SAM-pointed Domain Research Articles

Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC.

Cancer stem-like cells (CSCs) are critically involved in cancer metastasis and chemoresistance, acting as one major obstacle in clinical practice. While accumulating studies have implicated the metabolic reprogramming of CSCs, mitochondrial dynamics in such cells remain poorly understood. Here we pinpointed OPA1hi with mitochondrial fusion as a metabolic feature of human lung CSCs, licensing their stem-like properties. Specifically, human lung CSCs exerted enhanced lipogenesis, inducing OPA1 expression via transcription factor SAM Pointed Domain containing ETS transcription Factor (SPDEF). In consequence, OPA1hi promoted mitochondrial fusion and stemness of CSCs. Such lipogenesishi, SPDEFhi, and OPA1hi metabolic adaptions were verified with primary CSCs from lung cancer patients. Accordingly, blocking lipogenesis and mitochondrial fusion efficiently impeded CSC expansion and growth of organoids derived from patients with lung cancer. Together, lipogenesis regulates mitochondrial dynamics via OPA1 for controlling CSCs in human lung cancer.

Constraint-Based, Score-Based and Hybrid Algorithms to Construct Bayesian Gene Networks in the Bovine Transcriptome.

Simple SummaryIn this study, we investigated and compared six different Bayesian network algorithms from three different categories to identify hub genes critical to gene expression networks activated in response to progesterone in the bovine uterus. We observed many common hub genes identified between constraint-based algorithms (CBAs) and hybrid algorithms (HAs), while it appeared that score-based algorithm (SBA) methods led to more accurate and relevant predictions of core genes. The results revealed that the identification of hub genes was affected by the type of network reconstruction and by the subsequently used topological parameters. Two identified genes known to have roles during pregnancy are ISG15 and DGAT2. The identified hub genes are associated with biological processes such as amino acid metabolism, hormonal signaling pathways and the immune system. Our analysis revealed a role for miRNAs in the regulation of this system. The biological and physiological roles (enzymatic and hormonal effects) of unannotated identified hub genes should be functionally validated by further studies.Bayesian gene networks are powerful for modelling causal relationships and incorporating prior knowledge for making inferences about relationships. We used three algorithms to construct Bayesian gene networks around genes expressed in the bovine uterus and compared the efficacies of the algorithms. Dataset GSE33030 from the Gene Expression Omnibus (GEO) repository was analyzed using different algorithms for hub gene expression due to the effect of progesterone on bovine endometrial tissue following conception. Six different algorithms (grow-shrink, max-min parent children, tabu search, hill-climbing, max-min hill-climbing and restricted maximum) were compared in three higher categories, including constraint-based, score-based and hybrid algorithms. Gene network parameters were estimated using the bnlearn bundle, which is a Bayesian network structure learning toolbox implemented in R. The results obtained indicated the tabu search algorithm identified the highest degree between genes (390), Markov blankets (25.64), neighborhood sizes (8.76) and branching factors (4.38). The results showed that the highest number of shared hub genes (e.g., proline dehydrogenase 1 (PRODH), Sam-pointed domain containing Ets transcription factor (SPDEF), monocyte-to-macrophage differentiation associated 2 (MMD2), semaphorin 3E (SEMA3E), solute carrier family 27 member 6 (SLC27A6) and actin gamma 2 (ACTG2)) was seen between the hybrid and the constraint-based algorithms, and these genes could be recommended as central to the GSE33030 data series. Functional annotation of the hub genes in uterine tissue during progesterone treatment in the pregnancy period showed that the predicted hub genes were involved in extracellular pathways, lipid and protein metabolism, protein structure and post-translational processes. The identified hub genes obtained by the score-based algorithms had a role in 2-arachidonoylglycerol and enzyme modulation. In conclusion, different algorithms and subsequent topological parameters were used to identify hub genes to better illuminate pathways acting in response to progesterone treatment in the bovine uterus, which should help with our understanding of gene regulatory networks in complex trait expression.

Bee Venom Prevents Mucin 5AC Production through Inhibition of AKT and SPDEF Activation in Airway Epithelia Cells.

IL-13 induces mucus metaplasia, which causes airway obstruction in asthma. Bee venom (BV) and its components have shown anti-inflammatory effects in allergic diseases such as atopic dermatitis and asthma. In this study, we investigated the effect of BV on IL-13-induced mucus metaplasia through activation of the signal transducer and activator of transcription (STAT6), and regulation of SAM-pointed domain containing Ets-like factor (SPDEF) and forkhead box A2 (FOXA2) in the airway epithelia cell line A549. In A549 cells, BV (1.0 µg/mL) inhibited IL-13 (10 ng/mL)-induced AKT phosphorylation, increase in SPDEF protein expression, and decrease in FOXA2 protein expression—but not STAT6 phosphorylation. BV also prevented the IL-13-induced increase in mucin 5AC (MUC5AC) mRNA and protein expression. Moreover, we observed that inhibition of phosphoinositide 3 kinase (PI3K)/AKT using LY294002 (50 µM) could reverse the alterations in FOXA2 and MUC5AC expression -by IL-13 and BV. However, LY294002 did not affect IL-13- and BV-induced changes in SPDEF expression. These findings indicate that BV inhibits MUC5AC production through the regulation of SPDEF and FOXA2. The inhibition of MUC5AC production through FOXA2 is mediated via the suppression of PI3K/AKT activation by BV. BV may be helpful in the prevention of mucus metaplasia in asthma.

SPDEF targets EMT pathway to inhibit cell migration and invasion in triple‐negative breast cancer

Background Current treatments against triple-negative breast cancer (TNBC) are ineffective due to the high rate of metastasis and therapy resistance. There is, therefore, a pressing need for novel and efficacious therapies against TNBC. The basal-like TNBC has a poor prognosis and a high metastasis rate compared to the luminal-like breast cancer subtypes. Here, set to determine the role of Sam Pointed Domain Ets Transcription Factor also known as Prostate-Derived Ets Factor (SPDEF/PDEF), in TNBC. Methods Clinical data were extracted from the METABRIC breast cancer project, hosted at the TCGA database. MDA-MB-231 and MDA 231-LM2-4175 cells were stably transfected with PDEF/control pBABE retroviral vectors. Cell migration/invasion assays were done to explore the effects of PDEF expression on the motility of TNBC cells. Proliferation was measured under 2D and 3D culture conditions. qRT-PCR and immunoblot were performed to visualize gene and protein expression. Data were graphed and analyzed using Graph Pad Prism Software 8 using one-way ANOVA and the unpaired two-tailed Student's t-test. All data were evaluated in triplicate against vector control cells. Results Basal gene and protein expression screening revealed that the expression of PDEF is significantly lower in highly metastatic breast cancer cells compared to the non-metastatic ones. Data analysis from clinical breast cancer METABRIC cohort revealed the loss of PDEF is associated with tumor metastasis, tumor grade, and poor patient survival. PDEF is lost in highly metastatic and therapy resistant TNBC cells. Re-expression of PDEF diminished cell migration and invasion in TNBC cells. Decreased migration and invasion in response to PDEF was not a result of decreased cell growth and proliferation or increased cell death but a result of suppression of EMT markers in TNBC cells. Conclusions PDEF expression inhibits cell migration and invasion partially by down-regulating EMT-related protein markers, suggesting a critical role for PDEF in suppressing TNBC cell metastasis.

Neuroplastinβ-mediated upregulation of solute carrier family 22 member 18 antisense (SLC22A18AS) plays a crucial role in the epithelial-mesenchymal transition, leading to lung cancer cells' enhanced motility

Our recent study revealed an important role of the neuroplastin (NPTN)β downstream signal in lung cancer dissemination in the lung. The molecular mechanism of the signal pathway downstream of NPTNβ is a serial activation of the key molecules we identified: tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) adaptor, nuclear factor (NF)IA/NFIB heterodimer transcription factor, and SAM pointed-domain containing ETS transcription factor (SPDEF). The question of how dissemination is controlled by SPDEF under the activated NPTNβ has not been answered. Here, we show that the NPTNβ-SPDEF-mediated induction of solute carrier family 22 member 18 antisense (SLC22A18AS) is definitely required for the epithelial-mesenchymal transition (EMT) through the NPTNβ pathway in lung cancer cells. In vitro, the induced EMT is linked to the acquisition of active cellular motility but not growth, and this is correlated with highly disseminative tumor progression in vivo. The publicly available data also show the poor survival of SLC22A18AS-overexpressing lung cancer patients. Taken together, these data highlight a crucial role of SLC22A18AS in lung cancer dissemination, which provides novel input of this molecule to the signal cascade of NPTNβ. Our findings contribute to a better understanding of NPTNβ-mediated lung cancer metastasis.

Prostate-Derived ETS Factor (PDEF) Modulates Yes Associated Protein 1 (YAP1) in Prostate Cancer Cells: A Potential Cross-Talk between PDEF and Hippo Signaling.

PDEF (prostate-derived ETS factor, also known as SAM-pointed domain containing ETS transcription factor (SPDEF)) is expressed in luminal epithelial cells of the prostate gland and associates with luminal phenotype. The Hippo pathway regulates cell growth/proliferation, cellular homeostasis, and organ development by modulating phosphorylation of its downstream effectors. In previous studies, we observed decreased levels of PDEF during prostate cancer progression. In the present study, we evaluated the effects of the expression of PDEF on total/phosphoprotein levels of YAP1 (a downstream effector of the Hippo pathway). We observed that the PC3 and DU145 cells transfected with PDEF (PDEF-PC3 and PDEF-DU145) showed an increased phospho-YAP1 (Ser127) and total YAP1 levels as compared to the respective PC3 vector control (VC-PC3) and DU145 vector control cells (VC-DU145). We also observed an increased cytoplasmic YAP1 levels in PDEF-PC3 cells as compared to VC-PC3 cells. Moreover, our gene set enrichment analysis (GSEA) of mRNA expression in PDEF-PC3 and VC-PC3 cells revealed that PDEF resulted in inhibition of YAP1 target genes, directly demonstrating that PDEF plays a critical role in modulating YAP1 activity, and by extension in the regulation of the Hippo pathway. We also observed a decrease in YAP1 mRNA levels in prostate cancer tissues as compared to normal prostate tissues. Our analysis of multiple publicly available clinical cohorts revealed a gradual decrease in YAP1 mRNA expression during prostate cancer progression and metastasis. This decrease was similar to the decrease in PDEF levels, which we had reported earlier, and we observed a direct correlation between PDEF and YAP1 expression in CRPC data set. To the best of our knowledge, these results provide the first demonstration of inhibiting YAP1 activity by PDEF in any system and suggest a cross-talk between PDEF and the Hippo signaling pathway.

Abstract 1502: PDEF inhibits prostate cancer progression by promoting luminal differentiation

Abstract Background: The emergence of metastatic castration-resistant prostate cancer (PCa) is accompanied by the loss of prostate luminal cell identity. Cell identity is defined by key transcription factors by association with enhancer and super-enhancer elements. Our previous studies have shown that Sam Pointed Domain Ets Transcription Factor a.k.a. Prostate-Derived Ets Factor (SPDEF/PDEF), inhibits tumor metastasis in vivo. Recently, PDEF has been reported as one of the super enhancer related transcription factors in the luminal PCa cell line LNCaP and observed to be highly expressed in prostate luminal cells. We propose that PDEF functions as a putative tumor metastasis suppressor and inhibits tumor progression by restoring luminal cell identity. The present study was designed to investigate the role of PDEF in metastatic PCa progression. Methods: PC3 and DU145 cells were stably transfected with PDEF/control pBABE retroviral vectors. Global gene expression changes were probed using Affymetrix microarray. Microarray was analyzed with GSEA. qRT-PCR was performed to confirm the differential gene expression profile and immunohistochemistry, immunofluorescence and immunoblot were performed to visualize protein expression. ChIP-seq data were extracted from SRP002475 and aligned with Bowtie. Peaks were identified by MACS2. Clinical data were extracted from GSE16560, GSE21034 and TCGA database. Statistical analysis was performed with GraphPad and R. Results: Analysis of our microarray studies revealed that PDEF expression resulted in the negative enrichment of metastasis-related gene sets and the positive enrichment of gene sets involved in luminal differentiation. Especially, PDEF restores canonical AR signaling in PC3 cells. Confirmed with qPCR, IB and IF, PDEF also inhibits the expression of neuroendocrine-related genes, stemness-related genes while promoting the expression of prostate luminal differentiation-related genes. Furthermore, PDEF ChIP-seq analysis revealed a novel PDEF binding site at the putative promoter region of prostate luminal cell marker cytokeratin 18 while no significant peaks were identified within the regulatory elements of EMT related transcription factors. Our data suggest that PDEF promotes the expression of cytokeratin 18 in vitro and in vivo. Knockdown of cytokeratin 18 by siRNA increases the expression of Twist1 while decreases the expression of E-Cadherin while PDEF expression level is not affected. Moreover, data analysis from multiple clinical prostate cancer cohorts suggests the loss of PDEF is associated with tumor metastasis, tumor progression, and poor survival. Conclusions: PDEF expression leads to luminal differentiation phenotype in advanced prostate cancer cells. PDEF inhibits cell migration and metastasis in part by down-regulating EMT related transcription factors in part through directly promoting the expression of cytokeratin 18. Citation Format: Fengtian Wang, Sweaty Koul, Hari K. Koul. PDEF inhibits prostate cancer progression by promoting luminal differentiation [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 1502.

Abstract 3525: PDEF promote prostate cancer luminal/epithelial differentiation and inhibit tumor progression

Abstract Conventional therapies produce a high rate of cure for patients with localized prostate cancer (PCa), but there is no effective treatment for castration resistant metastatic PCa. Transcription factors by association with enhancer and super-enhancer elements are key drivers of cell identity. Our previous studies have shown that Sam Pointed Domain Ets Transcription Factor a.k.a. Prostate Derived Ets Factor (SPDEF/PDEF), inhibits cell migration, invasion and clonogenic activity in PCa cells and there is a graded loss of PDEF with increasing Gleason Score in PCa patient samples. PDEF has been reported to be one of the cell identity transcription factors in LNCaP cells. We proposed that PDEF functions as a putative tumor metastasis suppressor. Others have shown an increase in Twist1, a bHLH transcription factor, was positively correlated with Gleason Grading. Present study was designed to investigate the role of PDEF and interplay between PDEF and Twist1 in PCa. PC3 cells were stably transfected with PDEF/control PABABE vectors. Gene expression changes were monitored by Affymetrix microarray. Microarray was analyzed with GSEA. qRT-PCR was performed to confirm gene expression and IHC, IF and IB analysis were performed to visualize protein expression. Chip-seq data (SRP002475) was aligned with Bowtie. Peaks were identified by MACS and visualized using IGB. PDEF/Twist1 expression data was extracted from GSE16560. GraphPad was used to generate KM survival analysis of PCa patients. Our results show that PDEF expression is limited to epithelial/luminal cells of the prostatic glands. We observed that expression of Twist1 was down regulated in PC3 cells following PDEF expression. Analysis of gene expression data from our microarray studies revealed that PDEF re-expression was associated with negative enrichment of gene sets involved with cell migration and positive enriched of gene sets involved with epithelial/luminal differentiation. Chip-seq analysis revealed a binding of PDEF to KRT8/18 promoter region. These data suggest that PDEF inhibits core metastasis related genes through promoting a program of luminal/epithelial differentiation. In clinical samples of PCa, expression of PDEF was inversely associated, while expression of Twist1 was positively associated with Gleason grade. PDEF and Twist1 was able to predict patient survival, moreover integrated PDEF and Twist1 expression was able to better predict PCa patient survival as compared to PDEF or Twist1 alone. PDEF inhibits cell migration and metastasis in part by down-regulating Twist1 level and promoting luminal/epithelial differentiation. Loss of PDEF combined with gain of Twist1 expression may serve as a potential biomarker set for distinguishing lethal PCa from an indolent disease. Additional studies are underway to gain further insights into the role of PDEF in PCa progression and metastasis. Acknowledgement: FWCC support and Chair commitment funds (Koul H). Citation Format: Fengtian Wang, Sweaty Koul, Prakash Srinivasan Timiri Shanmugam, Qin Dong, Hari K. Koul. PDEF promote prostate cancer luminal/epithelial differentiation and inhibit tumor progression [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 3525. doi:10.1158/1538-7445.AM2017-3525

Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells.

Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor (SPDEF) plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing SPDEF using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the SPDEF promoter. All constructs were able to effectively suppress both SPDEF mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 (AGR2), mucin 5AC (MUC5AC)]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient SPDEF silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.

Abstract 2012: SPDEF enforces tumor quiescence by shifting the transcriptional targets of activated β-catenin

Abstract Background: Colorectal cancer (CRC) carcinogenesis is driven by a series of genetic and epigenetic changes that results in the oncogenic transformation of normal colonic mucosa. Canonical Wnt/β-catenin signaling pathway activation, with resultant high β-catenin transcriptional activity, is frequently implicated human CRC (∼ 90% of CRC); however, there are currently no treatments targeting this pathway. We have previously reported that SAM Pointed Domain Ets transcription Factor (SPDEF) is a colonic tumor suppressor that negatively regulates canonical Wnt/β-catenin signaling. In agreement with the tumor repressor role of SPDEF, the absence of SPDEF enhances intestinal tumor formation, while re-expression of SPDEF inhibits colon adenocarcinoma proliferation in both genetic (Apcmin/+) and chemically colitis-associated (AOM/DSS) CRC models. However, the molecular mechanism by which SPDEF mediates colorectal tumor repression is still largely unknown. Here we aim to elucidate the molecular mechanism by which SPDEF mediates repression of canonical Wnt/β-catenin signaling in CRCs. Material and Methods: To achieve our goal, we directly analyzed the effects of SPDEF expression in β-catenin-driven intestinal tumors in vivo using a new inducible mouse model (Lgr5CreERT2; β-cateninexon3; Rosa26rtta-ires-EGFP; TRE-Spdef) and human colon cancer xenografts. Moreover, wildtype or truncated SPDEF mutants were used for β-catenin transcriptional activity assay, co-immunoprecipitation, and chromatin immunoprecipitation in human colon cancer cell lines. Results: In this study, we find that SPDEF is sufficient to inhibit β-catenin-driven intestinal tumorigenesis and shrink established tumors in both transgenic mice and xenografted human colon cancer cells. SPDEF inhibits canonical β-catenin transcriptional activity through protein-protein interaction, independent of its DNA binding capacity. We find that SPDEF disrupts the binding between β-catenin and its DNA binding partners TCF1 and TCF3, but not LEF1 and TCF4, selectively displacing β-catenin from the promoter/enhancer regions of cell cycle genes without affecting stem cell signature genes. Consistent with this observation, re-expression of SPDEF enforces a quiescent state on β-catenin-driven tumor cells in vivo. Taken together, for the first time, we unveil a novel mechanism in which SPDEF shifts the transcriptional targets of activated β-catenin to regulate the active to quiescent switch in tumor initiating cells. These findings provide insights into the mechanisms that can regulate tumor cell quiescence, and offer a novel approach for targeting canonical Wnt/β-catenin transcriptional machinery as a therapeutic strategy. Citation Format: Yuan-Hung Lo, Taeko Noah, Min-Shan Chen, Winnie Zou, Noah Shroyer. SPDEF enforces tumor quiescence by shifting the transcriptional targets of activated β-catenin. [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 2012.

The Effect of Nrf2 Knockout on Ocular Surface Protection from Acute Tobacco Smoke Exposure: Evidence from Nrf2 Knockout Mice

Ocular surface mucosa is the first-line ocular tissue to be exposed to environmental stress. We evaluated tear functions and keratoconjunctival epithelial alterations after sidestream cigarette smoke (SCS) exposure and tried to clarify the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nfe2l2, also known as Nrf2), on the ocular surface. In wild-type and Nrf2(-/-) mice, tear volume did not change after SCS exposure. Tear film breakup time (tear stability) in Nrf2(-/-) mice was significantly shorter than that in wild-type mice after SCS exposure. Vital staining scores, including fluorescein and Rose Bengal staining, showed significantly higher values in Nrf2(-/-) mice than in wild-type mice after SCS exposure. Excessive oxidative stress accumulation was detected in Nrf2(-/-) mice after SCS exposure using immunohistochemical analysis. Immunohistochemical analysis also revealed decreased mucin 1 (Muc1) and Muc5ac staining in Nrf2(-/-) mice after SCS exposure. mRNA expression levels of Muc1, Muc4, and Muc5ac and of SAM-pointed domain epithelial-specific transcription factor in Nrf2(-/-) mice were lower than those in wild-type mice after SCS exposure. Mean tear IL-6 concentrations increased significantly in Nrf2(-/-) mice after SCS exposure. In conclusion, SCS exposure induced decreased tear stability, ocular surface damage, and altered conjunctival phenotype in Nrf2(-/-) mice. Nrf2 could play an important role in protection of the ocular surface against SCS exposure.

TGFβ signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.

The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor β receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFβ signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFβ restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFβ inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFβ in regulating goblet cell differentiation.

SPDEF: a molecular switch for E-cadherin expression that promotes prostate cancer metastasis

SPDEF: a molecular switch for E-cadherin expression that promotes prostate cancer metastasis

Spdef Null Mice Lack Conjunctival Goblet Cells and Provide a Model of Dry Eye

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.

SAM-pointed domain ETS factor mediates epithelial cell-intrinsic innate immune signaling during airway mucous metaplasia.

Airway mucus plays a critical role in clearing inhaled toxins, particles, and pathogens. Diverse toxic, inflammatory, and infectious insults induce airway mucus secretion and goblet cell metaplasia to preserve airway sterility and homeostasis. However, goblet cell metaplasia, mucus hypersecretion, and airway obstruction are integral features of inflammatory lung diseases, including asthma, chronic obstructive lung disease, and cystic fibrosis, which cause an immense burden of morbidity and mortality. These chronic lung diseases are united by susceptibility to microbial colonization and recurrent airway infections. Whether these twinned phenomena (mucous metaplasia, compromised host defenses) are causally related has been unclear. Here, we demonstrate that SAM pointed domain ETS factor (SPDEF) was induced by rhinoviral infection of primary human airway cells and that cytoplasmic activities of SPDEF, a transcriptional regulator of airway goblet cell metaplasia, inhibited Toll-like receptor (TLR) activation of epithelial cells. SPDEF bound to and inhibited activities of TLR signaling adapters, MyD88 and TRIF, inhibiting MyD88-induced cytokine production and TRIF-induced interferon β production. Conditional expression of SPDEF in airway epithelial cells in vivo inhibited LPS-induced neutrophilic infiltration and bacterial clearance. SPDEF-mediated inhibition of both TLR and type I interferon signaling likely protects the lung against inflammatory damage when inciting stimuli are not eradicated. Present findings provide, at least in part, a molecular explanation for increased susceptibility to infection in lung diseases associated with mucous metaplasia and a mechanism by which patients with florid mucous metaplasia may tolerate microbial burdens that are usually associated with fulminant inflammatory disease in normal hosts.

411 Developmental Regulation of Sam Pointed Domain Ets Factor (SPDEF) in Rat Ileum Cultured With Bile Acids

fibers as a percentage of total tissue area (μm2). The nerve volume densities were significantly decreased in the jejunum (23.7 vs. 31.3; p 0.05) and duodenum (30.1 vs. 28.8; p > 0.05). These findings suggest that endogenous microbiota are necessary for the normal patterning of the ENS in the early postnatal period, particularly in the mid to distal small intestine and colon. Further studies are needed to determine whether the mechanisms by which the endogenous microbiota act on the ENS involve a direct effect on neural cells.

Sam-Pointed Domain Containing Ets Transcription Factor in Luminal Breast Cancer Pathogenesis

We previously described frequent overexpression of Sam-pointed domain containing Ets transcription factor (SPDEF), also known as PDEF, in human breast cancer, and suggested a role for this transcription factor in breast tumor progression. To seek evidence in support of this hypothesis, the MCF-12A breast epithelial cell line was transfected with an SPDEF expression plasmid or with control vector plasmid and the transfected cells tested for their tumorigenic growth in vivo. The data showed that SPDEF expression in MCF-12A cells induced accelerated tumor growth in severe combined immune deficient mice compared with vector-transfected MCF-12A cells. Furthermore, Gene Expression Omnibus and Oncomine databases were mined to determine any correlation between SPDEF expression levels and clinical outcome. High SPDEF expression correlated with poor overall survival of patients with estrogen receptor+ breast cancer, in three independent data sets. In contrast, little correlation was observed between SPDEF expression and cancer relapse or remote metastases. SPDEF expression was further found to be restricted to tumors arising in the luminal epithelial lineage including estrogen receptor+ luminal subtype breast tumors, Her2/neu-positive tumors, and apocrine carcinomas. In contrast, little SPDEF expression was found in the basal subtype of breast tumors. Based on these results, we hypothesize that SPDEF has a function in the specification of the progenitor cells of the luminal epithelial lineage that become targets of oncogenesis in luminal breast cancer.