Molecularly Defined Subsets of Ewing Sarcoma Tumors Differ in Their Responses to IGF1R and WEE1 Inhibition.

PURPOSE: Targeted cancer therapeutics have not significantly benefited patients with Ewing sarcoma with metastatic or relapsed disease. Understanding the molecular underpinnings of drug resistance can lead to biomarker-driven treatment selection. EXPERIMENTAL DESIGN: Receptor tyrosine kinase (RTK) pathway activation was analyzed in tumor cells derived from a panel of Ewing sarcoma tumors, including primary and metastatic tumors from the same patient. Phospho-RTK arrays, Western blots, and IHC were used. Protein localization and the levels of key markers were determined using immunofluorescence. DNA damage tolerance was measured through PCNA ubiquitination levels and the DNA fiber assay. Effects of pharmacologic inhibition were assessed in vitro and key results validated in vivo using patient-derived xenografts. RESULTS: Ewing sarcoma tumors fell into two groups. In one, IGF1R was predominantly nuclear (nIGF1R), DNA damage tolerance pathway was upregulated, and cells had low replication stress and RRM2B levels and high levels of WEE1 and RAD21. These tumors were relatively insensitive to IGF1R inhibition. The second group had high replication stress and RRM2B, low levels of WEE1 and RAD21, membrane-associated IGF1R (mIGF1R) signaling, and sensitivity to IGF1R or WEE1-targeted inhibitors. Moreover, the matched primary and metastatic tumors differed in IGF1R localization, levels of replication stress, and inhibitor sensitivity. In all instances, combined IGF1R and WEE1 inhibition led to tumor regression. CONCLUSIONS: IGF1R signaling mechanisms and replication stress levels can vary among Ewing sarcoma tumors (including in the same patient), influencing the effects of IGF1R and WEE1 treatment. These findings make the case for using biopsy-derived predictive biomarkers at multiple stages of Ewing sarcoma disease management.

miR-149-PARP-2 Signaling Regulates E-cadherin and N-cadherin Expression in the Murine Model of Endometrium Receptivity.

Cadherins play an essential role in the attachment of the blastocyst to the endometrium, a process known as endometrial receptivity. Loss of E-cadherin expression is essential during the process, while the expression level of the other cadherin, N-cadherin, has been reported to be altered in cases of infertility. Both E-cadherin and N-cadherin can be regulated by members of the PARP family. Specifically, PARP-2, which is under the epigenetic control of miR-149, has been observed to promote E-cadherin expression in other human cells. We investigated the roles of E-cadherin and N-cadherin in endometrial receptivity using mouse models for normal endometrial receptivity, pseudopregnancy, and LPS-induced endometrial receptivity failure. E-cadherin and phosphorylated E-cadherin were predominantly expressed during pre-receptive stages as well as in the implantation site of the receptive stage, which were observed reduced during the later stages of implantation in both implantation and non-implantation regions, while N-cadherin was detected only at pre-receptive stages. E-cadherin and N-cadherin were also seen in the uterus during pseudopregnancy, showing a downregulation trend during receptive and post-receptive stages. LPS-induced failed endometrial receptivity showed upregulation of E-cadherin and downregulation of N-cadherin. The E-cadherin expression promoter, GSK-3, was lost and its suppressor, SLUG was upregulated during normal course of endometrial receptivity in mouse model, while GSK-3 was increased during LPS-induced failed embryo implantation. In an in vitro model of embryo implantation, E-cadherin expression is promoted by PARP-2 and regulated by miR-149 epigenetically in human endometrium epithelial cells. In conclusion, E-cadherin is predominantly expressed during pre-receptive stage and promoted by PARP-2, which is regulated by miR-149 in the endometrial epithelial cells.

The Eyes Absent proteins in development and in developmental disorders.

The Eyes Absent (EYA) transactivator-phosphatase proteins are important contributors to cell-fate determination processes and to the development of multiple organs. The transcriptional regulatory activity as well as the protein tyrosine phosphatase activities of the EYA proteins can independently contribute to proliferation, differentiation, morphogenesis and tissue homeostasis in different contexts. Aberrant EYA levels or activity are associated with numerous syndromic and non-syndromic developmental disorders, as well as cancers. Commensurate with the multiplicity of biochemical activities carried out by the EYA proteins, they impact upon a range of cellular signaling pathways. Here, we provide a broad overview of the roles played by EYA proteins in development, and highlight the molecular signaling pathways known to be linked with EYA-associated organ development and developmental disorders.

miRNA-149 targets PARP-2 in endometrial epithelial and stromal cells to regulate the trophoblast attachment process.

Embryo implantation is a highly complex process involving many regulatory factors, including several micro RNAs (miRNAs/miRs). One miRNA present in the stromal cells of normal endometrium is miR-149, which targets poly (ADP-ribose) polymerase 2 (PARP-2), a gene involved in endometrial receptivity for trophoblast implantation. However, the precise role of miR-149 in the endometrial receptivity during blastocyst implantation is still unknown. We studied miR-149-dependent PARP-2 regulation during trophoblast attachment to endometrial epithelial cells. Using FISH, we found that miR-149 is expressed in mouse endometrial epithelial and stromal cells at implantation and inter-implantation sites. Endometrial receptivity for embryo implantation and attachment is inhibited by the upregulation of miR-149 in the endometrium. Our RT-PCR analysis revealed downregulation of miR-149 in the implantation region of the uterus during the receptive stage (Day 5, 0500 h, p.c.) in the mouse. Under in-vitro conditions, miR-149 overexpression in human endometrial epithelial cells (hEECs) abrogated the human trophoblastic cells spheroid and mouse blastocyst attachment. Subsequently, miR-149 also regulates transformed human endometrial stromal cell (T-hESCs) decidualization by downregulating PARP-2 and upregulating caspase-8 proteins. Overexpression of miR-149 in hEECs and downregulated PARP-2 protein expression, reconfirming that PARP-2 is a downstream target of miR-149 in endometrial cells as well. miR-149 is also able to alter the expression of caspase-8, another PARP-2 regulator. In conclusion, our data indicate that miR-149 is one of the regulators of endometrial receptivity and decidualization for trophoblast implantation, and it exerts the effects by acting on the downstream targets PARP-2 and caspase-8.

Poly(ADP-ribose) polymerase-2 is essential for endometrial receptivity and blastocyst implantation, and regulated by caspase-8.

Embryo implantation is a very complex process and several factors play important roles. Using a mouse model, we investigated the functions of PARP-2 and caspase-8 during endometrial receptivity for blastocyst implantation. We found that PARP-2 was upregulated at the receptive stage's implantation region and predominantly expressed in the endometrial stromal region, but downregulated during pregnancy failure and pseudopregnancy. To reinforce the necessity of PARP-2 for embryo implantation, we pharmacologically inhibited PARP-2 'before' & 'after' embryo arrival and observed a reduction in blastocyst implantation. Conversely, elevated caspase-8 expression and activity during pseudopregnancy, delayed implantation, and embryo implantation failure conditions and decreased levels in the decidualization exhibited an inverse pattern with PARP-2, suggesting caspase-8 as a negative regulator for embryo implantation. In vitro caspase-8 downregulates the PARP-2 activity in the mouse endometrial epithelial and stromal cells. These data suggest that PARP-2 and its negative regulation by caspase-8 constitute a crucial step in embryo implantation.

A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasiveness†.

Endometriosis is a prevalent gynecological disorder that eventually gives rise to painful invasive lesions. Increased levels of transforming growth factor-beta 1 (TGF-B1) have been reported in endometriosis. However, details of the effects of high TGF-B1 on downstream signaling in ectopic endometrial tissue remain obscure. We induced endometriotic lesions in mice by surgical auto-transplantation of endometrial tissues to the peritoneal regions. We then treated endometriotic (ectopic and eutopic endometrial tissues) and nonendometriotic (only eutopic endometrial tissues) animal groups with either active TGF-B1 or PBS. Our results demonstrate that externally supplemented TGF-B1 increases the growth of ectopically implanted endometrial tissues in mice, possibly via SMAD2/3 activation and PTEN suppression. Adhesion molecules integrins (beta3 and beta8) and FAK were upregulated in the ectopic endometrial tissue when TGF-B1 was administered. Phosphorylated E-cadherin, N-cadherin, and vimentin were enhanced in the ectopic endometrial tissue in the presence of TGF-B1 in the mouse model, and correlated with epithelial-mesenchymal transition (EMT) in ovarian endometriotic cells of human origin. Furthermore, in response to TGF-B1, the expression of RHOGTPases (RAC1, RHOC, and RHOG) was increased in the human endometriotic cells (ovarian cyst derived cells from endometriosis patient) and tissues from the mouse model of endometriosis (ectopic endometrial tissue). TGF-B1 enhanced the migratory, invasive, and colonizing potential of human endometriotic cells. Therefore, we conclude that TGF-B1 potentiates the adhesion of ectopic endometrial cells/tissues in the peritoneal region by enhancing the integrin and FAK signaling axis, and also migration via cadherin-mediated EMT and RHOGTPase signaling cascades.

Integrin beta8 (ITGB8) activates VAV-RAC1 signaling via FAK in the acquisition of endometrial epithelial cell receptivity for blastocyst implantation.

Integrin beta8 (ITGB8) is involved in the endometrial receptivity. The blastocyst first interacts with the luminal endometrial epithelial cells during its implantation; therefore, we have investigated the signaling of ITGB8 via FAK and VAV-RAC1 in the endometrial epithelial cells. Integrin beta8 was found elevated in epithelial cells at late-pre-receptive (day4, 1600 h) and receptive (day5, 0500 h) stages of endometrial receptivity period in the mouse. Integrins downstream molecule FAK has demonstrated an increased expression and phosphorylation (Y397) in the endometrium as well as in the isolated endometrial epithelial cells during receptive and post-receptive stages. Integrin beta8 can functionally interact with FAK, VAV and RAC1 as the levels of phosphorylated-FAK, and VAV along with the RAC-GTP form was reduced after ITGB8 knockdown in the endometrial epithelial cells and uterus. Further, VAV and RAC1 were seen poorly active in the absence of FAK activity, suggesting a crosstalk of ITGB8 and FAK for VAV and RAC1 activation in the endometrial epithelial cells. Silencing of ITGB8 expression and inhibition of FAK activity in the Ishikawa cells rendered poor attachment of JAr spheroids. In conclusion, ITGB8 activates VAV-RAC1 signaling axis via FAK to facilitate the endometrial epithelial cell receptivity for the attachment of blastocyst.

RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model.

The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCK1, and RAC1 in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCK1, VAV, and RAC1 expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.

Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation.

Endoglin is a TGF-ß receptor that is expressed in uterine endothelial and stromal cells in addition to trophoblast expression. However, the functional importance of endoglin in the embryo implantation process is not clear. We observed endoglin expression in the endometrium throughout the stages of its receptivity; however, its expression was enhanced during the receptive stage. Endoglin expression was predominant in epithelial cells of the lumen and glands, but showed a milder expression in stromal cells. Endoglin expression was initially observed in the primary decidual zone and later extended to the secondary decidua zone. Knockdown of endoglin via siRNA reduced the implantation sites along with the blastocyst numbers. Mouse blastocyst with endoglin-silenced endometrial epithelial cells (human and mouse origin) showed poor trophoblast outgrowth, which suggests an essential role for endoglin during endometrial receptivity. In conclusion, our findings reveal the association of endoglin with endometrial receptivity, which is important for embryo attachment.

Gastro-protective and Anti-stress Efficacies of Monomethyl Fumarate and a Fumaria indica Extract in Chronically Stressed Rats.

Results of the very first experiments conducted to evaluate therapeutic potentials of a fumarate containing Fumaria indica extract and of fairly low daily oral doses of monomethyl fumarate for prevention of chronic unavoidable foot-shock stress-induced gastric ulcers, and possible involvement of diverse neuro-hormonal and oxidative process in their stress response desensitizing effects are reported and discussed in this article. Preventive effects of 21 daily oral 60, 120, and 240 mg/kg doses of a standardized 50 % methanolic F. indica extract (MFI) and 1.25, 2.50, and 5.00 mg/kg/day of pure monomethyl fumarate (MMF) were compared in rats subjected to one hour daily unavoidable foot-shocks. A pharmaceutically well-standardized Withania somnifera (WS) root extract was used as a reference herbal anti-stress agent in all experiments. Effects of the treatments on stress-induced alterations in body weight, adrenal and spleen weights, gastric ulcer and ulcer index, weight of glandular stomach, protective mucosal glycoprotein content, cellular proliferation, oxidative stress on stomach fundus, and brain tissues of male rats were quantified. Other parameters quantified were plasma corticosterone levels, brain monoamine levels, and expressions of the cytokines TNF-α, IL-10, and IL-1ß in blood and brain of stressed and treated rats. Most but not every observed stress-induced anomalies were suppressed or completely prevented by both MFI and pure MMF treatments in dose-dependent manner. Qualitatively, the observed activity profiles of both of them were similar to those of WS dose tested. These results reveal that both MFI and MMF are potent gastro-protective agents against chronic unavoidable stress-induced ulcers and strongly suggest that they act as regulators or modulators of monoamine, corticosterone, and cytokine homeostasis.

Trigger of autoimmune diseases (SLE): identification of LINE transposition based novel therapeutic molecular targets.

Autoimmune diseases are the highly heterogeneous at cellular and molecular level. The causes and consequences of most of the autoimmune diseases are not well explored. However the researches focusing on the development of biomarkers for the diagnosis of autoimmune diseases are seems to be inadequate and given treatment are insufficient to control or cure the disease properly. It is a big obstacle to develop any therapy without knowing the actual cause and molecular event playing role in disease onset. In this article we are raising the involvement of LINE or other transposition as a first trigger and cause for autoimmune disease. Further we are proposing a novel hybrid aptamers based biocapturing model which would help in the investigation of genome-wide LINE transposition in pristane induced SLE mice model. Importantly the effect of new LINE movements at the expression pattern of neighboring genes would be used as novel molecular prognostic biomarkers for onset of SLE and related autoimmune diseases. We are also proposing that the differential expression either inductive or suppressive pattern of expected several candidate genes would be implicated in the defective biochemical or cellular defects, and targeted therapy would be employed to such life threatening disease.

Protective effects of Andrographis paniculata extract and pure andrographolide against chronic stress-triggered pathologies in rats.

This study was designed to experimentally verify the possibility that Andrographis paniculata could be another medicinal herb potentially useful for prevention of diverse spectrums of pathologies commonly associated with chronic unavoidable environmental stress, and whether andrographolide could as well be its quantitatively major bioactive secondary metabolite. Preventive effects of 21 daily oral 50, 100 and 200 mg/kg doses of a therapeutically used extract of the plant (AP) and 30 and 60 mg/kg/day of pure andrographolide were compared in rats subjected to 1-h daily unavoidable foot-shocks. A pharmaceutically well-standardized Withania somnifera (WS) root extract was used as a reference herbal anti-stress agent in all experiments. Effects of the treatments on stress-induced alterations in body weight, gastric ulcer, adrenal and spleen weights, and depressive state and sexual behavior in male rats were quantified. Other parameters quantified were plasma cortisol levels, and expressions of the cytokines TNF-α, IL-10 and IL-1ß in blood and brain. All observed stress-induced pathological changes were less pronounced or completely prevented by both AP and pure andrographolide. Even the lowest tested doses of AP (50 mg/kg/day) or of andrographolide (30 mg/kg/day) suppressed almost maximally the blood IL-1ß and IL-10 as well as brain TNF-α and IL-10 expressions induced by chronic stress. Qualitatively, the observed activity profiles of both of them were similar to those of WS dose tested. These results reveal that both AP and andrographolide are pharmacologically polyvalent anti-stress agents, and that biological processes regulating corticosterone and cytokine homeostasis are involved in their modes of actions.