Serum/glucocorticoid Regulated Kinase 1 Research Articles

Estradiol Suppresses TLR4-triggered Apoptosis of Decidual Stromal Cells and Drives an Anti-inflammatory TH2 Shift by Activating SGK1.

A pro-inflammatory cytokine profile at the feto-maternal interface may predispose immune maladaptation notably in early miscarriages. We investigated the involvement of estradiol (E2)-activated serum-glucocorticoid regulated kinase 1 (SGK1) in preserving the tolerogenic and pro-survival intrauterine microenvironment beneficial to gestation maintenance. Decidual SGK1 was down-regulated in early miscarriage, consistent with the lower serum E2 concentration seen in pregnancy loss. Lipopolysaccharide (LPS)/Toll-like receptors 4 (TLR4) signaling induced apoptosis and the pro-inflammatory T helper type (TH) 1 response of decidual stromal cells (DSCs) were associated with miscarriage. SGK1 activation was suppressed by LPS/TLR4 signaling and would be rescued by E2 administration via the PI3K signaling pathway in DSCs. SGK1 activation attenuated TLR4-mediated cell apoptosis, while promoting cell viability of DSCs by up-regulating the pro-survival genes BCL2 and XIAP, and enhancing the phosphorylation of FOXO1. Furthermore, E2-induced SGK1 activation reduced the secretion of pro-inflammatory TH1 cytokines, and promoted the generation of TH2 cytokines and elevated IRF4 mRNA and protein levels in LPS-incubated DSCs. Pharmacologic inhibition of SGK1 or suppression by small interfering (si) RNA increased the phosphorylation and nuclear translocation of NF-κB to reverse the pro-TH2 and anti-inflammatory effects of E2 pretreatment, leading to compromised pregnancy. These findings suggest that the E2-mediated SGK1 activation suppressed LPS-mediated apoptosis and promoted the anti-inflammatory TH2 responses in DSCs, ultimately contributing to a successful pregnancy.

Involvement of serum glucocorticoid-regulated kinase 1 in reproductive success.

Reproductive processes, in particular events that concern pregnancy, are fine-tuned to produce offspring. Reproductive success is of prime importance for the survival of every species. The highly conserved and ubiquitously expressed serum glucocorticoid-regulated kinase 1 (SGK1) was first implicated in infertility as a regulator of a Na+ channel. In this review, we emphasize the prominent role of SGK1 during early pregnancy: 1) balancing uterine luminal fluid secretion and reabsorption to aid blastocyst adhesion and to import nutrients and energy; 2) transducing signals from the blastocyst to the receptive endometrium; 3) inducing multiple genes that are involved in uterine receptivity and trophoblast invasion; 4) regulating cell differentiation and antioxidant defenses at the fetomaternal interface; and 5) contributing to the proliferation and survival of decidual stromal cells. Accordingly, SGK1 coordinates many cellular processes that are crucial to reproductive activities. Aberrant expression or function of SGK1 results in implantation failure and early pregnancy loss. Further investigation of the molecular mechanisms of the function of SGK1 might provide novel diagnostic tools and interventions for reproductive complications.-Lou, Y., Hu, M., Mao, L., Zheng, Y., Jin, F. Involvement of serum glucocorticoid-regulated kinase 1 in reproductive success.

Potassium supplementation inhibits IL-17A production induced by salt loading in human T lymphocytes via p38/MAPK-SGK1 pathway

High salt intake contributes to the development of autoimmune/inflammatory diseases, while potassium supplementation antagonizes the effects. Interleukin (IL)-17A are tightly related with autoimmune/inflammatory diseases. Thus, we explored the effects and underlying molecular mechanism of high salt and potassium supplementation on IL-17A production in T lymphocytes. Forty-nine healthy participants received a low-salt, high-salt, followed by a high-salt diet plus potassium supplement for 7 days, respectively. Human T lymphocyte Jurkat cells were treated with different concentrations of NaCl and KCl. In the participants, IL-17A levels in plasma and in peripheral blood mononuclear cells (PBMC) were significantly increased after a high-salt diet, which was dramatically reversed when potassium was supplemented. In Jurkat cells, the addition of 40 mM NaCl markedly enhanced IL-17A production and the expression of phosphorylated p38/mitogen-activated protein kinase (MAPK) and its downstream target, serum/glucocorticoid-regulated kinase (SGK)1, whereas combined treatment with additional 2 mM KCl significantly decreased them. Respective inhibition of p38/MAPK and SGK1 suppressed IL-17A expression induced by NaCl, and KCl inhibited IL-17A production induced by specific activator of p38/MAPK. We conclude potassium supplementation has a blocking effect on IL-17A production in T lymphocytes induced by salt loading. This protective effect is mediated through the direct suppression of p38/MAPK-SGK1 pathway.

Association between chronic stress-induced structural abnormalities in Ranvier nodes and reduced oligodendrocyte activity in major depression.

Repeated stressful events are associated with the onset of major depressive disorder (MDD). We previously showed oligodendrocyte (OL)-specific activation of the serum/glucocorticoid-regulated kinase (SGK)1 cascade, increased expression of axon-myelin adhesion molecules, and elaboration of the oligodendrocytic arbor in the corpus callosum of chronically stressed mice. In the current study, we demonstrate that the nodes and paranodes of Ranvier in the corpus callosum were narrower in these mice. Chronic stress also led to diffuse redistribution of Caspr and Kv 1.1 and decreased the activity in white matter, suggesting a link between morphological changes in OLs and inhibition of axonal activity. OL primary cultures subjected to chronic stress resulted in SGK1 activation and translocation to the nucleus, where it inhibited the transcription of metabotropic glutamate receptors (mGluRs). Furthermore, the cAMP level and membrane potential of OLs were reduced by chronic stress exposure. We showed by diffusion tensor imaging that the corpus callosum of patients with MDD exhibited reduced fractional anisotropy, reflecting compromised white matter integrity possibly caused by axonal damage. Our findings suggest that chronic stress disrupts the organization of the nodes of Ranvier by suppressing mGluR activation in OLs, and that specific white matter abnormalities are closely associated with MDD onset.

Serum Glucocorticoid-Regulated Kinase 1 Blocks CKD-Induced Muscle Wasting Via Inactivation of FoxO3a and Smad2/3.

Muscle proteolysis in CKD is stimulated when the ubiquitin-proteasome system is activated. Serum glucocorticoid-regulated kinase 1 (SGK-1) is involved in skeletal muscle homeostasis, but the role of this protein in CKD-induced muscle wasting is unknown. We found that, compared with muscles from healthy controls, muscles from patients and mice with CKD express low levels of SGK-1. In mice, SGK-1-knockout (SGK-1-KO) induced muscle loss that correlated with increased expression of ubiquitin E3 ligases known to facilitate protein degradation by the ubiquitin-proteasome, and CKD substantially aggravated this response. SGK-1-KO also altered the phosphorylation levels of transcription factors FoxO3a and Smad2/3. In C2C12 muscle cells, expression of dominant negative FoxO3a or knockdown of Smad2/3 suppressed the upregulation of E3 ligases induced by loss of SGK-1. Additionally, SGK-1 overexpression increased the level of phosphorylated N-myc downstream-regulated gene 1 protein, which directly interacted with and suppressed the phosphorylation of Smad2/3. Overexpression of SGK-1 in wild-type mice with CKD had similar effects on the phosphorylation of FoxO3a and Smad2/3 and prevented CKD-induced muscle atrophy. Finally, mechanical stretch of C2C12 muscle cells or treadmill running of wild-type mice with CKD stimulated SGK-1 production, and treadmill running inhibited proteolysis in muscle. These protective responses were absent in SGK-1-KO mice. Thus, SGK-1 could be a mechanical sensor that mediates exercise-induced improvement in muscle wasting stimulated by CKD.

Abstract A2-13: Targeting genomic instability to identify molecular drivers of poor prognosis in cancer.

Abstract Background: Cancer is a disease of the genome whereby mutational events that confer a survival advantage to cells are selectively retained. Consequently, genome instability and evasion of cell death are fundamental hallmarks of cancer. Certain cancers, including epithelial ovarian cancers (EOC), pancreatic ductal adenocarcinomas (PDAC) and a subset of sarcomas with complex karyotypes are characterized as being very genomically unstable suggesting that such tumors are driven by defects in DNA damage recognition and repair. Additionally, these tumors are also characterized by a high frequency of p53 mutation, extensive intra-tumoral heterogeneity and resistance, acquired or intrinsic, to DNA damaging chemotherapeutic agents: these tumor types consequently carry a poor prognosis. This highlights the need for detailed molecular characterization to identify new therapeutic targets and stratification biomarkers. Aims: The aim of the study is to use a bioinformatic approach to identify commonly amplified genes, that function in DNA damage response and apoptotic processes, across the three tumor types, and which confer a poorer prognosis in these patients. Methods: For target identification, datasets for EOC and PDAC (obtained from TCGA) and sarcoma (obtained from GEO, NCBI) were analyzed for common copy number aberrations (CNA) in 734 genes relating to DNA damage response and apoptosis, and confer a poor prognosis in progression-free survival (PFS) data in EOC patients. Genes with prognostic significance (p <0.05) using the log rank test were picked from the top 100 most frequently amplified loci. Next, the targets were functionally validated by siRNA-mediated knockdown, overexpression and/or pharmacological inhibition using apoptosis, proliferation and migration assays. Results: Four genes, SGK3, c19orf40, MRPS12 and ZBTB32 were highlighted as being commonly amplified across all three tumor types, of which only SGK3 and c19orf40 were statistically significant in circular binary segmentation CNA calling when examining PFS data in EOC patients. SGK3, a member of the serum/glucocorticoid regulated kinase (SGK) family appeared interesting as it has similar functions and substrates to the AKT kinase family, which we have previously shown to have key roles in tumor cell survival in response to therapy. Intra-patient paired platinum sensitive (PEA1) and resistant (PEA2) and SKOV3 ovarian cancer cell lines, and Aspc1 and Panc-1 pancreatic cancer cell lines were used for validation studies. siRNA-mediated knockdown of SGK3 did not alter induction of caspase 3/7 activity in response to chemotherapy, relative to control treatments in all cell lines. To account for any compensatory effects by the other SGK members in the presence of SGK3 knockdown, all three SGK members (SGK1-3) were knocked down by siRNA and this also did not increase induction of caspase 3/7 activity. Wound healing migration assays however, revealed that over-expression of SGK3 increases cell motility suggesting SGK3's role in prognosis is via migration/tumor spread rather than response to therapy. The remaining targets identified in the bioinfomatic analysis are currently undergoing validation and preliminary results will be summarized. Conclusion: To conclude, bioinformatic analysis highlighted four genes related to DNA damage response/apoptosis that were commonly amplified across tumors characterized by genomic instability and resistance to chemotherapy. One of these genes, SGK3, appears to have a role in poor prognosis via migration. Citation Format: Karen A. Menezes, Paula Cunnea, Phillip Lawton, Ed Curry, Hani Gabra, Harpreet Wasan, Surinder K. Sharma, Euan A. Stronach. Targeting genomic instability to identify molecular drivers of poor prognosis in cancer.. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-13.

A high-throughput kinome screen reveals serum/glucocorticoid-regulated kinase 1 as a therapeutic target for NF2-deficient meningiomas.

Meningiomas are the most common primary intracranial adult tumor. All Neurofibromatosis 2 (NF2)-associated meningiomas and ~60% of sporadic meningiomas show loss of NF2 tumor suppressor protein. There are no effective medical therapies for progressive and recurrent meningiomas. Our previous work demonstrated aberrant activation of mTORC1 signaling that led to ongoing clinical trials with rapamycin analogs for NF2 and sporadic meningioma patients. Here we performed a high-throughput kinome screen to identify kinases responsible for mTORC1 pathway activation in NF2-deficient meningioma cells. Among the emerging top candidates were the mTORC2-specific target serum/glucocorticoid-regulated kinase 1 (SGK1) and p21-activated kinase 1 (PAK1). In NF2-deficient meningioma cells, inhibition of SGK1 rescues mTORC1 activation, and SGK1 activation is sensitive to dual mTORC1/2 inhibitor AZD2014, but not to rapamycin. PAK1 inhibition also leads to attenuated mTORC1 but not mTORC2 signaling, suggesting that mTORC2/SGK1 and Rac1/PAK1 pathways are independently responsible for mTORC1 activation in NF2-deficient meningiomas. Using CRISPR-Cas9 genome editing, we generated isogenic human arachnoidal cell lines (ACs), the origin cell type for meningiomas, expressing or lacking NF2. NF2-null CRISPR ACs recapitulate the signaling of NF2-deficient meningioma cells. Interestingly, we observe increased SGK1 transcription and protein expression in NF2-CRISPR ACs and in primary NF2-negative meningioma lines. Moreover, we demonstrate that the dual mTORC1/mTORC2 inhibitor, AZD2014 is superior to rapamycin and PAK inhibitor FRAX597 in blocking proliferation of meningioma cells. Importantly, AZD2014 is currently in use in several clinical trials of cancer. Therefore, we believe that AZD2014 may provide therapeutic advantage over rapalogs for recurrent and progressive meningiomas.

PP.42.01

Objective: Serum/Glucocorticoid Regulated Kinase 1 (SGK1) plays a significant role in the regulation of renal Na+ reabsorption and K+ secretion as well as blood pressure (BP). The aim of this study was to investigate whether common variants in SGK1 gene are involved in BP responses to dietary sodium intervention or potassium supplementation. Design and method: 344 subjects from 126 families were recruited from rural areas of Northern China. After a 3-day baseline observation, they were sequentially maintained on a 7-day low-sodium diet (3 g of NaCl or 51.3mmol of sodium per day), a 7-day high-sodium diet (18 g of NaCl or 307.8mmol of sodium per day) and a 7-day high-sodium plus potassium supplementation intervention (4.5 g of KCl or 60mmol/d potassium per day). Six single nucleotide polymorphisms (SNPs) were selected from the SGK1 gene. Results: SGK1 SNP rs3813344 was significantly associated with baseline systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial blood pressure (MAP) (P = 0.049, 0.015 and 0.018, respectively), and rs9389154 was associated with SBP at baseline (P = 0.040) after adjustment for multiple testing. In addition, rs9376026 was associated with DBP and MAP responses to low-sodium intervention significantly (P = 0.018 and 0.022, respectively). However, no associations between 6 SNPs in the SGK1 gene and BP responses to high-sodium or high-sodium plus potassium-supplementation intervention reached statistical significance. Conclusions: Our study indicates that SGK1 genetic variants may have an important role in BP response to dietary sodium intervention or potassium supplementation and BP regulation.

Sgk1 regulates desmoglein 1 expression levels in oligodendrocytes in the mouse corpus callosum after chronic stress exposure

Major depression, one of the most prevalent mental illnesses, is thought to be a multifactorial disease related to both genetic and environmental factors. However, the genes responsible for and the pathogenesis of major depression at the molecular level remain unclear. Recently, we reported that stressed mice with elevated plasma corticosterone levels show upregulation and activation of serum glucocorticoid-regulated kinase (Sgk1) in oligodendrocytes. Active Sgk1 causes phosphorylation of N-myc downstream-regulated gene 1 (Ndrg1), and phospho-Ndrg1 increases the expression of N-cadherin, α-catenin, and β-catenin in oligodendrocytes. This activation of the Sgk1 cascade results in morphological changes in the oligodendrocytes of nerve fiber bundles, such as those present in the corpus callosum. However, little is known about the molecular functions of the traditional and/or desmosomal cadherin superfamily in oligodendrocytes. Therefore, in this study, we aimed to elucidate the functions of the desmosomal cadherin superfamily in oligodendrocytes. Desmoglein (Dsg) 1, Dsg2, and desmocollin 1 (Dsc1) were found to be expressed in the corpus callosum of mouse brain, and the expression of a subtype of Dsg1, Dsg1c, was upregulated in oligodendrocytes after chronic stress exposure. Furthermore, Dsg1 proteins were localized around the plasma membrane regions of oligodendrocytes. A study in primary oligodendrocyte cultures also revealed that chronic upregulation of Sgk1 by dexamethasone administration is involved in upregulation of Dsg1c mRNA. These results may indicate that chronic stress induced Sgk1 activation in oligodendrocytes, which increases Dsg1 expression near the plasma membrane. Thus, Dsg1 upregulation may be implicated in the molecular mechanisms underlying the morphological changes in oligodendrocytes in response to chronic stress exposure.

Kinome Screen Reveals SGK1 as a Therapeutic Target for NF2: Inhibition of mTORC1/2 is More Effective than Rapamycin

Neurofibromatosis type 2 (NF2) is characterized by vestibular schwannomas and meningiomas (MN) due to bi-allelic NF2 inactivation with loss of NF2 tumor suppressor protein. Besides NF2-associated MNs, ~60% of sporadic MNs show loss of NF2, and these tumors are non-responsive to chemotherapeutic intervention. We previously showed activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling upon NF2 loss, leading to clinical trials with rapalogs. Here we carried out a high throughput kinome screen to identify kinases responsible for mTORC1 activation in NF2-null MN cells. Among the top candidates were the mTORC2-specific target serum/glucocorticoid-regulated kinase 1 (SGK1) and p21-activated kinase 1 (PAK1). In NF2-null MNs, SGK1 inhibition rescues mTORC1 activation, and SGK1 activation is sensitive to dual mTORC1/2 inhibitor AZD2014, but not to rapamycin. PAK1 inhibition also rescues mTORC1 activation, but in a manner independent of mTORC2/SGK1 signaling. Using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome editing we generated isogenic arachnoidal cell lines (ACs), the origin cell type for MNs, with or without NF2. NF2-null CRISPR ACs recapitulates the signaling of NF2-null MN cells. Interestingly, we observe increased SGK1 transcription and protein expression in NF2-CRISPR ACs and in primary NF2 MN lines.Moreover, AZD2014 treatment of NF2-null MN cells leads to decreased cell viability and shows greater efficacy than rapamycin. Therefore, inhibiting mTORC1 and mTORC2 signaling is a critical step for potential therapeutic interventions for NF2.

Serum/glucocorticoid‐regulated kinase (SGK) 1 and transient receptor potential vanilloid channel (TRPV) 4 mediate ventilation‐induced endothelial Ca 2+ influx and barrier failure

Study objective: Mechanical ventilation can damage the lung by causing endothelial barrier failure, edema formation and lung injury. Yet, the exact mechanisms underlying the mechanotransduction at the microvascular barrier are still unclear. In this context, the mechanosensitive Ca2+ channel TRPV4 represents an attractive candidate. Here, we tested the role of TRPV4 in VILI, and probed for a role of SGK1 as potential upstream regulator due to its ability to phosphorylate TRPV4, which facilitates its binding to F-actin. Methods: Mice were ventilated for 2 h with low (7 mL/kg) and high (20 mL/kg) tidal volumes (LVT/HVT) in vivo. Isolated-perfused lungs were inflated with 5 or 15 cmH2O (LPIP/HPIP) and [Ca2+]i was quantified by real-time imaging with or without pharmacological inhibition of SGK1 and TRPV4. Interactions between TRPV4, SGK1, and F-actin were analysed by co-immunoprecipitation analyses. Results: TRPV4 and SGK1 inhibition by HC-067047 or GSK650394 protected from HVT-induced lung injury in vivo, and attenuated the endothelial Ca2+ response to HPIP ex vivo. CoIP revealed interaction of TRPV4 with SGK1 and F-actin. Conclusions HVT promotes endothelial Ca2+ influx and barrier failure in a TRPV4 dependent manner via a signaling pathway that involves SGK1 and TRPV4 interaction with F-actin. SGK1 and TRPV4 may present new targets for the prevention or treatment of stretch-induced vascular pathologies.

Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1.

The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, we identified stromal interaction molecule 1 precursor (STIM1) as an auxiliary protein of this epithelial Ca2+channel using confocal microscopy analysis and GST pull-down assay. The STIM1 protein associates specifically with the C-terminal tail of TRPV4 to form a complex. In previous reports, we demonstrated that the serine824 residue of TRPV4 is one of the target phosphorylation sites of serum/glucocorticoid regulated kinase 1 (SGK1). In this report we further identified the role of serine 824 phosphorylation. The TRPV4 mutant S824D (not S824A) exhibited a diminished capacity to bind STIM1. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STIM1 is part of the TRPV4 protein complex. Our observations clearly suggest that the formation of a complex between TRPV4 and STIM1 and its plasma membrane localization are regulated through phosphorylation of serine824 of TRPV4, and that the STIM1-TRPV4 complex plays crucial roles in routing TRPV4 to the plasma membrane from the endoplasmic reticulum and in maintaining its function.

Restoration of Wnt/β-catenin signaling attenuates alcoholic liver disease progression in a rat model

Alcoholic liver disease (ALD) is characterized by the development of fatty liver, alcoholic hepatitis, fibrosis and cirrhosis. However, the underlying mechanism(s) associated with progression remains elusive. Pro-inflammatory cytokines have been implicated in ALD progression due to pro-apoptotic effects on hepatocytes. Wnt/β-catenin signaling recently has been shown to promote inflammation and apoptosis, suggesting that activation of this signaling pathway may modulate ALD progression. The current study was designed to test whether pharmacological activation of Wnt/β-catenin signaling altered ALD development and progression in a rat model. Adult male Long Evans rats were fed with isocaloric liquid diets containing 0% or 37% ethanol for 8 weeks, and also treated with Wnt agonist during the last 3 weeks of the feeding regimen. Liver and blood samples were subjected to histology, TUNEL assay, immunoblot analysis, real-time quantitative PCR, and alanine transaminase (ALT) assay. Wnt/β-catenin signaling was negatively correlated with Foxo3A expression and reduced steatosis, cellular injury and apoptosis in ALD rats. Mutation experiments demonstrated that Foxo3A was critical for modulating these effects. Activation of Wnt/β-catenin signaling suppressed Foxo3A-induced apoptosis through upregulation of serum/glucocorticoid regulated kinase 1 (SGK1). Moreover, pharmacological restoration of Wnt/β-catenin signaling reduced ALD progression in vivo. Wnt/β-catenin signaling plays a protective role in ALD progression via antagonizing Foxo3A-induced apoptosis, and activation of the Wnt/β-catenin signaling cascade attenuates ALD progression.

Common Variants in Serum/Glucocorticoid Regulated Kinase 1 (SGK1) and Blood Pressure Responses to Dietary Sodium or Potassium Interventions: A family-Based Association Study

Background/Aims: Serum/Glucocorticoid Regulated Kinase 1 (SGK1) plays a significant role in regulating renal Na⁺ reabsorption, K⁺ secretion, and blood pressure (BP). This study aimed to assess the association of common genetic variants in the SGK1 gene with BP responses to controlled dietary sodium or potassium interventions. Methods: A total of 334 subjects from 124 families were recruited from the rural areas of northern China. After a three-day baseline observation, they were sequentially maintained a seven-day low-sodium diet (3g/day of NaCl or 51.3mmol/day of sodium), a seven-day high-sodium diet (18g/day of NaCl or 307.8 mmol/day of sodium) and a seven-day high-sodium plus potassium supplementation intervention (4.5g/day of KCl or 60mmol/day of potassium). Six single-nucleotide polymorphisms (SNPs) in the SGK1 gene were selected. Results: After adjustment for multiple testing, SNP rs9376026 was significantly associated with diastolic BP (DBP) and mean arterial pressure (MAP) responses to low-sodium intervention (P = 0.018 and 0.022, respectively). However, the associations between selected SNPs in the SGK1 gene and BP responses to high-sodium or high-sodium plus potassium-supplementation intervention did not reach statistical significance. In addition, SNP rs9389154 and two other SNPs (rs1763509 and rs9376026) were associated respectively with systolic BP (SBP) and DBP at baseline (P = 0.040, 0.032, and 0.031, respectively). SNP rs3813344 was significantly associated with SBP, DBP, and MAP (P = 0.049, 0.015 and 0.018, respectively). Conclusion: Our study indicates that the genetic polymorphism in the SGK1 gene is significantly associated with BP responses to dietary sodium intervention.

Targeting serum glucocorticoid-regulated kinase-1 in squamous cell carcinoma of the head and neck: a novel modality of local control.

PurposeThe inhibition of serum glucocorticoid-regulated kinase-1 (SGK-1) has been found to decrease growth of colon and prostate cancer cells. The purpose of this study is to evaluate the therapeutic effect of SGK-1 inhibition in head and neck squamous cell carcinoma (SCC).Experimental DesignHuman head and neck tumors (HTB41/43) were established in athymic mice. Growth rates between mice treated with vehicle (PBS) injection (group 1, n = 5), SGK-1 Inhibitor GSK 650394 (group 2, n = 6), systemic cisplatin (group 3, n = 6), and a combination of SGK-1 Inhibitor and cisplatin (group 4, n = 6) were compared using repeated measures one-way ANOVA with Newman-Keuls Multiple Comparison Test. Tumor cells were subsequently submitted to further analyses.ResultsAt the end of the experiment mean tumor sizes were 122.33+/−105.86, 76.73+/−36.09, 94.52+/−75.92, and 25.76+/−14.89 mm2 (mean +/− SD) for groups 1 to 4. Groups 2 and 3 showed decreased tumor growth compared to controls (p<0.001). Group 4 displayed even greater growth suppression (p<0.0001). Importantly, group 4 fared better than group 3 (p<0.001). CD44 expression was reduced in group 2 (p<0.05), and to an even greater extent in groups 3 and 4 (p<0.0025). A trend towards reduction of HER 2 expression was noted in group 4.ConclusionsSGK-1 inhibition suppresses tumor growth, and in combination with systemic cisplatin exceeds the effect of cisplatin alone. Decreased expression of CD44 and HER 2 implies depletion of tumor stem cells, and less tumorigenicity. SGK-1 inhibition represents a potential modality of local control for palliation in advanced cases.

MTORC2 regulates renal tubule sodium uptake by promoting ENaC activity.

The epithelial Na+ channel (ENaC) is essential for Na+ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na+ and K+ transport in WT and Sgk1-/- mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na+ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K+ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na+ homeostasis through SGK1-dependent modulation of ENaC activity.

Abstract 1766: Growth modulation of head and neck cancer via Sgk-1 inhibition: a novel modality of local control

Abstract Introduction: Squamous cell carcinoma (SCC) of the skin is frequently aggressive in its biologic behavior, and the prognosis with metastatic disease is dismal. Nevertheless it is highly curable if detected early, usually via surgical excision. Large and repeat excision can cause considerable functional and cosmetic deformity, and in advanced stages may not be desirable. Serum glucocorticoid-regulated kinase-1 (SGK-1) is a serine-threonine kinase that has recently been found to activate β-catenin, part of intracellular components of the Wnt-signaling pathway. β-catenin has been implicated in promoting the stem cell compartment, as well as tumor growth. SGK-1 has been shown to inactivate glycogen synthase kinase-3β (GSK-3β). Importantly, GSK-3β deactivates β-catenin while also serving as a pivotal regulator of the mitochondrial permeability transition pore and cell fate determinant. In this study we tested the effects of local SGK-1 inhibition on tumor growth, as well as expression of CD44 and human epidermal growth factor receptor 2 (HER-2). CD44 is recognized as a key cell surface marker for stem cells in human head and neck cancer, while HER-2 is a trans-membrane protein linked to its migration and invasion. Methods: A human SCC cell line (HTB41) was established in vitro. Athymic mice (n=12) were then injected with HTB41(2x106 cells/animal), and tumor growth monitored. At 6mm, tumors were injected with an SGK-1 Inhibitor (Group 1) twice weekly (20 mg/mouse per injection). Controls (Group 2) received vehicle only (PBS), Group 3 weekly intraperitoneal Cisplatin (5mg/kg), and Group 4 both Cisplatin and SGK-1 inhibitor. Once the first tumor reached 12mm in size, the mice were sacrificed and tumor cells subjected to flow-cytometry, and statistical analysis. 2-tailed t-tests were performed, and p-values &amp;lt;0.05 considered significant. Results: The local inhibition of SGK-1 in combination with Cisplatin showed a significantly slower growth than the control group (p = 0.015). The growth rates were 1.80 +/- 0.12, 2.27 +/- 0.51, 0.89 +/- 0.87, 0.68 +/- 0.40 mm2/day (mean +/- sd) for Groups 1 to 4 respectively. Furthermore, Group 1 showed a right shift compared to controls. At 3%, CD44 expression was highest in Group 2 versus 1%, 0.2 % and 0.07% in Groups 1, 3, and 4. HER-2+ cells were 1% in Group 2, and 0.2%, 0.1% and 0.02% in Groups 1, 3 and 4. Conclusions: Decreased expression of CD44 in Group 4 suggests depletion of tumor stem cells, and may imply less metastatic potential. In addition, lower HER-2 expression in Group 4 may indicate less malignant behavior and perhaps better survival. It should be noted that other pathways might have quickly bypassed a preliminary growth suppressing effect of SGK-1 inhibition alone. Finally, SGK-1 inhibition via local injection added to systemic Cisplatin increases tumor growth suppression, and represents a potential modality of local control for palliation in advanced cases. Citation Format: Henrik O. Berdel, Hongyu Yin, Jun Liu, Chris Middleton, Nathan Yanasak, Rafik Abdelsayed, Wolfgang E. Berdel, Mahmood Mozaffari, Jack C. Yu, Babak Baban. Growth modulation of head and neck cancer via Sgk-1 inhibition: a novel modality of local control. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1766. doi:10.1158/1538-7445.AM2014-1766

Abstract 5313: Phosphoproteomic analysis with reverse phase protein array identifies N-myc downstream regulated gene 1 (NDRG1) as a biomarker of phosphatidylinositol-3-kinase (PI3K)α inhibitor activity in a PTEN-deficient glioma cell line

Abstract Aberrant activation of phosphoinositide 3-kinase (PI3K) signaling is commonly reported in cancer, and leads to deregulation of several intracellular processes, including cell survival, growth, proliferation and migration. PI3K activates AKT, serum/glucocorticoid regulated kinase (SGK), phosphoinositide -dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and several other molecules involved in cell progression and survival. Reports have also indicated that the deletion of the gene encoding the tumor suppressor phosphatase and tensin homologue (PTEN) is one of the key factors controlling activation of the PI3K pathway. Glioblastoma (GBM), the most common malignant brain tumor in adults, represents a compelling disease for kinase inhibitor therapy. Although many therapies to target the Ras-PI3K-mTOR axis in GBM have been attempted, their efficacies have not been pronounced, presumably because of the complexity of PI3K mediated signaling. To elucidate the role of PTEN in PI3K signaling, the phosphorylation status of two glioma cell lines, LN229 (wild type PTEN) and U-87 MG (PTEN deficient) was investigated with phosphoproteomic reverse phase protein array (RPPA). When basal phosphorylation was compared between both cell lines, the phosphorylation of N-myc downstream regulated gene 1 (NDRG1) (Thr346), a physiological substrate of SGK1, was increased in U-87 MG cells compared with levels in LN229 cells. These data suggest that PTEN deletion affects the phosphorylation status of NDRG1 in U-87 MG cells. Following treatment with PIK-90, a PI3Kα inhibitor, phosphorylation of Akt (Ser473) and its direct substrate PRAS40 (Thr246) were decreased in both cell lines, however, NDRG1 (Thr346) phosphorylation was strongly inhibited only in U-87 MG cells. These results suggest that the differential effects of PIK-90 on LN229 and U-87 MG cells is PTEN dependent, and that detecting NDRG1 (Thr346) phosphorylation could be a biomarker for PI3Kα inhibitor treatment in PTEN deleted cancer cells. Ongoing studies are focusing on elucidating the mechanism of action of PIK-90 in glioma cell lines. Citation Format: Takeomi Inoue, Naoko Iwata, Eiji Nishiwaki, Yasuyuki Kirii. Phosphoproteomic analysis with reverse phase protein array identifies N-myc downstream regulated gene 1 (NDRG1) as a biomarker of phosphatidylinositol-3-kinase (PI3K)α inhibitor activity in a PTEN-deficient glioma cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5313. doi:10.1158/1538-7445.AM2014-5313

Klotho gene deficiency causes salt-sensitive hypertension via monocyte chemotactic protein-1/CC chemokine receptor 2-mediated inflammation.

Klotho (KL) is a newly discovered aging suppressor gene. In mice, the KL gene extends the lifespan when overexpressed and shortens the lifespan when disrupted. This study investigated if KL deficiency affects BP and salt sensitivity using KL mutant heterozygous (+/-) mice and wild-type (WT) mice (9 weeks of age, 16 mice per group). Notably, systolic BP in KL(+/-) mice began to increase at the age of 15 weeks, reached a peak level at the age of 17 weeks, and remained elevated thereafter, whereas systolic BP remained consistent in WT mice. High salt (HS) intake further increased BP in KL(+/-) mice but did not affect BP in WT mice. Blockade of CC chemokine receptor 2 (CCR2), involved in monocyte chemotaxis, by a specific CCR2 antagonist (INCB3284) abolished the HS-induced increase in BP in KL(+/-) mice. Furthermore, HS loading substantially increased the expression of monocyte chemotactic protein-1 and the infiltration of macrophages and T cells in kidneys in KL(+/-) mice, and treatment with INCB3284 abolished these effects. Treatment of KL(+/-) mice with INCB3284 also attenuated the increased renal expressions of serum glucocorticoid-regulated kinase 1, thiazide-sensitive NaCl cotransporter, and ATP synthase β along with the renal structural damage and functional impairment induced by HS loading. In conclusion, KL deficiency caused salt-sensitive hypertension and renal damage by CCR2-mediated inflammation.

A gene-based analysis of variants in the serum/glucocorticoid regulated kinase (SGK) genes with blood pressure responses to sodium intake: the GenSalt Study.

BackgroundSerum and glucocorticoid regulated kinase (SGK) plays a critical role in the regulation of renal sodium transport. We examined the association between SGK genes and salt sensitivity of blood pressure (BP) using single-marker and gene-based association analysis.MethodsA 7-day low-sodium (51.3 mmol sodium/day) followed by a 7-day high-sodium intervention (307.8 mmol sodium/day) was conducted among 1,906 Chinese participants. BP measurements were obtained at baseline and each intervention using a random-zero sphygmomanometer. Additive associations between each SNP and salt-sensitivity phenotypes were assessed using a mixed linear regression model to account for family dependencies. Gene-based analyses were conducted using the truncated p-value method. The Bonferroni-method was used to adjust for multiple testing in all analyses.ResultsIn single-marker association analyses, SGK1 marker rs2758151 was significantly associated with diastolic BP (DBP) response to high-sodium intervention (P = 0.0010). DBP responses (95% confidence interval) to high-sodium intervention for genotypes C/C, C/T, and T/T were 2.04 (1.57 to 2.52), 1.79 (1.42 to 2.16), and 0.85 (0.30 to 1.41) mmHg, respectively. Similar trends were observed for SBP and MAP responses although not significant (P = 0.15 and 0.0026, respectively). In addition, gene-based analyses demonstrated significant associations between SGK1 and SBP, DBP and MAP responses to high sodium intervention (P = 0.0002, 0.0076, and 0.00001, respectively). Neither SGK2 nor SGK3 were associated with the salt-sensitivity phenotypes in single-maker or gene-based analyses.ConclusionsThe current study identified association of the SGK1 gene and BP salt-sensitivity in the Han Chinese population. Further studies are warranted to identify causal SGK1 gene variants.