Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. An alternative route for blocking RAS-driven oncogenic pathways is to target downstream effectors of RAS involved in promoting the oncogenic phenotype. One of the critical characteristics required for tumors to grow and progress is the ability of tumor cells to drive angiogenesis. Interestingly, oncogenic RAS promotes angiogenesis by upregulating the proangiogenic IL-8 cytokine, an NF-kB target gene; and recent studies have shown that oncogenic RAS also activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic or pharmacological inhibition of IKKβ or deletion of the RELA/p65 subunit of NF-κB. Therefore, we hypothesized that IKKβ inhibition would reduce KRAS-induced angiogenesis and tumor progression. To test this hypothesis, we used genetic and pharmacological approaches to inhibit IKKβ in K-Ras mutant lung cancer cell lines. Treatment of KRAS-positive A549 e H358 lung cancer cells with the highly specific IKKβ inhibitor Compound A (CmpdA) or siRNA-mediated knockdown of IKKβ in these cells reduced expression and secretion of the proangiogenic IL-8 cytokine. We found that IKKβ targeting also reduced expression and secretion of VEGF, a growth factor involved in promoting angiogenesis, which is also regulated by NF-κB. Moreover, conditioned media from A549 and H358 cells with siRNA-mediated IKKβ knockdown reduced endothelial cell (HUVECS) migration. In order to ascertain whether IKKβ inhibition can directly affect endothelial cell function, we treated HUVECs with CmpdA, which also resulted in reduced HUVEC migration. To evaluate how IKKβ affects endothelial cell function in vivo, we used a mouse model of neonatal retinopathy, where pathological retinal angiogenesis is induced by transient exposure of neonatal mice to hyperoxic conditions. Angiogenesis was significantly reduced in this model when neonatal mice were treated with 3 doses of 10 mg/Kg CmpdA. Finally, IKKβ knockdown in A549 cells also reduced expression of MMP-2 and MMP9 metalloproteases and reduced A549 cell invasion. Taken together, these results suggest that IKKβ inhibition therapy may reduce tumor angiogenesis, as well as invasive properties of KRAS-induced lung tumors, thereby having the potential to result in a sustained therapeutic effect, particularly if combined with other therapeutic approaches. Citation Format: Tatiana C. Lobo, Leila Magalhães, Laura Cardeal, Ricardo Giordano, Albert Baldwin, Daniela Bassères. IKKβ is a potential anti-angiogenic therapeutic target in KRAS-induced lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2015-1379

Vascular endothelial dysfunction as the mechanism for the development of obesity Raja'a Dalloul, Tatiana Lobo, Hamid Massaeli, Nasrin Mesaeli Weill Cornell Medicine- Qatar, Qatar Foundation, Doha, Qatar Background: Obesity is one of the major public health issues in the world with a rapid increase in its prevalence. According to the last public health report from supreme public health in Qatar in 2012, 71.8 % of the women were overweight compared to 68.3% of men. Among the gulf region, Qatar has the 6th highest rate of obesity in young boys. Moreover, the WHO survey in 2009 showed 70% of the Qatari children were obese because of the nutritional changes and unhealthy lifestyle. Adipocytes are considered the only cells where their size can vary in physiological conditions. Adipose tissues grow as they store excess energy intake. It's well established that adipose tissue is highly vascularized. These vascular networks play a vital role in the adipogenesis process. The vasculature of adipose tissue provide oxygen, growth factors, nutrients, and cytokines to the progenitor cells that are differentiated into pre-adipocytes and vascular endothelial cells. Vascular endothelial cells form the inner barrier of the vessel and is responsible for maintaining the vascular vasodilation and constriction. Defect in the endothelial cell function has been shown to result as a consequence of different diseases such as obesity, diabetes and high blood pressure. An increase or decrease in the generation of the reactive oxygen is one of the main cause of endothelium dysfunction. The fluctuation in the balance of these factors in the endothelial has an influence in the adipocyte cells which is responsible for the formation of fats or fatty tissues which cause an impairment of adipocytes and may lead to obesity. Calreticulin (CRT) is a multifunctional protein that is expressed in the endoplasmic reticulum of all mammalian cells. The main functions of CRT are regulation of intracellular Ca2+ hemostasis and lectin like chaperone. As part of ongoing research in our lab, we have developed a mouse model overexpressing CRT in endothelial cells. One of the phenotypes of this mouse is the development of obesity and type II diabetes overtime. Therefore, the current research was to examine the hypothesis that endothelial specific overexpression of CRT in mice leads to endothelial dysfunction leading to obesity and diabetes. Methodology: A cell targeted transgenic mouse model overexpressing CRT in endothelial cells [will be referred to as (ECCRT+)] was developed in our lab and used in our study. One of the major phenotype of these mice is the development of visceral obesity and diabetes. To characterize these mice phenotype, 4 weeks old wild-type (wt) and transgenic (ECCRT+) litter mate mice were fed with special diet containing either high fat (60%) or low fat (10%) diet for different time points (8-24 weeks). Body weight and blood glucose was measured bi-weekly. At the end time point glucose tolerance test (GTT) was performed to determine the state of diabetes. Epididymal adipose tissues were collected from the wt and ECCRT+ mice. The tissues were embedded in paraffin, sectioned and stained using histological and immunohistochemical techniques to examine the phenotypic changes (shape and size) of adipocyte in ECCRT+ and wildtype mice. Results: Our results illustrated that these mice suffered from endothelial dysfunction. The GTT assay illustrated that ECCRT+ mice developed diabetes at 16 weeks of age when on regular diet (10% fat diet) and high fat diet expedited the development of diabetes. Fat to body weight ratios, and histological analysis of the fat from these wt and ECCRT+ mice showed significant changes in the fat volume, adipocyte size and adipocyte number suggesting a possible association between endothelial dysfunction and adipogenesis which correlate to the obesity and diabetic developed in these mice. Conclusion: Many studies have focused on how obesity induces endothelial dysfunction. Our study is the first to show an important role of endothelial dysfunction in the process of adipogenesis leading to the development of obesity and diabetes. Our data also highlights the importance of an endoplasmic reticulum chaperone in this process. Acknowledge: This project has been funded by NPRP07-208-3-046.

Background: Lack of Schlafen family member 11 (SLFN11) expression has been recently identified as a dominant genomic determinant of response to DNA damaging agents in numerous cancer types. Thus, strategies aimed at increasing SLFN11 could be used to restore chemosensitivity of refractory cancers. As oncogenic downregulation is often driven by methylation of the promotor region, we explore the demethylation effect of 5-aza-2-deoxycytidine (decitabine), on the SLFN11 gene methylation. Since SLFN11 has been reported as an interferon inducible gene, and interferon is secreted during an active anti-tumor immune response, we investigated the in vitro effect of IFN-g; on SLFN11 expression in breast cancer cell lines. A second broader approach to show cross talk between immune cells and SLFN11 expression is indirect co-culture of breast cancer cells with activated PBMCs and evaluate if this can drive SLFN11 upregulation. Finally, as a definitive and specific way to modulate SLFN11 expression we implemented SLFN11 dCas9 (dead CRISPR associated protein 9) systems to specifically increase or decrease SLFN11 expression. Results: We first confirmed a correlation previously reported between methylation of SLFN11 promoter and its expression across multiple cell lines. We showed in-vitro that decitabine and IFN-g; could increase moderately the expression of SLFN11 in both BT-549 and T47D cell lines, but not in strongly methylated cell lines such as MDA-MB-231. Though, in-vitro, the co-culture of the same cell lines with CD8-CD25 activated PBMC failed to increase SLFN11 expression. On the one hand, the use of a CRISPR-dCas9 UNISAM system could increase SLFN11 expression significantly (up to 5-fold), stably and specifically in BT-549 and T47D cancer cell lines. Though, this system also failed to force a strong expression of SLFN11 in cell lines with robust SLFN11 promoter methylation such as MDA-MB-231. On the other hand, the use of CRISPR-dCas9 KRAB could significantly reduce the expression of SLFN11 in BT-549 and T47D. We then used the modified cell lines to confirm the alteration in chemo sensitivity of those cells to treatment with DNA Damaging Agents (DDAs) such as Cisplatin and Epirubicin or DNA Damage Response (DDRs) drugs like Olaparib. RNAseq was used to elucidate the mechanisms of action affected by the alteration in SLFN11 expression. Conclusion: To our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.

KRAS-induced lung cancer is a very common disease, for which there are currently no effective therapies. Direct targeting of KRAS has failed in clinical trials and intense efforts are underway to identify KRAS targets that play a crucial role in oncogenesis. One promising KRAS-regulated pathway that has so far been overlooked is the microRNA pathway. Even though many microRNAs that regulate expression of KRAS are known, microRNAs regulated by oncogenic KRAS remain largely unknown. Our goal was to identify microRNAs regulated by oncogenic KRAS in lung cells that could contribute to the oncogenic phenotype. Due to a reported positive correlation between microRNA486-5p (miR-486-5p) expression and the presence of KRAS mutations in colon cancer specimens, we decided to investigate in lung cells whether KRAS regulates miR-486-5p. For that purpose we used an immortalized human primary lung epithelial cell line (SALEB) and its isogenic KRAS-transformed counterpart (SAKRAS), as well as lung cancer cell lines with either gain-of-function or loss-of-function of KRAS. We found, in all models tested, a positive correlation between expression of oncogenic KRAS and expression of miR-486-5p. We also found a negative correlation between expression of oncogenic KRAS and expression of miR-486-5p targets, including FoxO1, a tumor suppressor. In order to evaluate how miR-486-5p affects Ras-induced oncogenic properties, we transfected miR-486-5p inhibitor oligonucleotides into KRAS-positive lung cancer cell lines H358 and A549. Inhibition of miR-486-5p expression leads to reduced growth in clonogenic assays and reduced MTT viability. This reduction is not associated with increased cell death, but, as measured by 3H-Thymidine incorporation, it is associated with decreased cell proliferation. Interestingly, transfection of miR-486-5p double-stranded RNA mimic oligonucleotides into KRAS negative lung cancer cell line H1703 or into H358 cells with loss of function of KRAS by RNA interference leads to enhanced proliferation and clonogenicity. Taken together, these results indicate, not only that miR-486-5p is a KRAS target in lung cancer, but also that miR-486-5p acts as an oncomiR contributing to KRAS-induced cell proliferation. Further understanding of miR-486-5p targets could uncover novel pathways for KRAS-induced lung cancer therapy design. Citation Format: Mateus N. Aoki, Amanda C. P. Salviatto, Tatiana C. C. Lobo, Ana Cláudia O. Carreira, Mari C. Sogayar, Daniela S. Basseres. MicroRNA486-5p is a KRas target involved in promoting cell proliferation in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2014-533

Carneiro-Lobo T.C., Dalloul R.S.D., Mesaeli N. Department of Biochemistry, Weill Cornell Medical College in Qatar, Doha, Qatar. Introduction: The incidence of diabetes and obesity has been rising world wide in the past few decades. In the Gulf Cooperation Council (GCC) a rapid increase in the prevalence of diabetes has been reposted in the past few years. In Qatar the incidence of diabetes is 16.7% in the Qatari population that is almost 3 times higher than the incidence of diabetes in UK. Type 2 diabetes mellitus is a growing public health problem and a major cause of cardiovascular disease in the United State. Type 2 diabetes is associated with systemic insulin resistance, which promotes hyperglycemia, and it has been proposed that these metabolic abnormalities account for increased cardiovascular risk. Endothelial dysfunction contributes to the pathogenesis and clinical expression of atherosclerosis and has been linked to type 2 diabetes mellitus and insulin resistance. Transport of insulin across the microvasculature is necessary to reach its target organs and is rate limiting in insulin action. The two possible mechanism of this movement is either through leaky tight junction in vascular endothelial cell layer or via transcytosis through these endothelial cells. Therefore, we hypothesized that overexpression of calreticulin would reduce insulin transport to the target tissue due to onset of endothelial dysfunction. Methods and Results: To test our hypothesis, we used genetic approaches to overexpress calreticulin (CRT). For in vitro experiments CRT was overexpressed using a Lentiviral-CRT-RFP to infect Human Umbilical Vein Endothelial Cells (HUVEC). For ex vivo studies we used endothelial cels isolated from our endothelial specific CRT overexpressing transgenic mice (ECCRT+). We examined the uptake of fluorescently labeled insulin in the endothelial cells. CRT-HUVEC and WT-HUVEC were incubated with 25 μg of FITC-insulin for 30 mins and 60 mins. We found that CRT-overexpressing cells reduce the transcytosis of insulin through the endothelial cells compared to control. The primary endothelial cells were isolated using columns containing PECAM (CD31) coated beads. Cells were then characterized by detection of CD31 (PECAM) and Von Willebrand factor (vWF) expression by immunofluorescence and western blot. After confirming the endothelial identity of our primary cells (ECCRT+) they were incubated with Alexa-647 insulin for 30 min or 60 min. Confocal microscopy was carried out to examine insulin uptake and transcytosis. Our data illustrate a reduction in insulin trancytosis in ECCRT+ endothelial cells as compared with WT cells. To evaluate how Calreticulin affects insulin trancytosis in vivo in whole animal, ECCRT+ mice and WT-mice were used. Alexaflour-647 insulin (1.2 μg/L) was injected via tail vein of ECCRT+ mouse and WT-mouse. To label the surface of endothelial cells, 20 μg/L FITC-isolectin was injected via tail vein following insulin injection. 30 mins after the injection mice were euthanized and tissue (lung, liver, heart and retina) were isolated, fixed and embedded for cryosections. Tissue sections were then mounted on slides and examined by confocal microscopy. We observed a lower concentration of insulin in cell membrane of endothelial cells of vascular wall in lung, liver, heart and retina of ECCRT+ mouse than WT-mice. Finally, we examined the leakiness of the vascular wall in our ECCRT+ mouse model. In these experiemnts we injected 2.5 mg/ml FITC-Dextran in the tail vein for 30 min. After euthaniasia, mice tissue was imaged under a Ziess flourescnet microscope to evaluate leakage of the flourescet signal in the tissue. Fluorescent images from the lung, liver and heart were collected and illustrated an increase in the leakiness of vascular wall of the small vessels in the ECCRT+ mice as compared to the WT mice. Conclusion: Taken together, our results illustrate that increased calreticulin expression in endothelial cells affect endothelial cell function, increase the vascular permeability and reduce the transcytosis of insulin through the endothelial cell of vasculature. The impaired insulin export from the vascular wall to the target tissue could contribute to the onset of insulin resistance and diabetes in the ECCRT+ mice which we have observed in our other studies on these mice. This is the first report to link calreticulin expression level and insulin transport in intact animal. Acknowledgment: This research was made possible by a grant by QNRF, NPRP07-208-3-046.