Downstream targets of let-60 Ras in C. elegans

Béatrice Romagnolo1, Min Jiang1, Moni Kiraly1, Carrie Breton1, Rebecca Begley1, John

Wang1, James Lund1, Charles M. Perou2 and Stuart K. Kim1,3

1Department of Developmental Biology and Genetics

Stanford University Medical School

Stanford California 94305

2Lineberger Comprehensive Cancer Center

Department of Genetics

Univ. North Carolina at Chapel Hill

Chapel Hill North Carolina 27599

3Correspondence should be addressed to SKK.

650-725-7671

650-725-7739 fax

kim@cmgm.stanford.edu

let-60 targets in C. elegans 8/29/01 2

Abstract

In C. elegans, let-60 Ras controls many cellular processes such as differentiation

of vulval epithelial cells, function of chemosensory neurons, and meiotic progression in

the germ line. Although much is known about the let-60 Ras signaling pathway,

relatively little is understood about the target genes induced by let-60 Ras signaling that

carry out terminal effector functions leading to morphological change. Here, we have

used DNA microarrays to identify 708 genes that change expression in response to

activated let-60 Ras. We find that a significant fraction of the worm let-60 targets have

human homologs that show altered expression in breast cancers, potentially identifying

genes that play an important and evolutionarily conserved role downstream of Ras.

Introduction

Ras is an important molecular switch that regulates diverse cellular responses,

such as tumor growth and neuronal differentiation1. In C. elegans, Ras is encoded by the

let-60 gene and is involved in many developmental processes, such as vulval induction,

migration of the sex myoblasts, function of chemosensory neurons, progression through

pachytene in meiosis I and differentiation of the excretory cell2,3. For example, extensive

studies on vulval induction have shown that activation of let-60 Ras causes the vulval

precursor cell P6.p to undergo three nuclear divisions (the 1° cell fate) instead of fusing

with the hypodermis after one nuclear division (the 3° cell fate)4. let-60 also causes P6.p

to express a lateral signal that induces its neighboring vulval precursor cells to adopt the

2° cell fate4-6. In addition to regulating the cell behavior of P6.p, let-60 may also direct

cell morphological events in the eight daughter cells generated from P6.p; these progeny

must detach from the hypodermis, undergo specific cell fusions amongst themselves,

let-60 targets in C. elegans 8/29/01 3

migrate dorsally (inversion), form new attachments with the anchor cell and the lateral

hypodermis, and then migrate ventrally (eversion)7. let-60 regulates diverse cell

morphological behaviors in other cell types as well, though less is known about these

tissues than the vulva. For example, let-60 mediates cell migration of the sex myoblasts,

the cell cycle of germ line nuclei, differentiation of the excretory cell, and function of the

chemosensory neurons2,3.

Little is known about how let-60 Ras directs cell morphological processes at the

molecular level. LET-60 Ras activates a protein kinase cascade that includes MPK-1

MAP kinase, which migrates into the nucleus and phosphorylates transcription factors

such as LIN-1 and LIN-318-10. However, the cascade of terminal effector molecules

acting downstream of the LET-60 Ras signaling pathway remain largely unknown.

Beyond C. elegans, one may ask whether Ras signaling regulates similar

downstream effectors in all animals. C. elegans LET-60 and mammalian Ras regulate

many similar kinds of cellular behavior, such as cell proliferation, adhesion, and

differentiation. An unanswered question is whether these cellular events might be

mediated by similar downstream effectors in worms and humans. Identification of these

conserved targets would help elucidate genes acting at the core of the Ras response.

In order to begin to understand how let-60 Ras signaling alters cellular behavior,

we have used DNA microarrays to determine the transcriptional response following LET-

60 Ras activation. These data constitute a molecular profile of the let-60 Ras response,

and identify many terminal effectors that may mediate let-60-induced morphological

changes. We compared our data to those from human breast cancers, and found that

let-60 targets in C. elegans 8/29/01 4

many let-60 targets in worms have human homologs that show altered expression in

breast cancers.

Results

Genome-wide scan for let-60(G12V) targets

We used DNA microarrays to identify genes acting downstream of let-60 Ras in

C. elegans. To accomplish this, we constructed a transgenic strain that expressed an

activated form of LET-60 Ras in every cell. In mammals, a glycine to valine missense

mutation at position 12 in Ras is commonly found in tumors and results in constitutive

activation of Ras11,12. We constructed the analogous mutation in let-60 Ras in order to

generate a strong gain-of-function allele, and then placed this gene under the control of

the heat-shock promoter hsp16-2. We generated worms that contain copies of transgenic

let-60(G12V) integrated into the genome, and then induced expression of let-60(G12V)

using heat shock in staged early L3 hermaphrodites. We chose to induce let-60(G12V)

expression during the early L3 stage because the let-60 signaling pathway normally acts

in the vulval precursor cells to induce vulval differentiation at this time4. Thus, some of

the targets induced by global expression of let-60(G12V) during the early L3 stage may

include those that are involved in vulval differentiation.

Following heat shock, these animals displayed three prominent phenotypes. First,

most animals died one to two days after heat shock (between 80 and 100%), either as L4

larvae or as adults. Second, almost all of the animals that survived the heat shock

treatment displayed a multivulval (Muv) phenotype, similar to the phenotype caused by

activation of the let-23 EGF receptor/let-60 Ras signaling pathway in the vulval precursor

cells (Fig. 1a)13. Third, most animals displayed a Clear (Clr) phenotype in which fluid

let-60 targets in C. elegans 8/29/01 5

accumulated within the pseudocoelom and cells were separated from each other (Fig. 1c).

Gain-of-function mutations in the egl-15 FGF receptor/let-60 Ras signaling pathway also

causes a Clr phenotype, suggesting that let-60(G12V) causes activation of this pathway14.

None of these phenotypes were observed in let-60(G12V) animals at 15°C, or when wild-

type worms were subjected to heat shock (Fig. 1b, 1d).

We used DNA microarrays containing 17,817 genes (94% of the genome) to

identify genes whose expression is altered by let-60(G12V)15. We synchronized a

population of let-60(G12V) hermaphrodites in the early L3 stage, and then subjected

them to heat-shock. In order to determine the kinetics of expression of the let-60 target

genes, we isolated mRNA from these worms immediately before heat-shock as well as

three times following heat-shock (0.5, 1 and 2 hours). As a control, we subjected staged

early L3 wild-type hermaphrodites to the same heat-shock treatment. In order to measure

the error in our DNA microarray experiments, we collected four to eight independent

samples of poly(A)+ RNA from each of the two strains at each of the four time points

(Materials and Methods).

The mRNA from each of the let-60(G12V) and wild-type time points was used to

prepare Cy3-labelled cDNA. Each of these labelled cDNA samples was then hybridized

on full genome microarrays against a common Cy5-labelled cDNA probe made from

reference mRNA, which was mRNA isolated from a single preparation of mixed stage

hermaphrodites. The Cy3-labelled samples and the Cy5-labelled references were

simultaneously hybridized to the full-genome DNA microarrays, and the relative levels of

Cy3 and Cy5 hybridization intensities were measured for every gene on the DNA

let-60 targets in C. elegans 8/29/01 6

microarray. Since every sample was hybridized to the same reference RNA, we could

compare one timepoint to any other.

We first calculated the log2 of Cy3/Cy5 ratios for every gene in each of the DNA

microarray hybridizations. For each time point (each repeated between 4 and 8 times),

we then calculated the average log ratio for each gene, resulting in wild-type and let-

60(G12V) time courses showing the average expression level for every gene following

heat-shock. The expression data are shown in Supplemental Table 1 as well as at

http://cmgm.stanford.edu/~kimlab/Ras/.

As expected, the heat-shock time courses for both strains showed induction of

genes encoding heat-shock proteins (Fig. 2a). There are 29 genes that encode heat-shock

proteins in C. elegans, and 13 of these showed strong induction (at least 4-fold induction

in one of the time courses) and 9 more showed weaker levels of activation (at least 2-fold

induction). Furthermore, we saw a strong increase in the expression of let-60 following

heat-shock of the let-60(G12V) strain (10.1 fold relative to the wild-type time

course)(Fig. 2b). let-60 was not significantly induced in wild-type animals following

heat-shock.

We showed that expression of LET-60(G12V) results in activation of MPK-1

MAP kinase (Fig. 2c). MAP kinase is a common downstream target of Ras in all

animals16, and mpk-1 MAP kinase is known to act downstream of let-60 Ras in many

cells in C. elegans8,9,17. We prepared a population of staged early L3 let-60(G12V)

animals, subjected them to heat-shock, and then prepared cell-free extracts at various

times following heat-shock. To detect activated MAP kinase, we used monoclonal

antibodies specific for the activated phosphorylated form of MAP kinase in Western

let-60 targets in C. elegans 8/29/01 7

blotting experiments. Figure 2c shows that MPK-1 MAP kinase first became activated

30 minutes after heat-shock, and remained activated for at least 2 hours.

Analysis of let-60 target genes

To identify targets of activated LET-60, we searched for genes that showed

expression changes in the let-60(G12V) time course that were different than those in the

wild-type time course. We used a mixed procedure analysis of variation (ANOVA) to

identify genes with expression levels that were significantly different between the two

time courses, given the measured variance in our experiments (Materials and Methods).

This analysis identified 708 genes (p <0.001); the data are shown in Supplemental Table

2 and at http://cmgm.stanford.edu/~kimlab/Ras/. 297 of these genes (42%) encode

proteins that show significant similarity to proteins that have been previously-

characterized in other organisms. Figure 3a and Supplemental Table 2 shows the types

of cellular roles represented by the let-60-regulated genes. 110 of the let-60-regulated

genes encode transcription factors or proteins involved in signaling pathways. These

genes may regulate the expression of other genes or processes, and could initiate a

secondary cascade of gene expression following LET-60 Ras activation. Most of the

remaining let-60-regulated genes encode proteins involved in terminal effector functions,

such as metabolism, energy generation or protein synthesis. These genes could be

immediate early genes directly regulated by the let-60 Ras signaling pathway, or they

could be genes that are induced as a secondary consequence of let-60 Ras activation.

We used hierarchical clustering to group the 708 let-60-regulated genes based on

similarities in their patterns of gene expression in the two heat-shock time courses. The

let-60 targets in C. elegans 8/29/01 8

hierarchical cluster shows three main nodes, denoted nodes A, B and C (Fig. 3b). Most

of the 411 genes in node A are activated by let-60(G12V) (1.4-237 fold) while most of

the 139 genes in node B are repressed by let-60(G12V) (1.8-77 fold). Node C contains

158 genes that show regulation in both time courses (1.4-11.6 fold), but whose expression

in the let-60(G12V) time course has an opposite profile or shows different kinetics than

the wild-type time course. Some of the genes in node C may be independently regulated

by let-60(G12V) and heat-shock. Node C may also include genes that are heat-shock-

regulated, but whose regulation can be modified by let-60(G12V). For example, in yeast

and mammalian cells, Ras activation can suppress the heat-shock response18,19.

Most of the genes in groups A and B show transient changes in expression, with

peak changes in expression at one hour (Fig. 3b). The decline in expression at two hours

is not due to transient let-60 Ras activation, since expression of transgenic let-60 and

activation of MAP kinase occurs throughout the two hour time course (Fig. 2b and 2c).

The heat shock-regulated genes also show expression changes throughout the time

course, indicating that the lack of regulation of the let-60 targets at 2 hours is not a

general property of all genes (Fig. 2a). Thus, the decline in expression of the let-60

targets at two hours suggests that there is a feedback inhibitory mechanism, which begins

after one hour and prevents activated LET-60 Ras and MPK-1 MAP kinase from

continuing to induce or repress gene expression.

Furthermore, there are relatively few genes that begin to be expressed at the two

hour time point in the let-60(G12V) time course. It seems likely that the 708 genes

identified in our experiments represent only the beginning of a cascade that is normally

induced by let-60 Ras activation, and hence we might expect to see new genes induced at

let-60 targets in C. elegans 8/29/01 9

two hours after the first wave of gene expression. One possibility is that the let-60

morphogenetic cascade is blocked by the heat shock response at two hours. Heat shock is

known to result in an inhibition of translation in Drosophila and mammalian cells20. In

C. elegans, LET-60 Ras protein might be expressed immediately following heat shock

but translation of RNA from immediate early genes might be inhibited by the heat shock

response, and thus might not be able to induce a second round of gene expression.

let-60 signaling in C. elegans

We investigated the set of 708 let-60-regulated genes (p<0.001) in order to gain

insight about how let-60 Ras might regulate developmental decisions and initiate

morphogenetic pathways in C. elegans. First, we found evidence for autoregulation of

the let-60 signaling pathway. Specifically, we found that let-60(G12V) strongly activated

expression of the sos-1 guanine nucleotide exchange factor (5 fold) (Fig. 4a). SOS-1

normally acts to convert Ras from its inactive GDP-bound form to its active GTP-bound

form21, so that increased expression of sos-1 would maintain or increase let-60 activity in

a feed-back loop.

We identified 28 let-60-regulated genes that encode putative transcription factors

that may regulate other genes in a secondary cascade (Fig. 4b). Of these 28 transcription

factor genes, only one (lin-29) was previously known to be regulated by let-60

signaling22. Regulation of two other transcription factor genes suggests that let-60 may

also have a role in controlling programmed cell death of neuronal cells in C. elegans.

ces-1 encodes a transcription factor similar to the Drosophila snail protein, and functions

to prevent programmed cell death in C. elegans23. ces-2 encodes a bZIP transcription

let-60 targets in C. elegans 8/29/01 10

factor and functions to promote programmed cell death by repressing expression of ces-

124. We observed decreased expression of ces-2 in the let-60(G12V) time course (4.3

fold) and increased expression of ces-1 (3.6 fold), possibly as a secondary consequence

of diminished ces-2 expression. These results suggest that expression of activated let-60

may inhibit programmed cell death in neurons by regulating expression of ces-2. These

results are consistent with results from flies and mammals, showing that Ras can inhibit

programmed cell death25. However, loss-of-function mutations and a weak gain-of-

function mutation (n1046) in the let-60 locus have not been shown to cause overt changes

in the types or numbers of cells that undergo programmed cell death. One possibility is

that let-60 may affect the cell death program only under certain environmental conditions

or only in certain cells.

In C. elegans, the LET-60 Ras signaling pathway has a dynamic role in regulating

perception and transmission of sensory signals from olfactory neurons3. We searched the

list of let-60-regulated genes for those known to be expressed in these cells in order to

shed light on how let-60 Ras signaling might control these neuronal processes (Fig. 4c-e).

osm-9 encodes a TRP-like channel and functions in olfactory neurons to regulate osmotic

avoidance26, and this gene shows an 8.1-fold increase in expression in the let-60(G12V)

time course. In addition, G protein-coupled receptors and transmembrane receptor-type

guanylyl cyclases are thought to function in signaling pathways for odor- and chemo-

sensation27. The let-60(G12V) time course shows altered expression of 37 G protein-

coupled receptors (2.1-40.5 fold) and 3 transmembrane receptor-type guanylyl cyclases

(4.8-15.5 fold).

let-60 targets in C. elegans 8/29/01 11

In addition to olfactory neurons, the let-60 time course showed changes in the

expression of genes expected to function in other neuronal types or at the neuromuscular

junction (Fig. 4c, d). For example, the let-60(G12V) time course showed altered

expression of five ligand-gated ion channels (2.1-12.6 fold), a neuropeptide Y receptor

(2.7 fold), and three nicotinic acetylcholine receptors (1.8-8.2 fold). The time course also

showed increased expression of genes encoding proteins involved in neurotransmitter

secretion or re-uptake: RAB-3 (3.6 fold), synaptotagmin (6.3 fold), and a serotonin

transporter (4 fold). There is 3.2-fold increased expression of cat-2, which encodes

tyrosine hydroxylase and is involved in dopamine synthesis28. Nicotinic acetylcholine

receptors and tyrosine hydroxylase are also regulated by Ras in mammalian neuronal

cells, indicating that this regulatory interaction has been conserved through evolution28,29.

Expression profiles of let-60(G12V) targets in let-60(gf) and let-23(lf) mutants

The list of 708 let-60(G12V) target genes could include genes that are

physiological targets regulated by let-60(+) during wild-type development of C.elegans.

It could also include non-physiological targets, possibly expressed only in response to

ubiquitous or high levels of expression of let-60(G12V). To provide further evidence

about which genes may be physiological targets, we performed a second set of

microarray experiments to determine whether expression of the let-60(G12V) targets was

affected in gain-of-function let-60(n1046) and loss-of-function let-23(sy1) mutants. The

let-60 allele n1046 is a G13E missense allele that results in weak activation of let-6030,31.

Genetic studies indicate that let-60(n1046) is hypermorphic rather than neomorphic,

indicating that let-60(n1046) causes constitutive activation of genetic pathways that are

let-60 targets in C. elegans 8/29/01 12

normally regulated by let-60(+)30. let-23 EGFR acts upstream of let-60 Ras, and would

be expected to turn on similar sets of genes as let-6032. The loss-of-function let-23 allele

sy1 shows a highly penetrant vulvaless phenotype but does not exhibit any other obvious

phenotype33.

We prepared populations of wild-type, let-60(n1046) and let-23(sy1) worms that

were synchronized in the L3 larval stage, and then prepared RNA every two hours from

32 hours to 44 hours after hatching. This age range spans the entire time from the

specification of vulval fates to the completion of the vulval lineages34. For most time

points, we isolated two independent RNA samples (Materials and Methods). As before,

we compared each sample in every time course to a single reference RNA on DNA

microarrays containing 11,917 genes (65% of the genome)35. Similar to before, we

measured the expression levels of the sample relative to the reference RNA, calculated

the log2(sample/reference), and then calculated the average of the repeats of

log2(sample/reference). We then calculated the difference in expression between the let-

60(n1046) and the wild-type time courses, between the let-23 and wild-type time courses,

and between the let-60(n1046) and let-23 time courses.

We used the data from the let-60(n1046) and let-23 time courses to further

analyze the 708 genes that were previously identified in the let-60(G12V) time course.

The DNA microarrays used in the vulval time course contained only 65% of the genome,

and 487 of the 708 let-60(G12V) target genes were present in the vulval time courses.

We selected genes that showed an average difference in expression between the let-60(gf)

and let-23(lf) time courses of about 1.65 fold in all seven time points, and in which the

expression change was similar to regulation by let-60(G12V) (induction or

let-60 targets in C. elegans 8/29/01 13

repression)(Materials and Methods). Many let-60 target genes may not show expression

changes in the vulval time course, most likely because regulation by Ras signaling in

only a few vulval cells in these mutants would be obscured if those genes are expressed

(but not regulated) in many other non-vulval cells. Regulation of other genes by let-60

during vulval induction may be transient, and may not show significant expression

changes when averaged across the entire 12 hour time course. Finally, expression

changes for some genes in the vulval time course might be obscured by experimental

noise because the microarray experiments were repeated only twice.

Although negative results in the vulval time course are difficult to interpret, genes

that do show changes in let-60(n1046) and let-23 are likely to be physiological targets of

let-60(+) during normal development. Of the 487 let-60(G12V) target genes, 34 met the

criteria for changes in expression levels between the let-60(n1046) and let-23(lf) time

courses (Fig. 5). Eleven of these genes are repressed and twenty-three are induced by let-

60. Most of the expression changes in transcript levels is found in the let-60 time course,

with little or no changes in the let-23 time course.

All 11 let-60-repressed genes encode novel proteins, and 11 of the 23 let-60-

induced genes encode novel proteins. Of the remaining 12 genes induced by let-60, three

encode signaling proteins that may be parts of secondary signaling pathways;

specifically, C18F10.4 and F35E2.7 both encode G-protein coupled receptors and

K03D3.9 encodes a Rho GTPase. Three genes encode proteins that are part of the cell

surface or that modify proteins on the cell surface, suggesting that these genes may be

involved in cell adhesion, migration or fusion. Specifically, R07C3.1 encodes a cell

surface glycoprotein, F30A10.4 encodes an enzyme involved in protein glycosylation (N-

let-60 targets in C. elegans 8/29/01 14

acetylglucosaminyltransferase), and K03B8.1 encodes an extracellular metalloprotease

that may cleave proteins on the cell surface or in the extracellular matrix. Three genes

(B0285.9, C12D5.7 and T19H12.1) encode proteins involved in lipid, fatty acid and

sterol metabolism. The remaining genes encode a polypeptide chain release factor

involved in translation (K02E7.3), a serine esterase (M110.7) and a protein with a CUB

domain (F16B12.1).

Similar types of Ras targets in worm development and human cancer

Ras is a potent oncogene and point mutations that result in its constitutive

activation occurs in about 30% of human tumors11. The mechanisms of how Ras

regulates cellular proliferation, differentiation or apoptosis remain elusive, in part

because terminal effector genes regulated by Ras are poorly understood. We examined

the list of 708 genes that are regulated by let-60 in C. elegans for those that might be

relevant to Ras signaling in mammalian tumors.

In mammals, Ras activation causes changes in cell adhesion and cell migration.

One of the let-60 Ras targets in C. elegans (C45G7.5) encodes a protein similar to

mammalian cadherins, which are proteins that mediate cell adhesion at adherens

junctions. C45G7.5 is induced 3 fold in the let-60(G12V) time course, suggesting that

there is an increase in cell adhesion in response to let-60(G12V) expression. In

mammals, one of the known mechanisms by which Ras affects cell adhesion and

migration involves alterations of surface carbohydrates and specific lectins36. For

example, Ras activation in NIH3T3 cells results in increased levels of N-

acetylglucosaminyltransferase (GlcNAc) and increased beta 1-6 branching, and these

same alterations in protein glycosylation are common in malignant tumors37,38. In C.

let-60 targets in C. elegans 8/29/01 15

elegans, twelve of the let-60 regulated genes encode lectins and three encode GlcNac

(Fig. 4f). The lectin genes show expression changes between 2.3-20.5 fold and the

GlcNac genes show 3.8-7.8 fold changes in transcript levels over the time course. These

15 genes might mediate changes in cell adhesion or cell migration in response to let-

60(G12V) expression.

Human tumors are known to express proteins that make them resistant to drugs.

For example, the human multidrug resistance gene MDR1 is a membrane transporter that

can export macromolecules (including drugs) from cells. MDR1 is a target for the Ha-

Ras oncogene and shows increased expression in many types of human cancers, even

before drug treatment has begun39. Besides MDR1, tumor cells can also express genes

such as UDP-glucuronosyltransferase or glutathione-S-transferase (GST) that modify and

inactivate exogenous compounds40. Among the list of let-60(G12V) target genes, there

are four that encode MDR-related proteins, and these genes show changes in expression

of 1.7-6.7 fold during the let-60 time course (Fig. 4g). Four genes encode UDP-

glucuronosyltransferase (showing induction levels of 2.4-6.2 fold) and two encode GST

(with 1.8-4.8 fold induction levels). Zinc metalloproteases commonly show increased

expression during invasion and metastasis in human cells41. In C. elegans, eleven

metalloprotease genes are induced by let-60(G12V) from 1.7 to 15.9 fold (Fig. 4h).

Ras expression causes mammalian tumor cells to proliferate. In C. elegans, LET-

60(G12V) did not cause a significant increase in the expression of any of the 12 cyclin

genes (data not shown). Since all cells require cyclin expression to divide, this

observation suggests that expression of LET-60(G12V) in L3 larvae is not likely to cause

let-60 targets in C. elegans 8/29/01 16

widespread cell proliferation throughout the animal, although it does cause certain cells

(such as the vulval precursor cells) to divide.

Another approach to find common targets of Ras in worms and humans is to start

with the C. elegans let-60-regulated genes, find human genes showing a high degree of

homology, and then determine whether these homologs show expression changes in

microarray experiments involving human tumors. The Ras signaling pathway is often

activated in human breast cell lines or tumors either by overexpression of wild-type Ras

or by activation of upstream receptors, such as by overexpression of the epidermal

growth factor receptor family member Erb-B2/HER2 or the estrogen receptor42-44. Thus,

breast tumors may activate the Ras signaling pathway by a variety of mechanisms and

might be expected to show expression changes of Ras-regulated genes.

We searched human cDNA and gene sequences for genes with a high degree of

similarity to one or more of the 708 let-60-regulated genes from C. elegans, and found

166 human homologs (Materials and Methods). Of these 166, 89 were represented on

human DNA microarrays used to profile gene expression of 78 surgical specimens of

human breast tumors and 4 normal breast specimens45. Of these 89 homologs, 43 showed

detectable expression levels in these breast cancer DNA microarray experiments. The

breast samples have been previously classified into normal breast-like samples and five

breast tumor subtypes (Erb-B2+, basal-like, and luminal subtypes A, B and C) based on

their gene expressions patterns. We examined the set of 43 human homologs to find

those that showed differences in expression between the normal breast-like group and the

breast tumor samples (Student's t-test; p<0.05). This analysis identified 21 genes (49%)

that showed increased expression in our let-60(G12V) time course and that also showed

let-60 targets in C. elegans 8/29/01 17

higher expression in at least one of the tumor types when compared to the normal breast-

like samples (Fig. 6). Eight of these showed increased expression in more than one

tumor subtype compared to normal breast-like samples. The simplest interpretation of

these data is that the tumor condition in these samples causes increased expression of

these 21 genes. However, since tumors and normal breast samples are a mixture of

adipose, epithelial, endothelial and blood cells, it could be that expression differences for

some of the genes might be caused by changes in the types of cells that make up the

sample instead of changes associated with malignant cells.

We observed increased expression of Ki-Ras in three distinct tumor subtypes

(Erb-B2+, luminal subtypes B and C) compared to normal samples, similar to previous

results showing increased expression of Ras in breast cancer46. The other breast tumor

types did not show increased levels of Ki-Ras expression. In these breast tumor types,

the activity of the Ras signaling pathway rather than Ras transcript level itself might be

increased possibly due to activation of upstream steps, such as increased expression of

growth factor receptors (e.g. HER2) or activation of estrogen receptors that might signal

through Ras. Alternatively, the basal-like and luminal sub-type A tumors may be

transformed by other oncogenic pathways instead of the Ras signaling pathway. In this

case, the 11 human homologs that show increased expression in these tumor types might

be predominantly regulated by these other oncogenic pathways in humans and by the let-

60 Ras pathway in worms.

One of the potential Ras-regulated breast cancer target genes encodes RNA

polymerase III. Expression of RNA polymerase III is repressed by the tumor suppressor

proteins Rb and p53, and many transformed cell types have elevated RNA polymerase III

let-60 targets in C. elegans 8/29/01 18

activity47,48. Furthermore, tumor cells generate more reactive oxidation products than

normal cells, and the list of human homologs includes 3 genes that encode proteins that

could modulate oxidative stress (peroxiredoxin 3, NADH-ubiquinone oxidoreductase and

cytochrome 450). Inhibition of NADH-ubiquinone oxidoreductase has been shown to

inhibit tumor proliferation and reduce cancer, indicating that this protein is important for

tumor function49.

Discussion

The experiments in this paper lay the foundation to understand how Ras signaling

can cause morphological change at the molecular level in C. elegans. Full-genome DNA

microarrays were used to profile transcript changes caused by expression of activated let-

60 Ras from a heat shock promoter, and identified 708 genes that showed significant

expression changes (P<0.001). Not only does this microarray analysis identify genes that

could mediate let-60 morphological change, but it can also identify certain gene classes

(such as cyclin genes) that are unlikely to be involved in the let-60 Ras response in all

cells because their expression levels do not change.

Since activated let-60(G12V) was expressed in essentially every cell following a

heat shock pulse, the let-60(G12V) time course experiments could identify not only

physiological targets regulated by let-60(+) during normal development but also non-

physiological ones. For example, some gene expression changes could occur in cells or

at times that do not normally involve let-60 activation. However, let-60(+) is normally

expressed in many cell types throughout development50, and let-60(lf) mutations affect a

diverse set of cell types (such as several types of hypodermal cells, neuronal cells, the

let-60 targets in C. elegans 8/29/01 19

excretory cell and the germ line)2,3. These results indicate a broad role for let-60 during

development, suggesting that many types of cells could be competent to respond to let-

60(G12V) in a physiologically relevant manner.

To help resolve this issue further, we performed a set of microarray experiments

comparing expression in wild-type animals to let-60(n1046) gain-of-function and let-

23(sy1) loss-of-function mutants. Genetic experiments showed that let-60(n1046) is a

hypermorphic rather than a neomorphic mutation30,31, indicating that this mutation is

likely to alter expression of genes that are regulated by let-60(+) during normal

development. A major limitation of the experiments comparing let-60(n1046) to let-

23(sy1) mutants is that RNA was prepared from whole worms, so that expression

differences in one cell would be obscured by expression in other cells. As expected, most

of the 708 genes identified in the let-60(G12V) time course experiment did not show

expression differences in the let-60(n1046)/let-23(lf) experiment, most likely because

these mutations affect expression in specific cells. However, 34 of the 708 let-60(G12V)

target genes also showed expression changes in the let-60(n1046)/let-23(sy1)

experiments, providing further evidence that these genes are downstream targets of let-

60.

An important issue is to understand how activation of the let-60 Ras signaling

pathway can result in different morphological outputs in different cell types. For

example, the vulval precursor cells and not other cell types express certain genes in

response to let-60 Ras activation. In part, this is because only the vulval precursor cells

express the LIN-31 transcription factor, which is phosphorylated by MPK-1 MAP kinase

in response to let-60 activation10. In the let-60(G12V) time course experiment, some

let-60 targets in C. elegans 8/29/01 20

gene expression changes may occur in only certain cell types, and thus could provide

important molecular insight about how let-60-mediated transcriptional changes are

dependent on cell context. For example, genes that were induced by let-60 in only one

cell type might be regulated by a transcription factor expressed only in that cell type and

under let-60 control.

In C. elegans as in other organisms, the Ras signaling pathway has been studied in

depth but the terminal effector genes that act downstream of this pathway and that

directly carry out morphological change are less well understood. Genetic and

microarray analysis are separate and complementary methods to identify genes acting

downstream of let-60 Ras. Genes that function in morphogenetic pathways downstream

of let-60 Ras need not show expression changes in response to Ras activation; for

example, these genes may be controlled at a post-transcriptional level. Genetic analysis

of vulval morphogenesis has previously identified three genes (sqv-3, sqv-7 and sqv-8)

that encode proteins in a glycosylation pathway and one (cog-2) that encodes a

transcription factor51-53. Mutations in these genes result in defective vulval differentiation

but none show significant expression changes in the let-60(G12V) microarray

experiments. Conversely, the 708 genes that are downstream targets of let-60 Ras need

not exhibit defective let-60 Ras-mediated morphogenesis in loss-of-function mutants; for

example, some genes could be functionally redundant. One advantage of using DNA

microarrays is that this approach can identify genes that act redundantly or in parallel

because the expression of nearly all of the genes are analyzed at the same time.

We have begun to compare the Ras response in worms to that in humans by

analyzing gene expression differences between normal breast and breast tumor samples.

let-60 targets in C. elegans 8/29/01 21

Overexpression of Ras or of receptors acting upstream of Ras (such as ErbB2/HER2)

frequently occurs in breast cancers, and would lead to activation of the Ras signaling

pathway. We found that a significant fraction of genes regulated by let-60 in C. elegans

have homologs that show expression changes in human breast cancer samples. These

results indicate that some of the morphological pathways acting downstream of Ras are

likely conserved from worms to humans. Conservation of the regulatory interaction

between these homologs and the Ras signaling pathway indicates that the homologs have

an important functional role in mediating morphological change downstream of Ras.

These homologs would be expected to function in basic cell biological processes shared

between worms and humans, rather than in biological processes specific to each species.

Genes that are regulated by Ras in all animals are attractive candidates to study as

important morphogenetic effectors, and to test as targets for cancer treatment.

let-60 targets in C. elegans 8/29/01 22

Materials and Methods

C. elegans genetics

Complementary DNA corresponding to the let-60(G12V) mutant (provided by M.

Han) was inserted into pPD49.78 (from A. Fire) which carries the heat-shock promoter

hsp16-2. Transgenic animals were generated by microinjection of HS::let-60(G12V)

DNA (50 ng/µl) and unc-119(+) DNA (100 ng/µl) into unc-119 mutants following the

method of Mello et al.54. An integrated line (gaIs143) was generated by gamma-

irradiation, and then back-crossed twice to wild-type.

Worms were grown and RNA was prepared as in Reinke et al.35. Synchronous

populations of let-60(G12V) worms were harvested 48 hours after feeding starved worms

synchronized in the mid-L1 stage. Developmental stage was verified by observing a

small sample of animals with Nomarski optics to score the size of the gonad and the

development of the vulva. To induce heat-shock, plates were transferred to a water bath

at 33oC for 35 minutes, followed by recovery at 15o for 30 minutes, 1 hour or 2 hours.

We isolated eight RNA samples for the 0 hour time point and four samples for time

points at 0.5, 1 and 2 hours.

Synchronous populations of wild-type (N2), let-60(n1046) or let-23(sy1) worms

were harvested from 32 hours to 44 hours after hatching, as in Jiang et al.15. Two

independent samples were isolated except for let-60(n1046) at 44 hours and let-23(sy1) at

32 or 40 hours, which have one sample each.

DNA microarrays

DNA microarrays used for the let-60(G12V) experiments are described in Jiang et

al.15, and for the let-60(n1046) and let-23(sy1) experiments in Reinke et al.35. RNA

let-60 targets in C. elegans 8/29/01 23

preparation, cDNA synthesis, microarray hybridization and microarray scanning were

performed as previously described35. Cy-3 dUTP was used to label experimental cDNA

and Cy5-dUTP was used to label cDNA from reference RNA made from a mixed stage

population of wild type worms. The reference used in the let-60(G12V) experiments was

a different preparation than that used for the let-60(n1046) and let-23(sy1) experiments.

Data analysis

In order to identify genes that are expressed differently following heat-shock

treatment of let-60(G12V) and wild-type animals, we employed a mixed-model ANOVA

analysis procedure as implemented by SAS. The model has a genotype factor (let-60 or

wild-type), a time factor (0, 0.5, 1, 2 hours), an interaction factor between genotype and

time, and a random factor from the repetitions. More explicitly, our statistical model is:

yijk=µ+Gi+Tj+(GT)ij+rk+eijk

where:

G, T, and GT are the effects due to genotype (G), time (T) and the interaction of

genotype and time (GT);

µ, Gi, Tj and (GT)ij, are fixed effects such that the mean for the ith mutant

(genotype) at time j is µij=µ+Gi+Tj+(GT)ij;

rk is the random effect associated with the kth repetition;

and e is random error associated with the kth repetition in genotype i at time j.

For implementation of mixed procedure analysis in SAS, fixed effects for genotype (G),

time (T), and genotype*time are placed in the MODEL statement, and random effects for

repetitions placed in the RANDOM statement. The interaction effect, GT, indicates that

the expression profile of one gene in the let-60(G12V) time course is different than in the

let-60 targets in C. elegans 8/29/01 24

wild-type time course. We analyzed genes that revealed significant values of GT

(p<0.001).

487 out of 708 target genes identified in the let-60(G12V) time course were

present on the microarrays used in the let-60(n1046) and let-23 time courses, and we

examined the expression for each of the 487 genes in these time courses. To calculate the

difference in expression between the two time courses, we calculated the log2(let-60/let-

23 expression ratio) for each replication of each gene at each time point. We then

determined the average log2(let-60/let-23 expression ratio) for each gene at each time

point, summed up the average expression change over the seven time points and then

selected 41 genes in which the cumulative expression change was more than 5. Out of

these 41, 34 display the same type of regulation (induction or repression) in the let-

60(G12V) and let-60(n1046) time courses.

BlastP was used to find human genes that are homologous to the 708 C. elegans

genes that are regulated by let-60(G12V), using the worm proteins dataset from ACeDB

and the human subset of Genbank. Human genes were selected if the blast match was

greater than p<10-10 over 80% of the length of the protein. The database is available at

http://cmgm.stanford.edu/~kimlab/Ras/.

To identify human homologs that are regulated in human breast tumors, we

performed a Student’s t-test analysis (two samples of unequal variance, p<0.05) between

each tumor subtype and the normal-breast like samples, and found 25 regulated genes.

From these genes, we selected 21 genes whose regulation (induction or repression) was

similar in the human breast samples and the worm let-60(G12V) time course.

let-60 targets in C. elegans 8/29/01 25

Western blot

Western blot analysis of staged L3 populations were performed as described in Lackner

et al., 1998. Polyclonal anti-Erk2 peptide antibody K-23 (SantaCruz Biotechnology,

Santa Cruz, CA) and monoclonal anti-Erk-1&2 M8159 (Sigma) were used for

immunostaining of MPK-1 and phospho-MPK-1 in Western blotting experiments.

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Legends

Figure 1 a. HS::let-60(G12V) hermaphrodite in the L4 stage following heat-shock

treatment, showing excess vulval induction. b. Wild-type hermaphrodite in the L4 stage

following heat-shock treatment. c. HS::let-60(G12V) hermaphrodite in the L3 stage

following heat-shock treatment, showing a Clr phenotype. d. Wild-type hermaphrodite

in the L3 stage following heat-shock treatment. Scale bars, 10µM.

Figure 2 a. Hierarchical cluster of predicted heat-shock proteins. Shown are expression

profiles of 29 genes encoding Heat-shock proteins in the let-60(G12V) and the wild-type

time courses, ordered by hierarchical clustering. Column headings refer to hours after

heat-shock. Expression levels at each time point are normalized to expression before

heat-shock. Single asterisks indicate genes that show maximum expression changes

greater than 2 fold, and double asterisks indicate genes that exhibit more than 4 fold

induction. Identities and expression levels of individual genes are available at

http://cmgm.stanford.edu/~kimlab/Ras/. Scale shows level of expression.

b. Expression of let-60 mRNA following heat-shock treatment of HS::let-60(G12V) and

wild-type strains. Shown are the average expression levels for four to eight repeats at

each time point (+/- SD), normalized to expression before heat-shock. The expression of

let-60 in the let-60(G12V) time course is significantly greater than in the wild-type time

course (Student's t-test, p<0.001). c. Western blot analysis of MAP kinase activation.

Top panel shows staining using monoclonal antibodies specific for the phosphorylated

form of mammalian MAP kinase. Bottom panel shows staining using monoclonal

antibodies against total mammalian MAP kinase protein. Columns refer to hours

let-60 targets in C. elegans 8/29/01 33

following heat-shock treatment in the time courses for HS::let-60(G12V) and wild-type

strains. Sizes of molecular weight standards (Kd) are indicated. mpk-1 expresses two

forms of MAP kinase protein by alternative splicing17, but only one form was stained by

the anti-phospho-MAP kinase monoclonal antibody.

Figure 3 a. Pie chart shows the distribution of 297 let-60-regulated genes. Gene

annotations were derived from Proteome, and were used to manually place genes into

functional classes. Numbers of genes in each class are shown, and do not include 411

genes that encode novel proteins.

b. Hierarchical clustering of 708 let-60(G12V)-regulated genes. Shown are the average

expression levels of 708 genes with significant changes in expression levels between the

let-60(G12V) and the wild-type time courses. Columns show hours after heat-shock

treatment in each of the time courses. Expression levels are normalized to expression

before heat-shock treatment. Hierarchical clustering was used to place the genes into

groups (A, B and C). Asterisk denotes expression of let-60. Scale shows level of

expression. Full data for A and B are available at the supplemental web site and at

http://cmgm.stanford.edu/~kimlab/Ras/.

Figure 4. Expression profiles of let-60-regulated genes. a. sos-1. b. Transcription

factors. c. Ion channels and neurotransmitter receptors. d. Neurotransmitter metabolic

proteins. e. G protein-coupled receptors. f. Cell surface glycoproteins. g. Drug resistance

proteins. h. Metalloproteases. Genes discussed in the paper are indicated. Full data are

available at http://cmgm.stanford.edu/~kimlab/Ras/. Scale shows level of expression.

let-60 targets in C. elegans 8/29/01 34

Figure 5. Shown are the expression of 34 genes in the let-60(G12V) time course, the let-

60(n1046) time course compared to the wild-type time course (let-60(n1046)/WT), the

let-23 time course compared to the wild-type time course (let-23/WT) and the let-60 time

course compared to the let-23(sy1) developmental time courses (let-60(n1046)/let-23).

These genes showed stronger regulation in the let-60(n1046)/WT time course than in the

let-23/WT time course. Full data for this figure are available at

http://cmgm.stanford.edu/~kimlab/Ras/. Scale shows level of expression.

Figure 6. Expression profile of 21 human genes across 78 human breast tumors, 3

fibroadenomas and 4 normal breast samples showing expression relative to their median

expression levels. Data are from Sørlie et al., 2001 (ref. 45). The 21 genes were selected

because they were 1) highly similar to C. elegans genes regulated by let-60(G12V), 2)

because they showed significant differences in expression between normal breast versus

tumor samples (Student's t-test, p<0.05), 3) because they were expressed in most of the

breast tissue samples and 4) because they were similarly induced or repressed in the

human and worm experiments. Shown are expression levels across normal breast-like

samples compared to the four or five distinct tumor sub-types described in 45: Erb-B2+,

basal-like, luminal subtype A, luminal subtype B/C. Colored dots indicate tumor types

showing significant expression differences when compared to normal breast. Scale

shows level of expression relative to the median. Identities and expression levels for all

of the genes are at http://cmgm.stanford.edu/~kimlab/Ras/ and all human breast tumor

data are available at http://genome-www.stanford.edu/breast_cancer/mopo_clinical/ .