Epidermal Growth Factor Family Research Articles

O-093 Endometrium-blastocyst cross talk: how far are we from understanding?

Abstract The early events characterising implantation of the human embryo represent a critical step in which proper activation of orchestrated molecular pathways regulate the success of pregnancy. Understanding this complex network is considered by many the last frontier of human reproduction. Although outstanding research advancements have provided clinicians tools to select the best quality embryo, identification of the optimal timing in which the endometrium is ready to accommodate it remains a challenge. Over the last decade several studies have identified many key players involved in the preparation of the endometrium to implantation and several diffusible signals guiding the embryo in its journey toward the maternal endometrial tissue have been uncovered (Massimiani et al. 2020). However, pieces of the puzzle are still missing. For convenience, the process of implantation is subdivided into several sequential processes, the completion of one being propaedeutic to the following. Once the blastocyst has arrived in close proximity to the uterine epithelium, the first event occurring is its escape from the zona pellucida. The molecular basis of blastocyst hatching remains poorly understood, and species-specific differences have been reported. Using the mouse model, we recently demonstrated that thyroid hormone up-regulates the expression of specific lytic enzymes (Isp1 and Isp2) known mediators of mouse embryo zona lysis. Interestingly, we observed that TH-mediated upregulation of Isp1 and Isp2 is strictly dependent on the presence of endometrial stromal cell feeder layer, clearly indicating that blastocyst–endometrium interaction is indispensable for TH-mediated increased expression of these proteases. So far, no molecular signals regulating hatching in humans have been identified, and only mechanical forces have been proposed to induce zona opening; however very recently the possibility that proteases may be involved also in humans has been suggested (Almagor et al., 2020), although further studies are needed to support this hypothesis. Once free from the zona, the embryo contacts the endometrial epithelium and starts its journey to further proceed in the consolidation of pregnancy. Hormones, pro- and anti-inflammatory mediators, growth factors and their receptors, adhesion molecules, and proteases and protease inhibitors have been reported in the regulation of the process of embryo apposition, adhesion, and invasion (Massimiani et al., 2020). Among the many molecular signals, the Notch pathway has been recognized key in embryo-endometrium cross talk (Cuman et al., 2014). Transient activation of the Notch1 receptor has been demonstrated in the process of decidualization (Afshar et al., 2012), a limiting step to implantation, and blastocyst-conditioned medium has been shown to induce the expression of Notch family members in decidual cells (Hess et al., 2007). Expression of notch receptors and ligands has been reported in the blastocyst trophectoderm as well (Cuman et al., 2014). We previously demonstrated that the secreted factor epidermal growth factor-like domain 7 (EGFL7) is a novel modulator of the Notch pathway, it is expressed by trophoblast cells of the mouse blastocyst (Fitch et al., 2004; Lacko et al., 2014) and it regulates human trophoblast migration and invasion ability by activating the Notch pathway (Massimiani et al., 2015). Data will be presented demonstrating that EGFL7 is expressed in the human endometrium and its expression is mainly localized in the glandular epithelium throughout the menstrual cycle. EGFL7 is significantly upregulated in whole endometrial tissue during the secretory phase, particularly in the stromal compartment; this observation is further supported by in vitro studies showing its upregulation in decidualized endometrial stromal cells, suggesting a role for EGFL7 in implantation. Further support is provided by the evidence that its levels are strongly downregulated in the endometrium of women with impaired fertility. We propose EGFL7 as a novel player involved in the embryo-endometrium cross talk.

Fibulin7 Mediated Pathological Cardiac Remodeling through EGFR Binding and EGFR-Dependent FAK/AKT Signaling Activation.

Adverse remodeling after myocardial infarction (MI) result in heart failure and sudden cardiac death. Fibulin7 (FBLN7) is an adhesion protein excreted into the extracellular matrix that functions in multiple biological processes. However, whether and how FBLN7 affects post-MI cardiac remodeling remains unclear. Here, the authors identify FBLN7 as a critical profibrotic regulator of adverse cardiac remodeling. They observe significantly upregulated serum FBLN7 levels in MI patients with left ventricular remodeling compared to those without MI. Microarray dataset analysis reveal FBLN7 is upregulated in human heart samples from patients with dilated and hypertrophic cardiomyopathy compared with non-failing hearts. The authors demonstrate that FBLN7 deletion attenuated post-MI cardiac remodeling, leading to better cardiac function and reduced myocardial fibrosis, whereas overexpression of FBLN7 results in the opposite effects. Mechanistically, FBLN7 binds to the epidermal growth factor receptor (EGFR) through its EGF-like domain, together with the EGF-like calcium-binding domain, and induces EGFR autophosphorylation at tyrosine (Y) 1068 and Y1173, which activates downstream focal adhesion kinase/AKT signaling, thereby leading to fibroblast-to-myofibroblast transdifferentiation. In addition, FBLN7-EGFR mediates this signal transduction, and the fibrotic response is effectively suppressed by the inhibition of EGFR activity. Taken together, FBLN7 plays an important role in cardiac remodeling and fibrosis after MI.

Endothelial TIE1 Restricts Angiogenic Sprouting to Coordinate Vein Assembly in Synergy With Its Homologue TIE2

BACKGROUND: Vascular growth followed by vessel specification is crucial for the establishment of a hierarchical blood vascular network. We have shown that TIE2 is required for vein development while little is known about its homologue TIE1 (tyrosine kinase with immunoglobulin-like and EGF [epithelial growth factor]-like domains 1) in this process. METHODS: We analyzed functions of TIE1 as well as its synergy with TIE2 in the regulation of vein formation by employing genetic mouse models targeting Tie1 , Tek , and Nr2f2 , together with in vitro cultured endothelial cells to decipher the underlying mechanism. RESULTS: Cardinal vein growth appeared normal in TIE1-deficient mice, whereas TIE2 deficiency altered the identity of cardinal vein endothelial cells with the aberrant expression of DLL4 (delta-like canonical Notch ligand 4). Interestingly, the growth of cutaneous veins, which was initiated at approximately embryonic day 13.5, was retarded in mice lack of TIE1. TIE1 deficiency disrupted the venous integrity, displaying increased sprouting angiogenesis and vascular bleeding. Abnormal venous sprouts with defective arteriovenous alignment were also observed in the mesenteries of Tie1 -deleted mice. Mechanistically, TIE1 deficiency resulted in the decreased expression of venous regulators including TIE2 and COUP-TFII (chicken ovalbumin upstream promoter transcription factor, encoded by Nr2f2 , nuclear receptor subfamily 2 group F member 2) while angiogenic regulators were upregulated. The alteration of TIE2 level by TIE1 insufficiency was further confirmed by the siRNA-mediated knockdown of Tie1 in cultured endothelial cells. Interestingly, TIE2 insufficiency also reduced the expression of TIE1. Combining the endothelial deletion of Tie1 with 1 null allele of Tek resulted in a progressive increase of vein-associated angiogenesis leading to the formation of vascular tufts in retinas, whereas the loss of Tie1 alone produced a relatively mild venous defect. Furthermore, the induced deletion of endothelial Nr2f2 decreased both TIE1 and TIE2. CONCLUSIONS: Findings from this study imply that TIE1 and TIE2, together with COUP-TFII, act in a synergistic manner to restrict sprouting angiogenesis during the development of venous system.

RNA-seq analysis of human trabecular endothelial cells after treatment with timolol maleate.

Timolol maleate (timolol), a β-receptor blocker, reduces intraocular pressure by decreasing aqueous humor production. Timolol reportedly also protects ganglion cells, decreases aqueous humor outflow facility, and destroys the extracellular matrix in the trabecular meshwork. In this study, we investigated the effects of timolol on cultured human trabecular endothelial cells purchased from ScienCell using next-generation sequencing. Experimental investigation. Total ribonucleic acid (RNA) was extracted after 24 h. More than 100 million RNAs in control and timolol-treated group were sequenced using a next-generation sequencer. The expression of 55,778 RNAs was analyzed. A total of 2,105 genes were significantly upregulated and 2,125 genes were downregulated, after the addition of timolol. VGF nerve growth factor inducible (VGF) (388-fold) had the maximum increase in expression, followed by amphiregulin (333-fold), a member of the epidermal growth factor family. Moreover, the expression of extracellular matrix-degrading enzymes, matrix metalloproteinases (MMPs) 1, 2, 3, 10, 12, and 14, increased. Timolol exerts various effects on human trabecular endothelial cells. The increase in MMP expression may contribute to the decrease in the aqueous humor outflow facility.

Mechanical Behavior of Octopus Egg Tethers Composed of Topologically Constrained, Tandemly Repeated EGF Domains.

Whether and how intramolecular crosslinks in polymeric materials contribute to mechanical properties is debated in both experimental and theoretical arenas. The tethering threads of Octopus bimaculoides egg cases provide a rare window to investigate this question in a biomaterial. The only detectable component of the load-bearing fibers in octopus threads is a 135 kDa protein, octovafibrin, comprising 29 tandem repeats of epidermal growth factor (EGF) each of which contains 3 intramolecular disulfide linkages. The N- and C-terminal C-type lectins mediate linear end-to-end octovafibrin self-assembly. Mechanical testing of threads shows that the regularly spaced disulfide linkages result in improved stiffness, toughness, and energy dissipation. In response to applied loads, molecular dynamics and X-ray scattering show that EGF-like domains deform by recruiting two hidden length β-sheet structures nested between the disulfides. The results of this study further the understanding of intramolecular crosslinking in polymers and provide a foundation for the mechanical contributions of EGF domains to the extracellular matrix.

Factor XII Structure-Function Relationships.

Factor XII (FXII), the zymogen of the protease FXIIa, contributes to pathologic processes such as bradykinin-dependent angioedema and thrombosis through its capacity to convert the homologs prekallikrein and factor XI to the proteases plasma kallikrein and factor XIa. FXII activation and FXIIa activity are enhanced when the protein binds to a surface. Here, we review recent work on the structure and enzymology of FXII with an emphasis on how they relate to pathology. FXII is a homolog of pro-hepatocyte growth factor activator (pro-HGFA). We prepared a panel of FXII molecules in which individual domains were replaced with corresponding pro-HGFA domains and tested them in FXII activation and activity assays. When in fluid phase (not surface bound), FXII and prekallikrein undergo reciprocal activation. The FXII heavy chain restricts reciprocal activation, setting limits on the rate of this process. Pro-HGFA replacements for the FXII fibronectin type 2 or kringle domains markedly accelerate reciprocal activation, indicating disruption of the normal regulatory function of the heavy chain. Surface binding also enhances FXII activation and activity. This effect is lost if the FXII first epidermal growth factor (EGF1) domain is replaced with pro-HGFA EGF1. These results suggest that FXII circulates in blood in a "closed" form that is resistant to activation. Intramolecular interactions involving the fibronectin type 2 and kringle domains maintain the closed form. FXII binding to a surface through the EGF1 domain disrupts these interactions, resulting in an open conformation that facilitates FXII activation. These observations have implications for understanding FXII contributions to diseases such as hereditary angioedema and surface-triggered thrombosis, and for developing treatments for thrombo-inflammatory disorders.

NRG1 fusions in solid tumors.

3132 Background: NRG1 fusions are rare but actionable genomic drivers that occur across a growing number of tumor types. NRG1 has an EGF-like domain that serves as a ligand for ErbB (HER3) receptors, thereby inducing heterodimerization, usually with HER2, and subsequent activation of relevant downstream signaling pathways. Agents targeting the HER2/HER3 pathway have shown early clinical promise in NRG1 fusion-positive cancers: currently zenocutuzumab has FDA Fast Track Designation for tumors harboring NRG1-fusions. Here, we update the incidence and characterization of NRG1 fusions across tumor types. Methods: Samples were submitted for clinical molecular profiling at Caris Life Sciences via Caris MI Tumor Seek (Phoenix, AZ). Gene fusion detection was performed on mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). All NRG1 fusions with ≥ 3 junction reads were identified for manual review and for characterization of fusion class, intact functional domains, domain prediction, breakpoints, frame retention and co-occurring alterations by next-generation sequencing. Results: In an analysis of 169,273 tumor specimens, 261 unique tumors with an NRG1 fusion were identified for an overall incidence of 0.154% across tumors. NRG1 fusions were most frequently detected in NSCLC, breast cancer, ovarian cancer, pancreatic ductal adenocarcinoma, colorectal cancer, cholangiocarcinoma, prostate cancer, bladder and urothelial cancers, esophagogastric cancers and less commonly, prostate cancer, soft tissue sarcoma, anal cancer, salivary gland cancers, head and neck cancer, small intestine cancer, small cell lung cancer, endometrial cancer, thyroid cancer, neuroendocrine cancer, high grade glioma, and melanoma. The highest incidence was in breast cancer (0.301%), followed by cholangiocarcinoma (0.263%), NSCLC (0.232%), carcinoma of unknown primary (0.215%), pancreatic ductal adenocarcinoma (0.190%), ovarian cancer (0.174%), bladder cancer (0.148%), esophageal cancer (0.145%) and vulvar cancers (0.145%). There were 153 unique NRG1 fusion partners. The most common fusion partner was CD74 (12.37%), followed by SLC3A2 (8.13%), ATP1B1 (4.59%), and RBPMS (4.24%). Conclusions: NRG1 fusions are rare but actionable genomic events with significant heterogeneity of tumor type and fusion partner.

A novel scavenger receptor (EcSRECII) as a lipopolysaccharide recognition molecule involved in regulating NF-κB activation through extracellular EGF-like cysteine-rich repeat domains with lysosomes in Epinephelus coioides

Scavenger receptors (SRs), as multifunctional pattern recognition receptors, play an important role in innate immunity in mammals, however, their function in fish is limited. Herein, scavenger receptor F2 in Epinephelus coioides (EcSRECII) induced an innate immune response to LPS in GS cells. EcSRECII markedly enhanced LPS-induced NF-κB and IFN-β signaling pathways, whereas knockdown of EcSRECII significantly inhibited LPS-induced NF-κB and IFN-β promoter activation. Interestingly, only retain of epidermal growth factor (EGF)/EGF-like domain in EcSRECII resulted in a punctate cytoplasmic distribution, while the C-terminal domain exhibited a distinct cytoskeletal cytoplasmic distribution. Moreover, devoid of this EGF/EGF-like domain fragment more sharply impaired its ability to activate EcSRECII-induced NF-κB activation than deletion of the C-terminal domain region, but both domains significantly induced IFN-β promoter activation. Full-length EcSRECII and the deletion mutant of C-terminal domain could partly colocalize with lysosomes by LPS derived from V. parahaemolyticus (V.p. LPS) in GS cells, but there was no similar distribution in the deletion mutant of EGF/EGF-like domain. This finding firstly suggested that the N-terminal EGF/EGF-like domain was necessary for the NF-κB signaling pathway to trigger resistance to vibrio infection and its functional exertion may be associated with lysosomes, thus providing insights into the regulation of resistance to vibrio infection in teleosts.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

BackgroundThe gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.MethodsImmortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.ResultsThe results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.ConclusionsBTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Structural changes in NOTCH3 induced by CADASIL mutations: Role of cysteine and non-cysteine alterations

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that results from mutations in NOTCH3. How mutations in NOTCH3 ultimately result in disease is not clear, although there is a predilection for mutations to alter the number of cysteines of the gene product, supporting a model in which alterations of conserved disulfide bonds of NOTCH3 drives the disease process. We have found that recombinant proteins with CADASIL NOTCH3 EGF domains 1 to 3 fused to the C terminus of Fc are distinguished from wildtype proteins by slowed mobility in nonreducing gels. We use this gel mobility shift assay to define the effects of mutations in the first three EGF-like domains of NOTCH3 in 167 unique recombinant protein constructs. This assay permits a readout on NOTCH3 protein mobility that indicates that (1) any loss of cysteine mutation in the first three EGF motifs results in structural abnormalities; (2) for loss of cysteine mutants, the mutant amino acid residue plays a minimal role; (3) the majority of changes that result in a new cysteine are poorly tolerated; (4) at residue 75, only cysteine, proline, and glycine induce structural shifts; (5) specific second mutations in conserved cysteines suppress the impact of loss of cysteine CADASIL mutations. These studies support the importance of NOTCH3 cysteines and disulfide bonds in maintaining normal protein structure. Double mutant analysis suggests that suppression of protein abnormalities can be achieved through modification of cysteine reactivity, a potential therapeutic strategy.

Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. Not applicable.

Molecular genetic characterization of sudden deaths due to thoracic aortic dissection or rupture

BackgroundSudden deaths due to thoracic aortic dissection or rupture (TADR) are often investigated by forensic pathologists in the United States. Up to a quarter of reported TADR result from a highly penetrant autosomal dominant single gene variant. Testing genes associated with familial TADR provides an underlying etiology for the cause of death and informs effective sudden death prevention for at-risk family members. At the New York City Office of Chief Medical Examiner (NYC-OCME), TADR cases are routinely tested by the in-house, CAP-accredited Molecular Genetics Laboratory. In this retrospective study, TADR and cardiovascular cases were reviewed to understand the burden of TADR in sudden deaths, value of molecular diagnostic testing in TADR, and genotype-phenotype correlations in a demographically diverse TADR cohort. MethodsBetween July 2019 and June 2022, cases with in-house cardiovascular genetic testing at NYC-OCME were retrospectively reviewed. Twenty genes associated with familial TADR were analyzed using high throughput massive parallel sequencing on postmortem tissues or bloodspot cards. Variant interpretation was conducted according to ACMG/AMP guidelines. ResultsA total of 1078 cases were tested for cardiovascular genetic conditions, of which 34 (3%) had TADR. Eight of those TADR cases had a pathogenic or likely pathogenic variant (P/LPV), 4 had a variant of uncertain significance (VUS), and 22 cases were negative for variants in TADR genes. The molecular diagnostic yield using the TADR subpanel was 23.5%. The genes with the greatest prevalence of P/LPV were FBN1 (6), followed by TGFBR2 (2), TGFBR1 (1), and MYLK (1). Highly penetrant P/LPV in TGFBR2, FBN1, and TGFBR1 were found in TADR individuals who died younger than 34 years old. Two P/LPV in FBN1 were secondary findings unrelated to cause of death. P/LPV in FBN1 included five truncating variants located in the N-terminal domains and one missense variant involved in the disulfide bonds of the EGF-like domain. All P/LPV in TGFBR1 and TGFBR2 were missense or in-frame deletion variants located in the protein kinase catalytic domain. Three variants were first reported in this study. ConclusionsMolecular testing of familial TADR-associated genes is a highly effective tool to identify the genetic cause of TADR sudden deaths and benefits surviving at-risk families.

Abstract 1238: Loss of HSPD1 induces cellular plasticity in triple-negative breast cancer cells

Abstract Abstract Background Unlike Breast cancer (BRCA) luminal subtypes, Triple negative breast cancer (TNBC) subtype lacks expression of estrogen receptor-α (ERα/ESR1), progesterone receptor (PGR) and/or epidermal growth factor (EGF) family receptor HER2/ERBB2 where patients do not benefit from endocrine and HER2 based therapies in addition to its poor differentiation that results in greater aggressiveness and poorer prognosis. The 60 KD Heat shock protein (HSP60) is a Mitochondrial protein that plays a role in stabilizing and refolding proteins and is regulated by HSPD1 gene. In normal cells, HSPD1 gene expression is controlled, however in TNBC, HSPD1 overexpression is common and is associated with poor cancer-specific survival. This study aims to understand downstream signaling pathways dependent on the function of HSPD1 in TNBC. Method To understand downstream signaling pathways dependent on the function of HSPD1, we analyzed the transcriptomic profiles of metastatic MDA-MB-231-WT and HSPD1-KD cells. Results Bioinformatics data analysis shows that HSPD1 is overexpressed in TNBC, particularly in basal subtype. High expression of HSPD1 related to low survival in breast cancer patients. HSPD1 KD in MDA-MB-231 cells identified several differentially regulated genes (DEG) as compared to WT MDA-MB-231 cells by RNA-seq analysis. Pathway analysis of DEG showed downregulation of mitochondrial respiration, thermogenesis, citric acid cycle, PD-1 signaling while upregulation of estrogen dependent gene expression, estrogen mediated signaling, nuclear receptor transcription pathway. These suggests that HSP60 overexpression in TNBC promotes basal type phenotype. Downregulation of HSP60 expression upregulated hormone receptors, particularly estrogen receptor (ESR1) and androgen receptor (AR) expression, thus inducing basal to luminal switch in MDA-MB-231 cells. These findings suggest that loss of HSPD1 significantly Induces plasticity in TNBC. Conclusion Our results suggest that loss of HSPDI in the context TNBC cells may promotes oncogenic transformation to an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the upregulation of Estrogen-receptors signaling pathways. Ongoing analyses will be performed to identify the possible links between loss of HSPD1 and key transcription factors that define mammary epithelial cell differentiation. Citation Format: Muhammad G. Khodary, Alehegne W. Yirsaw, Temesgen Samuel, Clayton Yates, Deepa Bedi. Loss of HSPD1 induces cellular plasticity in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1238.

Abstract 5486: Combining bromodomain and extraterminal (BET) protein inhibitor treatment with standard MAPK inhibitors improved MAPK signaling inhibition in colorectal cancer

Abstract Background: Overexpression and activation of the MAPK signaling pathway is known to play a key role in colorectal cancer (CRC) progression. Substantial efforts have been made to target MAPK signaling pathways in CRC, particularly blocking oncogenic mutations in KRAS or BRAF. However, standard treatments targeting MAPK signaling have demonstrated limited activities in CRC patients and are in strong need of improved efficacies. Thus, we investigated how the combination of BET inhibitor treatment could affect MAPK signaling activation. Methods: We performed a wide-scale drug combination study testing 15 drug combinations using CRC patient-derived xenograft (PDX) models. PDX models were treated for 21 days, and tumor volume and body weight changes were measured for assessment of treatment responses and toxicities. For the BET inhibitor (iBET-151) plus MEK inhibitor (binimetinib or trametinib) combination, RNA-seq and RPPA was performed on PDX tumor tissues collected after 7 days of treatment for pharmacodynamics study (n=3 per treatment condition). Tumors collected on day 21 were sequenced for H3K27Ac ChIP-seq and IHCs for understanding epigenomic changes in active enhancers and target validations. Results: Our preliminary screening of 15 drug combinations in CRC PDX models have shown the most promising and active responses in a combination of BETi with MEKi (5 out of 20 PDXs obtained tumor regression). Preclinical H3K27ac ChIP-seq data has shown lost enhancer peaks after BET inhibition were significantly associated with RAS signaling pathways (q<0.05). Gene set enrichment analysis on preclinical transcriptomic data also demonstrated downregulation of KRAS-signaling in BETi plus MEKi combination treatment compared to MEKi as monotherapy (p<0.05). MAPK activation scores (MPAS) were also significantly downregulated in transcriptomic and enhancer levels under BETi plus MEKi combinations compared to MEKi. Downstream transcription factors of MAPK signaling pathways, such as MYC and FOSL1, were synergistically downregulated in combination with BETi. Also, we found downregulation of EREG, a member of the epidermal growth factor (EGF) family, under BET inhibition which may play a role in MAPK downregulation together with standard therapy. Conclusions: Some studies have shown MAPK signaling activation as a resistance mechanism of BET inhibition in multiple cancer types. However, the understanding of the crosstalk between the activity of BET inhibition in a combination with MAPK signaling inhibition is limited in CRC. We found BET inhibition induces deeper inhibition of MAPK pathway activation together with standard targeted therapies. Our data demonstrated transcriptomic and epigenetic levels of the mechanism of BETi activity together with MAPK inhibition in CRC. Citation Format: Hey Min Lee, Alexey Sorokin, Preeti Kanikarla Marie, Saikat Chowdhury, Anand Singh, Amanda Anderson, Jumanah Yousef Alshenaifi, Funda Meric-Bernstam, Kunal Rai, Scott Kopetz. Combining bromodomain and extraterminal (BET) protein inhibitor treatment with standard MAPK inhibitors improved MAPK signaling inhibition in colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5486.

Cryo-electron microscopy structures of human thyroid peroxidase (TPO) in complex with TPO antibodies.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the human TPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92Å and 3.4Å for the 2G4 complex and 4F5 complex respectively) show TPO as a monomer with 4 domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein like (CCP) domain and the epidermal growth factor like (EGF) domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B (IDR-B) mainly via different residues. However the epitopes of the two antibodies contain three shared TPO residues. This is the first high resolution structure of TPO to be reported and it should help guide development of new inhibitors of TPO enzyme activity for therapeutic applications.

Fibroblast-derived EGF ligand neuregulin 1 induces fetal-like reprogramming of the intestinal epithelium without supporting tumorigenic growth.

Growth factors secreted by stromal fibroblasts regulate the intestinal epithelium. Stroma-derived epidermal growth factor (EGF) family ligands are implicated in epithelial regeneration and tumorigenesis, but their specific contributions and associated mechanisms remain unclear. Here, we use primary intestinal organoids modeling homeostatic, injured and tumorigenic epithelia to assess how the fibroblast-derived EGF family ligands neuregulin 1 (NRG1) and epiregulin (EREG) regulate the intestinal epithelium. NRG1 was expressed exclusively in the stroma, robustly increased crypt budding and protected intestinal epithelial organoids from radiation-induced damage. NRG1 also induced regenerative features in the epithelium, including a fetal-like transcriptome, suppression of the Lgr5+ stem cell pool and remodeling of the epithelial actin cytoskeleton. Intriguingly, unlike EGF and EREG, NRG1 failed to support the growth of pre-tumorigenic intestinal organoids lacking the tumor suppressor Apc, commonly mutated in human colorectal cancer (CRC). Interestingly, high expression of stromal NRG1 was associated with improved survival in CRC cohorts, suggesting a tumor-suppressive function. Our results highlight the power of stromal NRG1 in transcriptional reprogramming and protection of the intestinal epithelium from radiation injury without promoting tumorigenesis.

MP03-08 INCREASED EXPRESSION OF PRO-FIBROTIC AMPHIREGULIN IN RADIATION CYSTITIS

You have accessJournal of UrologyCME1 Apr 2023MP03-08 INCREASED EXPRESSION OF PRO-FIBROTIC AMPHIREGULIN IN RADIATION CYSTITIS Bernadette Zwaans, Sabrina Mota, Elijah Ward, Sarah Bartolone, and Michael Chancellor Bernadette ZwaansBernadette Zwaans More articles by this author , Sabrina MotaSabrina Mota More articles by this author , Elijah WardElijah Ward More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , and Michael ChancellorMichael Chancellor More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Radiation cystitis (RC) is a painful chronic bladder condition that results from radiation therapy for pelvic cancers and for which no safe and effective therapy exists. RC has a long latency; it can take years before We previously developed a pre-clinical model that closely mimics the human condition. We sought to use this model to identify and validate proteins that are altered after RT and that may drive RC. METHODS: Female C57Bl/6 mice received a single dose of 40Gy irradiation. Bladders were harvested at 1 week, 4 weeks, 3 months and 6 months after irradiation, RNA was harvested, mRNA sequencing was performed at paired-end 150bp on the Illumina NovaSeq6000 with a target of 30 million reads per sample, and bioinformatics analysis was performed. Findings of the RNA sequencing were validated using qPCR analysis in bladder tissues and human urothelial cells (SV-HUC-1). We subsequently assessed levels of our protein of interest in urine samples from prostate cancer survivors with history of external beam radiation therapy using ELISA. RESULTS: RNAseq analysis identified amphiregulin (AREG) to be significantly increased in mouse bladder tissues in response to irradiation (Figure 1). AREG expression in the bladder significantly increased over time starting at 1-week post-RT and continuing up to 6 months post-RT. AREG is a member of the Epidermal Growth Factor (EGF) family and AREG has been implicated in fibrosis in various organ systems including kidney, skin, heart, lung, and liver. These findings were validated with qPCR. Exposure of human urothelial cells to radiation also resulted in increased AREG RNA and protein expression in a dose dependent manner. In addition, AREG was detectable in the urine of prostate cancer survivors with history of pelvic radiation therapy and was significantly higher in the subset of patients with diagnosis of RC (Figure 1). CONCLUSIONS: We have identified increased AREG in bladder tissue and urothelial cells after radiation exposure. AREG has been implicated in pathological tissue fibrosis in various organ systems. As such, AREG may play an important role in mediating radiation cystitis in cancer survivors. AREG may be Further research is needed to determine if AREG could be a novel target to treat and detect bladder fibrosis. Source of Funding: Research reported in this abstract was supported by NIDDK of NIH by award K01DK114334 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e23 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bernadette Zwaans More articles by this author Sabrina Mota More articles by this author Elijah Ward More articles by this author Sarah Bartolone More articles by this author Michael Chancellor More articles by this author Expand All Advertisement PDF downloadLoading ...

A novel glycoprotein from earthworm extract PvE-3: Insights of their characteristics for promoting diabetic wound healing and attenuating methylglyoxal-induced cell damage

Diabetic chronic wound is a worldwide medical burden related to overdosed methylglyoxal (MGO) synthesis, which is the major precursor of glycation of proteins and DNA and is related to the dysfunction of dermal cells thus leading to chronic refractory wounds. Previous studies proved that earthworm extract accelerates diabetic wound healing and possesses cell proliferation and antioxidative effects. However, the effects of earthworm extract on MGO-damaged fibroblasts, the inner mechanisms of MGO-induced cell damage and the functional components in earthworm extract are still poorly understood. Firstly, we evaluated the bioactivities of the earthworm extract PvE-3 on the diabetic wound model and the diabetic related cell damage model. Then the mechanisms were investigated through transcriptomics, flow cytometry and fluorescence probe. The results revealed that PvE-3 promoted diabetic wound healing and protected fibroblast function in cell-damaged conditions. Meanwhile, the high-throughput screening implied the inner mechanisms of diabetic wound healing and PvE-3 cytoprotection effect were involved in the muscle cell function, the cell cycle regulation and the mitochondrial transmembrane potential depolarization. The functional glycoprotein isolated from PvE-3 possessed EGF-like domain which had a strong binding affinity with EGFR. The findings provided references to explore the potential treatments of diabetic wound healing.

DNA methylation profiling and transcriptome sequencing reveal the molecular mechanism of the high-carbohydrate diet on muscle growth of grass carp (Ctenopharyngodon idella)

To investigate the effects of a high-carbohydrate (HC) diet on the growth, serum biochemistry, and DNA methylation and gene transcription profiles of muscle tissue of grass carp (Ctenopharyngodon idella), fish with initial body weight (27.87 ± 0.40) g were fed with a control diet (CON, containing 330 g kg−1 carbohydrate) and an HC diet (containing 490 g kg−1 carbohydrate) for 8 weeks. The results showed that the HC diet had side-effects on muscle growth and health, and caused excessive fat accumulation in the tissues of grass carp. A total of 3135 differentially methylated regions (DMRs) and 530 differentially expressed genes (DEGs) in muscle were screened by bisulfite sequencing and transcriptome sequencing analysis. Twenty-seven DMRs were negatively correlated with DEGs, and eleven DMRs were located in the promoters of DEGs. The methylation level of promoter regions of seven target genes decreased and their transcription levels increased, including FOX protein, diacylglycerol kinase (DGKZ), proto-myosin regulatory protein (tmod1), Notch2, DnaJ homolog (dnajc1), von Willebrand factor D and EGF domains (vwde), and zgc:77838. The methylation level of promoter regions of four target genes increased and their transcription levels decreased, including epidermal growth factor-like domain 6 (EGFL6), estrogen receptor alpha (Erα), cohesin subunit SA-1-like, and LOC795573. These eleven essential genes were mainly involved in GO terms containing cell differentiation, muscle contraction, lipid phosphorylation, PI3K/Akt signaling pathway, and Notch signaling pathway. This study provides a new research direction for the molecular mechanism of the HC diet on muscle growth of grass carp.