Electronic cigarettes (e-cigs) are major contributors to inflammatory-mediated responses, which are implicated in a vast array of pathophysiological conditions, including cardiovascular disease (CVD). More recently e-cigs have been recognized as a source of thirdhand exposure (THEC); the process by which expelled toxins settle on materials (i.e., carpets, curtains, clothes etc.) and undergo chemical reactions, rendering them more harmful overtime. Herein, mice were exposed to THEC for four months, and platelet reactivity, systemic mediated effects on platelet function, and cytokine expression profiles were analyzed in both sexes. Our data revealed a hyperactive platelet phenotype as determined by shortened bleeding and occlusion times, enhanced platelet aggregation, and dense granule secretion with no significant difference between males and females. Cytokines, amongst other inflammatory molecules, are well documented mediators by which platelet function is modulated and they also enhance susceptibility to CVD. To this end, and to elucidate the mechanism by which platelet reactivity was augmented, washed platelets that were exposed only to clean air (CA) and resuspended in THEC exposed plasma, displayed significantly increased platelet aggregation, dense granule secretion, and p-selectin expression. Indeed, this data suggests that THEC exposure elicits a systemic effect, enhancing platelet response, and was further validated by a dysregulated cytokine profile using plasma, free of platelets, in a sex-dependent manner. Collectively and for the first time, we highlight that both males and females are at similar risk of THEC-mediated prothrombotic phenotype, which is underlined-at least in part- by an indirect systemic effect of exposure on platelet reactivity that involves changes in the cytokine profile. These findings underscore this form of exposure as a threat to cardiovascular health.

Although cigarette smoking is the most preventable cause of cardiovascular diseases, most researchers have focused on either direct/firsthand or secondhand smoke exposures. Recently though, attention has shifted to an emerging/indirect exposure trend-known as thirdhand smoke (THS)- which was previously “overlooked.” This phenomenon, which was/is thought to be harmless, has been identified as a serious health risk, including in the context of thrombogenesis/platelets. However, whether low dose THS exposure has the capacity to modulate platelets has not been investigated. Two sets of household materials were exposed to 20 cigarettes/day for a week on an alternating basis, with controls exposed to clean air. After the first set of exposed materials is placed in mice cages, exposure of the second set is initiated. The materials were interchanged weekly, for a total exposure duration of 1 month. Mice were then subjected to multiple platelet function assays. THS exposed mice exhibited shortened tail bleeding and occlusion times, indicating a prothrombotic phenotype. Moreover, we also observed that platelets from the exposed mice exhibited an enhanced aggregation response. However, we did not observe any gender differences in our in vivo as well as aggregation experiments; hence, subsequent characterization was carried out on male mice. It was also found that dense granules release, integrin activation, and PS exposure were also potentiated in the exposed platelets compared to the controls. Finally, we observed for the first time that the tobacco-specific nitrosamine and THS toxicant NNK enhanced platelet aggregation and thrombus formation. Collectively, we provide documentation that low dose of THS exposure is detrimental to health by increasing the risk of thrombosis through a hyperactive platelet phenotype that involves the toxicant NNK.

Cigarette smoking is acknowledged as the most preventable risk factor for thrombogenesis-associated cardiovascular disease. Mice prenatally exposed to the thirdhand smoke (THS) form of tobacco exhibited a higher tendency to develop occlusive thrombosis, along with enhancement of several platelet functional responses. Our objective was to investigate whether prenatal (in utero) THS exposure impacts the platelet transcriptome, resulting in enhanced platelet functional responses, thereby underlying THS-associated thrombogenicity. Blood samples obtained from twenty male mice prenatally exposed to THS, along with an equal number of age-matched male mice exposed to clean air (CA) as a control, were divided into pools of five animals and used to prepare leukocyte and red blood cell-depleted platelets. RNA sequencing for mRNA and microRNA (miRNA) was utilized to analyze and compare the platelet expression profiles of the two exposure groups. RNA seq analyses revealed distinct changes in both gene expression and miRNA profiles, with 448 coding genes and 18 miRNAs significantly altered between the two groups. miRNA–mRNA interaction analysis highlighted 14 differentially expressed miRNAs that potentially target 120 of the differentially expressed genes in our data set. Interestingly, altered genes in miRNA–mRNA pairs were functionally enriched into pathways associated with platelet physiology, including platelet activation, signaling and aggregation, and cellular response to chemical stimuli. Our findings establish—for the first time—that prenatal exposure to THS modifies the platelet transcriptome, thereby rendering platelets hypersensitive to stimuli and more prone to thrombogenicity. Additionally, we illuminate the coordinated function of platelet miRNA and mRNA targets in mediating this response.

Introduction It is well documented that cardiovascular disease (CVD) is the leading cause of death in the US and worldwide, with smoking being the most preventable cause. Additionally, most smokers die from thrombotic-based diseases, in which platelets play a major role. To this end, because of the proven harm of smoking, several novel tobacco products such as electronic(e)-waterpipe have been gaining popularity among different sectors of the population, partly due to their “false” safety claims. While many investigators have focused on the negative health effects of traditional cigarettes and e-cigarettes on the cardiovascular system, virtually little or nothing is known about e-waterpipes, which we investigated herein. Methods and Materials To investigate their occlusive CVD effects, we employed a whole-body mouse exposure model of e-waterpipe vape/smoke and exposed C57BL/6J male mice (starting at 7 weeks of age) for 1 month, with the controls exposed to clean air. Exposures took place seven times a week, according to the well-known Beirut protocol, which has been employed in many studies, as it mimics real-life waterpipe exposure scenarios; specifically, 171 puffs of 530 ml volume of the e-liquid at 2.6 s puff duration and 17 s puff interval. Results The e-waterpipe exposed mice had shortened bleeding and occlusion times, when compared to the clean air controls, indicating a prothrombotic phenotype. As for the mechanism underlying this phenotype, we found that e-waterpipe exposed platelets exhibited enhanced agonist-triggered aggregation and dense granule secretion. Also, flow cytometry analysis of surface markers of platelet activation showed that both P-selectin and integrin GPIIb-IIIa activation were enhanced in the e-waterpipe exposed platelets, relative to the controls. Finally, platelet spreading and Akt phosphorylation were also more pronounced in the exposed mice. Conclusion We document that e-waterpipe exposure does exert untoward effects in the context of thrombosis-based CVD, in part, via promoting platelet hyperreactivity.

INTRODUCTION:Thirdhand smoke (THS) is associated with many public health and disease concerns, such as respiratory illness, cancer, lipidemia, and cardiovascular disease (CVD). We have previously shown that a moderate to long-term exposure to THS increases the risk of thrombosis. However, whether short-term exposure to THS would produce any effects remains to be discovered. Therefore, this study investigated the impact of 1-month THS exposure on platelet function, in vivo and in vitro, and on cytokine response, in a sex-dependent manner. AIMS AND METHODS:Secondhand smoke or clean air (CA) exposed upholstery materials for 1 week were kept in cages housed with 5-6 mice, and the procedure was repeated for 4 weeks. These THS-exposed mice were evaluated for thrombogenesis and platelet function assays. In addition, cytokines expression was evaluated from pooled serum. RESULTS:Compared to the CA group, THS exposure significantly shortened the tail bleeding time and carotid artery thrombus formation. Moreover, the female mice appeared more sensitive to THS exposure than males. Furthermore, platelet aggregation, dense granule secretion, and P-selectin activation markers were significantly elevated due to THS exposure. In addition, high-throughput screening showed at least 30 cytokines differentially modulated by THS in females relative to 26 in male mice. CONCLUSIONS:Collectively, these results demonstrate that 1 month of THS exposure represents a high health risk, in part, by triggering a prothrombotic phenotype that appears to be more significant in females, who are at a much higher risk for occlusive CVD. Additionally, changes in cytokine levels mediate some of the THS-induced occlusive effects. IMPLICATIONS:This study revealed that THS exposure for 1 month is detrimental to the cardiovascular health of both sexes; however, females could be more aggressively affected than males. In addition, interleukins and chemokines could be critical factors for initiating prothrombotic activity due to THS exposure.

Introduction The use of e-cigarettes (ECs) has reached unprecedented levels, due to a variety of reasons, including the misconception regarding their safety. Thus, there have been efforts to characterize the effects of EC exposure, including in the context of thirdhand EC (THEC) on a host of disorders, such as cardiovascular disease (CVD). Methods To address this issue, we sought to characterize the effects of THEC on platelet function and thrombus formation, using a novel mouse exposure protocol that resembles real life scenarios. To assess these effects, a host of related in vivo (i.e. tail bleeding time, and ferric chloride injury induced thrombosis model) assays and in vitro platelet specific (e.g. aggregation, and dense granule secretion) investigative assays were conducted. Results Our in vivo characterization demonstrated that THEC exposed mice exhibited a prothrombotic phenotype reflected by their shortened tail bleeding (THEC: 37 ± 15 seconds, versus clean air: 183 ± 56 s) and occlusion times (THEC: 188 ± 39 s, versus clean air: 519 ± 70 s), relative to those exposed to clean air. Importantly, we found no difference in the platelet counts between the THEC and clean air mice. As for the underlying mechanism, separate experiments revealed significantly enhanced platelet aggregation, dense and alpha granule secretion, as well as integrin/GPIIb-IIIa activation and phosphatidylserine exposure in response to thrombin and ADP agonist stimulation. Conclusions Taken together, these results provide evidence that THEC does have the capacity to increase the risk of thrombotic disease, which should increase awareness regarding its underappreciated negative health effects.

The current work was designed to evaluate the anti-inflammatory and anti-arthritic potential of Coagulansin-A (Coag-A) using mouse macrophages and arthritic mice. In the LPS-induced RAW 264.7 cells, the effects of Coag-A on the release of nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines were analyzed. In addition, the mediators involved in the nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways were evaluated by the RT-qPCR and western blotting. Coag-A did not show significant cytotoxicity in the RAW 264.7 cells in the tested concentration range (1–100 µM). Coag-A significantly inhibited the production of NO, ROS, and key pro-inflammatory cytokines. The anti-inflammatory effects of Coag-A might be through inhibiting the NF-κB pathway and activating the Nrf2 pathway. In the arthritic mouse models, behavioral studies and radiological and histological analyses were performed. We found that the i.p. injection of Coag-A dose-dependently (1–10 mg/kg) reduced the Carrageenan-induced acute inflammation in the mice. In Complete Freund’s Reagent-induced arthritic mouse model, Coag-A (10 mg/kg) showed significant anti-inflammatory and anti-arthritic effects in terms of the arthritic index, hematological parameters, and synovium inflammation. After the Coag-A treatment, the bone and tissue damage was ameliorated significantly in the arthritic mice. Moreover, immunohistochemistry of mouse paw tissues revealed a significant reduction in the expression of pro-inflammatory cytokines in the NF-κB pathway, confirming Coag-A’s therapeutic potential and mechanism.

Tobacco smoking is a major public health threat and is associated with cardiovascular disease (CVD)-related morbidity and mortality. Being the single most preventable risk factor for CVD, a trend towards tobacco harm reduction started years ago. Thus, while tobacco usage has declined, that of Electronic Delivery Systems (ENDs) experienced widespread popularity, especially among pregnant women and women of childbearing age. This is alarming as pregnant women view them as safe(r) compared to cigarettes; which we now know not to be evidence-based. Thus, it is paramount to establish the health impact/CV safety of prenatal exposure to ENDs. This was addressed by employing a well-established ENDs inhalation system and performing whole-body exposures of 150 puffs/day on C57/BL6 mice, under prenatal/ in utero experimental settings throughout the gestational period. Our results show that platelets from the offspring of prenatally ENDs-exposed mice are hyperactive with enhanced: aggregation, dense and α granule secretion, activation of the αIIbβ3 integrin, clot retraction and phosphatidylserine expression, when compared to clean air exposed platelets, which suggests a prothrombotic state. Moreover, we observed that ENDs-exposed platelets are resistant to the inhibitory effects of prostacyclin, and that they exhibit increased activation of Akt and ERK. Furthermore, and consistent with these findings, ENDs were also found to shorten the thrombosis occlusion and bleeding times, relative to controls. Interestingly, we also observed alternations in the platelet miRNome, transcriptome and enrichment of platelet activation pathways, as follows: 1) miR-221 is differentially expressed, unlike mirR-146b; 2) Itgβ3, Src, and Rap2b transcripts are upregulated; and 3) oxidative phosphorylation, and platelet activation pathways are enriched. Taken together, our data demonstrate for the first time that prenatal exposure to ENDs modulates platelet reactivity and increase the risk of thrombogenesis, in part via impacting the platelet epigenetics and genetics. Thus, the negative health consequences of ENDs should not be underestimated, and warrant further investigation. These findings should also guide policy development for regulating exposure to ENDs.

It is well known that exposure to smoking during pregnancy has been associated with cardiovascular disease (CVD) in the offspring. It is important to note that direct exposure to smoking is not the only means of tobacco exposure during pregnancy. Indeed, there is a new form, termed thirdhand smoke (THS) that has been gaining attention as of late. THS refers to the residual tobacco smoke contamination that remains after a cigarette is extinguished, and which persists for months. This leads to the accumulation of chemicals over time, which undergo chemical reactions and aging, thereby becoming more toxic. To date, the negative effects of in utero THS on CVD are understudied. Since platelets are a major player in the genesis of CVD, we employed a transcriptomic (mRNA and miRNA), in vitro and in vivo-based approaches to investigate the effects of in utero THS exposure on platelet gene expression and function, as well as the risk of developing thrombosis in the adult offspring. Thus, C57 female mice were exposed to THS or clean air (control) one week before mating and throughout gestation, using a validated exposure protocol. The exposure was stopped at birth, and the offspring males were used for experimentation once they have reached at 8 - 10 weeks of age. RNA-Seq next generation sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of the in uteroTHS exposed mice. Pathway enrichment analysis revealed differential gene-expression changes in pathways associated with metabolism, oxidative stress, and platelet activation. Moreover, using miRNA-seq, we found 35 differentially expressed miRNAs in the in uteroTHS exposed platelets compared with controls. The miRNA-mRNA integrated interactions analysis showed 107 validated interactions including interesting interaction between downregulated micorRNAs (miR-101a-3p, miR-144-3p, miR-16-1-3p and miR-542-5p) and upregulated Cxcl12 protein coding gene, a known platelet activation cytokine. We next sough to confirm that changes in the platelet gene expression in the in uteroTHS exposed mice are accompanied by changes in platelet function. To this end, separate experiments showed that activation of Akt and ERK, key biochemical markers of platelet activation, is enhanced in the in uteroexposed mice, which was accompanied by an enhanced clot retraction response. Consistent with these findings, our in vivo studies showed that in uteroTHS exposed mice have significantly shortened (104 ± 10.21[sec]) survival in the pulmonary thromboembolism model in comparison to the clean air control (179 ± 20.6 [sec]). Collectively, our results show altered platelet gene expression, enhanced platelet function, and increased risk of thrombosis in mice that were subjected to the in utero THS exposure, relative to the control. These findings support the notion that in uteroTHS exposure is detrimental to human health and should increase public awareness and inform policy for managing exposure to this type of smoke.

Smoking is a risk factor for a variety of deleterious conditions, such as cancer, respiratory disease and cardiovascular disease. Thrombosis is an important and common aspect of several cardiovascular disease states, whose risk is known to be increased by both first- and secondhand smoke. More recently, the residual cigarette smoke that persists after someone has smoked (referred to as thirdhand smoke or THS) has been gaining more attention, since it has been shown that it also negatively affects health. Indeed, we have previously shown that 6-month exposure to THS increases the risk of thrombogenesis. However, neither the time-dependence of THS-induced thrombus formation, nor its sex dependence have been investigated. Thus, in the present study, we investigated these issues in the context of a shorter exposure to THS, specifically 3 months, in male and female mice. We show that the platelets from 3-month THS-exposed mice exhibited enhanced activation by agonists. Moreover, we also show that mice of both sexes exposed to THS have decreased tail bleeding as well as decreased thrombus occlusion time. In terms of the role of sex, intersex disparities in thrombus development and hemostasis as well as in platelet aggregation were, interestingly, observed. Together, our findings show that exposing mice to THS for 3 months is sufficient to predispose them to thrombosis; which seems to be driven, at least in part, by an increased activity in platelets, and that it does not manifest equally in both sexes.

Early diagnosis and treatment of patients with aggressive prostate cancer (PCa) remains a clinically unmet need. We aimed to determine the levels of small extracellular vesicle (sEV)-associated microRNAs (miRs); miR-4737, miR-6068, and miR-6076 in a large panel of PCa cells and delineate the biological significance of miR-6068 in promoting PCa cells. sEVs were isolated from the conditioned medium of PCa cells, followed by RNA extraction and quantitative Real-Time PCR analysis. Functional assays were performed, and the protein expression of hypermethylated in cancer 2 (HIC2), as a potential miR-6068 target gene, was evaluated in PCa tissues by immunohistochemistry. sEV-associated miR-6068, miR-4737, and miR-6076 levels displayed large and significant differences compared to normal cells. miR-6068 was explicitly upregulated in sEV of PC-3 and CWR-R1ca cells (P<0.010). Suppression of miR-6068 in CWR-R1ca cells decreased cell proliferation, colony formation, and cell migration. In contrast, upregulation of miR-6068 in RC77T/E cells decreased HIC2 levels and increased cell aggressive phenotypes. The overexpression of HIC2 in PCa tissues was primarily observed in the cytoplasm compared to benign prostatic hyperplasia (BPH) and normal tissues (P<0.0001). This study confirms the differential packaging of miR-4737, miR-6068, and miR-6076 in sEVs of PCa cells. MiR-6068 promotes PCa cells to acquire aggressive phenotypes by inhibiting the HIC2/Sirtuin 1 (SIRT1) axis.

Cigarette smoking is a major risk factor for acute coronary thrombosis. In fact, both active/first hand smoke (FHS) and passive/second hand smoke (SHS) exposure-including that which is in utero- are known to increase the risk of coronary thrombosis. While recently a new risk has been identified and termed third hand smoke (THS)-which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished- it remains to be determined whether in utero THS can also enhance the risk of thrombogenesis, much like FHS and SHS. Therefore, the present studies investigated the impact of in utero THS exposure in the context of platelet biology and related disease states. It was found that THS-exposed mice exhibited an enhanced platelet aggregation, and secretion responses. In utero THS was also found to enhance GPIIb/IIIa activation and phosphatidylserine exposure. Furthermore, and as for its in vivo impact, it was found that in utero THS exposure shortens the tail bleeding time, as well as the occlusion time in a model of thrombosis. Cytokine analysis revealed serum IL-9 downregulation and lung IL-5 upregulation in the THS exposed mice, which indicates impaired tissue repair and increased asthma risk, respectively. Thus, our data demonstrate for the first time that in utero THS exposure increases the risk of thrombosis-based disease states, which is attributed, at least in part, to their hyperactive platelets. Thus, the negative health consequences of THS should not be underestimated, and warrant further investigation. These findings should guide policy development for regulating exposure to this form of tobacco.

The utility of small extracellular vesicles (sEVs)-derived microRNAs (miRs) to segregate prostate cancer (PCa) patients according to tumor aggressiveness and ancestral background has not been fully investigated. Thus, we aimed to determine the diagnostic and prognostic utility of sEV-associated miRs in identifying aggressive PCa in African American (AA) and Caucasian (CA) men. Using a training cohort, miR profiling was performed on sEVs isolated from plasma of PCa patients. Top-ranked sEV-associated miRs were then validated in 150 plasma samples (75 AA and 75 CA) collected from two independent cohorts; NIH (n = 90) and Washington University (n = 60) cohorts. Receiver operating characteristic (ROC) curve, Kaplan–Meier and Cox proportional hazards regression were used to assess these miRs as clinical biomarkers. Among nine top-ranked sEV-associated miRs, miR-6068 and miR-1915-3p were enriched in sEVs collected from PCa patients compared to healthy volunteers. Moreover, miR-6716-5p and miR-3692-3p segregated AA from CA men and low from high Gleason score (GS), respectively. Upregulation of sEV-associated miR-1915-3p, miR-3692-3p and miR-5001-5p was associated with improved survival time, and only miR-1915-3p was associated with longer recurrence-free survival (RFS) as an independent prognostic marker. Taken together, we identified novel sEV-associated miRs that can differentiate PCa patients from normal, AA from CA and high from low GS and predicts RFS.

Abstract Background: The morbidity and mortality rates of prostate cancer (PCa) in African American (AA) are 2-3 times higher than European American (EuA) men. The molecular mechanisms underlying the aggressiveness of PCa have not fully identified. Thus, our aim was to evaluate the diagnostic/prognostic utility of exosomal microRNAs (miRs) to classify PCa patients according their race and aggressive phenotype in AA patients. Their functional role in tumor aggressiveness was also determined. Methods: Exosomes were isolated from the conditioned media of AA and EuA PCa cell lines. The expression of miRs was validated in exosomes, free-circulating plasma, and FFPE tissue specimens of forty AA and EuA patients using quantitative real-time PCR analysis. The sensitivity and specificity of exosomal miRs to classify prostate cancer patients according their race and aggressiveness were assessed using receiver operating characteristic (ROC) curve analysis. To study the functional significance of exosomal miRs, cell proliferation, clonogenic, cell cycle and migration assays were performed in PCa cells transfected with miR-3128. Results: Differential expression of exosomal miR-3613-3p, miR-3218, miR-3679, and miR-3680 was demonstrated in the plasma of AA versus EuA of PCa patients. While exosomal miR-3613 and miR-3679 (p<0.05) were upregulated, free-circulating miRs downregulated (p<0.05) in the plasma of AA versus EuA patients. The accuracy of miR-3679 to discriminate AA from EuA was improved when combined with the other three miRs (AUC jumped from 0.717 to 0.897). Intriguingly, miR-3128 showed a dual role in AA versus EuA cells. Overexpression of miR-3128 increased the cell growth in AA cells while it did the opposite in EuA cells. These data were recapitulated by migration, cell cycle and clonogenic assays. Conclusion: Our findings underline the role of exosomal miRs in health disparity of PCa. The differential expression of miRs in AA men demonstrates their reliability as biomarkers and their potential role in promoting tumor aggressiveness in AA men. Citation Format: Hamdy E.A. Ali, Rofaida Gaballa, Andrew S. Sholl, Mohamed Gaballah, Juan J. Bustamante, Preeti Zanwar, Hamed I. Ali, Zakaria Y. Abd Elmageed. Exosomal microRNAs are associated with prostate cancer aggressiveness in African American patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B045.

HER2 kinase as a well-established target for breast cancer (BC) therapy is associated with aggressive clinical outcomes; thus, herein we present structural optimization for HER2-selective targeting. HER2 profiling of the developed derivatives demonstrated potent and selective inhibitions (IC50: 5.4-12 nM) compared to lapatinib (IC50: 95.5 nM). Favorably, 17d exhibited minimum off-target kinase activation. NCI-5-dose screening revealed broad-spectrum activities (GI50: 1.43-2.09 μM) and 17d had a remarkable selectivity toward BC. Our compounds revealed significant selective and potent antiproliferative activities (∼20-fold) against HER2+ (AU565, BT474) compared to HER2(-) cells. At 0.1 IC50, 15i, 17d, and 25b inhibited pERK1/2 and pAkt by immunoblotting. Furthermore, 17d demonstrated potent in vivo tumor regression against the BT474 xenograft model. Notably, a metastasis case was observed in the vehicle but not in the test mice groups. CD-1 mice metabolic stability assay revealed high stability and low intrinsic clearance of 17d (T1/2 > 145 min and CLint(mic) < 9.6 mL/min/kg).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

Although integrin alpha 2 subunit (ITGA2) mediates cancer progression and metastasis, its transfer by exosomes has not been investigated in prostate cancer (PCa). We aimed to determine the role of exosomal ITGA2 derived from castration-resistant PCa (CRPC) cells in promoting aggressive phenotypes in androgen receptor (AR)-positive cells. Exosomes were co-incubated with recipient cells and tested for different cellular assays. ITGA2 was enriched in exosomes derived from CRPC cells. Co-culture of AR-positive cells with CRPC-derived exosomes increased their proliferation, migration, and invasion by promoting epithelial-mesenchymal transition, which was reversed via ITGA2 knockdown or inhibition of exosomal uptake by methyl-β-cyclodextrin (MβCD). Ectopic expression of ITGA2 reproduced the effect of exosomal ITGA2 in PCa cells. ITGA2 transferred by exosomes exerted its effect within a shorter time compared to that triggered by its endogenous expression. The difference of ITGA2 protein expression in localized tumors and those with lymph node metastatic tissues was indistinguishable. Nevertheless, its abundance was higher in circulating exosomes collected from PCa patients when compared with normal subjects. Our findings indicate the possible role of the exosomal-ITGA2 transfer in altering the phenotype of AR-positive cells towards more aggressive phenotype. Thus, interfering with exosomal cargo transfer may inhibit the development of aggressive phenotype in PCa cells.

Abstract Background: Although microRNA (miR) profiling has been widely used to predict clinical outcomes, differential miR expressions that can segregate prostate cancer (PCa) patients based on their races and tumor aggressiveness have not been fully investigated. We aimed to determine the diagnostic and prognostic abilities of exosomal miRs to identify the aggressive phenotypes of PCa in African American (AA) men. Methods: Exosomes were isolated from blood of twenty AA and European Americans (EuA) PCa patients at low and high Gleason scores and their aged-matched healthy subjects (n=20) as well as AA and EuA normal and PCa cells. miR profiling was performed on PCa exosomes derived from blood and PCa cells. The expression level was correlated with clinical outcomes of PCa patients. The sensitivity and specificity of exosomal miRs were assessed using receiver operating characteristic (ROC) curve. Results: Results from miR profiling showed a number of exosomal miRs that were able to differentiate normal from PCa, low from high Gleason scores and AA from EuA PCa patients. These dysregulated miRs were validated in another cohort of forty PCa patients in addition to a large panel of PCa cell lines. In the validation cohort, miR-5001, miR-3692 and miR-4529 were upregulated in the exosomes derived from blood of AA compared to EuA men. These miRs were correlated with age, T-stage, residual tumor, involvement of lymph nodes, Gleason score, and overall survival of AA patients. The combination of these miRs showed high discriminatory power (AUC=0.91) for segregation of PCa patients according to their clinical outcomes. Conclusion: miR profiling identified a new set of miRs that can differentiate PCa specimens based on their race and Gleason score. The differential expression of these miRs demonstrates their potential role as biomarkers in the context of racial disparity. Further studies are warranted to determine their role in PCa at advanced stages. Citation Format: Rofaida Gaballa, Mohamed Gaballah, Hamdy E.A. Ali, Andrew S. Sholl, Hamed I. Ali, Zakaria Y. Abd Elmageed. Exosomes-associated miR-5001, miR-3692 and miR-4529 are novel biomarkers for aggressive prostate cancer and associated with poor prognosis in African American patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C029.