Loss Of Sox2 Research Articles

Airway epithelial cell identity and plasticity are constrained by Sox2 during lung homeostasis, tissue regeneration, and in human disease

Maintenance of the cellular boundary between airway and alveolar compartments during homeostasis and after injury is essential to prohibit pathological plasticity which can reduce respiratory function. Lung injury and disease can induce either functional alveolar epithelial regeneration or dysplastic formation of keratinized epithelium which does not efficiently contribute to gas exchange. Here we show that Sox2 preserves airway cell identity and prevents fate changes into either functional alveolar tissue or pathological keratinization following lung injury. Loss of Sox2 in airway epithelium leads to a loss of airway epithelial identity with a commensurate gain in alveolar and basal cell identity, in part due to activation of Wnt signaling in secretory cells and increased Trp63 expression in intrapulmonary basal-like progenitors. In idiopathic pulmonary fibrosis, loss of SOX2 expression correlates with increased WNT signaling activity in dysplastic keratinized epithelium. SOX2-deficient dysplastic epithelial cells are also observed in COVID-19 damaged lungs. Thus, Sox2 provides a molecular barrier that suppresses airway epithelial plasticity to prevent acquisition of alveolar or basal cell identity after injury and help guide proper epithelial fate and regeneration.

Enhancer-promoter interactions can bypass CTCF-mediated boundaries and contribute to phenotypic robustness.

How enhancers activate their distal target promoters remains incompletely understood. Here we dissect how CTCF-mediated loops facilitate and restrict such regulatory interactions. Using an allelic series of mouse mutants, we show that CTCF is neither required for the interaction of the Sox2 gene with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs, between Sox2 and its distal enhancers, generated boundaries with varying degrees of insulation that directly correlated with reduced transcriptional output. However, in both epiblast and neural tissues, enhancer contacts and transcriptional induction could not be fully abolished, and insertions failed to disrupt implantation and neurogenesis. In contrast, Sox2 expression was undetectable in the anterior foregut of mutants carrying the strongest boundaries, and these animals fully phenocopied loss of SOX2 in this tissue. We propose that enhancer clusters with a high density of regulatory activity can better overcome physical barriers to maintain faithful gene expression and phenotypic robustness.

CRISPR engineered mouse embryonic stem cells with Sox2-tdTomato and Gata6-GFP knock-in for endoderm differentiation

An Embryonic stem line was engineered with CRISPR mediated knock-in to tag the endogenous locus of Sox2 with tdTomato and Gata6 with GFP. The site-specific knock-ins were genotyped by PCR and DNA sequencing. The timely expression of Gata6 and loss of Sox2 upon differentiation in cells and Embryoid bodies (EBs) were studied by microscopy. The GFP and tdtomato expressing population from day 4 EBs showed exclusive expression of GATA6 and SOX2 protein, confirming the appropriate expression of the fluorescent reporters in the cell line.

RARE-21Sox2 plays an important role in choroid plexus tumor development

Choroid plexus (CP) tumors are rare primary brain neoplasms found most commonly in children and are thought to arise from CP epithelial cells. Sox2 is a transcription factor that not only plays a role in development in the ventricular zone, CP, and roof plate, but also contributes to cancer stemness, tumorigenesis, and drug resistance. Gene expression studies demonstrate aberrant Sox2 expression in human CP tumors, suggesting a role in tumor development. A subset of CP tumors exhibit abnormal NOTCH pathway activity. Using animal models, we previously show that sustained NOTCH activity leads to CP tumors. Immunofluorescence, RT-qPCR, and RNA scope assays have revealed increased Sox2 levels in NOTCH-driven CP tumors compared to wild type CP in mice. To investigate the role of Sox2 in CP tumors, we eliminated Sox2 expression in NOTCH-driven CP tumors. Loss of Sox2 almost completely blocked NOTCH-driven CP tumor growth in these mice, supporting a role for Sox2 in these tumors. Ciliation regulation is one proposed functional pathway for tumorigenesis in CP tumors. Using immunofluorescence assays for cilia (ARL13b) and aquaporin transport protein 1 (AQP1) in combination with super resolution microscopy, we observe a stark contrast between wild type CP epithelial cells which are multiciliated and homogeneously express AQP1, indicative of normal epithelial differentiation, compared to NOTCH-driven CP tumors consisting of mono-ciliated cells with loss of AQP1 expression. In Sox2-deficient NOTCH-driven CP tumors, we observe tumor cells remain mono-ciliated and AQP1-negative, indicating that Sox2 loss does not affect the ciliation machinery. Together this warrants further study into the mechanisms of Sox2 functions in CP tumors. By unraveling the role of Sox2 in CP tumors, we may better understand their origin and biology to ultimately design improved treatment options.

Single-cell chromatin profiling of the primitive gut tube reveals regulatory dynamics underlying lineage fate decisions

Development of the gastrointestinal system occurs after gut tube closure, guided by spatial and temporal control of gene expression. However, it remains unclear what forces regulate these spatiotemporal gene expression patterns. Here we perform single-cell chromatin profiling of the primitive gut tube to reveal organ-specific chromatin patterns that reflect the anatomical patterns of distinct organs. We generate a comprehensive map of epigenomic changes throughout gut development, demonstrating that dynamic chromatin accessibility patterns associate with lineage-specific transcription factor binding events to regulate organ-specific gene expression. Additionally, we show that loss of Sox2 and Cdx2, foregut and hindgut lineage-specific transcription factors, respectively, leads to fate shifts in epigenomic patterns, linking transcription factor binding, chromatin accessibility, and lineage fate decisions in gut development. Notably, abnormal expression of Sox2 in the pancreas and intestine impairs lineage fate decisions in both development and adult homeostasis. Together, our findings define the chromatin and transcriptional mechanisms of organ identity and lineage plasticity in development and adult homeostasis.

Sox2 levels regulate the chromatin occupancy of WNT mediators in epiblast progenitors responsible for vertebrate body formation

WNT signalling has multiple roles. It maintains pluripotency of embryonic stem cells, assigns posterior identity in the epiblast and induces mesodermal tissue. Here we provide evidence that these distinct functions are conducted by the transcription factor SOX2, which adopts different modes of chromatin interaction and regulatory element selection depending on its level of expression. At high levels, SOX2 displaces nucleosomes from regulatory elements with high-affinity SOX2 binding sites, recruiting the WNT effector TCF/β-catenin and maintaining pluripotent gene expression. Reducing SOX2 levels destabilizes pluripotency and reconfigures SOX2/TCF/β-catenin occupancy to caudal epiblast expressed genes. These contain low-affinity SOX2 sites and are co-occupied by T/Bra and CDX. The loss of SOX2 allows WNT-induced mesodermal differentiation. These findings define a role for Sox2 levels in dictating the chromatin occupancy of TCF/β-catenin and reveal how context-specific responses to a signal are configured by the level of a transcription factor.

Sox2 regulates globose basal cell regeneration in the olfactory epithelium.

In the olfactory epithelium, mitotically active globose basal cells (GBCs) continuously replenish olfactory sensory neurons (OSNs) lost throughout life. Although an essential role of the transcription factor Sox2 in expanding olfactory progenitors/stem cells has been shown, its precise role in olfactory GBCs remain incompletely understood. We characterized the Sox2 expression in olfactory GBCs in normal conditions and in a lesion-regeneration model using a Lgr5EGFP-IRES-creERT2 strain. During GBC-mediated regeneration, genetic deletion of sox2 and lineage tracing experiments were performed to examine the function of Sox2 in the progeny of Lgr5-EGFP+ GBCs. Over 95% of Lgr5-EGFP+ GBCs express Sox2 in normal or regeneration conditions. Loss of Sox2 dramatically reduces the cell number in each lineage traced cluster. In the progeny of Lgr5-EGFP+ GBCs, loss of Sox2 significantly decreased the portion of OMP+ OSNs. However, the generation of sustentacular cells was unchanged. Our observations support an essential role of Sox2 in adult olfactory regeneration, likely acting on neuronal-lineage GBCs.

The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells.

Simple SummaryPrimary bone cancers like osteosarcoma (OS) are driven by bone cells that develop mutations, and behave in an uncontrolled manner. All bone cells, including defective ones, are sensitive to their physical environment. We tested the response of osteosarcoma cells, at the gene level, to two different kinds of mechanical stimulus, which are relevant to the tumor microenvironment. The first was fluid flowing over their surface, and the second was stiffness or rigidity of the surface beneath. In our study we found that fluid flow in particular has the ability to change the behavior of OS cancer cells, and could potentially be used to reduce their harmful effects. Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness—two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity—which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

Anti-oncogenic effects of SOX2 silencing on hepatocellular carcinoma achieved by upregulating miR-222-5p-dependent CYLD via the long noncoding RNA CCAT1

In this study, we determined the involvement of SOX2 and its downstream signaling molecules in hepatocellular carcinoma (HCC) progression. We carried out lentiviral transfection in HepG2 cells to determine the roles of SOX2, CCAT1, EGFR, miR-222-5p, and CYLD in HepG2 cells. We first determined the interaction between SOX2 and CCAT1 and that between miR-222-5p and CYLD and their effect on tumor growth in vivo was analyzed in HCC-xenograft bearing nude mice xenografts. SOX2 and CCAT1 were highly expressed in HCC tissues and HepG2 cells. SOX2 bound to the regulatory site of CCAT1. Silencing of SOX2 or CCAT1 inhibited HepG2 cell proliferation, migration, and invasion as well as decreased the expression of CCAT1 and EGFR. CCAT1 silencing reduced EGFR expression, but EGFR expression was increased in HCC tissues and HepG2 cells, which promoted proliferation, migration, and invasion in vitro. EGFR upregulated miR-222-5p, leading to downregulation of CYLD. miR-222-5p inhibition or CYLD overexpression repressed cell functions in HepG2 cells. SOX2 silencing decreased CCAT1, EGFR, and miR-222-5p expression but increased CYLD expression. Loss of SOX2 also reduced the growth rate of tumor xenografts. In summary, SOX2-mediated HCC progression through an axis involving CCAT1, EGFR, and miR-222-5p upregulation and CYLD downregulation.

TCam-2 Cells Deficient for SOX2 and FOXA2 Are Blocked in Differentiation and Maintain a Seminoma-Like Cell Fate In Vivo.

Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into seminomas and non-seminomas. While seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-seminomas, the embryonal carcinoma (EC), resembles malignant embryonic stem cells. Thus, ECs are able to differentiate into cells of all three germ layers (teratomas) and even extra-embryonic-tissue-like cells (yolk-sac tumor, choriocarcinoma). In the last years, we demonstrated that the cellular microenvironment considerably influences the plasticity of seminomas (TCam-2 cells). Upon a microenvironment-triggered inhibition of the BMP signaling pathway in vivo (murine flank or brain), seminomatous TCam-2 cells reprogram to an EC-like cell fate. We identified SOX2 as a key factor activated upon BMP inhibition mediating the reprogramming process by regulating pluripotency, reprogramming and epigenetic factors. Indeed, CRISPR/Cas9 SOX2-deleted TCam-2 cells were able to maintain a seminoma-cell fate in vivo for about six weeks, but after six weeks in vivo still small sub-populations initiated differentiation. Closer analyses of these differentiated clusters suggested that the pioneer factor FOXA2 might be the driving force behind this induction of differentiation, since many FOXA2 interacting genes and differentiation factors like AFP, EOMES, CDX1, ALB, HAND1, DKK, DLK1, MSX1 and PITX2 were upregulated. In this study, we generated TCam-2 cells double-deficient for SOX2 and FOXA2 using the CRISPR/Cas9 technique and xenografted those cells into the flank of nude mice. Upon loss of SOX2 and FOXA2, TCam-2 maintained a seminoma cell fate for at least twelve weeks, demonstrating that both factors are key players in the reprogramming to an EC-like cell fate. Therefore, our study adds an important piece to the puzzle of GCT development and plasticity, providing interesting insights in what can be expected in a patient, when GCT cells are confronted with different microenvironments.

SOX2 is required for inner ear growth and cochlear nonsensory formation before sensory development.

The transcription factor sex determining region Y-box2 (SOX2) is required for the formation of hair cells and supporting cells in the inner ear and is a widely used sensory marker. Paradoxically, we demonstrate via fate mapping that, initially, SOX2 primarily marks nonsensory progenitors in the mouse cochlea, and is not specific to all sensory regions until late otic vesicle stages. SOX2 fate mapping reveals an apical-to-basal gradient of SOX2 expression in the sensory region of the cochlea, reflecting the pattern of cell cycle exit. To understand SOX2 function, we undertook a timed-deletion approach, revealing that early loss of SOX2 severely impaired morphological development of the ear, whereas later deletions resulted in sensory disruptions. During otocyst stages, SOX2 shifted dramatically from a lateral to medial domain over 24-48 h, reflecting the nonsensory-to-sensory switch observed by fate mapping. Early loss or gain of SOX2 function led to changes in otic epithelial volume and progenitor proliferation, impacting growth and morphological development of the ear. Our study demonstrates a novel role for SOX2 in early otic morphological development, and provides insights into the temporal and spatial patterns of sensory specification in the inner ear.

Human Cytomegalovirus Immediate Early 1 Protein Causes Loss of SOX2 from Neural Progenitor Cells by Trapping Unphosphorylated STAT3 in the Nucleus.

The mechanisms underlying neurodevelopmental damage caused by virus infections remain poorly defined. Congenital human cytomegalovirus (HCMV) infection is the leading cause of fetal brain development disorders. Previous work has linked HCMV infection to perturbations of neural cell fate, including premature differentiation of neural progenitor cells (NPCs). Here, we show that HCMV infection of NPCs results in loss of the SOX2 protein, a key pluripotency-associated transcription factor. SOX2 depletion maps to the HCMV major immediate early (IE) transcription unit and is individually mediated by the IE1 and IE2 proteins. IE1 causes SOX2 downregulation by promoting the nuclear accumulation and inhibiting the phosphorylation of STAT3, a transcriptional activator of SOX2 expression. Deranged signaling resulting in depletion of a critical stem cell protein is an unanticipated mechanism by which the viral major IE proteins may contribute to brain development disorders caused by congenital HCMV infection.IMPORTANCE Human cytomegalovirus (HCMV) infections are a leading cause of brain damage, hearing loss, and other neurological disabilities in children. We report that the HCMV proteins known as IE1 and IE2 target expression of human SOX2, a central pluripotency-associated transcription factor that governs neural progenitor cell (NPC) fate and is required for normal brain development. Both during HCMV infection and when expressed alone, IE1 causes the loss of SOX2 from NPCs. IE1 mediates SOX2 depletion by targeting STAT3, a critical upstream regulator of SOX2 expression. Our findings reveal an unanticipated mechanism by which a common virus may cause damage to the developing nervous system and suggest novel targets for medical intervention.

Abstract 5001: Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization

Abstract Although significant progress has been made in understanding the molecular mechanisms that lead to metastatic breast cancer, it remains the overwhelming cause of death for patients. Current studies primarily focus on the prevention of early stages of metastasis, such as migration and invasion. But at the time of diagnosis tumor cells have likely left the primary tumor, suggesting that inhibition of the early stages of metastasis may not be the most effective means of inhibiting metastatic burden. Instead, the identification and targeting of molecules required for establishment and survival of cells at secondary sites is imperative for advancing therapies. We are examining the role of the developmental transcription factor Six2 in promoting metastatic burden. Six2 is a member of the Six family of transcription factors and is responsible for the maintenance of stem/progenitor cells during nephrogenesis. Using knockdown (KD) and overexpression (OE) models in murine mammary cells, we demonstrated that Six2 promotes specifically late-stage metastasis (growth at the secondary site). RNA-Seq analysis on control and Six2 OE cells showed a dramatic enrichment in stem cell transcriptional programs downstream of Six2 in mammary/breast cancer cells. Flow cytometry analyses and tumorsphere assays demonstrate that Six2 OE leads to an increase, whereas Six2 KD leads to a decrease, in the mammary stem cell population and tumorsphere formation, respectively. Using an immune competent mouse model, in vivo orthotopic limiting-dilution experiments reveal that Six2 regulates tumor initiation. In addition, we show that Six2 regulates late-stage metastasis of human breast cancer cells in vivo, as measured by metastatic burden after tail vein injection. Thus, our data demonstrate that Six2 similarly promotes stem phenotypes in breast cancer cells as it does during kidney development, and suggest that this attribute may be critical for its ability to promote metastatic outgrowth. To determine the molecular mechanism by which Six2 mediates stem phenotypes and metastasis in breast cancer, we interrogated our RNA-seq data, which suggested that Six2 may control master regulators of stemness. We demonstrate that both Sox2 and Nanog are regulated by Six2. In human breast cancer gene expression datasets, Six2 significantly positively correlates with both Sox2 and Nanog, and combined OE of Six2 with either Sox2 or Nanog results in poor prognosis. Preliminary data suggest that loss of Sox2 downstream of Six2 inhibits metastatic outgrowth, and that Sox2 is upstream of Nanog in the pathway. Together, our data demonstrate that Six2 regulates cancer stem phenotypes to promote metastatic outgrowth, uncovering a novel Six2/Sox2/Nanog axis that is critical for metastatic colonization. Citation Format: Michael U. Oliphant, Ahwan Pandey, Katherine Johnson, Rani Powers, Matthew Galbraith, James Costello, Heide L. Ford. Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization [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 5001.

Characterization of Patient‐Derived Glioblastoma Stem Cells for Marker Expression, Motility, and Invasiveness

No effective treatment exists for Glioblastoma Multiforme (GBM), the most common and deadly form of brain cancer. Many studies have increasingly pointed towards Glioblastoma Stem Cells (GSCs) as the main cause of GBM malignancy, but mechanisms controlling their invasiveness are not fully understood. Our lab has shown that the L1 Cell Adhesion Molecule (L1CAM) increases GBM cell motility and proliferation through autocrine/paracrine stimulation in vitro. Our objective was to determine whether or not L1CAM plays a similar role in GSCs derived from patient surgical specimens. Our recent focus has been on characterizing our GSC lines for L1CAM expression and on developing bright fluorescently labeled GSCs for in vivo invasion experiments. Characterization of GSC lines for two accepted GSC “markers” has revealed that GSC lines isolated by our adherent culture method resulted in cells almost exclusively positive for Sox‐2 and nestin by flow cytometry. When placed in “differentiation media” containing 10% FBS, one GSC line retained stable expression of GSC markers, while another showed total loss of Sox‐2, but kept nestin. Analysis via western blot showed several GSC cell lines expressed L1CAM as well as the ADAM10 protease that cleaves L1 from the cell surface to stimulate nearby cells. GSCs infected with the K72 lentiviral vector encoding Green Fluorescent Protein (GFP) resulted in bright green fluorescent labeling. Injection of labeled GSCs was done into chick embryo brains at E5 either by themselves or mixed with other cell lines labeled with mCherry. Tumors were allowed to form for 10 days, brains were dissected and fixed at E15, and 350 um vibratome sections were mounted on slides for analysis by confocal microscopy. Analyzed were tumor formation, extent of cell invasion, and deposition of “trails” of L1CAM that might guide GSCs to be more invasive. The GSC line that exhibited stable Sox‐2 expression was highly invasive into brain, kept its stem cell morphology, and tended to form a medulla in mixed tumors with other GBM cell lines. The GSC line that lost Sox‐2 expression was also highly invasive, but many cells exhibited long processes like axons, and cells tended to form the cortex of mixed tumors with other GBM cell lines. We conclude that our GSC lines show fundamentally different invasive behavior and propensities to remain as GSCs. Furthermore, the chick embryo brain tumor model appears to allow a range of individual cell phenotypes to develop from GSC and GBM cell lines that presumably reflect their origins and, thus, it is a good model system to characterize phenotypes of patient‐derived GSC lines.Support or Funding InformationWe thank the UD Summer Scholars Program, the UD Undergraduate Research Program, Dept. of Biological Sciences Undergraduate Research Fund, and graduate student Reetika Dutt.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sox2 regulates astrocytic and vascular development in the retina.

Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis.

Sox2 is dispensable for primary melanoma and metastasis formation.

Tumor initiation and metastasis formation in many cancers have been associated with emergence of a gene expression program normally active in embryonic or organ-specific stem cells. In particular, the stem cell transcription factor Sox2 is not only expressed in a variety of tumors, but is also required for their formation. Melanoma, the most aggressive skin tumor, derives from melanocytes that during development originate from neural crest stem cells. While neural crest stem cells do not express Sox2, expression of this transcription factor has been reported in melanoma. However, the role of Sox2 in melanoma is controversial. To study the requirement of Sox2 for melanoma formation, we therefore performed CRISPR-Cas9-mediated gene inactivation in human melanoma cells. In addition, we conditionally inactivated Sox2 in a genetically engineered mouse model, in which melanoma spontaneously develops in the context of an intact stroma and immune system. Surprisingly, in both models, loss of Sox2 did neither affect melanoma initiation, nor growth, nor metastasis formation. The lack of a tumorigenic role of Sox2 in melanoma might reflect a distinct stem cell program active in neural crest stem cells and during melanoma formation.

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.

SOX2 as a novel marker to predict neoplastic progression in Barrett's esophagus.

The value of Barrett's esophagus (BE) surveillance based on the histological diagnosis of low-grade dysplasia (LGD) remains debated given the lack of adequate risk stratification. The aim of this study was to evaluate the predictive value (PV) of SOX2 expression for neoplastic progression in BE patients. We conducted a case-control study within a prospective cohort of 720 BE patients. Patients with neoplastic progression, defined as the development of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), were classified as cases and patients without neoplastic progression were classified as controls. SOX2 expression was determined by immunohistochemistry in more than 12,000 biopsies from 635 patients; these results were combined with our previous p53 immunohistochemical data. Nondysplastic BE showed homogeneous nuclear staining for SOX2, whereas SOX2 was progressively lost in dysplastic BE. Loss of SOX2 was seen in only 2% of biopsy series without dysplasia, in contrast to 28% in LGD and 67% in HGD/EAC. Loss of SOX2 expression was associated with an increased risk of neoplastic progression in BE patients after adjusting for gender, age, BE length, and esophagitis (adjusted relative risk 4.8; 95% CI 3.2-7.0). The positive PV for neoplastic progression increased from 16% with LGD alone to 56% with concurrent loss of SOX2 and aberrant p53 expression. SOX2 expression is lost during transition from nondysplastic BE to HGD/EAC, and it is associated with an increased risk of neoplastic progression. The highest PV is achieved by concurrent loss of SOX2 and aberrant p53 expression in BE patients with LGD. The use of these markers has the potential to significantly improve risk stratification of Barrett surveillance.

QKI impairs self-renewal and tumorigenicity of oral cancer cells via repression of SOX2

Cancer stem cells (CSCs) may contribute to tumor initiation, distant metastasis and chemo-resistance. One of RNA-binding proteins, Quaking (QKI), was reported to be a tumor suppressor. Here we showed that reduced QKI levels were observed in many human oral cancer samples. Moreover further reduction of QKI expression in CSCs was detected compared with non-CSCs in oral cancer cell lines. Overexpressing QKI in oral cancer cells significantly reduced CSC sphere formation and stem cell-associated genes. In tumor implanting nude mice model, QKI significantly impeded tumor initiation rates, tumor sizes and lung metastasis rates. As a contrast, knocking down QKI enhanced the above effects. Among the putative CSC target genes, SOX2 expression was negatively affected by QKI, mechanism study revealed that QKI may directly regulate SOX2 expression via specific binding with its 3′UTR in a cis element-dependent way. Loss of SOX2 even completely reversed the sphere forming ability in QKI knockdown cell line. Taken together, these data demonstrated that SOX2 is an important CSC regulator in oral cancer. QKI is a novel CSC inhibitor and impaired multiple oral CSC properties via partial repression of SOX2. Therefore, reduced expression of QKI may provide a novel diagnostic marker for oral cancer.

Sox2 modulates the function of two distinct cell lineages in mouse skin

In postnatal skin the transcription factor Sox2 is expressed in the dermal papilla (DP) of guard/awl/auchene hair follicles and by mechanosensory Merkel cells in the touch domes of guard hairs. To investigate the consequences of Sox2 ablation in skin we deleted Sox2 in DP cells via Blimp1Cre and in Merkel cells via K14Cre. Loss of Sox2 from the DP did not inhibit hair follicle morphogenesis or establishment of the dermis and hypodermis. However, Sox2 expression in the DP was necessary for postnatal maintenance of awl/auchene hair follicles. Deletion of Sox2 via K14Cre resulted in a decreased number of Merkel cells but had no effect on other epithelial compartments or on the dermis. The reduced number of Merkel cells did not affect the number or patterning of guard hairs, nerve density or the interaction of nerve cells with the touch domes. We conclude that Sox2 is a marker of two distinct lineages in the skin and regulates the number of differentiated cells in the case of the Merkel cell lineage and hair follicle type in the case of the DP.