SOX2 In Cells Research Articles

Neurogenesis in the regenerating tail of tsinling dwarf skinks (Scincella tsinlingensis)

AbstractSpinal ganglia and peripheral nerves innervate the regenerating tail and muscles of lizards, which provide new opportunities to probe the mechanisms leading to successful functional recovery following spinal cord injury. The regenerated spinal cord and peripheral nerves were detected using immunohistochemistry in original and regenerating tails of Scincella tsinlingensis. Our results showed that positive PCNA, GFAP and SOX2 cells were observed in the ependymal ducts at 15 and 30 days after the autotomy (dpa), with a small number of immunopositive NSE neurons. GFAP and SOX2 positive cells were primarily localized along the regenerated spinal cord after 45 dpa, and the descending nerve of medulla tissue showed positive NSE. Peripheral axons distributed around the muscle and inside the connective tissue and muscle tissue at 15 dpa. The number of axons decreased after 30 dpa. The peripheral axons was mainly distributed between the connective tissue, muscle group and the muscle and epidermis, presenting a radial distribution centred on the cartilage tube at 120 dpa. MBP myelination labelling of the regenerated tail at 30 dpa revealed that the regenerated axons rapidly myelinated along the rostro‐caudal axis. The density of NMJ significantly increased at 120 dpa and 250 dpa. In the second regenerated tails, NMJ density at 250 dpa was still greater than original tails. Overall, these results indicated that neurogenesis was an early event and the ependymal cells were heterogeneous in the regenerated spinal cord of S. tsinlingensis. The regenerated peripheral axons presented a radial distribution centred on the cartilage canal, and the regenerated axons were rapidly myelinated along the rostro‐caudal axis. During the process of regeneration, there were a large number of regenerated neuromuscular joints with high density.

SOX9 negatively regulates the RLR antiviral signaling by targeting MAVS.

Mitochondrial virus-induced signal adaptor (MAVS), also known as VISA, IPS-1, and Cardif, is a crucial adaptor protein in the RIG-I-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes viral dsRNA and further transfers it to mitochondria, where it binds to MAVS through its CARD domain, generating a series of signal cascades. Transduction through this signaling cascade leads to phosphorylation and nuclear translocation of interferon regulatory factor 3/7 (IRF3/IRF7) and activation of NF-κB, which ultimately produces type I interferon (IFN) and proinflammatory cytokines. Here, our experiments demonstrated that overexpression of SRY-related high-mobility group protein 9 (SOX9) significantly inhibited Sendai virus (SeV)-induced and MAVS-mediated activation of the IFN-β promoter and ISRE. However, knocking out the expression of SOX9 in cells promoted SeV-induced IFN-β promoter and ISRE activation. Further studies have shown that SOX9 interacts with MAVS and targets MAVS to inhibit the association of MAVS-TRAF2, thereby inhibiting MAVS-mediated TRAF2 ubiquitination. Taken together, these results indicate that SOX9 downregulates IFN-β expression and antiviral signal transduction by targeting MAVS.

Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreERRosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-β) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

Abstract 1303: PRTN3, RGCC and SLCO4C1 are critical SOX9-regulated genes that control TNBC growth

Abstract Background: Breast cancer is the most common cancer in women, and about 12% U.S. women will develop invasive breast cancer over the lifetime. Triple-negative breast cancers (TNBCs) which lack expression of the estrogen and progesterone receptors and the HER2 protein.have a poor prognosis and are typically treated with chemotherapy. Our previous studies have demonstrated that SOX9 is essential for survival and metastasis of TNBCs via suppressing apoptosis and promoting epidermal to mesenchymal transition (EMT). Hypothesis: SOX9-regulated proteins control TNBC growth, and represent promising targets for the treatment of these potentially lethal breast cancers. Material and Methods: Several breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. RNA-Seq analysis was used to identify SOX9 downstream targets in TNBC cells (MDA MBA-232 and MDA MB-468 with SOX9 knockdown by siRNA transfection) or non-TNBC cells (MCF-7 with SOX9 overexpression by transfection of SOX9-expression plasmid), with support from MD Anderson's genomic biostatistics core. After identification of SOX9 downstream genes, we treated TNBC cells with or without siRNAs of these identified genes to evaluate their effect on TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. Significant genes are defined by using a false discovery rate (FDR) cut-off of 0.01 and log2 fold change of 2. Significant common genes are defined by using a FDR cut-off of 0.05 and log2 fold change of 1 among the cell lines. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student's t-test unless otherwise indicated. Results: TNBCs and late stage breast cancers showed higher SOX9 expression compared to non-TNBCs and early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell growth in vitro and in vivo. Using mRNA-seq analysis, we identified downstream genes regulated by SOX9 in breast cancer. 340 genes and 843 genes were suppressed in MDA MB-231 and MDA MB-468 respectively upon SOX9 knockdown, while 250 genes were upregulated in MCF-7 cells upon SOX9 overexpression. Between MDA MB-231 and MDA MB-468 cell line, there were 301 common genes were down-regulated upon SOX9 knockdown. 38 genes were down-regulated upon SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Among them, the top 10 genes that show largest difference between TNBCs and MCF-7 cells are MIOX, LAMB2P1, SLCO4C1, RGCC, PRTN3, LINC00173, UCN, LOC100129, VPREB3 and CTF1 genes. We then investigated the effect of these SOX9 downstream targets on cell growth in vitro. Loss of RGCC, PRTN3, and SLCO4C1 reduced cell growth in MDA MBA-231, and MDA MB-468, while having minimal effect on the growth of MCF-7 cells. Conclusion: Our results demostrate that the SOX9-regulated genes RGCC, PRTN3, and SLC04C1 are required for TNBC growth. These genes are critical regulators of TNBC growth and are potential novel targets for the treatment of these aggressive breast cancers. Grant Support: These studies were supported by a Breast Cancer Research Foundation (BCRF) grant (PB). Citation Format: Yanxia Ma, William M. Tahaney, Jing Qian, Jamal Hill, Yun Zhan, Abhijit Mazumdar, Powel H. Brown. PRTN3, RGCC and SLCO4C1 are critical SOX9-regulated genes that control TNBC growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1303.

SOX17, β-catenin and CyclinD1 expression in the endometrioid adenocarcinoma and influence of 5-AZA on expression.

The present study discusses the expression and effect of the SOX17 gene in endometrioid adenocarcinoma. MTT assay is performed to determine the growth inhibition ratio of the DNA methyltransferase inhibitor 5-AZA for endometrial carcinoma cells, and the real-time fluorescence quantification PCR (qRT-PCR) was used to detect the mRNA expression of SOX17, β-catenin, and CyclinD1 in endometrial carcinoma tissues before and after using 5-AZA to treat the endometrial carcinoma cell line. There were 30 cases on endometrioid adenocarcinoma tissues and 10 cases on normal endometrial tissues. The results revealed that the expression of SOX17 in endometrioid adenocarcinoma tissues was downregulated (P < 0.05), the expression of β-catenin and CyclinD1 was upregulated (P < 0.05), and the expression of SOX17, CyclinD1, and β-catenin was negatively correlated (r = -0.353, P > 0.05; R = -0.463, P < 0.05). The higher the histological grade and FIGO staging were, the lower the expression level of SOX17 was (P < 0.05). After HEC1A cells were treated by 5-AZA, the cell growth inhibition was most obvious (IC50 = 12.033) at 72 h, as determined by MTT assay. After cell treatment by 5-AZA, the genetic expression of SOX17 significantly increased, when compared with that before treatment (P < 0.05), while the genetic expression of β-catenin and CyclinD1 significantly declined (P < 0.05). These results indicate that the expression level of SOX17 in endometrioid adenocarcinoma declined, and the upregulated expression level of SOX17 in cells inhibited the growth of tumor cells.

Overexpression of SOX9 alleviates the progression of human osteoarthritis in vitro and in vivo.

PurposeRecent findings have identified that SOX9 served as a key role during the pathogenesis of osteoarthritis (OA). This study aimed to investigate the mechanisms by which SOX9 regulated the formation of OA in vitro and in vivo.Materials and methodsThe relative expressions of SOX9 in patients with OA and normal fracture of thighbone were analyzed by real-time-PCR. In vitro, IL-1β induced inflammatory response in human chondrocytes was used to evaluate the function of SOX9. The recombinant SOX9 lentivirus vector (Lenti-SOX9) was used to upregulate the expression of SOX9 in cells. ELISA was used to measure the concentration of tumor necrosis factor-α (TNF-α). The protein expressions of SOX9, matrix metalloproteinase-13 (MMP13), Collagen II, Aggrecan and Smad3 were analyzed by Western blot. Cell proliferation and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. In vivo, the effect of SOX9 on surgically induced OA mice was evaluated.ResultsThe gene level of SOX9 was remarkably downregulated in patients with OA compared with normal people, while the concentration of TNF-α was upregulated. In addition, IL-1β reduced the expressions of SOX9, Collagen II and Aggrecan and increased the level of MMP13 in chondrocytes. Moreover, Lenti-SOX9 notably inhibited IL-1β-induced growth inhibition and apoptosis in chondrocytes via increasing the expression of Smad3. Finally, Lenti-SOX9 markedly alleviated the symptoms of OA mice in vivo.ConclusionUpregulation of SOX9 inhibited IL-1β-induced inflammatory response via increasing the level Smad3 in human chondrocytes and exhibited therapeutic effect on surgically induced OA mice in vivo. Therefore, SOX9 may serve as a potential target in the treatment of OA in the future.

Abstract 2618: SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes

Abstract Background: There are few effective targeted therapies available for triple-negative breast cancers (TNBCs), the most aggressive form of breast cancer. A better understanding of the critical molecular regulators is necessary to develop effective targeted therapies for TNBCs. Our previous study focused on SOX9 transcription factor and determined that SOX9 is highly expressed in TNBCs which is required for TNBC cell survival and metastasis. Hypothesis: SOX9 controls TNBC cell growth and metastasis via directly regulating cell apoptosis and epithelial-mesenchymal transition (EMT) genes. Material and Methods: The breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. Cell growth was measured using an automated cell counting assay. Cell migration and invasion were detected by transwell migration and invasion assays. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. Xenograft mice model was established to evaluate the effect of SOX9 on TNBC cell growth in vivo. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. An EMT PCR array and the CHIP-seq enrichment analysis based on published SOX9-CHIP-seq datasets were performed to identify effect of SOX9 on downstream pathways. SOX9 binding peaks in gene promoter region were determined and confirmed by SOX9-CHIP-seq datasets and CHIP assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student’s t-test unless otherwise indicated. Results: Using available gene profiling datasets, we found that high expression of SOX9 was correlated with worse overall survival in Boersma breast dataset, but not in other datasets. Late stage breast cancers showed higher SOX9 expression compared to early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell survival and invasion in vitro and in vivo. To identify the molecular mechanisms of these functions, we used CHIP-seq data and CHIP assays to demonstrate that SOX9 directly binds to the promoter of several extrinsic apoptosis-inducing factors (such as fadd and tnfrsf10b) and represses their expressions. Loss of SOX9 caused up-regulation of the expression of these genes, consistent with the induction of apoptosis. We then investigated the effect of SOX9 on EMT-regulating genes. Loss of SOX9 induced down-regulation of most EMT genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of several EMT genes (such as vim and ctnnb1) and increases the expression of these genes. Conclusion: Our results demonstrate that by directly binding and regulating cell apoptosis and EMT gene expression, SOX9 controls TNBC survival and induces EMT resulting in aggressive and potentially lethal tumors. Grant Support: These studies were supported by a Breast Cancer Research Fund (BCRF) grant (PB). Citation Format: Yanxia Ma, Lakshmi Bollu, Jamal Hill, Yun Zhang, William M. Tahaney, Abhijit Mazumdar, Powel H. Brown. SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2618.

Abstract 3357: Genetic regulation of RUNX2 induce apoptotic cell death through regulating the expression of SOX2 in CRPC-NE cells

Abstract Although runt-related transcription factor 2 (RUNX2) is well known for its bone-specific transcriptional regulator in highly metastatic prostate cancer, there is little knowledge about its role of RUNX2 in castration-resistant neuroendocrine prostate cancer (it is named CRPC-NE). It is well known that expression of Androgen Receptor (AR) in CRPC-NE cases is lower than other types and AR suppresses RUNX2 expression by binding it and abrogating its recruitment to DNA. This fact suggests that in CRPC-NE, low expression of AR, the addiction of RUNX2 is relatively higher than that of other prostate cancer types. Extensive analysis of the clinical datasets revealed that RUNX2 is one of the most consistently up-regulated genes in CRPC-NE and the overexpression of SOX2 confers an accelerated disease progression and shortened overall survival periods to CRPC-NE patients. We also found that the silencing RUNX2 induced apoptotic cell death in CRPC-NE cells independent on p53, but through transcriptionally down-regulating SOX2 expressions. Mechanistically, ChIP-qPCR assay and luciferase reporter experiments confirmed that RUNX2 positively regulates SOX2 expression by binding to the consensus RUNX2 binding sequence (5'-TGTGGT-3') located in the proximal promoter region of SOX2. Short hairpin RNA (shRNA)-mediated knockdown of RUNX2 in the CRPC-NE cell line PC-3 induced the decreased expression of SOX2 and the increased expression of cleaved form of caspase-3 and PARP in a p53 independent manner. Silencing of SOX2 in PC-3 cells suppressed the proliferation of these cancer cells and induced apoptosis, which phenotypes were rescued by restoring SOX2 in the cells. These data indicates that inhibition of SOX2-apoptosis axis via RUNX2 could be a better therapeutic choice in CRPC-NE. Lastly, we examined the efficacy of our novel molecule Chlorambucil-conjugated Pyrrole Imidazole Polyamide (we named it as Chb-M'), which specifically binds to the consensus RUNX2 binding sequence and inhibits RUNX2 target gene. Surprisingly, Chb-M' had tremendous inhibitory effect on PC-3 cells (IC50 value at 620 nM) through effective SOX2 inhibition. Additionally, it was exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of CRPC-NE. Taken together, our work identified a novel interaction of RUNX2 and SOX2-apoptosis axis, offering a new strategy for the management of poor-prognostic advanced stage CRPC-NE cancer patients. Citation Format: Yuki Noguchi, Natsuki Wariishi, Shiina Iwai, Gengo Kashiwazaki, Junichi Taniguchi, Toshikazu Bando, Masaya Baba, Souichi Adachi, Hiroshi Sugiyama, Yasuhiko Kamikubo. Genetic regulation of RUNX2 induce apoptotic cell death through regulating the expression of SOX2 in CRPC-NE cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3357.

Abstract 3347: SOX9 is a critical regulator of triple-negative breast cancer growth and invasion

Abstract Background: Triple-negative breast cancers (TNBCs) have the worst prognosis of all breast cancers, and have few available therapies other than non-specific and toxic chemotherapy. To identify novel targets for TNBCs, we investigated expression levels of transcription factors (TFs) in TNBCs compared to those in Non-TNBCs. Our previous analyses identified the TFs highly expressed in TNBCs. Of these, six SRY (Sex Determining Region Y)-related HMG-box (SOX) TFs were highly expressed in TNBCs as compared to non-TNBCs. SOX genes belong to a superfamily of gene. There are approximately 20 SOX genes in humans and mice. Hypothesis: SOX TFs are important regulators of TNBC cell growth and metastasis. Material and Methods: We measured breast cancer cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell assays in non-TNBC (MCF7 and ZR75-1) and TNBC (MDA231 and MDA468) cells. DOX-inducible SOX9-knockout (KO) cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs were used to knockdown (KD) SOXs in the different breast cancer cells. Cell cycle phase and apoptosis were detected using flow cytometric analysis. Protein and mRNA levels of SOX9 in cell lines were examined by western blotting and qRT-PCR assays. SOX9 RNA expression data were obtained from the Oncomine Database. The Curtis dataset was used to analyze survival according to SOX9 expression using Kaplan-Meier survival curves and the statistical significance was determined using the log-rank test. Results: We performed a screen using specific siRNA targeting the 6 SOX transcription factors (SOX4, 6, 8, 9, 10 and 11) that are high-expressed in TNBC, and measured the effect of loss on SOX expression on TNBC cell growth and invasion. SOX4, 6, 8, 9, and 10-KD using siRNA caused decreased cell proliferation (by 30% or more) of MDA231 and MDA468 TNBC cells, but not of MCF7 and T47D cell lines (non-TNBC). SOX11 or 17-KD had no effect on breast cancer growth. SOX9-KD and SOX9-KO decreased cell migration and invasion of MDA231 and MDA468 cells. Reduced expression of SOX9 also inhibited the in vivo growth and metastasis of MDA MB-231 and LM2 cells in mice. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. Our studies also demonstrated that loss of SOX9 induced a G1 to S cell cycle arrest and apoptosis. In addition, high expression of SOX9 was correlated with worse overall survival and 5-year disease-free survival in patients with TNBC breast cancer. Conclusion: Our results demonstrate that the SOX9 acts as an essential molecule regulating TNBC growth and invasion. In the future, it may be possible to target SOX9 and its downstream genes to treat TNBC and prevent its metastasis. Grant Support: These studies were supported by a Susan G. Komen for the Cure Promise Grant (PB). Citation Format: Yanxia Ma, Jonathan Shepherd, Dekuang Zhao, Lakshmi Bollu, Jamal Hill, Yun Zhang, Abhijit Mazumdar, Powel H. Brown. SOX9 is a critical regulator of triple-negative breast cancer growth and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3347.

SOX9 activity is induced by oncogenic Kras to affect MDC1 and MCMs expression in pancreatic cancer.

SRY (sex determining region Y)-box 9 (SOX9) is required for oncogenic Kras-mediated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasias (PanINs) and ultimately pancreatic ductal adenocarcinoma (PDAC). However, how oncogenic Kras affects SOX9 activity is not yet understood, and SOX9-associated genes in PDAC are also unknown at all. Here, we investigated the mechanistic link between SOX9 and oncogenic Kras, studied biological function of SOX9, and identified SOX9-related genes and their clinical significance in patients with PDAC. Our studies reveal that oncogenic Kras induces SOX9 mRNA and protein expression as well as phosphorylated SOX9 expression in human pancreatic ductal progenitor cells (HPNE) and pancreatic ductal cells (HPDE). Moreover, oncogenic Kras promoted nuclear translocation and transcriptional activity of SOX9 in these cells. TAK1/IκBα/NF-κB pathway contributed to induction of SOX9 by oncogenic Kras, and SOX9 in turn enhanced NF-κB activation. SOX9 promoted the proliferation of HPNE and PDAC cells, and correlated with minichromosome maintenance complex components (MCMs) and mediator of DNA damage checkpoint 1 (MDC1) expression. The overexpressive MDC1 was associated with less perineural and lymph node invasion of tumors and early TNM-stage of patients. Our results indicate that oncogenic Kras induces constitutive activation of SOX9 in HPNE and HPDE cells, and Kras/TAK1/IκBα/NF-κB pathway and a positive feedback between SOX9 and NF-κB are involved in this inducing process. SOX9 accelerates proliferation of cells and affects MCMs and MDC1 expression. MDC1 is associated negatively with invasion and metastasis of PDAC.

ZNF606 interacts with Sox9 to regulate chondrocyte differentiation

SRY-related high-mobility-group box 9 (Sox9) gene is a transcription factor that plays an essential role in chondrocyte differentiation and cartilage formation. In this study, we identified the transcriptional factor ZNF606 as an interacting partner for Sox9 in cells. We further demonstrated that overexpression of ZNF606 inhibited the transcriptional activity of Sox9, while knockdown of ZNF606 increased Sox9-mediated transcription. Chromatin immunoprecipitation analysis revealed that ZNF606 prevents Sox9 binding to the enhancers of its target gene col2a1. Importantly, the interaction between ZNF606 and Sox9 was decreased during chondrocyte differentiation. Consistent with these findings, ZNF606 inhibited chondrocyte differentiation. Thus, our results demonstrate that ZNF606 acts as a novel Sox9 co-regulator that inhibits Sox9-mediated chondrocyte differentiation.

Role of the embryonic protein SOX2 in cholangiocarcinoma.

SOX2 overexpression correlates with aggressive behavior and poor overall survival in cholangiocarcinoma (CCA). However, the cellular functions and precise role of SOX2 in CCA have not been elucidated. Here, we inserted SOX2 coding sequence to establish a CAA cell line which stably overexpressed SOX2. In vitro experiments showed that overexpression of SOX2 in cells was associated with increased cell proliferation, suppressed cell apoptosis, as well as enhanced cell migration and invasion. Our findings may lead to a better understanding of the biological effect of SOX2 and provide mechanistic insights for developing potential therapeutic strategies for CCA treatment.

P38α Negatively Regulates Survival and Malignant Selection of Transformed Bronchioalveolar Stem Cells

Lung cancer is the cause of most cancer-related deaths in the Western world. Non-small cell lung cancer accounts for almost 80% of all lung cancers, and 50% of this type are adenocarcinomas. The cellular and molecular origin of this type of lung cancer remains elusive and the mechanisms are poorly known. It is known that K-Ras mutations appear in 25–30% of lung adenocarcinomas and it is the best known single mutation that can be related to lung cancers. Recently, it has been suggested that a putative population of mouse bronchioalveolar stem cells could be considered as the cell of origin of adenocarcinomas. These cells are expanded in the early stages of lung tumorigenesis. We have isolated a population of mouse bronchioalveolar stem cells and induced their transformation by oncogenic K-RasG12. Different approaches have shown that an intracellular network linking the p38α MAPK and the PI3K-Pdk1 pathways is involved in regulating the survival and malignant progression of the transformed cells. Absence of p38α catalytic activity leads to further Pdk1 activation (independent of Akt and Erk activity), enhancing the survival and proliferation of the more malignant lung cancer cells. This specifically selects high Sca-1/Sox9 cells that harbour a stronger colonizing potential, as they maintain their capacity to produce secondary tumors after serial transplantations.

Enhancer Profiling Reveals SOX9 As a Novel Transcription Regulator of B Cell Activation and DLBCL Transformation

Abstract 527Antigen stimulation of naïve B cells (NBC) induces differentiation with a phenotype characterized by robust proliferation and genomic instability tolerance to enable activated germinal center B cells (GCB) to undergo immunoglobulin affinity maturation. Aberrant genetic events resulting from this process lead to malignant transformation and diffuse large B cell lymphoma (DLBCL). Phenotypic progression from quiescent NBC to activated GCB and malignant DLBCL involves major shifts in gene expression. Recent studies suggest that enhancers play a key role in mediating cell type-specific gene regulation. We therefore postulated that enhancers are involved in dictating the gene expression programs that govern normal and malignant B cell phenotypes; and systematic discovery of enhancers coupled with bioinformatic analysis would uncover key enhancer-binding transcription factors (TFs) that regulate these cell states.To test this hypothesis, we performed ChIP-seq on enhancer histone marks, i.e. H3K4me2, H3K27Ac, and H3K4me3, in primary NBC and GCB, and in DLBCL cell lines in biological replicates. We defined enhancers by the criterion of H3K4me2hiH3K4me3low. We observed a striking pattern of enhancer re-organization between cell types. First, we found a larger number of enhancers in primary B-cells (∼20,000) than in DLBCL (∼12,000). Second, we confirmed that enhancers are cell type-specific. For example, 11,492 out of 20,173 NBC enhancers were lost during transition to GCB (loss of H3K4me2 enrichment), while 13,088 new enhancers were gained in GCB. A similar phenomenon was also observed in DLBCL when compared to either NBC or GCB. This re-organization of enhancers suggests that cells may have dynamic gene regulatory programs during differentiation or malignant transformation. To discover TFs that act through enhancers, we used bioinformatic analyses, including FIRE and MEME, to search for TF consensus binding sequences within enhancers. Over-represented DNA motifs included motifs of SPI1, RUNX1, STAT3, RELA and SOX9, etc. SOX9 motif was significantly enriched in GCB specific enhancers (p=3.07e-15). SOX9 belongs to the SOX family TFs and plays an important role in cartilage development, sex determination, and intestinal differentiation but has not been implicated in B cell development.To investigate the role of SOX9 in B cell activation and malignant transformation, we first examined the expression of SOX9 in these cells. RNA-seq performed on human tonsilar NBC and GCB showed more than 20-fold increase of SOX9 mRNA in GCB as compared to NBC (6.75±0.80 vs 0.29±0.14, RPKM, p=0.0002). In addition, SOX9 expression was maintained in plasma B cells (2.88±0.49, RPKM). To understand how SOX9 regulates transcriptional programming in GCB, we performed SOX9 ChIP-seq in GCB to look for its targets. We found that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb of TSS) associated with 963 genes. These target genes were significantly enriched in many important pathways including cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) (p<0.001). One of the SOX9 targets is PRMD1, a TF that controls the transition from GCB to plasma cells, suggesting that SOX9 may be involved in B cell terminal differentiation. To our surprise, we did not detect SOX9 mRNA in 10 out of 12 DLBCL cell lines by RNA-seq. Moreover, SOX9 was not expressed in the majority of primary malignant non-Hodgkin’s lymphoma cases studied by IHC in the Human Protein Atlas project. To examine whether reduced SOX9 expression could induce malignant transformation, we used shRNA to knockdown Sox9 in mouse BCL1 lymphoma cells and subjected them to colony forming assay in semi-solid methylcellulose. Knockdown of Sox9 increased BCL1 colony forming ability by 50% as compared to scramble, suggesting that loss of SOX9 expression maybe important for lymphomagenesis.In summary, we identified a novel germinal center TF, SOX9, by examining enrichment of TF motifs within enhancer regions uncovered by ChIP-seq. Our current data suggest that SOX9 may play an important role in germinal center reaction and subsequent terminal differentiation by regulating key factors, such as PRDM1, and that loss of SOX9 may contribute to DLBCL malignant transformation by potentially blocking the terminal differentiation of mature GCB. Disclosures:No relevant conflicts of interest to declare.

Disruption of a Sox9–β-catenin circuit by mutant Fgfr3 in thanatophoric dysplasia type II

Mutations in fibroblast growth factor (FGF) receptors are responsible for a variety of skeletal birth defects, but the underlying mechanisms responsible remain unclear. Using a mouse model of thanatophoric dysplasia type II in which FGFR3(K650E) expression was directed to the appendicular skeleton, we show that the mutant receptor caused a block in chondrocyte differentiation specifically at the prehypertrophic stage. The differentiation block led to a severe reduction in hypertrophic chondrocytes that normally produce vascular endothelial growth factor, which in turn was associated with poor vascularization of primary ossification centers and disrupted endochondral ossification. We show that the differentiation block and defects in joint formation are associated with persistent expression of the chondrogenic factor Sox9 and down-regulation of β-catenin levels and activity in growth plate chondrocytes. Consistent with these in vivo results, FGFR3(K650E) expression was found to increase Sox9 and decrease β-catenin levels and transcriptional activity in cultured mesenchymal cells. Coexpression of Fgfr3(K650E) and Sox9 in cells resulted in very high levels of Sox9 and cooperative suppression of β-catenin-dependent transcription. Fgfr3(K650E) had opposing effects on Sox9 and β-catenin protein stability with it promoting Sox9 stabilization and β-catenin degradation. Since both Sox9 overexpression and β-catenin deletion independently blocks hypertrophic differentiation of chondrocytes and cause chondrodysplasias similar to those caused by mutations in FGFR3, our results suggest that dysregulation of Sox9 and β-catenin levels and activity in growth plate chondrocytes is an important underlying mechanism in skeletal diseases caused by mutations in FGFR3.

A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

208 REPROGRAMMING OF BOVINE SOMATIC CELLS WITH CELLULAR EXTRACTS FROM ADIPOSE-DERIVED ADULT STEM CELLS AND HUMAN EMBRYONIC STEM CELLS

The differentiation potential of adult stem cells (ASC) has long been considered limited to the cell types present in the organ or tissue from which they are derived. Several studies have challenged this notion by demonstrating that ASC exhibit a high degree of plasticity; therefore, it is likely that these cells possess an epigenomic regulatory pattern more like pluripotent cells than somatic cells. In the present study, we used quantitative RT-PCR (qRT-PCR) to analyse bovine adipose-derived adult stem cells (ADAS) and bovine fetal fibroblasts (BFF) for the presence of Oct-4, Nanog and Sox-2. The ADAS cells were isolated from the stromal fraction of bovine adipose tissue from adult cows and BFF were isolated from 50-d-old fetuses. Both cell types were expanded through 3 passages in DMEM supplemented with 10% fetal bovine serum. Transcripts for these pluripotency-associated genes were detected in all samples, supporting the hypothesis that ADAS and BFF cells are less differentiated than other somatic cell types. To induce reprogramming we exposed BFF cells to ADAS cell extracts and human ESC (hESC) extracts. Cellular components of ADAS cells and hESC were extracted using cell lysis buffer and Dounce homogenization. Individual quanta of BFF cells were permeabilized with streptolysin O and allowed to incubate with 100 μL of ADAS cell extracts or hESC extracts containing an ATP regenerating system. BFF cells were then resealed with complete culture medium containing 2 mM CaCl2 and cultured for 72 h before mRNA isolation. Quantitative RT-PCR was performed to determine transcript levels for Oct4, Sox2, Nanog and poly adenylate polymerase (PAP) as a reference gene in untreated BFF cells, ADAS cells and treated BFF cells. Transcript levels were quantified by relative quantification using the ΔΔCT method and expressed as ratios of the target genes (Oct4, Sox2 and Nanog) to the reference gene (PAP) and normalized against a calibrator consisting of untreated BFF cells. Differences between normalized ratios were analysed by Student's t-test. We did not detect a difference in Oct-4 and Nanog transcript levels between untreated BFF cells and BFF cells exposed to ADAS cell extracts. Comparison between untreated BFF cells and BFF cells treated with ADAS cell extracts revealed a higher (P ≤ 0.05) Sox-2 expression in the extract-treated cells. The BFF cells were then exposed to extracts derived from hESC. Unlike other studies that have reported the up-regulation of Oct-4, Nanog and Sox-2 in cells treated with hESC extracts, we did not detect a difference in Oct-4, Nanog and Sox-2 expression levels between untreated BFF cells and BFF cells treated with hESC extracts. This may be due to expression differences between species. Currently, we are assessing the ability of mouse ESC extracts to reprogram bovine ADAS cells. This work was financed in part by a grant from the LSU System for the ACRES/LSU Collaborative Research Program.

235 EXPRESSION OF PLURIPOTENCY-ASSOCIATED GENES IN BOVINE FETAL FIBROBLAST CELLS AND ADIPOSE-DERIVED STEM CELLS

The extraordinary nature of embryonic stem cells (ESC) lends them the remarkable ability to give rise to all the cell types of a mammalian organism, an attribute known as pluripotency. The pluripotent state of ESC is dependent on the expression of the genes Oct-4, Nanog, and Sox-2, which have also been identified as the key transcriptional regulators of pluripotency. Until recently, Oct-4, Nanog, and Sox-2 were believed to be expressed solely in ESC; however, studies have reported the expression of these genes in some sources of adult stem cells (ASC) of both the mouse and pig. In the current study, we examined cells derived from bovine adipose tissue and fetal fibroblast for the expression of Nanog and Sox-2. Cells were isolated from the adipose tissue of an adult cow and the skin of 2 male fetuses, approximately 70 and 80 d old, and cultured through 6 passages. Total RNA was isolated from each of the 3 cell lines at passages 2, 4, and 6 using TRI Reagent®. Using the Bio-Rad iScript™ cDNA Synthesis Kit, the resulting RNA products were transcribed into cDNA 3 times for separate RT-PCR reactions. RT-PCR was performed with primers for Nanog, Sox-2, and Poly A Polymerase (PAP), as a reference gene for normalization. Primer sets for Nanog and PAP have previously been verified to amplify their respective transcripts in bovine embryos. Reaction products of PCR were subjected to electrophoresis and analyzed by Quantity One software (Bio-Rad). The number of pixels in each electrophoresis band was used to determine the relative transcript levels and was expressed as a ratio of Nanog or Sox-2 to PAP (Table 1). Both Nanog and Sox-2 were present in all cell lines at all 3 passages. Because Oct-4 is known to function synergistically with Nanog and Sox-2 to confer pluripotency, we are currently examining these samples for the presence of Oct-4. The presence of transcripts for the pluripotency-associated genes Nanog and Sox-2 in cells that are not inherently pluripotent suggests that there might be another level of regulation at the translational level for these genes. Table 1.Transcript abundance of pluripotency-associated genes relative to PA P in bovine fetal fibroblast (BFF) and adipose-derived adult stem cells (ADAS)

Transcription factor Sox-2 inhibits co-activator stimulated transcription.

Previous studies have shown that transcription of the fibroblast growth factor-4 (FGF-4) gene by early embryonic cells is dependent upon a powerful distal enhancer located 3 kb downstream of the transcription start site within the untranslated region of the last exon. The transcription factors Sox-2 and Oct-3 cooperatively bind to critical cis-regulatory elements within the enhancer to synergistically activate transcription. Moreover, the co-activator p300 can mediate the synergistic activity of Sox-2 and Oct-3, and p300 associates with the FGF-4 enhancer in vivo. Embryonal carcinoma (EC) cells have been used extensively as a model system to study the regulation of the FGF-4 gene during early development. Recently, it has been suggested that suboptimal levels of Sox-2 expression in F9 EC cells limit the transcription of the FGF-4 gene. The studies presented in this report argue that Sox-2 levels are not limiting in F9 EC cells. Moreover, overexpression of Sox-2 in F9 EC cells decreases FGF-4 promoter activity. In addition, overexpression of Sox-2 in these cells inhibits activation by the co-activators p300, CBP, and OCA-B in a manner that requires the transactivation domain of Sox-2. These findings suggest that Sox-2 levels in F9 EC cells are regulated carefully to avoid interference with the transcription of critical genes.