p90RSK Activation Research Articles

Targeting the p90RSK/MDM2/p53 Pathway Is Effective in Blocking Tumors with Oncogenic Up-Regulation of the MAPK Pathway Such as Melanoma and Lung Cancer

In most human tumors, the MAPK pathway is constitutively activated. Since p90RSK is downstream of MAPK, it is often hyperactive and capable of phosphorylating oncogenic substrates. We have previously shown that p90RSK phosphorylates MDM2 at S166, promoting p53 degradation in follicular thyroid carcinomas. Thus, the inhibition of p90RSK restores p53 expression, which in turn inhibits cell proliferation and promotes apoptosis. In the present study, we demonstrated that the p90RSK/MDM2/p53 pathway proved to be an excellent target in the therapy of tumors with MAPK hyperactivation. For this purpose, we selected p53wt melanoma, lung and medullary thyroid carcinoma cell lines with high activation of p90RSK. In these cell lines, we demonstrated that the p90RSK/MDM2/p53 pathway is implicated in the regulation of the cell cycle and apoptosis through p53-dependent transcriptional control of p21 and Bcl-2. Furthermore, with an immunohistochemical evaluation of primary melanomas and lung tumors, which exhibit highly activated p90RSK compared to corresponding normal tissue, we demonstrated that MDM2 stabilization was associated with p90RSK phosphorylation. The results indicate that p90RSK is able to control the proliferative rate and induction of apoptosis through the regulation of p53wt levels by stabilizing MDM2 in selected tumors with constitutively activated MAPKs, making p90RSK a new attractive target for anticancer therapy.

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts.

Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

Radiation therapy induces immunosenescence mediated by p90RSK.

Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated β-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.

Abstract 503: Multiparameter Mass Cytometry Reveals The Unique Response Of NaïVe B Cell Cd27 - Subset With The Increase Of T-bet And Cd38 Expression After Radiation Therapy In Thoracic Cancer Patients

Cancer Radiation therapy (RT) induces cardiovascular disease (CVD) even when the heart is shealed or not irradiated, but there is a paucity of available preventive measures for RT-induced CVD. Ionizing radiation (IR) induces senescence, which was originally discovered to suppress tumorigenesis by inducing cell cycle blockade and necrosis, and positioned IR as pro-senescence cancer therapy. IR-induced senescence cells secrete cytokines, growth factors, and reactive oxygen species (ROS), becoming so called senescence associated secretory phenotype (SASP), and we hypothesize that SASP induction in immune cells cause CVD after RT. Although the involvement of DNA damage response (DDR), efferocytosis, and clonal hematopoiesis drivers (CHD) to SASP induction has been suggested, the exact mechanisms through which RT induces SASP in a specific cell type remains unclear. We characterize most of the major human immune cell lineages in a single assay using mass cytometery (CyTOF). We generated a CyTOF panel which includes antibodies against various senescence phenotype, DDR, efferocytosis, and CHD. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 month after RT from 16 thoracic cancer patients. First, we found the frequency of only B cell subtype was decreased after RT. Second, we obtained 138 functional profiling subsets by unsupervised clustering with our antibody set, and found that T-bet expression was increased in the largest B cell subset of naïve B Cell (CD27 - ) Ki67 lo CD38 lo DNMT3a hi after RT, which showed the good correlation with p-p90RSK expression in the samples from pre-RT and post-RT. Lastly, the significant increase of CD38 expression in the subsets of naïve B cell (CD27 - ) and CD8 + T cell (EMRA) was detected. These data suggest the unique response of naïve B cell (CD27-) to RT with the increase of CD38 expression, and T-bet in the subset of B Cell (CD27 - ) Ki67 lo CD38 lo DNMT3a hi , and also the potential role of p90RSK activation in IR-induced T-bet expression. T-bet plays a role in developing the age-associated B cell (ABC), and the increase of CD38 expression promotes aging-related events. Therefore, the induction of T-bet and CD38 in naïve B (CD27 - ) cell after RT supports the novel role of naïve B cell in IR-induced SASP and subsequent CVD.

P90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway.

Previously, we reported that post-translational modifications (PTMs) of MAGI1, including S741 phosphorylation and K931 de-SUMOylation, both of which are regulated by p90RSK activation, lead to endothelial cell (EC) activation. However, roles for p90RSK and MAGI1-PTMs in regulating EC permeability remain unclear despite MAGI1 being a junctional molecule. Here, we show that thrombin (Thb)-induced EC permeability, detected by the electric cell-substrate impedance sensing (ECIS) based system, was decreased by overexpression of dominant negative p90RSK or a MAGI1-S741A phosphorylation mutant, but was accelerated by overexpression of p90RSK, siRNA-mediated knockdown of magi1, or the MAGI1-K931R SUMOylation mutant. MAGI1 depletion also increased the mRNA and protein expression of the large tumor suppressor kinases 1 and 2 (LATS1/2), which inhibited YAP/TAZ activity and increased EC permeability. Because the endothelial barrier is a critical mediator of tumor hypoxia, we also evaluated the role of p90RSK activation in tumor vessel leakiness by using a relatively low dose of the p90RSK specific inhibitor, FMK-MEA. FMK-MEA significantly inhibited tumor vessel leakiness at a dose that does not affect morphology and growth of tumor vessels in vivo. These results provide novel insights into crucial roles for p90RSK-mediated MAGI1 PTMs and the Hippo pathway in EC permeability, as well as p90RSK activation in tumor vessel leakiness.

Garcinia cambogia suppresses adipogenesis in 3T3-L1 cells by inhibiting p90RSK and Stat3 activation during mitotic clonal expansion.

Obesity is associated with an increase in adipose tissue, which is mediated by hyperplasia and hypertrophy. Therefore, inhibiting cell proliferation during mitotic clonal expansion (MCE) is one of the major strategies for preventing obesity. The antagonistic effects of Garcinia cambogia (G. cambogia) on obesity have been studied in animal experimental models. However, the effects of G. cambogia extract on MCE, and the underlying molecular mechanisms, are poorly understood. In this study, 3T3-L1 cells were used to investigate whether G. cambogia extract affected cell proliferation during MCE and to identify target molecules for any anti-adipogenic activity. G. cambogia extract suppressed isobutylmethylxanthine and dexamethasone-and-insulin (MDI)-induced adipogenesis at an early stage by attenuating MCE. In G. cambogia extract-treated preadipocytes, MDI-induced cell proliferation and cell cycle progression were inhibited by G0 /G1 arrest due to an increase in p21 and p27 expression, and inhibition of cyclin-dependent kinase 2, cyclin E1 expression, and retinoblastoma (Rb) phosphorylation. In addition, the MDI-induced phosphorylation and subsequent translocation into the nucleus of p90 ribosomal S6 kinase (p90RSK) and signal transducer and activator of transcription (Stat) 3 were suppressed. Specific inhibitors of p90RSK (FMK) and Stat3 (stattic) regulated cell proliferation and adipogenesis. In conclusion, this study demonstrated that G. cambogia extract inhibited MCE by regulating p90RSK, Stat3, and cell cycle proteins, leading to G0 /G1 arrest. These findings provide new insight into the mechanism by which G. cambogia suppresses adipocyte differentiation and show that p90RSK is critical for adipogenesis as a new molecular target.

Inhibition of p90RSK activation sensitizes triple-negative breast cancer cells to cisplatin by inhibiting proliferation, migration and EMT

Cisplatin (Cis-DDP) is one of the most widely used anti-cancer drugs. It is applicable to many types of cancer, including lung, bladder, and breast cancer. However, its use is now limited because of drug resistance. p90 ribosomal S6 kinase (p90RSK) is one of the downstream effectors in the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathway and high expression of p90RSK is observed in human breast cancer tissues. Therefore, we investigated the role of p90RSK in the Cis-DDP resistance-related signaling pathway and epithelial-mesenchymal transition (EMT) in breast cancer cells. First, we discovered that MDA-MB-231 cells exhibited more Cis-DDP resistance than other breast cancer cells, including MCF-7 and BT549 cells. Cis-DDP increased p90RSK activation, whereas the inactivation of p90RSK using a small interfering RNA (siRNA) or dominant-negative kinase mutant plasmid overexpression significantly reduced Cis-DDP-induced cell proliferation and migration via the inhibition of matrix metallopeptidase (MMP)2 and MMP9 in MDA-MB-231 cells. In addition, p90RSK activation was involved in EMT via the upregulation of mRNA expression, including that of Snail, Twist, ZEB1, N-cadherin, and vimentin. We also investigated NF-κB, the upstream regulator of EMT markers, and discovered that Cis-DDP treatment led to NF-κB translocation in the nucleus as well as its promoter activity. Our results suggest that targeting p90RSK would be a good strategy to increase Cis-DDP sensitivity in triple-negative breast cancers.

Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

BackgroundDisturbed flow (d-flow)–induced senescence and activation of endothelial cells (ECs) have been suggested to have critical roles in promoting atherosclerosis. Telomeric repeat-binding factor 2 (TERF2)-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, regulates the senescence-associated secretory phenotype (SASP), in which EC activation and senescence are engendered simultaneously by p90RSK-induced phosphorylation of TERF2IP S205 and subsequent nuclear export of the TERF2IP-TERF2 complex. In this study, we investigated TERF2IP-dependent gene expression and its role in regulating d-flow-induced SASP. MethodsA principal component analysis and hierarchical clustering were used to identify genes whose expression is regulated by TERF2IP in ECs under d-flow conditions. Senescence was determined by reduced telomere length, increased p53 and p21 expression, and increased apoptosis; EC activation was detected by NF-κB activation and the expression of adhesion molecules. The involvement of TERF2IP S205 phosphorylation in d-flow–induced SASP was assessed by depletion of TERF2IP and mutation of the phosphorylation site. ResultsOur unbiased transcriptome analysis showed that TERF2IP caused alteration in the expression of a distinct set of genes, including rapamycin-insensitive companion of mTOR (RICTOR) and makorin-1 (MKRN1) ubiquitin E3 ligase, under d-flow conditions. In particular, both depletion of TERF2IP and overexpression of the TERF2IP S205A phosphorylation site mutant in ECs increased the d-flow and p90RSK-induced MKRN1 expression and subsequently inhibited apoptosis, telomere shortening, and NF-κB activation in ECs via suppression of p53, p21, and telomerase (TERT) induction. ConclusionsMKRN1 and RICTOR belong to a distinct reciprocal gene set that is both negatively and positively regulated by p90RSK. TERF2IP S205 phosphorylation, a downstream event of p90RSK activation, uniquely inhibits MKRN1 expression and contributes to EC activation and senescence, which are key events for atherogenesis.

Signaling Cascade through DC-ASGPR Induces Transcriptionally Active CREB for IL-10 Induction and Immune Regulation

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However, how DCs display functional plasticity and control host immune responses have not been fully understood. In this study, we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR), a C-type lectin receptor (CLR) expressed on human DCs, resulted in rapid activation of Syk, followed by PLCγ2 and PKCδ engagements. However, different from other Syk-coupled CLRs, including Dectin-1, signaling cascade through DC-ASGPR did not trigger NF-κB activation. Instead, it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB, which consequently bound to IL10 promoter and initiated cytokine expression. In addition, DC-ASGPR ligation activated Akt, which differentially regulated the activities of GSK-3α/β and β-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway, which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses.

Senescent Phenotype Induced by p90RSK-NRF2 Signaling Sensitizes Monocytes and Macrophages to Oxidative Stress in HIV-Positive Individuals

The incidence of cardiovascular disease is higher in HIV-positive (HIV+) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. Peripheral whole blood from cART-treated HIV+ individuals and nontreated HIV-negative individuals was treated with H2O2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14+ monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. Monocytes from HIV+ patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H2O2-induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV+ individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV+ patients treated with cART.

Role of p90RSK in Kidney and Other Diseases.

The 90 kDa ribosomal s6 kinases (RSKs) are a group of serine/threonine kinases consisting of 4 RSK isoforms (RSK1-4), of which RSK1 is also designated as p90RSK. p90RSK plays an important role in the Ras-mitogen-activated protein kinase (MAPK) signalling cascade and is the direct downstream effector of Ras-extracellular signal-regulated kinase (ERK1/2) signalling. ERK1/2 activation directly phosphorylates and activates p90RSK, which, in turn, activates various signalling events through selection of different phosphorylation substrates. Upregulation of p90RSK has been reported in numerous human diseases. p90RSK plays an important role in the regulation of diverse cellular processes. Thus, aberrant activation of p90RSK plays a critical role in the pathogenesis of organ dysfunction and damage. In this review, we focus on the current understanding of p90RSK functions and roles in the development and progression of kidney diseases. Roles of p90RSK, as well as other RSKs, in cardiovascular disorders and cancers are also discussed.

Abstract 223: Monocytes/macrophages Are Sensitized to Secondary Oxidative Insult by Cardiovascular Toxic Drugs via the P90rsk-erk5 S496 Phosphorylation Pathway, Aggrandizing Atherosclerotic Plaque Formation

Increased cardiovascular (CV) events in HIV patients and cancer survivors and the fact that combination antiretroviral therapy (cART) and chemotherapy induce similar unwanted CV effects are becoming evident, but the reason for this is unclear. In this study, we examined the role of p90RSK in the CV toxicity of cART and anti-cancer drugs. The level of p90RSK activation by H2O2 was higher in peripheral monocytes from cART-treated patients than in those from untreated patients. Multiple linear regression analysis involving HIV+/-, Reynolds CV risk score, and basal p90RSK activation revealed HIV+/- and basal p90RSK activation to be strong determinants for plaque formation when adjusted for other independent variables. In murine macrophages most of the cART and several chemotherapy agents activated p90RSK and reduced antioxidant molecule expression and telomere (TL) lengths. p90RSK-mediated ERK5 S496 phosphorylation inhibited transcriptional activity of ERK5 and NRF2, decreasing efferocytosis, antioxidant production and TL lengths. NRF2 activator normalized cART-induced sensitization of p90RSK to H2O2. Lastly, we generated myeloid cells-specific wild type (WT) and dominant negative (DN) p90RSK transgenic mice, and ERK5 S496A knock-in mice, crossed with Ldlr-/- mice or treated with a single injection of adeno-associated virus vector (AAV) encoding a gain-of-function mutant of PCSK9 and fed a high fat diet. Our animal studies showed the crucial role of p90RSK-mediated ERK5 S496 phosphorylation in suppressing effecrocytosis and antioxidant production, and up-regulating senescence and inflammation-related molecules expression, leading to atherogenesis. Our results taken together show that p90RSK is activated by anti-HIV and anti-cancer drugs, and this activation sensitizes the monocyte/macrophages to the secondary oxidative insult by reducing NRF2 transcriptional activity and TL length. The p90RSK activation also reduces macrophage efferocytosis and antioxidant capacity, and increases inflammation and senescence via up-regulating ERK5 S496 phosphorylation, thereby accelerating atherosclerosis. Monocyte/macrophage p90RSK-ERK5 S496 phosphorylation could be a good target to prevent drugs-induced CV diseases.

Abstract 235: Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via P90rsk-mediated Erk5 S496 Phosphorylation

Background: Adverse cardiovascular events are a leading nonmalignant cause of morbidity and mortality among cancer survivors who have been exposed to ionizing radiation (IR), but the exact mechanism of the cardiovascular complications induced by IR remains unclear. In this study we investigated the potential role of the p90RSK-ERK5 module in regulating IR-induced endothelial cell inflammation and apoptosis. Methods and Results: Whole body radiation of mice with 2 Gy γ-ray significantly increased endothelial VCAM-1 expression; especially in the disturbed flow area in vivo . In vitro studies showed that IR increased p90RSK activation as well as subsequent ERK5 S496 phosphorylation in cultured human endothelial cells (ECs). A specific p90RSK inhibitor, FMK-MEA, significantly inhibited both p90RSK activation and ERK5 S496 phosphorylation, but it had no effect on IR-induced ERK5 TEY motif phosphorylation, suggesting that p90RSK regulates ERK5 transcriptional activity, but not its kinase activity. In fact, we found that IR-induced NF-kB activation and VCAM-1 expression in ECs were significantly inhibited by the over-expression of S496 phosphorylation site mutant of ERK5 (ERK5 S496A) compared to overexpression of wild type ERK5. Furthermore, when ECs were exposed to IR, the number of annexin V positive cells increased, and overexpression of ERK5 S496A, but not wild type ERK5, significantly inhibited this increase. Conclusions: Our results demonstrate that IR augmented disturbed flow-induced VCAM-1 expression in vivo. Endothelial p90RSK was robustly activated by IR and subsequently up-regulated ERK5 S496 phosphorylation, inflammation, and apoptosis in ECs. The EC p90RSK-ERK5 signaling axis can be a good target to prevent cardiovascular events after radiation therapy in cancer patients.

Abstract 234: Nuclear Export of Telomeric Repeat Binding Factor 2 (terf2)-interacting Protein (terf2ip) Expression, One of the Sherterin Complex Molecule, is Crucial for Disturbed Flow-induced Endothelial Inflammation and Senescence via Upregulating Terf2ip S205 Phosphorylation

Atherosclerosis is a multifactorial disease mainly caused by endothelial cell (EC) dysfunction resulting from EC senescence. Both EC inflammation and senescence are increased by disturbed flow (d-flow) but the exact mechanism is still unknown. The potential role of TERF2IP in regulating inflammation has been reported, but it has been controversial, especially in ECs. We found that d-flow-induced NF-kB activation was significantly inhibited by the depletion of TERF2IP, but TNF-induced NF-kB activation under laminar flow (l-flow) was not inhibited by TERF2IP depletion. TERF2IP S205 phosphorylation was increased by d-flow, but not by s-flow. This TERF2IP S205 phosphorylation was crucial for d-flow-induced TERF2IP nuclear export, which also caused nuclear export of its binding partner TERF2 in a piggy-back manner. Both the depletion of TERF2IP (TERF2IP siRNA) and overexpression of TERF2IP S205A mutant inhibited d-flow-induced TERF2 nuclear export as well as subsequent EC senescence. In addition, we found the key role of p90RSK activation in regulating EC inflammation and senescence via TERF2IP S205 phosphorylation. To determine the role of TERF2IP and TERF2 cellular localization in vivo, we performed in vivo en face immunostaining of mouse aortic arch in EC specific TERF2IP knock out (EC-TERF2IP-KO) and control mice using anti-TERF2IP or TEFR2 and anti-VE-cadherin antibodies. In control mice TERF2IP expression was very low in the l-flow area. In contrast we found a significant increase of TERF2IP expression, especially in the extra-nuclear region of ECs in the d-flow area. More nuclear localization of TERF2 was observed in EC-TERF2IP-KO mice than those in wild type, which supports our finding in vitro. Significant decreases of VCAM-1 expression and apoptosis were found in the d-flow area in EC-TERF2IP-KO mice compared to control mice. Lastly, we found a significant decrease in d-flow-induced atherosclerotic plaque formation after partial carotid ligation in EC-TERF2IP-KO mice crossed with Ldlr-/- mice compared to control Ldlr-/- mice. These data suggest the key role of nuclear export of TERF2IP in up-regulating EC inflammation and senescence via p90RSK-mediated TERF2IP S205 phosphorylation.

Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via p90RSK-Mediated ERK5 S496 Phosphorylation.

Adverse cardiovascular events are a leading nonmalignant cause of morbidity and mortality among cancer survivors who have been exposed to ionizing radiation (IR), but the exact mechanism of the cardiovascular complications induced by IR remains unclear. In this study we investigated the potential role of the p90RSK-ERK5 module in regulating IR-induced endothelial cell inflammation and apoptosis.Whole body radiation of mice with 2 Gy γ-ray significantly increased endothelial VCAM-1 expression; especially in the disturbed flow area in vivo. In vitro studies showed that IR increased p90RSK activation as well as subsequent ERK5 S496 phosphorylation in cultured human endothelial cells (ECs). A specific p90RSK inhibitor, FMK-MEA, significantly inhibited both p90RSK activation and ERK5 S496 phosphorylation, but it had no effect on IR-induced ERK5 TEY motif phosphorylation, suggesting that p90RSK regulates ERK5 transcriptional activity, but not its kinase activity. In fact, we found that IR-induced NF-kB activation and VCAM-1 expression in ECs were significantly inhibited by the over-expression of S496 phosphorylation site mutant of ERK5 (ERK5 S496A) compared to overexpression of wild type ERK5. Furthermore, when ECs were exposed to IR, the number of annexin V positive cells increased, and overexpression of ERK5 S496A, but not wild type ERK5, significantly inhibited this increase.Our results demonstrate that IR augmented disturbed flow-induced VCAM-1 expression in vivo. Endothelial p90RSK was robustly activated by IR and subsequently up-regulated ERK5 S496 phosphorylation, inflammation, and apoptosis in ECs. The EC p90RSK-ERK5 signaling axis can be a good target to prevent cardiovascular events after radiation therapy in cancer patients.

Abstract 21362: The Combination Antiretroviral Therapy (cart) Inhibits Efferocytosis-Related Gene Expression via Activating P90rsk and Accelerates Atherosclerosis

Background: Increased incidence of cardiovascular disease (CVD) in HIV patients has been established, but the consequence of anti-HIV therapies in CVD is unclear. The activation of p90RSK in endothelial cells is a critical event in atherosclerosis, but its role in macrophage is unknown. Hypothesis: macrophage p90RSK plays a role in anti-HIV drug-induced CVD and atherosclerosis. Methods and Results: In macrophages, efferocytosis-related signaling was upregulated and inflammatory gene expression was down-regulated in a p90RSK activity-dependent manner. Using Lysm-Cre dominant negative (DN) p90RSK transgenic mice in LDLR -/- background fed a high-cholesterol diet, we found that p90RSK activation increases plaque formation, inducing pro-atherogenic phenotypes and enhancing M1 polarization. Every cART regimen we tested, except backbone regimen, activated macrophage p90RSK, reduced efferocytosis, and increased M1-type related gene expression. In human, peripheral monocytes were isolated from HIV infected individuals on stable cART for at least 1 year and with viral load ≤50 copies/mL and also from HIV negative and non-cART treated controls matched for age, gender, environment and Reynolds CVD risk score. We found significantly increased p90RSK activation in the cART-treated group, and additional treatment of low dose of H 2 O 2 (200 nM) accelerated p90RSK activation more in the macrophage isolated from cART-treated patients than those in control patients in vitro . Conclusions: Our results demonstrate that p90RSK is robustly activated by anti-HIV drug treatments known to be a high CVD risk. This activation culminates in reduced activity of macrophage efferocytosis, thereby accelerating atherosclerotic plaque formation. Monocyte/macrophage p90RSK can be a good target and an excellent biological marker to prevent and predict drugs-induced CVD.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity.

The Na+/H+ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-β (PKC-β) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 μM glutamate or 20 mM NH4Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-β, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH4Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-β, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-β/ERK1/2/p90RSK pathway in neuronal cells.

Abstract 417: Trf2-Interacting Protein (Terf2ip) S205 Phosphorylation Mediates Atherosclerosis via Orchestrating Endothelial Senescence and Inflammation

Telomeric repeat-binding factor 2 (TRF2)-interacting protein (TERF2IP) S205 phosphorylation mediates atherosclerosis (AS) via orchestrating endothelial cell (EC) senescence (Sen) and inflammation. Nhat-Tu Le, Kyung-Sun Heo, Kyung Ae Ko, Yunting Tao,Tamlyn Thomas, Raymundo A Quintana Quezada, Keigi Fujiwara, and Jun-ichi Abe Department of Cardiology, UT MD Anderson Cancer Center Objective: Shortened telomere (TL) provokes EC Sen, which is associated with focalized EC inflammation and plaque formation in arteries exposed by disturbed blood flow (d-flow). Although d-flow is known to increase both EC inflammation and Sen, the exact mechanism by which d-flow induces these two different EC pathologies and subsequent AS is poorly understood. Methods & Results: TERF2IP, as a member of the sheltrin complex, is not only crucial for DNA double strand break repair at the TL, but also for activation of cytosolic IkB kinase (IKK) and subsequent NF-kB activity. TERF2IP and TRF2 form a stable heterodimer to protect the duplex region of TLs, while activating NF-kB, especially under the d-flow condition. We have discovered that inhibition of p90RSK reduces d-flow-induced EC Sen and inflammation via down-regulating TERF2IP S205 phosphorylation. The nuclear TERF2IP-TRF2 complex protects TLs by repairing DNA damages while the cytosolic complex plays a role in NF-kB activation. When we mutated the TERF2IP phosphorylation site (S205A) in ECs, the nuclear export of the TERF2IP-TRF2 complex was abolished, which inhibited d-flow-induced NF-kB activation and Sen. Both the depletion of TERF2IP by siRNA and S205A mutation inhibited d-flow-induced TERF2 nuclear export, and residual TRF2 at the TL inhibited d-flow-induced TL dysfunction. The mutation of TERF2IP S205A inhibited cytosolic IKK-NF-kB activation by eliminating the cytosolic TERF2IP-TERF2 complex. The inhibition of d-flow-induced inflammatory adhesion molecule expression and consequent AS were also confirmed in EC-specific TERF2IP knock out mice. Conclusion: These results suggest that S205 phosphorylation of TERF2IP induced by p90RSK activation is required for d-flow-induced TL shortening and NF-kB activation via regulating nuclear export of the TERF2IP-TERF2 complex.

Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch.

BackgroundMyocardial stretch increases force biphasically: the Frank‐Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]‐EGFR).Methods and ResultsSilencing capability of p‐shEGFR was assessed in EGFR‐GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA‐EGFR–injected hearts showed ≈60% reduction of EGFR protein expression compared with shRNA‐SCR–injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA‐SCR group, while it was blunted in shRNA‐EGFR–expressing muscles. Angiotensin II (Ang II)‐dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate‐free medium, demonstrating an increase in shRNA‐SCR–injected myocardium, an effect not observed in the silenced group. Ang II‐ or EGF‐triggered reactive oxygen species production was significantly reduced in shRNA‐EGFR–injected hearts compared with that in the shRNA‐SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox‐sensitive pathway, leading to p90RSK activation in shRNA‐SCR‐injected myocardium, an effect that was absent in the shRNA‐EGFR group.ConclusionsOur results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox‐sensitive kinase activation pathway, leading to SFR development.

Abstract 4662: Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis

Abstract Therapeutic strategies to target EGFR tyrosine kinase inhibitor (TKI) resistance mediated by mechanisms other than T790M are a major clinical challenge. Studies have implicated IL-6 as a mediator of EGFR TKI resistance, and IL-6 is known to be regulated by adrenergic receptors (AR) in some cancers. We investigated whether adrenergic pathways can promote T790M-independent EGFR TKI resistance in preclinical models and in clinical studies. We found that β2-AR was highly expressed in our panel of 119 cell lines and in NSCLC clinical specimens. Activation of β-ARs by stress hormones such as norepinephrine (NE) induced a dramatic rise in IL-6, and this occurred through β2-ARs. β-AR inhibitors (i.e. propranolol), but not α-AR inhibitors, blocked IL-6 induction. Analysis of downstream signaling pathways revealed that β2-ARs induced IL-6 expression through activation of adenylyl cyclase, p90RSK and CREB. To identify novel signaling pathways modulated by ARs, we stimulated NSCLC cell lines with NE and analyzed protein lysates by RPPA to detect expression and activation of >100 proteins. β-AR signaling inactivated the tumor suppressor LKB1 through phosphorylation of S428 and subsequently increased mTOR activity. LKB1 inactivation was critical for IL-6 induction. This finding is important as LKB1 loss is known to be a driver of NSCLC resistance and metastasis. Moreover, we found that β2-AR activation promoted EGFR TKI resistance in cell lines and in mouse models of EGFR mutant NSCLC. The effect of β-AR on EGFR TKI resistance was blocked by the addition of the beta blocker propranolol or IL-6 antibodies, in vitro and in vivo. Consistent with our preclinical studies, in the phase III ZEST clinical study testing erlotinib vs vandetanib, we found that high plasma levels of IL-6 was associated with a worse PFS and OS in the erlotinib arm. In addition, we found that circulating levels of IL-6 were significantly lower in NSCLC patients incidentally receiving beta blockers in the BATTLE trial. Finally, we analyzed the influence of incidental beta blocker use in the LUX-Lung3 study testing afatinib vs chemotherapy in EGFR mutant NSCLC patients. In patients not receiving beta blockers, the median PFS was 11.1 and 6.9 months for afatinib and chemotherapy, respectively, and afatinib improved PFS with a hazard ratio (HR) of 0.60. In patients receiving beta blockers, the median PFS was 13.6 and 2.5 for afatinib and chemotherapy, respectively, and afatinib improved PFS with a HR of 0.25. In conclusion, our preclinical and clinical data provide evidence that β2-AR activation can upregulate IL-6 in EGFR mutant+ NSCLC, modulate the LKB1/AMPK/mTOR axis, and promote EGFR TKI resistance. Moreover, EGFR mutant+ patients using beta blockers had greater relative PFS benefit from afatinib vs chemotherapy compared with those not using beta blockers, supporting future clinical testing of EGFR TKIs in combination with beta blockers. Citation Format: Monique B. Nilsson, Huiying Sun, Lixia Diao, Pan Tong, Youhong Fan, Hai Tran, Diane Liu, Guillermo Armaiz Pena, Jing Wang, Phil Rowe, Alan Webster, Jack Lee, Daniel Gomez, Waun Ki Hong, Ignacio Wistuba, Anil Sood, John Heymach. Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4662.