We thank Dr. Swathi Arur (MD Anderson Cancer Center) for providing the let-60(n1046). We also thank Dr. David Reiner (Texas A&#38;M Health Science Center Institute of Biosciences &#38; 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.

1. Nematode growth medium (NGM) plate preparation
<ol>
	<li>Add 2.5 g of peptone and 3 g of NaCl to 970 mL of deionized water contained in a 2 L Erlenmeyer flask. Stir contents using a magnetic stir bar. Thereafter, add 20 g of agar to the flask. Autoclave the contents of the flask at 121 &#176;C and a pressure of 15 lb/in2 for 30 min. After sterilization, place the flask on a stir plate and allow the medium to cool until the temperature reaches 50 &#176;C.</li>
	<li>To prepare the NGM plates add the followi...

<ol>
	<li>Marshall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Ras+and+Raf:+key+regulatory+proteins+in+cellular+transformation.">Interactions between Ras and Raf: key regulatory proteins in cellular transformation.</a> Molecular Reproduction and Development. 42 (4), 493-499 (1995).</li><li>Whelan, J. T., Hollis, S. E., Cha, D. S., Asch, A. S., Lee, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+the+Ras-ERK/MAPK+signaling+pathway.">Post-transcriptional regulation of the Ras-ERK/MAPK signaling pathway.</a> Journal of Cellular Physiology. 227 (3), 1235-1241 (2012).</li><li>Grandis, J. R., Tweardy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+transforming+growth+factor+alpha+and+epidermal+growth+factor+receptor+messenger+RNA+are+early+markers+of+carcinogenesis+in+head+and+neck+cancer.">Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer.</a> Cancer Research. 53 (15), 3579-3584 (1993).</li><li>Sasahira, T., Kirita, T., Kuniyasu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+molecular+pathobiology+in+oral+cancer:+a+review.">Update of molecular pathobiology in oral cancer: a review.</a> International Journal of Clinical Oncology. 19 (3), 431-436 (2014).</li><li>Stransky, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mutational+landscape+of+head+and+neck+squamous+cell+carcinoma.">The mutational landscape of head and neck squamous cell carcinoma.</a> Science. 333 (6046), 1157-1160 (2011).</li><li>Bos, J. 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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 &#176;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, t...

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 &#62; LET-60 &#62; LIN-45 &#62; MEK-2 &#62; 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&#176; and 2&#176; cell fates which give rise to a functional vulva12. Whereas distal VPCs differentiate into 3&#176; 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&#176; 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&#160;1&#176; and 2&#176; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Millipore Sigma&#160;</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0118-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco LB Broth</td>
			<td>Fisher Scientific</td>
			<td>DF0446-17-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP510-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>ICN10138201</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP213-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Acros organics</td>
			<td>AC455500050</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium pPhosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Fendiline</td>
			<td></td>
			<td></td>
			<td>Commercially Synthesized (Pharmaceutical grade)</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Millipore Sigma&#160;</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>8.25% Sodium Hypochlorite&#160;</td>
			<td></td>
			<td></td>
			<td>Bleach</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr>
			<td>(&#8722;)-Tetramisole Hydrochloride</td>
			<td>Millipore Sigma&#160;</td>
			<td>L9756</td>
			<td></td>
		</tr>
		<tr>
			<td>UO126 (MEK inhibitor)</td>
			<td>Millipore Sigma&#160;</td>
			<td>19-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical Sterile Polypropylene Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 10mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 25mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-575</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 1.5&#160; 18 mm X 18 mm Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12-541A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish with Clear Lid (60 x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid (100X15mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Glass Microscope Slides (75 x 25 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>12- Well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>50-197-4804</td>
			<td></td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strain</td>
			<td>Genotype</td>
			<td>Transgene</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>MT2124 &#160;</td>
			<td>let-60(n1046) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>MT7567</td>
			<td>lin-1(sy254) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>PS1839</td>
			<td>let-23(sa62) II.</td>
			<td></td>
			<td>CGC</td>
		</tr>
	</tbody>
</table>

identification of egfr and ras inhibitors using caenorhabditis elegans

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.

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...

Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

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.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

This protocol can be used in the initial phase of drug discovery to identify inhibitors that abolish the activities of EGFR and RAS proteins. The main advantage of this assay is that inhibitory effects of anti-EGFR and anti-RAS therapeutics can be easily visualized in C.egelans. Begin by spotting 100 microliters of overnight grown E.coli OP50 onto the center of each NGM plate.Allow the plates to dry for 24 hours in a laminar hood, then store them in a polystyrene container. Using a sterile worm pick, gather 10 to 12 gravid adult worms from a previously grown plate and transfer them to a fresh NGM plate seeded with E.coli OP50. Incubate the plate for 24 hours at 20 degrees Celsius.After 24 hours, use a sterile worm pick to remove adult worms from the plates. Incubate the plates at 20 degrees Celsius for approximately three days to allow the embryos to develop into gravid adult worms. Collect gravid adult worms into a 15 milliliter conical tube by washing two to four plates with M9W.Pellet the worms by centrifuging the tube at 450 times G for one minute, then decant the supernatant without disturbing the worm pellet. Prepare worm lysis solution by combining 400 microliters of 8.25%sodium hypochlorite and 100 microliters of five normal sodium hydroxide. Add this solution to the worm pellet and flick the tube to mix.Observe the lysis of the worms under a dissecting microscope to prevent overbleaching of the embryos. When 70%of the adult worms have lysed, add 10 milliliters of M9W to the conical tube to dilute the lysis mix. Centrifuge the tube at 450 times G for one minute, then replace the supernatant with 10 milliliters of M9W.After completing the washing steps, add three to five milliliters of M9W to resuspend the egg pellet. Rotate the tube overnight at a speed of 18 RPM on a tube rotator at room temperature. After overnight incubation, remove the tube from the rotator and pellet the L1 larvae by centrifuging the tube at 450 times G for one minute.Aspirate the M9W until 250 microliters is left in the tube. Begin by spotting 100 microliters of overnight grown E.coli OP50 onto the center of each NGM plate. Allow the plates to dry for 24 hours in the laminar hood, then store them in a polystyrene container.Grow 30 milliliters of E.coli OP50 in a 50 milliliter conical tube at 37 degrees Celsius overnight in an orbital shaker at 150 RPM. On the next day, spin the E.coli OP50 culture at 4, 000 times G for 10 minutes to pellet the cells, then remove the supernatant and resuspend the pellet in three milliliters of M9W to concentrate the culture. Prior to preparing the working solutions for the drug assay, add 0.1 milliliter of cholesterol into 100 milliliters of M9W.Prepare working solutions of each experimental drug by diluting the drug and vehicle in 4.8 milliliters of M9W supplemented with cholesterol. Dissolve DMSO in 4.8 milliliters of M9W supplemented with cholesterol to prepare the vehicle control. Add 200 microliters of concentrated E.coli OP50 culture to each tube containing the vehicle control or drugs and vortex the tubes to mix.Add two milliliters of each working drug solution or vehicle control to each well in a 12 well tissue culture plate. Test each drug concentration and vehicle control in duplicate. Add approximately 100 L1 larvae per well using a sterile micropipette, limiting the volume to 10 microliters.Incubate the plates at 20 degrees Celsius. Supplement wells with 50 microliters of 10X concentrated E.coli OP50 on day three of the assay if needed. Prepare a 2%weight to volume solution of agarose by adding 0.1 gram of agarose to five milliliters of deionized water.Heat the agarose in a microwave to dissolve it. Place strips of lab tape along two glass slides, which will act as spacers limiting the thickness of the agarose pads. Then place a third clean glass slide between the taped slides.To make an agarose pad, spot 100 microliters of molten agarose onto the center of the clean slide. Place another clean glass slide across the top of the agarose and gently press down to form a pad. Remove the top slide when the pad has solidified.When the appropriate stage of the lifecycle is reached, remove the plates from the incubator and collect the worms in 15 milliliter conical tubes. Centrifuge the tubes at 450 times G for one minute, then remove the M9W without disturbing the worm pellet. Wash the worms twice with five milliliters of fresh M9W.After removing all remaining M9W, add 500 microliters of two millimolar sodium azide or two millimolar tetramisole hydrochloride to anesthetize the worms. Allow tubes to incubate at room temperature for 15 minutes. Add 10 microliters of the anesthetized worm suspension onto the center of an agarose pad and place a number 1.5 cover slip gently over the suspension.If needed, fix the cover slip with nail polish to prevent drying. Use a DIC microscope at 10 and 20 times magnifications to observe the let-60(n1046)let-23(sa62)and lin-1(sy254)strains. For let-60(n1046)and let-23(sa62)score the adult worms based on the presence or absence of the Muv phenotype.For the lin-1 strain, count the number of VPCs that adopted one or two degrees cell fades on the ventral side of the L4 larvae. This protocol was used to demonstrate that R-fendiline is able to suppress the Muv phenotype in let-60(n1046)mutant strain in a dose dependent manner. Non-reversal of the Muv phenotype was observed in the lin-1 no mutant strain in response to increasing concentrations of R-fendiline, suggesting that R-fendiline blocks activated let-60 signaling at the level of RAS and C.elegans.Similarly, the Muv phenotype was significantly reduced in the let-23(sa62)strain in response to three, 10, and 30 micromolar R-fendiline treatments in comparison to the DMSO treated worms. During the bleach treatment, it is important to observe the lysis of the worms. The long incubation in the bleach mix will lead to death of the embryos, resulting in lower yields of L1 larvae.The localization of let-23 can be determined in the presence of anti-EGFR inhibitors using transgenic worms expressing GFP fused to let-23. This allows an investigator to determine if the anti-EGFR inhibitors cause the mislocalization of let-23 to the plasma membrane.

identification-of-egfr-and-ras-inhibitors-using-caenorhabditis-elegans

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JoVE Journal

Cancer Research

2.9K ビュー.  University of Texas Health Science Center. This protocol can be used in the initial phase of drug discovery to identify inhibitors that abolish the activities of EGFR and RAS proteins. The main advantage of this assay is that inhibitory effects of anti-EGFR and anti-RAS therapeutics can be easily visualized in C.egelans. Begin by spotting 100 microliters of overnight grown E.coli OP50 onto the center of each NGM plate.Allow the plates to dry for 24 hours in a laminar hood, then store them in a polystyrene container. Using a ste...