Georgia Dalagiorgou

Crosstalk between advanced glycation and endoplasmic reticulum stress: emerging therapeutic targeting for metabolic diseases.

CONTEXT Advanced glycation, the major posttranslational modification of proteins, DNA, and lipids, is accelerated under conditions of increased oxidative stress, hyperglycemia, and hypoxia contributing to a variety of metabolic diseases such as diabetes mellitus, obesity, inflammation, polycystic ovarian syndrome, ischemic cardiovascular disease, and neurodegenerative disorders. The potential role of advanced glycation in endoplasmic reticulum (ER) homeostasis is largely unknown. EVIDENCE ACQUISITION Basic and clinical peer-reviewed articles on advanced glycation and ER stress related to metabolic regulation were searched in PubMed from 2000-2011. The resulting articles as well as relevant cited references were reviewed. EVIDENCE SYNTHESIS Recent evidence indicates that hyperglycemia, hypoxia, and oxidative stress, apart of triggering advanced glycation, can also adversely affect ER function, leading to pathogenic ER stress, followed by the unfolded protein response. The concomitant presence of advanced glycation in the same conditions with ER stress suggests their crosstalk in the progression of diseases associated with hypoxic and oxidative stress. CONCLUSION Current data support the direct or indirect induction of ER stress response by advanced glycation end products or advanced glycation end product precursors in the pathogenesis of metabolic diseases. Inhibitors of advanced glycation acting as potent ER stress modulators with beneficial effects in restoring ER homeostasis and adjusting physiological unfolded protein response level present an emerging therapeutic approach with significant applications, especially in the context of metabolic dysfunction.

Polycystin-1: function as a mechanosensor.

Polycystin-1 (PC1), encoded by the Pkd1 gene, is a large transmembrane protein whose mutation is involved in autosomal dominant polycystic kidney disease. When expressed, PC1 activates a G-protein signaling pathway that subsequently modulates Ca(2+) channels. PC1 is highly expressed in developing tissue and via its C-terminus tail forms a complex with polycystin-2; this complex, found to be located at the primary cilia, seems to act as a mechanosensor that could affect proliferation, differentiation and apoptosis of cells. Also, loss of polycystins correlates with disruption of flow-dependent and steady-state intracellular Ca(2+) signaling. Despite the lack of clarity on the role of the polycystins as mechanosensor molecules, a new interest in this PCs/primary cilium complex is providing continuously new insights. In this review, some of the known features of PC1 such as structure, function, signaling pathways involved and its role as a possible therapeutic target are being discussed.

Is androgen receptor targeting an emerging treatment strategy for triple negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype. The absence of expression and/or amplification of estrogen and progesterone receptor as well as ERBB-2 prevent the use of currently available endocrine options and/or ERBB-2-directed drugs and indicates chemotherapy as the main current therapy. TNBC represents approximately 15% of breast cancer cases with high index of heterogeneity. Here, we review the role of androgen receptor in breast carcinogenesis and its association with alterations in the expression pattern and functional roles of regulatory molecules and signal transduction pathways in TNBC. Additionally, based on the so far preclinical and clinical published data, we evaluate the perspectives for using and/or developing androgen receptor targeting strategies for specific TNBC subtypes.

Polycystin-1 and polycystin-2 are involved in the acquisition of aggressive phenotypes in colorectal cancer.

The polycystins PC1 and PC2 are emerging as major players in mechanotransduction, a process that influences all steps of the invasion/metastasis cascade. We hypothesized that PC1 and PC2 facilitate cancer aggressiveness. Immunoblotting, RT‐PCR, semi‐quantitative and quantitative real‐time PCR and FACS analyses were employed to investigate the effect of polycystin overexpression in colorectal cancer (CRC) cells. The impact of PC1 inhibition on cancer‐cell proliferation was evaluated through an MTT assay. In vitro data were analyzed by Students t‐test. HT29 human xenografts were treated with anti‐PC1 (extracellular domain) inhibitory antibody and analyzed via immunohistochemistry to determine the in vivo role of PC1 in CRC. Clinical significance was assessed by examining PC1 and PC2 protein expression in CRC patients (immunohistochemistry). In vivo and clinical data were analyzed by non‐parametric tests, Kaplan‐Meier curves, log‐rank test and Cox model. All statistical tests were two‐sided. PC1 overexpression promotes epithelial‐to‐mesenchymal transition (EMT) in HCT116 cells, while PC2 overexpression results in upregulation of the mTOR pathway in SW480 cells. PC1 inhibition causes reduced cell proliferation in CRC cells inducing tumor necrosis and suppressing EMT in HT29 tumor xenografts. In clinical study, PC1 and PC2 overexpression associates with adverse pathological parameters, including invasiveness and mucinous carcinomas. Moreover, PC1 overexpression appears as an independent prognostic factor of reduced recurrence‐free survival (HR = 1.016, p = 0.03) and lowers overall survival probability, while aberrant PC2 expression predicts poor overall survival (p = 0.0468). These results support, for the first time, a direct link between mechanosensing polycystins (PC1 and PC2) and CRC progression.

Advanced glycation end products upregulate lysyl oxidase and endothelin-1 in human aortic endothelial cells via parallel activation of ERK1/2–NF-κB and JNK–AP-1 signaling pathways

Endothelial dysfunction involves deregulation of the key extracellular matrix (ECM) enzyme lysyl oxidase (LOX) and the vasoconstrictor protein, endothelin-1 (ET-1), whose gene expression can be modulated by the transcriptional activators nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). Advanced glycation end products (AGEs) present an aggravating factor of endothelial dysfunction which upon engagement to their receptor RAGE induce upregulation of mitogen-activated protein kinases (MAPKs), leading to NF-κB and AP-1 potentiation. We hypothesized that AGEs could induce NF-κΒ- and AP-1-dependent regulation of LOX and ET-1 expression via the AGE/RAGE/MAPK signaling axis. Western blot, real-time qRT-PCR, FACS analysis and electrophoretic mobility-shift assays were employed in human aortic endothelial cells (HAECs) following treatment with AGE-bovine serum albumin (AGE-BSA) to investigate the signaling pathway towards this hypothesis. Furthermore, immunohistochemical analysis of AGEs, RAGE, LOX and ET-1 expression was conducted in aortic endothelium of a rat experimental model exposed to high- or low-AGE content diet. HAECs exposed to AGE-BSA for various time points exhibited upregulation of LOX and ET-1 mRNA levels in a dose- and time-dependent manner. Exposure of HAECs to AGE-BSA also showed specific elevation of phospho(p)-ERK1/2 and p-JNK levels in a dose- and time-dependent fashion. AGE administration significantly increased NF-κΒ- and AP-1-binding activity to both LOX and ET-1 cognate promoter regions. Moreover, LOX and ET-1 overexpression in rat aortic endothelium upon high-AGE content diet confirmed the functional interrelation of these molecules. Our findings demonstrate that AGEs trigger NF-κΒ- and AP-1-mediated upregulation of LOX and ET-1 via the AGE/RAGE/MAPK signaling cascade in human endothelial cells, thus contributing to distorted endothelial homeostasis by impairing endothelial barrier function, altering ECM biomechanical properties and cell proliferation.

The role of CXC-chemokine receptor CXCR2 and suppressor of cytokine signaling-3 (SOCS-3) in renal cell carcinoma

BackgroundChemokine receptor signaling pathways are implicated in the pathobiology of renal cell carcinoma (RCC). However, the clinical relevance of CXCR2 receptor, mediating the effects of all angiogenic chemokines, remains unclear. SOCS (suppressor of cytokine signaling)-3 is a negative regulator of cytokine-driven responses, contributing to interferon-α resistance commonly used to treat advanced RCC with limited information regarding its expression in RCC.MethodsIn this study, CXCR2 and SOCS-3 were immunohistochemically investigated in 118 RCC cases in relation to interleukin (IL)-6 and (IL)-8, their downstream transducer phosphorylated (p-)STAT-3, and VEGF expression, being further correlated with microvascular characteristics, clinicopathological features and survival. In 30 cases relationships with hypoxia-inducible factors, i.e. HIF-1a, p53 and NF-κΒ (p65/RelA) were also examined. Validation of immunohistochemistry and further investigation of downstream transducers, p-JAK2 and p-c-Jun were evaluated by Western immunoblotting in 5 cases.ResultsBoth CXCR2 and IL-8 were expressed by the neoplastic cells their levels being interrelated. CXCR2 strongly correlated with the levels of HIF-1a, p53 and p65/RelA in the neoplastic cells. Although SOCS-3 was simultaneously expressed with p-STAT-3, its levels tended to show an inverse relationship with p-JAK-2 and p-c-Jun in Western blots and were positively correlated with HIF-1a, p53 and p65/p65/RelA expression. Neither CXCR2 nor SOCS-3 correlated with the extent of microvascular network. IL-8 and CXCR2 expression was associated with high grade, advanced stage and the presence/number of metastases but only CXCR2 adversely affected survival in univariate analysis. Elevated SOCS-3 expression was associated with progression, the presence/number of metastasis and shortened survival in both univariate and multivariate analysis.ConclusionsOur findings implicate SOCS-3 overexpression in RCC metastasis and biologic aggressiveness advocating its therapeutic targeting. IL-8/CXCR2 signaling also contributes to the metastatic phenotype of RCC cells but appears of lesser prognostic utility. Both CXCR2 and SOCS-3 appear to be related to transcription factors induced under hypoxia.

Expression of vascular endothelial factor-A, gelatinases (MMP-2, MMP-9) and TIMP-1 in uterine leiomyomas.

Abstract Background: Leiomyomas growth involves cellular hypertrophy, modulation of mitotic activity and upregulation of extracellular matrix (ECM). Vascular factors and matrix metalloproteinases (MMPs) play a coordinated role during neoplasia and tissue remodeling. The present study investigates the role of angiogenic factor vascular endothelial growth factor (VEGF)-A with the activity of main gelatinases, MMP-2/MMP-9 and their tissue inhibitor TIMP-1 in patients with leiomyomas. Methods: Peripheral blood of 46 women with uterine leiomyomas was obtained prior hysterectomy to assess VEGF-A, MMP-2, -9, TIMP-1 levels by enzyme-linked immunosorbent assay compared to 39 healthy controls. Protein expression levels of VEGF-A, MMP-2 and MMP-9 were evaluated by western immunoblotting and immunohistochemistry in leiomyomas tissue specimens after hysterectomy. Furthermore, the activity of gelatinases in leiomyoma tissue extracts and control myometrium was evaluated by semi-quantitative zymography. Results: Circulating levels of VEGF-A, MMP-2 and TIMP-1 were significantly elevated in leiomyoma patients compared to controls (p<0.001, p=0.004, p=0.003, respectively). A positive correlation was found between VEGF-A and MMP-2 (p=0.021) as well as MMP-9 (p=0.001) peripheral levels in the patient’s group. Furthermore, increased VEGF-A protein levels were detected in leiomyoma tissue compared to control myometrium, followed by increased localization of both VEGF-A and MMP-2 in the ECM embedding bundles of smooth muscle cells of leiomyomas. The activity of MMP-2 was significantly higher in leiomyomas than normal myometrium in all investigated tissues. Conclusions: This study demonstrates a possible coordinated role of VEGF-A and MMP-2 during uterine leiomyomas growth and angiogenesis with potential prognostic significance.

Mechanosensor polycystin-1 potentiates differentiation of human osteoblastic cells by upregulating Runx2 expression via induction of JAK2/STAT3 signaling axis

Polycystin-1 (PC1) has been proposed as a chief mechanosensing molecule implicated in skeletogenesis and bone remodeling. Mechanotransduction via PC1 involves proteolytic cleavage of its cytoplasmic tail (CT) and interaction with intracellular pathways and transcription factors to regulate cell function. Here we demonstrate the interaction of PC1-CT with JAK2/STAT3 signaling axis in mechanically stimulated human osteoblastic cells, leading to transcriptional induction of Runx2 gene, a master regulator of osteoblastic differentiation. Primary osteoblast-like PC1-expressing cells subjected to mechanical-stretching exhibited a PC1-dependent increase of the phosphorylated(p)/active form of JAK2. Specific interaction of PC1-CT with pJAK2 was observed after stretching while pre-treatment of cells with PC1 (anti-IgPKD1) and JAK2 inhibitors abolished JAK2 activation. Consistently, mechanostimulation triggered PC1-mediated phosphorylation and nuclear translocation of STAT3. The nuclear phosphorylated(p)/DNA-binding competent pSTAT3 levels were augmented after stretching followed by elevated DNA-binding activity. Pre-treatment with a STAT3 inhibitor either alone or in combination with anti-IgPKD1 abrogated this effect. Moreover, PC1-mediated mechanostimulation induced elevation of Runx2 mRNA levels. ChIP assays revealed direct regulation of Runx2 promoter activity by STAT3/Runx2 after mechanical-stretching that was PC1-dependent. Our findings show that mechanical load upregulates expression of Runx2 gene via potentiation of PC1–JAK2/STAT3 signaling axis, culminating to possibly control osteoblastic differentiation and ultimately bone formation.

176PHER-3 TARGETING AFFECTS THE DIMERIZATION PATTERN OF EGFR FAMILY MEMBERS IN BREAST CARCINOMAS (BC)

ABSTRACT Aim: Many BC patients present intrinsic or acquired resistance to anti-HER-2 directed therapies, mainly attributed to HER-3. Our goal was to evaluate the dimerization pattern of EGFR, HER2/3/4 with or without the addition of heregulin (Hg) and HER-3 targeting agents. Methods: MCF7 and SKBR3 cell lines were used. Dimerization patterns were studied using proximity ligation assay and immunofluorescence, where proteins in proximity can be visualized by combining classical immunocytochemistry with rolling circle amplification. Concentrations of used reagents were: HRG1-b1 10mM (R&D Systems), Pertuzumab (P) 30mM (Genetech), U3 (U) inhibitor 30mM (U3 Pharma). After blocking, cells were incubated with combination of primary antibodies (antiHER2/HER3, antiHER2/HER4, antiEGFR/HER3, anti-EGFR/HER4) in a preheated humidity chamber for 1 h at 37°C. Cells were then incubated with the PLA probes diluted 1:5 in antibody diluent (Olink Bioscience, Uppsala, Sweden) in a humidified chamber for 1 h at 37°C. Subsequent hybridization, ligation, amplification, and detection were performed as per manufacturers instruction. Fluorescence images were acquired using a Zeiss Axiovert microscope (Carl Zeiss Microscopy, Thornwood, NY). Data analysis was done using Duolink ImageTool Software that has been developed for quantification of PLA signals. Results: In MCF7 cells (PLA signals scale 0-12), Hg addition increased dimmers EGFR/HER4 (9,7 vs 5,13), EGFR/HER3 (8,07 vs 3,36) and less HER2/HER3 (4,12 vs 3,35). The addition of Hg and P resulted in EGFR/HER4 4,67, EGFR/HER3 2,46, HER2/HER3 3,0 and Hg and U resulted in EGFR/HER4 0,6, EGFR/HER3 0,6, HER2/HER3 0,6. In SKBR3 cells (PLA signals scale 0-60), Hg addition increased dimmers EGFR/HER4 (60 vs 40), HER2/HER3 (26,93 vs 15,92) and less EGFR/HER3 (16 vs 10,6). The addition of Hg and P resulted in EGFR/HER4 17,0, HER2/HER3 19,9, EGFR/HER3 12,5 and Hg and U resulted in EGFR/HER4 0,7, HER2/HER3 0,7, EGFR/HER3 0,7. All images and data analysis will be presented in full detail. Conclusions: The addition of Hg enhances HER3-based dimmer formation, while HER-3 targeting agents lower HER3-containing dimmer formation. The technique can be used in paraffin-embedded BC specimens and may identify patient sub-groups who benefit by HER-3 targeting agents. Disclosure: All authors have declared no conflicts of interest.