Models of Melanoma Metastasis: Using a Transient siRNA‐Based Protein Inhibition Strategy in Mice to Validate the Functional Relevance of Pharmacological Agents

Arati Sharma1, Gavin P. Robertson2

1 The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, 2 PSU Melanoma Therapeutics Program, 500 University Drive, Hershey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.6
DOI:  10.1002/0471141755.ph1406s38
Online Posting Date:  September, 2007
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While a pharmacological agent may inhibit the activity of a protein in cultured cells by triggering a particular biological process, it may function differently in intact animals. Thus, an assay is needed to rapidly assess whether a drug candidate displays the same mechanism of action in vivo as in vitro. The experimental approach described in this unit utilizes synthetic siRNA in a transient animal assay to define the action of a drug candidate when inhibiting the activity of a particular gene. Commercially available synthetic siRNA is introduced into cancer cells by nucleofection to reduce protein expression. Cells are then introduced into animals and the mechanism responsible for tumor inhibition assessed. The action of a compound identified in vitro is then compared to that noted in vivo following siRNA‐mediated inhibition to determine whether it reduces tumor development in the same manner in both systems. Curr. Protoc. Pharmacol. 38:14.6.1‐14.6.15. © 2007 by John Wiley & Sons, Inc.

Keywords: melanoma; metastases; siRNA; pharmacological agents; GFP‐tagged cells; cancer; protein inhibition

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • GFP‐tagged metastatic human melanoma cell lines:
    • 1205 Lu (can be obtained from Dr. Meenhard Herlyn, Wistar Institute)
    • C8161.Cl9 (can be obtained from Dr. Danny R. Welch, University of Alabama)
    • UACC 903 M (can be obtained from Dr. Gavin Robertson, Penn State University College of Medicine)
  • DMEM (Invitrogen)
  • Fetal bovine serum (Hyclone)
  • Stealth siRNA for targeted genes and controls (Invitrogen); scrambled siRNA controls as well as siRNA to a non‐involved gene should be included (see recipe)
  • DEPC‐treated water (Invitrogen)
  • Fluorescently tagged siRNA (Alexa Fluor 546 labeled nonsilencing duplex siRNA; Qiagen, no. P1027098)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI)
  • Phosphate‐buffered saline (PBS)
  • 4% paraformaldehyde
  • Mounting medium with DAPI (Vectashield, Vector Laboratories)
  • Protein lysis buffer (see recipe)
  • BCA Protein Assay (Pierce)
  • Primary antibodies:
    • Anti–pErk and anti–pMek (Cell Signaling Technologies)
    • Antibodies to B‐Raf, C‐Raf, Erk 2, Cyclin D1, p27 and α‐enolase (Santa Cruz Biotechnology)
  • Horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology)
  • Blocking solution: TBS‐T (see recipe) containing 5% (w/v) nonfat dry milk
  • TBS‐T (see recipe)
  • Trypsin
  • Hanks' balanced salt solution without calcium, magnesium, and phenol red (Mediatech)
  • Nude mice: Female, age between 3‐ and 4‐weeks‐old upon arrival (Harlan)
  • UO126 (see recipe) or BAY 43‐9006 (see recipe), a nonspecific Raf kinase inhibitor
  • 4% paraformaldehyde in PBS (see recipe)
  • 0.5 M EDTA (Mediatech)
  • 70% ethanol
  • 0.01 M citrate buffer
  • 3% hydrogen peroxide (H 2O 2)
  • 1% bovine serum albumin (BSA)
  • Biotinylated anti–rabbit IgG (Vector Labs)
  • Peroxidase‐labeled streptavidin (BD Pharmingen)
  • AEC substrate kit (Zymed laboratories)
  • Hematoxylin
  • 37°C humidified incubator, 5% CO 2
  • Nucleofector (Amaxa)
  • Nucleofection kits (Amaxa); e.g., Cell Line Nucleofector Kit R (with Solution R) and program K‐17 for 1205 Lu, C8161.Cl9 and UACC 903 M cell lines
  • Cell culture dishes
  • 6‐well plates
  • Glass coverslips
  • Clear nail polish
  • Cell scraper (Sarstedt, no. 83.1830)
  • 200‐µl pipet
  • NuPage gel (Life Technologies)
  • Polyvinylidene difluoride (PVDF) membrane (Pall Corporation)
  • Novex western transfer apparatus (Invitrogen, no. EI0001 or no. EI9051)
  • Enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech)
  • 120‐W bulb
  • Mouse restrainer
  • 1‐ml syringes (Becton Dickinson)
  • 27‐G, 1/2‐in. needles (Becton Dickinson)
  • Alcohol prep pads (isopropyl alcohol, 70% v/v; NovaPlus)
  • Standard dissecting instruments, sterile
  • Microscopes
    • Nikon Eclipse E600 microscope with attached Cool SNAP Digital camera
    • Nikon SMZ 1500 dissecting microscope with a Plan Apo 1.6× objective and fluorescence detection capabilities
  • ImagePro analysis software (Scanalytics)
  • 1/2‐ml insulin syringes with 27‐G, 1/2‐in. needles (Becton Dickinson)
  • Slides (e.g., Fisherbrand Superfrost/plus microscope slides or similar product, Fisher Scientific)
  • 95°C water bath
  • Additional reagents and equipment for quantitation of total protein ( appendix 3A), counting cells using a hemacytometer (Phelan, ), euthanizing animals using CO 2 asphyxiation (Donovan and Brown, ), electrophoresis ( appendix 3B) and embedding and sectioning tissue ( appendix 3D)
NOTE: Duplicate experiments consisting of 8 to 10 animals per group should be performed.
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Literature Cited

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   Bollag, G., Freeman, S., Lyons, J.F., and Post, L.E. 2003. Raf pathway inhibitors in oncology. Curr. Opin. Investig. Drugs 4:1436‐1441.
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   Donovan J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
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   Hilger, R.A., Scheulen, M.E., and Strumberg, D. 2002. The Ras‐Raf‐MEK‐ERK pathway in the treatment of cancer. Onkologie 25:511‐518.
   Hingorani, S.R., Jacobetz, M.A., Robertson, G.P., Herlyn, M., and Tuveson, D.A. 2003. Suppression of BRAF(V599E) in human melanoma abrogates transformation. Cancer Res. 63:5198‐5202.
   Kramer, B.W., Gotz, R., and Rapp, U.R. 2004. Use of mitogenic cascade blockers for treatment of C‐Raf induced lung adenoma in vivo: CI‐1040 strongly reduces growth and improves lung structure. BMC Cancer 4:24.
   Kumar, R., Angelini, S., Czene, K., Sauroja, I., Hahka‐Kemppinen, M., Pyrhonen, S., and Hemminki, K. 2003. BRAF mutations in metastatic melanoma: A possible association with clinical outcome. Clin. Cancer Res. 9:3362‐3368.
   Lyons, J.F., Wilhelm, S., Hibner, B., and Bollag, G. 2001. Discovery of a novel Raf kinase inhibitor. Endocr. Relat. Cancer. 8:219‐225.
   Mercer, K.E. and Pritchard, C.A. 2003. Raf proteins and cancer: B‐Raf is identified as a mutational target. Biochim. Biophys. Acta. 1653:25‐40.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Reifenberger, J., Knobbe, C.B., Sterzinger, A.A., Blaschke, B., Schulte, K.W., Ruzicka, T., and Reifenberger, G. 2004. Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas. Int. J. Cancer 109:377‐384.
   Satyamoorthy, K., Li, G., Gerrero, M.R., Brose, M.S., Volpe, P., Weber, B.L., Van Belle, P., Elder, D.E., and Herlyn, M. 2003. Constitutive mitogen‐activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res. 63:756‐759.
   Sharma, A., Trivedi, N.R., Zimmerman, M.A., Tuveson, D.A., Smith, C.D., and Robertson, G.P. 2005. Mutant V599EB‐raf regulates growth and vascular development of malignant melanoma tumors. Cancer Res. 65:2412‐2421.
   Sharma, A., Tran, M.A., Liang, S., Sharma, A.K., Amin, S., Smith, C.D., Dong, C., and Robertson, G.P. 2006. Targeting mitogen‐activated protein kinase/extracellular signal‐regulated kinase in the mutant (V600E) b‐raf signaling cascade effectively inhibits melanoma lung metastases. Cancer Res. 66:8200‐8209.
   Smalley, K.S. 2003. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int. J. Cancer 104:527‐532
   Stahl, J.M., Sharma, A., Cheung, M., Zimmerman, M., Cheng, J.Q., Bosenberg, M.W., Kester, M., Sandirasegarane, L., and Robertson, G.P. 2004. Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res. 64:7002‐7010.
   Tuveson, D.A., Weber, B.L., and Herlyn, M. 2003. BRAF as a potential therapeutic target in melanoma and other malignancies. Cancer Cell 4:95‐98.
   Webb, C.P., Van Aelst, L., Wigler, M.H., and Woude, G.F. 1998. Signaling pathways in Ras‐mediated tumorigenicity and metastasis. Proc. Natl. Acad. Sci. U.S.A. 95:8773‐8778.
   Wilhelm, S., Carter, C., Lynch, M., Lowinger, T., Dumas, J., Smith, R.A., Schwartz, B., Simantov, R., and Kelley, S. 2006. Discovery and development of sorafenib: A multikinase inhibitor for treating cancer. Nat. Rev. Drug Discov. 5:835‐844.
Key References
   Sharma et al., 2006. See above.
  This is the original reference for this protocol. While it does not include the detail presented here it is recommended that this reference be read prior to undertaking this assay.
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