Blockading RAS Signalling Controls Tumour Growth

Blockading RAS signalling controls tumour growth

Supplementary Information

MATERIALS AND METHODS

Expression vectors

A vector for expression of oncogenic mutant H-RASG12V (pcDNA3-HRASV12) was made by sub-cloning full-length human H-RASG12V cDNA into the EcoRI site of pcDNA3 (Invitrogen). The iDab#6-memb expression vector (pEF-iDabVH6) was made by sub-cloning the anti-RAS iDab#6-memb (Tanaka et al., 2003) into the SfiI and NotI sites of pEF-FLAG-Memb (Tanaka and Rabbitts, 2003).

Transgenic mice

CC10-Cre and CC10-iDab#6-memb mice: A CC10 genomic transgenic cassette was constructed by sub-cloning the NotI and blunt-ended BamHI digested fragment comprising human growth hormone polyA signal from pLck-hGH plasmid (Wildin et al., 1995) into the NotI and blunt-ended XhoI sites of pMG2 (Forster et al., 2005) to give pMG2-pA. The HindIII fragment of Rat Clara cell 10kDa promoter from pCC10CAT-2300 (Stripp et al., 1992) was sub-cloned into the HindIII site of pMG2-pA to give pCC10-pA. The final CC10-Cre transgene was made by sub-cloning the blunt-ended XbaI/XhoI fragment containing the Cre cDNA from pPGK-Cre (a gift from Kurt Fellenberg) into the EcoRV site of pCC10-pA. The CC10-iDab#6-memb transgene was made by PCR amplification of the anti-RAS iDab#6-memb with an N-terminal FLAG-tag and a C-terminal farnesylation signal peptide from pGCiG-anti-RAS VH#6 (Tanaka et al., 2007), cleaving the PCR fragment with BamHI and cloning into the BamHI site of pCC10-pA. Transgene fragments (prepared by excision with SceI) were injected into pronuclei of fertilised eggs and transgenic strains were developed. The lines with high copy number of the transgene were selected for further use and called CC10-Cre and CC10-iDab#6-memb.

Conditional K-RasV12 mice: The mice bearing the Cre-dependent oncogenic K-Ras transgene (Lox-GFP-polyA-Lox K-Ras driven by the b-actin promoter) were kindly provided by Prof. A. Berns (Meuwissen et al., 2001).

Genotyping of the adult mice was performed by Southern filter hybridization of genomic DNA prepared from mouse tissues including tail biopsies by general phenol/chloroform methods. Ten µg DNA were digested to completion with EcoRI, separated on 0.8% agarose gels and transferred to positive-charged nylon membranes after denaturation. The membranes were hybridized with anti-RAS iDab#6-memb cDNA (for genotyping of the CC10-iDab#6-memb allele), Cre cDNA (for genotyping CC10-Cre) or EGFP cDNA (for genotyping the conditional transgenic K-RasV12) using randomly 32P-dCTP labeled probes.

To confirm Cre recombinase activation in lung Clara cells of CC10-Cre transgenic mice, the mouse line was crossed with the lacZ reporter mouse line ROSA26 (Soriano, 1999). After genotyping by filter hybridization, lungs of the mice heterozygous for the two alleles (CC10-Cre; ROSA26) were extracted and stained with b-galactosidase by perfusing X-gal staining solution (0.4 mg/ml X-gal, 5 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide in PBS) through the trachea and immersing the whole lung in excess X-gal solution overnight at 37 ˚C.

Expression of anti-RAS iDab#6-memb in lungs of CC10-iDab#6-memb transgenic mice was confirmed by Western blotting. Lungs were removed from the founder mice and snap-frozen on dry ice. They were homogenized in NP-40 lysis buffer and protein samples were immunoprecipitated with agarose-conjugated anti-FLAG monoclonal antibody (anti-FLAG M2 affinity gel, Sigma), fractionated on 15% SDS-PAGE and stained with the same anti-FLAG antibody (M2, sigma).

Tumour group mice were observed for between 50 and 350 days (or until appearance of any adverse symptoms) and humanely sacrificed, followed by dissection and macroscopic plus microscopic examination. Sections were made after fixation in 10% neutral buffered formaldehyde, embedded in paraffin and 4mm sections stained with haematoxylin and eosin.

Tetracycline-inducible cell lines.

The iDab#6-memb conditional expression clone pTet0-iDab#6-memb-ires-GFP was generated by sub-cloning the fragment containing anti-RAS iDab#6-memb with an in-frame FLAG-tag at the N-terminus and membrane localization signal peptide at C-terminus, an internal ribosomal entry site (IRES) and GFP from pGC-IRES-iDab#6-memb (Tanaka et al., 2007) into the SacII and BamHI sites of the plasmid pUHD10-3 (Gossen et al., 1995).

The human cancer cell lines HT1080, HCT116 and SW480 and their derivatives were grown in DMEM with 10 % FCS and penicillin and streptomycin (P/S), the cell line DLD1 grown in RPMI with 10% FCS and P/S. To establish cells with the tetracycline-inducible (TET-ON) system in which tetracycline/doxycycline can induce gene expression in the cells, the parental cell lines were transfected with pUHD172-1neo (Gossen et al., 1995) and selected with 1 mg/ml of G418 (Geneticin, Invitrogen). Colonies obtained after selection were individually tested by transient transfection with pTet0-iDab#6-memb-ires-GFP that allows GFP expression in a reverse tetracycline transactivator (rtTA)-dependent fashion after treatment of cells with 1 mg/ml doxycycline 24 hours after transfection. Analysis of fluorescence was by flow cytometry after an additional 24 hrs.

One clone from each parental cell line (viz. HT1080-tetON, DLD1-tetON, HCT116-tetON and SW480-tetON) were chosen to generate inducible cell lines for further analysis. The pTet0-iDab#6-memb-ires-GFP construct were linearized with ScaI and co-transfected into each of the TET-ON clones with linearized pPGK-hygro (te Riele et al., 1990) at a 20 to 1 molar ratio. Single colonies were obtained by selection with 1 mg/ml G418 and 0.3 mg/ml hygromycin B (Calbiochem) for 1 week. The clones were tested by growth with and without 1 mg/ml doxycycline and flow cytometric detection of green fluorescence after 48 hrs. Clones showing low background GFP expression in the absence of doxycycline and the highest percentage of GFP-positivity in the presence of doxycycline were selected. Doxycycline-dependent expression of iDab#6-memb in the chosen clones (HT1080-tetON-iDabVH, DLD1-tetON-iDabVH, HCT116-tetON-iDabVH and SW480-tetON-iDabVH) was confirmed by Western blot analysis with anti-FLAG antibody (M2, Sigma).

To establish these lines with ubiquitous Firefly luciferase expression for bioluminescence imaging, pEF-FLuc was generated by sub-cloning the fragment of Firefly luciferase cDNA with a polyA signal into the NcoI and XbaI sites of pEF/myc/cyto (Invitrogen). The plasmid were linearized with XmnI and co-transfected into each TET-ON-iDab#6-memb cell together with pPGK-puro (Tucker et al., 1996) at a 20 to 1 molar ratio. Single colonies were obtained by selection with 1 mg/ml G418, 0.3 mg/ml hygromycin B (Calbiochem) and 1-5 mg/ml puromycin for about 1 week. The luciferase expression in each clone was assayed with the Dual-luciferase assay (Promega) according to Manufacture’s instructions and the clones HCT116-tetON-iDab#6-memb-luc and SW480-tetON-iDab#6-memb-luc were used for the mouse transplantation assays.

Animal studies

All animal studies including the transgenic mouse model, the xeno-transplant tumourigenecity assay in immunodeficient mice and mouse imaging were performed under a licence complying with Home Office Animals (Scientific Procedures) Act 1986 (UK) and approved by the University of Leeds, Leeds Institute of Molecular Medicine Institutional Ethical Committee.

CC10-Cre and CC10-iDab#6-memb mice were generated and validated as described. Conditional K-RasV12 mice were kindly provided by Prof. A. Berns (Meuwissen et al., 2001). These mice were crossed with CC10-Cre transgenic mice to generate mice carrying both alleles (conditional KRasV12; CC10-Cre). Mice with both transgenes were also crossed to CC10-iDab#6-memb transgenic mice to generate mice heterozygous for the three alleles (conditional KRasV12; CC10-Cre; CC10-iDab#6-memb).

Tumourigenecity assays using human cancer cell xeno-grafts in mice.

Cell lines carrying a tetracycline-responsive anti-RAS iDab#6-memb conditional gene (described above) were used for xeno-grafts. 106 HCT116-tetON-iDab#6-memb-luc cells were injected subcutaneously into each flank of 6 to 8 week-old CB17/Lcr-Prkdcscid/Crl SCID mice (Charles River) or 106 SW480-tetON-iDab#6-memb-luc or parental SW480-tetON cells were injected into each flank of 6 to 8 week-old CD-1 nude mice. The mice were fed with normal diet and water until their subcutaneous tumour reached palpable size (> 4 mm diameter, approximately 5 days after injection). The mice were then divided two groups and one of which was supplied with Doxycyclin via drinking water (2 mg Dox/ml in 10% black-currant juice) and the Dox Diet (200 mg/kg, BIO-serve). Subcutaneous tumour growth was monitored by measuring tumour size twice a week with callipers and by bioluminescence as described below. The assay was terminated when the tumour sizes reached a maximum of 17 mm. After humane sacrifice, the mice were dissected and pathological and biological examinations were performed.

Mouse tumour bioluminescence imaging (BLI).

Bioluminescence was measured in transplanted subcutaneous tumours non-invasively using the IVIS imaging system (Xenogen Corp) following injection of the luciferase substrate luciferin as described (Tanaka and Rabbitts, 2008). All of the images were taken after intraperitoneal injection of luciferin (140 mg/kg body weight; Synchem). During image acquisition, mice were sedated continuously by inhalation of 3% isofluorane. Image analysis and bioluminescence quantification was performed using Living Image software (Xenogen).

Protein analysis and kinase assays

NIH3T3 cells were grown in DMEM medium with 10% foetal calf serum (FCS). 24 hours before transfection, the cells were washed with PBS and DMEM without FCS was added for serum starvation. pcDNA3-HRASGV12 and pEF-iDab#6-memb were transiently co-transfected into these serum-starved cells using Lipofectamine 2000 (Invitrogen) according to Manufacture’s instructions. Forty-eight hours after transfection, the cells were harvested and Western blots were performed to detect specific proteins described below.

Western blotting

For culture cells, the cells were harvested and lysed in 1 x SDS sample buffer (62.5mM Tris-Cl pH 6.8 at 25 ˚C, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue). For xeno-transplanted tumours, the tissues were removed and snap-frozen on dry ice, before homogenization in 0.5ml NP-40 lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 1% NP-40 and 10 % glycerol), followed by addition of equal volume of 2x SDS-PAGE loading buffer. The prepared protein samples were boiled and fractionated on 12 or 15% SDS-PAGE, transferred to PVDF membranes (Millipore) and incubated with the appropriate antibodies including anti-ERK1/2, anti-phospho ERK1/2 (Thr202/Tyr204), anti-Akt, anti-phospho Akt (Ser473) (all polyclonal antibodies were from Cell Signaling Technology), anti-FLAG (M2, Sigma) or anti-pan Ras (Ab-3, Oncogene Research). Antibodies were detected with the appropriate Horseradish Peroxidase-linked secondary antibody and visualized with a chemiluminescence (ECL) kit (Amersham).

Histology and Immunohistochemistry

Mouse organs were fixed in 10 % neutral buffered formaldehyde, embedded in paraffin and 4 mm sections stained with haematoxylin and eosin. For immunohistochemistry, 4 mm thick slides were stained with anti-CC10 monoclonal antibody as follows. The tissue sections were de-paraffinized and hydrated and antigen retrieval by microwave heating in sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0). After incubating with 3% hydrogen peroxide in PBS and blocking with normal rabbit serum, the sections were stained with goat anti-CC10 monoclonal antibody (1: 100; T-18, Santa-Cruz) and biotinylated rabbit anti-goat (1:1000; DAKO) were used as the secondary antibody. Visualization was achieved using streptavidin-coupled horse radish peroxidase (HRP) (Vector) and diaminobenzidine as chromogen.

References

Forster A, Pannell R, Drynan LF, Codrington R, Daser A, Metzler M et al (2005). The invertor knock-in conditional chromosomal translocation mimic. Nature Methods 2: 27-30.

Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H (1995). Transcriptional activation by tetracycline in mammalian cells. Science 268: 1766-1769.

Meuwissen R, Linn SC, van der Valk M, Mooi WJ, Berns A (2001). Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-Ras oncogene. Oncogene 20: 6551-8.

Soriano P (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70-1.

Stripp BR, Sawaya PL, Luse DS, Wikenheiser KA, Wert SE, Huffman JA et al (1992). cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem 267: 14703-12.

Tanaka T, Lobato MN, Rabbitts TH (2003). Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J. Mol. Biol. 331: 1109-1120.

Tanaka T, Rabbitts TH (2003). Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J 22: 1025-1035.

Tanaka T, Rabbitts TH (2008). Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds. J Mol Biol 376: 749-757.

Tanaka T, Williams RL, Rabbitts TH (2007). Tumour prevention by a single antibody domain inhibiting binding of signal transduction molecules to activated RAS. EMBO J 26: 3250-3259.

te Riele H, Maandag ER, Clarke A, Hooper M, Berns A (1990). Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature 348: 649-51.

Tucker KL, Beard C, Dausmann J, Jackson-Grusby L, Laird PW, Lei H et al (1996). Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev 10: 1008-20.

Wildin RS, Wang HU, Forbush KA, Perlmutter RM (1995). Functional dissection of the murine lck distal promoter. J Immunol 155: 1286-95.

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