Regulates Sox2 Expression Research Articles

Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.

SummaryNon-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

Long non-coding RNA PVT1 contributes to cell growth and metastasis in non-small-cell lung cancer by regulating miR-361-3p/SOX9 axis and activating Wnt/β-catenin signaling pathway

Lung cancer is the most frequent cause of cancer-related mortality in men, and 85 % of lung cancer is diagnosed as non-small-cell lung cancer (NSCLC). Plasmacytoma variant translocation1 (PVT1) serves as an oncogenic factor in NSCLC. However, the pathogenesis of PVT1 in NSCLC is still vague. The expression levels of PVT1, sex determining region Y (SRY)-related high mobility group (HMG)-box9 (SOX9), and microRNA (miR)-361-3p in NSCLC tissues and cells were detected by quantitative real-time polymerase chain reaction (RT-qPCR). The protein levels of SOX9, β-catenin, and c-Myc were detected by western blot assay. Cell proliferation, apoptosis, migration and invasion were measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, transwell assays, severally. The interaction between miR-361-3p and PVT1 or SOX9was predicted by starBase, and then verified by the dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. PVT1 and SOX9 was highly expressed in NSCLC tumor tissues and cells. PVT1 facilitated proliferation, migration, invasion and hindered apoptosis of NSCLC cells. MiR-361-3p was a target of PVT1 in NSCLC cells. SOX9 acted as a target of miR-361-3p. PVT1 worked as a miR-361-3p sponge to regulate SOX9 expression. PVT1 modulate the Wnt/β-catenin Signaling Pathway by miR-361-3p/SOX9 axis. Our studies disclosed that PVT1 boosted proliferation, migration, invasion and curbed apoptosis by miR-361-3p/SOX9 axis, providing the possible therapeutic strategy for NSCLC.

Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

BackgroundContinuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS.MethodsHuman bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting.ResultscLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2.ConclusionsCollectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

Regulation of the Sox3 Gene in an X0/X0 Mammal without Sry, the Amami Spiny Rat, Tokudaia osimensis

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.

SOX9 Immunohistochemistry in the Distinction of Angiomatoid Fibrous Histiocytoma From Histologic Mimics: Diagnostic Utility and Pitfalls.

Angiomatoid fibrous histiocytoma (AFH) can be diagnostically difficult because of its varied histologic appearance and potential to occur at unusual sites. The identification of recurrent rearrangements (EWSR1-CREB1, EWSR1-ATF1, and FUS-ATF1) is a helpful diagnostic tool. Additional immunohistochemical markers in AFH could aid in restricting the differential diagnosis and selecting appropriate cases for targeted molecular studies. SOX9 is a transcription factor that is crucial for chondrogenesis and is expressed in neoplasms with chondroid differentiation, and other malignant bone and soft tissue tumors. Recently a role of EWS in regulation of SOX9 expression has been reported, the rearrangements typical of AFH may play a role in SOX9 expression. In this study, we analyzed SOX9 expression in 13 pediatric AFH with varying histology, and an additional 80 cases of other myofibroblastic or fibrohistiocytic lesions. SOX9 expression was present in 11 of 13 AFH, 2 of 53 dermatofibroma (1 aneurysmal and 1 cellular) and 1 calcifying aponeurotic fibroma. The remaining tumors were negative. SOX9 is selectively expressed in AFH and may be a useful maker in combination with desmin, CD99, CD68, and EMA in small biopsies, especially in cases with unusual morphologic features. SOX9 appears to be highly specific for AFH, being weakly expressed in a subset of aneurysmal dermatofibroma and absent in other myofibroblastic lesions, except calcifying aponeurotic fibroma. It should be used with caution when differentiating AFH from malignant neoplasms such as Ewing sarcoma.

MiR-190 enhances endocrine therapy sensitivity by regulating SOX9 expression in breast cancer

BackgroundBreast cancer is the most common cancer among women worldwide, and approximately 70% of breast cancers are hormone receptor-positive and express estrogen receptor-α (ERα) or/and progesterone receptor. Therapies targeting ERα have been successfully used in patients with ERα+ breast cancer. However, intrinsic or acquired resistance to anti-estrogen therapy presents a major challenge. The Wnt/β-catenin signaling pathway regulates various processes that are important for cancer progression, and emerging evidences have shown a close interaction between Wnt/β-catenin and ERα signaling. miR-190 is also involved in ER signaling and our previous study indicated that miR-190 suppresses breast cancer metastasis.MethodsThe effect of miR-190 on breast cancer anti-estrogen sensitivity was investigated both in vitro and in vivo. The protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively. Chromatin immunoprecipitation and dual-luciferase reporter assays were used to validate the regulation of the zinc-finger E-box binding homeobox 1/ ERα-miR-190-SRY-related high mobility group box 9 (ZEB1/ERα-miR-190-SOX9) axis.ResultsmiR-190 increased the anti-estrogen sensitivity of breast cancer cells both in vitro and in vivo. miR-190 inhibited Wnt/β-catenin signaling by targeting SOX9, and its expression inversely correlated with that of SOX9 in breast cancer samples. Furthermore, ERα and ZEB1 competitively regulated miR-190 expression.ConclusionsOur data uncover the ZEB1/ERα-miR-190-SOX9 axis and suggest a mechanism by which the Wnt/β-catenin signaling pathway is involved in breast cancer anti-estrogen therapy.

HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo.

During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway in controlling cell positioning and expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.

Abstract 4092: Glycosyltransferase ST6Gal-I promotes pancreatic ductal adenocarcinoma progression and metastasis

Abstract Sialyltransferase ST6Gal-I adds α2-6 sialic acids to select N-glycosylated cell surface receptors, thereby modulating receptor function and intracellular signaling. ST6Gal-I is upregulated in multiple human cancers, including >70% of pancreatic ductal adenocarcinoma (PDAC) samples. ST6Gal-I confers cancer stem cell (CSC) properties evidenced by tumorspheroid growth, chemoresistance and tumor initiating potential. As an example, we previously reported that ST6Gal-I imparts gemcitabine resistance by dampening DNA damage. One possible mechanism by which ST6Gal-I promotes a CSC phenotype is by increasing the expression of the master stem cell transcription factor, Sox-9. Forced overexpression or knockdown of ST6Gal-I directly regulates Sox9 expression in a wide array of established cancer lines. Sox9 is known to be a potent driver of acinar to ductal metaplasia (ADM), a key PDAC initiating event. To interrogate the role of ST6Gal-I and Sox9 in ADM, ST6Gal-I was overexpressed in the nontransformed acinar cell line, 266-6. ST6Gal-I overexpression was found to induce expression of Sox9 and other ductal markers. Furthermore, ST6Gal-I overexpressing cells spontaneously formed foci in adherent culture, a hallmark of malignant transformation. Contrarily, ST6Gal-I knockdown in 266-6 cells decreased cell viability. Post ADM, PDAC progresses through the formation of PanINs. This process of progressive lesion formation leading to frank PDAC can be replicated in murine models of pancreatic cancer utilizing pancreas specific oncogenic K-Ras (KC). To investigate the role of ST6Gal-I pertaining to PDAC in vivo we generated ST6Gal-I knock-in (KI) mice with oncogenic K-Ras (KC-ST6Gal-I KI). The KC-ST6Gal-I KI mice presented with significantly decreased overall survival (median = 4.3 months) compared to the KC mice (median = 14 months). This was coupled with a marked increase in liver and lung metastasis in KC-ST6Gal-I KI mice compared to KC mice. To better understand the molecular events underlying ST6Gal-I's PDAC-promoting function, we generated organoids from KC, KC-ST6Gal-I KI, ST6Gal-I KI and WT mice. Preliminary data indicate that there is increased Sox-9 expression in ST6Gal-I KI organoids compared to WT controls. As well, the number of organoids formed in culture is increased in ST6Gal-I KI mice compared to the control. Since organoids are derived from the resident stem cells, this data further indicates increased stemness with enhanced ST6Gal-I expression. In ongoing studies we are defining the signaling mechanisms underpinning a ST6Gal-I/Sox9 axis in PDAC initiation events and other aspects of CSC behavior. These collective studies highlight a novel role for tumor glycans in PDAC pathogenesis, and implicate ST6Gal-I as a promising therapeutic target. Citation Format: Asmi Chakraborty, Robert B. Jones, Christopher A. Klug, Lesley E. Smythies, Susan L. Bellis. Glycosyltransferase ST6Gal-I promotes pancreatic ductal adenocarcinoma progression and metastasis [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 4092.

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.

Tumor-derived exosomal lnc-Sox2ot promotes EMT and stemness by acting as a ceRNA in pancreatic ductal adenocarcinoma.

Long noncoding RNAs (lncRNAs) or exosomes have recently been shown to play vital regulatory or communication roles in cancer biology. However, the roles and mechanisms of exosomal lncRNAs in tumor invasion or metastasis of pancreatic ductal adenocarcinoma (PDAC) remain unknown. In this study, we aimed to investigate the detailed roles and mechanisms of tumor-generated exosomes in progression and metastasis of PDAC in vitro and in vivo. We identified a lncRNA-Sox2ot from exosomes of highly invasive PDAC cells, and analyzed the expression of Sox2ot in the plasma samples and found that the plasma exosomal Sox2ot expression was high and correlated with TNM stage and overall survival rate of PDAC patients. Further research showed that Sox2ot promotes epithelial-mesenchymal transition (EMT) and stem cell like properties by regulating Sox2 expression. Sox2ot competitively binds to the miR-200 family to regulate the expression of Sox2, thus promoting invasion and metastasis of PDAC. We also confirmed the transmission of the exosomes from producer cells to recipient PDAC cells, exosomal Sox2ot can promote tumor invasion and metastasis in vitro and in vivo. We further confirmed tumor generated exosomes could excrete to tumor cell or blood circulation in vivo condition. Finally, we observed a decreased exosomal Sox2ot expression in postoperative blood samples of PDAC patients. The exosomal lncRNA Sox2ot plays important roles in tumor progression and may be a useful maker for pancreatic cancer prognosis.

Sense-Antisense lncRNA Pair Encoded by Locus 6p22.3 Determines Neuroblastoma Susceptibility via the USP36-CHD7-SOX9 Regulatory Axis

Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.

Abstract 3518: Brg1 plays a critical role in PanIN formation through regulating Sox9 expression

Abstract Background: Mutations of SWI/SNF chromatin remodeling complexes are one of the hallmarks of human pancreatic ductal adenocarcinoma (PDA). Brahma related gene 1 (Brg1) is a catalytic ATPase subunit of SWI/SNF complexes and Brg1 is silenced in about 10% of human PDA. Recent study revealed that Brg1 inhibits formation of intraductal pancreatic mucinous neoplasm (IPMN) and IPMN-derived PDA in the context of oncogenic Kras and that IPMNs originate from adult pancreatic ductal cells, demonstrating a tumor suppressive role of Brg1. However, the role of Brg1 in the formation of pancreatic intraepithelial neoplasia (PanIN) from acinar cells and PanIN-derived PDA is not fully understood. Aim: To investigate the functional role of Brg1 in the formation of PanIN Methods and Results: We generated Ptf1a-CreER; KrasG12D; Brg1f/f (KBC) mice, in which Brg1 is deleted exclusively in adult pancreatic acinar cells along with expression of oncogenic Kras upon tamoxifen induction of Cre activity and assessed spontaneous acinar-to-ductal metaplasia (ADM) and PanIN formation. We found that ADM and PanIN formation was significantly decreased in KBC mice compared with littermate controls. Next, we induced caerulein-acute pancreatitis to KBC mice and assessed for pancreatitis-induced tumorigenesis. We found that PanINs were decreased in KBC mice compared with littermate controls, although pancreatitis-induced ADMs were widely observed in KBC mice. Since Brg1 is known to critical regulator of p53, mutant p53 allele was crossed with KBC mice. Unexpectedly, even in the context of mutant p53, loss of Brg1 significantly deceased PanIN formation. These results suggest that Brg1 plays a tumor-supportive role in acinar cell-derived PanIN formation in various contexts. Primary acinar cell culture experiments revealed that ADM formation was markedly reduced in the absence of Brg1 and that Sox9 expression was downregulated in Brg1-depleted acinar cells. Furthermore, we confirmed that Sox9 expression is downregulated in Brg1-depleted ADMs/PanINs compared with Brg1-retained ADMs/PanINs by immunohistochemistry. To determine the functional relationship between Brg1 and Sox9, we generated Ptf1a-CreER; KrasG12D; Brg1f/f; Sox9OE mice, in which Sox9 is constitutively overexpressed. Remarkably, Sox9 overexpression canceled the phenotype of decrease in PanIN formation by Brg1 deletion. Furthermore, to investigate whether Brg1/Sox9 axis is observed in human PanIN-derived PDAs, we performed immunohistochemistry of Brg1 and Sox9 in human PanIN-derived PDAs. In 27 human PDAs, all of 5 PDAs with low Brg1 expression had low Sox9 expression. Conclusions: Our results suggest that Brg1 plays a critical role in acinar cell-derived PanIN formation in part through regulating Sox9 expression positively. Our data underscore a cell-type specific, context-dependent role of Brg1 in the initiation of PDA formation. Citation Format: Motoyuki Tsuda, Akihisa Fukuda, Satoshi Ogawa, Kenji Masuo, Norihiro Goto, Yukiko Hiramatsu, Yu Muta, Kozo Ikuta, Yoshito Kimura, Yoshihide Matsumoto, Yutaka Takada, Takuto Yoshioka, Takahisa Maruno, Haruhiko Akiyama, Kyoichi Takaori, Shinji Uemoto, Tsutomu Chiba, Hiroshi Seno. Brg1 plays a critical role in PanIN formation through regulating Sox9 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2017-3518

Dual roles of Akirin2 protein during Xenopus neural development

To ensure correct spatial and temporal patterning, embryos must maintain pluripotent cell populations and control when cells undergo commitment. The newly identified nucleoprotein Akirin has been shown to modulate the innate immune response through epigenetic regulation and to play important roles in other physiological processes, but its role in neural development remains unknown. Here we show that Akirin2 is required for neural development in Xenopus and that knockdown of Akirin2 expands the expression of the neural progenitor marker Sox2 and inhibits expression of the differentiated neuronal marker N-tubulin. Akirin2 acts antagonistically to Geminin, thus regulating Sox2 expression, and maintains the neural precursor state by participating in the Brg1/Brm-associated factor (BAF) complex mediated by BAF53a. Additionally, Akirin2 also modulates N-tubulin expression by acting upstream of neuronal differentiation 1 (NeuroD) and in parallel with neurogenin-related 1 (Ngnr1) during terminal neuronal differentiation. Thus, our results reveal a novel model in which Akirin2 precisely coordinates and temporally controls Xenopus neural development.

Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

Tooth development is initiated by epithelial-mesenchymal interactions via basement membrane (BM) and growth factors. In the present study, we found that nephronectin (Npnt), a component of the BM, is highly expressed in the developing tooth. Npnt localizes in the BM on the buccal side of the tooth germ and shows an expression pattern opposite that of the dental epithelial stem cell marker Sox2. To identify the roles of Npnt during tooth development, we performed knockdown and overexpression experiments using ex vivo organ and dental epithelial cell cultures. Our findings showed that loss of Npnt induced ectopic Sox2-positive cells and reduced tooth germ size. Over expression of Npnt showed increased proliferation, whereas the number of Sox2-positive cells was decreased in dental epithelial cells. Npnt contains 5 EGF-like repeat domains, as well as an RGD sequence and MAM domain. We found that the EGF-like repeats are critical for Sox2 expression and cell proliferation. Furthermore, Npnt activated the EGF receptor (EGFR) via the EGF-like repeat domains and induced the PI3K-Akt signaling pathway. Our results indicate that Npnt plays a critical scaffold role in dental epithelial stem cell differentiation and proliferation, and regulates Sox2 expression during tooth development.

Mesoderm-specific Stat3 deletion affects expression of Sox9 yielding Sox9-dependent phenotypes.

To date, mutations within the coding region and translocations around the SOX9 gene both constitute the majority of genetic lesions underpinning human campomelic dysplasia (CD). While pathological coding-region mutations typically result in a non-functional SOX9 protein, little is known about what mechanism(s) controls normal SOX9 expression, and subsequently, which signaling pathways may be interrupted by alterations occurring around the SOX9 gene. Here, we report the identification of Stat3 as a key modulator of Sox9 expression in nascent cartilage and developing chondrocytes. Stat3 expression is predominant in tissues of mesodermal origin, and its conditional ablation using mesoderm-specific TCre, in vivo, causes dwarfism and skeletal defects characteristic of CD. Specifically, Stat3 loss results in the expansion of growth plate hypertrophic chondrocytes and deregulation of normal endochondral ossification in all bones examined. Conditional deletion of Stat3 with a Sox9Cre driver produces palate and tracheal irregularities similar to those described in Sox9+/- mice. Furthermore, mesodermal deletion of Stat3 causes global embryonic down regulation of Sox9 expression and function in vivo. Mechanistic experiments ex vivo suggest Stat3 can directly activate the expression of Sox9 by binding to its proximal promoter following activation. These findings illuminate a novel role for Stat3 in chondrocytes during skeletal development through modulation of a critical factor, Sox9. Importantly, they further provide the first evidence for the modulation of a gene product other than Sox9 itself which is capable of modeling pathological aspects of CD and underscore a potentially valuable therapeutic target for patients with the disorder.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme

ABSTRACTSyndromic craniosynostosis caused by mutations in FGFR2 is characterised by developmental pathology in both endochondral and membranous skeletogenesis. Detailed phenotypic characterisation of features in the membranous calvarium, the endochondral cranial base and other structures in the axial and appendicular skeleton has not been performed at embryonic stages. We investigated bone development in the Crouzon mouse model (Fgfr2C342Y) at pre- and post-ossification stages to improve understanding of the underlying pathogenesis. Phenotypic analysis was performed by whole-mount skeletal staining (Alcian Blue/Alizarin Red) and histological staining of sections of CD1 wild-type (WT), Fgfr2C342Y/+ heterozygous (HET) and Fgfr2C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (Sox9, Shh, Fgf10 and Runx2) was studied by in situ hybridisation and protein expression (COL2A1) by immunohistochemistry. Our analysis has identified severely decreased osteogenesis in parts of the craniofacial skeleton together with increased chondrogenesis in parts of the endochondral and cartilaginous skeleton in HOM embryos. The Sox9 expression domain in tracheal and basi-cranial chondrocytic precursors at E13.5 in HOM embryos is increased and expanded, correlating with the phenotypic observations which suggest FGFR2 signalling regulates Sox9 expression. Combined with abnormal staining of type II collagen in pre-chondrocytic mesenchyme, this is indicative of a mesenchymal condensation defect. An expanded spectrum of phenotypic features observed in the Fgfr2C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon–Pfeiffer syndrome. FGFR2 mutation results in impaired skeletogenesis; however, our findings suggest that many phenotypic aberrations stem from a primary failure of pre-chondrogenic/osteogenic mesenchymal condensation and link FGFR2 to SOX9, a principal regulator of skeletogenesis.

Response letter: “epigenetic modulation of a miR-296-5p:HMGA1 axis regulates Sox2 expression and glioblastoma stem cells”

In this issue of Translational Cancer Research Ferrucci and Zollo provide a thoughtful commentary discussing our recent Oncogene publication titled “Epigenetic Modulation of a miR-296-5p:HMGA1 Axis Regulates Sox2 expression and Glioblastoma Stem Cells”. The experts examined our results with a critical eye and comment on the strengths and weaknesses of our study while at the same time providing very thoughtful and considerate suggestions for future studies and new directions.

Abstract 2482: The Calcineurin/NFAT pathway mediates tumor initiating cell and drug resistance in lung cancer through SOX2 enhancer binding and ALDH upregulation

Abstract Tumor initiating cells (TIC) are a small population of cancer cells with enhanced tumorigenicity and drug resistance. Their stemness properties could be activated by stress-induced cell plasticity but the mechanisms remain to be studied. Calcium signaling is important for integrating cell responses to intra- and extracellular stresses. The role of its downstream pathway, calcineurin/NFAT, in lung TIC has not been explored. This study investigated the role and mechanisms of calcineurin/NFAT in regulating TIC and drug resistance by analyzing NFATc2 expression in multiple TIC populations and functional evaluation using in vitro and in vivo models. In tumor spheres and TIC isolated by marker-based flow cytometry, NFATc2 showed overexpression and higher luciferase reporter activities. Knockdown of NFATc2 reduced TIC marker proportions and suppressed TIC properties including pluripotency genes expression, sphere formation, cell mobility and drug resistance in vitro. Xenograft models and limiting dilution assay showed shNFATc2 effectively suppressed tumorigenesis and reduced TIC frequency. Stable NFATc2 knockdown sensitized tumors to cisplatin and gefitinib treatment. In contrast, TIC-supportive effects were observed in vitro and in vivo when NFATc2 was overexpressed. Further, NFATc2 was activated in stable gefitinib or cisplatin resistant cell lines, and knockdown of NFATc2 sensitized them to drug treatment. These data implicated NFATc2 is involved in lung TIC maintenance and drug resistance. To further investigate the molecular mechanisms of how NFATc2 mediate these roles, we analyzed pluripotency genes and observed a significant correlation between NFATc2 and SOX2 expression in lung cancer cell lines and human lung carcinomas. Computational analysis predicted NFATc2 binding sequences in SOX2 regulatory regions. Detailed studies including luciferase reporter assay, site-directed mutagenesis and CHIP-qRCR confirmed NFATc2 could regulate SOX2 expression through binding to its enhancer. Furthermore, immunohistochemistry on human lung cancers showed significant correlation between expressions of SOX2 and the anti-oxidative stem cell marker ALDH, suggesting SOX2 regulates ALDH expression. In vitro, NFATc2 knockdown suppressed ALDH1A1 expression but it was rescued by SOX2 overexpression. Vice versa, ALDH1A1 was upregulated in NFATc2 overexpressing cells but this was prevented by SOX2 knockdown. Functionally, reactive oxidative species (ROS) levels assessed by flow cytometry were suppressed by activated NFATc2/SOX2/ALDH1A1 axis, while treatment with the ROS inhibitor N-acetylcysteine reversed the sensitization of shNFATc2 on cisplatin treatment. Together, the data showed NFATc2 is involved in the regulation of TIC phenotypes in lung cancer. It increases drug resistance by ROS scavenging mediated by the SOX2/ALDH1A1 axis. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky PC Tin, Maria P. Wong. The Calcineurin/NFAT pathway mediates tumor initiating cell and drug resistance in lung cancer through SOX2 enhancer binding and ALDH upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2482.

Abstract 3345: Targeting the YAP1/COX2/SOX2 signaling axis eliminates cancer stem cells in urothelial carcinoma

Abstract Urothelial cancer stem cells (CSCs) contribute to tumor maintenance and resistance to therapy and accumulated evidence suggest that chronic carcinogen exposure induce “stemness”. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. Here we used our recently published in vitro chronic arsenic (As) exposed models to characterize the property of urothelial CSCs due to arsenic exposure. We hypothesized that urothelial stem cells have a survival selection advantage during carcinogen exposure such as As, and it facilitates their malignant transformation and in acquiring selective phenotypes similar to CSC. Arsenic-exposed cells displayed more aggressive phenotype than arsenic unexposed cells in a time dependent manner as determined by MTT, invasion, and wound healing assay. In gene set enrichment analysis of expression array of chronic arsenic exposed and unexposed cells; EGFR, COX2 and YAP1 were top-ranked oncogenic signature based on enrichment score in As-exposed cells. Further analysis indicated that several known basal cell markers such as KRT5, KRT6A, and KRT6C etc were overexpressed in As exposed cells in comparison with As unexposed cells. Because the presence of urothelial CSCs in basal-type provides a biological explanation for their aggressive behaviors, we assessed the influence of As on generating CSC phenotypes. As exposure was associated with overabundance of potential CSCs characterized by sphere formation, self-renewal capacity, redifferentiation, and chemotherapy resistance. To explore global association of As exposure and CSC generation, we used the Human Stem Cell RT2 Profiler™ PCR Array and found that SOX2 has been gradually overexpressed in line with acquired spheroid formation and self-renewal capacities. SOX2 is frequently overexpressed in numerous urothelial carcinoma (UC) cell lines as well as in arsenic-exposed cells, especially in the spheroid cells. Stable silencing of SOX2 reduces in vitro CSCs properties with reduce expression of CSCs associated molecules such as NANOG,OCT4 etc and also in vivo tumorigenicity. COX2 and YAP1 are also frequently overexpressed in UC cell lines, and inhibition of COX2 or YAP1 reduced SOX2 expression. Interestingly, the inhibition of COX2 and YAP1 expression inversely induced YAP1 and COX2 expression, respectively. Combination treatment with COX2 and YAP1 inhibitors reduced SOX2 expression and suppressed spheroid formation significantly than either inhibitor alone. In xenograft model, the combination treatment suppressed the tumor growth rate, but not either inhibitor alone. In conclusion, chronic As exposure induces CSCs with SOX2 overexpression, one of the most important CSC factor for UC. COX2 and YAP1 coordinately regulate SOX2 expression, and mutually compensate for the reduction of expression of SOX2 to maintain CSCs. Thus targeting the COX2/YAP/SOX2 signaling axis eliminates urothelial CSCs. Citation Format: Akira Oki, Mohammad Hoque. Targeting the YAP1/COX2/SOX2 signaling axis eliminates cancer stem cells in urothelial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3345.