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Inducible BRAF Suppression Models for Melanoma Tumorigenesis
C H A P T E R
T H R E E
Inducible BRAF Suppression Models for Melanoma Tumorigenesis Klaus P. Hoeflich,* Bijay Jaiswal,* David P. Davis,* and Somasekar Seshagiri* Contents 1. Introduction 2. Materials and Methods 2.1. Inducible shRNA vector system 2.2. Generation of BRAF-inducible shRNA cell clones 2.3. In vitro characterization of BRAF-inducible shRNA cell clones 2.4. Role of BRAF in tumor maintenance and progression 2.5. In vivo mechanism of action 2.6. Assessment of metastatic tumor development by BRAF knockdown 3. Conclusion Acknowledgement References
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Abstract Somatic mutations in BRAF have been reported in 50 to 70% of melanomas. The most common mutation is a valine to glutamic acid substitution at codon 600 (V600E). V600EBRAF constitutively activates ERK signaling and promotes proliferation, survival, and tumor growth. However, although BRAF is mutated in up to 80% of benign nevi, they rarely progress into melanoma. This implicates the BRAF mutation to be an initiating event that requires additional lesions in the genome for full-blown progression to melanoma. Even though the mutations appear early during the pathogenesis of melanoma, targeted BRAF knockdown using inducible shRNA in melanoma cell lines with BRAF mutations shows that BRAF is required for growth and maintenance of tumor in xenograft models.
*
Genentech Inc., Department of Molecular Biology, South San Francisco, CA
Methods in Enzymology, Volume 439 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)00403-X
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2008 Elsevier Inc. All rights reserved.
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1. Introduction RAF serine/threonine kinases, ARAF, BRAF, and CRAF/Raf-1, are part of a conserved three-component kinase signaling module that regulates various cellular processes, including proliferation, apoptosis, and differentiation (Wellbrock et al., 2004). RAF is activated by phosphorylation of residues in its catalytic loop upon binding the small G protein RAS through its RAS-binding domain. RAF then phosphorylates and activates the dualspecificity protein kinases MEK1 and MEK2. Active MEK1/2 in turn activates mitogen-activated protein kinases (MAPK) ERK1 and ERK2. ERK activation, depending on the cellular context, regulates a number of biological processes, including cell growth, survival, and differentiation. The occurrence of oncogenic RAS mutations and hyperactivation of ERK in cancer is well established (Allen et al., 2003; Downward, 2003). Activating oncogenic mutations in BRAF have been described in various cancers, particularly in melanoma (70%), thyroid (30%), colorectal (15%), and ovarian cancers (30%) (Davies et al., 2002; Dhomen et al., 2007; Wellbrock et al., 2004). Among the various BRAF mutations, 95% of the missense mutations result in the substitution of valine at position 600 by a glutamic acid (V600EBRAF). Further, 98% of all BRAF mutations are in the activation segment and the remaining mutations occur in the G loop. Mutant V600EBRAF has elevated kinase activity and is a potent activator of ERK (Wan et al., 2004). Furthermore, it can transform cells and promote proliferation and anchorage-independent growth (Mercer et al., 2005; Wellbrock et al., 2004). Studies on the occurrence of BRAF mutations during melanoma pathogenesis show that the mutations are present in over 80% of nevi (Pollock et al., 2003; Yazdi et al., 2003), suggesting that it is an initiating event. However, only a small proportion of these nevi progress into melanoma, suggesting the need for additional genome alterations for melanoma progression (Gray-Schopfer et al., 2007). To understand the requirement of BRAF postinitiation in tumor growth and maintenance, as well as its validity as a bona fide melanoma target, we developed a doxycycline (Dox)-inducible short hairpin RNA (shRNA) system to enable conditional BRAF silencing in V600EBRAF mutant melanoma cell lines (Fig. 3.1). Induction of BRAF-specific shRNA results in knockdown of BRAF by 72 h postinduction, leading to reduced phosphorylated ERK (Fig. 3.2A) and decreased proliferation (Hoeflich et al., 2006). These cell lines form tumors in immunodeficient nu/nu mice when implanted subcutaneously. When treated with 1 to 2 mg/ml Dox plus 5% sucrose to induce the BRAF shRNA, tumor bearing mice show tumor regression in the LOX-IMVI model or stasis in the A375 model. In contrast, mice bearing tumors treated with 5% sucrose alone continued to grow
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A
H1-TetO2
attL1
attL2 BRAF shRNA GatewayTM mediated recombination β -actin
B 5⬘LTR
attR1/2
TetRopt
IRES
Puro
3⬘sinLTR
Figure 3.1 Vectors: Schematic representation of the retroviral vector system for tetracycline/doxycycline-inducible synthesis of shRNA.This vector system is composed of a shuttle plasmid for shRNA cloning (A) and a retroviral destination vector (B).To generate BRAF targeting shRNA constructs, the sense and antisense oligonucleotides listed in Table 3.1 are annealed and cloned into the shuttle plasmid using BglII and HindIII. The resulting sequence-verified H1 promoter-shRNA cassette is then transferred to the viral destination vector via Gateway-mediated recombination (Invitrogen, Carlsbad CA).TheTet-repressor is constitutively expressed from the b-actin promoter.Therefore, in the absence of Dox the TetR binds to TetO2 within the H1 promoter and prevents shRNA expression. Addition of Dox results in dissociation of the TetR protein from TetO2 and derepression of the shRNA transcriptional unit. TetR, Tet-repressor protein; TetO2, tetracycline operator 2; IRES, internal ribosomal entry site; PURO, puromycin resistance gene; LTR, long terminal repeat; attL/R- recombination sites.
(see Fig. 3.2B and C). Further, control lines expressing an inducible shRNA targeting luciferase or GFP showed no effect on tumor growth in either the presence or the absence of Dox (see Fig. 3.2B and C). These studies show the requirement for BRAF signaling for in vivo tumor growth in these lines and confirm BRAF to be an important therapeutic target. Given the ability to regulate BRAF expression at will during tumor development, we sought to exploit this to understand the requirement of BRAF in tumor maintenance by cycling Dox treatment. In order to do this, we subcutaneously implanted in nude mice the engineered LOX-IVMI line described earlier. When the tumors reached 1500-mm3 volume, Dox was then added to the drinking water of mice bearing these large tumors to induce expression of the BRAF shRNA. These mice showed a visible decrease in tumor volume 5 days post-BRAF shRNA induction, and by 2 weeks there was a significant reduction in tumor volume (Fig. 3.3A). This suggested the requirement of BRAF for tumor maintenance, even after the tumors were effectively very large. In another experiment, mice with subcutaneous LOX-IMVI/BRAF-shRNA tumors were placed on Dox when tumors reached 200 mm3. The tumors regressed while under Dox treatment; however, when these mice were taken off Dox, thereby restoring BRAF expression, mice with a palpable tumor showed tumor regrowth (see Fig. 3.3B). This study indicates that restoration of BRAF signaling in these cells is important and sufficient for tumor formation.
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a-B-Raf a-A-Raf a-C-Raf a-pERK
a-pERK a-ERK2
shBRAF shBRAF + Dox shLuc shLuc + Dox
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C A375 tumor volume (mm3)
a-B-Raf
0 0.9 2.7 8 24 74 222 670 2000
A375 B-Raf-shRNA1
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0 0.9 2.7 8 24 74 222 670 2000
a-ERK2
0 0.9 2.7 8 24 74 222 670 2000
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shGFP shGFP + Dox
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Figure 3.2 Inducible knockdown of B-Raf expression prevents melanoma tumor growth. (A) Experimental validation of B-Raf knockdown in melanoma cell lines. LOX-IMVI and A375 cell clones stably expressing B-Raf shRNA or control GFP and luciferase (Luc) shRNAs were treated with the indicated Dox concentrations for 72 h. Lysates were then analyzed by immunoblotting. (B^D) B-Raf shRNA knockdown demonstrates antitumor efficacy in xenograft models. LOX-IMVI and A375 inducible shRNA cells were implanted subcutaneously in the flank of athymic mice as described in the text.Treatment in each experiment was initiated on the day when mice had tumors
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Figure 3.3 B-Raf knockdown is reversible and tightly regulated in vivo. (A) BRAFdependent LOX-IMVI neoplasias were allowed to grow for 14 days before administration of Dox was initiated to knockdown BRAF-dependent signaling and tumorigenesis. (B) Dox-treated mice with regressing subcutaneous tumors that are subsequently removed from Dox at day 14 undergo tumor recurrence (adapted from Hoeflich et al., 2006).
The conditional ablation of BRAF allowed us to examine the mechanism of tumor regression through the immunohistochemical analysis of tumors. We determined that cells in the LOX-IMVI model undergo apoptotic cell death following BRAF knockdown, as evidenced by increased caspase-3 levels, leading to tumor regression (Hoeflich et al., 2006).
ranging in size from 100 to 150 mm3. Administration of 2 mg/ml Dox via drinking water produced regression in (B) LOX-IMVI or stasis in (C) A375 tumors expressing an inducible B-Raf-specific shRNA. (D) GFP or (B) Luciferase control shRNAs did not affect tumor growth kinetics. No lethality or weight loss was observed (adapted from Hoeflich et al., 2006).
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In an effort to understand the role of BRAF in metastatic melanoma, we engineered A375M, a metastatic melanoma line, to inducibly express BRAF using the Dox-regulated shRNA vector system (Fig. 3.4A). This line B 100 A
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Figure 3.4 Reduction of A375M systemic tumor growth by B-Raf shRNA knockdown. (A) Western blot analysis showing expression of B-Raf and phosphorylation of Mek1 in uninduced cells (lane 1) and cells treated with 2 mg/ml Dox for 72 h (lane 2). Total Mek1 serves as an internal control to show equal loading. (B) Kaplan^Meier survival data of scid-beige mice injected intravenously with 4 105 A375M-luc/shRNAB-Raf cells and receiving drinking water containing 5% sucrose only (control) or sucrose with 1 mg/ml Dox. Animals were monitored for tumor onset and illness until they reached a terminal stage and were euthanized. Each group consisted of at least 10 mice.The reduction in tumor growth conferred by Dox-mediated B-Raf knockdown is significant according to the log-rank test, p < 0.0001. (C) Representative in vivo bioluminescence imaging of mice and (D) quantification of tumor burden of mice receiving Dox versus sucrose-treated control mice. Homogeneous cohorts of mice with established tumor lesions were divided into treatment groups 2 weeks after injection of A375M-luc/shRNA-B-Raf cells. Bioluminescence is represented relative to intensity at day 14 for each animal (adapted from Hoeflich et al., 2006).
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also carried a luciferase reporter gene to facilitate in vivo imaging. Following tail vein injection of the engineered A375M cells, mice were placed on Dox to induce BRAF shRNA and were observed for tumor formation by imaging. The overall survival of Dox-treated animals showed a statistically significant increase compared to untreated animals, indicating the requirement of BRAF in the establishment of metastastis (see Fig. 3.4B). In studies where BRAF knockdown was induced after establishment of systemic tumors, a delayed progression in metastatic tumors was evident by bioluminescent imaging and quantification (see Fig. 3.4C and D). These studies show anti-BRAF therapy as an effective strategy for treating BRAF mutant melanomas.
2. Materials and Methods 2.1. Inducible shRNA vector system To study the effect of BRAF knockdown on the in vivo growth of melanoma, we created a Dox-regulated shRNA expression system (Gray et al., 2007; Hoeflich et al., 2006). Briefly, this vector system is composed of a shuttle plasmid for shRNA cloning (see Fig. 3.1A) and a retroviral destination vector (see Fig. 3.1B). The shuttle plasmid contains a H1 promoter (Brummelkammp et al., 2002; Myslinski et al., 2001) modified with a tetracycline operator 2 (TetO2) (Hillen et al., 1984) inserted between the TATA box and the transcriptional start site. To generate BRAF-targeting shRNA constructs, the sense and antisense oligonucleotides listed in Table 3.1 are annealed and cloned into the shuttle plasmid using BglII and HindIII. The resulting sequence-verified H1 promoter-shRNA cassette is then transferred to the appropriate viral destination vector via Gateway-mediated recombination (Invitrogen, Carlsbad, CA). As illustrated in Fig. 3.1B, the destination vector contains a second expression cassette consisting of a human b-actin promoter driving a wild-type tetracycline repressor (TetR) (Yao et al., 1998) upstream of an internal ribosomal entry site (IRES) and the puromycin marker for selection. The retroviral vector backbone is derived from pQXCIP (Clontech, Palo Alto, CA). In the ‘‘off’’ state, the TetR, expressed from the human b-actin promoter, binds the modified H1 promoter, thereby preventing shRNA expression. However, in the presence of a tetracycline analog (Dox), the TetR protein is released from the TetO2 within the H1 promoter (Hillen et al., 1984), resulting in shRNA transcription and knockdown of endogenous BRAF expression.
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Table 3.1 BRAF and control RNAi oligonucleotides
Viral system
Retrovirus
Oligonucleotide name
BRAF shRNA-1 (sense) BRAF shRNA-1 (antisense) BRAF shRNA-2 (sense) BRAF shRNA-2 (antisense) Luciferase shRNA (sense) Luciferase shRNA (antisense) EGFP shRNA (sense)
EGFP shRNA (antisense)
Oligonucleotide sequence
50 -GAT CCC CAG AAT TGG ATC TGG ATC ATT TCA AGA GAA TGA TCC AGA TCC AAT TCT TTT TTT GGA AA-30 0 5 -AGC TTT TCC AAA AAA AGA ATT GGA TCT GGA TCA TTC TCT TGA AAT GAT CCA GAT CCA ATT CTG GG-30 0 5 -GAT CCC CGC TAC AGA GAA ATC TCG ATT TCA AGA GAA TCG AGA TTT CTC TGT AGC TTT TTT GGA AA-30 50 -AGC TTT TCC AAA AAA GCT ACA GAG AAA TCT CGA TTC TCT TGA AAT CGA GAT TTC TCT GTA GCG GG-30 0 5 -GAT CCC CCT TAC GCT GAG TAC TTC GAT TCA AGA GAT CGA AGT ACT CAG CGT AAG TTT TTT GGA AA-30 50 -AGC TTT TCC AAA AAA CTT ACG CTG AGT ACT TCG ATC TCT TGA ATC GAA GTA CTC AGC GTA AGG GG-30 0 5 -GAT CCC CAG ATC CGC CAC AAC ATC GAT TCA AGA GAT CGA TGT TGT GGC GGA TCT TGT TTT TTG GAA A-30 0 5 -AGC TTT TCC AAA AAA CAA GAT CCG CCA CAA CAT CGA TCT CTT GAA TCG ATG TTG TGG CGG ATC TGG G-30
2.2. Generation of BRAF-inducible shRNA cell clones Melanoma A375, A375M (American Type Culture Collection, Manassas, VA), and LOX-IMVI (NCI-60) cells are maintained at 37 and 5% CO2 in Dulbecco’s modified Eagle’s medium or RPMI 1640 media, respectively, with 10% tetracycline-free fetal bovine serum, 4 mM L-glutamine, and penicillin/streptomycin. Both LOX-IMVI and A375 cell lines carry
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activated BRAFV600E alleles and show strong BRAF/MEK/ERK signaling. Retrovirus infection is performed using Phoenix packaging cells according to the manufacturer’s instructions (Orbigen, San Diego, CA). As the puromycin resistance gene encoded in the vector is under the control of a constitutive b-actin promoter, 2 to 5 mg/ml puromycin can be used to select infected cells expressing shRNA. Aliquots of the isolated stable clones should be stored in liquid nitrogen. To ensure that any observed phenotypes result from gene silencing of the BRAF target gene and not of unintended off-target transcripts, two distinct BRAF-specific shRNAs are incorporated into these experiments to increase the confidence with which the observed changes in melanoma tumor growth can be directly linked to BRAF silencing. The first shRNA we validated for BRAF knockdown (see Fig. 3.2) corresponds to the translated sequence just following the G loop of the kinase domain (amino acids 461 to 467) in which no oncogenic mutations have been described to date. Accordingly, another hairpin specific to another region of the BRAF transcript (encoding amino acids 597 to 603 and the V600E mutation) was also selected and characterized (Hoeflich et al., 2006). Equivalent results were obtained using either shRNA construct.
2.3. In vitro characterization of BRAF-inducible shRNA cell clones Stable clones are plated in 96-well tissue culture plates and are treated with 1 mg/ml Dox (BD Clontech) for 3 days. RNA is prepared using Turbocapture mRNA Geneplate (Qiagen, Valencia, CA), and endogenous BRAF knockdown is assessed by a quantitative reverse transcriptase polymerase chain reaction. Clones are characterized for the level of endogenous BRAF mRNA expression and extent of Dox regulation. Cell clones showing greater than 70% knockdown upon Dox treatment are further characterized by Western blot for changes in BRAF protein expression and phosphorylated ERK1/2. Cell lysates are prepared using modified RIPA buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Brij-35, 0.1% deoxycholate, protease inhibitors (Roche Molecular Biochemicals), and a phosphatase inhibitor cocktail (Sigma). SDS-PAGE (4 to 12% gel) is used to resolve the proteins in the lysate. After electrophoresis, the proteins are electrotransferred onto a polyvinylidene fluoride microporous membrane and immunodetected using standard procedures. Antibodies used for Western blotting are as follows: anti-ERK2, anti-p-ERK1/2 (Thr202/Tyr204), anti-MEK1, anti-p-MEK1 (Ser217/221), and anti-ARAF (Cell Signaling Technology); anti-BRAF (F-7; Santa Cruz Biotechnology); anti-RAF1 (BD Transduction Labs); anti-h-actin (Sigma Life Science); and horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology).
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For our studies, densitometry quantification of immunoblots revealed an effective BRAF protein knockdown of 80% and 98% for LOX-IMVI and A375 cell clones, respectively. The suppression of BRAF protein levels is dose dependent with a cellular IC50 of approximately 5 ng/ml. BRAF knockdown is reversible and time dependent, with maximal mRNA depletion detected 2 days postinduction and the corresponding protein depletion occurring at day 3. Several independent LOX-IMVI and A375 clones should be characterized to ensure against a clonal selection bias. Following biochemical characterization of inducible shRNA clones, phenotypic cell culture assays can be performed. Upon Dox addition, LOX-IMVI and A375 cells lacking BRAF show consistent changes in two-dimensional properties as compared with control shRNA-infected cells. These phenotypes include reduced cell proliferation and a flattened epithelial-like cell morphology change. To determine the effect of BRAF ablation on cell cycle progression, A375 cells expressing either BRAF or control GFP shRNAs are cultured in 0.1% serum in the presence or absence of 1 mg/ml Dox. At 2-day intervals, viable cell counts are determined by the Trypan blue exclusion method using a Vi-Cell analyzer (Beckman Coulter) (Hoeflich et al., 2006).
2.4. Role of BRAF in tumor maintenance and progression To examine whether ablation of BRAF function in LOX-IMVI and A375 melanoma cells might affect their ability to form tumors in vivo, subcutaneous tumor models were established for these cell lines. In our studies, 6- to 8-week-old female nu/nu mice (Charles River Laboratories) are injected in the right flank with either 3 106 human LOX-IMVI or 1 107 human A375 shRNA-containing cell clones resuspended into 200 ml phosphatebuffered saline. When tumors reach a mean volume of approximately 150 mm3, mice with similarly sized tumors are grouped into treatment cohorts. Tumor volumes are measured in two dimensions (length and width) using Ultra Cal-IV calipers (Fred V. Fowler Company) using the formula of tumor volume (mm3) ¼ (length width2) 0.5. Between 7 and 10 mice are used for each treatment group, and results are presented as mean tumor volume SEM. At the end of the dosing study, or as indicated, appropriate tumor samples can be taken. In our studies, Dox-mediated knockdown of BRAF completely inhibited LOX-IMVI tumorigenesis in vivo and led to tumor regression (see Figs. 3.2B and 3.3), even despite the incomplete depletion of BRAF as shown in vitro for the selected clone. Complete responses, defined as 100% tumor regression from the initial starting tumor volume at any day during the study, were observed in 6/10 animals in the Dox treatment cohort. In A375 xenografts, BRAF shRNA induction also halted tumor progression (see Fig. 3.2C); however, the tumors did not regress as observed in studies involving the LOX-IMVI
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cell line. There was no discernible effect on tumor growth observed with cells expressing control shRNAs. For oral administration of Dox, mice receive 5% sucrose only or 5% sucrose plus 1 mg/ml Dox for control and knockdown cohorts, respectively. Dox is prepared in 1-gallon carboys and stored at 4 for up to 1 month. All drinking water bottles are changed three times per week. Because Dox is light sensitive, dark-colored bottles should be used. A variety of Dox administration regimens have been tested throughout the course of these BRAF studies and we have found this to the optimal way to deliver Dox to mice for short-term xenograft studies. In-house comparison with a Dox chow diet (6 g/kg Dox in solid pellets; Bio-Serv) showed that Dox administration in drinking water results in more rapid in vivo knockdown and is more cost effective (data not shown). To further explore whether inactivation of oncogenic signaling is sufficient for the elimination of well-established tumors, moribund mice with very large (1500 mm3) tumor volumes can be switched to Dox. An example of such a study is shown in Fig. 3.3. Within 5 days post-BRAF shRNA induction, the LOX-IMVI tumor volume had decreased visibly and after 2 weeks the tumors had grossly regressed (see Fig. 3.3). Furthermore, the effect of restoring BRAF expression in regressing tumors by discontinuing treatment in a Dox cohort can be tested. Upon Dox withdrawal, tumor recurrence was observed in mice that still had palpable subcutaneous tumors (see Fig. 3.3). This approach is useful to show that knockdown of BRAF does not lead to an irreversible cascade of molecular events in tumor cells and that prolonged BRAF suppression is necessary to eliminate tumors in preclinical models.
2.5. In vivo mechanism of action Because understanding the mechanism of action of a drug target is a key step in the drug development cycle, tumor histological analysis is performed to define the spectrum of cellular responses that can be caused by targeted BRAF inhibition. LOX-IMVI tumors from mice treated with Dox for 1 to 7 days are harvested, and formalin-fixed, paraffin-embedded specimens are collected. Following routine hematoxylin and eosin evaluation of the slides, immunohistochemical staining is performed on 5-mm-thick paraffinembedded sections using anti-Ki-67 (MIB-1, DakoCytomation), anticleaved caspase-3 (Cell Signaling Technology), and antipanendothelial cell marker (MECA-32, Pharmingen) antibodies with a standard avidin–biotin horseradish peroxidase detection system according to the manufacturer’s instructions. Tissues are counterstained with hematoxylin, dehydrated, and mounted. In all cases, antigen retrieval is performed with the DAKO target retrieval kit as per the manufacturer’s instructions.
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Compared to xenografts from control animals, tumors from Dox-treated mice exhibited a profound decrease in Ki67-positive proliferating tumor cells and an increase in scattered apoptotic cells as determined by immunochemical staining for activated caspase-3 (Hoeflich et al., 2006). The magnitude of these phenotypes reaches a stable maximum following 4 days for in vivo Dox treatment. BRAF signaling does not play a pivotal role in regulating tumor vascularization as determined by staining of endothelial cells (Hoeflich et al., 2006). In summary, data obtained from this approach support the current view that BRAF signaling is important for mediating cellular proliferation and survival in tumorigenesis.
2.6. Assessment of metastatic tumor development by BRAF knockdown Given that metastases are the predominant cause of melanoma-associated death, it is useful to provide preclinical validation for targeting BRAF in the context of an experimental metastasis model. To address this, A375M cells that have been selected for high metastatic ability can be utilized (Collisson et al., 2003). Tail vein injection of 4 105 A375M cells (total volume of 50 ml) into female scid-beige mice leads to pulmonary, ovarian, and adrenal tumors after a relatively short latency. After engineering A375M cells to express BRAF-specific shRNA for Dox-regulatable knockdown of BRAF protein and signaling, mice can be monitored longitudinally for tumor onset, progression, survival, and response to BRAF knockdown. Using this approach, BRAF ablation significantly slowed tumor growth and prolonged the survival of mice ( p < 0.0001, log-rank test), and the progression of disease was still partially inhibited when the induction of knockdown was delayed until systemic tumors were well established (see Fig. 3.4). These experiments provide evidence supporting anti-BRAF therapy as a promising strategy to inhibit certain metastatic tumors. In our metastatic tumor model experiments, A375M cell clones were used that had been previously engineered to constitutively express a firefly luciferase protein (Ray et al., 2004) and bioluminescence images could then be acquired using a cooled intensified charge-coupled device camera. Tumor progression was monitored by weekly bioluminescence imaging for luciferase and mice were monitored daily for survival. However, an alternate and more accessible method is ex vivo staining of tumor-bearing lungs via tracheal injection of 15% India ink. Whole lungs are soaked in water for 5 min and are fixed in Fekete’s solution (70% ethanol, 10% formaldehyde, 5% glacial acetic acid) for 24 h. This method allows for bleaching of tumor colonies against the black background of the stained lungs. Superficial tumor burden can then be scored by inspection.
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3. Conclusion The inducible shRNA gene knockdown system can be used both in vitro and in vivo to understand the role of BRAF in melanoma.
ACKNOWLEDGEMENT Parts of this work were adapted from our work published in Hoeflich et al. (2006).
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Ray, P., De, A., Min, J. J., Tsien, R. Y., and Gambhir, S. S. (2004). Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 64, 1323–1330. Wan, P. T., Garnett, M. J., Roe, S. M., Lee, S., Niculescu-Duvaz, D., Good, V. M., Jones, C. M., Marshall, C. J., Springer, C. J., Barford, D., and Marais, R. (2004). Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867. Wellbrock, C., Karasarides, M., and Marais, R. (2004). The RAF proteins take centre stage. Nat. Rev. Mol. Cell. Biol. 5, 875–885. Wellbrock, C., Ogilvie, L., Hedley, D., Karasarides, M., Martin, J., Niculescu-Duvaz, D., Springer, C. J., and Marais, R. (2004). V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, 2338–2342. Yao, F., Svensjo¨, T., Winkler, T., Lu, M., Eriksson, C., and Eriksson, E. (1998). Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum. Gene Ther. 9, 1939–1950. Yazdi, A. S., Palmedo, G., Flaig, M. J., Puchta, U., Reckwerth, A., Rutten, A., Mentzel, T., Hugel, H., Hantschke, M., Schmid-Wendtner, M. H., Kutzner, H., and Sander, C. A. (2003). Mutations of the BRAF gene in benign and malignant melanocytic lesions. J. Invest. Dermatol. 121, 1160–1162.
T H R E E
Inducible BRAF Suppression Models for Melanoma Tumorigenesis Klaus P. Hoeflich,* Bijay Jaiswal,* David P. Davis,* and Somasekar Seshagiri* Contents 1. Introduction 2. Materials and Methods 2.1. Inducible shRNA vector system 2.2. Generation of BRAF-inducible shRNA cell clones 2.3. In vitro characterization of BRAF-inducible shRNA cell clones 2.4. Role of BRAF in tumor maintenance and progression 2.5. In vivo mechanism of action 2.6. Assessment of metastatic tumor development by BRAF knockdown 3. Conclusion Acknowledgement References
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Abstract Somatic mutations in BRAF have been reported in 50 to 70% of melanomas. The most common mutation is a valine to glutamic acid substitution at codon 600 (V600E). V600EBRAF constitutively activates ERK signaling and promotes proliferation, survival, and tumor growth. However, although BRAF is mutated in up to 80% of benign nevi, they rarely progress into melanoma. This implicates the BRAF mutation to be an initiating event that requires additional lesions in the genome for full-blown progression to melanoma. Even though the mutations appear early during the pathogenesis of melanoma, targeted BRAF knockdown using inducible shRNA in melanoma cell lines with BRAF mutations shows that BRAF is required for growth and maintenance of tumor in xenograft models.
*
Genentech Inc., Department of Molecular Biology, South San Francisco, CA
Methods in Enzymology, Volume 439 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)00403-X
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1. Introduction RAF serine/threonine kinases, ARAF, BRAF, and CRAF/Raf-1, are part of a conserved three-component kinase signaling module that regulates various cellular processes, including proliferation, apoptosis, and differentiation (Wellbrock et al., 2004). RAF is activated by phosphorylation of residues in its catalytic loop upon binding the small G protein RAS through its RAS-binding domain. RAF then phosphorylates and activates the dualspecificity protein kinases MEK1 and MEK2. Active MEK1/2 in turn activates mitogen-activated protein kinases (MAPK) ERK1 and ERK2. ERK activation, depending on the cellular context, regulates a number of biological processes, including cell growth, survival, and differentiation. The occurrence of oncogenic RAS mutations and hyperactivation of ERK in cancer is well established (Allen et al., 2003; Downward, 2003). Activating oncogenic mutations in BRAF have been described in various cancers, particularly in melanoma (70%), thyroid (30%), colorectal (15%), and ovarian cancers (30%) (Davies et al., 2002; Dhomen et al., 2007; Wellbrock et al., 2004). Among the various BRAF mutations, 95% of the missense mutations result in the substitution of valine at position 600 by a glutamic acid (V600EBRAF). Further, 98% of all BRAF mutations are in the activation segment and the remaining mutations occur in the G loop. Mutant V600EBRAF has elevated kinase activity and is a potent activator of ERK (Wan et al., 2004). Furthermore, it can transform cells and promote proliferation and anchorage-independent growth (Mercer et al., 2005; Wellbrock et al., 2004). Studies on the occurrence of BRAF mutations during melanoma pathogenesis show that the mutations are present in over 80% of nevi (Pollock et al., 2003; Yazdi et al., 2003), suggesting that it is an initiating event. However, only a small proportion of these nevi progress into melanoma, suggesting the need for additional genome alterations for melanoma progression (Gray-Schopfer et al., 2007). To understand the requirement of BRAF postinitiation in tumor growth and maintenance, as well as its validity as a bona fide melanoma target, we developed a doxycycline (Dox)-inducible short hairpin RNA (shRNA) system to enable conditional BRAF silencing in V600EBRAF mutant melanoma cell lines (Fig. 3.1). Induction of BRAF-specific shRNA results in knockdown of BRAF by 72 h postinduction, leading to reduced phosphorylated ERK (Fig. 3.2A) and decreased proliferation (Hoeflich et al., 2006). These cell lines form tumors in immunodeficient nu/nu mice when implanted subcutaneously. When treated with 1 to 2 mg/ml Dox plus 5% sucrose to induce the BRAF shRNA, tumor bearing mice show tumor regression in the LOX-IMVI model or stasis in the A375 model. In contrast, mice bearing tumors treated with 5% sucrose alone continued to grow
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A
H1-TetO2
attL1
attL2 BRAF shRNA GatewayTM mediated recombination β -actin
B 5⬘LTR
attR1/2
TetRopt
IRES
Puro
3⬘sinLTR
Figure 3.1 Vectors: Schematic representation of the retroviral vector system for tetracycline/doxycycline-inducible synthesis of shRNA.This vector system is composed of a shuttle plasmid for shRNA cloning (A) and a retroviral destination vector (B).To generate BRAF targeting shRNA constructs, the sense and antisense oligonucleotides listed in Table 3.1 are annealed and cloned into the shuttle plasmid using BglII and HindIII. The resulting sequence-verified H1 promoter-shRNA cassette is then transferred to the viral destination vector via Gateway-mediated recombination (Invitrogen, Carlsbad CA).TheTet-repressor is constitutively expressed from the b-actin promoter.Therefore, in the absence of Dox the TetR binds to TetO2 within the H1 promoter and prevents shRNA expression. Addition of Dox results in dissociation of the TetR protein from TetO2 and derepression of the shRNA transcriptional unit. TetR, Tet-repressor protein; TetO2, tetracycline operator 2; IRES, internal ribosomal entry site; PURO, puromycin resistance gene; LTR, long terminal repeat; attL/R- recombination sites.
(see Fig. 3.2B and C). Further, control lines expressing an inducible shRNA targeting luciferase or GFP showed no effect on tumor growth in either the presence or the absence of Dox (see Fig. 3.2B and C). These studies show the requirement for BRAF signaling for in vivo tumor growth in these lines and confirm BRAF to be an important therapeutic target. Given the ability to regulate BRAF expression at will during tumor development, we sought to exploit this to understand the requirement of BRAF in tumor maintenance by cycling Dox treatment. In order to do this, we subcutaneously implanted in nude mice the engineered LOX-IVMI line described earlier. When the tumors reached 1500-mm3 volume, Dox was then added to the drinking water of mice bearing these large tumors to induce expression of the BRAF shRNA. These mice showed a visible decrease in tumor volume 5 days post-BRAF shRNA induction, and by 2 weeks there was a significant reduction in tumor volume (Fig. 3.3A). This suggested the requirement of BRAF for tumor maintenance, even after the tumors were effectively very large. In another experiment, mice with subcutaneous LOX-IMVI/BRAF-shRNA tumors were placed on Dox when tumors reached 200 mm3. The tumors regressed while under Dox treatment; however, when these mice were taken off Dox, thereby restoring BRAF expression, mice with a palpable tumor showed tumor regrowth (see Fig. 3.3B). This study indicates that restoration of BRAF signaling in these cells is important and sufficient for tumor formation.
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a-B-Raf a-A-Raf a-C-Raf a-pERK
a-pERK a-ERK2
shBRAF shBRAF + Dox shLuc shLuc + Dox
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shGFP shGFP + Dox
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Figure 3.2 Inducible knockdown of B-Raf expression prevents melanoma tumor growth. (A) Experimental validation of B-Raf knockdown in melanoma cell lines. LOX-IMVI and A375 cell clones stably expressing B-Raf shRNA or control GFP and luciferase (Luc) shRNAs were treated with the indicated Dox concentrations for 72 h. Lysates were then analyzed by immunoblotting. (B^D) B-Raf shRNA knockdown demonstrates antitumor efficacy in xenograft models. LOX-IMVI and A375 inducible shRNA cells were implanted subcutaneously in the flank of athymic mice as described in the text.Treatment in each experiment was initiated on the day when mice had tumors
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Figure 3.3 B-Raf knockdown is reversible and tightly regulated in vivo. (A) BRAFdependent LOX-IMVI neoplasias were allowed to grow for 14 days before administration of Dox was initiated to knockdown BRAF-dependent signaling and tumorigenesis. (B) Dox-treated mice with regressing subcutaneous tumors that are subsequently removed from Dox at day 14 undergo tumor recurrence (adapted from Hoeflich et al., 2006).
The conditional ablation of BRAF allowed us to examine the mechanism of tumor regression through the immunohistochemical analysis of tumors. We determined that cells in the LOX-IMVI model undergo apoptotic cell death following BRAF knockdown, as evidenced by increased caspase-3 levels, leading to tumor regression (Hoeflich et al., 2006).
ranging in size from 100 to 150 mm3. Administration of 2 mg/ml Dox via drinking water produced regression in (B) LOX-IMVI or stasis in (C) A375 tumors expressing an inducible B-Raf-specific shRNA. (D) GFP or (B) Luciferase control shRNAs did not affect tumor growth kinetics. No lethality or weight loss was observed (adapted from Hoeflich et al., 2006).
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In an effort to understand the role of BRAF in metastatic melanoma, we engineered A375M, a metastatic melanoma line, to inducibly express BRAF using the Dox-regulated shRNA vector system (Fig. 3.4A). This line B 100 A
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Figure 3.4 Reduction of A375M systemic tumor growth by B-Raf shRNA knockdown. (A) Western blot analysis showing expression of B-Raf and phosphorylation of Mek1 in uninduced cells (lane 1) and cells treated with 2 mg/ml Dox for 72 h (lane 2). Total Mek1 serves as an internal control to show equal loading. (B) Kaplan^Meier survival data of scid-beige mice injected intravenously with 4 105 A375M-luc/shRNAB-Raf cells and receiving drinking water containing 5% sucrose only (control) or sucrose with 1 mg/ml Dox. Animals were monitored for tumor onset and illness until they reached a terminal stage and were euthanized. Each group consisted of at least 10 mice.The reduction in tumor growth conferred by Dox-mediated B-Raf knockdown is significant according to the log-rank test, p < 0.0001. (C) Representative in vivo bioluminescence imaging of mice and (D) quantification of tumor burden of mice receiving Dox versus sucrose-treated control mice. Homogeneous cohorts of mice with established tumor lesions were divided into treatment groups 2 weeks after injection of A375M-luc/shRNA-B-Raf cells. Bioluminescence is represented relative to intensity at day 14 for each animal (adapted from Hoeflich et al., 2006).
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also carried a luciferase reporter gene to facilitate in vivo imaging. Following tail vein injection of the engineered A375M cells, mice were placed on Dox to induce BRAF shRNA and were observed for tumor formation by imaging. The overall survival of Dox-treated animals showed a statistically significant increase compared to untreated animals, indicating the requirement of BRAF in the establishment of metastastis (see Fig. 3.4B). In studies where BRAF knockdown was induced after establishment of systemic tumors, a delayed progression in metastatic tumors was evident by bioluminescent imaging and quantification (see Fig. 3.4C and D). These studies show anti-BRAF therapy as an effective strategy for treating BRAF mutant melanomas.
2. Materials and Methods 2.1. Inducible shRNA vector system To study the effect of BRAF knockdown on the in vivo growth of melanoma, we created a Dox-regulated shRNA expression system (Gray et al., 2007; Hoeflich et al., 2006). Briefly, this vector system is composed of a shuttle plasmid for shRNA cloning (see Fig. 3.1A) and a retroviral destination vector (see Fig. 3.1B). The shuttle plasmid contains a H1 promoter (Brummelkammp et al., 2002; Myslinski et al., 2001) modified with a tetracycline operator 2 (TetO2) (Hillen et al., 1984) inserted between the TATA box and the transcriptional start site. To generate BRAF-targeting shRNA constructs, the sense and antisense oligonucleotides listed in Table 3.1 are annealed and cloned into the shuttle plasmid using BglII and HindIII. The resulting sequence-verified H1 promoter-shRNA cassette is then transferred to the appropriate viral destination vector via Gateway-mediated recombination (Invitrogen, Carlsbad, CA). As illustrated in Fig. 3.1B, the destination vector contains a second expression cassette consisting of a human b-actin promoter driving a wild-type tetracycline repressor (TetR) (Yao et al., 1998) upstream of an internal ribosomal entry site (IRES) and the puromycin marker for selection. The retroviral vector backbone is derived from pQXCIP (Clontech, Palo Alto, CA). In the ‘‘off’’ state, the TetR, expressed from the human b-actin promoter, binds the modified H1 promoter, thereby preventing shRNA expression. However, in the presence of a tetracycline analog (Dox), the TetR protein is released from the TetO2 within the H1 promoter (Hillen et al., 1984), resulting in shRNA transcription and knockdown of endogenous BRAF expression.
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Table 3.1 BRAF and control RNAi oligonucleotides
Viral system
Retrovirus
Oligonucleotide name
BRAF shRNA-1 (sense) BRAF shRNA-1 (antisense) BRAF shRNA-2 (sense) BRAF shRNA-2 (antisense) Luciferase shRNA (sense) Luciferase shRNA (antisense) EGFP shRNA (sense)
EGFP shRNA (antisense)
Oligonucleotide sequence
50 -GAT CCC CAG AAT TGG ATC TGG ATC ATT TCA AGA GAA TGA TCC AGA TCC AAT TCT TTT TTT GGA AA-30 0 5 -AGC TTT TCC AAA AAA AGA ATT GGA TCT GGA TCA TTC TCT TGA AAT GAT CCA GAT CCA ATT CTG GG-30 0 5 -GAT CCC CGC TAC AGA GAA ATC TCG ATT TCA AGA GAA TCG AGA TTT CTC TGT AGC TTT TTT GGA AA-30 50 -AGC TTT TCC AAA AAA GCT ACA GAG AAA TCT CGA TTC TCT TGA AAT CGA GAT TTC TCT GTA GCG GG-30 0 5 -GAT CCC CCT TAC GCT GAG TAC TTC GAT TCA AGA GAT CGA AGT ACT CAG CGT AAG TTT TTT GGA AA-30 50 -AGC TTT TCC AAA AAA CTT ACG CTG AGT ACT TCG ATC TCT TGA ATC GAA GTA CTC AGC GTA AGG GG-30 0 5 -GAT CCC CAG ATC CGC CAC AAC ATC GAT TCA AGA GAT CGA TGT TGT GGC GGA TCT TGT TTT TTG GAA A-30 0 5 -AGC TTT TCC AAA AAA CAA GAT CCG CCA CAA CAT CGA TCT CTT GAA TCG ATG TTG TGG CGG ATC TGG G-30
2.2. Generation of BRAF-inducible shRNA cell clones Melanoma A375, A375M (American Type Culture Collection, Manassas, VA), and LOX-IMVI (NCI-60) cells are maintained at 37 and 5% CO2 in Dulbecco’s modified Eagle’s medium or RPMI 1640 media, respectively, with 10% tetracycline-free fetal bovine serum, 4 mM L-glutamine, and penicillin/streptomycin. Both LOX-IMVI and A375 cell lines carry
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activated BRAFV600E alleles and show strong BRAF/MEK/ERK signaling. Retrovirus infection is performed using Phoenix packaging cells according to the manufacturer’s instructions (Orbigen, San Diego, CA). As the puromycin resistance gene encoded in the vector is under the control of a constitutive b-actin promoter, 2 to 5 mg/ml puromycin can be used to select infected cells expressing shRNA. Aliquots of the isolated stable clones should be stored in liquid nitrogen. To ensure that any observed phenotypes result from gene silencing of the BRAF target gene and not of unintended off-target transcripts, two distinct BRAF-specific shRNAs are incorporated into these experiments to increase the confidence with which the observed changes in melanoma tumor growth can be directly linked to BRAF silencing. The first shRNA we validated for BRAF knockdown (see Fig. 3.2) corresponds to the translated sequence just following the G loop of the kinase domain (amino acids 461 to 467) in which no oncogenic mutations have been described to date. Accordingly, another hairpin specific to another region of the BRAF transcript (encoding amino acids 597 to 603 and the V600E mutation) was also selected and characterized (Hoeflich et al., 2006). Equivalent results were obtained using either shRNA construct.
2.3. In vitro characterization of BRAF-inducible shRNA cell clones Stable clones are plated in 96-well tissue culture plates and are treated with 1 mg/ml Dox (BD Clontech) for 3 days. RNA is prepared using Turbocapture mRNA Geneplate (Qiagen, Valencia, CA), and endogenous BRAF knockdown is assessed by a quantitative reverse transcriptase polymerase chain reaction. Clones are characterized for the level of endogenous BRAF mRNA expression and extent of Dox regulation. Cell clones showing greater than 70% knockdown upon Dox treatment are further characterized by Western blot for changes in BRAF protein expression and phosphorylated ERK1/2. Cell lysates are prepared using modified RIPA buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Brij-35, 0.1% deoxycholate, protease inhibitors (Roche Molecular Biochemicals), and a phosphatase inhibitor cocktail (Sigma). SDS-PAGE (4 to 12% gel) is used to resolve the proteins in the lysate. After electrophoresis, the proteins are electrotransferred onto a polyvinylidene fluoride microporous membrane and immunodetected using standard procedures. Antibodies used for Western blotting are as follows: anti-ERK2, anti-p-ERK1/2 (Thr202/Tyr204), anti-MEK1, anti-p-MEK1 (Ser217/221), and anti-ARAF (Cell Signaling Technology); anti-BRAF (F-7; Santa Cruz Biotechnology); anti-RAF1 (BD Transduction Labs); anti-h-actin (Sigma Life Science); and horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology).
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For our studies, densitometry quantification of immunoblots revealed an effective BRAF protein knockdown of 80% and 98% for LOX-IMVI and A375 cell clones, respectively. The suppression of BRAF protein levels is dose dependent with a cellular IC50 of approximately 5 ng/ml. BRAF knockdown is reversible and time dependent, with maximal mRNA depletion detected 2 days postinduction and the corresponding protein depletion occurring at day 3. Several independent LOX-IMVI and A375 clones should be characterized to ensure against a clonal selection bias. Following biochemical characterization of inducible shRNA clones, phenotypic cell culture assays can be performed. Upon Dox addition, LOX-IMVI and A375 cells lacking BRAF show consistent changes in two-dimensional properties as compared with control shRNA-infected cells. These phenotypes include reduced cell proliferation and a flattened epithelial-like cell morphology change. To determine the effect of BRAF ablation on cell cycle progression, A375 cells expressing either BRAF or control GFP shRNAs are cultured in 0.1% serum in the presence or absence of 1 mg/ml Dox. At 2-day intervals, viable cell counts are determined by the Trypan blue exclusion method using a Vi-Cell analyzer (Beckman Coulter) (Hoeflich et al., 2006).
2.4. Role of BRAF in tumor maintenance and progression To examine whether ablation of BRAF function in LOX-IMVI and A375 melanoma cells might affect their ability to form tumors in vivo, subcutaneous tumor models were established for these cell lines. In our studies, 6- to 8-week-old female nu/nu mice (Charles River Laboratories) are injected in the right flank with either 3 106 human LOX-IMVI or 1 107 human A375 shRNA-containing cell clones resuspended into 200 ml phosphatebuffered saline. When tumors reach a mean volume of approximately 150 mm3, mice with similarly sized tumors are grouped into treatment cohorts. Tumor volumes are measured in two dimensions (length and width) using Ultra Cal-IV calipers (Fred V. Fowler Company) using the formula of tumor volume (mm3) ¼ (length width2) 0.5. Between 7 and 10 mice are used for each treatment group, and results are presented as mean tumor volume SEM. At the end of the dosing study, or as indicated, appropriate tumor samples can be taken. In our studies, Dox-mediated knockdown of BRAF completely inhibited LOX-IMVI tumorigenesis in vivo and led to tumor regression (see Figs. 3.2B and 3.3), even despite the incomplete depletion of BRAF as shown in vitro for the selected clone. Complete responses, defined as 100% tumor regression from the initial starting tumor volume at any day during the study, were observed in 6/10 animals in the Dox treatment cohort. In A375 xenografts, BRAF shRNA induction also halted tumor progression (see Fig. 3.2C); however, the tumors did not regress as observed in studies involving the LOX-IMVI
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cell line. There was no discernible effect on tumor growth observed with cells expressing control shRNAs. For oral administration of Dox, mice receive 5% sucrose only or 5% sucrose plus 1 mg/ml Dox for control and knockdown cohorts, respectively. Dox is prepared in 1-gallon carboys and stored at 4 for up to 1 month. All drinking water bottles are changed three times per week. Because Dox is light sensitive, dark-colored bottles should be used. A variety of Dox administration regimens have been tested throughout the course of these BRAF studies and we have found this to the optimal way to deliver Dox to mice for short-term xenograft studies. In-house comparison with a Dox chow diet (6 g/kg Dox in solid pellets; Bio-Serv) showed that Dox administration in drinking water results in more rapid in vivo knockdown and is more cost effective (data not shown). To further explore whether inactivation of oncogenic signaling is sufficient for the elimination of well-established tumors, moribund mice with very large (1500 mm3) tumor volumes can be switched to Dox. An example of such a study is shown in Fig. 3.3. Within 5 days post-BRAF shRNA induction, the LOX-IMVI tumor volume had decreased visibly and after 2 weeks the tumors had grossly regressed (see Fig. 3.3). Furthermore, the effect of restoring BRAF expression in regressing tumors by discontinuing treatment in a Dox cohort can be tested. Upon Dox withdrawal, tumor recurrence was observed in mice that still had palpable subcutaneous tumors (see Fig. 3.3). This approach is useful to show that knockdown of BRAF does not lead to an irreversible cascade of molecular events in tumor cells and that prolonged BRAF suppression is necessary to eliminate tumors in preclinical models.
2.5. In vivo mechanism of action Because understanding the mechanism of action of a drug target is a key step in the drug development cycle, tumor histological analysis is performed to define the spectrum of cellular responses that can be caused by targeted BRAF inhibition. LOX-IMVI tumors from mice treated with Dox for 1 to 7 days are harvested, and formalin-fixed, paraffin-embedded specimens are collected. Following routine hematoxylin and eosin evaluation of the slides, immunohistochemical staining is performed on 5-mm-thick paraffinembedded sections using anti-Ki-67 (MIB-1, DakoCytomation), anticleaved caspase-3 (Cell Signaling Technology), and antipanendothelial cell marker (MECA-32, Pharmingen) antibodies with a standard avidin–biotin horseradish peroxidase detection system according to the manufacturer’s instructions. Tissues are counterstained with hematoxylin, dehydrated, and mounted. In all cases, antigen retrieval is performed with the DAKO target retrieval kit as per the manufacturer’s instructions.
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Compared to xenografts from control animals, tumors from Dox-treated mice exhibited a profound decrease in Ki67-positive proliferating tumor cells and an increase in scattered apoptotic cells as determined by immunochemical staining for activated caspase-3 (Hoeflich et al., 2006). The magnitude of these phenotypes reaches a stable maximum following 4 days for in vivo Dox treatment. BRAF signaling does not play a pivotal role in regulating tumor vascularization as determined by staining of endothelial cells (Hoeflich et al., 2006). In summary, data obtained from this approach support the current view that BRAF signaling is important for mediating cellular proliferation and survival in tumorigenesis.
2.6. Assessment of metastatic tumor development by BRAF knockdown Given that metastases are the predominant cause of melanoma-associated death, it is useful to provide preclinical validation for targeting BRAF in the context of an experimental metastasis model. To address this, A375M cells that have been selected for high metastatic ability can be utilized (Collisson et al., 2003). Tail vein injection of 4 105 A375M cells (total volume of 50 ml) into female scid-beige mice leads to pulmonary, ovarian, and adrenal tumors after a relatively short latency. After engineering A375M cells to express BRAF-specific shRNA for Dox-regulatable knockdown of BRAF protein and signaling, mice can be monitored longitudinally for tumor onset, progression, survival, and response to BRAF knockdown. Using this approach, BRAF ablation significantly slowed tumor growth and prolonged the survival of mice ( p < 0.0001, log-rank test), and the progression of disease was still partially inhibited when the induction of knockdown was delayed until systemic tumors were well established (see Fig. 3.4). These experiments provide evidence supporting anti-BRAF therapy as a promising strategy to inhibit certain metastatic tumors. In our metastatic tumor model experiments, A375M cell clones were used that had been previously engineered to constitutively express a firefly luciferase protein (Ray et al., 2004) and bioluminescence images could then be acquired using a cooled intensified charge-coupled device camera. Tumor progression was monitored by weekly bioluminescence imaging for luciferase and mice were monitored daily for survival. However, an alternate and more accessible method is ex vivo staining of tumor-bearing lungs via tracheal injection of 15% India ink. Whole lungs are soaked in water for 5 min and are fixed in Fekete’s solution (70% ethanol, 10% formaldehyde, 5% glacial acetic acid) for 24 h. This method allows for bleaching of tumor colonies against the black background of the stained lungs. Superficial tumor burden can then be scored by inspection.
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3. Conclusion The inducible shRNA gene knockdown system can be used both in vitro and in vivo to understand the role of BRAF in melanoma.
ACKNOWLEDGEMENT Parts of this work were adapted from our work published in Hoeflich et al. (2006).
REFERENCES Allen, L. F., Sebolt-Leopold, J., and Meyer, M. B. (2003). CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK). Semin. Oncol. 30, 105–116. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553. Collisson, E. A., Kleer, C., Wu, M., De, A., Gambhir, S. S., Merajver, S. D., and Kolodney, M. S. (2003). Atorvastatin prevents RhoC isoprenylation, invasion, and metastasis in human melanoma cells. Mol. Cancer Ther. 2, 941–948. Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W., Davis, N., Dicks, E., et al. (2002). Mutations of the BRAF gene in human cancer. Nature 417, 949–954. Dhomen, N., and Marais, R. (2007). New insight into BRAF mutations in cancer. Curr. Opin. Genet. Dev. 17, 31–39. Downward, J. (2003). Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22. Gray, D. C., Hoeflich, K. P., Peng, L., Gu, Z., Gogineni, A., Murray, L. J., Eby, M., Kljavin, N., Seshagiri, S., Cole, M. J., and Davis, D. P. (2007). pHUSH: A single vector system for conditional gene expression. BMC Biotechnol. 7, 61. Gray-Schopfer, V., Wellbrock, C., and Marais, R. (2007). Melanoma biology and new targeted therapy. Nature 445, 851–857. Hillen, W., Schollmeier, K., and Gatz, C. (1984). Control of expression of the Tn10encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region. J. Mol. Biol. 172, 185–201. Hoeflich, K. P., Gray, D. C., Eby, M. T., Tien, J. Y., Wong, L., Bower, J., Gogineni, A., Zha, J., Cole, M. J., Stern, H. M., Murray, L. J., Davis, D. P., et al. (2006). Oncogenic BRAF is required for tumor growth and maintenance in melanoma models. Cancer Res. 66, 999–1006. Mercer, K., Giblett, S., Green, S., Lloyd, D., DaRocha Dias, S., Plumb, M., Marais, R., and Pritchard, C. (2005). Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts. Cancer Res. 65, 11493–11500. Myslinski, E., Ame, J. C., Krol, A., and Carbon, P. (2001). An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acids Res. 29, 2502–2509. Pollock, P. M., Harper, U. L., Hansen, K. S., Yudt, L. M., Stark, M., Robbins, C. M., Moses, T. Y., Hostetter, G., Wagner, U., Kakareka, J., Salem, G., Pohida, T., et al. (2003). High frequency of BRAF mutations in nevi. Nat. Genet. 33, 19–20.
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