Role Of SOX2 Research Articles

Incomplete and delayed Sox2 deletion defines residual ear neurosensory development and maintenance.

The role of Sox2 in neurosensory development is not yet fully understood. Using mice with conditional Islet1-cre mediated deletion of Sox2, we explored the function of Sox2 in neurosensory development in a model with limited cell type diversification, the inner ear. In Sox2 conditional mutants, neurons initially appear to form normally, whereas late- differentiating neurons of the cochlear apex never form. Variable numbers of hair cells differentiate in the utricle, saccule, and cochlear base but sensory epithelium formation is completely absent in the apex and all three cristae of the semicircular canal ampullae. Hair cells differentiate only in sensory epithelia known or proposed to have a lineage relationship of neurons and hair cells. All initially formed neurons lacking hair cell targets die by apoptosis days after they project toward non-existing epithelia. Therefore, late neuronal development depends directly on Sox2 for differentiation and on the survival of hair cells, possibly derived from common neurosensory precursors.

Abstract P194: A New Role of Sox6 in Blood Pressure Through Renin Regulation

Hypertension afflicts 33% of the U.S. adult population. Despite current treatments, approximately 50% of people are unresponsive to treatment. There is a critical need to develop new therapies to treat this disease and its complications. The Renin Angiotensin Aldosterone System plays a key role in regulating blood pressure in humans. Renin controls the rate-limiting step in the conversion of angiotensinogen to angiotensin I. In adults, renin is produced and stored by Juxtaglomerular (JG) cells in the kidney. However, the transcriptional mechanisms that govern formation of renin expressing cells under normal or pathophysiological conditions remain poorly understood. During blood pressure changes the number of adult renal cells expressing renin increase through a process known as JG cell expansion. We found that this process involves differentiation of mesenchymal stromal-like cells (MSC) to renin expressing cells among others. We aim to determine new regulators of renin expression and blood pressure control. Renin expression in vitro was induced by treatment of MSCs with 3-isobutyl-1-methylxanthine (IBMX) and Forskolin. MSCs were transduced with lentivirus carrying vectors Sox6 shRNA or control shRNA. A new transgenic mouse, in which Sox6 is deleted specifically in renin expressing cells (Ren1dCre/Sox6 fl/fl ), was used to study the impact of Sox6 in renin expression in vivo . Gene array comparing renal MSC and JG cells identified potential genes that control MSC differentiation, including Sox6. In vitro silencing of Sox6 by shRNA decreased differentiation of renal MSCs into renin producing cells (3.5 fold, n=4, P= 0.01). Preliminary IHC data showed that the transcription factor Sox6 is expressed by renin producing cells in the adult kidney during JG cell expansion. Plasma renin concentration (PRC) increased during JG cell expansion (induced with low sodium diet and furosemide) in wild type mice (27-fold), whereas in mice lacking Sox6 in JG cells PRC was as low as non-treated mice. We conclude that Sox6 has a novel role in modulating renin expression and thereby can contribute to renal physiology, opening new possibilities of addressing questions regarding physiological regulation of renin and hypertension.

Sox2 Suppresses Gastric Tumorigenesis in Mice

Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model. Moreover, Sox2 loss enhances, rather than impairs, tumor formation in Apc-deficient gastric cells invivo and invitro by inducing Tcf/Lef-dependent transcription and upregulating intestinal metaplasia-associated genes, providing a mechanistic basis for the observed phenotype. Together, these data identify Sox2 as a context-dependent tumor suppressor protein that is dispensable for normal tissue regeneration but restrains stomach adenoma formation through modulation of Wnt-responsive and intestinal genes.

Abstract 2538: The emerging role of SOX2 in cancer cell plasticity and EGFR-TKI resistance

Abstract Activating mutations in the epidermal growth factor receptor (EGFR) lead to the aberrant growth of lung adenocarcinoma while endowing cancer cells with sensitivity to EGFR-tyrosine kinase inhibitors (TKIs). Nonetheless, EGFR-TKI resistance often occurs in less than one year. The intriguing phenomenon that cancer cells have biological features similar to stem cells suggests that cancer and stem cells may share the same regulatory signaling pathway, but the involvement of stem cell signaling in EGFR-TKI resistance is not clear. Herein, we observed that SOX2, a master transcriptional factor controlling self-renewal of lung stem cells, was highly expressed in lung adenocarcinoma and inversely correlated with epithelial-to-mesenchymal transition (EMT). Clonogenic analysis showed that SOX2-silenecing attenuated cell growth in EGFR-mutated lung cancer cells. The selection of EGFR-TKI resistant HCC827 (delE746_A750) cells induced EMT while attenuating the expression of SOX2 and spheroid forming ability. Preselection of HCC827 (delE746_A750) cells with EMT features endowed cells with TKI resistance but suppressed SOX2 expression. Pharmacological inhibition of HDACs in EGFR-mutated lung cancer cells attenuated SOX2 expression but induced EMT, thus enhancing the development of EGFR-TKI resistance. Our findings support the notion that the switching expression between SOX2 and EMT plays a critical role in EGFR-TKI resistance, with the potential as biomarkers for lung cancer prediction. Citation Format: Yu-Fan Chiu, Ming-Han Kuo, An-Chun Lee, Yu-Ting Chou. The emerging role of SOX2 in cancer cell plasticity and EGFR-TKI resistance. [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 2538.

Abstract 2910: Overcoming therapy resistance in SOX2-expressing head and neck squamous cell carcinoma cells by targeting the downstream BCL2-pathway

Abstract SOX2 (SRY (sex determining region Y)-box 2) is a transcriptional master regulator that confers “stemness” in early embryogenesis and acts as an oncogene in various cancers. Recently, SOX2 expression has been shown in several solid tumor types. However, the SOX2 expression pattern and the correlation with histopathologic status and clinical outcome are highly variable among tumors, suggesting distinct roles of SOX2 in individual tumors. In head and neck squamous cell carcinoma (HNSCC), we reported that expression of SOX2 is detectable and associates with high-risk disease in non-HPV human HNSCC. Functionally, SOX2 overexpression enhanced apoptosis resistance in response to treatments with cisplatin and other apoptosis-inducing agents, indicating SOX2 as a mediator of therapy resistance in human HNSCC. On the molecular level, induction of the anti-apoptotic protein BCL2 was observed following SOX2-induction. Here we explore if the anti-apoptotic effects of SOX2 are functionally mediated through up-regulation of BCL2 expression and explore BCL2 as a potential direct downstream target of SOX2. We tested SOX2 and BCL2 expression in different human HNSCC cell-lines by qPCR and respectively immunoblot. To analyse the functional effect of SOX2, SOX2 overexpressing and control SCC25 cells generated via transduction with lentiviral particles were analysed in in vitro apoptosis assays after treatments with staurosporine, TRAIL or cisplatin. Furthermore, to analyse the influence of BCL2 in SOX2-overexpressing cells, treatment with the BCL2 inhibitor ABT-199 was used. We could confirm the strong inductive effect of SOX2 overexpression on BCL2 gene and protein levels in the human SCC25 cell line. In line, cells treated with SOX2 overexpressing lentiviruses showed enhanced apoptotic resistance as compared to control-transduced cells. Notably, HNSCC cells were less sensitive to ABT-199 as previously reported for lymphatic leukemic cells (5-10 μM versus 0.1-1 μM for EC50). However, ABT-199 treatment in low dosage (1 μM) was indeed able to sensitize SOX2-overexpressing human HNSCC cells to apoptosis, indicating up-regulation of BCL2 as an important mechanism by which SOX2 confers treatment resistance in these cells. Moreover, analysis of the BCL2 promoter revealed a potential SOX2-binding site, and a direct association of SOX2 protein with this site could be confirmed by chromatin immunoprecipitation assays (CHIP). We are currently further exploring the effect of ABT-199 on the therapy resistance of SOX2-overexpressing cells using in vivo models. Taken together, our findings indicate that, in human HNSCC cells, SOX2 plays an important role in drug resistance by elevating BCL2 expression. In patients with SOX2-expressing HNSCC, combinatorial treatment with ABT-199 may enhance response to conventional chemotherapies by re-sensitizing cells to apoptosis induction. Citation Format: Hui Wang, Angela Quiesser, Andreas Schröck, Sven Perner, Claudia Lengerke. Overcoming therapy resistance in SOX2-expressing head and neck squamous cell carcinoma cells by targeting the downstream BCL2-pathway. [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 2910.

Abstract 300: Inhibition of oncogenic MAPK signaling in melanoma triggers SOX2-dependent adaptive drug resistance

Abstract Treatment of metastatic melanoma with BRAFV600E-specific small molecule inhibitors (BRAFi) provides dramatic clinical relief response but fails to suppress long-term tumor growth in the long-term. Extensive studies have uncovered identified a number of resistance-causing genetic alterations that, which are acquired upon continued drug exposure. However, little is known about the changes in gene expression programs changes during the critical time before drug resistance is manifested on at the genomic level. These changes may lead to adaptive drug resistance and hence, represent vital mechanisms by which tumor cells survive the acute drug insult prior to acquisition of resistance-causing mutations. Using a combination of RNAseq and molecular analyses, we find that acute inhibition of oncogenic MAPK signaling by BRAFi or MEKi in established and primary patient-derived melanoma cell cultures, leads to a rapid induction of the stem cell maintenance and pluripotency factor SOX2. Immunohistochemical analysis of Sox2 expression in melanoma sections derived from BrafV600E/Pten−/−-mice treated for 36 hours with BRAFi/MEKi confirmed the emergence of a Sox2-positive melanoma subpopulation in vivo. SOX2 Ectopic ectopic expression of SOX2 protected melanoma cells from the anti-proliferative effects of BRAFi indicative ofindicating a role for SOX2 in mediating drug resistance. Furthermore, siRNA-mediated depletion or overexpression experiments of SOX2 unveiled revealed that it is necessary and sufficient for the expansion of a drug-tolerant SOX2+CD24+ cell population. This subpopulation is characterized by increased migratory and invasive behavior as well as an increased proliferative potential in the presence of BRAFi when compared to SOX2+CD24− cells. These results imply that BRAFi treatment induces a SOX2-dependent cellular program with features of adaptive drug resistance. Suppression of drug-dependent induction of SOX2 or its downstream effector pathways could potentially extend the clinical efficacy of BRAFi. Citation Format: Stefanie Flueckiger, Andrea Aloia, Lukas Frischknecht, Amanda Tuozzo, Davide Croci, Olga Shakhova, Christian Britschgi, Reinhard Dummer, Mitchell Paul Levesque, Wilhelm Krek. Inhibition of oncogenic MAPK signaling in melanoma triggers SOX2-dependent adaptive drug resistance. [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 300.

Abstract 2913: The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival

Abstract Introduction: Basal-like breast cancers (BLBCs) are aggressive breast cancers that are associated with poor survival and have distinctive mRNA expression profiles compared to non-BLBC tumors. Through an integrated screen of transcription factors (TFs) we identified SOX11 as uniquely critical for growth in BLBC, but not essential in estrogen-receptor (ER)-positive breast cancer cells. In this study we examine the role of SOX11 regulating growth and other BLBC phenotypes, including increased migration, invasion and poor survival. Methods: SOX11 was identified through an integrated primary screen comparing mRNA expression of TFs, DNA sequences of differentially expressed BLBC genes, and differential binding of nuclear proteins to oligonucleotide sequences of transcription factor binding sites, and a secondary siRNA screen of growth in a panel of breast cancer cell lines. Further growth studies were performed using siRNA to SOX11 and measuring 2D-growth, or 3D-growth of cell lines grown in soft-agar. Immuno-fluorescent staining of phosphorylated histone H3 (pH3) was used to measure the effect of SOX11 depletion on proliferation. Migration and invasion assays were performed with membranes coated in Matrigel (invasion) or uncoated (migration). Survival analyses were performed with patients dichotomized by median SOX11 expression level, and log rank statistical comparisons as well as a multivariable Cox Proportional Hazards model to investigate the prognostic value of SOX11. Results: SOX11 depletion resulted in reduced growth of ER-negative breast cancer cell lines, including ER-negative/ Her2-positive cells; whereas, growth of ER-positive breast cancer cells or ER-negative, non-transformed breast cells was not affected. In MDA-MB-468 cells, SOX11 depletion resulted in a 60% reduction of pH3-positive cells. Additionally, depletion of SOX11 in MDA-MB-231 or MDA-MB-468 cells reduced migration and invasion 40-60%, while over-expression of SOX11 in MCF7 cells increased migration nearly 12-fold. High SOX11 is correlated with worse prognosis, whether analyzed for all patients or subsets of patients divided by ER or HER2 status. In the multivariable analysis accounting for tumor size, grade, lymph node status, and PAM50 class, high SOX11 expression was associated with increased risk of breast cancer related death with a hazard ratio of 1.42 and p-value < 0.001. Conclusions: SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. Growth reduction of BLBC cells following SOX11 depletion is a result of decreased proliferation. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer. Citation Format: Jonathan Shepherd, Abhijit Mazumdar, Anna Tsimelzon, Susan G. Hilsenbeck, Powel H. Brown. The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival. [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 2913.

TGF-β induces SOX2 expression in a time-dependent manner in human melanoma cells.

The sry-related high-mobility box (SOX)-2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF-β1 stimulation in a time-dependent manner. Moreover, the knockdown of SOX2 impaired TGF-β-induced invasiveness. This phenotype switch can be explained by SOX2-mediated cross talk between TGF-β and non-canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF-β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF-β signaling in melanoma, which may have further implications in the clinic.

SOX10-MITF pathway activity in melanoma cells.

Melanoma is one of the most dangerous and lethal skin cancers, with a considerable metastatic potential and drug resistance. It involves a malignant transformation of melanocytes. The exact course of events in which melanocytes become melanoma cells remains unclear. Nevertheless, this process is said to be dependent on the occurrence of cells with the phenotype of progenitor cells – cells characterized by expression of proteins such as nestin, CD-133 or CD-271. The development of these cells and their survival were found to be potentially dependent on the neural crest stem cell transcription factor SOX10. This is just one of the possible roles of SOX10, which contributes to melanomagenesis by regulating the SOX10-MITF pathway, but also to melanoma cell survival, proliferation and metastasis formation. The aim of this review is to describe the broad influence of the SOX10-MITF pathway on melanoma cells.

Sox2: regulation of expression and contribution to brain tumors

Tumors of the CNS are composed of a complex mixture of neoplastic cells, in addition to vascular, inflammatory and stromal components. Similar to most other tumors, brain tumors contain a heterogeneous population of cells that are found at different stages of differentiation. The cancer stem cell hypothesis suggests that all tumors are composed of subpopulation of cells with stem-like properties, which are capable of self-renewal, display resistance to therapy and lead to tumor recurrence. One of the most important transcription factors that regulate cancer stem cell properties is SOX2. In this review, we focus on SOX2 and the complex network of signaling molecules and transcription factors that regulate its expression and function in brain tumor initiating cells. We also highlight important findings in the literature about the role of SOX2 in glioblastoma and medulloblastoma, where it has been more extensively studied.

Mouse Sox17 haploinsufficiency leads to female subfertility due to impaired implantation.

Embryonic implantation comprises a dynamic and complicated series of events, which takes place only when the maternal uterine endometrium is in a receptive state. Blastocysts reaching the uterus communicate with the uterine endometrium to implant within a narrow time window. Interplay among various signalling molecules and transcription factors under the control of ovarian hormones is necessary for successful establishment of pregnancy. However, the molecular mechanisms that allow embryonic implantation in the receptive endometrium are still largely unknown. Here, we show that Sry-related HMG box gene-17 (Sox17) heterozygous mutant female mice exhibit subfertility due to implantation failure. Sox17 was expressed in the oviduct, uterine luminal epithelium, and blood vessels. Sox17 heterozygosity caused no appreciable defects in ovulation, fertilisation, blastocyst formation, and gross morphology of the oviduct and uterus. Another group F Sox transcription factor, Sox7, was also expressed in the uterine luminal and glandular epithelium relatively weakly. Despite uterine Sox7 expression, a significant reduction in the number of implantation sites was observed in Sox17 heterozygous mutant females due to haploinsufficiency. Our findings revealed a novel role of Sox17 in uterine receptivity to embryo implantation.

Sox2-Deficient Müller Glia Disrupt the Structural and Functional Maturation of the Mammalian Retina.

PurposeMüller glia (MG), the principal glial cells of the vertebrate retina, display quiescent progenitor cell characteristics. They express key progenitor markers, including the high mobility group box transcription factor SOX2 and maintain a progenitor-like morphology. In the embryonic and mature central nervous system, SOX2 maintains neural stem cell identity. However, its function in committed Müller glia has yet to be determined.MethodsWe use inducible, MG-specific genetic ablation of Sox2 in vivo at the peak of MG genesis to analyze its function in the maturation of murine MG and effects on other cells in the retina. Histologic and functional analysis of the Sox2-deficient retinas is conducted at key points in postnatal development.ResultsAblation of Sox2 in the postnatal retina results in disorganization of MG processes in the inner plexiform layer and mislocalized cell bodies in the nuclear layers. This disorganization is concurrent with a thinning of the neural retina and disruption of neuronal processes in the inner and outer plexiform layers. Functional analysis by electroretinography reveals a decrease in the b-wave amplitude. Disruption of MG maturation due to Sox2 ablation therefore negatively affected the function of the retina.ConclusionsThese results demonstrate a novel role for SOX2 in glial process outgrowth and adhesion, and provide new insights into the essential role Müller glia play in the development of retinal cytoarchitecture. Prior to this work, SOX2 was known to have a primary role in determining cell fate. Our experiments bypass cell fate conversion to establish a new role for SOX2 in a committed cell lineage.

Sox2 contributes to tooth development via Wnt signaling.

The transcription factor Sox2 is a stem cell marker that dictates cell lineage. It has been shown to mark the epithelial stem cells of the continuously growing mouse incisors. Sox2 also interferes with Wnt signaling by binding to β-catenin, a central mediator of the Wnt pathway. We show that these functions of Sox2 are essential for mouse molar development. Sox2 has previously been shown to play a role in the formation of new teeth from the existing dental epithelium. To assess Sox2 function related to cell migration within a tooth, we monitored cell movement by using a DiI system and observed that DiI moves from molar 1 to molar 2 during tooth development. However, upon temporal knockdown of Sox2, DiI remains in the molar 1 region. This study also provides novel insights into the role of Sox2 and the important validation of Sox2 as a potent target in Wnt signaling during tooth development. Our data reveal that the degradation of Wnt signaling caused by the knockdown of Sox2 results in a lack of cell migration during tooth development.

Role of SOX7 in Hematopoietic System Development and Hematological Malignancies--Review

The sex-determining region Y-box 7 (Sox7) is a important member of SOX family containing high mobi- lity group (HMG), mapped to human chromosome 8p23.1. Wnt/β-catenin signaling pathway plays an important role in cell survival, differentiation, self-renewal, proliferation and apoptosis, and is closely related with carcinogenesis. SOX7 gene is likely to be a tumor suppressor gene in MDS and other hematological malignancies. As a negative regulator of the WNT/β-catenin signaling pathway, the function loss of this gene can lead to carcinogenesis. The methylation of SOX7 gene leads to the silence of this gene, resulting in tumorigenesis. The decision of hematopoietic stem cells to self-renew or differentiate is a stochastic process, but SOX7 can promote the differentiation into all blood cell types. This review focuses on the role of SOX7 in hematopoietic system development and hematological malignancies.

SOX2 is sequentially required for progenitor proliferation and lineage specification in the developing pituitary.

Sox2 mutations are associated with pituitary hormone deficiencies and the protein is required for pituitary progenitor proliferation, but its function has not been well characterized in this context. SOX2 is known to activate expression of Six6, encoding a homeodomain transcription factor, in the ventral diencephalon. Here, we find that the same relationship likely exists in the pituitary. Moreover, because Six6 deletion is associated with a similar phenotype as described here for loss of Sox2, Six6 appears to be an essential downstream target of SOX2 in the gland. We also uncover a second role for SOX2. Whereas cell differentiation is reduced in Sox2 mutants, some endocrine cells are generated, such as POMC-positive cells in the intermediate lobe. However, loss of SOX2 here results in complete downregulation of the melanotroph pioneer factor PAX7, and subsequently a switch of identity from melanotrophs to ectopic corticotrophs. Rescuing proliferation by ablating the cell cycle negative regulator p27 (also known as Cdkn1b) in Sox2 mutants does not restore melanotroph emergence. Therefore, SOX2 has two independent roles during pituitary morphogenesis; firstly, promotion of progenitor proliferation, and subsequently, acquisition of melanotroph identity.

Structural and conformational insights into SOX2/OCT4-bound enhancer DNA: a computational perspective

The roles of SOX2 and OCT4 are critical in stem cell maintenance either in the context of iPSCs generation or cancer stem cell growth; therefore, it is imperative to study their cooperative binding and SOX2/OCT4-induced DNA conformational switching.

Identification of the neural crest-specific enhancer of Seraf gene in avian peripheral nervous system development

In vertebrate embryos, trunk neural crest cells give rise to Schwann cells, along with other derivatives. In this study, to elucidate the molecular mechanisms for the Schwann cell specification, we aimed to identify enhancer elements responsible for the expression of the Seraf gene, the earliest marker for the Schwann cell precursors in the avian embryos. We first compared the genomic structure around the Seraf locus in various vertebrates, and found that, while mammals do not have a Seraf homolog, teleost fish species have it. However, the intergenic sequences around the Seraf locus are not conserved between zebrafish and chicken, consistent with the fact that fish Seraf expression is not Schwann cell precursor-specific. We thus compared the intergenic sequences around the Seraf locus among avian species, and identified a potential enhancer containing a cluster of Sox10-binding sites. Accordingly, the identified enhancer is activated in a neural crest-specific manner in transfected quail embryos. We also found that Sox10 activated the enhancer in cultured cells. Thus, our results revealed a new role of Sox10 in the earliest phase of the Schwann cell fate specification.

Abstract 5057: Single-cell RNA-seq analysis of heterogeneous populations within gastroenteropancreatic neuroendocrine tumors: the role of Sox transcription factors

Abstract Gastroenteropancreatic neuroendocrine tumors (GPNETs) are rare, with 8,000-estimated cases/year in the United States. Several transcriptome analyses have been performed at the population level, but the heterogeneity of these tumors has never been assessed at the single cell level. Moreover, few genetic mutations have been consistently linked to the origin and progression of GPNETs and prognostic biomarkers are greatly lacking. The most commonly reported familial mutations in GPNETS are found in the Multiple Endocrine Neoplasia type 1 gene (MEN1) or Von Hippel-Lindau's gene (VHL). Similar to other neoplasias, we would expect that a combination of mutations drive the malignant transformation in GPNETs. We are interested in assessing the role of Sox factors in GPNET initiation and progression, particularly Sox4 and Sox9. These Sox factors are expressed in stem and progenitor cells in the gastrointestinal tract. Using a Sox9EGFP mouse, we have shown that Sox9 is expressed at differential levels in stem and progenitor cells, demonstrating high expression in a cell population consistent with long-lived secretory progenitor cells with endocrine and stem cell like properties. Other studies from our lab and others point at the role of Sox4 and Sox9 in differentiation of intestinal/pancreatic progenitors and endocrine cells. Based on these observations, we hypothesize that differential levels of Sox factors mark cells with different potential to initiate carcinoids with an underlying secretory signature, consistent with that of GPNET. Using histological analysis, we assessed the expression of Sox 4/9 in samples from 20 GPNETs patients and we found that the majority exhibited heterogeneity within the tumor. We further confirmed that these tumors expressed putative GPNET markers such as CHGA, serotonin and substance P among others. To gain a more global understanding of the genetic heterogeneity present in these tumors, we performed single-cell RNA-seq analysis from a GPNET patient. Consistent with histological analysis, we also observed heterogeneous levels of several Sox Transcription Factors, including Sox4 and Sox9. These data provided insight into the pathways that may be responsible for GPNET initiation and progression. Notable genes highly expressed include markers of stemness, endocrine progenitor cells, endocrine secretion/differentiation, immune subpopulations and cytokine production. These data also elucidated some potential targets for immunotherapies such as IL-10. Through collaborations with 3 different hospitals, we now have access to at least 24 more fixed/fresh GPNET tissues and we are currently focusing our efforts on the confirmation of our findings. Our results suggest the potential use of single-cell RNA-seq analysis to untangle the pathways that drive the cell populations present in the invasive and proliferative front of GPNETs. Citation Format: Haydee Lara, Tamara Matysiak-Budnik, Scott T. Magness. Single-cell RNA-seq analysis of heterogeneous populations within gastroenteropancreatic neuroendocrine tumors: the role of Sox transcription factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5057. doi:10.1158/1538-7445.AM2015-5057

Sox4 participates in the modulation of Schwann cell myelination.

In order to identify new regulators of Schwann cell myelination potentially playing a role in peripheral nervous system (PNS) pathologies, we analysed gene expression profiling data from three mouse models of demyelinating neuropathies and from the developing PNS. This analysis revealed that Sox4, which encodes a member of the Sry-related high-mobility group box protein family, was consistently upregulated in all three analysed models of neuropathy. Moreover, Sox4 showed a peak in its expression during development that corresponded with the onset of myelination. To gain further insights into the role of Sox4 in PNS development, we generated a transgenic mouse that specifically overexpresses Sox4 in Schwann cells. Sox4 overexpression led to a temporary delay in PNS myelination without affecting axonal sorting. Importantly, we observed that, whereas Sox4 mRNA could be efficiently overexpressed, Sox4 protein expression in Schwann cells was strictly regulated. Finally, our data showed that enforced expression of Sox4 in the mouse model for Charcot-Marie-Tooth 4C aggravated its neuropathic phenotype. Together, these observations reveal that Sox4 contributes to the regulation of Schwann cell myelination, and also indicates its involvement in the pathophysiology of peripheral neuropathies.

Hepatitis B virus replication and sex-determining region Y box 4 production are tightly controlled by a novel positive feedback mechanism.

Hepatitis B virus (HBV) infection is a major cause of liver diseases. However, the mechanisms underlying HBV infection and pathogenesis remain largely unknown. The sex-determining region Y box 4 (Sox4) is a transcriptional factor, which preferentially regulates the development of various organs, tissues, and cancers. But, the role of Sox4 in viral infection and pathogenesis has not been elucidated. Here, we demonstrated that Sox4 is up-regulated by HBV, and revealed the mechanism by which HBV regulates Sox4 expression. First, HBV stimulates Sox4 expression through transcriptional factor Yin Yang 1 (YY1), which binds to Sox4 promoter to activate Sox4 transcriptional activity. Second, miR-335, miR-129-2 and miR-203 inhibit Sox4 expression by targeting its mRNA 3’UTR, while HBV suppresses the microRNAs expression, resulting in up-regulating Sox4 post-transcriptionally. Third, Sox4 protein is degraded by proteasome, while HBV surface protein (HBsAg) prevents Sox4 from degradation by directly interacting with the protein, thereby enhancing Sox4 production post-translationlly. More interestingly, HBV-activated Sox4 in turn facilitates HBV replication by direct binding to the viral genome via its HMG box. Thus, this study revealed a novel positive feedback mechanism by which Sox4 production and HBV replication are tightly correlated.