahn, Nilay S. Sethi. Sox9 drives an aberrant transcriptional program impeding intestinal differentiation in colorectal cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B023.

601 Overexpression of the transcription factor SOX2 in keratinocytes inhibits pressure ulcer formation after cutaneous I/R injury

It is empirically known that wounds on the oral mucosa heal faster than those on the skin, and comparative molecular profiling using human subject from oral mucosa and skin tissue has revealed that the transcription factor SOX2, which is highly expressed in the oral mucosa but not in the skin, activates the mucosal epithelium in the steady state, creating an environment conducive to wound healing. Recent study demonstrated that cutaneous wound healing in epidermal specific SOX2-overexpresing mice was promoted compared with control mice.

SOX4 Mediates ATRA-Induced Differentiation in Neuroblastoma Cells.

Neuroblastoma (NB), which is considered to be caused by the differentiation failure of neural crest cells, is the most common extracranial malignant solid tumor in children. The degree of tumor differentiation in patients with NB is closely correlated with the survival rate. To explore the potential targets that mediate NB cell differentiation, we analyzed four microarray datasets from GEO, and the overlapping down- or upregulated DEGs were displayed using Venn diagrams. SOX4 was one of the overlapping upregulated DEGs and was confirmed by RT-qPCR and Western blot in ATRA-treated NGP, SY5Y, and BE2 cells. To clarify whether SOX4 was the target gene regulating NB cell differentiation, the correlation between the expression of SOX4 and the survival of clinical patients was analyzed via the R2 database, SOX4 overexpression plasmids and siRNAs were generated to change the expression of SOX4, RT-qPCR and Western blot were performed to detect SOX4 expression, cell confluence or cell survival was detected by IncuCyte Zoom or CCK8 assay, immunocytochemistry staining was performed to detect cells' neurites, and a cell cycle analysis was implemented using Flow cytometry after PI staining. The results showed that the survival probabilities were positively correlated with SOX4 expression, in which overexpressing SOX4 inhibited NB cell proliferation, elongated the cells' neurite, and blocked the cell cycle in G1 phase, and that knockdown of the expression of SOX4 partially reversed the ATRA-induced inhibition of NB cell proliferation, the elongation of the cells' neurites, and the blocking of the cell cycle in the G1 phase. These indicate that SOX4 may be a target to induce NB cell differentiation.

Sox9 is required in regeneration of pancreatic β cells following injury

The reduction of insulin secretion due to pancreatic β cell injury caused by autoimmune reaction is the pathological basis of Type 1 diabetes mellitus (T1DM). Therefore, seeking new molecular targets for alleviating pancreatic β cell injury will provide experimental basis for the prevention and treatment of T1DM. SRY-box 9 (Sox9) is not only an important molecule regulating the development of various organs, but also its high expression can aggravate the pathological process of various diseases. In addition, Sox9+ cells are also pancreatic progenitor cells, participating in pancreatic repair reaction induced by injury. In our study, elevated blood glucose and lack of pancreatic β cells almost returned to normal over time after streptozotocin (STZ)-induced pancreatic β cell damage, implying that pancreatic β cells were regenerated after STZ-induced injury. In particular, the expression of Sox9 was significantly elevated during pancreatic β cell regeneration. On this basis, we conducted in vitro experiments to verify whether overexpression of Sox9 could inhibit the damage of pancreatic β cells by inflammatory factors. Our results showed that overexpression of Sox9 alleviated the damage of pancreatic β cells by inflammatory factors and improved the inhibitory effect of inflammatory factors on insulin secretion of pancreatic β cells. Unsurprising, blood glucose levels, insulin content and pancreatic β cell number failed to return to near-normal levels timely after pancreatic β cells specific knockout Sox9 mice were treated with STZ, further confirming the importance of Sox9 in facilitating pancreatic β cell repair or regeneration. Our study indicate that enhanced Sox9 activity might protect pancreatic β cells from autoimmune induced damage and thus improve the pathological process of T1DM.