c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been reported to be involved in tumorigenesis and metastatic progression. We synthesized a novel triazolopyridazine derivative KRC-327 which selectively targets the c-Met. When we performed receptor tyrosine kinases (RTKs) array with 42 different phosphorylated-RTKs, KRC-327 strongly inhibited expression of activated c-Met in MKN-45 cancer cells. This was confirmed by immunofluorescence staining. Also, KRC-327 decreased the expression of Gab1, Akt, signal transducer and activator of transcription 3 (STAT3) and Erk, down-stream signals of c-Met. KRC-327 strongly suppressed the growth of c-Met over-expressed cancer cells (MKN-45, SNU-638, SNU-5), while not in c-Met absent cancer cell lines (MKN-1, SNU-1). Furthermore, KRC-327 effectively induced cell cycle arrest, especially G0/G1 arrest by increasing expression of p21, p27 and decreasing that of cyclin D1. In the ligand-induced functional studies, KRC-327 inhibited proliferation of HGF-stimulated BxPC-3 cells, the migration of HGF-stimulated AGS cancer cells, and suppressed colony formation in HGF-stimulated U-87MG cells. In xenograft animal models, KRC-327 significantly not only delayed tumor growth but also suppressed phosphorylation of c-Met and its signaling cascades as well as proliferation. Taken together, these results demonstrate that KRC-327 selectively targets c-Met, resulting in inhibition of cell growth and proliferation. Therefore, we suggest that KRC-327 may be a novel drug candidate with the therapeutic potential of targeting c-Met in human cancer.

Oligodendrocytes (OLs) myelinate axons and provide electrical insulation and trophic support for neurons in the central nervous system (CNS). Platelet-derived growth factor (PDGF) is critical for steady-state number and differentiation of oligodendrocyte precursor cells (OPCs), but its downstream targets are unclear. Here, we show for the first time that Gab1, an adaptor protein of receptor tyrosine kinase, is specifically expressed in OL lineage cells and is an essential effector of PDGF signaling in OPCs in mice. Gab1 is down-regulated by PDGF stimulation and up-regulated during OPC differentiation. Conditional deletions of Gab1 in OLs cause CNS hypomyelination by affecting OPC differentiation. Moreover, Gab1 binds to downstream GSK3β and regulated its activity, and thereby affects the nuclear accumulation of β-catenin and the expression of a number of transcription factors critical to myelination. Our work uncovers a novel downstream target of PDGF signaling, which is essential to OPC differentiation and CNS myelination.

A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation.

The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site docking protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.

Molecular and clinical characteristics of pediatric meningiomas are poorly defined. Therefore, we analyzed clinical, morphological and molecular profiles of pediatric meningiomas. Forty pediatric meningiomas from January 2002 to June 2015 were studied. 1p36, 14q32 and 22q-deletion were assessed by fluorescent in situ hybridization and mutations of most relevant exons of AKT, SMO, KLF4, TRAF and pTERT using sequencing. Expression of GAB1, stathmin, progesterone receptor (PR), p53 along with MIB-1 LI was examined using immunohistochemistry. There were 36 sporadic and four NF2 associated meningiomas. Among sporadic meningiomas, the majority (72.2%) of cases harbored 22q-deletion. Difference in frequency of combined 1p/14q deletion in Grade-I versus Grade-II/III tumors was not significant (13.7% vs 28.5%, P = 0.57). PR immunoreactivity was seen in 65.5% of Grade-I and 14.2% of Grade-II/III tumors (P = 0.03). The majority (97.2%) of meningiomas were immunonegative for p53. Stathmin and GAB co-expression was observed in 58.3% of cases. Notably, AKT, SMO, KLF4, TRAF7 (exon 17) and pTERT mutations were seen in none of the cases analyzed. 1p/14q codeletion was frequent in skull base as compared to non-skull base meningiomas (23% vs 11.1%, P = 0.37). All NF2 meningiomas harbored 22q-deletion and showed GAB and stathmin co-expression while none showed 1p/14q loss. Pediatric meningiomas share certain phenotypic and cytogenetic characteristics with adult counterparts, but GAB and stathmin co-expression in the majority of cases and non-significant difference in frequency of 1p/14q co-deletion between low- and high-grade meningiomas indicate an inherently aggressive nature. Characteristic AKT/SMO, KLF4/TRAF7 and pTERT genetic alterations seen in adults are distinctly absent in pediatric meningiomas.

Objective Growth hormone (GH) deficiency (GHD) is commonly treated with recombinant human GH (rhGH). Individual response to rhGH therapy varies widely and there is evidence that variations in growth-related genes, e. g. the GH receptor (GHR) gene, may impact treatment response. We aimed to identify genetic polymorphisms which could serve as predictive markers of response to rhGH therapy. Methods We conducted a genetic analysis of single nucleotide polymorphisms (SNPs) and the GHR exon 3 deletion in 101 paediatric GHD patients receiving rhGH. Patients were analysed for 13 known SNPs in 11 genes of the GH axis (SOS1, IGFR1, GAB1, LHX4, IGFBP3, GRB10, GHRHR, GHSR), growth plate (VDR, ESR1) and cell cycle (CDK4). Individual index of responsiveness (IoR) values were compared by genotype. We also analysed the potential association between the IoR and the GHR exon 3 deletion. IoRs were analysed by genotype by one-way analysis of variance and unpaired t-test. Results Variations in two SNPs, rs2888586 (SOS1) and rs2069502 (CDK4), and the GHR exon 3 deletion were significantly associated with response to rhGH treatment. Conclusions Genetic variations are potentially suitable as predictive markers of rhGH treatment response in GHD. Genetic analysis provides a starting point for individualised treatment of GHD.

Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp-2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates, including Rasa1 and Cortactin with increased pTyr and Gab1 and Erk1/2 with decreased pTyr. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks.

The protein tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors.Mutations in the SHP2gene have beenlinked to many different types of human diseases, including developmental disorders, leukemia and solid tumors.Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the wild-type enzyme, the interactions between its N-SH2 and PTP domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enables the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

The hepatocyte growth factor (HGF) signaling pathway was examined in human normal melanocytes and three malignant melanoma cell lines. HGF-induced activation of c-Met, its receptor-tyrosine kinase, was observed in both melanocytes and melanoma cells, whereas phosphatidylinositol 3-kinase (PI3K), a downstream target of c-Met, was not activated in the melanocytes but enhanced in the melanoma cell lines. The electrophoretic mobility of Gab1, the scaffolding adapter protein that couples activated c-Met and PI3K, was slower in the melanocytes than that in the melanoma cells, and the mobility shifted to that of the melanoma cells after treatment with alkaline phosphatase, indicating that Gab1 is highly phosphorylated on serine and threonine in the melanocytes. Introduction of protein kinase C (PKC)-betaII into the melanoma cells, which is expressed in melanocytes but absent in melanoma cells, resulted in serine and threonine phosphorylation of Gab1 and also prevented tyrosine phosphorylation of Gab1 and its association with PI3K. Furthermore, the introduction of PKC-betaII suppressed HGF-induced activation of PI3K, and attenuated the in vitro invasion activity of the melanoma cells. These results indicate that the HGF signaling process from Gab1 to PI3K is negatively regulated by PKC-betaII, and its loss is critical for melanoma cells to gain invasive potential.

Certain pathological changes, including angiogenesis, actively contribute to the pathogenesis of splenomegaly in portal hypertension (PH), although the detailed molecular and cellular mechanisms remain elusive. In this study, we demonstrated that endothelial Grb-2-associated binder 1 (Gab1) plays a negative role in PH-associated splenomegaly independent of angiogenesis. PH, which was induced by partial portal vein ligation, significantly enhanced Gab1 expression in endothelial cells in a time-dependent manner. Compared with controls, endothelium-specific Gab1 knockout (EGKO) mice exhibited a significant increase in spleen size while their PH levels remained similar. Pathological analysis indicated that EGKO mice developed more severe hyperactive white pulp and fibrosis in the enlarged spleen but less angiogenesis in both the spleen and mesenteric tissues. Mechanistic studies showed that the phosphorylation of endothelial nitric oxide synthase (eNOS) in EGKO mice was significantly lower than in controls. In addition, the dysregulation of fibrosis and inflammation-related transcription factors [e.g., Krüppel-like factor (KLF) 2 and KLF5] and the upregulation of cytokine genes (e.g., TNF-α and IL-6) were observed in EGKO mice. We thus propose that endothelial Gab1 mediates multiple pathways in inhibition of the pathogenesis of splenomegaly in PH via prevention of endothelial dysfunction and overproduction of proinflammatory/profibrotic cytokines.

S100a8 serves an important role in cell differentiation and is abnormally expressed in common tumors, but there are few studies on the association between S100a8 and brain I/R injury. The present study aimed to investigate the role of S100a8 in oxygen‑glucose deprivation and reoxygenation (OGD/R)‑induced BV2 microglia cell injury, and to elucidate the potential underlying molecular mechanisms. BV2 cells were exposed to OGD/R to mimic ischemia/reperfusion (I/R) injury in vitro. S100a8 expression was detected via reverse transcription‑quantitative PCR and western blot analyses. Following transfection with short hairpin RNAs targeting S100a8, the levels of inflammatory cytokines and oxidative stress‑related factors were determined using commercial kits. Apoptosis was assessed using flow cytometric analysis and the expression levels of apoptosis‑related proteins were determined using western blot analysis. Subsequently, the mRNA and protein levels of Grb2‑associated binder 1 (GAB1) were assessed following S100a8 silencing. Immunoprecipitation (IP) was performed to verify the association between S100a8 and GAB1. The levels of inflammation, oxidative stress and apoptosis were assessed following GAB1 silencing, along with S100a8 silencing in BV2 cells subjected to OGD/R. The results indicated that exposure to OGD/R markedly upregulated S100a8 expression in BV2 cells. S100a8 silencing inhibited inflammation, oxidative stress and apoptosis, accompanied by changes in the expression of related proteins. The IP assay revealed a strong interaction between GAB1 and S100a8. In addition, GAB1 silencing reversed the inhibitory effects of S100a8 silencing on inflammation, oxidative stress and apoptosis in OGD/R‑stimulated BV2 cells. Taken together, the results of the present study demonstrated that S100a8 silencing alleviated inflammation, oxidative stress and the apoptosis of BV2 cells induced by OGD/R, partly by upregulating the expression of GAB1. Thus, these findings may potentially provide a novel direction to develop therapeutic strategies for cerebral I/R injury.

BACKGROUND

Gab1 (Grb2-associated binder 1) is a key coordinator that belongs to the insulin receptor substrate-1 like family of adaptor molecules and is tyrosine phosphorylated in response to various growth factors, cytokines, and numerous other molecules. Tyrosine phosphorylated Gab1 is able to recruit a number of signaling effectors including PI3K, SHP2 and PLC-γ. In this study, we characterized the localization and regulation of tyrosine phosphorylation of Gab1 in the retina.

RESULTS

Our immuno localization studies suggest that Gab1 is expressed in rod photoreceptor inner segments. We found that hydrogen peroxide activates the tyrosine phosphorylation of Gab1 ex vivo and hydrogen peroxide has been shown to inhibit the protein tyrosine phosphatase PTP1B activity. We found a stable association between the D181A substrate trap mutant of PTP1B and Gab1. Our studies suggest that PTP1B interacts with Gab1 through Tyrosine 83 and this residue may be the major PTP1B target residue on Gab1. We also found that Gab1 undergoes a light-dependent tyrosine phosphorylation and PTP1B regulates the phosphorylation state of Gab1. Consistent with these observations, we found an enhanced Gab1 tyrosine phosphorylation in PTP1B deficient mice and also in retinas treated ex vivo with a PTP1B specific allosteric inhibitor.

CONCLUSIONS

Our laboratory has previously reported that retinas deficient of PTP1B are resistant to light damage compared to wild type mice. Since Gab1 is negatively regulated by PTP1B, a part of the retinal neuroprotective effect we have observed previously in PTP1B deficient mice could be contributed by Gab1 as well. In summary, our data suggest that PTP1B regulates the phosphorylation state of retinal Gab1 in vivo.

Grb2-associated binder 1 (Gab1) is often aberrant in cancerous cells and tissues, whose alteration is responsible for aggressive phenotypes. In this study, we examined the Gab1 expression in human oral squamous cell carcinoma (OSCC) tissues and investigated the cellular and molecular effect of Gab1 on migration, invasion, and cell growth of the OSCC cell lines SCC15 and SCC25. We found that Gab1 was overexpressed in OSCC tissues and cells, which is related to the protein levels of various molecules associated with cellular proliferation, migration, and invasion. Functional assays identified that Gab1 overexpression promoted cell proliferation and invasion of OSCC cells and inhibited cell apoptosis in the SCC15 and SCC25 cell lines. On the other hand, Gab1 silencing affected the proliferation and invasion of OSCC cells and induced cell apoptosis. Western blot assay identified that Gab1 overexpression suppressed the expression of Cdc20 homolog 1 (Cdh1) and then promoted cell invasion in OSCC cells. Furthermore, Gab1-mediated Cdh1 downregulation was significantly reversed when the cells were subjected to an inhibitor of p-Akt. In conclusion, these results suggested that Gab1 induced malignant progression of OSCC cells probably via activation of the Akt/Cdh1 signaling pathway. Thus, Gab1 may be a potential therapeutic target in the treatment of OSCC patients.

Grb2-associated binder 1 (Gab1) plays an important role in the regulation of cell growth and transformation. A single nucleotide polymorphism (SNP) (rs3805246) in the Gab1 gene has been suggested to be related to the risk of Helicobacter pylori infection and chronic atrophic gastritis (CAG) in a study from Japan. We aimed to assess the associations in a population-based study from Germany. In the baseline examination of ESTHER, a population-based study conducted in Saarland, serum pepsinogen I and II and H. pylori serostatus were measured by ELISA. The Gab1 SNP (rs3805246) was genotyped in 351 serologically defined CAG cases and 351 age- and sex-matched non-CAG controls. A nonsignificant association was observed between the Gab1 SNP and CAG, with an adjusted odds ratio of 1.15 (0.85-1.55) for AA/AG carriers compared to GG carriers. The magnitude of the association did not change when the analysis was restricted to H. pylori seropositive subjects. Furthermore, no significant relation was found between the SNP and H. pylori seropositivity among non-CAG controls. We could not confirm a major association between Gab1 SNP (rs3805246) and the predisposition to H. pylori infection and CAG in this study population from Germany. Further studies with larger sample size are needed to clarify a potential modest effect of Gab1 genetic polymorphisms.

<AbstractText>Fibroblast growth factors (FGFs) are growth factors that have diverse biological activities including broad mitogenic and cell survival activities. They function through the activation of a specific tyrosine kinase receptor that transduces the signal by activating several intracellular signaling pathways.</AbstractText><AbstractText>To identify the different signaling pathways involved in the mechanism of action of FGF8 and FGF18 on ovine granulosa cells using mass spectrometry.</AbstractText><AbstractText>Ovine ovarian granulosa cells were harvested from adult sheep independently at the stage of the estrous cycle and were cultured at a density of 500,000 viable cells in 1 ml DMEM/F12 medium for five days. The cells were then treated on day 5 of culture with 10 ng/mL FGF8 and FGF18 for 30 minutes, and total cell protein was collected for mass spectrometry.</AbstractText><p><div><b>Results</b></div>Mass spectrometry showed that both FGF8 and FGF18 significantly induce simultaneous upregulation of several proteins, including ATF1, STAT3, MAPK1, MAPK3, MAPK14, PLCG1, PLCG2, PKCA, PIK3CA, RAF1, <em>GAB1</em>, and BAG2 ( <mml:math>></mml:math> 1.5-fold; p <mml:math><</mml:math> 0.01).</p><AbstractText>ATF1 and STAT3 are important transcription factors involved in cell growth, proliferation and survival, and consequently can hamper or rescue the normal ovine reproductive system function.</AbstractText>

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis.

Ovarian cancer is the most lethal gynecological malignancy and molecular mechanisms of its progression and metastasis are not completely understood. Some members of GAB (GRB2-associated binding) protein family have been reported to be involved in tumor cell proliferation and metastasis in various cancer types. In the present study, we analyzed the expression of GAB proteins (GAB1, GAB2 and GAB3) in ovarian cancer compared to normal ovarian tissue, in terms of tumor stage, tumor grade and histological type. Differential expression analyses performed in R programming environment using multiple transcriptome datasets (n = 1449) showed that GAB1 expression is decreased in ovarian cancer independently of tumor stage, grade and histotype. Unlike GAB1, expression of GAB2 and GAB3 are increased from early stage to late stage and from low grade to high grade in epithelial ovarian cancer. GAB2 and GAB3 also showed histotype-dependent expression. GAB3 was computed as a top gene whose expression most significantly changed between tumor cells from primary tumor, metastases and ascites. High expression of GAB2 and GAB3 was shown to be associated with shorter progression-free survival in ovarian cancer. This study shows that GAB2 and GAB3 can be important regulators of tumor progression and metastasis in ovarian cancer.

Keywords: Cancer progression; GAB2; GAB3; Metastasis; Ovarian cancer, Transcriptomics.

BACKGROUND

In a previous microarray analysis, GRB2-associated binding protein 1 (GAB1), a docking protein closely related to the insulin receptor substrate, was down-regulated in endometrium of women with polycystic ovary syndrome (PCOS).

OBJECTIVE

The objective of the study was to characterize the cyclic expression of endometrial GAB1 in vivo in normal women and those with PCOS as well as investigate the possible mechanisms of endometrial regulation of GAB1 expression and action in vitro.

METHODS

This was an experimental and case-control study.

METHODS

The study was conducted at a tertiary university hospital.

METHODS

Normal proven fertile women (controls; n = 31) and women with PCOS (cases; n = 26) participated in the study.

METHODS

INTERVENTIONS included timed endometrial biopsies at different phases of the menstrual cycle. Ishikawa cells were cultured with β-estradiol (E2), medroxyprogesterone acetate, and E2 + medroxyprogesterone acetate. Transfection of small interfering RNA for GAB1 in Ishikawa cells incubated with or without insulin.

METHODS

GAB1 mRNA expression in Ishikawa cells and in endometrium of cases and controls was measured. Protein expression of phosphorylated MAPK by Western blot was also measured. Immunohistochemical localization and expression of phosphorylated GAB1 in endometrium was also measured, using a digital histological score.

RESULTS

In endometrial tissue, GAB1 mRNA was reduced in the proliferative phase of PCOS women, compared with controls (P = .003; ANOVA). When all the phases of the menstrual cycle were grouped, GAB1 protein expression was reduced in endometrium of PCOS women (P < .0001; Student t test). E2 increases GAB1 mRNA expression in Ishikawa cells (P = .001; ANOVA). Phosphorylated MAPK is reduced in cells transfected with small interfering RNA for GAB1 (P = .008; ANOVA) and incubated with insulin.

CONCLUSIONS

GAB1 mRNA expression is positively modulated by E2. Endometrial GAB1 protein and mRNA expression are reduced in women with PCOS, suggesting that the endometrium of PCOS women have a defect in insulin signaling due to GAB1 down-regulation.

IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth and tumorigenesis. Whereas both growth factors increase DNA synthesis in MECs, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. In the present study, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5-100 ng/ml). IGF-I activated AKT via insulin receptor substrate-1 and p85, the regulatory subunit of phosphatidylinositol-3 kinase. Treatment with EGF had no effect on insulin receptor substrate-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2 and Gab2 with phospho-SHC, phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA1⁷⁰⁻²⁴⁷ and yGPAA1⁷⁰⁻³³⁹ of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70-247 and 70-339 respectively. The secondary structural content of the stable and monodisperse yGPAA1⁷⁰⁻²⁴⁷ has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA1⁷⁰⁻²⁴⁷ has an R(g) (radius of gyration) of 2.72±0.025 nm and D(max) (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA1⁷⁰⁻²⁴⁷. The large elliptical shape of yGPAA1⁷⁰⁻²⁴⁷ is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA1⁷⁰⁻²⁴⁷ will be discussed together with the recently determined low-resolution structures of yPIG-K²⁴⁻³³⁷ and yPIG-S³⁸⁻⁴⁶⁷ from S. cerevisiae in the GPI transamidase complex.

Scaffolding molecules bind simultaneously and link together various components of signal-transduction pathways. Grb2-associated binder 2 (Gab2) is a scaffolding protein required for FcgammaR-initiated allergic responses in mast cells and FcgammaR-mediated phagocytosis in macrophages, where it links IgE and IgG receptors to the phosphatidylinositol-3 kinase (PI-3K) pathway. The FcgammaR expressed by natural killer (NK) cells triggers antibody-dependent cellular cytotoxicity (ADCC). We show here that mouse NK cells express Gab2 and that although PI-3K was required for ADCC, this FcgammaR-mediated function was normal in Gab2-/- NK cells. Moreover, NK cell development, spontaneous cytotoxicity, and responses to and production of cytokines were not perturbed in Gab2-/- mice. Considering the striking differences between the signaling requirements of FcgammaR in macrophages and NK cells, our findings suggest that the organization of signal transduction downstream of the same FcR can be cell type-specific. Conversely, Gab family members Gab1, Gab2, and Gab3 may play specific roles in different leukocytes. As pharmacological targeting of Gab2 in mast cells is a potential strategy to treat allergy, our results suggest prudence, as NK cells may participate in IgE-mediated anaphylaxis in a Gab2-independent manner.

Background: MicroRNAs (miRNAs) target genes involved in the hyperproliferation of keratinocytes or immune dysfunction of psoriasis. This study prospectively determined the involvement of miR-183-3p in the pathogenesis of psoriasis.

Methods: Differentially expressed miR-183-3p between psoriatic lesional and non-lesional skin were determined by quantitative RT-PCR and in situ hybridization (ISH). CCK8 and wound healing assays were performed to assess cell viability and migration of human keratinocyte cell line (HaCaT). The target of miR-183-3p was validated by luciferase activity assay.

Results: Lower miR-183-3p expression was observed in psoriatic lesional skin compared to psoriatic non-lesional skin. MiR-183-3p over-expression inhibited the viability and migration of HaCaT cells, while inhibition of miR-183-3p promoted the viability and migration of HaCaT cells. Moreover, miR-183-3p could bind to the 3' UTR of GAB1 (growth factor receptor binding 2-associated binding protein 1) and decrease the mRNA and protein expression of GAB1 in HaCaT cells. In addition, higher GAB1 expression was observed in psoriatic lesional skin than psoriatic non-lesional skin.

Conclusion: MiR-183-3p exhibited inhibition property in the proliferation and migration of HaCaT cells via down-regulation of GAB1, suggesting the potential therapeutic strategy for psoriasis.

Keywords: GAB1; Keratinocytes; Migration; Proliferation; Psoriasis; miR-183-3p.

Approximately 10% of non-small cell lung cancer (NSCLC) patients in the United States and 40% of NSCLC patients in Asia have activating epidermal growth factor receptor (EGFR) mutations and are eligible to receive targeted anti-EGFR therapy. Despite an extension of life expectancy associated with this treatment, resistance to EGFR tyrosine kinase inhibitors and anti-EGFR antibodies is almost inevitable. To identify additional signaling routes that can be cotargeted to overcome resistance, we quantified tumor-specific molecular changes that govern resistant cancer cell growth and survival. Mass spectrometry-based quantitative proteomics was used to profile in vivo signaling changes in 41 therapy-resistant tumors from four xenograft NSCLC models. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third-generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase (SFK) substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as SFK signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth in vitro This result suggests that residual SFK signaling mediates therapeutic resistance and that elimination of this signal through combination therapy may delay onset of resistance. Overall, analysis of individual resistant tumors captured unique in vivo signaling rewiring that would have been masked by analysis of in vitro cell population averages. Mol Cancer Ther; 16(11); 2572-85. ©2017 AACR.