Kinase Suppressor Of Ras 1 Research Articles

Nucleobindin-2 Is a Positive Modulator of EGF-Dependent Signals Leading to Enhancement of Cell Growth and Suppression of Adipocyte Differentiation

Nucleobindin-2 is a 420-amino-acid EF-hand calcium-binding protein that undergoes proteolytic processing to generate an 82-amino-acid amino-terminal peptide termed nesfatin-1. To determine whether nucleobindin-2 has any biological function, nucleobindin-2 was either overexpressed or knocked down by short hairpin RNA in cultured CHO cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IE) and in 3T3-L1 cells. Reduction in nucleobindin-2 expression inhibited EGF-stimulated MAPK kinase (S217/S221) and Erk phosphorylation (T202/Y204). In contrast, there was no significant effect on EGF-stimulated EGF receptor phosphorylation, EGF receptor internalization, or 52-kDa Shc and c-Raf phosphorylation. Although kinase suppressor of Ras-1 and protein phosphatase 2A expression was not changed, intracellular calcium concentrations and PP2A activity was significantly increased in nucleobindin-2 knocked-down cells. Concomitant with these alterations in EGF-stimulated signaling, cell proliferation was significantly reduced in nucleobindin-2 knocked-down cells. Moreover, reduced nucleobindin-2 expression in 3T3-L1 preadipocytes resulted in a greater extent of 3T3-L1 cell adipocyte differentiation. Taken together, these data indicate that nucleobindin-2 regulates EGF-stimulated MAPK kinase/Erk signaling, cell proliferation, and adipocyte differentiation.

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.

Src inhibitors, PP2 and dasatinib, increase retinoic acid-induced association of Lyn and c-Raf (S259) and enhance MAPK-dependent differentiation of myeloid leukemia cells

All-trans-retinoic-acid (ATRA)-induced differentiation of human myeloid leukemia cells is characterized by persistent MAPK signaling. Fragmentary data suggests Src family kinase (SFK) inhibitors enhance differentiation and thus have potential therapeutic value. The present study shows that SFK inhibitors PP2 and dasatinib enhance aspects of MAPK signaling and regulate a panel of differentiation markers including CD11b and p47phox. HL-60 and NB4 myeloid leukemia cells show accelerated ATRA-induced G1/0 arrest/differentiation with inhibitor co-treatment. We also identified components of a Lyn- and c-Raf-containing MAPK signaling complex augmented by the inhibitors. PP2 and dasatinib increased ATRA-induced expression of Lyn and c-Raf (total and c-RafpS259) and their interaction. The Lyn-associated serine/threonine kinase CK2 also complexed with c-Raf and c-RafpS259, and the KSR1 scaffold protein bound c-Raf, Lyn, and ERK. c-Raf/ERK association was increased by the inhibitors, which is significant since ERK may cause c-Raf C-terminal domain (CTD) phosphorylation in a putative feedback mechanism. Consistent with this, inhibitor treatment caused more CTD phosphorylation. Lyn knockdown decreased c-Raf CTD and S259 phosphorylation. This is the first evidence suggesting SFK inhibitors enhance ATRA-induced differentiation through a possible feedback loop involving KSR1-scaffolded c-Raf and ERK complexed with Lyn and CK2.

Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

Genetic disruption of the scaffolding protein, Kinase Suppressor of Ras 1 (KSR1), differentially regulates GM-CSF-stimulated hyperproliferation in hematopoietic progenitors expressing activating PTPN11 mutants D61Y and E76K

Activating PTPN11 mutants promote hematopoietic progenitor hyperactivation of Erk and hypersensitivity to GM-CSF. We hypothesized that Kinase Suppressor of Ras 1 (KSR1) contributes to activating PTPN11-induced GM-CSF hypersensitivity. Bone marrow progenitors from WT and KSR1−/− mice expressing WT Shp2, Shp2E76K, or Shp2D61Y were evaluated functionally and biochemically. KSR1 activation and interaction with phospho-Erk was enhanced in Shp2D61Y- and ShpE76K-expressing cells. Genetic disruption of KSR1 partially normalized Shp2E76K-induced GM-CSF hypersensitivity, but failed to correct Shp2D61Y-induced GM-CSF hypersensitivity. Collectively, these studies suggest that cells expressing Shp2E76K have a greater dependence on KSR1 for GM-CSF hypersensitivity than cells expressing Shp2D61Y.

Compartmentalization of the MAPK scaffold protein KSR1 modulates synaptic plasticity in hippocampal neurons.

ERK1/2 is required for certain forms of synaptic plasticity, including the long-term potentiation of synaptic strength. However, the molecular mechanisms regulating synaptically localized ERK1/2 signaling are poorly understood. Here, we show that the MAPK scaffold protein kinase suppressor of Ras 1 (KSR1) is directly phosphorylated by the downstream kinase ERK1/2. Quantitative Western blot analysis further demonstrates that expression of mutated, feedback-deficient KSR1 promotes sustained ERK1/2 activation in HEK293 cells in response to EGF stimulation, compared to a more transient activation in control cells expressing wild-type KSR1. Immunocytochemistry and confocal imaging of primary hippocampal neurons from newborn C57BL6 mice further show that feedback phosphorylation of KSR1 significantly reduces its localization to dendritic spines. This effect can be reversed by tetrodotoxin (1 μM) or PD184352 (2 μM) treatment, further suggesting that neuronal activity and phosphorylation by ERK1/2 lead to KSR1 removal from the postsynaptic compartment. Consequently, electrophysiological recordings in hippocampal neurons expressing wild-type or feedback-deficient KSR1 demonstrate that KSR1 feedback phosphorylation restricts the potentiation of excitatory postsynaptic currents. Our findings, therefore, suggest that feedback phosphorylation of the scaffold protein KSR1 prevents excessive ERK1/2 signaling in the postsynaptic compartment and thus contributes to maintaining physiological levels of synaptic excitability.

KSR1 Is Overexpressed in Endometrial Carcinoma and Regulates Proliferation and TRAIL-Induced Apoptosis by Modulating FLIP Levels

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.

KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1 −/− colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

Pharmacologic Inactivation of Kinase Suppressor of Ras1 Sensitizes Epidermal Growth Factor Receptor and Oncogenic Ras-Dependent Tumors to Ionizing Radiation Treatment

Selective enhancement of tumor response to radiation therapy is a highly attractive objective, but it has not been met clinically. Gain-of-function Ras (gf) signaling via hyperactivation of receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), or via oncogenic mutation of Ras is shown to confer radioresistance and requires the engagement of the Raf/MEK/ERK pathway. However, upstream mediators of such interaction in cancer cells that could be targeted for radiosensitization have not been identified and characterized. Here, we provide original observations both in vitro and in vivo that kinase suppressor of Ras1 (KSR1) is a new target for reversing gf Ras-mediated radioresistance. We employed EGFR-dependent A431 squamous cell carcinoma (SCC) and genetically defined the molecular function of KSR1 in irradiation-induced Raf/MEK/ERK activation. In vitro KSR1 inactivation via genetic inhibition of its expression or kinase function abrogated ionizing radiation-induced activation of the Raf/MEK/ERK2 cascade, enhanced the cytotoxic effect of radiation, and achieved radiosensitization associated with inhibition of DNA damage repair and enhancement of clonogenic death. In vivo pharmacologic inactivation of KSR1 by KSR1 AS-ODN infusion leads to radiosensitization in EGFR-dependent A431 SCC and in oncogenic K-Ras-driven A549 human non-small cell lung carcinoma. These observations collectively establish KSR1 as a novel target for radiosensitization and show the feasibility of using KSR1 AS-ODN as a radiosensitizer for treating gf Ras-dependent human malignancies. Identification of such mediators of gf Ras signaling in response to irradiation holds promises for improving the therapeutic efficacy of radiation therapy and our ability to eradicate tumor.

VRK2 Inhibits Mitogen-Activated Protein Kinase Signaling and Inversely Correlates with ErbB2 in Human Breast Cancer

The epidermal growth factor (EGF)-ErbB-mitogen-activated protein kinase (MAPK) transcription signaling pathway is altered in many types of carcinomas, and this pathway can be regulated by new protein-protein interactions. Vaccinia-related kinase (VRK) proteins are Ser-Thr kinases that regulate several signal transduction pathways. In this work, we study the effect of VRK2 on MAPK signaling using breast cancer as a model. High levels of VRK2 inhibit EGF and ErbB2 activation of transcription by the serum response element (SRE). This effect is also detected in response to H-Ras(G12V) or B-Raf(V600E) oncogenes and is accompanied by a reduction in phosphorylated extracellular signal-regulated kinase (ERK) levels, p90RSK levels, and SRE-dependent transcription. Furthermore, VRK2 knockdown has the opposite effect, increasing the transcriptional response to stimulation with EGF and leading to increased levels of ERK phosphorylation. The molecular mechanism lies between MAPK/ERK kinase (MEK) and ERK, since MEK remains phosphorylated while ERK phosphorylation is blocked by VRK2A. This inhibition of the ERK signaling pathway is a consequence of a direct protein-protein interaction between VRK2A, MEK, and kinase suppressor of Ras 1 (KSR1). Identification of new correlations in human cancer can lead to a better understanding of the biology of individual tumors. ErbB2 and VRK2 protein levels were inversely correlated in 136 cases of human breast carcinoma. In ErbB2(+) tumors, there is a significant reduction in the VRK2 level, suggesting a role for VRK2A in ErbB2-MAPK signaling. Thus, VRK2 downregulation in carcinomas permits signal transmission through the MEK-ERK pathway without affecting AKT signaling, causing a signal imbalance among pathways that contributes to the phenotype of breast cancer.

KSR1 Protects From Interleukin-10 Deficiency-Induced Colitis in Mice by Suppressing T-Lymphocyte Interferon-γ Production

Immunological disorders of the gastrointestinal tract such as inflammatory bowel disease often result in recurrent and persistently elevated levels of proinflammatory cytokines. Kinase suppressor of Ras 1 (KSR1) is involved in tumor necrosis factor-mediated colon epithelial cell survival, yet its role in chronic inflammation has not been defined. In this study, we tested the hypothesis that KSR1 is protective against spontaneous experimental colitis. KSR1(-/-)Interleukin-10 (Il10)(-/-) mice were generated and histolopathologic parameters of intestinal inflammation were scored. Bone marrow transplants performed on wild-type and KSR1(-/-)Il10(-/-) mice determined the contribution of KSR1 in hematopoietic lineages. Mucosal T helper (Th) 1 and Th17 cytokine were also examined. In vitro Th1 and Th17 polarization assays were conducted and interleukin (IL)-17A and interferon-γ (IFN-γ) production analyzed by flow cytometry. Neutralizing antibodies against IgG, IL-17A, or IFN-γ were administered to 3-week-old KSR1(-/-)Il10(-/-) mice for 3 weeks and scored for colitis. KSR1(-/-)Il10(-/-) mice developed accelerated and severe spontaneous colitis by 4 weeks of age. KSR1 expression in hematopoietic lineages was protective against colitis. Both IFN-γ and IL-17A transcripts were elevated in colons of KSR1(-/-) and KSR1(-/-)Il10(-/-) mice. IFN-γ production was increased in lamina propria T cells isolated from KSR1(-/-) and KSR1(-/-)Il10(-/-) mice. Additionally, in vitro Th1 polarization was increased while Th17 polarization was impaired in KSR1-deficient naïve T cells. Finally, administration of IFN-γ neutralizing antibodies attenuated colitis in KSR1(-/-)Il10(-/-) mice. Mice lacking both KSR1 and IL-10 develop exacerbated colitis due to dysregulated IFN-γ production in T lymphocytes.

Kinase suppressor of Ras 1 is required for full ERK activation in thymocytes but not for thymocyte selection

The scaffold protein kinase suppressor of Ras 1 (KSR1) is critical for efficient activation of ERK in a number of cell types. Consistent with this, we observed a defect in ERK activation in thymocytes that lack KSR1. Interestingly, we found that the defect was much greater after PMA stimulation than by CD3 activation. Since ERK activation is believed to be important for thymocyte development, we analyzed thymocyte selection in KSR1-deficient (KSR1(-/-) ) mice. We found that positive selection in two different TCR transgenic models, HY and AND, was normal. On the other hand, negative selection in the HY model was slightly impaired in KSR1(-/-) mice. However, a defect in negative selection was not apparent in the AND TCR model system or in an endogenous superantigen-mediated model of negative selection. These results suggest that, despite a requirement for KSR1 for full ERK activation in thymocytes, full and efficient ERK activation is not essential for the majority of thymocyte selection events.

Modulation of the oncogene-dependent tissue factor expression by kinase suppressor of ras 1

Introduction Tissue factor (TF) is the key trigger of the coagulation cascade and the membrane signalling receptor for coagulation protease, factor VIIa. In cancer, TF has been implicated in tumor cell survival, growth, and angiogenesis, and is upregulated as a result of oncogenic transformation. Materials and Methods We assayed TF expression and tumourigenicity in mice in the case of human cancer cell lines expressing oncogenic receptor tyrosine kinases. These cells were also subjected to genetic modulation of the kinase suppressor of ras 1 (KSR1), and treated with oncoprotein inhibitors in vitro and in vivo. Results Here we show that herceptin, AG1478 and CI-1033, inhibitors of two different members of the ErbB family of oncogenes (HER-2 and EGFR), reduce TF levels in epithelial cancer cells. In EGFR-driven A431 cells, TF upregulation is diminished upon genetic targeting of KSR1, the scaffolding protein involved in EGFR signalling. Conversely, upregulation of KSR1 in A431 cells increases their TF expression and tumourigenicity in mice. The latter property remains dependent on EGFR, as pan-Erb (EGFR) inhibitor, CI-1033, blocks TF promoter activity and inhibits tumour formation by the parental and KSR1 overexpressing A431 cells. Conclusions KSR1 emerges, as an important modulator of TF expression in EGFR-driven cancer cells, which also impacts their aggressiveness in vivo

Kinase suppressor of Ras transphosphorylates c-Raf-1

Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr 269, the putative target for KSR1 kinase activity. We show that this antibody detects Thr 269-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr 269 to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr 269. These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1.

A Preformed Signaling Complex Mediates GnRH-Activated ERK Phosphorylation of Paxillin and FAK at Focal Adhesions in LβT2 Gonadotrope Cells

Most receptor tyrosine kinases and G protein-coupled receptors (GPCRs) operate via a limited number of MAPK cascades but still exert diverse functions, and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however, the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is preformed, and exposure of L beta T2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, protein kinase C delta, -epsilon, and -alpha, Ras, MAPK kinase 1/2, ERK1/2, tubulin, focal adhesion kinase (FAK), paxillin, vinculin, caveolin-1, kinase suppressor of Ras-1, and the GnRHR. Exposure to GnRH (5 min) causes MAPK kinase 1/2, ERK1/2, tubulin, vinculin, and the GnRHR to detach from c-Src, but they reassociate within 30 min. On the other hand, FAK, paxillin, the protein kinase Cs, and caveolin-1 stay bound to c-Src, whereas kinase suppressor of Ras-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin, and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. Receptor tyrosine kinases and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multiprotein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.

KSR1 Is Required for Cell Cycle Reinitiation Following DNA Damage

KSR1 (kinase suppressor of Ras 1) is a molecular scaffold and positive regulator of the Raf/MEK/ERK phosphorylation cascade. KSR1 is required for maximal ERK activation induced by growth factors and by some cytotoxic agents. We show here that KSR1 is also required for maximal ERK activation induced by UV light, ionizing radiation, or the DNA interstrand cross-linking agent mitomycin C (MMC). We further demonstrate a role for KSR1 in the reinitiation of the cell cycle and proliferation following cell cycle arrest induced by MMC. Cells lacking KSR1 underwent but did not recover from MMC-induced G(2)/M arrest. Expression of KSR1 allowed KSR1(-/-) cells to re-enter the cell cycle following MMC treatment. However, cells expressing a mutated form of KSR1 unable to bind ERK did not recover from MMC-induced cell cycle arrest, demonstrating the requirement for the KSR1-ERK interaction. In addition, constitutive activation of ERK was not sufficient to promote cell cycle reinitiation in MMC-treated KSR1(-/-) cells. Only cells expressing KSR1 recovered from MMC-induced cell cycle arrest. Importantly, MMC-induced DNA damage was repaired in KSR1(-/-) cells, as determined by resolution of gamma-H2AX-containing foci. These data indicate that cell cycle reinitiation is not actively signaled in the absence of KSR1, even when DNA damage has been resolved. These data reveal a specific role for the molecular scaffold KSR1 and KSR1-mediated ERK signaling in the cellular response to DNA interstrand cross-links.

Activation of the ERK signal transduction pathway by Epstein–Barr virus immediate-early protein Rta

BRCA1-associated protein 2 (BRAP2) is known to interact with the kinase suppressor of Ras 1 (KSR1), inhibiting the ERK signal transduction cascade. This study found that an Epstein-Barr virus (EBV) immediate-early protein, Rta, is a binding partner of BRAP2 in yeast and confirmed the binding in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation in 293(maxi-EBV) cells. Binding studies also showed that Rta and KSR1 interacted with the C-terminal 202 aa region in BRAP2. Additionally, Rta appeared to prevent the binding of KSR1 to BRAP2, activating the ERK signal transduction pathway and the transcription of an EBV immediate-early gene, BZLF1. Activation of the ERK signal transduction pathway by Rta may be critical for the maintenance of the lytic state of EBV.

Downregulation of KSR1 in pancreatic cancer xenografts by antisense oligonucleotides correlates with tumor drug uptake

While antisense oligonucleotide (AS-ODN) technology holds promise for the treatment of cancer, to date there have been no clinical successes. Unfortunately, current assays are not sufficiently sensitive to measure tissue ODN levels. Hence it has not been possible to ascertain whether treatment failures result from failure of drug delivery. To investigate the relationship between drug uptake and therapeutic effect, we developed an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay (NCHL-ELISA) to quantify Kinase Suppressor of Ras1 (KSR1) AS-ODN drug uptake in plasma and tumor tissues. In mice harboring PANC-1 pancreatic cancer xenografts and continuously infused with AS-ODN, our ELISA detects plasma and tumor KSR1 AS-ODN levels over an extended range, from 0.05 nM to 20 nM. Using this sensitive assay, we demonstrate that KSR1 repression in pancreatic cancer xenografts correlates highly with AS-ODN uptake into tumor tissues. In contrast, plasma drug levels do not correlate with tumor drug content or target downregulation. These studies indicate the efficacy of our ELISA, and suggest that tumor biopsy material will need to be procured to estimate the potential of this antisense technology.

HKSR-2, a vitamin D–regulated gene, inhibits apoptosis in arabinocytosine-treated HL60 leukemia cells

Ras signaling can be modulated by the scaffolding activity of kinase suppressor of Ras-1 (KSR-1) and by the hKSR-2 protein, resulting in diverse phenotypic outcomes. The mitogen-activated protein kinase cascade downstream from Ras and KSRs includes Raf-1 and extracellular signal-regulated kinase 1/2 kinases, known to enhance survival potential of a range of cell types. Because the molecular events that increase survival of HL60 cells induced to differentiate toward monocytic phenotype by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are not known, we investigated if KSR proteins provide a survival function in these cells. We found that whereas kinase suppressor of Ras-1 had no detectable effect on cell survival in the system studied here, 1,25-(OH)2D3-induced up-regulation of hKSR-2 enhanced the resistance of HL60 cells to arabinocytosine. Knockdown of hKSR-2 by either small interfering RNA or antisense oligonucleotides increased arabinocytosine-induced apoptosis, which was accompanied by reduced Bcl-2/Bax and Bcl-2/Bad ratios, and increased caspase-3 activating cleavage. In contrast, up-regulation of Mcl-1 was not abrogated by anti-sense (AS) AS-hKSR-2, pointing to a specific role of Bcl-2 in control of 1,25-(OH)2D3-induced increased cell survival. These findings are consistent with the previously shown lack of fully differentiated monocytic cells in HL60 cultures exposed to 1,25-(OH)2D3 in which hKSR-2 was knocked down, suggesting that optimal differentiation of these cells requires enhanced antiapoptotic mechanisms provided, at least in part, by hKSR-2. Collectively, these results suggest that hKSR-2 may offer a new target for novel therapies of acute myelogenous leukemia.

Characterization of kinase suppressor of Ras-1 expression and anticancer drug sensitivity in human cancer cell lines

Previous studies have indicated that the ERK1/2 MAP kinase signaling pathway plays an important role not only in cell growth, cell cycle regulation, and differentiation, but also in determining the sensitivity of cells to anticancer agents as well. Furthermore, expression of kinase suppressor of Ras-1 (KSR1), a molecular scaffold that modulates signaling through the ERK1/2 MAP kinase pathway, has been shown to influence the cellular sensitivity to the anticancer agent cisplatin. To further define the role of KSR1 expression on drug sensitivity, the expression of KSR1 was examined in the NCI60 anticancer drug screen, a panel of cancer cell lines representing nine tissue types, established by the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI). The expression of thousands of molecular targets has been examined in the NCI60 panel as well as the cellular toxicity for greater than 400,000 compounds. KSR1 expression varied almost 30-fold difference between the highest and lowest expressing cell lines in the NCI60. Using the COMPARE analysis algorithm, KSR1 expression was correlated with sensitivity of the compounds screened by DTP and several novel agents were identified whose sensitivity correlated with KSR1 expression in the NCI60 panel. Cytotoxicity of two agents, cytochalasin H and tunicamycin, identified through the COMPARE analysis of KSR1 expression and drug sensitivity, was also examined in wild type (KSR(+/+)) mouse embryo fibroblasts (MEFs) and MEFs deficient in KSR1 expression (KSR1(-/-)). These studies demonstrated enhanced sensitivity, as well as increased ERK activation, in KSR(-/-) MEFs following exposure to tunicamycin or cytochalasin H compared to KSR(+/+) MEFs. Furthermore, restoration of KSR1 expression in KSR(-/-) MEFs following stable transduction of cells with a KSR1 expression vector, enhanced sensitivity of cells to tunicamycin and cytochalasin H and decreased ERK1/2 activation following exposure to these drugs. In addition, the sensitivity to cytochalasin H and tunicamycin of breast cancer cell lines with low KSR1 expression, (HS578T and MDA-MB-231/ATCC), was increased relative to the sensitivity of breast cancer cells with higher levels of KSR1 (MCF7). These studies indicate that KSR1 may play an important role in the determination of cellular sensitivity to anticancer agents.