Nodal Expression Research Articles

On the Enigma of the Human Neurenteric Canal

Existence and biomedical relevance of the neurenteric canal, a transient midline structure during early neurulation in the human embryo, have been controversially discussed for more than a century by embryologists and clinicians alike. In this study, the authors address the long-standing enigma by high-resolution histology and three-dimensional reconstruction using new and historic histological sections of 5 human 17- to 21-day-old embryos and of 2 marmoset monkey embryos of the species Callithrix jacchus at corresponding stages. The neurenteric canal presents itself as the classical vertical connection between the amniotic cavity and the yolk sac cavity and is lined (a) craniolaterally by a horseshoe-shaped “hinge of involuting notochordal cells” within Hensen’s node and (b) caudally by the receding primitive streak epiblast dorsally and by notochordal plate epithelium ventrally, the latter of which covered the (longitudinal) notochordal canal on its ventral side at the preceding stage. Furthermore, asymmetric parachordal nodal expression in Callithrix and morphological asymmetries within the nodes of the other specimens suggest an early non-cilium-dependent left-right symmetry breaking mode previously postulated for other mammals. We conclude that structure and position of the mammalian neurenteric canal support the notion of its homology with the reptilian blastopore as a whole and with a dorsal segment of the blastopore in amphibia. These new features of the neurenteric canal may further clarify the aetiology of foetal malformations such as junctional neurulation defects, neuroendodermal cysts, and the split notochord syndrome.

Antagonistic interaction between Nodal and insulin modulates pancreatic \u03b2-cell proliferation and survival

BackgroundInsulin signaling pathway in β-cell is essential to promote β-cells proliferation and survival, while Nodal–ALK7–Smad3 signaling involves β-cells apoptosis. We attempted to address inter-relationship between Nodal and insulin in modulating β-cell proliferation and apoptosis.MethodsUsing INS-1 β-cells and isolated rat islets, we examined the effects of Nodal, insulin, or the two combined on β-cell proliferation and/or apoptosis.ResultsThe β-cells under high-glucose or palmitate conditions showed significant up-regulation of Nodal expression and activation of its downstream signaling pathway resulted in increased cleaved caspase-3. Insulin treatment led to significantly attenuated Nodal-induced cell apoptotic pathway. Similar results were found in directly Nodal-treated β-cell that insulin could partially block Nodal-induced up-regulation of ALK7–Smad3–caspase-3 signaling pathways with significantly attenuated β-cell apoptosis. Interestingly, we found that insulin-induced Akt activation and downstream molecules including GSK-3β, β-catenin and ERK1/2 was significantly attenuated by the co-treatment with Nodal, resulted in decreased cell proliferation. Furthermore, Nodal decreased glucose-evoked calcium influx and played a negative role during glucose-stimulated insulin secretion in the β-cells. Immunocytochemistry studies showed that Nodal treatment translocated Smad3 from cytosol mostly to the nucleus; however, co-treatment with insulin significantly decreased Smad3 nuclear localization. Co-immunoprecipitation experiments showed a directly interaction between Smad3 and Akt, and this interaction was enhanced by co-treatment with insulin.ConclusionsOur data suggest that the antagonistic interaction between Nodal and insulin has a role in the regulation of β-cell mass and secretion.

Nodal induces epithelial-mesenchymal transition in gallbladder carcinoma via the Smad2/4 pathway

Objective To study the expressions of Nodal in normal gallbladder, and gallbladders with cholelithiasis, cholecystitis and carcinoma; and to study the impact of inhibiting or promoting Nodal expressions in gallbladder carcinoma on the Smad2/4 pathway and epithelial-mesenchymal transition. Methods Immunohistochemistry was used to detect the expressions and distributions of Nodal protein in 30 normal gallbladders, 96 simple cholecystitis/calculous cholecystitis specimens and 42 gallbladder carcinoma specimens. The mRNA and protein expressions of Nodal in normal and malignant gallbladder mucosal epithelium cells and breast cancer cells were detected by RT-PCR, western blotting and wound healing tests. The impact of activating agents and inhibitors on the expression levels of Nodal and its signaling pathway Smad2/4 and EMT-related proteins were analyzed. Results Immunohistochemistry showed that the positive rates of Nodal in the gallbladder cancer group was significantly higher than that in the gallbladder stone group and the normal gallbladder group. The results were 83.3% (35/42), 44.8% (43/96), 6.7% (2/30) respectively (P<0.01). RT-PCR and Western blotting showed the expressions of Nodal in gallbladder carcinoma cells were higher than normal gallbladder cells (P<0.05). After using rhNodal to up regulate the Nodal expression, the Smad2 protein phosphorylation was promoted and the EMT associated proteins were up-regulated. After using the inhibitor SB431542 to suppress the Nodal expression, the Smad2 protein phosphorylation decreased and the EMT associated proteins were down-regulated. Conclusions The expression of Nodal was closely related to cell proliferation and metastasis in gallbladder carcinoma. Tumor progression was promoted via the smad2/4 pathway through epithelial-mesenchymal transition. Key words: Gallbladder carcinoma, Gallstone; Transforming growth factor-beta, Nodal; Smad2/4, signal conduction; Epithelial-mesenchymal transition

MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo.

In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-β ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a β-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a β-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.

The effect of maternal NODAL on STOX1 expression in extravillous trophoblasts is mediated by IGF1

The number of molecules identified to be involved in communication between placenta and decidua is fast expanding. Previously, we showed that NODAL expressed in maternal endometrial stromal cells is able to affect NODAL and STOX1 expression in placental extravillous trophoblasts. The effect of maternal NODAL on placental NODAL expression is achieved via Activin A, while preliminary data suggests that maternal NODAL affects STOX1 expression in trophoblasts potentially via IGF1. In the current study, T-HESC endometrial stromal cells were treated with siRNAs against NODAL after which IGF1 mRNA expression was determined by quantitative RT-PCR, while IGF1 secretion was measured by ELISA. Recombinant IGF1 and inhibitors of the MAPK and PI3K/AKT pathways were added to SGHPL-5 extravillous trophoblasts after which the effects on STOX1 mRNA and STOX1 protein expression were determined. The effect of IGF1 and the MAPK and PI3K/AKT inhibitors on the invasive capacity of SGHPL-5 cells was investigated by performing invasion assays. We found that T-HESC cells treated with NODAL siRNAs showed significant upregulation of IGF1 mRNA expression and IGF1 protein secretion. Addition of IGF1 to SGHPL-5 cell media significantly upregulated STOX1 mRNA and protein expression. Using inhibitors of the PI3K/AKT and MAPK pathway showed that the effect of IGF1 on STOX1 expression is accomplished via MAPK signaling. Secondly, PI3K inhibition independently leads to reduced STOX1 expression which can be rescued by adding IGF1. IGF1 was unable to influence the invasive capacity of SGHPL-5 cells, while inhibiting the PI3K/AKT pathway did reduce the invasion of these cells. To conclude, here we show that downregulated NODAL expression in endometrial stromal cells, previously associated with pre-eclampsia like symptoms in mice, increases IGF1 secretion. Increased levels of IGF1 lead to increased expression levels of STOX1 in extravillous trophoblasts via the MAPK pathway, hereby identifying a novel signaling cascade involved in maternal-fetal communication.

Nodal and Hedgehog synergize in gill slit formation during development of the cephalochordate Branchiostoma floridae.

The larval pharynx of the cephalochordate Branchiostoma (amphioxus) is asymmetrical. The mouth is on the left, and endostyle and gill slits are on the right. At the neurula, Nodal and Hedgehog (Hh) expression becomes restricted to the left. To dissect their respective roles in gill slit formation, we inhibited each pathway separately for 20 min at intervals during the neurula stage, before gill slits penetrate, and monitored the effects on morphology and expression of pharyngeal markers. The results pinpoint the short interval spanning the gastrula/neurula transition as the critical period for specification and positioning of future gill slits. Thus, reduced Nodal signaling shifts the gill slits ventrally, skews the pharyngeal domains of Hh, Pax1/9, Pax2/5/8, Six1/2 and IrxC towards the left, and reduces Hh and Tbx1/10 expression in endoderm and mesoderm, respectively. Nodal auto-regulates. Decreased Hh signaling does not affect gill slit positions or Hh or Nodal expression, but it does reduce the domain of Gli, the Hh target, in the pharyngeal endoderm. Thus, during the neurula stage, Nodal and Hh cooperate in gill slit development - Hh mediates gill slit formation and Nodal establishes their left-right position.

Wavy movements of epidermis monocilia drive the neurula rotation that determines left–right asymmetry in ascidian embryos

Tadpole larvae of the ascidian, Halocynthia roretzi, show morphological left–right asymmetry in the brain structures and the orientation of tail bending within the vitelline membrane. Neurula embryos rotate along the anterior–posterior axis in a counterclockwise direction, and then this rotation stops when the left side of the embryo is oriented downwards. Contact of the left-side epidermis with the vitelline membrane promotes nodal gene expression in the left-side epidermis. This is a novel mechanism in which rotation of whole embryos provides the initial cue for breaking left–right symmetry. Here we show that epidermal monocilia, which appear at the neurula rotation stage, generate the driving force for rotation. A ciliary protein, Arl13b, fused with Venus YFP was used for live imaging of ciliary movements. Although overexpression of wild-type Arl13b fusion protein resulted in aberrant movements of the cilia and abrogation of neurula rotation, mutant Arl13b fusion protein, in which the GTPase and coiled-coil domains were removed, did not affect the normal ciliary movements and neurula rotation. Epidermis cilia moved in a wavy and serpentine way like sperm flagella but not in a rotational way or beating way with effective stroke and recovery stroke. They moved very slowly, at 1/7 Hz, consistent with the low angular velocity of neurula rotation (ca. 43°/min). The tips of most cilia pointed in the opposite direction of embryonic rotation. Similar motility was also observed in Ciona robusta embryos. When embryos were treated with a dynein inhibitor, Ciliobrevin D, both ciliary movements and neurula rotation were abrogated, showing that ciliary movements drive neurula rotation in Halocynthia. The drug also inhibited Ciona neurula rotation. Our observations suggest that the driving force of rotation is generated using the vitelline membrane as a substrate but not by making a water current around the embryo. It is of evolutionary interest that ascidians use ciliary movements to break embryonic left–right symmetry, like in many vertebrates. Meanwhile, ascidian embryos rotate as a whole, similar to embryos of non-vertebrate deuterostomes, such as echinoderm, hemichordate, and amphioxus, while swimming.

Lefty promotes the proliferation and invasion of trophoblast cells by inhibiting nodal expression.

Trophoblast cells play a vital role in embryo implantation. Exploring cytokines secreted by trophoblast cells will be significant in revealing the molecular mechanism of embryo implantation. In vitro-cultured trophoblast cells treated with Lefty revealed that Lefty could downregulate the expression of Nodal while promoting the expression of MMP-2 and MMP-9. Thus, Lefty may promote the proliferation and invasion of trophoblast cells, thereby reducing the amount of apoptosis of these cells. Lefty siRNA knockdown or overexpression of Nodal showed opposite effects to treatment with Lefty. When trophoblast cells overexpressing Nodal were treated with Lefty, Nodal expression increased, and MMP-2 and MMP-9 expression was still promoted. Additionally, the proliferation and invasion of trophoblast cells were upregulated, and trophoblast apoptosis was reduced. Together, these data reveal that Lefty can promote the proliferation and invasion of trophoblast cells and inhibit their apoptosis by downregulating Nodal expression while promoting the expression of MMP-2 and MMP-9. This finding provides new proof for the mechanism of trophoblast cell invasion in decidua.

A Requirement for Zic2 in the Regulation of Nodal Expression Underlies the Establishment of Left-Sided Identity

ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.

Abstract 4492: Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment

Abstract Clinical studies indicate that obesity is correlated with poor outcomes in breast cancer patients. Obese individuals have a high proportion of adipose tissue, which is abundant in stem and progenitor cells. Human adipose-derived stromal cells (hADSCs) secrete several cytokines including CCL5, an inflammatory chemokine shown to increase tumor metastatic ability in vitro. We previously identified that CCL5 is secreted into the microenvironment when hADSCs are co-cultured with breast tumor cells. When the clinical relevance of CCL5 was assessed using cBioPortal, an open-source genomics database, we found that CCL5 mRNA overexpression was correlated with increased overall survival in breast cancer patients, which counters what we and others have observed in vitro. In this study, we compared the expression of CCL5 and Nodal, a potent embryonic morphogen re-expressed by aggressive cancer cells, in either hADSC or MDA-MB-231 breast cancer cells in response to three-dimensional collagen matrices conditioned by hADSCs or breast cancer cells. After preconditioning a three-dimensional collagen matrix with either hADSCs or MDA-MB-231 cells (and following removal of the cells), the denuded three-dimensional matrices were reseeded with the opposite cell type. We found that the expression of both CCL5 and Nodal was significantly reduced in the breast cancer cells plated on the hADSC-preconditioned matrix. However, hADSCs did not express Nodal or alter CCL5 protein expression when grown on a MDA-MB-231-preconditioned matrix. MDA-MB-231 cells plated onto a hADSC-preconditioned matrix appeared more mesenchymal and stem-like, while the adipose stem cells placed into a tumor-preconditioned matrix were induced into a more aggressive cancer cell phenotype by engaging in vasculogenic mimicry. Furthermore, we found there was no change in cell proliferation of any cells grown under these different conditions. These observations suggest a relationship between hADSCs and the breast tumor microenvironment where human adipose-derived stem cells appear to inhibit the expression of CCL5 and Nodal by the aggressive tumor cells without altering tumor cell proliferation. Citation Format: Daniel Berrebi, Abhishek Yadav, Eric Lundstrom, Nicole Werwie, Dominic Muttillo, Richard E. Seftor, Elisabeth A. Seftor, Linda Vona-Davis. Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment [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 4492.

Abstract 2647: Bromodomain-containing protein 7 (BRD7) is a candidate tumor suppressor in breast carcinoma and prognostic biomarker

Abstract Background Breast cancer (BC) is a multifactorial disorder in which multiple genetic and epigenetic factors changes drive tumor initiation and progression. Bromodomain-containing protein 7 (BRD7) is a member of the bromodomain-containing protein family mainly localized in the nuclear compartment of the cell. BRD7 is a transcriptional co-activator for p53 modulating histone acetylation, p53 acetylation and promoter activity in p53 target genes. Previous studies show that BRD7 is down-regulated in a number of cancer types and this may correlate with clinical outcomes. However, the potential involvement of changes in BRD7 and prognostic importance in BC are poorly understood. Purpose of the study We have investigated the expression, CpG island methylation and sub-cellular localization of BRD7 in BC cell lines and clinical cases and thereby assessed its prognostic significance by correlating with clinicopathologic features and time-dependent clinical outcomes. Materials and methods BRD7 expression was analysed by qRT-PCR and IHC in 13 BC cancer cell lines. Expression was also assessed by IHC in 50 cases of primary breast cancer and 14 paired metastatic lesions. CpG island methylation was determined quantitatively by pyrosequencing. BC cases were divided for nuclear and non-nuclear BRD7 antigen localization and then stratified for high and low BRD7 expression. The effects of BRD7 expression and BC clinical characteristics data on OS and PFS were evaluated using univariate and multivariate Cox regression analyses. Results BRD7 was expressed at levels similar to normal breast epithelium and the CpG island unmethylated in all 13 cell lines. Methylation analysis was successful in 42 FFPE BC samples collected, with 26,2% of them methylated (11/42). IHC analysis on primary tissues revealed that BDR7 is mainly localized in the nucleus (34/50: 68%). Patients with BRD7 nuclear localization (N=34) showed smaller tumors (p=0.012), a longer OS (p=0.002) and a lower relapse rate (p=0.03). Moreover, nuclear BRD7 expression is associated with wild-type TP53 (p=0.04) and unmethylated BRD7 (p=0.04), compared to the non-nuclear cases (N=16). No difference was seen in nodal status, grading, clinical stage, histological type, ER, PR, HER2 and Ki-67 expression. Patients (N=12) showing both nuclear localization and high BRD7 expression presented the smallest tumors (10/12 = 83%) and both a longer OS (p=0,005) and PFS (p=0,001). Among the 4/14 patients with nuclear localization in their primary tumors, 3 (75%) presented a diffuse or negative BRD7 in the paired metastasis. Conclusion BRD7 is a strong candidate for breast cancer tumor suppressor and prognostic biomarker. Citation Format: Cristiana Lo Nigro, Daniela Vivenza, Ornella Garrone, Rodolfo Brizio, Fiamma Mantovani, Giannino Del Sal, Marco Carlo Merlano, Tim Crook. Bromodomain-containing protein 7 (BRD7) is a candidate tumor suppressor in breast carcinoma and prognostic biomarker [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 2647.

Pitx2 and nodal as conserved early markers of the anterior-posterior axis in the rabbit embryo

Attaining molecular and morphological axial polarity during gastrulation is a fundamental early requirement for normal development of the embryo. In mammals, the first morphological sign of the anterior–posterior axis appears anteriorly in the form of the anterior marginal crescent (or anterior visceral endoderm) while in the avian the first such sign is the Koller’s sickle at the posterior pole of the embryonic disc. Despite this inverse mode of axis formation many genes and molecular pathways involved in various steps of this process seem to be evolutionarily conserved amongst amniotes, the nodal gene being a well-known example with its functional involvement prior and during gastrulation. The pitx2 gene, however, is a new candidate described in the chick as an early marker for anterior–posterior polarity and as a regulator of axis formation including twinning. To find out whether pitx2 has retained its inductive and early marker function during the evolution of mammals this study analyses pitx2 and nodal expression at parallel stages during formation of the anterior–posterior polarity in the early rabbit embryo using whole-mount in situ hybridization and serial light-microscopical sections. At a late pre-gastrulation stage a localized reduction of nodal expression presages the position of the anterior pole of the embryonic disc and thus serves as the earliest molecular marker of anterior–posterior polarity known so far. Pitx2 is expressed in a polarized manner in the anterior marginal crescent and in the posterior half of the embryonic disc during further development. In the anterior segment of the posterior pitx2 expression domain, the anterior streak domain (ASD) is defined by nodal expression as a hypothetical progenitor region of the anterior half of the primitive streak. The expression patterns of both genes thus serve as signs of a conserved involvement in early axis formation in amniotes and, possibly, in twinning in mammals as well.

Nodal induced by hypoxia exposure contributes to dacarbazine resistance and the maintenance of stemness in melanoma cancer stem‑like cells

Nodal signaling has a critical role in the processes of embryogenesis and is necessary for maintaining cell stemness. However, its upregulation in melanoma is positively correlated with malignant potential, including cancer progression, metastasis and recurrence, and a recent report has revealed its role in promoting self‑renewal capacity in melanoma. Our study aimed to explore the effects of hypoxia exposure, which is one of the main causes of chemoresistance in melanoma, on the physiological processes of melanoma cancer stem‑like cells (CSCs) via regulating Nodal. A375 CSCs were enriched by culturing in serum‑free medium (SFM) and were analyzed for the expression levels of Nodal and its correlated proteins by semi‑quantitative western blotting. Lentiviral‑packaged Nodal coding sequence or short‑hairpin RNA (shRNA) was employed. After hypoxia exposure, the effects on glucose uptake, ATP production and O2 consumption were detected, and whether Nodal contributed to the proliferation, invasion, colony formation, self‑renewal capacity and chemoresistance was evaluated. We demonstrated that hypoxia exposure induced Nodal expression and activated the Smad2/3 pathway in A375 CSCs. Hypoxic‑induced Nodal partially promoted dacarbazine resistance, promoted invasion and self‑renewal capacity, but not proliferation which was further confirmed using Nodal knockdown. Blockage of Nodal signaling activity with the small‑molecule inhibitor SB431542 partially reversed Nodal‑induced chemoresistance. Nodal knockdown further sensitized A375 CSCs to dacarbazine after SB431542 pretreatment, indicating the involvement of proNodal in dacarbazine resistance. The introduction of mut‑proNodal induced chemoresistance further confirming the role of proNodal in this process. Taken together, our results demonstrated that Nodal induced by hypoxia exposure induced a malignant phenotype and chemoresistance in A375 CSCs, and proNodal also contributed to these processes, indicating that Nodal may be a potential therapeutic target for melanoma.

Simultaneous effects of IGF1 and Fadrozole on parthenogenesis and pluripotency markers in chicken embryo

So far, a synergistic effect was detected between insulin-like growth factor-I (IGF1) and anti-aromatase for sex reversal and pre-/post-natal growth of fertilized chicken embryo. Here, we hypothesized whether the growth and sexual female-to-male reversal effects of IGF1 and an anti-aromatase, Fadrozole, could improve the development of unfertilized, parthenogenetic chicken embryos. Simultaneous administration of IGF1 and Fadrozole increased the percentage of grade A embryos from 1.7% (no injection group) to 70.6%. The expression profile of parthenotes and newly laid fertilized embryos showed that IGF1 and Fadrozole increased SOX2 and NANOG expression, while decreased the TBX3 expression in the parthenogenetic embryos. However, a considerably higher expression of PRDM16, IGF2, NODAL and HDAC2 was observed in the fertilized group compared to the parthenogenetic embryos. In conclusion, chicken sexual determination is initiated at the earliest stage of embryonic development before gonadal differentiation. Combined administration of IGF1 and Fadrozole increased the developmental rate of parthenogenetic embryos. Also, simultaneous supplementation of IGF1 and Fadrozole induced the expression of pluripotency genes with no effect on the expression of growth and differentiation factors.

Abstract P4-09-09: Correlation of breast cancer index (BCI) predictive (HoxB13/IL17BR) results to nodal status and hormone receptor expression in early stage HR+ breast cancer

Abstract Background: The HoxB13/IL17BR (H/I) ratio, the endocrine response component of the Breast Cancer Index (BCI), was initially discovered in patients with LN- breast cancer, and validated as a predictive biomarker of benefit from extended endocrine therapy (EET) in a cohort from MA.17 that included both LN- and LN+ patients. Here, correlative analyses were performed to further characterize BCI Predictive (H/I) results with nodal status and quantitative hormone receptor expression. Methods: Analyses were performed using data from the BCI Clinical Database for Correlative Studies, an IRB-approved de-identified database that contains &amp;gt;50 clinicopathologic and molecular variables from cases submitted for BCI in clinical practice (N=19,126). Clinicopathologic variables were abstracted from pathology reports, and were available for a subset of these cases. Cases from patients with confirmed nodal status were analyzed. Chi-squared tests and ANOVA were used to compare results between subgroups. Results: Analyses included 13,114 patients (median age 58.9y; range 23-92y); 9562 were LN- and 3552 LN+. The distribution of individual H/I scores and proportion of patients classified as High H/I were generally similar in LN- and LN+ patients, though a modestly greater proportion of LN+ patients (46.2%) were classified as High H/I compared to LN- patients (42.0%; P&amp;lt;0.01). In both LN- and LN+ patients, median quantitative ER (qER) expression was slightly higher in patients with Low H/I than with High H/I; qPR showed the same trend but with a larger magnitude (P&amp;lt;0.0001 for both). Similar results were observed for percent positive staining by IHC (P&amp;lt;0.0001). In both LN- and LN+ patients, H/I showed a weak negative correlation with qER (LN-, 0.227; LN+, 0.192) and qPR expression (LN-, 0.311; LN+, 0.311). Conclusion: In this study to evaluate potential biological correlates of BCI, results showed that H/I biomarker activity did not appear to be dependent on nodal status. Secondly, although ER expression is an established biomarker for endocrine sensitivity, High H/I status did not correlate with increased quantitative ER and PR expression. The H/I ratio may be an independent new marker for endocrine sensitivity independent of the strength of ER expression. Citation Format: Elias A, Israel MA, Zhang Y, Schnabel CA, Mayordomo J. Correlation of breast cancer index (BCI) predictive (HoxB13/IL17BR) results to nodal status and hormone receptor expression in early stage HR+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-09-09.

Abstract OT3-06-04: A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC)

Abstract Primary Objective: To determine pathological complete response (pCR) rate in patients with primary TNIBC treated with PaCT in comparison with CT, followed by AC. To explore if the pCR rate correlates with reduced nodal expression status; and with arginine methylation status of epidermal growth factor receptor (EGFR). We will identify molecular biomarkers predictive of the pCR rate by analysis of multiplexed immunohistochemical (IHC) staining, identify molecular biomarkers predictive of the pCR rate by genomic and proteomic analysis, and determine whether the inhibition of the EGFR pathway down regulates the COX-2 pathway and mesenchymal marker. Background: EGFR is overexpressed in triple negative breast cancer (TNBC) and inflammatory breast cancer (IBC). Therefore, EGFR targeted therapy may have a promising role in TNBC and IBC. A study showed that EGFR-targeted therapy may enhance the initial chemosensitivity of TNBC cells. Panitumumab blocks epidermal growth factor ligands and transforming growth factor EGFá (TGFá) binding to EGFR, inhibits tumor growth, and elicits both tumor regression and eradication of established tumors in murine xenograft tumor models. Panitumumab, a fully humanized anti-EGFR antibody, has been shown to be active in a breast cancer preclinical model using human breast cancer cell line MDA-MB-468, which has been shown to overexpress EGFR by both IHC and fluorescence in situ hybridization (FISH). Furthermore, EGFR tyrosine kinase inhibitors such as erlotinib have antitumor activity against human IBC cell lines. Thus, EGFR targeted therapy may have a promising role in TNBC and IBC. Study Design: In this open label randomized phase II trial, up to 72 patients with primary IBC, have no HER2 overexpression, and have &amp;lt;10% expression of ER and PgR, who also meet other criteria will be randomized to PaCT arm - receiving panitumumab single agent in window study and 4 cycles PaCT, or CT arm - receiving 4 cycles of CT. All patients will receive 4 cycles of AC before surgery. Statistical Considerations: A sample size of 36 patients per arm will achieve 84% power to detect a difference of 0.24 in pCR rate between 0.2 in the CT arm and 0.44 in the PaCT arm with a type I error rate of 10% using one-sided Z test. Based on historical data, we expect that the pCR rate of a PaCT regimen to achieve 24% additional efficacy compared with the CT regimen. Status of the study: Activation date: Oct. 2016. So far 6 patients have been enrolled. Enrollment continues. Sponsor: Amgen. State of Texas appropriation for rare and aggressive breast cancer research. NIH grant 1R01CA205043-01A1 Citation Format: Willey JS, Parker CA, Lim B, Valero V, Le-Petross HT, Krishnamurthy S, Woodward WA, Lucci A, Wood AL, Sun H, Babiera GV, Song J, Shen Y, Valero V, Wang X, Ueno NT. A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-06-04.

Relation of Nodal expression to the specification of the dorsal-ventral axis and tissue patterning in the starfish Patiria pectinifera.

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid-blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left-right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF-beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.

Canine invasive mammary carcinomas as models of human breast cancer. Part 1: natural history and prognostic factors

PurposeDogs have been proposed as spontaneous animal models of human breast cancer, based on clinicopathologic similarities between canine and human mammary carcinomas. We hypothesized that a better knowledge of the natural history and prognostic factors of canine invasive mammary carcinomas would favor the design of preclinical trials using dogs as models of breast cancer.MethodsThe 2-year outcome of 350 female dogs with spontaneous invasive mammary carcinoma was studied. The investigated prognostic factors included age at diagnosis, pathologic tumor size, pathologic nodal stage, lymphovascular invasion, histological grade, and expression of Estrogen Receptor alpha (ERα), Progesterone Receptor, Ki-67, Human Epidermal Growth Factor Receptor 2, basal cytokeratins 5/6, and Epidermal Growth Factor Receptor. Multivariate survival analyses were performed using the Cox proportional hazards model.ResultsThe overall survival after mastectomy was 11 months. Within 1 year post mastectomy, 41.5% of dogs (145/350) died from their mammary carcinoma. By multivariate analysis, the significant prognostic factors for overall survival included a pathologic tumor size larger than 20 mm [HR 1.47 (95% confidence interval 1.15–1.89)], a positive nodal stage [pN+, HR 1.89 (1.43–2.48)], a histological grade III [HR 1.32 (1.02–1.69)], ERα negativity [HR 1.39 (1.01–1.89)], a high Ki-67 proliferation index [HR 1.32 (1.04–1.67)], and EGFR absence [HR 1.33 (1.04–1.69)].ConclusionThe short natural history of spontaneous canine invasive mammary carcinomas and high rate of cancer-related death allow for rapid termination of preclinical investigations. The prognostic factors of invasive mammary carcinomas are remarkably similar in dogs and humans, highlighting the similarities in cancer biology between both species.

ALDH enzyme activity is regulated by Nodal and histamine in the A549 cell line.

The present study aimed to examine whether the enzyme activity of aldehyde dehydrogenase (ALDH) was regulated by Nodal and histamine in the human alveolar adenocarcinoma A549 cell line. The regulated enzyme activity of ALDH was analyzed by flow cytometry in the A549 cell line. ALDH1 and Nodal expression was investigated by immunohistochemistry in28 cases of lung mixed adenocarcinoma. The enzyme activity of ALDH was upregulated by histamine and agonists of histamine H1 receptor (H1R) and histamine H2 receptor (H2R). ALDH activity was also downregulated by recombinant human Nodal and antagonists of H1R and H2R in the A549 cell line. In addition, expression of Nodal and ALDH1 were inversely correlated in lung mixed adenocarcinoma. ALDH enzyme activity was regulated by Nodal and histamine in lung adenocarcinoma.

Persistence of RSV promotes proliferation and epithelial-mesenchymal transition of bronchial epithelial cells through Nodal signaling.

Nodal may play an important role in the development of cancers. The present study was designed to determine the effects of Nodal induced by respiratory syncytial virus (RSV) infection on the occurrence and development of lung cancer and the underlying mechanisms. After verification of RSV infection by observation of cytopathic effect and indirect immunofluorescence, real-time PCR, Western blot and methylation assays were used to verify the influence of RSV on Nodal expression. Then, a Nodal overexpressed vector was constructed and the effects of Nodal on the proliferation and apoptosis of bronchial epithelial cells (BECs) and epithelial-mesenchymal transition (EMT) were assayed by flow cytometry and Western blot, respectively. Moreover, Lefty and pSmad2/3 were assayed by Western blot and Cyclin D1, CDK4, c-myc and Bcl-2 induced by Nodal overepression or RSV infection were also assayed by real-time PCR. The results showed that Nodal over expression and demethylation of the promoter were observed in BECs after RSV infection. Activation of Nodal promoted proliferation, colony formation and EMT and inhibited apoptosis of BECs. Nodal also promoted malignant change by promoting expression of cyclin D1 and related-dependent kinase and inhibiting apoptosis. Besides, RSV infection inhibited Lefty expression and promoted the activation of pSmad2/3. RSV also promoted Cyclin D1, CDK4, c-myc and Bcl-2 expression through the activation of pSmad2/3. Our data showed that persistence of RSV promoted the proliferation, epithelial-mesenchymal transition and expression of oncogenes through Nodal signaling, which may be associated with the occurrence and development of lung cancers.