Combination of arsenic trioxide and apatinib synergistically inhibits small cell lung cancer by down-regulating VEGFR2/mTOR and Akt/c-Myc signaling pathway via GRB10.

BACKGROUND: Small cell lung carcinoma (SCLC) is characterized by -poor prognosis, -high predilection for -metastasis, -proliferation, and -absence of newer therapeutic options. Elucidation of newer pathways characterizing the disease may allow for development of targeted therapies and consequently favorable outcomes. METHODS: The current study explored the combinatorial action of arsenic trioxide (ATO) and apatinib (APA) in vitro and in vivo. In vitro models were tested using -H446 and -H196 SCLC cell lines. The ability of drugs to reduce -metastasis, -cell proliferation, and -migration were assessed. Using bioinformatic analysis, differentially expressed genes were determined. Gene regulation was assessed using gene knock down models and confirmed using Western blots. The in vivo models were used to confirm the resolution of pathognomic features in the presence of the drugs. Growth factor receptor bound protein (GRB) 10 expression levels of human small cell lung cancer tissues and adjacent tissues were detected by IHC. RESULTS: In combination, ATO and APA were found to significantly reduce -cell proliferation, -migration, and -metastasis in both the cell lines. Cell proliferation was found to be inhibited by activation of Caspase-3, -7 pathway. In the presence of drugs, it was found that expression of GRB10 was stabilized. The silencing of GRB10 was found to negatively regulate the VEGFR2/Akt/mTOR and Akt/GSK-3ß/c-Myc signaling pathway. Concurrently, absence of metastasis and reduction of tumor volume were confirmed in vivo. The immunohistochemical results confirmed that the expression level of GRB10 in adjacent tissues was significantly higher than that in human small cell lung cancer tissues. CONCLUSIONS: Synergistically, ATO and APA have a more significant impact on inhibiting cell proliferation than each drug independently. ATO and APA may be mediating its action through the stabilization of GRB10 thus acting as a tumor suppressor. We thus, preliminarily report the impact of GRB10 stability as a target for SCLC treatment.

Genetic variation near GRB10 associated with bone growth and osteosarcoma risk in canine and human populations.

BACKGROUND: Canine and human osteosarcoma are similar in clinical presentation and tumor genomics. Giant breed dogs experience elevated osteosarcoma incidence, and taller stature remains a consistent risk factor for human osteosarcoma. Whether evolutionarily conserved genes contribute to both human and canine osteosarcoma predisposition merits evaluation. METHODS: A multi-center sample of childhood osteosarcoma patients and controls underwent genome-wide genotyping and imputation. Ancestry-adjusted SNP associations were calculated within each dataset using logistic regression, then meta-analyzed across the three datasets, totaling 1091 patients and 3026 controls. Ten regions previously associated with canine osteosarcoma risk were mapped to the human genome, spanning ∼6 Mb. We prioritized association testing of 5985 human SNPs mapping to candidate osteosarcoma risk regions detected in Irish wolfhounds, the largest dog breed studied. Secondary analyses explored 6289 additional human SNPs mapping to candidate osteosarcoma risk regions identified in Rottweilers and greyhounds. RESULTS: Fourteen SNPs were associated with human osteosarcoma risk after adjustment for multiple comparisons, all within a 42 kb region of human Chromosome 7p12.1. The lead variant was rs17454681 (OR=1.25, 95 %CI: 1.12-1.39; P=4.1×10-5), and independent risk variants were not observed in conditional analyses. While the associated region spanned 2.1 Mb and contained eight genes in Irish wolfhounds, associations were localized to a 50-fold smaller region of the human genome and strongly implicate GRB10 (growth factor receptor-bound protein 10) in canine and human osteosarcoma predisposition. PheWAS analysis in UK Biobank data identified noteworthy associations of the rs17454681 risk allele with varied measures of height and pubertal timing. CONCLUSIONS: Our comparative oncology analysis identified a novel human osteosarcoma risk allele near GRB10, a growth inhibitor that suppresses activated receptor tyrosine kinases including IGF1R, PDGFRB, and EGFR. Epidemiologists may benefit from leveraging cross-species comparisons to identify haplotypes in highly susceptible but genetically homogenous populations of domesticated animals, then fine-mapping these associations in diverse human populations.

GRB10 is a novel factor associated with gastric cancer proliferation and prognosis.

GRB10 and its family members GRB7 and GRB14 were important adaptor proteins. They regulated many cellular functions by interacting with various tyrosine kinase receptors and other phosphorus-containing amino acid proteins. More and more studies have shown that the abnormal expression of GRB10 is closely related to the occurrence and development of cancer. In our current research, expression data for 33 cancers from the TCGA database was downloaded for analysis. It was found that GRB10 was up-regulated in cholangiocarcinoma, colon adenocarcinoma, head and neck squamous carcinoma, renal chromophobe, clear renal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous carcinoma, gastric adenocarcinoma and thyroid carcinoma. Especially in gastric cancer, the high GRB10 expression was closely associated with poorer overall survival. Further research showed that the knockdown of GRB10 inhibited proliferation and migration ability in gastric cancer. Also, there was a potential binding site for miR-379-5p on the 3'UTR of GRB10. Overexpression of miR-379-5p in gastric cancer cells reduced GRB10-regulated gastric cancer proliferation and migration capacity. In addition, we found that tumor growth was slower in a mice xenograft model with knock down of GRB10 expression. These findings suggested that miR-379-5p suppresses gastric cancer development by downregulating GRB10 expression. Therefore, miR-379-5p and GRB10 were expected to be potential targets for the treatment of gastric cancer.

GRB10 sustains AR activity by interacting with PP2A in prostate cancer cells.

Prostate cancer (PCa) progression is driven by androgen receptor (AR) signaling. Unfortunately, androgen-deprivation therapy and the use of even more potent AR pathway inhibitors (ARPIs) cannot bring about a cure. ARPI resistance (ie, castration-resistant PCa, CRPC) will inevitably develop. Previously, we demonstrated that GRB10 is an AR transcriptionally repressed gene that functionally contributes to CRPC development and ARPI resistance. GRB10 expression is elevated prior to CRPC development in our patient-derived xenograft models and is significantly upregulated in clinical CRPC samples. Here, we analyzed transcriptomic data from GRB10 knockdown in PCa cells and found that AR signaling is downregulated. While the mRNA expression of AR target genes decreased upon GRB10 knockdown, AR expression was not affected at the mRNA or protein level. We further found that phosphorylation of AR serine 81 (S81), which is critical for AR transcriptional activity, is decreased by GRB10 knockdown and increased by its overexpression. Luciferase assay using GRB10-knockdown cells also indicate reduced AR activity. Immunoprecipitation coupled with mass spectrometry revealed an interaction between GRB10 and the PP2A complex, which is a known phosphatase of AR. Further validations and analyses showed that GRB10 binds to the PP2Ac catalytic subunit with its PH domain. Mechanistically, GRB10 knockdown increased PP2Ac protein stability, which in turn decreased AR S81 phosphorylation and reduced AR activity. Our findings indicate a reciprocal feedback between GRB10 and AR signaling, implying the importance of GRB10 in PCa progression.

Novel visualized quantitative epigenetic imprinted gene biomarkers diagnose the malignancy of ten cancer types.

BACKGROUND: Epigenetic alterations are involved in most cancers, but its application in cancer diagnosis is still limited. More practical and intuitive methods to detect the aberrant expressions from clinical samples using highly sensitive biomarkers are needed. In this study, we developed a novel approach in identifying, visualizing, and quantifying the biallelic and multiallelic expressions of an imprinted gene panel associated with cancer status. We evaluated the normal and aberrant expressions measured using the imprinted gene panel to formulate diagnostic models which could accurately distinguish the imprinting differences of normal and benign cases from cancerous tissues for each of the ten cancer types. RESULTS: The Quantitative Chromogenic Imprinted Gene In Situ Hybridization (QCIGISH) method developed from a 1013-case study which provides a visual and quantitative analysis of non-coding RNA allelic expressions identified the guanine nucleotide-binding protein, alpha-stimulating complex locus (GNAS), growth factor receptor-bound protein (GRB10), and small nuclear ribonucleoprotein polypeptide N (SNRPN) out of five tested imprinted genes as efficient epigenetic biomarkers for the early-stage detection of ten cancer types. A binary algorithm developed for cancer diagnosis showed that elevated biallelic expression (BAE), multiallelic expression (MAE), and total expression (TE) measurements for the imprinted gene panel were associated with cell carcinogenesis, with the formulated diagnostic models achieving consistently high sensitivities (91-98%) and specificities (86-98%) across the different cancer types. CONCLUSIONS: The QCIGISH method provides an innovative way to visually assess and quantitatively analyze individual cells for cancer potential extending from hyperplasia and dysplasia until carcinoma in situ and invasion, which effectively supplements standard clinical cytologic and histopathologic diagnosis for early cancer detection. In addition, the diagnostic models developed from the BAE, MAE, and TE measurements of the imprinted gene panel GNAS, GRB10, and SNRPN could provide important predictive information which are useful in early-stage cancer detection and personalized cancer management.

Role of Grb10 in mTORC1-dependent regulation of insulin signaling and action in human skeletal muscle cells.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that binds to the insulin receptor, upon which insulin signaling and action are thought to be inhibited. Grb10 is also a substrate for the mechanistic target of rapamycin complex 1 (mTORC1) that mediates its feedback inhibition on phosphatidylinositide 3-kinase (PI3K)/Akt signaling. To characterize the function of Grb10 and its regulation by mTORC1 in human muscle, primary skeletal muscle cells were isolated from healthy lean young men and then induced to differentiate into myotubes. Knockdown of Grb10 enhanced insulin-induced PI3K/Akt signaling and glucose uptake in myotubes, reinforcing the notion underlying its function as a negative regulator of insulin action in human muscle. The increased insulin responsiveness in Grb10-silenced myotubes was associated with a higher abundance of the insulin receptor. Furthermore, insulin and amino acids independently and additively stimulated phosphorylation of Grb10 at Ser476. However, acute inhibition of mTORC1 with rapamycin blocked Grb10 Ser476 phosphorylation and repressed a negative-feedback loop on PI3K/Akt signaling that increased myotube responsiveness to insulin. Chronic rapamycin treatment reduced Grb10 protein abundance in conjunction with increased insulin receptor protein levels. Based on these findings, we propose that mTORC1 controls PI3K/Akt signaling through modulation of insulin receptor abundance by Grb10. These findings have potential implications for obesity-linked insulin resistance, as well as clinical use of mTORC1 inhibitors.

Proproliferative function of adaptor protein GRB10 in prostate carcinoma.

Growth factor receptor-binding protein 10 (GRB10) is a well-known adaptor protein and a recently identified substrate of the mammalian target of rapamycin (mTOR). Depletion of GRB10 increases insulin sensitivity and overexpression suppresses PI3K/Akt signaling. Because the major reason for the limited efficacy of PI3K/Akt-targeted therapies in prostate cancer (PCa) is loss of mTOR-regulated feedback suppression, it is therefore important to assess the functional importance and regulation of GRB10 under these conditions. On the basis of these background observations, we explored the status and functional impact of GRB10 in PCa and found maximum expression in phosphatase and tensin homolog (PTEN)-deficient PCa. In human PCa samples, GRB10 inversely correlated with PTEN and positively correlated with pAKT levels. Knockdown of GRB10 in nontumorigenic PTEN null mouse embryonic fibroblasts and tumorigenic PCa cell lines reduced Akt phosphorylation and selectively activated a panel of receptor tyrosine kinases. Similarly, overexpression of GRB10 in PTEN wild-type PCa cell lines accelerated tumorigenesis and induced Akt phosphorylation. In PTEN wild-type PCa, GRB10 overexpression promoted mediated PTEN interaction and degradation. PI3K (but not mTOR) inhibitors reduced GRB10 expression, suggesting primarily PI3K-driven regulation of GRB10. In summary, our results suggest that GRB10 acts as a major downstream effector of PI3K and has tumor-promoting effects in prostate cancer.-Khan, M. I., Al Johani, A., Hamid, A., Ateeq, B., Manzar, N., Adhami, V. M., Lall, R. K., Rath, S., Sechi, M., Siddiqui, I. A., Choudhry, H., Zamzami, M. A., Havighurst, T. C., Huang, W., Ntambi, J. M., Mukhtar, H. Proproliferatve function of adaptor protein GRB10 in prostate carcinoma.

The in vitro functional analysis of single-nucleotide polymorphisms associated with growth hormone (GH) response in children with GH deficiency.

Response to recombinant human growth hormone (r-hGH) in the first year of therapy has been associated with single-nucleotide polymorphisms (SNPs) in children with GH deficiency (GHD). Associated SNPs were screened for regulatory function using a combination of in silico techniques. Four SNPs in regulatory sequences were selected for the analysis of in vitro transcriptional activity (TA). There was an additive effect of the alleles in the four genes associated with good growth response. For rs3110697 within IGFBP3, rs1045992 in CYP19A1 and rs2888586 in SOS1, the variant associated with better growth response showed higher TA with r-hGH treatment. For rs1024531 in GRB10, a negative regulator of IGF-I signalling and growth, the variant associated with better growth response had a significantly lower TA on r-hGH stimulation. These results indicate that specific SNP variants have effects on TA that provide a rationale for their clinical impact on growth response to r-hGH therapy.

Levels of miR-31 and its target genes in dermal mesenchymal cells of patients with psoriasis.

BACKGROUND: Psoriasis is characterized by chronic inflammatory dermatosis, and the pathogenesis of psoriasis is associated with mesenchymal stem cells (MSCs) and deregulation of the expression of miR-31. This study aimed to clarify the function of miR-31 in dermal MSCs (DMSCs) in the pathogenesis of psoriasis. METHODS: The expression of miR-31 was assayed by a microarray and that of target genes of miR-31 was tested by quantitative PCR. RESULTS: The expression of miR-31 in the psoriasis group was 0.2677 folds that of the control group. The expression of EMP1 and EIG121L genes, whose products are located on the cell membrane, in the psoriasis group was 4.095579 and 5.367017 folds that in the control group, respectively. The expression of GRB10, PTPN14, QKI, RNF144B, and TACC2 genes, whose products are located in the cytoplasm, in the psoriasis group was 1.440428, 1.198335, 1.737285, 7.379546, and 1.531947 folds that of the control. The expression of PRELP, whose products are secreted in the extracellular space, in the psoriasis group was 1.351684 folds that of the control. The expression of RBMS1, KHDRBS3, and SATB2, whose products play a role in the nucleus, in the psoriasis group was 2.237199, 1.277159, and 1.005742 folds that of the control, respectively. CONCLUSIONS: Our results suggest that the low expression of miR-31 in DMSCs in patients with psoriasis causes an increase in the expression of some of its target genes, which in turn facilitates T lymphocyte activation by inhibiting the proliferation of DMSCs and therefore participates in the pathogenesis of psoriasis.

Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant Prostate Cancer.

BACKGROUND: Although androgen deprivation therapy is initially effective in controlling growth of hormone-naive prostate cancers (HNPCs) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. OBJECTIVE: To identify CRPC driver genes that may provide new targets to enhance CRPC therapy. DESIGN, SETTING, AND PARTICIPANTS: Patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The functionality of a potential CRPC driver was validated via its knockdown in cultured prostate cancer cells; its clinical relevance was established using data from prostate cancer patient databases. RESULTS AND LIMITATIONS: Eighty genes were found to be significantly upregulated at the CRPC stage, while seven of them also showed elevated expression prior to CRPC development. Among the latter, growth factor receptor bound protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, elevated GRB10 expression in clinical prostate cancer samples correlated with more aggressive tumor types and poorer patient treatment outcome. GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. A positive correlation between AKT activity and GRB10 expression was also found in clinical cohorts. CONCLUSIONS: GRB10 acts as a driver of CRPC and sensitizes androgen receptor pathway inhibitors, and hence GRB10 targeting provides a novel therapeutic strategy for the disease. PATIENT SUMMARY: Development of castration-resistant prostate cancer (CRPC) is a major problem in the management of the disease. Using state-of-the-art patient-derived hormone-naive prostate cancer xenograft models, we found and validated the growth factor receptor bound protein 10 gene as a driver of CRPC, indicating that it may be used as a new molecular target to enhance current CRPC therapy.

Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways.

Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of Grb10 expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmt1 overexpression dramatically reversed Grb10 up-regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that Grb10 expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future.

Maternal GRB10 microdeletion is a novel cause of cystic placenta: Spectrum of genomic changes in the etiology of enlarged cystic placenta.

INTRODUCTION: The genetics and pathology of diploid complete and triploid partial hydatidiform moles have been well established. Enlarged cystic placenta often indicates an underlying etiology and is frequently associated with adverse pregnancy outcome. Several imprinted genes are strongly expressed in placental tissues and essential for normal placental growth and development. Disruption of these imprinted genes can lead to abnormal placental pathology and placental stunting or overgrowth. We present the genetic etiologies of five unusual mosaic cases of enlarged cystic placentas and report a novel etiology, mosaicism for deletion of the maternal GRB10 gene. METHODS: Five mosaic placental mesenchymal dysplasia cases with discrete populations of "cystic" and "normal" villi and/or atypical p57KIP2 immunostaining were evaluated by genetic analysis; including G-banded karyotyping, fluorescence in situ hybridization (FISH), whole genome CGH + SNP microarray, conventional Sanger sequencing, and STR microsatellite analysis. RESULTS: Genetic etiologies ranged from genome-wide changes, including mosaic androgenetic isodisomy and mosaic diandric triploidy, to a novel microdeletion of the maternally-expressed GRB10 gene. An abnormal mosaic population of cells was also detected in the fetus in two cases. DISCUSSION: Four cases were mosaic for either diandric triploidy or an androgenetic cell population, and the enlarged cystic placentas were likely due to an excess of paternally-expressed growth promoting genes and also the absence of maternally-expressed growth restricting genes. Also we identified mosaicism for a novel microdeletion of the maternal GRB10 allele, a potent growth inhibitor, which resulted in placental overgrowth in the cystic area of one placenta. We advocate the use of ancillary techniques to investigate complex mosaic cases of enlarged cystic placentas to discover atypical genetic etiologies and to increase our understanding of the placental genome.

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization.

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.

Placental imprinting variation associated with assisted reproductive technologies and subfertility.

Infertility affects one in 6 couples in developed nations, resulting in an increasing use of assisted reproductive technologies (ART). Both ART and subfertility appear to be linked to lower birth weight outcomes, setting infants up for poor long-term health. Prenatal growth is, in part, regulated via epigenetically-controlled imprinted genes in the placenta. Although differences in DNA methylation between ART and control infants have been found, it remains unclear whether these differences are due to the ART procedures or to the underlying parental subfertility and how these methylation differences affect imprinted gene expression. In this study, we examined the expression of 108 imprinted genes in placental tissues from infants born to subfertile parents (n = 79), matched naturally-conceived controls (n = 158), and infants conceived using in vitro fertilization (IVF, n = 18). Forty-five genes were identified as having significantly different expression between the subfertile infants and controls, whereas no significant differences were identified between the IVF and control groups. The expression of 4 genes-IGF2, NAPIL5, PAX8-AS1, and TUBGCP5-was significantly downregulated in the IVF compared with the subfertile group. Three of the 45 genes significantly dysregulated between subfertile and control placentae-GRB10, NDN, and CD44 -were found to have a significant positive correlation between expression and birth weight. Methylation levels for these 3 genes and 4 others-MKRN3, WRB, DHCR24, and CYR61-were significantly correlated with expression. Our findings indicate that epigenetic differences in placentas resulting from IVF pregnancies may be related to the underlying subfertility in parents using IVF rather than the IVF procedure itself.

The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2.

Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.

FGFR3 gene mutation plus GRB10 gene duplication in a patient with achondroplasia plus growth delay with prenatal onset.

BACKGROUND: Achondroplasia is a well-defined and common bone dysplasia. Genotype- and phenotype-level correlations have been found between the clinical symptoms of achondroplasia and achondroplasia-specific FGFR3 mutations. RESULT: A 2-year-old boy with clinical features consistent with achondroplasia and Silver-Russell syndrome-like symptoms was found to carry a mutation in the fibroblast growth factor receptor-3 (FGFR3) gene at c.1138G > A (p.Gly380Arg) and a de novo 574 kb duplication at chromosome 7p12.1 that involved the entire growth-factor receptor bound protein 10 (GRB10) gene. Using quantitative real-time PCR analysis, GRB10 was over-expressed, and, using enzyme-linked immunosorbent assays for IGF1 and IGF-binding protein-3 (IGFBP3), we found that IGF1 and IGFBP3 were low-expressed in this patient. CONCLUSIONS: We demonstrate that a combination of uncommon, rare and exceptional molecular defects related to the molecular bases of particular birth defects can be analyzed and diagnosed to potentially explain the observed variability in the combination of molecular defects.

Imprinted survival genes preclude loss of heterozygosity of chromosome 7 in cancer cells.

The genomes of a wide range of cancers, including colon, breast, and thyroid cancers, frequently show copy number gains of chromosome 7 and rarely show loss of heterozygosity. The molecular basis for this phenomenon is unknown. Strikingly, oncocytic follicular thyroid carcinomas can display an extreme genomic profile, with homozygosity of all chromosomes except for chromosome 7. The observation that homozygosity of chromosome 7 is never observed suggests that retention of heterozygosity is essential for cells. We hypothesized that cell survival genes are genetically imprinted on either of two copies of chromosome 7, which thwarts loss of heterozygosity at this chromosome in cancer cells. By employing a DNA methylation screen and gene expression analysis, we identified six imprinted genes that force retention of heterozygosity on chromosome 7. Subsequent knockdown of gene expression showed that CALCR, COPG2, GRB10, KLF14, MEST, and PEG10 were essential for cancer cell survival, resulting in reduced cell proliferation, G1 -phase arrest, and increased apoptosis. We propose that imprinted cell survival genes provide a genetic basis for retention of chromosome 7 heterozygosity in cancer cells. The monoallelically expressed cell survival genes identified in this study, and the cellular pathways that they are involved in, offer new therapeutic targets for the treatment of tumours showing retention of heterozygosity on chromosome 7. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mediation analysis for survival data using semiparametric probit models.

Causal mediation modeling has become a popular approach for studying the effect of an exposure on an outcome through mediators. Currently, the literature on mediation analyses with survival outcomes largely focused on settings with a single mediator and quantified the mediation effects on the hazard, log hazard and log survival time (Lange and Hansen 2011; VanderWeele 2011). In this article, we propose a multi-mediator model for survival data by employing a flexible semiparametric probit model. We characterize path-specific effects (PSEs) of the exposure on the outcome mediated through specific mediators. We derive closed form expressions for PSEs on a transformed survival time and the survival probabilities. Statistical inference on the PSEs is developed using a nonparametric maximum likelihood estimator under the semiparametric probit model and the functional Delta method. Results from simulation studies suggest that our proposed methods perform well in finite sample. We illustrate the utility of our method in a genomic study of glioblastoma multiforme survival.

Monoallelic loss of the imprinted gene Grb10 promotes tumor formation in irradiated Nf1+/- mice.

Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.

Carrageenan Inhibits Insulin Signaling through GRB10-mediated Decrease in Tyr(P)-IRS1 and through Inflammation-induced Increase in Ser(P)307-IRS1.

Inflammation induced by exposure to the common food additive carrageenan leads to insulin resistance by increase in Ser(P)(307)-insulin receptor substrate 1 (IRS1) and subsequent decline in the insulin-stimulated increase in Ser(P)(473)-AKT. Inhibition of carrageenan-induced inflammation reversed the increase in Ser(P)(307)-IRS1 but did not completely reverse the carrageenan-induced decline in Ser(P)(473)-AKT. To identify the additional mechanism responsible for the decrease in Ser(P)(473)-AKT, studies were performed in human HepG2 cells and in C57BL/6J mice. Following carrageenan, expression of GRB10 (growth factor receptor-bound 10 protein), an adaptor protein that binds to the insulin receptor and inhibits insulin signaling, increased significantly. GRB10 silencing blocked the carrageenan-induced reduction of the insulin-stimulated increase in Tyr(P)-IRS1 and partially reversed the decline in Ser(P)(473)-AKT. The combination of GRB10 silencing with BCL10 silencing and the reactive oxygen species inhibitor Tempol completely reversed the decline in Ser(P)(473)-AKT. After carrageenan, GRB10 promoter activity was enhanced because of activation by GATA2. A direct correlation between Ser(P)(473)-AKT and Ser(P)(401)-GATA2 was evident, and inhibition of AKT phosphorylation by the PI3K inhibitor LY294002 blocked Ser(401)-GATA2 phosphorylation and the increase in GRB10 expression. Studies indicated that carrageenan inhibited insulin signaling by two mechanisms: through the inflammation-mediated increase in Ser(P)(307)-IRS1, a negative regulator of insulin signaling, and through a transcriptional mechanism leading to increase in GRB10 expression and GRB10-inhibition of Tyr(P)-IRS1, a positive regulator of insulin signaling. These mechanisms converge to inhibit the insulin-induced increase in Ser(P)(473)-AKT. They provide internal feedback, mediated by Ser(P)(473)-AKT, Ser(P)(401)-GATA2, and nuclear GATA2, which links the opposing effects of serine and tyrosine phosphorylations of IRS1 and can modulate insulin responsiveness.