IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers

Abstract

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

Figure 1.

Development and validation of DLD-1 βcatenin-DEADPOOL cells. (A) Schematic of the βcatenin-dependent iCasp9 system. (B) AP20187 (AP) dose–response curve in DLD-1 βcatenin-DEADPOOL cells. Mean ± SD, n = 3 independent experiments. (C) Schematic of genome-wide suppressor screen to identify genes required for βcatenin signaling in CRC. (D) Crystal violet stain of DLD-1 iCasp9/Cas9 line transduced with lentiviral vectors enabling expression of LacZ, CTNNB1 #1, or CTNNB1 #2 gRNA and treated with EtOH or 1 nM AP20187. Representative of three independent experiments.

Figure 2.

Genome-wide CRISPR-Cas9 DEADPOOL screen identifies IPO11 as a gene required for βcatenin signaling. (A) Scatter plot of normalized read counts for each gRNA in 1 nM AP20187 versus EtOH. (B) Top genes identified from the screen, ranked by the number of unique guides with a Z score >3. (C) Crystal violet stain of DLD-1 βcatenin-DEADPOOL cells expressing FLAG-IPO11 cDNA (IPO11 gRNA #1 and #2 insensitive) expressing LacZ, IPO11 #1, or IPO11 #2 gRNA and treated with EtOH or 1 nM AP20187. (D) DLD-1 expressing Cas9, pBAR βcatenin-dependent luciferase reporter, and LacZ, IPO11 #1, and IPO11 #2 gRNA. n = 3 independent experiments. (E) RT-qPCR of βcatenin target genes in monoclonal DLD-1 IPO11 KO lines. n = 3 independent experiments. (F) Crystal violet stain of DLD-1 βcatenin-DEADPOOL cells expressing FLAG-LEF1-VP16 and LacZ, IPO11 #1, or IPO11 #2 gRNA and treated with EtOH or 1 nM AP20187. Bars represent mean fold change ± SD. Statistical analyses were performed by one-way ANOVA followed by Dunnett’s multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure S1.

IPO11 KO does not affect βcatenin transcription. (A) DLD-1 Cas9 cells were infected with the indicated lentivirus, and RT-qPCR was performed to determine relative levels of βcatenin mRNA. Bars represent mean fold change ± SD. Statistical analyses were performed by one-way ANOVA followed by Dunnett’s multiple comparison test of three replicates. n.s, not-significant; P ≥ 0.05. (B) TIDE analysis for DLD-1 IPO11 clone #1 KO (https://tide.nki.nl/). Representative plot of two replicates. Statistical analysis was performed by two-tailed t test of the variance–covariance matrix of the standard errors, P > 0.001. Total effect = 79.9%. (C) TIDE analysis for DLD-1 IPO11 clone #2 KO (https://tide.nki.nl/). Representative plot of two replicates. Statistical analysis was performed by two-tailed t test of the variance–covariance matrix of the standard errors, P > 0.001. Total effect = 81.2%.

Figure 3.

IPO11 regulates nuclear βcatenin protein levels in APC mutant CRC cells. (A) DLD-1 control and IPO11^−/− cells fractionated into cytoplasmic and nuclear compartments and immunoblotted for the indicated proteins. Representative image of three independent experiments. (B) Immunofluorescence detection of βcatenin subcellular localization in SW480 Cas9 cells expressing LacZ, IPO11 #1, and IPO11 #2 gRNA. Quantification is mean intensity of fluorescence ± SD of three replicates. Statistical analysis was performed by one-way ANOVA Dunnett’s test. ****, P < 0.0001. (C) HCT116 cells expressing LacZ, IPO11 #1, and IPO11 #2 gRNA were fractionated into cytoplasmic and nuclear compartments and immunoblotted for the indicated proteins. Representative image of three replicates. (D) RKO control and IPO11^−/− cells were stimulated with WNT3A conditioned media, fractionated into cytoplasmic and nuclear compartments, and immunoblotted for the indicated proteins. Representative image of three independent experiments. (E) Dot plot of Bayes Factor (BF) for IPO11 in APC wild-type (wt; n = 6) and APC mutated cell lines (n = 17). Mann-Whitney U test, **, P < 0.001. Retrieved from PICKLES (Lenoir et al., 2018). BF > 5 represents high-confidence essential gene.

Figure 4.

IPO11 binds βcatenin to mediate its nuclear transport. (A) GST-pulldown assay using purified IPO11 and βcatenin proteins. (B) HEK293T cells were transfected with the indicated expression plasmids, and lysates were subjected to coimmunoprecipitations followed by Western blot. (C) Crystal violet stain of DLD-1 βcatenin-DEADPOOL cells expressing FLAG-ΔN-IPO11 and treated with EtOH or 1 nM AP20187. (D) Schematic of eGFP-βgalactosidase-βcatenin fusion constructs. FL, full length. (E) Confocal microscopy–based quantification of nuclear to cytoplasmic eGFP intensity ratios for the indicated constructs expressed in control or IPO11^−/− clones. Each point represents a single cell, with data collected over two independent experiments. n > 25 for each group. Two-way ANOVA Sidak’s multiple comparison test. **, P < 0.01; ****, P < 0.0001. (F) Representative images of E. (G) DLD-1 cells overexpressing FLAG-βcatenin or FLAG-NLS-βcatenin and LacZ, IPO11 #1, and IPO11 #2 gRNA were fractionated into cytoplasmic and nuclear compartments and immunoblotted for indicated proteins. (H) FITC-βcatenin nuclear import assay in semipermeabilized HeLa cells, imaged by confocal microscopy. Each point represents a single cell, pooled from three independent experiments; representative images of each condition are presented. Ordinary one-way ANOVA with Sidak’s multiple comparisons test, ****, P < 0.0001, mean ± SD. βcatenin alone, n = 148; βcatenin + Importin-11, n = 109; βcatenin + IPO11 + Ran, n = 128; βcatenin + IPO11 + RanQ69L, n = 191. ns, not significant.

Figure S2.

IPO11 expression and gene dependency in CRCs. (A) Expression levels of IPO11 mRNA in several types of CRCs (https://www.oncomine.org). (B) IPO11 gene dependency correlated to CTNNB1 gene dependency in CRC cell lines containing APC mutation (https://depmap.org). (C) HEK293T cells transfected with eGFP-βgal fusions to βcatenin truncation plasmids and immunoblotted for the indicated proteins. Representative image of two independent replicates.

Figure S3.

Purification and conjugation of His-βcatenin and FLAG-IPO11 and patient-derived CRC organoid growth assay. (A) Coomassie and fluorescent imaging of SDS-PAGE gels showing purity and FITC conjugation of βcatenin. (B) Coomassie gel showing purification and purity of FLAG-IPO11. (C) Fluorescence-based growth assay in CRC organoids. (D) CSC171C CRC organoids were infected with indicated fluorescent guides, and growth was monitored over time. (E) T7 assay of CSC171C organoids at indicated genomic loci. Representative image of three independent replicates.

Figure 5.

IPO11 is required for CRC organoid growth. (A) Crystal violet stain of colony formation of DLD-1 βcatenin-DEADPOOL cells transduced with LacZ, IPO11 #1, and IPO11 #2 gRNA. (B) RT-qPCR of ASCL2 in CSC171C organoid line transduced with LacZ, IPO11 #1, or IPO11 #2 gRNA. Mean fold change ± SD, representative of n = 3 independent experiments. Statistical analysis was performed by one-way ANOVA Dunnett’s test. *, P ≤ 0.05; **, P ≤ 0.01. (C) Fluorescence images of CSC171C organoid line expressing Cas9 and gRNA for LacZ, CTNNB1 #1, IPO11 #1, or IPO11 #2. (D) Points represent mean fold change ± SD of fluorescence of C representative of n = 3 independent experiments. Statistical analysis was performed by one-way ANOVA Dunnett’s test. ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Proposed model of βcatenin nuclear translocation through direct binding of βcatenin armadillo repeats and nuclear pore proteins via a Ran-independent mechanism. IPO11 directly binds βcatenin in the cytoplasm to mediate βcatenin nuclear import. In the nucleus, IPO11 binds to Ran-GTP, which leads to the dissociation of the cargo–importin-β complex. βcatenin accumulates in the nucleus.