Sox2 Target Research Articles

Inhibition of the Wnt/β‑catenin signaling pathway and SOX9 by XAV939 did not alleviate inflammation in a dextran sulfate sodium‑induced ulcerative colitis model.

The Wnt/β-catenin signaling pathway has been reported to be hyperactivated during the pathogenesis of ulcerative colitis (UC). The present study aimed to explore the therapeutic efficacy of the Wnt/β-catenin signaling inhibitor XAV939 in mitigating UC symptoms. Utilizing a dextran sulfate sodium (DSS)-induced UC mouse model, the present study aimed to evaluate the impact of XAV939 on intestinal morphology through hematoxylin and eosin staining and to measure the expression levels of critical proteins in the Wnt/β-catenin signaling cascade. XAV939 did not exert a significant influence on the morphological features and inflammatory status of the intestinal epithelium. However, XAV939 was found to effectively suppress the Wnt/β-catenin signaling pathway and its downstream target SOX9. This suppression implied a reduction in the differentiation of intestinal stem cells into secretory cell progenitor cells. Additionally, XAV939 was ineffective at reversing the DSS-induced decrease in expression levels of Villin and peroxisome proliferator-activated receptor γ, which suggested that it did not facilitate the differentiation of intestinal absorptive cells. The present findings indicated that the Wnt/β-catenin signaling pathway may not be the predominant mechanism in the pathogenesis of DSS-induced UC.

Identification and verification of KEAP1‐related genes and targets regulated by potential ingredients in KRAS mutant colorectal cancer

AbstractAs one of the most prevalent tumors, colorectal cancer (CRC) owns complex pathogenesis and the high recurrence rate significantly influence the prognosis of patients with KRAS mutant CRC, to discovery the crosstalk among KEAP1‐NFE2L2 and oncogenic KRAS as the potential prognostic biomarkers in CRC, which might be used to develop novel and effective therapeutics. By using bioinformatics analysis, we explored and identified key genes as the predicted targets in the crosstalk among KEAP1 oncogenic signatures of KRAS mutation that were linked with development, metastasis, and poor prognosis in CRC. We further investigated the correlations between the clinical characteristics and expressions of prognostic genes among the KRAS and KEAP1‐related key hub genes in CRC, which as predicted targets to find the potential herbal ingredients in HERB database, further to verify the active herbal ingredients could regulate the key targets that related to KEAP1‐NFE2L2 in KRAS mutant CRC. The newest TCGA data reveals that the mutation of KRAS is found in 44% of CRC patients. In total, 28 genes were identified as differentially expressed genes (DEGs), and the hub genes such as CDKN2A, SPP1, FOS, BCL2L11, and HPSE were verified. Results indicated that key genes (SOX9, SPP1) significant correlation with the target prediction of the active herbal ingredients by molecular docking analysis. Furthermore, KEAP1, NFE2L2, SOX9 expression were decreased significantly with the treatment of potential ingredients. Furthermore, cyclopamine could enhance the sensitivity of HCT116 cells, downregulated the expression of SPP1, and induced activation of KEAP1‐NFE2L2 pathway, which cell death are characteristic features of apoptosis, and enhanced anticancer effect. Therefore, KEAP1‐related genes might be important oncogenic signatures in KRAS mutant CRC cells and cyclopamine was identified as a potential ingredient and regulated the predict targets of SOX9 and SPP1, may be expand the efficacy and range of novel and effective therapeutics.

Sox10 Activity and the Timing of Schwann Cell Differentiation Are Controlled by a Tle4-Dependent Negative Feedback Loop.

The HMG-domain containing transcription factor Sox10 plays a crucial role in regulating Schwann cell survival and differentiation and is expressed throughout the entire Schwann cell lineage. While its importance in peripheral myelination is well established, little is known about its role in the early stages of Schwann cell development. In a search for direct target genes of Sox10 in Schwann cell precursors, the transcriptional co-repressor Tle4 was identified. At least two regions upstream of the Tle4 gene appear involved in mediating the Sox10-dependent activation. Once induced, Tle4 works in tandem with the bHLH transcriptional repressor Hes1 and exerts a dual inhibitory effect on Sox10 by preventing the Sox10 protein from transcriptionally activating maturation genes and by suppressing Sox10 expression through known enhancers of the gene. This mechanism establishes a regulatory barrier that prevents premature activation of factors involved in differentiation and myelin formation by Sox10 in immature Schwann cells. The identification of Tle4 as a critical downstream target of Sox10 sheds light on the gene regulatory network in the early phases of Schwann cell development. It unravels an elaborate regulatory circuitry that fine-tunes the timing and extent of Schwann cell differentiation and myelin gene expression.

Abstract 7574: Exploring the therapeutic potential of proteasome inhibitors in targeting SOX2-driven LUSC and SCLC

Abstract This study addresses the current lack of small molecules targeting SOX2, a pivotal protein in cancer, through an examination of the effectiveness of FDA-approved drugs and proteasome inhibitors. Building on prior research demonstrating the proteasome inhibitor CFZ's capacity to diminish the expression of ITGB4 and PXN in LUAD effectively, our investigation highlights CFZ and IXA as potent inhibitors at nanomolar concentrations. Notably, CFZ treatment enhances sensitivity to cisplatin by suppressing the expression of ITGB4 and the stemness-associated gene SOX2.At a mechanistic level, CFZ hinders histone acetylation at the SOX2 promoter site, leading to rapid and specific downregulation. RNA-seq analysis of COH2 cells treated with CFZ unveils substantial downregulation of genes related to transcription and DNA binding. Z score analysis further reveals a noteworthy inhibition of key transcription factors (TFs), including SOX2, NANOG, POU5F1, SALL4, and KLF4. This TF inhibition extends to neuroendocrine cell lines, identifying 16 overlapping targets of SOX2 associated with stemness. These findings suggest a potential therapeutic approach for targeting cisplatin-resistant cancer stem cells across various cancer types. Moreover, the study proposes a synergistic effect of cisplatin and CFZ in mitigating the growth of neuroendocrine phenotype, SCLC. Given the limited medical options currently available for this phenotype, this insight holds particular significance. In conclusion, our study unveils CFZ as a potent transcriptional suppressor of SOX2, providing a novel strategy to sensitize cisplatin-resistant cancer stem cells. The results underscore the interplay of genetic and non-genetic mechanisms in drug resistance, emphasizing the significance of repurposing drugs for other indications. Citation Format: Atish Mohanty, Linlin Guo, Sravani Ramisetty, Sharad Singhal, Erminia Massarelli, Prakash Kulkarni, Ravi Salgia. Exploring the therapeutic potential of proteasome inhibitors in targeting SOX2-driven LUSC and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7574.

Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

Trk Inhibition Reduces Tumorsphere Formation and Changes Expression of Stemness Markers in SK-ES-1 Ewing Sarcoma Cells

Introduction: Ewing sarcoma (ES) is a highly aggressive type of childhood cancer characterized by a chromosomal translocation resulting in fusions between the gene encoding EWS RNA Binding Protein 1 (EWSR1) and one gene of the ETS family, most frequently FLI-1, resulting in the EWS-FLI1 aberrant transcription factor. ES tumors can contain a subpopulation of cells showing cancer stem cell (CSC) features, which express stemness markers including CD133, OCT4 (Octamer-binding transcription factor 4), and NANOG, and display capacity to form tumorspheres likely enriched in CSCs. Neurotrophin (NT) receptors of the tropomyosin receptor kinase (Trk) family (TrkA, TrkB, and TrkC) may play a role in stimulating ES progression, but their possible role in CSCs remains unknown. Objective: To verify the effect of Trks inhibition on the formation of tumorspheres as well as the gene expression of stem markers. Method: The cells were dissociated and the formation of spheres was induced with supplemented culture medium and the K252a treatment was performed. After RNA extraction, mRNA expression levels of target genes Prom1 (CD133), OCT4 (POU5F1), SOX2, and Musashi-1 (MSI1) were analyzed by qPCR. Results: The pan-Trk inhibitor K252a (100 or 500 mM) hindered tumorsphere formation in human SK-ES-1 ES cell cultures. K252a also reduced mRNA expression of Prom1 (CD133-coding gene) while enhancing expression of OCT4. No changes in mRNA levels of SOX2 or Musashi-1 were observed. Conclusion: These findings provide the first evidence suggesting that Trk activity can influence stemness in ES cells.

Sry-Box Transcription Factor 9 (SOX9)-Driven Mesenchymal Stromal Cell Differentiation Signature As a Determinant of Induction Chemotherapy Responsiveness in Acute Myeloid Leukemia Core Bone Marrow Biopsies

Leukemic clones in acute myeloid leukemia (AML) alter the bone marrow (BM) microenvironment so that the normal functions of hematopoietic stem cells (HSCs) are interrupted, aiding malignant progression. Accumulating evidence points to a protective role that the microenvironment provides for the AML clone (Colmone et al., Science, 2008). It is therefore crucial to detail the mechanisms by which the BM stroma promotes leukemia progression to inform the development of more effective targeted therapies. Most studies focused on detailing the role of BM stroma in AML etiology use BM aspirates, which have a limited ability to represent the physical structure of the BM microenvironment. To accurately capture BM composition, our group used the core bone marrow biopsies (cBMBs) of 15 AML patients who responded to and 14 AML patients who did not respond to 7+3 induction chemotherapy (Treaba et al., British Journal of Haematology, 2022). RNA-sequencing data of these biopsies (GEO GSE213210) indicated significant differences in the composition of the stromal component of responders and non-responders. Responder cBMBs were enriched in undifferentiated CXCL12-positive mesenchymal stem cells (MSCs), with elevated expression of platelet derived growth factor beta (PDGFRB), snail family transcriptional repressor 2 (SNAI2), or vimentin (VIM). CXCL12-positive MSCs in non-responder cBMBs had increased expression of osteo-committed markers such as osteopontin (SPP1), osterix (SP7), and collagens (Figure 1B, osteogenesis). Based on these findings, we formulated the hypothesis that the differentiation state of the BM stroma at diagnosis may indicate treatment outcomes, specifically at the initial 14-day post-induction therapy assessment. First, we determined the adipocyte- and chondrocyte-commitment states of BM MSCs in responder and non-responder AML cBMBs before and after the 14-day induction therapy. We found significant increases in transcripts linked to adipogenesis in responder cBMBs, suggesting an increase in frequencies of adipo-committed MSCs that represent the early differentiation state of these cells. This observation was consistent with our already reported findings (Treaba et al., British Journal of Haematology, 2022) that less differentiated MSCs are prevalent in responder cBMBs post-treatment. Intriguingly, we also noted significant differences between expression of SOX9-regulated transcripts in responder compared to non-responder cBMBs. As a master regulator of chondrogenesis, SOX9 is required for MSCs to differentiate towards a chondrogenic fate. SOX9 expression was significantly upregulated in non-responder post-treatment versus diagnostic cBMBs, with a log2 fold change of 3.6. Collagen 2 alpha 1 (COL2a1) and collagen 9 alpha 1 (COL9a1), two targets of SOX9 important in chondrogenesis, were also notably elevated in non-responder cBMBs with a log2 fold change of 6.1 and 7, respectively. In contrast, SOX6, which also regulates chondrogenesis, and COL2a1 were downregulated in responder post-treatment versus diagnostic cBMBs, with a log2 fold change of -2.7 and -4.5, respectively. Another striking change was seen when directly comparing the transcriptomes of responder to non-responder cBMBs at both diagnosis and post-treatment stages. Non-responder log2 expression of COL2a1 relative to responder log2 expression increased from -4.7 at diagnosis to 5.9 at post-treatment. Importantly, SOX9-regulated or -interacting genes were the most downregulated chondrogenic genes in responder cBMBs and the most upregulated in non-responder cBMBs, which is consistent with the contrasting differentiated state of MSCs between responders and non-responders initially observed with osteogenic genes. In summary, for the first time, we report that expression levels of SOX9 and SOX9-regulated targets in AML patients are indicative of the differentiated state of MSCs in AML patients who responded to or did not respond to 7+3 induction therapy. These data represent compelling support for our hypothesis correlating mesenchymal differentiation capacity with treatment response and warrant in-depth mechanistic studies.

Decreased expression of GEM in osteoarthritis cartilage regulates chondrogenic differentiation via Wnt/β-catenin signaling

BackgroundGEM (GTP-binding protein overexpressed in skeletal muscle) is one of the atypical small GTPase subfamily members recently identified as a regulator of cell differentiation. Abnormal chondrogenesis coupled with an imbalance in the turnover of cartilaginous matrix formation is highly relevant to the onset and progression of osteoarthritis (OA). However, how GEM regulates chondrogenic differentiation remains unexplored.MethodsCartilage tissues were obtained from OA patients and graded according to the ORASI and ICRS grading systems. The expression alteration of GEM was detected in the Grade 4 cartilage compared to Grade 0 and verified in OA mimic culture systems. Next, to investigate the specific function of GEM during these processes, we generated a Gem knockdown (Gem-Kd) system by transfecting siRNA targeting Gem into ATDC5 cells. Acan, Col2a1, Sox9, and Wnt target genes of Gem-Kd ATDC5 cells were detected during induction. The transcriptomic sequencing analysis was performed to investigate the mechanism of GEM regulation. Wnt signaling pathways were verified by real-time PCR and immunoblot analysis. Finally, a rescue model generated by treating Gem-KD ATDC5 cells with a Wnt signaling agonist was established to validate the mechanism identified by RNA sequencing analysis.ResultsA decreased expression of GEM in OA patients’ cartilage tissues and OA mimic chondrocytes was observed. While during chondrogenesis differentiation and cartilage matrix formation, the expression of GEM was increased. Gem silencing suppressed chondrogenic differentiation and the expressions of Acan, Col2a1, and Sox9. RNA sequencing analysis revealed that Wnt signaling was downregulated in Gem-Kd cells. Decreased expression of Wnt signaling associated genes and the total β-CATENIN in the nucleus and cytoplasm were observed. The exogenous Wnt activation exhibited reversed effect on Gem loss-of-function cells.ConclusionThese findings collectively validated that GEM functions as a novel regulator mediating chondrogenic differentiation and cartilage matrix formation through Wnt/β-catenin signaling.

SOX9 is a key component of RUNX2-regulated transcriptional circuitry in osteosarcoma

BackgroundThe absence of prominent, actionable genetic alternations in osteosarcomas (OS) implies that transcriptional and epigenetic mechanisms significantly contribute to the progression of this life-threatening form of cancer. Therefore, the identification of potential transcriptional events that promote the survival of OS cells could be key in devising targeted therapeutic approaches for OS. We have previously shown that RUNX2 is a transcription factor (TF) essential for OS cell survival. Unfortunately, the transcriptional network or circuitry regulated by RUNX2 in OS cells is still largely unknown.MethodsThe TFs that are in the RUNX2 transcriptional circuitry were identified by analyzing RNAseq and ChIPseq datasets of RUNX2. To evaluate the effect of SOX9 knockdown on the survival of osteosarcoma cells in vitro, we employed cleaved caspase-3 immunoblotting and propidium iodide staining techniques. The impact of SOX9 and JMJD1C depletion on OS tumor growth was examined in vivo using xenografts and immunohistochemistry. Downstream targets of SOX9 were identified and dissected using RNAseq, pathway analysis, and gene set enrichment analysis. Furthermore, the interactome of SOX9 was identified using BioID and validated by PLA.ResultOur findings demonstrate that SOX9 is a critical TF that is induced by RUNX2. Both in vitro and in vivo experiments revealed that SOX9 plays a pivotal role in the survival of OS. RNAseq analysis revealed that SOX9 activates the transcription of MYC, a downstream target of RUNX2. Mechanistically, our results suggest a transcriptional network involving SOX9, RUNX2, and MYC, with SOX9 binding to RUNX2. Moreover, we discovered that JMJD1C, a chromatin factor, is a novel binding partner of SOX9, and depletion of JMJD1C impairs OS tumor growth.ConclusionThe findings of this study represent a significant advancement in our understanding of the transcriptional network present in OS cells, providing valuable insights that may contribute to the development of targeted therapies for OS.

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.

Different chromatin-scanning modes lead to targeting of compacted chromatin by pioneer factors FOXA1 and SOX2

Pioneer transcription factors interact with nucleosomes to scan silent, compact chromatin, enabling cooperative events that modulate gene activity. While at a subset of sites pioneer factors access chromatin by assisted loading with other transcription factors, the nucleosome-binding properties of pioneer factors enable them to initiate zygotic genome activation, embryonic development, and cellular reprogramming. To better understand nucleosome targeting invivo, we assess whether pioneer factors FoxA1 and Sox2 target stable or unstable nucleosomes and find that they target DNase-resistant, stable nucleosomes, whereas HNF4A, a non-nucleosome binding factor, targets open, DNase-sensitive chromatin. Despite FOXA1 and SOX2 targeting similar proportions of DNase-resistant chromatin, using single-molecule tracking, we find that FOXA1 uses lower nucleoplasmic diffusion and longer residence times while SOX2 uses higher nucleoplasmic diffusion and shorter residence times to scan compact chromatin, while HNF4 scans compact chromatin much less efficiently. Thus, pioneer factors target compact chromatin through distinct processes.

LncRNA XIST Inhibits Gastric Cancer Progression Through the Wnt Pathway by Regulating the miR-30a-5p/SOX9 Axis

We aimed to investigate the impact of XIST on the pathogenesis of gastric cancer (GC). We used quantitative real-time polymerase chain reaction (RT-qPCR) to determine the expression levels of XIST, SOX9, and miR-30a-5p in GC cells. Western blot assay was used to measure the protein levels of SOX9, β-catenin, and c-Myc. We also performed MTT and transwell assays to assess proliferation, apoptosis, and invasion. Dual-luciferase reporter assays and RNA immunoprecipitation (RIP) assays were used to confirm the interaction between miR-30a-5p and XIST or SOX9. We found that GC tumor cells express high levels of XIST and SOX9, which promote cell proliferation, invasion, and resistance to apoptosis. Specifically, we observed that XIST acts as a sponge for miR-30a-5p in GC cell line SGC-7901, while miR-30a-5p targets SOX9. SOX9 expression is regulated by XIST via miR-30a-5p acting as a competitive endogenous RNA. Furthermore, we identified a miR-30a-5p/SOX9 axis that modulates the Wnt/β-catenin signaling pathway. In conclusion, our study demonstrates that LncXIST interacts with miR-30a-5p as a competitive endogenous RNA to promote GC cell proliferation, migration, invasion, and inhibition of apoptosis through the regulation of SOX9 expression.

MP01-11 IN VITRO PROPAGATION OF UNDIFFERENTIATED SPERMATOGONIA ISOLATED FROM A 46, XX MALE TESTIS

You have accessJournal of UrologyCME1 Apr 2023MP01-11 IN VITRO PROPAGATION OF UNDIFFERENTIATED SPERMATOGONIA ISOLATED FROM A 46, XX MALE TESTIS Mark Xu, Aaron Bradshaw, Harpreet Gosal, Abinav Udaiyar, Omar Abdelaal, Kimberly Stogner-Underwood, Stuart Howards, Tamer Yalcinkaya, Anthony Atala, and Hooman Sadri-Ardekani Mark XuMark Xu More articles by this author , Aaron BradshawAaron Bradshaw More articles by this author , Harpreet GosalHarpreet Gosal More articles by this author , Abinav UdaiyarAbinav Udaiyar More articles by this author , Omar AbdelaalOmar Abdelaal More articles by this author , Kimberly Stogner-UnderwoodKimberly Stogner-Underwood More articles by this author , Stuart HowardsStuart Howards More articles by this author , Tamer YalcinkayaTamer Yalcinkaya More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Hooman Sadri-ArdekaniHooman Sadri-Ardekani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The SRY-positive 46, XX genotype is a testicular disorder of sex development found in 1/20,000 males. Patients are phenotypically male with small testes, variable hypogonadism, and azoospermia. Current literature has not yet documented spermatogonia in 46 XX male patients. This study aimed to identify and propagate spermatogonia in vitro in a 46, XX male. METHODS: A 28-year-old Caucasian male was initially suspected of having Klinefelter Syndrome (XXY), but karyotype and FISH analysis found a 46, XX genotype with a Y chromosome translocation containing the SRY gene onto the short distal arm of the X chromosome. He underwent experimental bilateral microscopic testicular biopsy and tissue preservation under IRB protocol. Fresh tissue was sent for histopathologic analysis using H&E staining and immunostaining of undifferentiated spermatogenic markers, including PGP 9.5/UCHL1 and ZBTB16. Biopsy portions from bilateral testes were used for characterization. Digital PCR and Real-time Quantitative (RT-Q) PCR were performed with ZBTB16 (undifferentiated spermatogonia), STAR (Leydig cell), CD34 (Peritubular cell), and SOX9 (Sertoli cell) target primers on testis samples from both sides. Isolated cells from the right testicular biopsy were cultured in the StemPro medium for four weeks. RT-QPCR was performed with target primers for undifferentiated spermatogonia (ZBTB16, UCHL1, THY1, CD9, ITGA6, ITGB1), Sertoli (SOX9, GATA4, CYP19a1), Leydig (STAR, CALB2), and peritubular cells (CD34). Digital PCR was performed using ZBTB16 target primer to quantify undifferentiated spermatogonia. Flow cytometry on cultured cells identified the spermatogonia stem cell (SSC) population as negative for HLA-ABC and positive for CD9 and CD49f. RESULTS: Testes exhibited Leydig cell hyperplasia, seminiferous tubule hyalinization like in Klinefelter Syndrome, and tubules filled mainly with Sertoli cells. Rare cells in seminiferous tubules were weakly positive for PGP9.5 and ZBTB16, representing undifferentiated spermatogonia. Digital and RT-QPCRs of testis tissue and cultured cells found evidence of all major testicular somatic cells and undifferentiated spermatogonia. Digital PCR for ZBTB16 identified 7.61% of cultured cells as undifferentiated spermatogonia and 0.71% as SSCs. Flow cytometry identified 2.52% of cultured cells as SSCs. CONCLUSIONS: This is the first study suggesting the presence of SSCs in 46 XX male testes. Future work will focus on further characterization of cultured testicular cells and In Vitro or In Vivo differentiation of SSCs from XX male testis. Source of Funding: WFIRM © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e6 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mark Xu More articles by this author Aaron Bradshaw More articles by this author Harpreet Gosal More articles by this author Abinav Udaiyar More articles by this author Omar Abdelaal More articles by this author Kimberly Stogner-Underwood More articles by this author Stuart Howards More articles by this author Tamer Yalcinkaya More articles by this author Anthony Atala More articles by this author Hooman Sadri-Ardekani More articles by this author Expand All Advertisement PDF downloadLoading ...

Dysregulated YAP1/Hippo Pathway Contributes to Doxorubicin (ADM) Resistance in Acute Myeloid Leukemia (AML).

Dysregulated Yes-associated protein 1 (YAP1) is closely associated with cancer progression and chemo-resistance. We aim to explore the role of YAP1/Hippo pathway in regulating doxorubicin (ADM)-resistance in acute myeloid leukemia (AML). In this study, we established two ADM-resistant cell lines (THP-1/ADM and K562/ ADM). Then, cell viability and apoptosis were detected by MTT assay and FCM assay, respectively. Real-time PCR was performed to examine the expression of genes in the AML/ADM cells and the clinic BM samples. The levels of all related proteins were examined by Western blot. We found that the YAP1 and its downstream target genes, including EGFR, SOX2, and OCT4, were associated with ADM resistance, evidenced by the increased expression in ADMresistant AML/ADM cells and clinical BM specimens. Additionally, YAP1 ablation enhanced the promoting effects of ADM treatment on cell death in AML/ADM cells. Conversely, YAP1 increased ADM-a resistance in the original ADM-sensitive AML cells. These results may provide important new insights into understanding the role of YAP1 in regulating AML resistance by affecting CSCs characteristics. In summary, we evidenced that the dysregulated YAP1/Hippo pathway influenced ADM resistance in AML. YAP1 might be a novel biomarker for the treatment of drug resistance in AML.

Microenvironmental Stiffness Directs Chondrogenic Lineages of Stem Cells from the Human Apical Papilla via Cooperation between ROCK and Smad3 Signaling.

Cell-based cartilage tissue engineering faces a great challenge in the repair process, partly due to the special physical microenvironment. Human stem cell from apical papilla (hSCAP) shows great potential as seed cells because of its versatile differentiation capacity. However, whether hSCAP has potent chondrogenic differentiation ability in the physical microenvironment of chondroid remains unknown. In this study, we fabricated poly(dimethylsiloxane) (PDMS) substrates with different stiffnesses and investigated the chondrogenic differentiation potential of hSCAPs. First, we found that hSCAPs cultured on soft substrates spread more narrowly accompanied by cortical actin organization, a hallmark of differentiated chondrocytes. On the contrary, stiff substrates were favorable for cell spreading and stress fiber formation. More importantly, the increased chondrogenic differentiation of hSCAPs seeded on soft substrates was confirmed by characterizing increased extracellular proteoglycan aggregation through Alcian blue staining and Safranin O staining and enhanced markers toward chondrogenic differentiation including SRY-box transcription factor 9 (Sox9), type II collagen (Col2), and aggrecan in both normal α-minimum essential medium (αMEM) and specific chondrogenic medium (CM) culture conditions. Then, we investigated the mechanosensing/mechanotransduction governing the chondrogenic differentiation of hSCAPs in response to different stiffnesses and found that stiffness-sensitive integrin β1 and focal adhesion kinase (FAK) were essential for mechanical signal perception and were oriented at the start of mechanotransduction induced by matrix stiffness. We next showed that the increased nuclear accumulation of Smad3 signaling and target Sox9 facilitated the chondrogenic differentiation of hSCAPs on the soft substrates and further verified the importance of Rho-associated protein kinase (ROCK) signaling in regulating chondrogenic differentiation and its driving factors, Smad3 and Sox9. By using SIS3, the specific inhibitor of p-Smad3, and miRNA targeting Rho-associated protein kinase 1 (ROCK-1), we finally confirmed the importance of ROCK/Smad3/Sox9 axis in the chondrogenic differentiation of hSCAPs in response to substrate stiffness. These results help us to increase the understanding of how microenvironmental stiffness directs chondrogenic differentiation from the aspects of mechanosensing, mechanotransduction, and cell fate decision, which will be of great value in the application of hSCAPs in cartilage tissue engineering.

Identification of potential target genes of honokiol in overcoming breast cancer resistance to tamoxifen.

Honokiol (HON) inhibits epidermal growth factor receptor (EGFR) signaling and increases the activity of erlotinib, an EGFR inhibitor, in human head and neck cancers. In this study, using a bioinformatics approach and in vitro experiments, we assessed the target genes of HON against breast cancer resistance to tamoxifen (TAM). Microarray data were obtained from GSE67916 and GSE85871 datasets to identify differentially expressed genes (DEGs). DEGs common between HON-treated and TAM-resistant cells were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and protein-protein interaction (PPI) networks were constructed. Selected genes were analyzed for genetic alterations, expression, prognostic value, and receiver operating characteristics (ROC). TAM-resistant MCF-7 (MCF-7 TAM-R) cells were generated and characterized for their resistance toward TAM. A combination of HON and TAM was used for cytotoxicity and gene expression analyses. Molecular docking was performed using the Molecular Operating Environment software. PPI network analysis revealed that FN1, FGFR2, and RET were the top three genes with the highest scores. A genetic alteration study of potential target genes revealed MMP16 and ERBB4 as the genes with the highest alterations among the breast cancer samples. Pathway enrichment analysis of FGFR2, RET, ERBB4, SOX2, FN1, and MMP16 showed that the genetic alterations herein were likely to impact the RTK-Ras pathway. The expression levels of RET, MMP16, and SOX2 were strongly correlated with prognostic power, with areas under the ROC curves (AUC) ​​of 1, 0.8, and 0.8, respectively. The HON and TAM combination increased TAM cytotoxicity in MCF-7 TAM-R cells by regulating the expression of potential target genes ret, ERBB4, SOX2, and FN1, as well as the TAM resistance regulatory genes including HES1, VIM, PCNA, TP53, and CASP7. Molecular docking results indicated that HON tended to bind RET, ErbB4, and the receptor protein Notch1 ankyrin domain more robustly than its native ligand. HON could overcome breast cancer resistance to TAM, potentially by targeting FGFR2, RET, ERBB4, MMP16, FN1, and SOX2. However, further studies are required to validate these results.

CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

SOX10 is a key regulator of melanoma progression and promotes a melanocytic/differentiated state. Here we identified melanoma cell lines lacking SOX10 expression which retain their invivo growth capabilities. More importantly, we find that SOX10 can regulate T-cell infiltration in melanoma while also decreasing common cancer stem cell (CSC) properties. We show that SOX10 regulates CEACAM1, a surface protein with immunomodulatory properties. SOX10 directly binds to a distal CEACAM1 promoter region approximately 3-4kbps from the CEACAM1 transcriptional start site. Furthermore, we show that a SOX10-CEACAM1 axis can suppress CD8+ T-cell infiltration as well as reduce CSC pool within tumors, leading to reduced tumor growth. Overall, these results identify SOX10 as a direct regulator of CEACAM1, and uncover both a pro- and anti-tumorigenic roles for SOX10 in melanoma.

Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair

Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair