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. 2009 Dec;20(24):5306-13.
doi: 10.1091/mbc.e09-08-0699.

Synthetic lethal genetic interactions that decrease somatic cell proliferation in Caenorhabditis elegans identify the alternative RFC CTF18 as a candidate cancer drug target

Jessica McLellan  1 , Nigel O'Neil, Sanja Tarailo, Jan Stoepel, Jennifer Bryan, Ann Rose, Philip Hieter
Affiliations

Synthetic lethal genetic interactions that decrease somatic cell proliferation in Caenorhabditis elegans identify the alternative RFC CTF18 as a candidate cancer drug target

Jessica McLellan et al. Mol Biol Cell. 2009 Dec.

Abstract

Somatic mutations causing chromosome instability (CIN) in tumors can be exploited for selective killing of cancer cells by knockdown of second-site genes causing synthetic lethality. We tested and statistically validated synthetic lethal (SL) interactions between mutations in six Saccharomyces cerevisiae CIN genes orthologous to genes mutated in colon tumors and five additional CIN genes. To identify which SL interactions are conserved in higher organisms and represent potential chemotherapeutic targets, we developed an assay system in Caenorhabditis elegans to test genetic interactions causing synthetic proliferation defects in somatic cells. We made use of postembryonic RNA interference and the vulval cell lineage of C. elegans as a readout for somatic cell proliferation defects. We identified SL interactions between members of the cohesin complex and CTF4, RAD27, and components of the alternative RFC(CTF18) complex. The genetic interactions tested are highly conserved between S. cerevisiae and C. elegans and suggest that the alternative RFC components DCC1, CTF8, and CTF18 are ideal therapeutic targets because of their mild phenotype when knocked down singly in C. elegans. Furthermore, the C. elegans assay system will contribute to our knowledge of genetic interactions in a multicellular animal and is a powerful approach to identify new cancer therapeutic targets.

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Figures

Figure 1.
Figure 1.
Synthetic genetic interactions in yeast. (A) The human orthologues of the genes on the outside of the wheel are mutated in colon tumors. Genes on the inside were identified as being SL with a subset of the genes mutated in colon tumors. Solid lines and dashed lines, SL and SS interactions, respectively. Circles shaded in gray represent essential genes in yeast. The temperature indicated next to the allele indicates the temperature at which genetic interactions were tested. (B and C) Examples of growth curves performed that yielded a SS interaction and no interaction, respectively. (D). The scal parameter extracted from the ensembles of growth curves in B and C are shown. The growth profile of each curve can be summarized by scal, a value inversely proportional to growth rate. Double mutant strains with a scal value statistically greater than the predicted scal of a neutral genetic interaction are considered SS. scal values for the double mutant, the associated single mutants, and the WT strain are shown on a horizontal line and slower growing strains have a greater scal value.
Figure 2.
Figure 2.
Paradigms for C. elegans genetic interaction assays. (A) Typical screening method using RNAi. RNAi treatment is initiated during the L1–L4 stages of development to deplete maternal, and hence embryonic, stores of mRNA. A frequent readout is enhanced embryonic lethality in the subsequent generation. (B) The Pvl screening method where RNAi treatment begins at the L1 stage, once animals have completed the embryonic cell divisions. This experimental approach is designed to identify phenotypes caused by defects in the postembryonic cell divisions, such as Pvl. Black arrows denote when RNAi treatment is initiated. Gray arrows indicate when phenotype scoring takes place. Black wedges depict the phenotypes caused by RNAi knockdown and the associated phenotypic lag. (C) C. elegans cell lineage diagram showing the embryonic and postembryonic cell divisions and highlighting the vulval and seam cell divisions (Sulston and Horvitz, 1977). Source: Adapted from the C. elegans server (www.umdnj.edu/mobioweb/moss/celegansonline.html/).
Figure 3.
Figure 3.
him-1(RNAi) causes Pvl. (A) N2 (wild type) worms and him-1(e879) worms were treated with both him-1(RNAi) and no RNAi and then scored for the presence of Pvl. N2 normally shows a low frequency of Pvl that is enhanced when treated with him-1(RNAi). The him-1 hypomorphic mutant has a low frequency of Pvl when treated with no RNAi. Treatment of him-1 worms with him-1(RNAi) produced a more penetrant phenotype (79.3 ± 3.6% compared to 3.9 ± 1.0%). Error bars, SEM; n > 500 for each experiment. (B) DIC image of N2; (no RNAi). (C) DIC image of N2; him-1(RNAi). (D) zmp-1::GFP was used to score the number of GFP-positive foci in worms with and without Pvl. Wild type typically has four GFP-positive foci, whereas 90.4 ± 1.6% of worms with a Pvl have fewer than four GFP-positive foci. Error bars, SEM; n > 120 for each experiment. (E) Image of the four GFP foci seen in worms with a wild-type vulva. (F) Image of a Pvl worm with two GFP foci. (G and H) In the Pvl screening assay worms are visually scored on a Zeiss dissecting microscope at ×160 for the presence of a small protrusion, a protruding vulva. (G) Wild-type worms treated with no RNAi. (H) him-1 worms treated postembryonically with him-1(RNAi). Black arrows, protruding vulva.
Figure 4.
Figure 4.
him-1(RNAi) causes defects in cell division in an independent lineage. (A) ajm-1::GFP worms were treated with no RNAi or him-1(RNAi) and adults were scored for gaps in their lateral seam syncytia. Error bars, SEM; n > 120 for each experiment. (B) ajm-1::GFP;(no RNAi). (C) ajm-1::GFP; him-1(RNAi). (D) Worms carrying the SCM::GFP marker were treated with either no RNAi or him-1(RNAi). (E) SCM::GFP;(no RNAi). 15 nuclei, not including H0, can be identified (F) SCM::GFP; him-1(RNAi). Fewer than 15 nuclei are seen in 65% of worms including irregular nuclear bodies (arrowhead). (G) Example of a typical nuclear body. (H and I) Examples of irregular nuclear bodies. n = 30 for each experiment.
Figure 5.
Figure 5.
Genetic interactions of cohesin can be recapitulated in C. elegans. (A) An increase in Pvl frequency is seen when him-1 worms are treated with RNAi against the C. elegans orthologues of RAD27, CTF4, CTF18, CTF8, and DCC1 but not with control RNAi. (B and C) smc-3(ok1703) and scc-1(ok1017) homozygotes show a similar profile of genetic interactions. Error bars, SEM; n > 500 for each experiment.
Figure 6.
Figure 6.
Genetic interactions are specific rather than general. (A) The frequency of Pvl does not synergistically increase when him-1 is treated with randomly chosen RNAi clones. (a) RNAi clones shown to interact with the MAD1/mdf-1 checkpoint component. (b) Genes involved in different aspects of DNA metabolism. (c) Randomly chosen genes from chromosome I. RNAi against RAD27/crn-1 was included as a positive control. Error bars, SEM; n > 150 for each experiment. (B) him-6 worms show a synthetic interaction when treated with RNAi against RAD27 and CTF4, whereas no interaction is detected with the components of the alternative RFCCTF18. Error bars, SEM; n > 500 for each experiment.
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Comment in

  • Mol Biol Cell. 20:5037.

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