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. 2016 Apr;16(2):164-72.
doi: 10.1038/tpj.2015.50. Epub 2015 Jul 7.

Genetic screening reveals a link between Wnt signaling and antitubulin drugs

A H Khan  1 , J S Bloom  2 , E Faridmoayer  1 , D J Smith  1
Affiliations

Genetic screening reveals a link between Wnt signaling and antitubulin drugs

A H Khan et al. Pharmacogenomics J. 2016 Apr.

Abstract

The antitubulin drugs, paclitaxel (PX) and colchicine (COL), inhibit cell growth and are therapeutically valuable. PX stabilizes microtubules, while COL promotes their depolymerization. But, the drug concentrations that alter tubulin polymerization are hundreds of times higher than their clinically useful levels. To map genetic targets for drug action at single-gene resolution, we used a human radiation hybrid panel. We identified loci that affected cell survival in the presence of five compounds of medical relevance. For PX and COL, the zinc and ring finger 3 (ZNRF3) gene dominated the genetic landscape at therapeutic concentrations. ZNRF3 encodes an R-spondin regulated receptor that inhibits Wingless/Int (Wnt) signaling. Overexpression of the ZNRF3 gene shielded cells from antitubulin drug action, while small interfering RNA knockdowns resulted in sensitization. Further a potent pharmacological inhibitor of Wnt signaling, Wnt-C59, protected cells from PX and COL. Our results suggest that the antitubulin drugs perturb microtubule dynamics, thereby influencing Wnt signaling.

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Figures

Figure 1
Figure 1
Statistical power; means±s.e.m.
Figure 2
Figure 2
Survival curves and drug correlations. (a) Survival curves for RH23; means±s.e.m. (b) P-values for drug correlations across RH panel on a negative logarithmic scale. The thin ends of the wedges correspond to lowest drug concentrations. 6 MP, 6-mercaptopurine; COL, colchicine; DMSO, dimethyl sulfoxide; MTX, methotrexate; PX, paclitaxel; RH, radiation hybrid.
Figure 3
Figure 3
Growth variations and broad-sense heritabilities. (a) Normalized growth variation among RH clones in paclitaxel, 25 nm; means±s.e.m. (b) Normalized growth variation among RH clones in colchicine, 25 nm; means±s.e.m. (c) Broad-sense heritabilties, H2, at various drug concentrations; means±s.e.m. RH, radiation hybrid.
Figure 4
Figure 4
Chromosome 22 locus for paclitaxel and colchicine. (a) Paclitaxel, 25 nm. (b) Colchicine, 25 nm. (c) Closeup of locus. University of California, Santa Cruz (UCSC) genes at top. Red horizontal lines, FWER <5% threshold. FWER, family-wise error rate.
Figure 5
Figure 5
ZNRF3. (a) Effect of ZNRF3 on cell survival in paclitaxel. Proliferation relative to vehicle-treated control. (b) Effect of ZNRF3 on cell survival in colchicine. (c) ZNRF3 copy number. Comparing classified copy number with array comparative genomic hybridization (aCGH) copy number.
Figure 6
Figure 6
Suggestive QTLs. (a) Paclitaxel, 76 nm. (b) Methotrexate, 0.7 μm. (c) DMSO, 10%. Red horizontal lines, FWER <10% threshold. DMSO, dimethyl sulfoxide; FWER, family-wise error rate; QTL, quantitative trait locus.
Figure 7
Figure 7
Validating ZNRF3 in the action of the antitubulin drugs. (a) Verification of ZNRF3 overexpression and small interfering RNA (siRNA) knockdown in HEK293 cells using quantitative PCR. (b) ZNRF3 overexpression diminishes the effects of the antitubulin drugs in HEK293 cells. (c) ZNRF3 overexpression also decreases the effects of the drugs in A23 cells. (d) Knockdown of ZNRF3 expression with an siRNA sensitizes HEK293 cells to paclitaxel and colchicine. (e) Pharmacological blockade of Wnt signaling with Wnt-C59 protects HEK293 cells from the actions of paclitaxel and colchicine. Growth curves normalized to zero drug concentrations; means±s.e.m.
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