Mitotic Vulnerability in Triple-Negative Breast Cancer Associated with LIN9 Is Targetable with BET Inhibitors

Abstract

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR.

Figure 1. Sustained BET activity is necessary for timely progression through mitosis

HCC38 and MDA-MB-231 cells were treated with vehicle or 1000 nM JQ1 and observed via live-cell imaging for 96 hours. (A) Average percent confluency of HCC38 (left) and MDA-MB-231 (right) cells over time. Data are means ± SEM. (B) Quantitation of the length of time required by individual TNBC cells to complete mitosis six hours after the addition of vehicle or JQ1. Data are means ± SEM (*=p<0.05 compared to vehicle). (C and D) Representative live-cell images (20×) of HCC38 (C) and MDA-MB-231 (D) cells treated with vehicle or 1000 nM JQ1. Numbers indicate minutes following the initiation of mitosis.

Figure 2. BET inhibitors promote mitosis-associated death or prolonged interphase

(A) Pie chart showing the percentage of vehicle- and JQ1-treated HCC38 cells that underwent different mitosis-associated cell fates: exit and divide (blue), exit and die (black), or die in mitosis (white). Vehicle versus JQ1 for all three outcomes, p<<0.001. (B) Quantitation of the length of time for HCC38 cells to divide again (blue dots) or die (black dots) following mitotic exit. p<0.05 compared to vehicle. (C) Comparison of the duration of mitosis of individual JQ1-treated HCC38 cells that die in mitosis, exit mitosis and die, or exit mitosis and divide. Die in Mitosis versus Exit and Die, p<0.05. (D) Pie chart showing the percentage of vehicle- and JQ1-treated MDA-MB-231 cells that underwent different post-mitotic cell fates: exit and divide (blue), exit and die (black), or prolonged interphase (grey). Vehicle versus JQ1 for all three outcomes, p<<0.001. (E) Quantitation of the length of time for MDA-MB-231 cells to divide again (blue dots) or die (black dots) following mitotic exit. p<0.05 compared to vehicle. For all graphs, each dot represents an individual cell, and red lines are mean ± SEM.

Figure 3. BET activity is necessary for sustained expression of cell cycle-associated genes

(A–C) MDA-MB-231 and HCC70 cells were treated for 72 hours with vehicle or 500 nM JQ1 and transcriptomes were analyzed using Affymetrix Human Gene 2.0 ST expression microarrays. (A) Venn diagram showing the number of genes whose expression significantly changed in each cell line as well as the number of genes commonly altered in both. (B) Top 5 non-overlapping Reactome terms for MDA-MB-231 (left) and HCC70 (right) cells. (C) GSEA of cell cycle-classifying genes whose expression was altered by JQ1 in MDA-MB-231 and HCC70 cells. (D-F) RT-qPCR analysis of three cell cycle/mitosis genes (CCNB1, KIF20A, and PLK1) in four TNBC cell lines treated with vehicle or 500 nM JQ1 for 24 hours. (G) RT-qPCR analysis of cell cycle and mitosis genes (CCNB1, KIF20A, PLK1, and KIF2C) in MDA-MB-231 (n=5) and MDA-MB-468 (n=10) tumors from orthotopically xenografted mice treated with vehicle or JQ1. (H) Volcano plots depicting mRNA log2 fold changes versus the corresponding log₁₀ p-values for genes whose expression significantly changes in response to JQ1 in MDA-MB-231 (right) and HCC70 (left) cells after 72 hours. Red dots indicate genes that are critical for mitosis. For all bar graphs, data are presented as means ± SD (*=p<0.05 compared to vehicle).

Figure 4. BET proteins directly modulate the mitotic transcriptional program

(A) RT-qPCR quantitation of the expression of selected mitosis-controlling transcription factors in MDA-MB-231 cells treated with vehicle or 500 nM JQ1 for 6 hours. (B and C) RT-qPCR analysis of BETi-repressed genes in tumors from vehicle- or JQ1-treated mice harboring (B) MDA-MB-231 (n=5) or (C) MDA-MB-468 (n=10) tumors. (D) Representative gene-specific ChIP-PCR analysis of MDA-MB-231 cells assessing binding of BRD4 to selected genes encoding mitosis-controlling transcription factors following treatment with vehicle or 500 nM JQ1 for 24 hours. For all graphs, data are presented as means ± SD (*=p<0.05 compared to vehicle or siNS).

Figure 5. BETi suppress mitosis transcription factors in the absence of super-enhancers

Binding of H3K27Ac in MDA-MB-231 cells was analyzed using ChIP-seq. These data were compared with data generated from JQ1-treated MDA-MB-231 cells analyzed by gene expression microarray. (A) Number of genes in MDA-MB-231 cells that are associated with super-enhancers (SE genes) or lack super-enhancers (Non-SE genes). Total genes = all genes expressed in MDA-MB-231 cells based on microarray data. Mitosis genes = 599 genes identified by SuperPath as critical for mitosis. Percentages indicate the percent of genes that contain super-enhancers. (B) Violin plots depicting fold change in gene expression of SE-associated genes and non-SE-associated genes for all genes expressed in MDA-MB-231 cells (p=4.2×10⁻¹⁵). (C) Violin plots depicting fold change in gene expression of SE-associated genes and non-SE-associated genes for mitosis genes. (D) Heatmap showing log2 fold change in expression of all JQ1-regulated genes in MDA-MB-231 cells ranked according to enhancer strength. The black line indicates the cut-off between SEs and typical enhancers. Four of the five BETi-regulated mitosis-regulating transcription factors are listed. MYBL2 is absent because it lacks a specific enrichment of H3K27Ac binding within 10 kbp of its promoter. (E) ChIP-seq binding profiles for H3K27Ac at the promoter regions for five mitosis-regulating transcription factors. MYC is shown as a representative SE-associated gene.

Figure 6. LIN9 mediates the effects of BET inhibition

(A) Confirmation of siRNA-mediated knockdown of LIN9 in MDA-MB-231 cells after five days. Inset is a representative western blot showing suppression of LIN9 substantially reduces LIN9 protein. (B) RT-qPCR analysis of BETi-target genes in MDA-MB-231 cells following LIN9 silencing. (C) Representative images (20×) of MDA-MB-231 cells following LIN9 silencing that were stained with DAPI (blue, nuclei) and Texas Red-X phalloidin (red, actin cytoskeleton). MDA-MB-231 cells treated with 500 nM JQ1 are included as a comparison. Arrows indicate multinucleated cells. (D) Kaplan-Meier curve of relapse-free survival for breast cancer patients with high and low expression of LIN9. (E) Violin plots depicting BETi-induced expression (log2 fold change) of genes that are not correlated (r<0.5) or are correlated (r≥0.5) with LIN9 expression. The right side of the panel depicts subdivision of genes that were correlated with LIN9 (r≥0.5) according to residence on chromosome 1q versus their responsiveness to JQ1 (p-value for r<0.5 vs. r≥0.5 = 1.8×10⁻⁴⁷; p-value for r≥0.5 on 1q vs. not on 1q = 1.2×10⁻⁵). r = Pearson’s coefficient. (F) Absolute fold change in expression following JQ1 treatment of genes that are highly correlated with LIN9 expression (r≥0.5) and those that are moderately or not correlated (r<0.5) in the TCGA and METABRIC datasets. (G) Percent of genes changed with JQ1 that are bound by LIN9 or not bound by LIN9 (p<<0.001). For panels A, B, and C, data are presented as means ± SD (*=p<0.05 compared to vehicle or siNS).