NUP93 is highly essential for survival of mammary tumor cell lines, and its abundance associates with poor prognosis of patients with breast cancer
To identify genes most relevant to progression of breast cancer, we performed computational analyses integrating RNA sequencing data from both METABRIC 14 and The Cancer Genome Atlas, and incorporated both somatic copy number alterations (CNA) and patient survival data (> 3000 patients). In parallel, we added gene essentiality scores derived from whole-genome shRNA screens that used 138 cancer lines 15, 16. Next, we compared essentiality scores of the top 500 clinically relevant genes and the bottom 500 genes. As expected, the top 500 genes were found to be more essential (p-value < 0.01 for pan-cancer cell lines and < 0.02 for breast cancer cell lines). Combining the top 500 clinically relevant genes and the 500 most essential genes, we obtained a ranked list of 20 genes, which are both clinically relevant and phenotypically essential for breast cancer (Supplementary Table 1). Interestingly, two tubulin genes topped the list of 20 genes, in line with reports linking overexpression of tubulins to survival of patients with breast cancer 17. The scores received by nucleoporin 93 (NUP93) were close to those received by the tubulins. Figure 1A presents the essentiality scores received by NUP93 in a series of breast cancer cell lines. Because anti-tubulin drugs are widely used in chemotherapy, but no approved anti-cancer drugs target NUP93 18, we focused on the nucleoporin. Additional analyses confirmed that the over-expression of this gene leads to poor prognosis (logrank P < 3.61E-12). Importantly, this trend remained significant when age, metastasis to lymph nodes and disease subtypes were controlled (Cox hazard ratio = 1.22, P < 1.96E-4). As an example, we divided the METABRIC dataset into three groups according to NUP93 expression levels. The Kaplan-Meier survival analyses separately performed for each group revealed that NUP93 overexpression is independently and significantly associated with shorter disease-free patient survival (Fig. 1B). The same dataset was stratified also according to the status of estrogen receptor (ER; Fig. 1C), which revealed that the more aggressive group of tumors, which lack ER expression, displayed relatively high NUP93.
An association between NUP93 and more virulent mammary tumors emerged from analysis of the 10 integrative clusters (IC) of breast cancer 2. IC10, which incorporates mostly triple negative tumors, showed highest NUP93 expression. Likewise, NUP93’s transcripts were relatively high in IC5, which identifies almost all cases with ERBB2/HER2 amplification. In addition, we found that high NUP93 was significantly associated with CNA (Fig. 1E). As expected, the highest gains of the gene were found in the basal subtype, and this was followed by the HER2-enriched subtype. Furthermore, high NUP93 expression was typical to high-grade tumors (Fig. 1F). In conclusion, by developing a novel computational pipeline, we learned that a component of the NPC is highly essential for survival of breast cancer cells and can predict shorter survival of a subset of patients with breast cancer.
Experimental strategies applying induced overexpression and downregulation reveal involvement of NUP93 in proliferation, migration and invasiveness
Because NUP93 is overexpressed in the basal subtype, we selected two basal models: (i) MDA-MB-231 basal B breast cancer cells, and (ii) MCF10A, a non-tumorigenic immortalized basal B line. Firstly, we expressed an inducible allele of NUP93 in MDA-MB-231 cells and verified relatively high expression on induction with tetracycline (Tet; Fig. 2A). Reciprocally, we established sublines expressing different doxycycline (DOX) inducible shRNAs (iSh; Fig. 2B). As predicted, exposing cells to Tet enhanced their ability to incorporate a radioactive nucleoside into DNA (Fig. 2C), whereas DOX-induced downregulation reduced DNA synthesis (Fig. 2D). Next, we applied a cell viability assay, which supported the ability of ectopic NUP93 to enhance viability (Figs. S1A and S1E). Similar conclusions were derived from experiments that used subclones constitutively expressing shNUP93 (Fig. S1B and S1F; left panels) and cells expressing siRNAs specific to NUP93 (Fig. S1B and S1F; right panels). A parallel set of experiments, which made use of genetically modified MCF10A cells (Figs. S1C and S1D) confirmed association of higher viability signals with overexpression (Fig. S1G) and lower signals in NUP93-depleted cells (Fig. S1H).
According to a recent breast cancer study, predictors of migration, rather than proliferation, are strongly associated with patient survival 19. When we placed inducible MDA-MB-231 cells on the upper compartment of cell culture inserts and treated them with the inducer, they migrated significantly faster than untreated cells (Fig. 2E). The reciprocal approach, which used DOX-inducible shRNAs, confirmed lower migration rates relative to control shRNA (Fig. 2F). Moreover, when the intervening membrane was coated with an extracellular matrix we similarly observed increased and decreased invasion rates with cells exposed to Tet (overexpression) or DOX (knockdown), respectively (Figs. 2G and 2H). Additional migration (Fig. S1I) and invasion assays (Fig. S1J) using MDA-MB-231 cells, as well as a similar set of MCF10A cell experiments (Figs. S1K-S1N) provided further support to the notion that NUP93 increases cellular motility. In conclusion, in line with the high essentiality and clinical significance, high abundance of NUP93 associated in vitro with increased viability, mitosis, migration and matrix invasion.
NUP93 overexpression enhances trans-endothelial migration and 3D invasion, as well as remodels focal adhesion sites
According to a recent report, NUP93 is involved in 3D migration and this correlates with an altered actin cytoskeleton 20. In line with this report, when pre-formed spheroids were induced with tetracycline to elevate NUP93, many cells migrated outward to form invasion zones (Fig. S2A). Reciprocally, NUP93 silencing reduced zone area (Fig. S2B). Increased invasiveness emerged also from TEM (trans-endothelial migration) assays that used endothelial monolayers overlaid by cells expressing inducible shNUP93 (Fig. S2C). Because VEGF might be involved, we used ELISA and found that siNUP93 reduced VEGF secretion (Fig. S2D). Interestingly, depletion of NUP93 also increased cell area (Fig. S2E) and reduced by 4-fold the ability of cells to form invadopodia (Fig. S2F). Notably, invadopodia contain microdomains with spatiotemporal dynamics of actin-rich adhesion sites 21. Indeed, we found that depletion of NUP93 was associated with remodeling of the actin cytoskeleton (Fig. S2G) and up-regulation of integrin alpha5 and integrin beta1 (Fig. S2H). Next, we extended the analysis to additional cell adhesion molecules, such as two paxillin family members—paxillin and Hic-5, which have been widely implicated in turnover of adhesion sites 22. Imaging and quantification of the respective adhesion areas unveiled redistribution along with up-regulation of the respective areas in siNUP93-treated cells (Fig. S2I). In summary, the overexpressed NUP93 increases 3D cell invasion, as well as enhances TEM, invadopodium assembly and remodels adhesion sites containing molecules like paxillin, fascin and zyxin.
High NUP93 abundance associates with increased rates of tumor growth and metastasis, along with matrix reorganization
Next, we implanted MDA-MB-231 cells, expressing a Tet-inducible NUP93 allele, in the fat pad of female mice and added the inducer to the drinking water of one of 2 groups of mice. Both the volumes and weights of tumors we harvested 3 weeks later were significantly larger in the treated group (Fig. S3A). A reciprocal experiment that made use of cells expressing a DOX-inducible shNUP93 reinforced the ability of NUP93 to accelerate tumor growth (Fig. S3B). This conclusion was independently supported by using cells stably expressing shNUP93 and determining tumor growth (Fig. S3C). As an initial test of metastasis, lungs were excised and metastases were quantified (Fig. S3D). The results reflected strong inhibitory effects, which prompted studies employing inducible expression and two different metastasis assays. Cells inducibly overexpressing NUP93 were injected either into the tail vein (Fig. 3A, left panel) or into the subaxillary mammary fat pad (Fig. 3A; right panel). Seven days later, we added tetracycline to the drinking water and mice were sacrificed 3 weeks later. The results indicated that NUP93 overexpressors are endowed with > 3-fold stronger capacity to colonize lungs. Further, when the same protocol was applied on tumors inducibly expressing shNUP93, we observed remarkably fewer lung metastases (Fig. 3B). Together, these observations assigned to NUP93 an important role in metastasis.
To resolve the identity of protein mediators, we sequenced RNA from MDA-MB-231 cells that were pretreated with siNUP93. The major differentially expressed (DE) genes are shown in Fig. 3C and Supplementary Excel File 1. Along with up-regulation of the cysteine-rich angiogenic inducer 61 23, we observed downregulation of several mitochondrial genes, such two subunits of the respiratory chain I, which drives ATP generation, and RNR1, a subunit of ribonucleotide reductase, which catalyzes dNTP production. These alterations might reflect the metabolically active state of NUP93-overexpressing cells. We also noted TGF-beta, keratins and extracellular matrix (ECM) components 24. The heatmap shown in Fig. 3D lists the major matrisome DE genes, including several types of collagen. These observations were supported using PCR (Fig. S3E) and immunoblotting for collagen IVa6 (Fig. S3F). Because collagen enables cell-to-matrix adhesion through binding with integrins, we predicted accompanying changes in substrate adhesion. Experiments using DOX-inducible shNUP93 (Fig. S3G), or an inducible NUP93 (Fig. S3H), confirmed that NUP93 reduces adhesion to substrate. To examine collagen involvement, we stained collagen IVa6 (Fig. S3I). Importantly, collagen fibrils were observed only in NUP93-depleted cells. For in vivo assays, tumors excised from DOX-treated mice were stained with either a collagen dye, picrosirius red (Fig. 3E), or an anti-collagen IVa6 antibody (Fig. S3J). Evidently, tumors from mice treated with shNUP93 showed strong staining only if the animals were pre-treated with Doxycycline. Since collagen is induced by TGF-beta 25, we used various methods to probe for the ligand and the receptor, TGFBRII. The results indicated co-induction, along with localization of the receptor to lamellipodia (Figs. 3F-3G, S3K-S3M). In conclusion, high abundance of NUP93 confers accelerated growth and metastatic spread upon mammary tumor cells. The underlying mechanism involves weakening ECM adhesion due to inhibition of collagen deposition, lower expression of ECM-modifying enzymes and reduced secretion of TGF-beta.
Overexpression of NUP93 enhances nuclear translocation of SMAD, ERK, STAT3, GR and p105 (NF-kB) in response to TGF-beta, EGF, glucocorticoid and TNF-alpha, respectively
Steroid resistant nephrotic syndrome is caused by NUP93 mutations, which interfere with BMP7-induced SMAD transcriptional activity 12. In addition, it was shown that insect NUP93 is needed for nuclear import of active SMADs 26. Hence, we assumed that NUP93 overexpression enhances signals initiated by BMP7/TGF-beta and other stimuli. Probing endogenous importin7 and NUP93 revealed a perinuclear ring of importin7, similar to the pattern of endogenous NUP93 (Fig. 4A). Importantly, importin7’s perinuclear localization was lost in mammary cells pre-treated with siNUP93. To validate the prediction that NUP93, by recruiting specific importins, mediates TGF-beta induced nuclear translocation of SMADs, we depleted NUP93 in MCF10A cells and followed the kinetics of nuclear import (Fig. 4B). While SMAD2/3 translocated to the nucleus of control cells within 20 minutes, we observed only limited translocation in NUP93-depleted cells. Assuming that NUP93 controls translocation of additional importin7’s cargos, we examined the extracellular signal-regulated kinase (ERK). Upon stimulation with EGF, ERK undergoes phosphorylation that exposes a nuclear translocation signal (NTS), which facilitates binding to importin7 27. Accordingly, when we stimulated control cells with EGF the phosphorylated form of ERK (pERK) clearly translocated to the nucleus, but cells depleted of NUP93 displayed much weaker translocation (Fig. 4C). This difference was verified by fractionation of cell extracts into cytoplasmic and nuclear fractions (Fig. 4D). Consistent with these results, we observed reciprocal changes in MCF10A cells engineered to inducibly overexpress NUP93 (Fig. 4E).
In similarity to ERK, STAT3 undergoes phosphorylation and nuclear translocation upon stimulation with EGF, but alpha importins, rather than importin7, have been implicated 28, 29. Nevertheless, analyses of both total STAT3 (Fig. S4A) and pSTAT3 (Fig. S4B) indicated that NUP93 can regulate EGF-induced import of STAT3. These observations raised the possibility that NUP93 anchors different importins and translocates them via the NPC, along with the respective cargos. Hence, we followed two additional cargos, the glucocorticoid receptor (GR) and the p105 subunit of the nuclear factor kappa B (NF-κB; Figs. S4C and S4D). Using NUP93-depleted cells, in both cases we observed strong inhibition of ligand-induced translocation following stimulation with either dexamethasone (DEX), a synthetic GR ligand, or with the tumor necrosis alpha (TNFa). Notably, importin alpha/beta, as well as importin7, translocate GR 30, whereas NF-κB is translocated by importin alpha3 and alpha4 31. In conclusion, apart from the known functions of NUP93 as a scaffold nucleoporin involved in NPC assembly, this molecule emerges as a broad-spectrum transporter of the active forms of signaling proteins.
Myristoylated peptides corresponding to the NTS of ERK and SMAD inhibit progression of NUP93-overexpressing tumors
Next, we attempted blocking nuclear translocation of ERK by preventing binding of importin7 to ERK’s NTS. The strategy used a previously described NTS-derived peptide fused to myristic acid 32. As expected, the ERK-derived peptide inhibited nuclear accumulation of ERK, but a control peptide exerted no effect (Fig. 5A). In addition, the peptide reduced incorporation of radioactive thymidine into DNA, only in cells overexpressing NUP93 (Figs. S5A and S5B). Likewise, the S-phase fraction observed with cells treated with the peptide was severely reduced (Fig. S5C). Similarly, the ERK peptide reduced the ability of cells to form colonies, migrate and invade (Figs. S5D-S5F). Hence, we implanted NUP93-overexpressing cells in the subaxillary mammary fat pad of mice, and once tumors became palpable intravenously treated animals with the ERK-derived peptide. RFP-fluorescence and size of the primary tumors were measured (Figs. 5C and 5D), along with tumor weight (Fig. 5E). In addition, we cut out the lungs to assay metastases (Fig. 5F). Evidently, tumors overexpressing NUP93 grew faster and colonized lungs better than the control tumors. Moreover, while the ERK peptide weakly inhibited growth and metastasis of control tumors, the inhibitory effects observed upon treatment with the ERK peptide were significantly stronger, implying that NUP93-overexpressing tumors acquire dependence on ERK’s nuclear transport.
Because NUP93 mutations causing a renal disease fail SMAD signaling 12 and tumors frequently corrupt the TGF-beta pathway 33, we synthesized a similar SMAD peptide, based on a previously identified Ser-Pro-Ser triad 27. The peptide nearly completely inhibited TGF-beta-induced nuclear import of SMAD2/3 (Fig. 5B). In addition, it reduced incorporation of radioactive thymidine (Fig. S5B), markedly lowered the S-phase fraction (Fig. S5C), as well as inhibited the ability of cells to form colonies, migrate and invade (Figs. S5D-S5F). As with the ERK peptide, we implanted NUP93-overexpressing cells in the fat pad of mice and delivered the SMAD peptide three times per week. RFP-fluorescence, as well as tumor volumes (Figs. S5G and S5H) and weights were determined (Fig. S5I) and lungs metastases were counted (Fig. S5J). We observed weak inhibition of control tumors, but stronger effects were observed when treating NUP93-overexpressing tumors. Moreover, treatment with the SMAD peptide only weakly inhibited metastasis of control tumors, but the numbers of micro-metastases formed by NUP93-overexpressing cells were reduced by 75–85% (Fig. S5J). In conclusion, the ERK and SMAD peptides exemplify the potential therapeutic scenario offered by targeting the cargo-importin-NUP93 axis, specifically in NUP93-overexpressing breast cancers. Importantly, it has recently been reported that inhibition of NPC formation causes selective cancer cell death, while normal cells undergo a reversible cell cycle arrest 34. In conclusion, the myristoylated peptides we tested might effectively and selectively inhibit cancer cells overexpressing NUP93.
Natural gain- and loss-of-function mutations confirm NUP93’s roles in metastasis
Three NUP93 mutations, E14K, Q15X and R327C, were identified by a screen aimed at putative driver mutations escalating the risk of metastasis 13. Hence, we stably expressed two mutant alleles in MCF10A (Fig. S6A) and in MDA-MB-231 cells (Fig. 6A). Both mutants, especially R327C, superseded the ability of wildtype NUP93 to seed colonies (Figs. 6B and S6B). In addition, we performed DNA synthesis (Figs. 6C and S6C), migration and invasion assays (Fig. 6D, 6E, S6D and S6E), which indicated that the mutants were more active than the wildtype form. Next, we implanted the respective MDA-MB-231 cells in the fat pad of animals and followed tumor growth (Figs. 6F and 6G). Both mutations enhanced tumor growth relative to WT and control (EV) cells. Similarly, quantification of metastatic lung nodules indicated that both mutant alleles were consistently more active than WT in either metastasis format (Fig. S6F). Notably, we occasionally observed metastases in other organs. Focusing on liver metastases, we observed striking differences (Fig. 6H): Whereas cells overexpressing either mutant colonized livers, we were unable to detect any lesion in livers from mice injected with WT cells. In conclusion, the gain-of-function NUP93 mutations enhance oncogenic attributes and confer organ-specific metastatic colonization.
Analyses of patients with nephrotic syndromes detected homozygous missense NUP93 mutations 12. To study oncogenic effects, we firstly disrupted the endogenous NUP93 gene using Crispr-CAS9. Several knockout (KO) clones were established (Fig. 6I). Next, we generated a series of KO cells expressing WT or individual mutants (Fig. S6G). As expected, all KO sublines exhibited reduced proliferation DNA synthesis (Figs. S6H and S6I), as well as relatively low rates of migration/invasion, and high adhesion (Figs. S6J-S6L). Unlike WT, neither mutant recovered normal migration/invasion rates when expressed in KO cells (Figs. S6K and S6L). In vivo tests of tumor growth (Figs. 6J and 6K) and metastasis (Figs. 6L and S6M) supported the LOF phenotypes: stable expression of neither mutant was able to reconstitute the relatively high rates of tumor growth/metastasis displayed by KO cells re-expressing WT. In summary, by studying GOF and LOF mutations of NUP93 we obtained independent evidence in support of the critical roles played by NUP93 in breast cancer progression.
NUP93 overexpression increases GTP-loading onto RHO GTPases and enhances transcriptional outcome of the WNT and other signaling pathways
RHO family GTPases are switches involved in mammary tumorigenesis and metastasis 35. Hence, we utilized the ability of effectors to bind with the active, GTP-bound forms. Overexpression of NUP93 increased CDC42-GTP and RAC1-GTP, but decreased RHOA-GTP (Fig. S7A). Congruently, siNUP93 reduced active RAC1 and CDC42, but increased RHOA-GTP. Notably, RAC1 and CDC42 collaborate with KRAS. Accordingly, we observed significant downregulation of RAS-GTP in siNUP93-treated cells (Fig. S7B). Because RAS is activated upon stimulation of numerous pathways, we undertook a multiple promoter-reporter strategy. HEK293 cells were co-transfected with a NUP93 expression vector and various luciferase plasmids containing different DNA response elements (REs), including the glucocorticoid RE (GRE; Fig. 7A). This revealed that NUP93 overexpression activated several promoters, including GRE, SMAD4 and FLI1. Yet, the strongest signal (> 50-fold) was observed with, a beta-catenin responsive reporter containing the binding site for T cell factor (TCF)/lymphoid enhancer factor (LEF). Nuclear accumulation of beta-catenin is induced by WNT; once in the nucleus, beta-catenin associates with TCF/LEF and activates target genes 36. As expected, co-transfection of the reporters and siNUP93 decreased several signals, but two collagen promoters were activated (Fig. S7C). In addition, co-transfecting NUP93’s oncogenic mutants increased reporter activity beyond the WT signal (Fig. 7B). Accordingly, transfection of NUP93, either WT or mutants, strongly elevated the beta-catenin protein (Fig. 7C). This translated to increased expression of multiple beta-catenin target genes (three are shown in Fig. S7D). Because EGFR can transactivate the beta-catenin pathway 37 and SMAD3 can occupy WNT-responsive elements 38, we examined transactivation by EGF and TGF-beta. The results confirmed transactivation (Fig. 7D), which might enhance the relatively large effect of WNT. Next, we addressed stability of beta-catenin. Cell treatment with cycloheximide, a protein synthesis inhibitor, followed by immunoblotting, confirmed short half-life of beta-catenin (Fig. 7E). In contrast, when overexpressed, both WT NUP93 and the oncogenic mutants strongly prolonged the half-life, implying multiple NUP93/beta-catenin interactions.
Using WNT3A, we found that R327C-NUP93, more than WT, enhanced ligand-stimulated promoter activation (Fig. 7F). To test if this cooperative effect was due to nuclear transport, we firstly fractionated control cells and detected only a small fraction of beta-catenin in the nucleus (Fig. S7E). Importantly, this fraction was erased by NUP93 knockdown. Moreover, immunofluorescence indicated that treatment with WNT3A induced relatively weak nuclear translocation of beta-catenin, which was enhanced by wildtype NUP93, and further increases were observed in cells expressing oncogenic NUP93 (Fig. 7G). Thus, both fractionation and immunofluorescence supported the cooperative effect of WNT and NUP93. To validate functionality, we knocked-down LEF1 and stimulated cells with WNT3A (Figs. 7H and 7I). While siLEF1 inhibited cell migration/invasion by approximately 30% in WT-expressing cells, this increased to 90% in cells expressing R327C-NUP93, in support of cooperativity. In conclusion, when overexpressed, NUP93 enhances loading of GTP onto RAS, RAC1 and CDC42, molecular switches of cell migration. In line with this, several signaling pathways were found to be constitutively active in NUP93-overexpressing cells, including the WNT pathway, hyperactivation of which has been implicated in metastasis 39. WT NUP93 elevated expression and inhibited degradation of beta-catenin, while the oncogenic mutants, better than WT, enhanced WNT-induced nuclear transport of beta-catenin.
Proteome- and transcriptome-wide analyses identify podocalyxin and multiple RNA transcripts as potential mediators of NUP93-induced metastasis
To complement the promoter analyses, we applied liquid chromatography-mass spectrometry (LC-MS/MS) on cytoplasmic and nuclear fractions. As expected, siNUP93 significantly reduced beta-catenin in the nucleus (Figs. 7J and S7F; Supplementary Excel File 2). Alongside, the cytoplasmic fraction showed downregulation of specific MAPK pathways, whereas the nuclear fraction displayed elevated SP1, which up-regulates collagen 40, bystin, which regulates cell adhesion 41, and cullin-3, which is essential for collagen export 42. All three proteins were up-regulated in NUP93-depleted cells and underwent nuclear-cytoplasm translocations (Figs. S7G and S7H). The proteomic analyses also indicated that the mucin podocalyxin (PODXL) underwent up-regulation in the cytoplasm of siNUP93-treated cells. We confirmed elevated PODXL levels in NUP93-deficient cells, as well as downregulation upon re-expression of wildtype NUP93 (Figs. 7K and S7I). Notably, neither G591V- nor Y629C-NUP93 were able to downregulate PODXL. In addition, downregulating PODXL, in similarity to knocking-down cullin3 and bystin, decreased cell adhesion (Fig. 7L), implying involvement of all three proteins in NUP93-mediated weakening of mammary cell adhesion.
Along with transport of proteins, the NPC mediates selective exchange of RNA molecules between the nucleus and cytoplasm 43. To explore transport of specific RNAs, we depleted NUP93 in MCF7 breast cancer cells and examined the subcellular localization of polyadenylated transcripts (Fig. S7I), essentially as previously described 44. NUP93 depletion significantly affected the localization of hundreds of RNAs, led to a 3-fold increase in cytoplasmic enrichment of transcripts from 200 genes, and conversely, to 3-fold increase in nuclear enrichment of transcripts from 153 other genes (see Supplementary Excel File 3). Because the lincRNA called NORAD (LINC00657) was identified also by the RNAseq analysis presented in Fig. 3C, we focused on this molecule. Notably, NORAD enhances TGF-beta signaling and metastasis 45. Hence, we employed single-molecule FISH (smFISH) to examine sub-cellular distribution of this intronless, mostly cytoplasmic lncRNA. In line with the RNA-seq analysis, upon NUP93 depletion NORAD exhibited strong nuclear enrichment in both MCF7 and MDA-MB-231 cells (Fig. S7J). Conceivably, nuclear retention inactivates NORAD, along with similar RNAs, to retard metastasis of NUP93-low breast cancers.
In summary, by integrating clinical and laboratory lines of evidence, we concluded that high abundance of NUP93 is associated with highly aggressive breast cancers. Correspondingly, the overexpressed NUP93 enhanced TEM and matrix invasion, while reorganizing the matrisome. These attributes translated to increased rates of tumor growth and metastasis in animal models. Mechanistically, overexpression of NUP93 boosts the ultimate nuclear transport step activated by diverse extracellular signals, including WNT, EGF and TGF-beta. Collectively, these lines of evidence identify NUP93 as a driver of metastasis, which hijacks the beta-catenin and other signaling pathways.