DAB2IP loss in luminal a breast cancer leads to NF-κB–associated aggressive oncogenic phenotypes
Angana Mukherjee, Rasha T. Kakati, Sarah Van Alsten, Tyler Laws, Aaron L. Ebbs, Daniel P. Hollern, Philip M. Spanheimer, Katherine A. Hoadley, Melissa A. Troester, Jeremy M. Simon, Albert S. Baldwin
Angana Mukherjee, Rasha T. Kakati, Sarah Van Alsten, Tyler Laws, Aaron L. Ebbs, Daniel P. Hollern, Philip M. Spanheimer, Katherine A. Hoadley, Melissa A. Troester, Jeremy M. Simon, Albert S. Baldwin
View: Text | PDF
Research Article Oncology

DAB2IP loss in luminal a breast cancer leads to NF-κB–associated aggressive oncogenic phenotypes

Abstract

Despite proven therapy options for estrogen receptor–positive (ER+) breast tumors, a substantial number of patients with ER+ breast cancer exhibit relapse with associated metastasis. Loss of expression of RasGAPs leads to poor outcomes in several cancers, including breast cancer. Mining the The Cancer Genome Atlas (TCGA) breast cancer RNA-Seq dataset revealed that low expression of the RasGAP DAB2IP was associated with a significant decrease in relapse-free survival in patients with Luminal A breast cancer. Immunostaining demonstrated that DAB2IP loss occurred in grade 2 tumors and higher. Consistent with this, genes upregulated in DAB2IP-low Luminal A tumors were shared with more aggressive tumor subtypes and were associated with proliferation, metastasis, and altered ER signaling. Low DAB2IP expression in ER+ breast cancer cells was associated with increased proliferation, enhanced stemness phenotypes, and activation of IKK, the upstream regulator of the transcription factor NF-κB. Integrating cell-based ChIP-Seq with motif analysis and TCGA RNA-Seq data, we identified a set of candidate NF-κB target genes upregulated with loss of DAB2IP linked with several oncogenic phenotypes, including altered RNA processing. This study provides insight into mechanisms associated with aggressiveness and recurrence within a subset of the typically less aggressive Luminal A breast cancer intrinsic subtype.

Authors

Angana Mukherjee, Rasha T. Kakati, Sarah Van Alsten, Tyler Laws, Aaron L. Ebbs, Daniel P. Hollern, Philip M. Spanheimer, Katherine A. Hoadley, Melissa A. Troester, Jeremy M. Simon, Albert S. Baldwin

×

Figure 5

DAB2IP loss activates the IKK/NF-κB signaling pathway in Luminal A breast cancer cells.

Options: View larger image (or click on image) Download as PowerPoint
DAB2IP loss activates the IKK/NF-κB signaling pathway in Luminal A breas...
(A) After transfection with DAB2IP or control siRNA, MCF10A cells were transfected with WT or mutant 3×κB luciferase reporter plasmids and pRL-TK Renilla plasmid. Cell incubation for 24 hours was followed by dual luciferase assay (****P < 0.0001) (n = 4). (B) Immunoblot showed increased phospho-IKKαβ expression (arrow) in siDAB2IP MCF10A cells (n = 3). (C) After transfection, siDAB2IP and siControl MCF10A cell proliferation was determined by MTS assay (****P < 0.0001) (n = 9). (D) shDAB2IP and shControl T47D cells were transfected with WT or mutant 3×κB luciferase reporter constructs and pRL-TK Renilla construct for dual luciferase assay (***P = 0.0001) (n = 4). (E) Cytoplasmic and nuclear extracts from siDAB2IP and siControl T47D cells were used for immunoblotting to show an increase in the cytoplasmic phospho-IKKαβ levels (arrows) in siDAB2IP cells (n = 3). (F) Transfected T47D cells were treated with 5 μM compound A or DMSO every 8 hours, followed by MTS assay at 24 and 48 hours to assess proliferation (*P = 0.0282, **P = 0.0034, ***P = 0.0005) (n = 3). (G) After treatment with 5 μM compound A or DMSO, siDAB2IP and siControl T47D cells were subjected to scratch-wound assays (**P = 0.008, ***P = 0.0002, ****P < 0.0001) (n = 3). (H) shDAB2IP and shControl T47D cells were treated with 5 μM compound A or DMSO for tumorsphere assay, with images taken on days 1, 4, and 7 (**P = 0.004, **P = 0.0063, **P = 0.0018 ****P < 0.0001) (n = 3, seeded in 3 wells per replicate). Unpaired Student’s t test and multiple comparisons corrected with Dunnett’s test were used to analyze the data. Magnification, 40×.