Qatar Foundation Annual Research Forum Volume 2012 Issue 1

Stromal interaction molecule 1 (STIM1), the ER calcium sensor, couples to Orai1, the calcium channel, and mediates store-operated calcium entry (SOCE). STIM1 localizes to the ER membrane. Following Ca²+ store depletion, STIM1 forms puncta that localize to the cortical ER and binds Orai1 to allow Ca²+ influx. The egg's competency to activate at fertilization is dependent on its ability to generate a fertilization-specific Ca2+ transient. This is achieved through remodeling of its calcium signaling machinery during oocyte maturation. Oocyte maturation is driven by kinase cascade including MOS- mitogen-activated protein kinase (MAPK) - maturation promoting factor (MPF). Coordinately, SOCE inactivates during maturation. This inactivation is due to internalizing of Orai1 and the inability of STIM1 to form puncta. Here we show that MPF phosphorylates STIM1. Through molecular and pharmacological manipulation we are able to differentially activate the kinases along the kinase cascade that drives oocyte maturation. STIM1 phosphorylation in these studies was monitored through a mobility shift on SDS-PAGE. When MPF is activated a mobility shift is observed. However, this is not the case when only Mos or the MAPK cascade were active. Our next step is to determine the role of MPF phosphorylation of STIM1, because previous studies have argued that phosphorylation does not inhibit STIM1 clustering during meiosis. Our data argue that MPF is the primary kinase that phosphorylates STIM1 during meiosis.

Background: The use of the serum prostate-specific antigen (PSA) test for prostate cancer screening has resulted in a diagnostic dilemma: PSA is not prostate cancer specific and could be found in the normal prostate at equal or higher levels than is found in prostate cancer. Prostate cancer gene 3 (PCA3) encodes a prostate-specific mRNA with a median 66-fold up-regulation compared to adjacent benign tissue. In contrast, PSA gene expression is similar in cancerous and benign cells; PSA mRNA levels may therefore be used to normalize for the amount of prostate-specific ribonucleic acid (RNA) in molecular test samples. This report describes the characterization of a prototype quantitative PCA3-based test for whole urine. Methods: First-catch urine specimens were collected after digital rectal examination. The total RNA was isolated with Trizol, amplified, and quantified by use of a real-time PCR method. PSA mRNA concentrations were used to normalize PCA3 signals and confirm the yield of prostate-specific RNA. PCA3 score is calculated by one thousand times of PCA3/PSA mRNA ratio. ROC curve analysis is applied to determine the cutoff value of PCA3 score according to prostate biopsy results. Results: We have collected urine samples of 163 patients from uro-oncology clinic at Hamad Hospital. Due to the mixture of the blood cells and prostate cells in the urine, we failed to obtain RNA with proper quality for 29 samples. The specimen informative rate (fraction of specimens yielding proper RNA for analysis) was 82.2%. From ROC curve with the known diagnostic data, the cut off of PCA3 score is 1232 with a sensitivity of 70% and specificity of 68%. Conclusion: The established PCA3 assay could increase specificity to the current prostate cancer diagnosis. Comparing to the commercial kit, it provides equivalently useful information and it is much cheaper and faster. The challenge is to avoid the interference from the blood cells in the urine.

Background & Objectives: Charcot neuroarthropathy (CN) is a devastating complication of diabetes. It has two-fold higher rate of major amputation compared to those without CN. Unfortunately, to date, there is no pathological marker or diagnostic criterion for it and diagnosis relies on pattern recognition and clinical intuition. Not surprisingly, the diagnosis can be missed up to 95% of the time and the average diagnostic delay has been estimated at almost 7 months. Recent studies reported asymmetric plantar temperature differences secondary to inflammation as a hallmark for the diagnosis and treatment response of Charcot foot syndrome. However, little attention has been given to temperature response to activity. Methods: Fifteen individuals with acute CN and 17 age-matched non-CN participants with type 2 diabetes and peripheral neuropathy were recruited. All participants walked for two predefined paths of 50 and 150 steps. A thermal image was acquired at baseline after acclimatization and immediately after each walking trial. The plantar temperature (PT) response as a function of number of steps was examined using a validated wearable sensor technology. A custom image processing toolbox was designed to characterize the plantar temperature response to activity. Results: During initial activity, the PT was reduced in all participants, but the temperature drop for the non-affected foot was 1.9 times greater than the affected side in CN group (p=0.04). Interestingly, the PT in CN was sharply increased after 50 steps for both feet, while no difference was observed in non-CN between 50 and 200 steps. Conclusions: The observed differential thermal response to walking initiation between Charcot and non-Charcot feet warrants future investigation to provide further insight into the correlation between activity dosing and thermal response. It may also lead to a valuable insight into identifying an "inflammatory trigger" that may ultimately provide an early warning sign or increased sensitivity for subsequent unilateral or bilateral CN development or clinical expression of foot ulcer. These results also support that managing modulating duration of continuous steps and or prolonged standing during daily activity could be helpful for reducing the trauma in patients with CN or patients at risk of diabetic foot ulcer.

Background and Objectives: Oncogene induced senescence is a tumor suppressor mechanism that limits progression of pre-malignant lesions. Identifying the molecular mechanisms by which cells can escape senescence may provide novel cancer therapeutic and preventive targets. Two tumor suppressors, p53 and Rb, are involved in senescence, but their specific roles are still unclear. Our aim is to investigate the role of the Rb and p53 pathways in cellular senescence, to identify mechanisms of cellular escape from senescence, and to evaluate human tumor samples for disruption of the identified pathways. Methods: We are using a transgenic mouse with pineal-cell specific cyclin D1 expression (Irbp-Cyclin D1 mouse). These mice develop premalignant pineal hyperplasia with features of cellular senescence, and tumors progress only when the p53 or the RB pathway is disrupted. Results: We found that p53 was activated early in the senescence response, and led to cell cycle exit. RB activation occurred days later, and coincided with the senescence phenotype that included formation of heterochromatin foci. Disruption of either pathway alone was sufficient for pineal tumor progression. Using genetically engineered mice with regulatable expression of p53, we are now investigating the specific role of p53 in the induction versus maintenance of cell cycle exit. In addition, we are using primary cultures of Irbp-Cyclin D1 pineal cells to examine the effects of turning the p53 and RB pathways "on" and "off" during cellular senescence, to dissect their roles during induction and maintenance of senescence. We are also analyzing premalignant versus malignant tumors from these transgenic mice, to identify recurring genetic lesions that may be important targets for tumor prevention. Finally, we are evaluating human tumor samples for genetic lesions that overlap with the identified pathways. Conclusion: Our preliminary results suggest that the p53 and RB pathways have distinct and complementary effects in the senescence response, where the p53 pathway is important for induction of senescence, while the RB pathway may be responsible for its maintenance. Studies are currently ongoing to verify these observations. These findings will have direct implications on novel approaches to cancer therapy and prevention, by activating cellular senescence and tumor suppression.

Background and Objectives: The prevalence of type 2 diabetes (T2D) in Qatar is the highest in the world. It is estimated that about one quarter of the T2D patients in Qatar are still undiagnosed. We set out to examine determinants of pre-diabetes (PD) or undiagnosed T2D (UT2D). Furthermore, we examined risk factors for glucose regulation in patients with known T2D. Methods: We examined 178 patients with known T2D and 196 controls. Anthropometrics and HbA1c were measured. Socio-demographic (age, gender, ethnicity and educational level) and health information were assessed through questionnaires. Results: Twenty-six (13.3%) and twelve (6.1%) participants in the control group were PD and UT2D, respectively. Control participants from South Asian descent were at a greater risk of being PD or UT2D than Arab controls (Odds Ratio: 5.27 (95% confidence interval: 2.10, 13.22). Being obese was also associated with an increased risk of PD or UT2D (Odds Ratio: 2.94 (95% confidence interval: 1.31, 6.59). Of control participants from South Asian descent, 38% were actually PD or UT2D. In patients with known T2D, insulin use and a longer duration of T2D were both related to higher HbA1c levels (both p<0.0001). No socio-demographic factors were associated with glucose control in T2D. Conclusions: In a nation with a large South Asian immigrant population, we found a strong increased risk of being PD or UT2D in these subjects. Focus should be on the prevention and early identification in South Asian individuals. Larger studies are necessary to further examine the determinants of T2D and glucose regulation in Qatar.

Many tumors are regulated by complex interactions with cellular components of the microenvironment including mesenchymal stem cells (MSC) or endothelial cells (ECs). While the role of receptor-ligand interaction at the cell surface is well documented, direct cell-to-cell contact has not been clearly established. Recently, tunneling nanotubes (TnTs) have been shown to support cell-to-cell transfer of organelles, various plasma membrane components and cytoplasmic molecules in several cell lines. We thus investigated the formation of TnTs between stromal cells and cancer cells supported by them. We demonstrate that TnTs occur between different cancer and stromal cell lines. TnTs formation seemed to be dependent of large membrane adhesion. We show that intercellular transfers of cytoplasmic content can occur by TnTs. However, we demonstrate that the exchange of mitochondria occurs preferentially between endothelial cells and cancer cells and contributes to chemoresistance. Our results point out the role of direct endothelial to cancer cell exchange, which emphasize our hypothesis that this angiogenesis-independent role of the endothelium plays a role in the constitution of the metastatic niche.

Objectives: To investigate the incidence, characteristics and patterns of football injuries at club level in Qatar. Design: Prospective cohort study. Methods: Data were prospectively collected from the first division football league clubs in Qatar, in accordance with the international consensus statement on football injury epidemiology. An injury was defined as any physical complaint sustained during football activity resulting in the inability to participate fully in the next training or match. Individual injuries and exposure of each player were recorded by the medical staff of each team over one season. Results: A total of 217 injuries were recorded, with an injury rate during matches of 14.5/1000 h (95% CI: 11.6-18.0) compared with 4.4/1000 h during training sessions (95% CI: 3.7-5.2). More than one third of all injuries were muscle strains (36.4%). Hamstring strains (54.4% of all muscle strains) exhibited a higher incidence than all other injury types (p < 0.001). The thigh was the most frequent injury location (41.9%, p < 0.001). Re-injuries (15% of total injuries) were mainly comprised of muscle strains associated with a higher severity compared with new injuries. Conclusions: Despite the different environmental, social and cultural setting, our findings are comparable with previous data from a European club football, confirming the previous finding at national team level that there are no regional peculiarities of football injuries in this part of the Asiatic continent. The relatively high overuse injury incidence rate and the high recurrence rate for (severe) thigh muscle strains, especially during games, warrants prevention strategies.

Eight consanguineous Arab families with novel autosomal recessive disorders were mapped with illumina 700K SNP. All relevant positional candidate genes were screened for pathogenic mutations. None were identified. Multiple homozygosity intervals were obtained for each family since no significant LOD scores were possible. Whole exome sequencing was on ABI SOLiD4 for 1 affected individual from each family. Mapping and annotation was on LifeScope software. Data validation was done manually for each linkage interval, by visual inspection of read depth and bead number coverage. On average 30,000 sequence variations were detected in each sample including novel variants, known polymorphisms & exome sequencing errors. For each chromosome with a linkage interval, data was isolated and filtered by exportation to Excel spreadsheets and visual inspection to exclude non-linkage interval data. The number of variants in the linkage intervals for each family was between 400 and 1300. Homozygous sequence variations within the linkage intervals were between 50 and 300 with 15-30 novel variants. Determination if a variant was homozygous or heterozygous, novel or annotated was done manually upon visual inspection of data on Excel spreadsheets. For each novel variant it was manually determined if it were exonic, splice site specific or intronic. For each annotated variation it was manually determined if it is associated to a disease phenotype relevant to the family disease. Minor genotype frequency was investigated for annotated variants if they represent disease states. All novel exonic variants were tested in silico with PolyPhen and Sift Protein Modeling software to access the effect on protein function. All damaging variants (novel or annotated exonic, and splice site) were validated by Sanger sequencing and tested for co-segregation to disease. An identical approach is essential to access pathogenic effects of insertion/deletion variants within each linkage interval. This approach is tedious, involves a tremendous amount of manual work and is prone to oversight errors. Software tools development for automating next-generation sequencing data analysis is essential to eliminate manual work and identify pathogenic mutations among the plethora of existing variants. Such automation is applicable in cases without linkage intervals to limit the number of variants under consideration.

Background and Objectives: There is a high prevalence of obesity and its co-morbidities within the Arab population, especially in Qatar. Furthermore, the younger age of onset of obesity and its preponderance amongst females have seen an increase in type 2 diabetes and cardiovascular disease in this cohort. Adipose tissue dysfunction preceding frank obesity may underlie the increased risk of metabolic syndrome (MeS). Therefore aims of this study were to characterize the relationship between components of MeS and adipokines in a Qatari population. Methods: Non-diabetic subjects were recruited from the general Qatari population and patients awaiting weight reduction surgery (Al-Emadi Hospital). Lipid profiling and liver function were determined by conventional techniques (HMC). Insulin resistance was measured by HOMA. Adipokines were measured by ELISA. Results: In the whole study population (n=56, 13 males/43 females, 30.5+/-7.5 years old, BMI of 37.5+/-11kg/m-2) significant positive correlations between plasma leptin and BMI were confirmed. However, after controlling for BMI, partial correlations showed that leptin was associated negatively with all measures of blood pressure (all p≤0.01), triglycerides, total- and LDL-cholesterol (all p≤0.01), with liver enzymes (SGPT, AST and Billirubin, p=0.009, p≤0.01 and p=0.02 respectively), and positively with HDL cholesterol (p=0.05). Furthermore, leptin, was also negatively correlated with fasting blood glucose and HOMA (p=0.03 and p=0.01 respectively). In order to investigate this further, the population was dichotomized into age-matched normal weight (n=14) and obese (n=42) sub-groups. The obese group had significantly higher SBP (p=0.006), triglycerides (p=0.03), leptin (p≤0.001) and lower HDL (p≤0.001). Obesity was also associated with deteriorating liver functions manifested as elevation in SGPT (p =0.02), ALP (p= 0.01) and AST (p =0.02). Conclusions: Leptin was elevated in obesity. After correcting for BMI, an inverse relationship between leptin and risk factors for MeS was apparent, suggesting a protective role for leptin in this population, perhaps through its vasodilatory capability. However, when dichotomized into lean and obese groups, chronic elevation in leptin appeared to be associated with increased risk of MeS and deterioration of liver function, as previously described. These novel data are currently being confirmed in a larger ethnicity matched cohort.

A global survey of cancer has shown that lung cancer is the most common cause of the new cancer cases and cancer deaths in both men and women worldiwide. In Qatar a recent retrospective study based on a cohort of patient registry of Al Amal Cancer Hospital from 1991-2006, showed that lung cancer and lumph node cancer are the major cancers in men (5.9%, incident rate per 100,000) while in women breast cancer had the highest incidence. To study the mechanisms of lung cancer development we recently developed a transgenic mouse model overexpressing CRT under the control of Tie2-promotor. The main phenotype of these mice is the development of metastatic lung adenocarcinoma similar to human patients. The objectives of the current study was to identify the molecular pathways involved in the metastatic behavior of lung adenocarcinoma. To examine our objectives we used anchorage independent and adherent culture of lung adenocarcinoma cells isolated from mouse and human cell lines. Using microarray and bioinformatic analysis we have identified several pathways and genes that are altered in mouse cells. The microarray analysis demonstrates that 90 genes are up-regulated and 66 genes are down regulated under anchorage-independent condition compared to adherent conditions. The bioinformatic analyses demonstrate that the up-regulated genes are implicated in cellular development, cellular growth and proliferation pathways. These pathways include: VEGF signaling, bladder cancer signaling, VEGF family ligand-receptor interaction and Ephrin-receptor signaling. The bioinformatic analyses also demonstrate that the downregulated genes were implicated in cell cycle regulation and cell-to-cell signaling and interaction. The analysis of these genes by canonical pathawy demonstrate that these genes are involved in the regulation of very relevant pathaway implicated in cancer essentially : Hematopoeisis from multipotent stem cells, TGF-β signaling and tight junction signaling. We are currently examining these pathways in human adenocarcinoma cell line. In conclusion, our data illustrate that although calreticulin is an endoplasmic reticulum calcium binding chaperone, it is involved in the process of tumor development and metastasis. Further work is needed to decipher the molecular mechanism of involvement of calreticulin in tumor development. This research was funded by QNRF grants UREP09-084-3-016 and NPRP4-043-3-016.