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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Departments of 1 Molecular Biology, 2 Molecular Oncology, 3 Tumor Biology and Angiogenesis, and 4 Pathology, Genentech, South San Francisco, California
Requests for reprints: Somasekar Seshagiri, Genentech, Inc., 1 DNA Way MS224, South San Francisco, CA 94080. Phone: 650-225-3351; Fax: 650-225-1762; E-mail: sekar{at}gene.com.
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Introduction |
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Recently, activating somatic mutations in BRAF have been reported in 70% of melanomas (5, 13). These mutations are also present in premalignant atypical or dysplastic nevi and may thereby implicate BRAF activation as an initiating event in tumorigenesis (14). BRAF mutations also occur with high frequency in papillary thyroid carcinomas, serous ovarian cancers, and colorectal serrated adenocarcinomas whereas mutations in liver, pancreas, non–small-cell lung cancer, glioma, and acute myelogenous leukemia have been detected at lower frequency. Almost 90% of BRAF mutations are a T1799A transversion in exon 15 that results in a Val600Glu (V600E) amino acid substitution leading to constitutive kinase activation (15, 16). Interestingly, fewer than 1% of melanoma samples with this prevalent BRAF mutation also contain activating RAS mutations (which occur in more than 10% of melanomas), providing strong genetic evidence for a direct RAS/BRAF signaling pathway in this tumor type. Furthermore, in those cancers where neither RAS nor BRAF is mutated, sustained RAF/MEK/ERK signaling can arise from other mechanisms (17). These include both deregulated upstream activators, such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (ERBB2 or HER2), platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (MET), and vascular endothelial growth factor receptor (VEGFR), and loss of downstream negative regulators such as MAPK phosphatase 3 (DUSP6) and RAF kinase inhibitor protein. It is thereby thought that the majority of human tumors, not just those with RAS or BRAF mutations, engage the RAF/MEK/ERK signaling cascade. Taken together, this makes BRAF an attractive therapeutic target.
In this context, several small-molecule BRAF inhibitors have been reported, with BAY 43-9006 being furthest along in clinical testing (18, 19). BAY 43-9006 was discovered through a combinatorial chemistry approach aimed at studying structural modifications to a bis-aryl urea chemical series (20). Although initially developed as a RAF kinase inhibitor, it has since been shown to have activity towards several additional kinases, including VEGFR2, VEGFR3, PDGFRß, FLT3, c-KIT, and p38 MAPK (21). Whereas the antitumor activity of this inhibitor is thought to result from the combined effect of targeting angiogenesis (via receptor tyrosine kinases) and proliferation (via RAF/MEK/ERK–associated signaling), any correlation between BAY 43-9006 in vivo efficacy and BRAF mutation has yet to be established in ongoing clinical trials. For instance, in a phase II study of stage IV refractory melanoma patients receiving BAY 43-9006, Ahmad et al. (22) did not detect BRAF mutations in the majority of patients with stable disease. Flaherty et al. (23) also assessed BRAF mutational status in a phase I/II pharmacokinetic and pharmacodynamic trial of BAY 43-9006 in patients with metastatic melanoma and reported that BRAFV600E mutation did not confer an advantage in terms of partial responses and stabilized disease. Hence, off-target receptor tyrosine kinase inhibition and antiangiogenic activity may be crucial to the therapeutic potential of BAY 43-9006.
Whereas efforts to design kinase inhibitors have exploited structural differences in the vicinity of ATP-binding pockets to achieve considerable molecular selectivity, there is still a great potential for cross-reactivity and rigorous experimental testing of compounds commonly used in the clinic have revealed numerous off-target interactions (24, 25). As shown for BAY 43-9006, mitigating the function of certain combinations of kinases with a single inhibitor may have a beneficial effect in particular disease states; however, interpreting biological results and elucidating the primary disease targets becomes challenging. Coupled to this, the most important consideration for successful therapy design remains the choice of a suitable target. This is highlighted by the clinical success of imatinib (Gleevec) which targets the key molecular drivers of chronic myeloid leukemia and gastrointestinal stromal tumors, Bcr-Abl and c-Kit/PDGFR
kinases, respectively (26). Taken together, the development of new tools to validate candidate kinases in a disease context is highly desired.
Here we describe tetracycline-inducible short-hairpin RNA (shRNA) interference method to directly test the antitumorigenic effect of in vivo BRAF inhibition in melanoma tumor progression. With this RNA interference–based approach, we show that oncogenic BRAF is causally involved in tumor growth and address key questions about the contribution of this oncogene to tumor growth, maintenance, and resistance to apoptosis.
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Materials and Methods |
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Inducible shRNA constructs. Hairpin oligonucleotides used in this study are as follows: BRAF shRNA-1 (sense) 5'-GATCCCCAGAATTGGATCTGGATCATTTCAAGAGAATGATCCAGATCCAATTCTTTTTTTGGAAA-3', BRAF shRNA-1 (antisense) 5'-AGCTTTTCCAAAAAAAGAATTGGATCTGGATCATTCTCTTGAAATGATCCAGATCCAATTCTGGG-3'; BRAF shRNA-2 (sense) 5'-GATCCCCGCTACAGAGAAATCTCGATTTCAAGAGAATCGAGATTTCTCTGTAGCTTTTTTGGAAA-3', BRAF shRNA-2 (antisense) 5'-AGCTTTTCCAAAAAAGCTACAGAGAAATCTCGATTCTCTTGAAATCGAGATTTCTCTGTAGCGGG-3'; luciferase shRNA (sense) 5'-GATCCCCCTTACGCTGAGTACTTCGATTCAAGAGATCGAAGTACTCAGCGTAAGTTTTTTGGAAA-3', luciferase shRNA (antisense) 5'-AGCTTTTCCAAAAAACTTACGCTGAGTACTTCGATCTCTTGAATCGAAGTACTCAGCGTAAGGGG-3'; and enhanced green fluoroescent protein (EGFP) shRNA (sense) 5'-GATCCCCAGATCCGCCACAACATCGATTCAAGAGATCGATGTTGTGGCGGATCTTGTTTTTTGGAAA-3', EGFP shRNA (antisense) 5'-AGCTTTTCCAAAAAACAAGATCCGCCACAACATCGATCTCTTGAATCGATGTTGTGGCGGATCTGGG-3'. The complementary double-stranded shRNA oligos were inserted into our tetracycline-inducible retrovirus gene transfer vector using BglII and HindIII restriction enzyme sites as outlined for pTER (27). Our vector system is composed of a kanamycin-resistant, H1 promoter–driven shRNA expression shuttle plasmid and an ampicillin-resistant retroviral vector backbone that contains a codon-optimized Tet repressor-internal ribosomal entry site-puromycin cassette to enable Tet-regulated shRNA expression (Fig. 1A). Knockdown vectors are constructed by cloning shRNA oligos into the shuttle vector followed by a Gateway recombination reaction (Invitrogen) to transfer the shRNA cassette in the retroviral vector. All constructs were verified by sequencing. Gene knockdown using these shRNAs was first verified in transient assays.
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Western blot analysis. Cells were lysed in modified radioimmunoprecipitation assay buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Brij-35, 0.1% deoxycholate, protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN), and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). SDS-PAGE (4-12% gel) was used to resolve the proteins in the lysate. After electrophoresis, the proteins were electrotransferred onto a polyvinylidene fluoride microporous membrane and immunodetected using standard procedures.
Xenograft models. Six- to eight-week-old female athymic nu/nu mice or scid-beige mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in the conventional animal facility of our institute. For s.c. tumor models, mice were injected in the right flank with either 3 x 106 human LOX-IMVI or 1 x 107 human A375 shRNA-containing cell clones resuspended in 200 µL PBS. When tumors reached a mean volume of 100 to 150 mm3, the mice with similarly sized tumors were grouped into treatment cohorts. Mice received 5% sucrose only or 5% sucrose plus 1 to 2 mg/mL doxycycline for control and knockdown cohorts, respectively. All water bottles were changed thrice a week. Tumors were measured with calipers and mice weighed twice a week. Mice whose tumors reached 2,000 mm3 were euthanized. At the end of the dosing study, or as indicated, appropriate tumor samples were taken. Between 7 and 10 mice were used for each treatment group and results are presented as mean tumor volume ± SE.
For metastatic tumor models, female scid-beige mice were injected i.v. with 50 µL PBS containing 4 x 105 A375M-luc shRNA cell clones. Tumor progression was monitored by weekly bioluminescence imaging for luciferase and mice were monitored daily for survival. At least nine mice were used for each cohort.
Bioluminescence imaging. Scid-beige mice were injected i.p. with 250 µL 200 mg/kg D-luciferin (Molecular Probes, Carlsbad, CA) and anesthetized using isoflurane. During image acquisition in a light-tight box, the animal was maintained on isoflurane via nose cone and body temperature was maintained using a warming pad. Bioluminescence images were acquired using a cooled intensified charge-coupled device camera. Image acquisition times were typically 1 minute. Images were processed by coregistering a reference image with the bioluminescence data image.
Immunohistochemistry. Formalin-fixed, paraffin-embedded specimens were collected and a routine H&E slide was first evaluated. Immunohistochemical staining was done on 5-µm-thick paraffin-embedded sections using anti-Ki-67 (clone MIB-1, mouse anti-human with the DAKO ARC Kit), anti–panendothelial cell marker (clone MECA-32, monoclonal rat anti-mouse), and anti–cleaved caspase-3 (rabbit anti-human and anti-mouse) antibodies with a standard avidin-biotin HRP detection system according to the instructions of the manufacturer. Tissues were counterstained with haematoxylin, dehydrated, and mounted. In all cases, antigen retrieval was done with the DAKO Target Retrieval Kit as per instructions of the manufacturer.
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Results |
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To address the question of whether BRAF specifically could serve as a therapeutic target for melanoma, we employed this inducible-shRNA method to deplete the expression of BRAF in cultured melanoma tumor cells. LOX-IMVI cells, derived from a lymph node melanoma metastasis, and A375 cells from a malignant cutaneous melanoma were genotyped for BRAF. PCR amplification and sequencing of exon 15 of BRAF revealed the presence of activated BRAFV600E alleles in both LOX-IMVI and A375 cell lines. Consistent with this, strong BRAF/MEK/ERK signaling is observed in these cells. This suggests a role for BRAF in their tumorigenic potential. We generated stable cells that express either BRAF or control shRNA using previously published oligonucleotide sequences (28). Several independent LOX-IMVI and A375 clones were characterized to ensure against a clonal selection bias. In the uninduced state, cells expressing the inducible hairpins were not altered in their baseline growth properties and no discernable background expression could be detected (data not shown). As shown in Fig. 1B, dramatic BRAF suppression was observed in both cell lines by doxycycline-mediated hairpin targeting of BRAF but not GFP or luciferase. Densitometry quantitation of immunoblots revealed an effective BRAF protein knockdown of 
80% and 98% for LOX-IMVI and A375, respectively (Supplementary Fig. S1). The suppression of BRAF protein levels was achieved in a dose-dependent manner with a doxycycline IC50 of 5 ng/mL. BRAF knockdown was reversible and time dependent, with the maximal mRNA depletion detected 2 days post induction and the corresponding protein depletion occurring at day 3 (data not shown). Induced BRAF-directed shRNA did not diminish the expression of ARAF and RAF1, as has been shown previously for these shRNAs (29). Consistent with the known BRAF phosphorylation-dependent activation of ERK1/2 via MEK1/2, increasing doxycycline concentration resulted in a reduction of p-ERK1/2 whereas total ERK1/2 levels remained unchanged (Fig. 1B). This indicates that BRAF-mediated ERK1/2 activation and signaling can be abrogated in these cells by doxycycline-mediated induction of BRAF shRNA.
In phenotypic analyses, we first investigated the effects of BRAF knockdown on the in vitro growth of these melanoma cells. On doxycycline addition, LOX-IMVI and A375 cells lacking BRAF show consistent changes in two-dimensional properties as compared with control shRNA-infected cells. These include a delay in cell cycle entry and reduced proliferation (Supplementary Fig. S2) and a flattened epithelial-like cell morphology change (data not shown). Of greater interest, we next tested whether ablation of BRAF function in LOX-IMVI and A375 cells might affect their ability to form tumors in vivo. Inoculation of 3 x 106 LOX-IMVI cells into immunodeficient nu/nu mice produced large tumors at 2 weeks (Fig. 1C), consistent with the reported strong tumorigenicity of this cell line. Mice bearing these inducible-shRNA xenografted tumors were given either 5% sucrose or 2 mg/mL doxycycline plus 5% sucrose and monitored for tumor progression. Strikingly, knockdown of BRAF completely inhibited LOX-IMVI tumorigenesis in vivo and led to tumor regression (Fig. 1C) even despite the incomplete depletion of BRAF as shown in vitro for the selected clone. Complete responses were observed in 6 of 10 animals in the doxycycline treatment cohort. In contrast, sucrose-treated LOX-IMVI/BRAF-shRNA tumors or LOX-IMVI tumors expressing a control shRNA directed against luciferase continued to increase in size. Similar results were obtained in a second LOX-IMVI study with the same dosing paradigm (data not shown). There was no difference in body weight between treatment groups and significant doxycycline-related toxicity was not observed.
We did a similar in vivo study with A375 melanoma cell lines to further substantiate the role of BRAF in tumorigenesis. In A375 xenografts, BRAF-shRNA induction also halted tumor progression (Fig. 1D); however, the tumors did not regress as observed in the studies involving the LOX-IMVI cell line. There was no discernable effect on tumor growth observed with A375 cells expressing GFP control shRNA (Fig. 1E). Taken together, these results suggest that gain-of-function BRAF signaling is strongly associated with the in vivo tumorigenic ability of melanoma cells and confirm BRAF as an important therapeutic target.
Effect of conditional and dose-responsive BRAF gene suppression on tumorigenesis. Recent reports using inducible transgenic mouse models have begun to address the consequences of oncogene inactivation for the maintenance of an established neoplastic phenotype (30, 31). In some instances, even brief inactivation of a single oncogene can be sufficient to induce sustained tumor regression (30). To further explore the possibility of whether inactivation of oncogenic BRAF signaling may be effective in treating melanoma and to examine the mechanism by which oncogenic BRAF inactivation induces tumor regression, moribund mice with 1,500-mm3 LOX-IMVI/BRAF-shRNA s.c. tumors (Fig. 1C) were switched to doxycycline. Within 5 days post BRAF shRNA induction, the tumor volume had visibly decreased, and after 2 weeks, the tumors had grossly regressed (Fig. 2A), showing that impairment of BRAF can result in rapid elimination of tumor cells. Despite the large starting tumor volume, growth inhibition was equally efficient as compared with early-onset BRAF knockdown in smaller volume tumors. Furthermore, we tested the effect of restoring BRAF expression in regressing tumors by discontinuing treatment in a doxycycline cohort (Fig. 1C). On doxycycline withdrawal, tumor recurrence was observed in two of eight mice (Fig. 2B). In cases where the tumor did not regrow, by the time shRNA induction was stopped by doxycycline removal the animals no longer had a palpable tumor, indicating that all the implanted tumor cells in these animals had already been eliminated. This indicates that whereas BRAF knockdown does not lead to an irreversible cascade of molecular events in these tumor cells, prolonged BRAF suppression is sufficient to promote elimination of well-established tumors.
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LOX-IMVI tumor regression is specific to BRAF knockdown. Although RNA interference methods have great potential to elucidate gene function, it remains necessary to validate that the observed phenotypes result from gene silencing of the target gene and not of unintended, off-target transcripts (32). Thus, incorporation of multiple BRAF-specific shRNAs into these assays increases the confidence with which the observed changes in melanoma tumor growth can be directly linked to BRAF silencing. The first shRNA used for BRAF knockdown (Fig. 1) corresponds to the translated sequence just following the G-loop of the kinase domain (amino acids 461-467) in which no oncogenic mutations have been described to date. Accordingly, another hairpin specific to a distinct region of the BRAF transcript (encoding amino acids 597-603 and the V600E mutation) was also selected (28). Using this second retroviral construct (BRAF-shRNA2), we infected LOX-IMVI cells and clones were analyzed by quantitative PCR and Western blotting (Fig. 3A). Equally efficient doxycycline-mediated BRAF knockdown and attenuated downstream signaling were observed as compared with that of BRAF-shRNA1 depicted in Fig. 1B. Moreover, in xenograft models, shRNA2-mediated knockdown of BRAF also showed robust tumor regression on doxycycline induction (Fig. 3B). Taken together, these results further support that the elicited phenotypes reflect silencing of the BRAF target gene and affirm the role of oncogenic BRAF in tumor growth.
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3-fold in vitro (data not shown), BRAF signaling does not play a pivotal role in regulating tumor vascularization as determined by staining of endothelial cells (Fig. 4). In summary, these results support the current view that BRAF signaling is important for mediating cellular proliferation and survival in tumorigenesis.
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Discussion |
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Many of the challenges associated with conventional RNA interference methods are circumvented by use of a tetracycline-inducible shRNA system that can be introduced into tumor cell lineages (40). For instance, whereas numerous tissue culture applications are done using only transient siRNA knockdown, elucidating the role of an oncogene in transformation necessitates long-term shRNA induction for thorough in vivo analysis (Fig. 1C). Although some published studies have combined xenograft tumor experiments with transient knockdown using very optimal siRNA sequences (39), these very short studies are limited by variability in standard measurement of tumor growth (as with caliper measurement of s.c. tumors <100 mm3) and the dearth of tissues amenable to further biochemical or histologic analyses. Moreover, the standard plasmid- and viral-based methods for constitutive shRNA expression may unavoidably introduce significant selection pressure on the cells when targeting genes essential for cell viability or proliferation. Long-term suppression of gene expression can also result in compensatory or even nonphysiologic responses. Furthermore, as most tumor cell lines have undergone multiple genetic alterations and may have unstable genomes, stable cell lines generated by drug selection may have additional genetic damage. For this reason, an inducible gene silencing system allows for internal controls, thereby confirming the altered tumor growth phenotype as the result of the target gene loss-of-function rather than additional genetic hits. One caveat to consider for all RNA interference methods is that loss of a gene product leading to a molecular knockdown may not be equivalent to inhibiting only its catalytic function. To this end, cells depleted for endogenous proteins could be reconstituted with knockdown-resistant target genes rendered functionally inactive by point mutations to better mimic a chemical knockdown achieved through small-molecule inhibitors. Nevertheless, shRNA-mediated molecular knockdown provides a useful readout for consequences of target inhibition before a drug candidate becomes available. In addition, given the recent advances in in vivo delivery of siRNAs (41), it is quite conceivable that molecular knockdown studies will help address the feasibility of developing targeted siRNA-based therapies.
Having established the value of this inducible system for target validation in preclinical cancer research, it will be of interest to further examine inhibition of both oncogenic and wild-type forms of BRAF in cell lines that also include other indications. In addition, inducible BRAF knockdown may be combined with current standard of care agents to assess synergistic and adverse effects. This could potentially aid in developing an appropriate clinical trial design to test BRAF small-molecule inhibitors. Global analysis of the BRAF-regulated melanoma transcriptome or signaling pathways in inducible shRNA cells may also yield novel diagnostic or surrogate biomarkers for pathway activity. Taken together, this method allows for extensive interpretation of the biological outcome resulting from frequent BRAF activating mutations in melanoma.
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Acknowledgments |
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We thank Sanjiv Gambhir for providing A375M-luc cells; Sheila Bheddah, Linda Hall, Bijay Jaiswal, Angela McNutt, Sandy Adams, Kanan Pujara, Karen Toy, Mark Vasser, William Forrest, and members of the Seshagiri lab for their technical assistance; and Vishva Dixit and Fred de Sauvage for helpful discussions.
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Footnotes |
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5 B.P. Davis and D.C. Gray, unpublished observations. 
Received 8/ 1/05. Revised 10/ 7/05. Accepted 11/ 3/05.
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