Kinase Suppressor Of Ras Research Articles

7447 Rare KSR2 Variants: The Effect of Glycine and N-acetylcysteine (GlyNAC) Supplementation

Abstract Disclosure: J. Youn: None. Y. Carcamo-Bahena: None. E. Lartigue: None. S. Sisley: None. Kinase suppressor of ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Of note, disruption of the kinase domain of KSR2 in humans and mice causes obesity by reducing metabolic rate, suggesting its important role in energy homeostasis. In the present study, we identified two variants (P189L, T290A) with mutations located in the proximal CA2 region from children with severe, early-onset obesity. Since variants in this region have not been confirmed to cause obesity, we generated plasmid constructs containing these KSR2 mutations by site directed mutagenesis. The HEK293 cells transfected with KSR2 plasmid construct containing P189L or T290A had reduced binding affinity with AMPK and phosphorylation of AMPK without affecting KSR2 protein integrity. These variants also significantly reduced cellular metabolic rates measured by Seahorse analysis in C2C12 cells, suggesting that KSR2 variants in the proximal CA2 region can result in impaired KSR2 function, subsequently leading to a decrease in cellular metabolic rate. In addition, these KSR2 variants decreased protein levels of NRF-2, a transcription factor of cytoprotective genes regulating the antioxidant glutathione (GSH) pathway compared to wild type KSR2 which augmented NRF2 levels. We also observed lower levels of total GSH and GSH/Glutathione disulfide (GSSG) ratio and higher levels of thiobarbituric acid reactive substances (TBARS), an indicator of oxidative stress, indicating an imbalance of antioxidant and oxidant with proximal KSR2 variants. However, treatment of cells with Glycine and N-acetylcysteine (GlyNAC) reversed the detrimental effects of KSR2 variants by normalizing protein expression levels of NRF-2, GSH/GSSG ratios and TBARS levels. Taken together, we show strong evidence that two new KSR2 variants, P189L and T290A, cause disruption of KSR2 function. Additionally, we also report a novel role of KSR2 in maintaining redox homeostasis suggesting that increased oxidative stress following GSH deficiency may play an important role in the development of obesity from KSR2 variants. Presentation: 6/1/2024

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status.

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells.

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.

MAPK Signaling Pathway Is Essential for Female Reproductive Regulation in the Cabbage Beetle, Colaphellus bowringi.

The mitogen-activated protein kinase (MAPK) signaling pathway is a well-conserved intracellular signal transduction pathway, and has important roles in mammalian reproduction. However, it is unknown whether MAPK also regulates insect reproductive mechanisms. Therefore, we investigated the role of the MAPK signaling pathway in ovarian growth and oviposition in the cabbage beetle Colaphellus bowringi, an economically important pest of Cruciferous vegetables. As an initial step, 14 genes from the extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (P38) cascades were knocked down using RNA interference (RNAi). The results revealed that RNAi knockdown of MAPK-ERK kinase (MEK), ERK, Kinase suppressor of RAS 2 (KSR2), and P38 induced ovarian development stagnation, low fecundity, and decreased longevity, which indicate that ERK and P38 signaling pathways are important for female C. bowringi survival and reproduction. The potential regulatory role of ERK and P38 pathways in the female reproductive process was investigated using quantitative real-time PCR. We found that ERK pathway possibly regulated ecdysone biosynthesis and P38 pathway possibly involved in the germline stem cell (GSC) development and differentiation. Our findings demonstrated the importance of the MAPK signaling pathway in the female reproduction of insects, and further enhanced the molecular mechanism of female reproductive regulation in insects.

An Antagonist of KSR1‐Driven Adaptive Resistance to Clinical RAS‐MAPK Inhibitors

Kinase Suppressor of RAS (KSR), RAF and MEK function downstream of RAS and upstream of ERK to mediate growth factor and receptor tyrosine kinase (RTK) signaling. Significant clinical breakthroughs have occurred in targeting mutant oncogenes and vulnerabilities in the RAS‐MAPK pathway, including against KRAS‐G12C, BRAF‐V600E, and MEK1/2. However, these drugs have revealed that the inhibition of individual RAS‐MAPK signaling molecules can lead to relief of negative feedback signaling and paradoxically amplify RAS‐MAPK output over time. Indeed this mechanism, sometimes referred to as ‘rebound signaling’, has arose as a key driver of adaptive drug resistance to several classes of therapies. We hypothesize that KSR1, an isoform implicated in several Ras‐driven cancers, may function in the resistance mechanisms to Ras‐MAPK inhibition therapy. To test this hypothesis, we evaluate KSR1 as a driver of resistance to RAS‐MAPK inhibition and developed a selective KSR1 antagonist, K‐X1. We discovered strong synergy between K‐X1 and MEK inhibition in models of adaptive resistance. In vivo target engagement assays suggest that K‐X1 operates through alterations in the stability and interactions of KSR1‐mediated complexes. Our work highlights a potential strategy to overcome adaptive resistance to therapies targeting the MAPK pathway via KSR1.

Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions.

Pseudokinases often operate through functionally related enzymes and receptors. A prime example is the pseudokinase KSR (Kinase Suppressor of RAS), which can act as both an amplifier and inhibitor of members in the RAS-MAPK (Mitogen Activated Protein Kinase) signaling pathway. KSR is structurally related to the active RAF kinases over multiple domains; moreover, the pseudokinase domain of KSR forms physical and regulatory complexes with both RAF and MEK through distinct interfaces. Characterization of small molecule interactions on KSR has been used to uncover novel chemical tools and understand the mechanism of action of clinical drugs. Here, we elaborate on assays and structural methods for measuring binding at orthosteric and interfacial binding sites on KSR. These distinct small molecule pockets provide therapeutic paths for targeting KSR1 and KSR2 pseudokinases in disease, including in RAS and RAF mutant cancers.

ELK4 promotes the development of gastric cancer by inducing M2 polarization of macrophages through regulation of the KDM5A-PJA2-KSR1 axis

BackgroundWe tried to elaborate the molecular mechanism of ETS-like transcription factor 4 (ELK4) affecting gastric cancer (GC) progression through M2 polarization of macrophages mediated by lysine-specific demethylase 5A (KDM5A)-Praja2 (PJA2)-kinase suppressor of ras 1 (KSR1) axis.MethodsGC expression dataset was obtained from GEO database, and the downstream regulatory mechanism of ELK4 was predicted. Tumor-associated macrophages (TAMs) were isolated from GC tissues. The interaction among ELK4, KDM5A, PJA2 and KSR1 was analyzed by dual luciferase reporter gene, ChIP and Co-IP assays. The stability of KSR1 protein was detected by cycloheximide (CHX) treatment. After TAMs were co-cultured with HGC-27 cells, HGC-27 cell biological processes were assessed through gain- and loss-of function assays. Tumorigenicity was detected by tumorigenicity test in nude mice.ResultsIn GC and TAMs, ELK4, KDM5A and KSR1 were highly expressed, while PJA2 was lowly expressed. M2 polarization of macrophages promoted the development of GC. ELK4 activated KDM5A by transcription and promoted macrophage M2 polarization. KDM5A inhibited the expression of PJA2 by removing H3K4me3 of PJA2 promoter, which promoted M2 polarization of macrophages. PJA2 reduced KSR1 by ubiquitination. ELK4 promoted the proliferative, migrative and invasive potentials of GC cells as well as the growth of GC xenografts by regulating KSR1.ConclusionELK4 may reduce the PJA2-dependent inhibition of KSR1 by transcriptional activation of KDM5A to promote M2 polarization of macrophages, thus promoting the development of GC.

Structural basis for the action of the drug trametinib at KSR-bound MEK.

The MAPK/ERK Kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and aging4. However, many MEK inhibitors (MEKi) are limited due to on-target toxicities5–7 and drug resistance8–10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (Kinase Suppressor of Ras) with various MEKi, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. Through complexation, KSR remodels the prototypical MEKi allosteric pocket thereby impacting binding and kinetics, including drug residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these subcomplexes. Based on these insights we created trametiglue, which limits adaptive resistance to MEKi through enhanced interfacial binding. Together, our results reveal the plasticity of an interface pocket within MEK subcomplexes that has implications for the design of next generation drugs targeting the RAS pathway.

0016 Genetic Basis of Daytime Napping and Consequence on Cardiometabolic Health

Abstract Introduction Although daytime napping is a common, evolutionarily conserved behavior, its genetic basis is unknown. Elucidating its genetic basis may clarify relevant underlying biological pathways and determine causal links with cardiometabolic health. Methods We performed a genome-wide association study of self-reported daytime napping using linear regression in adults of European ancestry in the UK Biobank (n=452,633) and assessed robustness of signals with accelerometer-derived daytime inactivity duration (n=84,671). Next, we conducted a phenome-wide association study in a hospital-based clinical biobank (n=30,683) using napping genome-wide polygenic score (GPS), and Mendelian randomization (MR) with cardiometabolic traits. To deconstruct the napping genetic variants, we applied a novel “soft clustering” Bayesian nonnegative matrix factorization method and generated partitioned cluster-specific polygenic risk scores (PRS). Results We identified 121 distinct genome-wide significant loci for daytime napping, with lead signals at or near genes KSR2 (kinase-suppressor of ras 2), HCRTR1/HCRTR2 (hypocretin-receptor 1/2), SKOR2 (SKI family transcriptional-corepressor 2), and MAPT (microtubule-associated protein tau), among others. The loci associated with accelerometer-derived daytime inactivity duration. Gene enrichment analyses pointed to pathways involved in neurogenesis and others including nervous system development and opioid signaling. Genetic overlaps were evident in a clinical biobank where highest, compared to lowest, decile of napping GPS associated with 30%, 40%, and 50% higher odds for essential hypertension, obesity, and nonalcoholic liver disease, respectively (P<0.0001). In MR, potential causal links were identified with higher diastolic blood pressure (2.67 mmHg per napping category-increase, 95% CI 1.62–3.23, P=6.80e-07), systolic blood pressure (3.65mmHg, 1.86–5.44, P=6.40e-05), and waist circumference (0.28 SD-units, 0.11–0.45, P=0.0015). The clustering of variants identified 3 robust clusters (cluster-1: “higher sleep propensity”; cluster-2: “more fragmented/inefficient night sleep”; cluster-3: “early sleep timing”). Only clusters 2 and 3 PRSs were associated with worse cardiometabolic health outcomes, including higher BMI, waist circumference, CRP, and triglycerides (all P<0.05). Conclusion These findings expand our understanding of the genetic architecture of napping implicating multiple biological pathways, indicating possible genetic overlap and causal links to cardiometabolic traits, and suggesting distinct nap-promoting mechanisms with differential associations with health outcomes. Support This work is supported by grants NIH-F32DK102323, NIH-4T32HL007901, NIH-R01DK107859, NIH-R35HL135818, and MGH Research Scholar Fund.

Lymphocyte Specific Protein‐1 suppresses TCPOBOP induced hepatocellular proliferation

Lymphocyte specific protein‐1 (LSP‐1), an f‐actin binding protein expressed in hematopoietic cells as well as hepatocytes, is the gene with the most copy number variations detected in human HCC. Our previous work has demonstrated that LSP‐1 suppresses hepatocellular proliferation following partial hepatectomy as well as in primary hepatocyte cultures. LSP‐1 acts as a scaffold for the Ras/MEK/ERK MAP kinase pathway and along with kinase suppressor of Ras (KSR) inhibits ERK phosphorylation and thus its activation preventing downstream signaling to induce cellular proliferation and migration. To determine if LSP‐1 functions to attenuate hepatocellular growth in models which induce proliferation without loss of tissue or liver injury, we aimed to explore if LSP‐1 can modulate TCPOBOP induced hepatocellular proliferation and hepatomegaly. TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene), a constitutive androstane receptor (CAR) agonist, has been shown to induce hepatocellular hyperplasia through a CAR dependent mechanism. Since LSP‐1 has been shown to suppress hepatocyte proliferation during liver regeneration following partial hepatectomy and in culture, we hypothesized that LSP‐1 will attenuate TCPOBOP driven hepatocellular proliferation. In this study, we administered TCPOBOP to both a global LSP1 knockout mouse model as well as a hepatocyte specific LSP‐1 transgenic (TG) mouse model. Livers were harvested at various time point after TCPOBOP gavage and proliferation was measured using Ki67 and PCNA immunohistochemistry as well as liver to body weight ratios. Increased LSP‐1 expression in the transgenic livers caused decreased Ki67 staining on day 2 after TCPOBOP administration as well as no measurable change in liver to body weight ratios as compared to baseline. However, by day 5, the TG liver to body weight ratios increased above those of the WT but the percentage of Ki67 positive hepatocytes was decreased in the TG as compared to the wild type controls. Loss of LSP‐1 expression led to a statistically significant difference in the liver to body weight ratios between the KO and WT mice on day 2 after TCPOBOP administration as well as increased hepatocellular proliferation on both days 2 and 5 after gavage in the KO livers. CAR target genes and downstream signaling pathways that are modulated by LSP‐1 expression will be analyzed.ConclusionLSP‐1 attenuates TCPOBOP induced hepatocellular proliferation and loss of expression leads to increased hyperplasia supporting the role of LSP‐1 as a growth suppressor.Support or Funding InformationSupported by the Department of Defense grant CA160119, the University of Pittsburgh Menten Endowment, and the Cleveland Foundation Morningstar Fund.

MiR-199a Inhibits Tumor Growth and Attenuates Chemoresistance by Targeting K-RAS via AKT and ERK Signalings.

Glioma is the most malignant brain tumors in the world, the function and molecular mechanism of microRNA-199a (miR-199a) in glioma is not fully understood. Our research aims to investigate miR-199a/K-RAS axis in regulation of glioma tumor growth and chemoresistance. The function of miR-199a in glioma was investigated through in vitro and in vivo assays. We found that miR-199a in tumor tissues of glioma patients was significantly downregulated in this study. Kinase suppressor of ras 1 (K-RAS), was indicated as a direct target of miR-199a, as well as expression levels of K-RAS were inversely correlated with expression levels of miR-199a in human glioma specimens. Forced expression of miR-199a suppressed AKT and ERK activation, decreased HIF-1α and VEGF expression, inhibited cell proliferation and cell migration, forced expression of K-RAS restored the inhibitory effect of miR-199a on cell proliferation and cell migration. Moreover, miR-199a renders tumor cells more sensitive to temozolomide (TMZ) via targeting K-RAS. In vivo experiment validated that miR-199a functioned as a tumor suppressor, inhibited tumor growth by targeting K-RAS and suppressed activation of AKT, ERK and HIF-1α expression. Taken together, these findings indicated that miR-199a inhibits tumor growth and chemoresistance by regulating K-RAS, and the miR-199a/K-RAS axis is a potential therapeutic target for clinical intervention in glioma.

A8864 Two novel polymorphisms of the CNKSR3 gene is associated with essential hypertension in Han population

Objectives: Kinase suppressor of ras 3 (CNKSR3) was a direct mineralocorticoid receptor (MR) target gene that is up-regulated in response to physiological concentrations of aldosterone. The aim of the present study was to assess the association between the human CNKSR3 gene polymorphism and essential hypertension in a Han population of China. Methods: We use 422 control subjects and 1868 confirmed essential hypertension subjects. All patients and controls were genotyped for the same three single nucleotide polymorphisms SNPs (rs11541340, rs4870285 and rs7757161) of CNKSR3 gene by a real-time PCR instrument. Results: In the Han population, the distribution of rs11541340 and rs7757161 genotypes showed a significant difference between essential hypertension and control subjects (P = 0.044 and p = 0.008). There was a significant difference between essential hypertension and control participants (P = 0.013 and P = 0.002) in rs11541340 recessive model (GGvs CG +), even after adjustment for covariates (OR: 2.22, 95% confidence interval [CI]: 1.157–4.259, P = 0.016). The distribution of rs7757161 C alleles showed a difference between essential hypertension and control participants (for alleles P = 0.042). Conclusion: The GG genotype of rs11541340 in CNKSR3 gene could be a protective genetic marker while the C alleles of rs7757161 in CNKSR3 gene could be a protective genetic marker of essential hypertension of Han population in China.

Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression

BackgroundSmurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Methods: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation.ResultsCNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines.ConclusionsAltogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.

MEK drives BRAF activation through allosteric control of KSR proteins.

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

The KSR2-rs7973260 Polymorphism is Associated with Metabolic Phenotypes, but Not Psychological Phenotypes, in Chinese Elders.

To examine the associations between genetic variants of KSR2 (kinase suppressor of RAS)-rs7973260, RAPGEF6 (guanine nucleotide exchange factor 6)-rs3756290, LOC105377703-rs4481363, and subjective well-being (SWB) and depressive symptoms (DSs) in Chinese elders, which were recently associated in a genome-wide association study conducted in Caucasians. The pleiotropic effects of KSR2-rs7973260 on metabolic phenotypes were also explored. We used data from 1788 older individuals aged 70-84 years from the aging arm of the Rugao Longevity and Aging Study, a population-based cohort study conducted in the Jiangsu province of China. No significant distributions of genotype frequencies were observed between life-satisfied and -unsatisfied groups across those with the three polymorphisms. The level of SWB components (positive affect, negative affect, and affect balance) and DSs did not differ among genotypes of the three variants. However, the presence of GA+AA of KSR2-rs7973260 was significantly higher in the metabolic syndrome (MetS), severe hypertriglyceridemia (HTG), and diabetes groups than in control groups (43.7% vs. 37.6%, 46.4% vs. 37.6%, 45.8% vs. 37.9%, respectively). The A allele of rs7973260 was associated with increased risk of MetS, severe HTG, and diabetes with an odds ratios (95% confidence intervals) of 1.289 (1.002-1.658), 1.438 (1.076-1.921), and 1.384 (1.022-1.875), which remained significant after multiple adjustments. Rs7973260, rs3756290, and rs4481363 were not associated with SWB and DSs in Chinese elders. However, the KSR2-rs7973260 A allele exhibited pleiotropic effects on some metabolic phenotypes in Chinese elders. These effects should be validated in future studies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling

Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies1. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes2. Kinase suppressor of Ras (KSR) is a MAPK scaffold3–5 that is subject to allosteric regulation through dimerization with RAF6,7. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

MicroRNA-497 inhibits tumor growth and increases chemosensitivity to 5-fluorouracil treatment by targeting KSR1.

Colorectal cancer (CRC) is one of the leading cancer-related causes of death in the world. Recently, downregulation of microRNA-497 (miR-497) has been observed in CRC tissues. In this study, we found that miR-497 expression levels were downregulated in human CRC specimens compared to the adjacent normal tissues. MiR-497 expression levels were strongly correlated with clinical stages and lymph node metastases. Furthermore, kinase suppressor of ras 1 (KSR1), a known oncogene, was a direct target of miR-497, and KSR1 expression levels were inversely correlated with miR-497 expression levels in human CRC specimens. Overexpression of miR-497 inhibited cell proliferation, migration, invasion and increased chemosensitivity to 5-fluorouracil treatment, whereas forced expression of KSR1 had the opposite effect. Taken together, these results revealed that lower miR-497 levels in human CRC tissues induce KSR1 expression which is associated with CRC cancer occurrence, advanced stages, metastasis and chemoresistance. Lower miR-497 levels may be a potential biomarker for CRC advanced stages and treatment response.

PP.42.16

Objective: Kinase suppressor of ras 3 (CNKSR3) was a direct mineralocorticoid receptor (MR) target gene that is up-regulated in response to physiological concentrations of aldosterone. Aldosterone is the principal hormonal regulator of sodium homeostasis. Thus, the regulation of CNKSR3 gene is correlated to essential hypertension. The aim of the present study was to assess the association between the human CNKSR3 gene polymorphism and essential hypertension in a Han population of China.Design and method: We use 422 control subjects and 1868 confirmed essential hypertension subjects. All patients and controls were genotyped for the same three single nucleotide polymorphisms SNPs (rs11541340, rs4870285 and rs7757161) of CNKSR3 gene by a real-time PCR instrument. Results: In the Han population, for total, the distribution of rs11541340 and rs7757161 genotypes showed a significant difference between essential hypertension and control subjects (P = 0.044 and p = 0.008). The distribution of rs4870285 genotypes showed no significant difference between essential hypertension and control subjects. The distribution of rs11541340 and rs7757161 recessive model (GT vs GG + TT and CT vs CC + TT, respectively) showed a significant difference between essential hypertension and control participants (P = 0.013 and P = 0.002). The distribution of rs7757161 alleles and dominant model(CC vs CT+TT) showed a significant difference between essential hypertension and control participants (for alleles P = 0.042, P < 0.001). The significant difference of rs11542340 and rs7757161 in recessive model was retained after adjustment for covariates (OR: 2.22, 95% confidence interval [CI]: 1.157–4.259, P = 0.016; OR: 3.125, 95% CI: 1.590–6.143, P = 0.001, respectively). Conclusions: The GG genotype of rs11541340 in CNKSR3 gene could be a protective genetic marker of essential hypertension and C allele may be a protective genetic marker of essential hypertension of Han population in China. The TT genotype of rs7757161 in CNKSR3 gene could be a protective genetic marker of essential hypertension and C allele may be a risk genetic marker of essential hypertension of Han population in China.

Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed form of liver cancer with high morbidity and mortality. Copy number variation (CNV) analysis of human HCC revealed that leukocyte-specific protein 1 (LSP1) had the highest number of cases with CNV. LSP1, a F-actin-binding protein, is expressed in hematopoietic cells and interacts with kinase suppressor of Ras (KSR), a scaffold for the extracellular signal-related kinase/mitogen-activated protein kinase pathway. Expression of LSP1 in liver, and its role in normal hepatocellular function and carcinogenesis, remains unknown. Therefore, LSP1 messenger RNA and protein levels were analyzed in normal hepatocytes in culture, rat liver following partial hepatectomy (PHx), and hepatoma cell lines. In culture and after PHx, LSP1 increased after the termination of hepatocyte proliferation. To investigate LSP1 function in HCC, short hairpin RNA was utilized to stably knock down LSP1 expression in the JM1 rat hepatoma cell line. Loss of LSP1 in JM1 cells resulted in dramatic up-regulation of cyclin D1 and phosphorylated ERK2, increased cell proliferation, and migration. Coimmunoprecipitation and immunofluorescence analysis displayed an interaction and colocalization between LSP1, KSR, and F-actin in JM1 cells and liver during regeneration. Conversely, expression of LSP1 in the JM2 rat hepatoma cell line led to decreased proliferation. Enhanced expression of LSP1 in mouse hepatocytes during liver regeneration after injection of an LSP1 expression plasmid also led to decreased hepatocyte proliferation. LSP1 is expressed in normal hepatocytes and liver after PHx after termination of proliferation. In rat hepatoma cell lines and mouse liver in vivo, LSP1 functions as a negative regulator of proliferation and migration. Given the high frequency of LSP1 CNV in human HCC, LSP1 may be a novel target for diagnosis and treatment of HCC.