Expression Of Lefty Research Articles

Pioneer Factors FOXA1 and FOXA2 Assist Selective Glucocorticoid Receptor Signaling in Human Endometrial Cells.

Successful pregnancy relies on dynamic control of cell signaling to achieve uterine receptivity and the necessary biological changes required for endometrial decidualization, embryo implantation, and fetal development. Glucocorticoids are master regulators of intracellular signaling and can directly regulate embryo implantation and endometrial remodeling during murine pregnancy. In immortalized human uterine cells, we have shown that glucocorticoids and estradiol (E2) coregulate thousands of genes. Recently, glucocorticoids and E2 were shown to coregulate the expression of Left-right determination factor 1 (LEFTY1), previously implicated in the regulation of decidualization. To elucidate the molecular mechanism by which glucocorticoids and E2 regulate the expression of LEFTY1, immortalized and primary human endometrial cells were evaluated for gene expression and receptor recruitment to regulatory regions of the LEFTY1 gene. Glucocorticoid administration induced expression of LEFTY1 messenger RNA and protein and recruitment of the glucocorticoid receptor (GR) and activated polymerase 2 to the promoter of LEFTY1. Glucocorticoid-mediated recruitment of GR was dependent on pioneer factors FOXA1 and FOXA2. E2 was found to antagonize glucocorticoid-mediated induction of LEFTY1 by reducing recruitment of GR, FOXA1, FOXA2, and activated polymerase 2 to the LEFTY1 promoter. Gene expression analysis identified several genes whose glucocorticoid-dependent induction required FOXA1 and FOXA2 in endometrial cells. These results suggest a molecular mechanism by which E2 antagonizes GR-dependent induction of specific genes by preventing the recruitment of the pioneer factors FOXA1 and FOXA2 in a physiologically relevant model.

Identification of LEFTY as a molecular marker for ovarian clear cell carcinoma.

To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-β superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTY1 being predominant compared to that of LEFTY2. OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21waf1 pathway or increased p27kip1 expression, directly or indirectly. Moreover, the treatment of stable cell lines with cisplatin led to increased apoptotic cells, together with the inhibition of protein expression of a pSmad2-mediated X-linked inhibitor of apoptosis and a decreased bcl2/bax ratio. Blocking LEFTY1 expression with a specific short hairpin RNA inhibited cisplatin-induced apoptosis, probably through the increased expression of both XIAP and bcl2, but not bax. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in OCCCas with a high LEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.

Cerberus–Nodal–Lefty–Pitx signaling cascade controls left–right asymmetry in amphioxus

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus, Nodal, Lefty, and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal, Lefty, and Pitx, whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal, whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.

Nodal and BMP expression during the transition to pentamery in the sea urchin Heliocidaris erythrogramma: insights into patterning the enigmatic echinoderm body plan

BackgroundThe molecular mechanisms underlying the development of the unusual echinoderm pentameral body plan and their likeness to mechanisms underlying the development of the bilateral plans of other deuterostomes are of interest in tracing body plan evolution. In this first study of the spatial expression of genes associated with Nodal and BMP2/4 signalling during the transition to pentamery in sea urchins, we investigate Heliocidaris erythrogramma, a species that provides access to the developing adult rudiment within days of fertilization.ResultsBMP2/4, and the putative downstream genes, Six1/2, Eya, Tbx2/3 and Msx were expressed in the earliest morphological manifestation of pentamery during development, the five hydrocoele lobes. The formation of the vestibular ectoderm, the specialized region overlying the left coelom that forms adult ectoderm, involved the expression of putative Nodal target genes Chordin, Gsc and BMP2/4 and putative BMP2/4 target genes Dlx, Msx and Tbx. The expression of Nodal, Lefty and Pitx2 in the right ectoderm, and Pitx2 in the right coelom, was as previously observed in other sea urchins.ConclusionThat genes associated with Nodal and BMP2/4 signalling are expressed in the hydrocoele lobes, indicates that they have a role in the developmental transition to pentamery, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. We suggest that the Nodal and BMP2/4 signalling cascades might have been duplicated or split during the evolution to pentamery.

Hedgehog participates in the establishment of left-right asymmetry during amphioxus development by controlling Cerberus expression.

Correct patterning of left-right (LR) asymmetry is essential during the embryonic development of bilaterians. Hedgehog (Hh) signaling is known to play a role in LR asymmetry development of mouse, chicken and sea urchin embryos by regulating Nodal expression. In this study, we report a novel regulatory mechanism for Hh in LR asymmetry development of amphioxus embryos. Our results revealed that Hh-/- embryos abolish Cerberus (Cer) transcription, with bilaterally symmetric expression of Nodal, Lefty and Pitx In consequence, Hh-/- mutants duplicated left-side structures and lost right-side characters, displaying an abnormal bilaterally symmetric body plan. These LR defects in morphology and gene expression could be rescued by Hh mRNA injection. Our results indicate that Hh participates in amphioxus LR patterning by controlling Cer gene expression. Curiously, however, upregulation of Hh signaling failed to alter the Cer expression pattern or LR morphology in amphioxus embryos, indicating that Hh might not provide an asymmetric cue for Cer expression. In addition, Hh is required for mouth opening in amphioxus, hinting at a homologous relationship between amphioxus and vertebrate mouth development.

Abstract 2891: High throughput single cell gene expression profiling by multiplex qPCR

Abstract Single cell gene expression analysis is a powerful technique that provides a unique and insightful perspective on biological pathways and processes. Here we present a robust workflow that enables fast and accurate analysis of up to 100 genes in isolated single cells. Our workflow is highly sensitive, by assessing RNA reference standards we find that a single RNA transcript is detected with about 80% efficiency. We used this workflow to study differentiation in cultured NTera2 cells (NT2), a human embryonic stem cell model system. We analyzed untreated NT2 cells, and NT2 cells treated with low and high doses of retinoic acid (RA) for 10 days to initiate differentiation to a neuronal lineage. The expression levels of 16 genes were quantified in 164 single cells by multiplex real-time qPCR with two technical replicates. The entire experiment, from cultured cells to results, can be completed in 2-3 days and requires four 384-well qPCR plates for gene expression quantification. We find that control cells and cells treated with a high dose of RA (10 uM) are relatively homogeneous in the expression levels of the targeted genes. However cells treated with a low dose of RA (0.25 uM) exhibit significant heterogeneity with respect to gene expression; about half of the cells are similar to the high-dose RA cells, the other cells exhibit a wide range of partial differentiation. Interestingly, we find that LEFTY2 expression is almost exclusive to the low dose RA cells and strongly correlates with partial differentiation. A time-course study analyzing cell populations reveals that LEFTY2 is only transiently expressed in differentiating NT2 cells with peak expression at 3 days of high dose RA treatment. These findings imply that, in NT2 cells, LEFTY2 is a potential biomarker of early differentiation. In summary, we present an accurate, sensitive and robust single cell analysis procedure that uses standard reagents and platforms. We envision that this workflow will enable researchers to investigate cell heterogeneity in biological pathways in a cost-effective way. Citation Format: Steven T. Okino, Michelle Kong, Jason Ma, Joshua Fenrich, Luca Boveri, Yann Jouvenot, Yan Wang. High throughput single cell gene expression profiling by multiplex qPCR. [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 2891.

Ino80 is essential for proximal-distal axis asymmetry in part by regulating Bmp4 expression

BackgroundUnderstanding how embryos specify asymmetric axes is a major focus of biology. While much has been done to discover signaling pathways and transcription factors important for axis specification, comparatively little is known about how epigenetic regulators are involved. Epigenetic regulators operate downstream of signaling pathways and transcription factors to promote nuclear processes, most prominently transcription. To discover novel functions for these complexes in axis establishment during early embryonic development, we characterized phenotypes of a mouse knockout (KO) allele of the chromatin remodeling Ino80 ATPase.ResultsIno80 KO embryos implant, but fail to develop beyond the egg cylinder stage. Ino80 KO embryonic stem cells (ESCs) are viable and maintain alkaline phosphatase activity, which is suggestive of pluripotency, but they fail to fully differentiate as either embryoid bodies or teratomas. Gene expression analysis of Ino80 KO early embryos by in situ hybridization and embryoid bodies by RT-PCR shows elevated Bmp4 expression and reduced expression of distal visceral endoderm (DVE) markers Cer1, Hex, and Lefty1. In culture, Bmp4 maintains stem cell pluripotency and when overexpressed is a known negative regulator of DVE differentiation in the early embryo. Consistent with the early embryo, we observed upregulated Bmp4 expression and down-regulated Cer1, Hex, and Lefty1 expression when Ino80 KO ESCs are differentiated in a monolayer. Molecular studies in these same cells demonstrate that Ino80 bound to the Bmp4 promoter regulates its chromatin structure, which correlates with enhanced SP1 binding. These results in combination suggest that Ino80 directly regulates the chromatin structure of the Bmp4 promoter with consequences to gene expression.ConclusionsIn contrast to Ino80 KO differentiated cells, our experiments show that undifferentiated Ino80 KO ESCs are viable, but fail to differentiate in culture and in the early embryo. Ino80 KO ESCs and the early embryo up-regulate Bmp4 expression and down-regulate the expression of DVE markers Cer1, Hex and Lefty1. Based on this data, we propose a model where the Ino80 chromatin remodeling complex represses Bmp4 expression in the early embryo, thus promoting DVE differentiation and successful proximal-distal axis establishment. These results are significant because they show that epigenetic regulators have specific roles in establishing embryonic axes. By further characterizing these complexes, we will deepen our understanding of how the mammalian embryo is patterned by epigenetic regulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0238-5) contains supplementary material, which is available to authorized users.

Lefty1 alleviates renal tubulointerstitial injury in mice with unilateral ureteral obstruction.

Lefty is a member of the transforming growth factor (TGF) β superfamily, which is implicated in left‑right patterning during embryogenesis. Previous studies revealed that lefty attenuates the epithelial‑mesenchymal transition in tubular epithelial cells. In the present study, the protective effect of lefty1 on renal interstitial injury was further assessed. Mice with a unilateral ureteral obstruction (UUO) were sacrificed on days 3, 5 and 7 following surgery, and the association between the expression of lefty1 and the degree of interstitial fibrosis was investigated. Subsequently, mice with a UUO were administered recombinant lefty1 (300 µg/kg body weight) or vehicle (0.9% saline solution; 100 µl) through tail‑vein injection every other day for 6 days. The effects of lefty1 were assessed by measuring the degree of tubulointerstitial fibrosis, tubular injury and atrophy, and also by monitoring the expression levels of α‑smooth muscle actin (α‑SMA), TGF‑β1, phosphorylated (p)‑Smad2/3, kidney injury molecular‑1 and endogenous lefty1. The expression of lefty1 in the kidney decreased in a time‑dependent manner in mice with a UUO, which was inversely correlated with the degree of renal interstitial fibrosis. Furthermore, compared with vehicle treatment, lefty1 attenuated renal interstitial fibrosis. Ureteral ligation induced increased expression levels of α‑SMA, TGF‑β1 and p‑Smad2/3. However, these effects were reduced following treatment with lefty1. The UUO also induced tubular injury and atrophy, whereas lefty1 treatment exerted a marked suppressive effect on tubular injury. In addition, exogenous lefty1 administered to mice restored the endogenous expression levels of lefty1. The present study demonstrated that lefty1 attenuated renal interstitial injury by inhibiting the Smad‑dependent TGF‑β1 signaling pathway. Lefty1 may therefore by a putative therapeutic agent in the treatment of renal injury.

Characterization and genomic structure of Dnah9, and its roles in nodal signaling pathways in the Japanese flounder (Paralichthys olivaceus).

The nodal signaling pathway has been shown to play crucial roles in inducing and patterning the mesoderm and endoderm, as well as in regulating neurogenesis and left-right axis asymmetry. Here, we present the first complete cDNA and genomic sequences as well as the promoter predication of the Dnah9 gene in the Japanese flounder. The 15,558-bp-long cDNA is divided into 96 exons and spread over 138 kb of genomic DNA. Protein sequence comparison showed that it shares higher identity with other vertebrate orthologs, with an ATP binding dynein motor, AAA domain and microtubule binding stalk of dynein motor. Dnah9 exhibited maternal and ubiquitous expression in all cells of the early development stages, but became concentrated in the head at 1 DAH, as identified by qRT-PCR and in situ hybridization methods. Furthermore, after nodal signaling was inhibited, the level of Southpaw did not change significantly at early development stage (50 % epiboly) but increased significantly at late stages (27-somite stages and 1 DAH), as well as the expression of Lefty, an inhibitor of nodal signaling, increased continuously. On the other hand, the expression level of Dnah9 decreased. The transcription factor binding site of FAST-1 (SMAD interacting protein) was identified in the transcription region of Dnah9 by the promoter analysis, which might format the complexes of SMADs, FAST-1 and the transcription region of Dnah9 served as a bridge of Dnah9 and nodal signaling. All evidences indicated that Dnah9 might be downstream of nodal during the early development stages, and an indirect function through SMADs for nodal signaling pathway.

AVE protein expression and visceral endoderm cell behavior during anterior–posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression

The initial landmark of anterior–posterior (A–P) axis formation in mouse embryos is the distal visceral endoderm, DVE, which expresses a series of anterior genes at embryonic day 5.5 (E5.5). Subsequently, DVE cells move to the future anterior region, generating anterior visceral endoderm (AVE). Questions remain regarding how the DVE is formed and how the direction of the movement is determined. This study compares the detailed expression patterns of OTX2, HHEX, CER1, LEFTY1 and DKK1 by immunohistology and live imaging at E4.5-E6.5. At E6.5, the AVE is subdivided into four domains: most anterior (OTX2, HHEX, CER1-low/DKK1-high), anterior (OTX2, HHEX, CER1-high/DKK1-low), main (OTX2, HHEX, CER1, LEFTY1-high) and antero-lateral and posterior (OTX2, HHEX-low). The study demonstrates how this pattern is established. AVE protein expression in the DVE occurs de novo at E5.25-E5.5. Neither HHEX, LEFTY1 nor CER1 expression is asymmetric. In contrast, OTX2 expression is tilted on the future posterior side with the DKK1 expression at its proximal domain; the DVE cells move in the opposite direction of the tilt.

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

Lefty inhibits in vitro decidualization by regulating P57 and cyclin D1 expressions.

Endometrial decidualization is highly important for successful construction and maintenance of embryo implantation and pregnancy. Lefty gene at different menstrual cycle phases has different expressions, indicating its regulatory significance. To study the mechanism of Lefty in decidualization, human endometrial stromal cells (hESCs) were cultured and induced with medroxyprogesterone acetate (MPA) and 8-bromoadenosine-cAMP (8-Br-cAMP) in vitro as a research model. Our results showed that Lefty1 overexpression inhibited MPA- and 8-Br-cAMP-induced hESC decidualization and significantly reduced the secretion of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP-1). With the inhibition of Lefty1 expression, hESC decidualization induced by MPA and 8-Br-cAMP became more remarkable, and the secretions of PRL and IGFBP-1 were higher too. Further tests indicated that during the process of decidualization, P57 expression increased, whereas cyclin D1 expression decreased. Although Lefty1 overexpression did not significantly change the expressions of P57 and cyclin D1, inhibition of Lefty1 expression resulted in more evident changes in P57 and cyclin D1 expressions. Meanwhile, cell cycle examination showed that Lefty1 overexpression reduced the cell cycle arrest at G1/S phase in the in vitro hESC decidualization model. Therefore, Lefty1 could regulate the cell cycle via modulating the expressions of P57 and cyclin D1 and then inhibit the decidualization in vitro.

FGF signaling is required for brain left–right asymmetry and brain midline formation

Early disruption of FGF signaling alters left–right (LR) asymmetry throughout the embryo. Here we uncover a role for FGF signaling that specifically disrupts brain asymmetry, independent of normal lateral plate mesoderm (LPM) asymmetry. When FGF signaling is inhibited during mid-somitogenesis, asymmetrically expressed LPM markers southpaw and lefty2 are not affected. However, asymmetrically expressed brain markers lefty1 and cyclops become bilateral. We show that FGF signaling controls expression of six3b and six7, two transcription factors required for repression of asymmetric lefty1 in the brain. We found that Z0-1, atypical PKC (aPKC) and β-catenin protein distribution revealed a midline structure in the forebrain that is dependent on a balance of FGF signaling. Ectopic activation of FGF signaling leads to overexpression of six3b, loss of organized midline adherins junctions and bilateral loss of lefty1 expression. Reducing FGF signaling leads to a reduction in six3b and six7 expression, an increase in cell boundary formation in the brain midline, and bilateral expression of lefty1. Together, these results suggest a novel role for FGF signaling in the brain to control LR asymmetry, six transcription factor expressions, and a midline barrier structure.

Lefty1 and Lefty2 Control the Balance Between Self-Renewal and Pluripotent Differentiation of Mouse Embryonic Stem Cells

Lefty expression has been recognized as a stemness marker because Lefty is enriched both in undifferentiated embryonic stem cells (ESCs) and in blastocysts. Here, we examined the function of Lefty1 and Lefty2 in the maintenance of self-renewal and pluripotency of mouse ESCs (mESCs). Suppression of Lefty1 or Lefty2 expression in mESCs did not alter the self-renewal properties of mESCs under nondifferentiating conditions, but suppression of these genes did affect Smad2 phosphorylation and differentiation. Lefty1 knockdown mESCs showed enhanced phosphorylation of Smad2 and increased differentiation potential, whereas Lefty2 knockdown mESCs exhibited reduced phosphorylation of Smad2 and enhanced self-renewal in the presence of a differentiation signal. In vivo, teratomas developed from Lefty2 knockdown mESCs contained massive expansions of immature neuroepithelium, a marker of malignant teratomas. Taken together, these results suggest that optimal expression of Lefty1 and Lefty2 is critical for the balanced differentiation of mESCs into three germ layers.

Abstract 2650: Age-dependent association between protein expression of the embryonic stem cell marker Cripto-1 and survival of glioblastoma patients.

Abstract Aggressive tumor cells can show characteristics similar to normal embryogenic progenitor cells, and de-regulated embryonic developmental signaling networks have been linked to cancer initiation and progression. Thus, cell fate regulation in embryonic development and cell transformation during oncogenesis may share common signaling pathways. Several tumor types have been suggested to be initiated from stem-like cells. Exploring in tumors, the re-emergence of embryonic signaling pathways involved in stem cell self-renewal can produce new insight into the initiation and progression of cancer and may lead to development of new therapeutic strategies. The Nodal pathway is required for maintenance of undifferentiated embryonic stem cells. Nodal is a morphogen that belongs to the TGF-beta family. Its signaling can be regulated by co-factors such as the Epidermal Growth Factor - like member, Cripto-1 and another TGF-beta member, Lefty. Nodal and Cripto-1 are critical in embryonic development and is generally poorly detected in differentiated tissues. However, an increased expression has been detected in different human tumor types. The dismal prognosis of glioblastomas calls for increased biological knowledge and novel therapeutic targets. Gene expression studies in a glioblastoma xenograft model, where multicellular spheroids from glioblastoma patient biopsies were generated and implanted intracranially in nude rats and where highly infiltrative non-angiogenic tumors can switch to angiogenic tumors upon in vivo intracranial passaging, has revealed importance of gene ontology categories connected to development and negative regulators of differentiation. In this model we detected several significant overrepresented morphogenesis categories, and we hypothesized that increased activity in Nodal signaling also could be found in human glioblastomas. We assessed the gene expression of Nodal, Cripto-1 and Lefty in microarrays of infiltrative and angiogenic xenograft samples developed from four glioblastoma patients. The genes were expressed both in the infiltrative and angiogenic xenografts. Further we examined Nodal, Cripto-1 and Lefty expression in 199 primary glioblastomas by immunohistochemistry, and assessed their potential clinical relevance by analyzing the association between their protein expression and patient survival. A weak to moderate protein expression of Nodal and Lefty and a stronger expression of Cripto-1 was found in the patient samples. Interestingly a significant association between patient age, Cripto-1 expression and survival was demonstrated, where higher Cripto-1 scores were associated with poorer survival in younger patients. These findings suggest that Cripto-1 may represent a novel negative prognostic marker in younger glioblastoma patients and also a potential therapeutic target in this patient group. Citation Format: Berit B. Tysnes, Hege AA Sætran, Sverre J. Mørk, Naira V. Margaryan, Geir E. Eide, Rolf Bjerkvig, Luigi Strizzi, Mary J.C. Hendrix. Age-dependent association between protein expression of the embryonic stem cell marker Cripto-1 and survival of glioblastoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2013-2650

Suppression ofLeftyexpression in induced pluripotent cancer cells

Cancer and stem cells share the ability to silence tumor suppressors. We focused on Lefty, which encodes one of the most abundant tumor suppressors in embryonic stem (ES) cells and is not expressed in somatic cancer cells. We found that transforming growth factor β (TGF-β) induced demethylation of the Lefty B cytosine-phosphate-guanine (CpG) island and increased Lefty expression (10-200 times) in human pancreatic cancer cells and human liver cancer cells (PLC/PRF/5 and HLF). Expression of Cripto, another important factor in Nodal-Lefty signaling, was not increased after adding TGF-β. We generated reprogrammed cancer cells that revealed high expression of immature marker proteins, high proliferation, and the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, suggesting that these cells may have cancer stem cell-like phenotypes. We investigated Lefty and found that reprogrammed human liver cancer cells (induced pluripotent cancer cells) displayed a much lower ability to express Lefty, although less Lefty B CpG methylation was also observed. We also found that a MEK inhibitor dramatically enhanced Lefty expression in human pancreatic cancers with mutated ras, whereas Lefty B CpG methylation was not decreased. These observations indicate that despite the demethylation of DNA strands in promoter regions of pluripotency-associated genes, including Lefty gene, Lefty expression was not induced well in reprogrammed cells. Of note was the fact that Lefty is abundantly expressed in human ES cells but not in induced pluripotent stem (iPS) cells. We thus think that reprogrammed cancer cells share the mechanism for expression of Lefty with iPS cells. This shared mechanism may contribute to the cancerous transformation of iPS cells.

Spatial Restriction of Bone Morphogenetic Protein Signaling in Mouse Gastrula through the mVam2-Dependent Endocytic Pathway

The embryonic body plan is established through positive and negative control of various signaling cascades. Late endosomes and lysosomes are thought to terminate signal transduction by compartmentalizing the signaling molecules; however, their roles in embryogenesis remain poorly understood. We showed here that the endocytic pathway participates in the developmental program by regulating the signaling activity. We modified the mouse Vam2 (mVam2) locus encoding a regulator of membrane trafficking. mVam2-deficient cells exhibited abnormally fragmented late endosomal compartments. The mutant cells could terminate signaling after theremoval of the growth factors including TGF-β and EGF, except BMP-Smad1/Smad5 signaling. mVam2-deficient embryos exhibited ectopic activation of BMP signaling and disorganization of embryo patterning. We found that mVam2, which interacts with BMP type I receptor, is required for the spatiotemporal modulation of BMP signaling, via sequestration of the receptor complex in the late stages of the endocytic pathway.

Nodal activates smad and extracellular signal-regulated kinases 1/2 pathways promoting renal cell carcinoma proliferation.

Expression of the nodal gene is high in a number of tumor cell types and may promote tumor growth. The expression of lefty, an inhibitor of nodal is often reduced in tumor cells. To the best of our knowledge, few studies have investigated the expression of nodal and lefty in renal cell carcinoma (RCC) cells. In the present study, quantitative polymerase chain reaction assays demonstrated that the level of nodal expression in RCC cells was high compared with that of adjacent non-tumor tissue cells, while the opposite pattern was observed for the level of lefty expression. Furthermore, lefty overexpression in RCC cells inhibited the expression of nodal. Nodal overexpression promoted RCC cell proliferation and invasion, and inhibited RCC cell apoptosis. Nodal downregulation and lefty overexpression led to similar observations: The inhibition of RCC cell proliferation and invasion, and the promotion of RCC cell apoptosis. The results of the present study suggested that the expression of nodal promoted RCC growth by activating the smad and extracellular signal-regulated kinases 1/2 pathways. The expression of lefty in RCC cells was lower than that in adjacent non-tumor cells, which may result in the overexpression of nodal, thereby promoting the growth of RCC. The results of the present study may therefore be useful for the development of novel biomarkers for RCC tumor diagnosis, and suggest a potential target gene for the treatment of RCC.

Loss of plakophilin 2 disrupts heart development in zebrafish

The desmosomal armadillo protein plakophilin 2 is the only plakophilin expressed in the heart, and mutations in the human plakophilin 2 gene result in arrhythmogenic right ventricular cardiomyopathy. To investigate loss of function, we knocked down plakophilin 2 by morpholino microinjection in zebrafish. This resulted in decreased heart rate, cardiac oedema, blood pooling, a failure of the heart to pattern correctly and a twisted tail. Co-injection of plakophilin 2 mRNA rescued the morphant phenotype, indicating the specificity of the knockdown. Desmosome numbers were decreased in morphant hearts and the plaque and midline structures of the desmosomes in the intercalated discs were disrupted when examined by electron microscopy. cmlc2 and vmhc expression at 48 hours post-fertilization (hpf) showed incomplete looping of the heart in morphant embryos by whole mount in situ hybridization, and bmp4 expression was expanded into the ventricle. The domain of expression of the heart marker nkx2.5 at 24 hpf was expanded. At the 18 somite stage, expression of the cardiogenic gene lefty2 was abolished in the left cardiac field, with concomitant increases in bmp4, spaw and lefty1 expression, likely resulting in the looping defects. These results indicate that plakophilin 2 has both structural and signalling roles in zebrafish heart development.

BMP inhibition by DAN in Hensen's node is a critical step for the establishment of left–right asymmetry in the chick embryo

During left–right (L–R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L–R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L–R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L–R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L–R axis by inhibiting BMP signaling around the node.