The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.
The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.
The cellular pathways that regulate developmental events within organisms are highly conserved among all metazoans. One such pathway is the EGFR-RAS-ERK mitogen activated protein kinase (MAPK) signaling cascade which is a critical pathway that governs cell proliferation, differentiation, migration and survival1,2. Defects in this signaling pathway can lead to pathological or disease states such as cancer. The epidermal growth factor receptor (EGFR) has shown to be highly expressed in human tumors, including 50% of oral squamous cell carcinomas, and contributes to the development of malignant tumors3,4,5. Whereas mutations in the three RAS isoforms H-, K- and N-RAS are major drivers for malignant transformation in multiple human cancers. Amongst these three RAS isoforms, oncogenic mutations in K-RAS are most prevalent6,7,8. For EGFR and RAS to function, they must localize to the plasma membrane (PM). Preventing the localization of these molecules to the PM can completely abrogate the biological activity of this signal pathway9,10. Hence the inhibition of the localization of these proteins to the PM is a therapeutic strategy to block the downstream signaling and the resulting adverse outcomes. Using a high-content screening assay, fendiline, an L-type calcium channel blocker, was identified as an inhibitor of K-RAS activity11. Nanoclustering of K-RAS to the inner leaflet of the PM is significantly reduced in the presence of fendiline. Furthermore, K-RAS is redistributed from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. More importantly, the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-RAS is blocked by the inhibition of downstream signaling by fendiline11. These data suggest fendiline functions as a specific K-RAS anticancer therapeutic that causes the mis-localization of the RAS protein to the PM.
The nematode Caenorhabditis elegans has been extensively studied in the context of development. Many of the signal pathways that govern development in the worm are evolutionary and functionally conserved. For example, the EGFR mediated activation of RAS and the subsequent activation of the ERK MAPK signal cascade is conserved in the worm12. The cascade is represented by the following proteins: LET-23 > LET-60 > LIN-45 > MEK-2 > MPK-1. LET-60 is homologous to RAS, while LET-23 is homologous to EGFR. In the worm, this pathway regulates the development of the vulva13. The vulva is an epithelial aperture on the ventral body wall of the worm that allows fertilized eggs to be laid. The formation of the vulva in the worm is dependent on the exposure of the vulval precursor cells (VPC) to a gradient of activation of the EGFR-RAS-MAPK signal cascade. During the normal development, the proximal VPCs receive strong signals from the gonadal anchor cells to differentiate into 1° and 2° cell fates which give rise to a functional vulva12. Whereas distal VPCs differentiate into 3° cell fates that fuse to the hypodermal syncytium and do not form vulva due to depleted signaling. In the absence of signaling, all VPCs differentiate into 3° cell fates resulting in the formation of no vulva. However, constitutive signaling leads to the formation one or more non-functional vulva due to the induction of all VPCs to assume 1° and 2° cell fates.
Mutations that cause defective or excessive vulval induction have been identified for many of the genes that encode for proteins representing this pathway. Defective vulval induction results in a vulvaless (Vul) phenotype, while excessive vulval induction results in a multivulva (Muv) phenotype that is represented by the development of numerous nonfunctional ectopic pseudovulvae throughout the ventral body wall. The Muv phenotype expressed by the let-60(n1046) strain is due to a gain of function mutation in RAS, while in the let-23(sa62) strain it is due an activating mutation in EGFR14,15. The strong Muv phenotype in these mutant strains has been shown to be perturbed by pharmacological interventions as demonstrated by the treatment of let-60(n1046) worms with the MEK-1 inhibitor U012616,17. Interestingly, we have shown that R-fendiline and inhibitors that affect sphingomyelin metabolism suppress the Muv phenotype in the worm18. To demonstrate these inhibitors block let-60 signaling at the level of RAS, the lin-1 null strain has been utilized17. Lin-1 is an Ets-like inhibitory transcription factor that functions as a repressor in the development of the vulva19. Strong reversion of the Muv phenotype in let-60(n1046) worms and no effect on lin-1 null worms suggest that these inhibitions occur at the level of RAS.
In this protocol, we demonstrate the use of C. elegans as a model to identify inhibitors of RAS and EGFR proteins. Using a liquid-based assay, we demonstrate the inhibitory effects of R-fendiline by suppressing the Muv phenotypes in the let-60(n1046) and let-23(sa62) mutant strains of C. elegans. This assay validates the use of C. elegans as a tool in the initial phase of drug discovery for anticancer therapeutics.
1. Nematode growth medium (NGM) plate preparation
2. Propagation of C. elegans
3. Preparation of a synchronous C. elegans culture
4. Preparation of drug assays
NOTE: The steps in this assay are shown in Figure 1.
5. Agarose pad preparation for microscopy
6. Observation of the Muv phenotype in the let-60, let-23 and lin-1 strains
NOTE: Only candidate drugs that suppress the Muv phenotypes in let-23 and let-60 strains will be assayed using the lin-1 strain to determine if the inhibition occurs at the level of RAS or EGFR.
We first demonstrate that R-fendiline is able to suppress the Muv phenotype in the let-60(n1046) mutant strain compared to the DMSO treated worms. Our data shows that R-fendiline is able to block the Muv phenotype in the let-60(n1046) in a dose-dependent manner (Figure 2A,B). However, non-reversal of the Muv phenotype was observed in the lin-1 null mutant strain in response to increasing concentrations of R-fendiline (Figure 2B). The data suggests that R-fendiline blocks activated let-60 signaling at the level of RAS in C. elegans. Similarly, we observed the Muv phenotype was significantly reduced in the let-23(sa62) strain in response to 3, 10 and 30 µM R-fendiline treatment relative to the DMSO treated worms (Figure 2C,D). In all experiments, Students t-test was used to determine the statistical significance.
Figure 1: Flowchart representing the steps involved in preparing the drug assays using let-60(n1046), let-23(sa62) and lin-1(sy254) strains. Please click here to view a larger version of this figure.
Figure 2: R-fendiline alters let-60 and let-23 function in C. elegans in a dose-dependent manner. (A) Representative images of let-60(n1046) worms in the presence of vehicle (DMSO) or 30 μM R-fendiline. (B) Quantification of Muv phenotype in let-60(n1046) worms treated in the presence of DMSO, 3, 10 and 30 µM R-fendiline, or 30 μM U0126. (C) Representative images of let-23(sa62) worms in the presence of vehicle (DMSO) or 30 µM R-fendiline. (D) Quantification of Muv phenotype in let-23(sa62) worms treated in the presence of DMSO, 3, 10 and 30 µM R- fendiline, or 30 μM U0126. In all images the pseudovulva are indicated by white arrows and normal vulva by white asterisks. A total of 60 worms were imaged for each treatment. The experiment was repeated 3 times. (*** P<0.001 and ** P<0.01 were considered significant) Please click here to view a larger version of this figure.
The assays we describe using the worm are simple and inexpensive to identify inhibitors of EGFR and RAS function. C. elegans is an attractive model for drug discovery because it is easy to grow in the lab due to the short life cycle (3 days at 20 °C) and the ability to generate large numbers of larvae. More importantly, the EGFR-RAS-ERK MAPK pathway is evolutionarily and functionally conserved with mammals providing a genetically tractable system to analyze the effects of EGFR and RAS inhibitors. Further, the transparent nature of the worms enables an investigator to visualize distinct structures and the localization of Green fluorescent Protein (GFP) or other fluorophore fused to proteins of interest by DIC and fluorescent microscopy.
The protocols we used to propagation and maintain the various C. elegans used in this study were previously established20,21. However, in the preparation of NG plates we incorporated streptomycin and nystatin to prevent bacterial and fungal contamination. The addition of these antimicrobial agents did not impede the development of the worms and the induction of the Muv phenotype.
There are several advantages for obtaining L1 larvae by the worm synchronization protocol. Many larvae can be obtained from 2 or more plates containing gravid adults and the larvae collected are all age synchronized. This ensures development of the worms is consistent within the population. Some mutant strains displaying the Muv phenotype are poor egg layers resulting in low yields of larvae as seen in the null mutation harbored in the Ets family transcription factor lin-119. Bleach treatment of the lin-1 gravid adults will significantly increase the number of larvae required for the assay.
It is vital to observe the lysis of the worms during the preparation of a synchronous C. elegans culture. The thick eggshell partially protects the embryos from the action of the bleach-sodium hydroxide mix even as the cuticle of the larvae and adults dissolve. However, prolonged exposure to the lysis solution will penetrate this protective casing leading to the death of the embryos. Hence it is important to stop the action of the bleach-sodium hydroxide mix when 70% of the adult worms have lysed. This is achieved by diluting the lysis solution with M9W. The maintenance of the arrested L1 larvae is another step to consider in the synchronous C. elegans culture protocol. Prolonged incubation of the L1 arrested larvae in M9W can cause them to transform into the dauer stage due to accumulation of the dauer pheromone. To avoid the formation of the dauer stage, it is suggested to use the L1 arrested larvae within 1–2 days of collecting the embryos.
The basal Muv phenotype is 60%–90% and 90% for the let-60(n1046) and let-23(sa62) worms, respectively. This suggests the let-60(n1046) worms are subject to phenotypic drift and, therefore, it is important to report the basal levels of expression of the Muv phenotype. Treatment of let-60(n1046) and let-23(sa62) worms with R-fendiline and other K-RAS inhibitors reduce the percentage of worms expressing the Muv phenotype. However, it is also reported that some inhibitors of the EGFR-RAS-ERK MAPK pathway may reduce the number of pseudovulvae per worm alone or may affect both the expression and the number of pseudovulvae17. Hence it is important to count the number of pseudovulvae in the Muv worms in both drug and DMSO treated worms. The Muv phenotype expressed in let-60(n1046) and let-23(sa62) adult worms is clearly visible under a dissecting microscope. However, the lin-1 (null) strain is relatively unhealthy, developmentally impaired and the vulval protrusions are poorly distinguished in the adult worms. Therefore, instead of the ectopic vulval protrusions, in lin-1 (null) worms, VPCs that adopt a adopted 1° or 2° cell fates on the ventral side in the L4 stage, can be counted using a high resolution DIC microscope.
The assay is inexpensive, easy, and not time consuming to setup. To further improve the processing time, the worms can be anesthetized to a final concentration of 2 mM sodium azide within the wells of tissue culture plate and imaged using a dissecting microscope equipped with a camera. Another modification of the assay would be to use heat killed E. coli OP50 instead of live bacteria23. It has been shown that bacteria can metabolize certain small molecules leading to reduced bioactivities24.
Previous studies have shown that the induction of the vulva in the worm is dependent on certain environmental cues. The vulvaless phenotypes of lin-3(n378) and let-23(n1045) have shown to be partially suppressed by starvation and exiting the dauer stage14. Furthermore, a study by Moghal et al., showed that vulvaless lin-3(n378), let-23(sy1) and let-60(sy95dn) mutants grown in M9 buffer had a higher number of VPCs assuming vulval cell fates compared with animals grown on standard NG plates24. The data suggests worms grown in a liquid environment influences vulval induction. However, in our studies we did not observe the suppression of the Muv phenotype in a liquid environment.
In this protocol we demonstrate the use of C. elegans to evaluate the anti-RAS properties of fendiline. In a previous study, we have shown that multiple acid sphingomyelinase inhibitors, including tricyclic antidepressants such as desipramine, imipramine, and amitriptyline inhibit the Muv phenotype18. Furthermore, inhibitors of the sphingomyelin and ceramide biosynthetic pathways suppress the Muv phenotype expressed in the let-60(n1046) worms. These findings using the worm were validated in mammalian cell lines.
In conclusion, we demonstrate the use of C. elegans to identify inhibitors of EGFR and RAS activity in a liquid-based assay. Furthermore, the worm provides another system to identify and characterize the mechanisms of action of anti-RAS and EGFR therapeutics.
The authors have nothing to disclose.
We thank Dr. Swathi Arur (MD Anderson Cancer Center) for providing the let-60(n1046). We also thank Dr. David Reiner (Texas A&M Health Science Center Institute of Biosciences & Technology in Houston) for the lin-1 strain. Finally, we thank Dr. Danielle Garsin and her lab (The University of Texas, McGovern Medical School) for providing some of the reagents. Some worm strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This Research was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) grant RP200047 to JF Hancock.
Media and chemicals | |||
Agarose | Millipore Sigma | A9539-50G | |
Bacto Peptone | Fisher Scientific | DF0118-17-0 | |
BD Difco Agar | Fisher Scientific | DF0145-17-0 | |
BD Difco LB Broth | Fisher Scientific | DF0446-17-3 | |
Calcium Chloride | Fisher Scientific | BP510-500 | |
Cholesterol | Fisher Scientific | ICN10138201 | |
Magnesium Sulfate | Fisher Scientific | BP213-1 | |
Nystatin | Acros organics | AC455500050 | |
Potassium Phosphate Dibasic | Fisher Scientific | BP363-500 | |
Potassium pPhosphate Monobasic | Fisher Scientific | BP362-500 | |
R-Fendiline | Commercially Synthesized (Pharmaceutical grade) | ||
Sodium Azide | Millipore Sigma | S2002-25G | |
Sodium chloride | Fisher Scientific | BP358-1 | |
Sodium Hydroxide | Fisher Scientific | SS266-1 | |
8.25% Sodium Hypochlorite | Bleach | ||
Sodium Phosphate Dibasic | Fisher Scientific | BP332-500 | |
Streptomycin Sulfate | Fisher Scientific | BP910-50 | |
(−)-Tetramisole Hydrochloride | Millipore Sigma | L9756 | |
UO126 (MEK inhibitor) | Millipore Sigma | 19-147 | |
Consumables | |||
15mL Conical Sterile Polypropylene Centrifuge Tubes | Fisher Scientific | 12-565-269 | |
50mL Conical Sterile Polypropylene Centrifuge Tubes | Fisher Scientific | 12-565-271 | |
Disposable Polystyrene Serological Pipettes 10mL | Fisher Scientific | 07-200-574 | |
Disposable Polystyrene Serological Pipettes 25mL | Fisher Scientific | 07-200-575 | |
No. 1.5 18 mm X 18 mm Cover Slips | Fisher Scientific | 12-541A | |
Petri Dish with Clear Lid (60 x 15 mm) | Fisher Scientific | FB0875713A | |
Petri Dishes with Clear Lid (100X15mm) | Fisher Scientific | FB0875712 | |
Plain Glass Microscope Slides (75 x 25 mm) | Fisher Scientific | 12-544-4 | |
12- Well Tissue Culture Plates | Fisher Scientific | 50-197-4804 | |
Software | |||
Prism | Graphpad | ||
Bacterial Strains | |||
E. coli OP50 | |||
Worm Strains | |||
Strain | Genotype | Transgene | Source |
MT2124 | let-60(n1046) IV. | CGC | |
MT7567 | lin-1(sy254) IV. | CGC | |
PS1839 | let-23(sa62) II. | CGC |