Cancer-associated exportin-6 upregulation inhibits the transcriptionally repressive and anticancer effects of nuclear profilin-1

Abstract

Aberrant expression of nuclear transporters and deregulated subcellular localization of their cargo proteins are emerging as drivers and therapeutic targets of cancer. Here, we present evidence that the nuclear exporter exportin-6 and its cargo profilin-1 constitute a functionally important and frequently deregulated axis in cancer. Exportin-6 upregulation occurs in numerous cancer types and is associated with poor patient survival. Reducing exportin-6 level in breast cancer cells triggers antitumor effects by accumulating nuclear profilin-1. Mechanistically, nuclear profilin-1 interacts with eleven-nineteen-leukemia protein (ENL) within the super elongation complex (SEC) and inhibits the ability of the SEC to drive transcription of numerous pro-cancer genes including MYC. XPO6 and MYC are positively correlated across diverse cancer types including breast cancer. Therapeutically, exportin-6 loss sensitizes breast cancer cells to the bromodomain and extra-terminal (BET) inhibitor JQ1. Thus, exportin-6 upregulation is a previously unrecognized cancer driver event by spatially inhibiting nuclear profilin-1 as a tumor suppressor.

Keywords: BET bromodomain; ENL; MYC; epigenetics; exportin-6; nucleocytoplasmic transport; profilin-1; super elongation complex; transcription; tumor suppressor.

Figure 1.. XPO6 upregulation occurs in cancer and associates with poor patient survival

(A) Pan-cancer XPO6 mRNA levels in the TCGA cohorts. Whiskers represent min-max. Mann-Whitney U non-parametric test was used to compare between normal and tumor samples for each cancer type. RSEM, RNA-seq by expectation maximization. (B) XPO6 mRNA levels of 112 breast tumors in the TCGA dataset with adjacent normal tissues. p value was based on Wilcoxon Signed Rank non-parametric test. (C) qRT-PCR of XPO6 mRNA levels in breast epithelial cell lines. One-way ANOVA and Dunnett’s multiple comparisons tests were used to compare between MCF-10A and breast cancer cell lines. Data are mean ± SEM. **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Western blot of XPO6 in untransformed and transformed breast epithelial cell lines, with different cytoplasmic and nuclear proteins as controls. (E) Univariate Kaplan-Meier analysis of the association between XPO6 mRNA levels and the overall survival (OS) of TCGA patients with bladder, renal clear cell, hepatocellular, and breast carcinomas. (F) Univariate Kaplan-Meier analysis of the association between XPO6 mRNA levels and the OS of stage II breast cancer patients within the TCGA cohort. (G) Univariate Kaplan-Meier analysis of the association between XPO6 protein levels and the OS of a cohort of 65 breast cancer patients. p values for (E)–(G) were based on log-rank tests. See also Figures S1 and S2 and Tables S1A–S1C.

Figure 2.. XPO6 is required for breast cancer cell growth in vitro

(A) Human breast epithelial cell lines were infected with a small hairpin targeting luciferase (shLUC) and two different XPO6 shRNAs. (B) Relative growth effects of XPO6 knockdown by dividing normalized Alamar blue values (day 9/1) of shXPO6 versus shLUC cells. Data are mean ± SEM of a representative experiment (sextuples per condition). p values were based on one-way ANOVA and Dunnett’s multiple comparison tests. (C) Colony formation assay using cells in (A). (D) Colony formation assay using MCF-10A cells infected with XPO6 or luciferase. Colony areas were expressed as percentages. Data are mean ± SEM of a representative experiment (triplicates per condition). (E) MCF-7 cells expressing WT or RNAi-Res XPO6 were infected with shLUC or shXPO6 #3 and subjected to Western blot analysis. Lanes for the E2F1 blot were cropped and rearranged from the same blot (indicated by the black line). Relative growth effects of XPO6 knockdown were calculated by normalizing colony areas of shXPO6 versus shLUC cells. Data are mean ± SD of one representative experiment (triplicates per condition). (F) MCF-7 cells infected with shLUC or shXPO6 #3 were labeledwithBrdU and stained for BrdU or with propidium iodide (PI). Fifteen random fields were quantified. Percent BrdU positivity of cells in all images is shown. Data are mean ± SEM. Scale bars, 40 μm. (G) MCF-7 cells from (F) were synchronized by double thymidine block, released for different hours, and analyzed for DNA contents. p values for (D)–(F) were based on unpaired t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. All results were confirmed by three independent experiments. See also Figures S3 and S4.

Figure 3.. XPO6 loss inhibits breast cancer growth in vivo

(A) MCF-7 cells expressing XPO6(WT) or XPO6(RNAi-Res) were infected with shLUC or shXPO6 #3 and inoculated in female nude mice (n = 6). Caliper measurement of tumors began at day 15. Two-way ANOVA and Sidak’s multiple comparison tests were used to compare tumor volumes between shLUC and shXPO6 groups. (B) Endpoint tumor weights. p values were based on one-way ANOVA and Tukey’s multiple comparison tests. (C and D) Three randomly selected tumors per group were immunostained for p-Rb(Ser⁷⁹⁵) (C) and Ki67 (D). Positive tumor cells from random fields of each image were normalized against total tumor cells stained by hematoxylin. Each dot represents 300–450 tumor cells. Scale bars, 100 μm. p values were based on one-way ANOVA and Tukey’s multiple comparison tests. All data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4.. Nuclear Pfn1 is required for the growth inhibitory effect of XPO6 loss

(A) MCF-7 cells expressing XPO6(WT) or XPO6(RNAi-Res) were infected with shLUC or shXPO6 #3 and subjected to immunofluorescence staining for endogenous Pfn1 and nuclear staining by DAPI. Representative images and quantitative analysis of nuclear versus cytoplasmic fluorescence intensity are shown. Over 500 cells per condition were analyzed. Data are mean ± SEM. p values were based on unpaired t tests. Scale bars, 20 μm. (B) MCF-7 cells were infected individually or simultaneously with shPFN1 (#2) (controlled by shCTRL) and shXPO6 #3 (controlled by shLUC). Relative cell growth rates were expressed as day 11/day 1 ratios as described in Figure 2B. (C and D) Pfn1-null mouse chondrocytes were first infected with untagged Pfn1 (controlled by empty vector; C) or YFP-Pfn1 with or without an NES tag (controlled by YFP; D), followed by further infection with shLUC or shXPO6 #2 (recognizing mouse XPO6). Cells were subjected to western blot (Pfn1 antibody [C] and GFP antibody [D]) and growth analyses as described in (B). For (B)–(D), relative growth of shLUC cells in each subgroup was arbitrarily set to 1. Data are mean ± SEM of a representative experiment (sextuplicates per condition). p values were based on unpaired t tests. **p < 0.01; ****p < 0.0001. Results were confirmed by three independent experiments. See also Figures S5 and S6 and Table S1D.

Figure 5.. Nuclear Pfn1 interacts with the SEC

(A) IP of YFP or YFP-NLS-Pfn1(WT or S137D) from nuclear extracts of MDA-MB-231 stable cells by a GFP antibody, followed by silver staining. (B) Eluates from (A) were analyzed by liquid chromatography-MS. Proteins specifically bound to NLS-Pfn1(WT) were identified using label-free quantification (LFQ) intensity and a threshold of >1.5-fold-higher intensity over those bound to YFP and NLS-Pfn1(S137D). LFQ intensities of detectable SEC components are shown. (C) Confirmation of the interaction between NLS-Pfn1(WT) and SEC by anti-GFP co-IP as in (B) using nuclear extracts of stable MCF-7 cells, followed by western blot. (D) Interaction between the SEC and endogenous Pfn1 in MCF-7 cells. ENL and cyclin T1 were pulled down followed by western blot. (E) Cyclin T1 pulldown from shLUC- versus shENL-infected MCF-7 cells, followed by western blot for endogenous Pfn1 and other proteins. In total, 50-fold-less input was used for the Pfn1 blot given the small fraction of nuclear Pfn1 (most in the cytoplasm) interacting with the SEC. (F) Size-exclusion chromatography and western blot analyses of nuclear extracts of MDA-MB-231 cells for endogenous Pfn1 and SEC components. Results in (C)–(F) were confirmed by three independent experiments. Lanes in (A), (D), and (E) were cropped and rearranged from the same blots for clarity of presentation (indicated by the black lines). See also Table S2.

Figure 6.. Nuclear Pfn1 inhibits SEC function

(A) Western blot of NLS- or NES-tagged YFP-Pfn1 relative to endogenous Pfn1 by GFP or Pfn1 antibodies. S137D, residing in the epitope of the Pfn1 antibody, abolishes the detection. (B) qRT-PCR of MYC in MCF-7 cells in (A). p values were based on unpaired t test relative to YFP control. (C) qRT-PCR of XPO6 and MYC in MCF-7 cells expressing XPO6(WT) or XPO6(RNAi-Res) and infected with shLUC or shXPO6 (#3). p values were based on unpaired t test by comparing shLUC versus shXPO6 #3. (D) qRT-PCR of MYC in XPO6 KD/rescue MCF-7 xenograft samples (10 tumors/group, 2 technical replicates/tumor) from Figure 3. p values were based on unpaired t test. (E) qRT-PCR of MYC in Pfn1-null chondrocytes infected first with vector or Pfn1 and subsequently with shLUC or shXPO6 #2 (recognizing mouse XPO6). p values were based on unpaired t test. (F) qRT-PCR of MYC and Pfn1 in MCF-7 cells expressing Pfn1(WT) or Pfn1(RNAi-Res) and infected with scrambled shCTRL or shPFN1 #1 or #2. p values were based on unpaired t test by comparing shCTRL versus shPFN1. (G) Correlations of MYC mRNA levels with XPO6, BRD4, and NCL in different TCGA datasets. The y axis represents Spearman’s correlation coefficients (ρ). Fifteen cancer types in which statistically significant positive correlations between XPO6 and MYC expression are shown (ρ > 0; FDR q values < 0.05). (H) ChIP using antibodies for ENL, cyclin T1, Cdk9, p-Rpb1(Ser²), and H3K36me3 from MCF-7 cells expressing YFP or YFP-NLS-Pfn1(WT or S137D) followed by MYC qPCR. p values were based on unpaired t test. (I) ChIP using antibodies for ENL, p-Rpb1(Ser²), and H3K36me3 from MCF-7 cells infected with shLUC or shXPO6 #3 followed by MYC qPCR. p values were based on unpaired t test. All data (except A and G) represent mean ± SEM of representative experiments, which were confirmed at least three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S7 and Table S3.

Figure 7.. XPO6 loss triggers anticancer transcriptomic effects and sensitization to BET inhibitor JQ1

(A) RNA-seq using shLUC- or shXPO6-#3-infected MCF-7 cells expressing XPO6(WT) or XPO6(RNAi-Res). GSEA evaluating changes in the 50 hallmark gene sets induced by XPO6 knockdown. Shown are MYC targets V1 and V2 gene sets. (B and C) qRT-PCR validation of DE genes induced by XPO6 knockdown specifically in MCF-7 cells expressing XPO6(WT) but not XPO6(RNAi-Res). (B) XPO6 and MYC target genes NCL (activated) and HMOX1 (repressed) were analyzed. (C) Various known SEC target genes were analyzed. p values were based on unpaired t test by comparing shLUC versus shXPO6 for each gene. (D) Representative gene sets significantly enriched by XPO6 knockdown in MCF-7 cells expressing XPO6(WT) but not XPO6(RNAi-Res). Analysis was performed using R/GAGE against GO molecular functions and multiple curated MSigDB databases (hallmark, KEGG, Reactome, Chemical and Genetic Perturbation) and graphed by R/ggplot2. (E) MDA-MB-231 cells infected with control or sgXPO6 viruses were treated with DMSO or JQ1 in colony formation assays for 10 days. p values were based on one-way ANOVA and Dunnett’s multiple comparisons by comparing control and sgXPO6 cells at different JQ1 concentrations. Data in (B), (C), and (E) are mean ± SEM of representative experiments (triplicates per condition) and were confirmed three times. (F) MDA-MB-231 cells from (E) were orthotopically injected into female nude mice and treated with vehicle or JQ1 for 3 weeks. Mice in the sgXPO6 #1 and #3 groups were combined for analysis. Arrows indicate treatment start dates. p values were based on two-way ANOVA and Sidak’s multiple comparison tests to compare vehicle versus JQ1 groups. (G) Relative growth rates of individual tumors during the dosing period were first calculated by dividing tumor volumes at various time points by day 1. The calculated tumor growth rates in the JQ1 groups of control or sgXPO6 mice were subsequently divided by the averaged growth rates of the corresponding vehicle groups, giving rise to the relative JQ effect (y axis). p values were based on two-way ANOVA and Sidak’s multiple comparison tests. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figures S8–S10 and Table S4.