US20040058359A1 - Erbin as a negative regulator of Ras-Raf-Erk signaling - Google Patents

Erbin as a negative regulator of Ras-Raf-Erk signaling Download PDF

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US20040058359A1
US20040058359A1 US10/446,640 US44664003A US2004058359A1 US 20040058359 A1 US20040058359 A1 US 20040058359A1 US 44664003 A US44664003 A US 44664003A US 2004058359 A1 US2004058359 A1 US 2004058359A1
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erbin
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ras
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Lin Mei
Wem Xiong
Yang Huang
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Definitions

  • This invention relates to use of Erbin as a novel suppressor of Ras for inhibition of cell, particularly malignant cell, proliferation.
  • Erbin has now been found to be a negative regulator of the Ras-Raf-Erk signaling pathway.
  • Extracellular signal regulated kinases are a subfamily of mitogen-activated protein kinases (MAPK) that play important roles in a great array of cell programs, including proliferation, differentiation and apoptosis.
  • MAPK mitogen-activated protein kinases
  • receptor typrosine kinases are activated and undergo autophophorylation on tyrosine residues.
  • the phosphorylated tyrosine residues recruit adaptor proteins to plasma membrane by directly interacting with modules including Sre homology 2 (SH2) or phosphotyrosine binding domain (PTB).
  • SH2 Sre homology 2
  • PTB phosphotyrosine binding domain
  • Grb2 one such adaptor, brings guanyl nucleotide exchange factor (SOS) to the plasma membrane in proximity with Ras and expedites exchange of GDP and GTP on Ras.
  • SOS guanyl nucleotide exchange factor
  • the activated Ras (GTP-bound) then directly binds to Raf and allows the latter to be activated at the plasma membrane.
  • Active Raf triggers sequential activation of MEK, a MAPK kinase, and Erk, leading to phosphorylation of various regulatory proteins, including nuclear transcription factors such as Erk-1 and Myc as well as many cytoplasmic proteins.
  • Ras-Raf-Erk pathway Several modulators have been identified that positively influence the pathway at different levels.
  • MEK partner 1 MP1
  • MP1 MEK partner 1
  • a second protein is the Kinase Suppresor of Ras (KSR) that is believed to act as a scaffold for Raf-1, MEK and Erk instead of a Raf kinase although it contains a kinase domain.
  • KSR Kinase Suppresor of Ras
  • a third such regulator is the Connector Enhancer of KSR (CNK) with multiple functional domains that directly binds to Raf and is involved in activation of the Raf/MEK/Erk pathway.
  • CNK Connector Enhancer of KSR
  • Sur-8 Another interesting protein is Sur-8 that contains multiple leucine-rich regions (LRR) and binds to both Ras and Raf-1. Although Ras can directly associate with Raf when it is activated by charging with GTP, the presence of Sur8 increases the interaction between Ras and Raf and the activation of downstream signaling events. These non-enzymatic factors are important regulators for normal cell proliferation and differentiation.
  • Ras pathway there are negative regulators of the Ras pathway in cells.
  • Sprouty a Ras suppressor in Drosophila and its mammalian homologue Spred (Sprouty-related EVH1 domain-containing protein) appear to serve as physiological negative feedback regulators of growth factor-mediated Erk pathway.
  • the Ras effector RINI has been shown to inhibit Ras-induced activation of Raf by competitively binding to active Ras.
  • the Raf kinase inhibitory protein (RKIP) initially isolated as a phosphatidylethanolamine binding protein, binds directly to the kinase domains of both Raf and MEK and inhibits MEK phosphorylation.
  • These negative regulators function to ensure that all programs are adequately executed through autonomous turn-on and turn-off mechanisms. They may also counterbalance over-amplified proliferative signals caused by active mutation of Ras frequently occurring in human cancers or mutation of upstream components leading to the increased activity of the wild-type Ras which is found as well in human cancers. Such an inhibitory mechanism is key to maintain normal cell growth rate.
  • Erbin belongs to the LAP (LRR and PDZ) protein family of PDZ domain-containing proteins.
  • the family members include LET-413 in C. elegans, Scribble, a Drosophila protein essential for epithelial integrity, Densin-180, and Lano in mammals. Genetic studies in non-vertebrate have demonstrated that LAP proteins play a role in cell polarity and cell morphology of epithelial cells.
  • Erbin was initially identified as a binding partner for ErbB2 and delta-catenin and ARVCF. This 180-kDa protein contains two domains: LRR and PDZ.
  • the sequence of Erbin has been deposited in GenBank and assigned No. AF 263744. The biological function of Erbin and its impact on ErbB2-mediated signaling have yet to be addressed.
  • the instant invention provides a method of inhibiting proliferation of maligant cells by administration of a Ras-activity inhibitory effective amount of DNA which encodes the protein Erbin into the maligant cells.
  • the amount of DNA administered is sufficient to provide a blood concentration of 1 ⁇ M to 100 ⁇ M of DNA which encodes Erbin into the cells.
  • the DNA encoding Erbin may be administered via viral vectors.
  • the invention also provides methods for evaluating proliferative properties and progression of proliferative activities in tumor cells by measurement of Erbin in tumor tissue.
  • This invention relates to use of Erbin, a known protein whose sequence is known and a DNA sequence encoding Erbin is deposited in GenBank as No. AF 263744, which is: aaagatcttt tttttttttttttttttttttttttttggcgga gatcctcgtt (SEQ ID. No.
  • Erbin is a negative regulator of the Ras-Raf-Erk signaling pathway. Expression of Erbin decreases neuregulin-induced transcription of AChR ⁇ -subunit gene, an event that requires Erk activation. Although it interacts with the ErbB2 C-terminus through the PDZ domain, Erbin has no effect on ErbB2 tyrosine phosphorylation and binding to adaptor proteins Shc or Grb2. In contrast, expression of Erbin greatly impairs Erk activation by ligands that activate receptor tyrosine kinases, without causing a significant change in AKT activity. It is now shown herein that Erbin diminishes the ability of Ras but not Raf-1 to activate Erk.
  • Erbin binds only to active Ras, as opposed to inactive Ras or Raf, resulting in inhibition of the interaction between Ras and Raf both in vivo and in vitro.
  • Erbin acts to suppress tumorigenesis of cancer cells, especially breast and prostate cancer cells.
  • the control of tumorigenesis by increasing level of Erbin in malignant tissue by various means, especially through gene therapy, to obtain a blood or target tissue cell concentration of about 1 ⁇ M to 100 ⁇ M concentration would be appropriate for control of malignancies.
  • Plasmid Construction The human Erbin N-terminal domain (aa 1-391) consisting of 16 LRRs was generated by PCR amplification using sense primer containing BamHI and antisense primer containing XhoI. The resulting 1.2 kb-fragment was digested with BamHI and XhoI, and subcloned in the BamHI-SalI sites of yeast vector pGBT9 downstream of the Gal4 DNA binding domain (Clontech). Myc-Erbin LRR, Myc-ErbinD965 and Myc-DPDZ were generated by introducing a stop codon after the LRR domain following aa 965 or aa 1279 in pRK5-Myc-Erbin.
  • N-terminal deletion mutant (pRK5-Erbin965) was described previously (Huang, Y. Z., Wang, Q., Xiong, W. C., and Mei, L. (2001) J Biol Chem 276, 19318-19326); and Borg, J. P., Marchetto, S., Le Bivic, A., Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Sydney, J., Margolis, B., and Birnbaum, D. (2000) Nat Cell Biol 2, 407-414).
  • pCMV-Flag-Erk1 was generously provided by Dr. Mike Weber (University of Virginia).
  • Flag-Ras, Flag-RasV12, Flag-RasN17, GST-Raf-C4, and GST-Raf-BXB were described as previously (Zang, M., Hayne, C., and Luo, Z. (2002) J Biol Chem 277, 4395-4405).
  • HEK 293 cells and COS-7 cells were cultured as described previously (Huang, supra).
  • the C2C2 cells were maintained as undifferentiated myoblasts in DMEM with high glucose supplemented with 20% fetal bovine serum, and 0.5% chicken embryo extract. Fusing of myoblasts into myotubes was induced by culturing myoblasts for 48 hours in differentiation medium DM (DMEM plus 4% horse serum).
  • DMEM fetal bovine serum
  • Rat lung epithelial Mv1Lu cells were maintained in DMEM plus 10% fetal bovine serum (FBS).
  • Rat pheochromocytoma-derived PC12 cells were grown in DMEM supplemented with 10% FBS and 5% horse serum.
  • HEK 293, COS-1 and C2C12 cells were transfected with the standard calcium phosphate technique.
  • PC12 cells and Mv1Lu cells were transfected with SuperFect regents (Qiagen).
  • Two days after transfection cells were washed with PBS and lysed in the modified RIPA buffer containing 20 mM sodium phosphate, pH 7.4, 50 mM sodium fluoride, 40 mM sodium pyrophosphate, 1% Triton X-100, 2 mM sodium vanadate, 10 mM p-nitrophenyl phosphate, and protease inhibitors. Lysed cells were incubated on ice for 20 min and centrifuged at 13,000 ⁇ g for 10 min at 4 C. The clear supernatant was designated as cell lysates.
  • Immunoprecipitation and Immunoblottina Cell lysates ( ⁇ 400 ⁇ g of protein) were incubated without or with indicated antibodies one hour at 40° C. and subsequently with protein A- or protein G-agarose beads overnight at 4° C. on a rotating platform. After centrifugation, beads were washed five times with the modified RIPA buffer. Bound proteins were eluted with the SDS sample buffer, resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher and Schuell).
  • Nitrocellulose membranes were incubated at room temperature for one hour in the blocking buffer containing Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% milk or 5% BSA followed by an incubation with indicated antibodies in the blocking buffer. After washing 3 times for 5 min each with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit IgG (Amersham Pharmacia Biotech) followed by washing. Immunoreactive bands were visualized with enhanced chemiluminescence substrate (Pierce).
  • the nitrocellulose filter was incubated in a buffer containing 62.5 mM Tris/HCl, pH 6.7, 100 mM ⁇ -mer-captoenthanol, and 2% SDS at 50° C. for 30 min, and washed with 0.1% Tween 20 in 50 mM TBS at room temperature for 1 hr, and reblotted with different antibodies.
  • the following antibodies were used: Flag (M2, Sigma), Myc (9E10, Santa Cruz), phospho-MAPK (Promega), phospho-Akt (Ser473, New England Biolab) and Erbin.
  • Luciferase Assay Myoblasts were co-transfected with or without Myc-Erbin, plus the ⁇ -subunit promoter-luciferase transgene that contains 416 nucleotides of the 5′UTR of the ⁇ -subunit gene (25) and a control plasmid pRL-SV40 (Promega). Twenty four hours after transfection, the myoblasts were incubated in DM to induce myotube formation. Myotube formation was complete 48 hours after switch to DM. The C2C12 myotubes were stimulated with neuregulin at a final concentration of 10 Nm at 37° C. for 24 hours.
  • Mv1Lu cells were transiently transfected with the promoter reporter construct p3TP-Lux, which promoter contains three AP-1 sites and the plasminogen activator inhibitor-1 (PAI-1) promoter and firefly luciferase.
  • pRL-SV40 that express Renilla luciferase under the control of SV40 promoter was cotransfected as a control to monitor the transfection efficiency.
  • Fourty eight hours after transfection cells were lysed and activities of the two different luciferases were assayed with respective substrates with a dual luciferase assay kit (Promega).
  • PC12 cells were cotransfected pEGFP with empty vector pRK5-Myc, Myc-Erbin or its mutants, or Erbin RNAi duplex. Forty eight hours after transfection, PC12 cells were stimulated by 100 ng/ml or 20 ⁇ g/ml NGF for 2 days. Cells were examined by fluorescence microscopy. Cells with processes 1.5 times longer than the diameter of the cell body were considered to be differentiated.
  • RNAi Inhibition of Erbin expression by RNAi—The target region of siRNA was 100 nt downstream of the start codon, which contained approximately 50% G/C content.
  • the nucleotide sequence was 5′ UAG ACU GAC CCA GCU GGA A dTdT 3′ (nt 866-884) (Borg, J. P., Marchetto, S., Le Bivic, A., Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Sydney, J., Margolis, B., and Birnbaum, D. (2000) Nat Cell Biol 2, 407-414).
  • the NCBI sequence bank was searched against this segment Cof DNA using the blast program, which revealed no match, suggesting of the specificity of target recognition by siRNA.
  • the 21-nucleotide RNAs were chemically synthesized by Dharmacon Research Inc. Synthetic oligonucleotides were deprotected and gel-purified.
  • To demonstrate the silencing effect of endogenous Erbin expression by siRNA cells in 60 mm-culture dish were co-transfected with empty vector pEGFP and with siRNA duplex using the SuperFect kit (Qiagen). Briefly, 2 mg of pEGFP and 30 ml of 20 mM RNAi duplex were mixed with 300 ml of Opti-MEM (GIBCO-BRL).
  • Opti-MEM was added to obtain a final volume of 1 ml.
  • Cells were incubated with the mixture for 2-3 hours at 37° C. and 5% CO before the addition of 5 ml of growth medium. Seventy two hours after transfection, cells were resuspended in PBS buffer.
  • GFP-positive cells were collected by fluorescence-activated cell sorting (FACS) analysis. Cells were lysed in modified RIPA buffer and lysates were subjected to immunoblotting for expression of Erbin. In parallel experiments, GFP-positive PC12 cells were scored for differentiation.
  • Protein Assay Protein was assayed with Coomassie Protein Assay Regent (Pierce) using bovine serum albumin as a standard.
  • Erbin inhibits neuregulin-induced transcription of the AChR ⁇ -subunit gene and Erk activation—It was found that transcription of AChR subunit genes is increased by neuregulin, a ligand that activates the ErbB2 receptor tyrosine kinase. Previous studies in this laboratory and by others have demonstrated that neuregulin-induced AChR expression requires ErbB2 tyrosine phosphorylation and activation of the Ras-Raf-Erk signaling pathway. It now is seen that Erbin, interacting with ErbB2, plays a role in regulating neuregulin signaling.
  • Erbin inhibits Erk activation by Ras—An attempt was made to dissect the position for Erbin action by walking upstream of Erk. Since ErbB2 directly interacts with the cytoplasmic domain of tyrosine kinase receptors, it is possible that Erbin interferes with the tyrosine kinase activation and/or subsequent binding to adaptor proteins. To test these hypotheses, it was decided to take the advantage of NeuT, an active form of ErbB2 (Bargmann, C. I., Hung, M. C., and Weinberg, R. A. (1986) Cell 45, 649-657).
  • Erbin disrupts the Ras-Raf interaction—In considering the mechanism of the Erbin-induced inhibition of the Erk pathway, it was postulated that Erbin inhibits the interaction between active Ras and Raf by competitive binding to either of them, or diminishing the GTP-bound form of Ras. To test this, an active mutant of Ras (Flag-RasV12) was coexpressed with GST-Raf1 into HEK293 cell. Flag-RasV12 was found to copurify with GST-Raf1. In a reciprocal study, Raf1 was detected in the immunoprecipitates of active Ras. Remarkably, coexpression of Erbin decreased the interaction between active Ras and Raf.
  • the Ras pull-down assay developed by Rooij and Bos in which they exploited the nature of high affinity of GTP-Ras for the Ras-binding domain (RBD) of Raf-1, as compared with GDP-Ras was employed.
  • the RBD (aa 50-150) was expressed as a GST-fusion protein and used to pull down GTP-Ras that had been activated inside cells cotransfected with or without Erbin.
  • GTP-Ras was recovered by the GST-Raf-RBD beads.
  • Expression of Erbin inhibited the interaction of Raf with growth factor-activated Ras, accompanied by a decrease in phospho-Erk1.
  • RNA interference (RNAi) techniques were used for this purpose.
  • sequence-specific double-stranded RNA are first employed in evolutionarily diverse organisms including plants, fungi, and metazoans (Hammond, S. M., Caudy, A. A., and Hannon, G. J. (2001) Nat Rev Genet 2, 110-119).
  • RNAi has been shown to specifically suppress the expression of endogenous and heterologous genes in mammalian cell lines.
  • RNAi duplexes directed against Erbin were synthesized and transfected in PC12 cells to suppress the expression of endogenous Erbin.
  • cells expressing co-transfected EGFP were sorted out and analyzed for Erbin expression by Western blot.
  • Expression of Erbin in RNAi-transfected cells was significantly decreased in comparison with missense RNAi-transfected cells (control).
  • the suppressing effect by Erbin RNAi appeared to be specific since it had no effect on expression of endogenous Erk1.
  • expression of cotransfected EGFP was unaffected.
  • RNAi-transfected PC12 cells were challenged with 20 ng/ml NGF to induce differentiation.
  • NGF a sub-maximal effective concentration of NGF was used, as at this concentration, NGF caused differentiation of only 25 ⁇ 2% PC12 cells.
  • suppressing Erbin expression by sense RNAi enhanced NGF-mediated cell differentiation (52 ⁇ 2%), while missense RNAi had no significant effect (18 ⁇ 2%).
  • Ad-Erbin or Erbin-LRR and control AdGFP adenoviruses are generated in BJ5183 (available from American Type Culture Collection (ATCC) as deposit JHU-18) bacterial cells by homologous recimbination of the viral pAdEasy-1 and pAdTrack CMV-Erbin or Erbin-LRR and pAdTrack-CMV, respectively.
  • ATCC American Type Culture Collection
  • Cells available from ATCC under deposit number CRL-1573 known as 293 cells are used as hosts for viral productions by known methods. (See Neuron. (2002), 35:489-505)
  • the adenoviruses expressing Erbin or Erbin-LRR are injected into the tumor tissue to infect cancer cells, particularly breast or prostate cancer cells.
  • mice are anesthetized with phenobarbital sodium.
  • Tumor tissues are injected with 100 ⁇ g of closed circular DNA (pcDNA3-Erbin or Erbin-LRR) at concentration of 1.0 ⁇ g/ ⁇ l in saline using a syringe with a 27-gauge needle.
  • injected tumor tissues are subjected to electroporation with a pair of tungsten needles inserted into the tumor encompassing DNA injection sites. Electric pulses are delivered using an electric pulse generator.
  • Erbin expression in tumor tissues and cells may be monitored by two methods: Western blot and immunohitochemical analysis, both using anti-Erbin antibody.
  • Tumor tissues are rapidly dissected and frozen in isopentane cooled with dry ice. Ten ⁇ M sections are prepared using a cryostat, thaw mounted on gelatin-coated slides and stored at ⁇ 80°C. Sections of tumor tissue sections are incubated with 2% normal goat serum (Vector Laboratories, Burlingame, Calif.) in PBS for 1 hour at room temperature to reduce background staining and then incubated with the affinity-purified and anti-Erbin antibody in 2% normal goat serum in PBS overnight at 4° C.
  • 2% normal goat serum Vector Laboratories, Burlingame, Calif.
  • the sections After wiashing the sections five times with PBS, each for 30 minutes, the sections are incubated with a flourescein istothiocyanate-conjugated anti-rabbit antibody (Zymed Laboratories Inc., San Francisco, Calif.) or visualized by the established DAB method. Fluorescent images are captured on Sony CCD camera mounted on a Nikon E600 microsope using Photoshop imaging software. The amount of Erbin is then determined.
  • a flourescein istothiocyanate-conjugated anti-rabbit antibody Zymed Laboratories Inc., San Francisco, Calif.
  • Fluorescent images are captured on Sony CCD camera mounted on a Nikon E600 microsope using Photoshop imaging software. The amount of Erbin is then determined.
  • the amount of Erbin as measured is measured against standards and against the amount of Erbin in normal tissues. It is also possible to evaluate progression of disease by measuring the amount of Erbin in samples from target tissue repeatedly.
  • Erbin functions to kick Raf off the plasma membrane by competing with Raf in binding to active Ras.
  • overexpression of Erbin diminishes the accessibility of Ras-GTP to Raf-1.
  • Erbin may compete with Sur-8 for binding to Ras and then dissociate the Sur 8/Ras/Raf ternary complex, resulting in down-regulation of the Erk pathway. It is conceivable that Erbin may act more quickly and efficiently than Sur 8, as it is enriched at the plasma membrane. Active mutations of the Ras gene renders it the most frequent oncogene found in human cancers and even more, many other oncogenes exploit Ras and its downstream cohorts to execute their functions.
  • Erbin for inhibitory mechanisms and blockers for this pathway is indicted as a means of inhibiting growth of malignant cells.
  • the method involved administration of a proliferation inhibiting effective amount of Erbin or the administration of a vector which gives rise to production of a proliferation inhibiting effective amount of Erbin.

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