Kinase Suppressor Of Ras Research Articles

Differential Expression of MicroRNA in Non-tumoral Liver Tissue of Patients With Non-alcoholic Steatohepatitis Cirrhosis and Hepatocellular Cancer

Introduction: Patients with NASH cirrhosis are at risk for developing hepatocellular cancer (HCC). Due to unpredictable risk of progression, all cirrhosis patients are recommended to undergo surveillance. MicroRNAs (miRNAs) play important roles in the development and progression of HCC, and it is quite possible that certain miRNAs in the non-tumor cirrhotic tissue may serve as a biomarker for subsequent development of HCC. The aim of the current study is to identify differentially expressed miRNAs in nontumor cirrhotic tissue of patients with NASH cirrhosis with and without HCC. Methods: miRNA expression profile using the Thermo Scientific Dharmacon RNAi Discovery platform was obtained from non-tumoral liver explant tissue of NASH cirrhosis with HCC (n=5), without HCC (n=12), and control (n=24; 12 tumor adjacent normal tissue with no chronic liver disease +12 normal liver biopsies of patients suspected with NAFLD). Differentially expressed miRNAs between 2 set of conditions (ex: HCC vs. NASH cirrhosis) were determined by performing student t-test and filtered at a p<0.01. Real-time PCR was performed to confirm the differentially expressed miRNAs microarray data. Results: NASH cirrhosis patients with and without HCC were similar in age, gender, race, BMI (36±2 vs. 30±7 kg/m2), and MELD score (13±5 vs. 13±6). Several miRNAs were statistically differentially expressed in HCC when compared to cirrhosis (miRNAs=8) and controls (miRNA =144). The 8 miRNAs that were significantly different between HCC and NASH cirrhosis with fold change are shown Table 1. A decrease in expression was seen with HCC compared to NASH cirrhosis or normal liver. Gene prediction analysis identified several genes controlled by these differentially expressed miRNA. Notable among these genes was kinase suppressor of RAS 2 (KSR2) gene, known to be associated with hepatocellular cancer.Table 1Conclusion: Eight miRNAs are differentially expressed in non-tumoral cirrhotic tissue in patients with HCC. Recognition of these hepatic miRNAs in patients with NASH cirrhosis may help identify the risk of subsequent HCC development.

Targeting cysteine rich C1 domain of Scaffold protein Kinase Suppressor of Ras (KSR) with anthocyanidins and flavonoids - a binding affinity characterization study.

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, ERK to provide spatial and temporal regulation of Ras-dependent ERK cascade signaling. Interruption of this mechanism can have a high influence in inhibiting the downstream signaling of the mutated tyrosine kinase receptor kinase upon ligand binding. Still none of the studies targeted to prevent the binding of Raf, MEK binding on kinase suppressor of RAS. In that perspective the cysteine rich C1 domain of scaffold proteins kinase suppressor of Ras-1 was targeted rather than its ATP binding site with small ligand molecules like flavones and anthocyanidins and analyzed through insilico docking studies. The binding energy evaluation shows the importance of hydroxyl groups at various positions on the flavone and anthocyanidin nucleus. Over all binding interaction shows these ligands occupied the potential sites of cysteine rich C1 domain of scaffold protein KSR.

The metastasis suppressor Nm23 as a modulator of Ras/ERK signaling

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR’s function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR’s discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs’ effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase

In metazoans, the highly conserved MAPK signaling pathway regulates cell fate decision. Aberrant activation of this pathway has been implicated in multiple human cancers and some developmental disorders. KSR1 functions as an essential scaffold that binds the individual components of the cascade and coordinates their assembly into multiprotein signaling platforms. The mechanism of KSR1 regulation is highly complex and not completely understood. In this study, we identified Tyr(728) as a novel regulatory phosphorylation site in KSR1. We show that Tyr(728) is phosphorylated by LCK, uncovering an additional and unexpected link between Src kinases and MAPK signaling. To understand how phosphorylation of Tyr(728) may regulate the role of KSR1 in signal transduction, we integrated structural modeling and biochemical studies. We demonstrate that Tyr(728) is involved in maintaining the conformation of the KSR1 kinase domain required for binding to MEK. It also affects phosphorylation and activation of MEK by RAF kinases and consequently influences cell proliferation. Moreover, our studies suggest that phosphorylation of Tyr(728) may affect the intrinsic kinase activity of KSR1. Together, we propose that phosphorylation of Tyr(728) may regulate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.

A pickup in pseudokinase activity

Kinases catalyse the phosphorylation of target substrates on hydroxy group-containing residues as a means to nucleate multi-component complexes or to stabilize unique conformational states. Through this biochemical activity, kinases play critical roles in many signal transduction and disease pathways. Pseudokinases constitute a subclass of these enzymes that were originally predicted as inactive on the basis of mutations of key conserved active-site residues. However, recent biochemical and structural analyses have revealed several enzymatically active pseudokinases, suggesting either that novel mechanisms of phosphorylation are at play or that the constraints for highly conserved active-site residues are looser than originally anticipated. The purpose of the present review is to summarize several of the active pseudokinases, and one in particular termed KSR (kinase suppressor of Ras), which was recently found to possess a kinase activity that can become accelerated through an allosteric mechanism. Utilization of catalytic activity or structural features of the kinase fold may be key to the function of many pseudokinases.

MiR-378 Controls Cardiac Hypertrophy by Combined Repression of Mitogen-Activated Protein Kinase Pathway Factors

Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth. Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378: MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus-mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction-induced cardiac hypertrophy and improved cardiac function. Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus-miR-378 may prove to be an effective therapeutic strategy in myocardial disease.

Abstract 4316: A dynamic interplay of KSR1, p53 and BRCA1 in breast cancer.

Abstract Background: We have previously performed a human kinome screen and identified kinase suppressor of ras 1 (KSR1) as a potential regulator of estrogen receptor α (ERα) in breast cancer. Recent studies have suggested that KSR1, originally known as a positive modulator interacting with RAS and RAF in the MAPK pathway, may have dual functions as a scaffold protein and a kinase. Although KSR1 has been implicated in Ras-dependent cancers, its role in breast cancer has not been studied. Methods: The clinical significance of KSR1 levels and single nucleotide polymorphisms (SNPs) were assessed by analyzing its expression in tissue microarrays (n=1000) by immunohistochemistry in univariate and multivariate analyses. Expression of KSR1 in a panel of breast cancer cell lines were measured. A KSR1 stable over-expression breast cancer cell line (MCF7-KSR1) was generated to study its effects on growth and colony formation by 3D matrigel and soft agar assays. A xenograft mouse model was used to examine the effects of KSR1 on tumor development in vivo. Stable isotope labelling by amino acids in cell culture (SILAC) was used to analyse KSR1-regulated proteins and pathways. Additional molecular biology techniques (including immunoprecipitation, luciferase reporter and uibiquitination assay) were performed to study the action of KSR1 on p53 and BRCA1. Results: KSR1 was differentially expressed in breast cancer patients. High KSR1 expression correlated significantly with longer DFS (p=0.014) and OS (p=0.012) in &amp;gt; 20 yrs follow-up, independent of tumor stage, grade and size. KSR1 was associated positively with BRCA1 (p=0.002) and inversely with p53 levels (p=0.038). Two KSR1 SNPs’ were identified with no correlations with either DFS or OS. RT-qPCR and western blotting showed that KSR1 is ubiquitously expressed in breast cancer cell lines. In vitro 3D matrigel and soft agar assays revealed that MCF7-KSR1 had a significant decreased number of colonies and formed smaller size colonies compared to MCF7-parental cells. In vivo breast cancer mouse model showed tumor volumes of xenografts over-expressing KSR1 were significantly reduced compared to controls. SILAC analysis revealed various novel KSR1-regulated proteins and phospho-proteins in breast cancer. Our results showed over-expression of KSR1 decreases the transcriptional activity of p53 by reducing DBC1 phosphorylation, which leads to a reduced interaction of DBC1-SIRT1 and therefore allowing SIRT1 to deacetylate p53. Finally, KSR1 stabilizes BRCA1 by up-regulating BARD1 and promoting the interaction of BRCA1-BARD1. Conclusion: KSR1 is an independent prognostic factor in breast cancer; high KSR1 correlates with longer DFS and OS. KSR1 over-expression inhibits tumor formation and growth in vitro and in vivo. KSR1 is part of a dynamic interplay with p53 and BRCA1; it reduces p53 transcriptional activity by abolishing the interaction of SIRT1-DBC1, while it stabilizes BRCA1 by enhancing BRCA1-BARD1 interaction. Citation Format: Hua Zhang, Justin Stebbing, Aleksandra Filipovic, Ian O. Ellis, Heinz-Josef Lenz, Georgios Giamas. A dynamic interplay of KSR1, p53 and BRCA1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4316. doi:10.1158/1538-7445.AM2013-4316

Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas.

Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and (V600E)BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like (V600E)BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.

Mitogen-Activated Protein (MAP) Kinase Scaffolding Proteins: A Recount

The mitogen-activated protein kinase (MAPK) pathway is the canonical signaling pathway for many receptor tyrosine kinases, such as the Epidermal Growth Factor Receptor. Downstream of the receptors, this pathway involves the activation of a kinase cascade that culminates in a transcriptional response and affects processes, such as cell migration and adhesion. In addition, the strength and duration of the upstream signal also influence the mode of the cellular response that is switched on. Thus, the same components can in principle coordinate opposite responses, such as proliferation and differentiation. In recent years, it has become evident that MAPK signaling is regulated and fine-tuned by proteins that can bind to several MAPK signaling proteins simultaneously and, thereby, affect their function. These so-called MAPK scaffolding proteins are, thus, important coordinators of the signaling response in cells. In this review, we summarize the recent advances in the research on MAPK/extracellular signal-regulated kinase (ERK) pathway scaffolders. We will not only review the well-known members of the family, such as kinase suppressor of Ras (KSR), but also put a special focus on the function of the recently identified or less studied scaffolders, such as fibroblast growth factor receptor substrate 2, flotillin-1 and mitogen-activated protein kinase organizer 1.

RGS19 inhibits Ras signaling through Nm23H1/2-mediated phosphorylation of the kinase suppressor of Ras

Besides serving as signal terminators for G protein pathways, several regulators of G protein signaling (RGS) can also modulate cell proliferation. RGS19 has previously been shown to enhance Akt signaling despite impaired Ras signaling. The present study examines the mechanism by which RGS19 inhibits Ras signaling. In HEK293 cells stably expressing RGS19, serum-induced Ras activation and phosphorylations of Raf/MEK/ERK were significantly inhibited, while cells expressing RGS2, 4, 7, 8, 10, or 20 did not exhibit this inhibitory phenotype. Conversely, siRNA-mediated knockdown of RGS19 enabled partial recovery of serum-induced ERK phosphorylation. Interestingly, two isoforms of the tumor metastasis suppressor Nm23 (H1 and H2) were upregulated in 293/RGS19 cells. As a nucleoside diphosphate kinase, Nm23H1 can phosphorylate the kinase suppressor of Ras (KSR). Elevated levels of phosphorylated KSR were indeed detected in the nuclear fractions of 293/RGS19 cells. Co-immunoprecipitation assays revealed that Nm23H1/2 can form complexes with RGS19, Ras, or KSR. siRNA-mediated knockdown of Nm23H1/2 allowed 293/RGS19 cells to partially recover their ERK responses to serum treatment, while overexpression of Nm23H1/2 in HEK293 cells suppressed the serum-induced ERK response. This study demonstrates that expression of RGS19 can suppress Ras-mediated signaling via upregulation of Nm23.

Abstract P1-04-04: KSR1 is involved in functional interaction between p53 and BRCA1 and is an independent predictor of overall survival in breast cancer

Abstract Background: Our recent human kinome screening to identify new regulators of estrogen receptor α (ERα) suggested an important role of kinase suppressor of ras 1 (KSR1) in breast cancer. KSR1 was originally identified as a positive regulator interacting with both RAS and RAF in the mitogen-activated protein kinase (MAPK) pathway. Recent studies have suggested that KSR1 may have dual functions as a scaffolding protein and also a protein kinase. Although KSR1 has been implicated in Ras-dependent cancers, its clinical significance and function in breast cancer have not been elucidated. Methods: The clinical significance of KSR1 was assessed by analyzing its expression in breast cancer tissue microarrays (TMAs, n=1000) by immunohistochemistry. Pearson chi2 and Fisher's Exact Test were used to correlate the expression levels with various tumor biomarkers; Kaplan Meier analyses were used to investigate impact on disease-free (DFS) and overall survival (OS). Multivariate Cox-proportional hazards analysis was also performed to evaluate the significant of KSR1 as an independent factor in breast cancer-specific survival (BCSS) and disease-free interval (DFI). The expression levels of KSR1 in breast cancer cell lines were measured by RT-qPCR and western blotting. A KSR1 stable over-expression breast cancer cell line (MCF7-KSR1) was generated to study its effect on growth and colony formation by 3D matrigel assay and soft agar assay in vitro. Furthermore, MCF7-KSR1 stable cells were also used in nude mice xenograft model in vivo. Luciferase reporter assay was employed to examine the effect of KSR1 on p53 transcriptional activity. Further immunofluorescence staining, western blotting and reporter assays were performed to elucidate the interaction between KSR1, BRCA1 and p53. Results: KSR1 was expressed with low (60%) and high levels (40%) in breast cancer patients. High expression correlated significantly with longer DFS (p = 0.014) and longer OS (p = 0.012) in more than 20 yrs follow-up. Interestingly, KSR1 was positively associated with BRCA1 (p = 0.002) and reversely with p53 (p = 0.038). High KSR1 levels were a significant predictor of longer breast cancer-specific survival BCSS (p = 0.001) and longer disease free interval DFI (p = 0.002) independent of tumour stage, grade and size. RT-qPCR and western blotting demonstrated that KSR1 is ubiquitously expressed in breast cancer cell lines. In vitro 3D matrigel and soft agar assay showed that MCF7-KSR1 cells had a significant decreased number of colonies and formed smaller size colonies compared to MCF7-vector cells. Furthermore, over-expression of KSR1 (MCF7-KSR1) significantly suppressed the growth of breast cancer xenografts in nude mice. Finally, KSR1 was involved in the functional interaction between BRCA1 and p53 and elevated KSR1 potentially resulted in up-regulated BRCA1. KSR1 also inhibited p53-dependent transcriptional activity through suppression of p53 acetylation and promotion of p53 neddylation. Conclusion: KSR1 is an independent prognostic factor in breast cancer and high KSR1 expression correlates with longer DFS and OS. Furthermore, it is potential that KSR1 is important in tumour suppression by its involvement in functional feedback regulation of BRCA1 and p53. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-04.

Pravastatin normalizes ET-1-induced contraction in the aorta of type 2 diabetic OLETF rats by suppressing the KSR1/ERK complex

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. In the present study, we hypothesized that treatment with pravastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) would normalize the ET-1-induced contraction in aortas isolated from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Contractile responses were examined by measuring isometric force in endothelium-denuded aortic helical strips from four groups: Long-Evans Tokushima Otsuka (LETO; genetic control), OLETF (type 2 diabetic), pravastatin-treated LETO, and pravastatin-treated OLETF rats. Both immunoblot analysis and immunoprecipitation assays were used to examine Src, protein phosphatase (PP)2A, kinase suppressor of Ras (KSR)1, and ERK signaling pathway protein levels and activities. In endothelium-denuded aortas isolated from OLETF rats at the chronic stage of diabetes (56-60 wk) (vs. those from age-matched LETO rats), we found the following: 1) ET-1-induced contraction was enhanced, 2) ERK1/2 phosphorylation was increased, 3) phosphorylations of KSR1 and PP2A were reduced (i.e., enhancement of the kinase active state), 4) ERK1/2-KSR1 complexes were increased, and 5) Src tyrosine kinase activity was diminished. Endothelium-denuded aortas isolated from OLETF rats treated with pravastatin (10 mg/kg po, daily for 4 wk) exhibited normalized ET-1-induced contractions and suppressed ET-1-stimulated ERK phosphorylation, with the associated phosphorylated KSR1 and phosphorylated PP2A levels being increased toward normal levels. These results suggest that in type 2 diabetic rats, pravastatin normalizes ET-1-induced contraction in aortic smooth muscle via a suppression of PP2A/KSR1/ERK activities after an enhancement of Src kinase activity.

PD-0016 KSR1 Gene Polymorphism in Mcrc Patients Treated with First-Line Folfiri and Bevacizumab

ABSTRACT Introduction Kinase suppressor of Ras (KSR) 1 is a scaffolding protein regulating the Raf/Mek/ERK cascade. Preclinical data showed that KSR1 could increase estrogen receptor transcriptional activity after exposure to estrogens. Recent retrospective analyses suggested a possible role for KSR1 rs2241906 C/T polymorphism in predicting the outcome of patients with metastatic colorectal cancer (mCRC). Methods MCRC patients receiving first-line FOLFIRI + bevacizumab prospectively enrolled in a translational research program were selected on the basis of KRAS and BRAF mutational status availability. KSR1 rs2241906 polymorphism was analyzed on DNA extracted from peripheral blood by means of PCR and direct sequencing. Taking into account the connection between estrogen pathway and KSR1, subgroup analyses according to gender were pre-planned. Results 287 patients were included. Main patients’ characteristics were the following: M/F = 175/112; median age = 62 (range 26-79); ECOG-PS 0/1-2 = 240/47; synchronous/metachronous disease = 213/74; Kohne score (low/intermediate/high/data missing) = 122/129/22/14; KRAS-BRAF mutational status (wt-wt/mut-wt/wt-mut)= 123/146/18. In the overall KRAS-BRAF wt population, KSR1 polymorphism did not affect the outcome. The analysis performed according to gender showed that in the KRAS-BRAF wt population females with KSR1 rs2241906 T/- achieved a significantly better PFS (median 15.9 mos) in comparison to C/C variant carriers (median 8.8 mos) (HR = 0.44 [95%CI 0.21-0.91], p = 0.010); meanwhile males with T/- showed a worse PFS in comparison to those carrying the C/C variant (HR = 1.92 [95%CI 1.05-3.53], p = 0.021). These results were significant also in a multivariate model and a significant interaction of gender with PFS according to KSR1 allelic variants was demonstrated (p = 0.004). Conclusion This prospectively conceived pharmacogenetic study confirms the role of KSR1 polymorphisms in affecting the outcome of mCRC KRAS-BRAF wt patients. In particular, these results indirectly indicate that estrogenic stimulation could affect the proliferation of KRAS-BRAF wt CRC cells through an interaction with KSR1. Preclinical investigations to further explore this preliminary hypothesis are ongoing.

Genetic variants of kinases suppressors of ras in KRAS-BRAF wild-type metastatic colorectal cancer patients treated with cetuximab and irinotecan.

3597 Background: Scaffold proteins kinase suppressor of ras (KSRs) have emerged as critical modulators of the EGFR pathway that binds to RAS promoting ERK activation. KSR1 and KSR2 have been shown to be over expressed in various solid tumours including colon cancer. In-vitro, the down-regulation of KSR results in reduction of cells proliferation and tumour growth independently to KRAS status. Moving from this biological rational we investigated the correlation between functional polymorphisms in the KSR genes and clinical outcomes in patients with KRAS and BRAF wild-type mCRC treated with cetuximab and irinotecan. Methods: Germline DNA was obtained from peripheral blood of patients with mCRC KRAS and BRAF wild- type resistant to irinotecan treated with a combination of cetuximab and irinotecan. Seven functionally relevant SNP were selected on the basis of public literature resources and databases, 4 in the KSR1 and 3 in the KSR2 genes and analyzed by PCR followed by direct sequencing. All candidate SNPs were evaluated for association with response rate, PFS and OS. Results: A total of 66 patients were included.After a median follow-up of 13.3 months, median PFS and OS were 4.7 and 11.3 months respectively. Seventeen out of 66 patients (26%) achieved a RECIST response. In univariate analysis, KSR2 rs11068551 any G(AG/GG) genotype was significantly associated with shorter PFS compared to that of A/A carriers (median 3.5 vs. 7.4 months, HR=1.74 [95%CI: 1-3.04] p=0.049, log-rank test). This result remained significant in a multivariate model (HR=2.13 [95%CI: 1.15-3.95], p=0.017, log-rank test). None of the other tested SNPs were significantly associated with outcomes. Conclusions: Beyond KRAS and BRAF, some proteins including KSR are involved in modulating the level of activation of the EGFR pathway. Our results show that the functionally relevant KSR2 rs11068551 could affect PFS in advanced mCRC patients treated with cetuximab and irinotecan. Preclinical studies to confirm and further explain these preliminary findings are ongoing.

KSR1 gene polymorphism in mCRC patients treated with first-line FOLFIRI and bevacizumab.

3546 Background: Kinase suppressor of Ras (KSR) 1 is a scaffolding protein regulating the Raf/Mek/ERK cascade. Preclinical data showed that KSR1 could increase estrogen receptor transcriptional activity after exposure to estrogens. Recent retrospective analyses suggested a possible role for KSR1 rs2241906 C/T polymorphism in predicting the outcome of patients with mCRC. Methods: MCRC patients receiving first-line FOLFIRI+bevacizumab prospectively enrolled in a translational research program were selected on the basis of KRAS and BRAF mutational status availability. KSR1 rs2241906 polymorphism was analyzed on DNA extracted from peripheral blood by means of PCR and direct sequencing. Taking into account the connection between estrogen pathway and KSR1, subgroup analyses according to gender were pre-planned. Results: 287 patients were included. Main patients’ characteristics were the following: M/F=175/112; median age=62 (range 26-79); ECOG-PS 0/1-2=240/47; synchronous/metachronous disease=213/74; Köhne score (low/intermediate/high/data missing)=122/129/22/14; KRAS-BRAF mutational status (wt-wt/mut-wt/wt-mut)= 123/146/18. In the overall KRAS-BRAF wt population, KSR1 polymorphism did not affect the outcome. The analysis performed according to gender showed that in the KRAS-BRAF wt population females with KSR1 rs2241906 T/- achieved a significantly better PFS (median 15.9 mos) in comparison to C/C variant carriers (median 8.8 mos) (HR=0.44 [95%CI 0.21-0.91], p=0.010); meanwhile males with T/- showed a worse PFS in comparison to those carrying the C/C variant (HR=1.92 [95%CI 1.05-3.53], p=0.021). These results were significant also in a multivariate model and a significant interaction of gender with PFS according to KSR1 allelic variants was demonstrated (p=0.004). Conclusions: This prospectively conceived pharmacogenetic study confims the role of KSR1 polymorphisms in affecting the outcome of mCRC KRAS-BRAF wt patients. In particular, these results indirectly indicate that estrogenic stimulation could affect the proliferation of KRAS-BRAF wt CRC cells through an interaction with KSR1. Preclinical investigations to further explore this preliminary hypothesis are ongoing.

Genetic variants of kinases suppressors of ras (KSR1) to predict survival in patients with ER-alpha positive metastatic breast cancer.

e11018 Background: In addition to its activating role of MAPK pathway, the protein kinase suppressor of ras 1 (KSR1) has been identified as a novel modulator of estrogen receptor alpha (ERα), as silencing of KSR1 in ERα+ breast cancer (BC) cells lines resulted in dowregulation of ERα transcriptional activity in the presence of oestradiol. Our previously data revealed the existence of single nucleotide polymorphisms (SNPs) in EGFR and ERα pathway genes that may predict clinical outcome in ERα positive metastatic BC (mBC) patients. With respect to those findings we aimed to evaluate the clinical implication of KSR1 SNPs on survival in patients with primary ERα+ mBC treated with tamoxifen. Methods: Tumoral DNA was obtained from 222 patients treated with tamoxifen withERα+ invasive BC who had undergone surgery between 1981 and 2003 (median age 56 [28-90]). Three relevant KSR1 SNPs were selected based on public literature resources and databases, including 2 SNPs localised in the regulating 3’untranslated region (rs224190, rs 1075952) and 1 non-synonymous SNP localised in the coding exon 7 region (rs2293180). All SNP were analysed using DNA sequencing with OS and DFS as primary endpoint. Results: The overall median follow-up was 6.4 years.In univariate analysis the rs2241906 SNP was significantly associated with DFS and OS and patients with the TT genotypedemonstrated shorter DFS (2.1 vs. 7.1 months, p=0.005) and OS ( 2.6 vs. 8.4 months p=0.002) than patients with the x/C genotypes. The associations remained significant in the multivariate analysis adjusting age, lymph node status and HER2 status (HR (95%CI): 4.81 (2.00-11.59) and 5.74 (2.29-14.43), for DFS and OS, respectively). The same significant correlation was retrieved with the SNP rs1075952 in linkage disequilibrium with rs2241906 that was used as a control. No relationship was shown between rs2293180 and survival. Conclusions: Our findings demonstrated that KSR1 polymorphisms could arise as a potential marker to predict survival in patients with mBC treated with tamoxifen. The putative role of KSR1 as a modulator of ERα activity could functionally explain our results yielding insight for further studies.

Abstract 1842: PHT-782 an inhibitor of the KRAS signaling nanocluster protein CNKSR1 blocks mutant KRAS signaling and cell growth but does not inhibit wild type KRAS cell growth

Abstract KRAS is the predominant form of mutated RAS (mut-KRAS) found in 25% of patient tumors across many cancer types. Mut-KRAS plays a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. Using a global siRNA screen and an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth. A top hit in the screen was CNKSR1 (connector enhancer of kinase suppressor of RAS 1), a protein associated with KRAS in the RAS membrane signaling nanocluster. siRNA knockdown of CNKSR1 inhibited the growth of mut-KRAS non small cell lung cancer (NSCLC) cell lines but not NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein with a pleckstrin homology (PH) domain, a 100 to 120 amino acid, highly conserved but low sequence identity 3D superfold. PH-domains are found in a number of signaling proteins where they are responsible for binding to membrane phosphatidylinositols-3-phosphates (PIP2/3). Over expression of the PH-domain of CNKSR1 in H1373 mut-KRAS NSCLC cells inhibits cell growth suggesting it has a dominant negative effect over native CNKSR1, and that the PH-domain of CNKSR1 is necessary for the effects on mut-KRAS activity. We used in-silico docking of large compound libraries and identified 7 small molecule compounds with predicted low micromolar binding affinity for the PH-domain of CNSKR1. Surface plasmon resonance spectroscopy confirmed the in-silico predictions with binding to the recombinant PH-domain of CNKSR1. The most active compound was PHT-782 (Kd=1.8 μM). PHT-782 inhibited mut-KRAS signaling and the growth of mut-KRAS isogenic and NSCLC cell lines (IC50 ∼ 30 μM) as effectively as siRNA knockdown of KRAS. Importantly, PHT-782 did not block the growth of wt-KRAS cell lines at concentrations up to 100 μM. In mice PHT-782 exhibited a plasma t12 &amp;gt; 4 hr, and had moderate antitumor activity against a mut-KRAS xenograft model without observable toxicity at doses up to 200 mg/kg. To improve potency and selectivity we have undertaken further iterative in-silico lead optimization using a proprietary computational modeling platform PHuDock®. We have identified several candidates with improved binding potency and optimal pharmacodynamic and pharmacokinetic properties. The results of our studies show that the PH domain of CNKSR1 can be targeted by small molecule inhibitors and that the agents selectively block mut-KRAS signaling and cell growth, thus creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1842. doi:1538-7445.AM2012-1842

Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.

KSR1 (kinase suppressor of ras 1)

Review on KSR1 (kinase suppressor of ras 1), with data on DNA, on the protein encoded, and where the gene is implicated.

The oncogenetic role of microRNA-31 as a potential biomarker in oesophageal squamous cell carcinoma

miR-31 (microRNA-31) is frequently altered in numerous cancers. The aim of the present study was to investigate the role of miR-31 in ESCC (oesophageal squamous cell carcinoma). We measured miR-31 in 45 paired ESCC tissues and 523 serum samples using real-time RT (reverse transcription)-PCR. The serum samples were divided into a discovery group (120 ESCCs and 121 normal controls), a validation group (81 ESCCs and 81 controls), and a final group comprising six other common tumours (colorectal, liver, cervical, breast, gastric and lung cancers; total n=120). A Mann-Whitney U test and Wilcoxon matched-pairs test were used for the statistics. miR-31 was up-regulated in 77.8% of the ESCC tissues. Serum miR-31 levels in ESCC patients were significantly higher than in normal controls (P<0.001). It yielded an ROC (receiver operating characteristic) AUC (area under the curve) of 0.902 [95% CI (confidence interval), 0.857-0.936] in the discovery group and a similar result in the validation group [ROC AUC, 0.888 (95% CI, 0.819-0.939)]. Patients with high-levels of serum miR-31 also had a poorer prognosis in relapse-free survival (P=0.001) and tumour-specific survival (P=0.005). In vitro studies showed that miR-31 promoted ESCC colony formation, migration and invasion. Luciferase reporter and Western blot assays confirmed that three tumour suppressor genes, namely EMP1 (epithelial membrane protein 1), KSR2 (kinase suppressor of ras 2) and RGS4 (regulator of G-protein signalling 4), were targeted by miR-31. We conclude that miR-31 plays oncogenetic functions and can serve as a potential diagnostic and prognostic biomarker for ESCC.