1. DAB2IP expression is selectively lost in luminal B breast cancers and it functions as a tumor suppressor by affecting multiple RAS isoforms.
DAB2IP expression is selectively lost in luminal B breast cancers and it functions as a tumor suppressor by affecting multiple RAS isoforms. A,DAB2IP mRNA expression across the different subtypes of human breast cancer. DAB2IP is specifically low in the luminal B subtype (P < 6.69 × 109, t test comparing LumA vs. LumB). B, Tukey box plot showing the median DAB2IP expression per subtype (P = 1.5 × 1014, ANOVA). C, Methylation of the CpG island in the DAB2IP promoter (chr9: 123,331,678) across subtypes. The DAB2IP promoter is specifically methylated in the luminal B subtype (P < 0.005, t test comparing LumA vs. LumB). D, Methylation at the CpG island in the DAB2IP promoter inversely correlates with expression. E, Kaplan–Meier curve showing relapse-free survival of luminal B tumors with high or low DAB2IP expression (log rank P = ). F, DAB2IP mutations in human tumor samples. Each triangle represents a nonsynonymous mutation, and each diamond represents a nonsense mutation. Blue triangles indicate breast cancer mutations, and red triangles indicate breast cancer mutations in the catalytic RasGAP domain. The table shows DAB2IP mutations in human breast tumors and the domain in which they occur. G, DAB2IP expression in lysates of basal (Ba, HCI-004) and luminal B (HCI-011, -013, -017, -003, -005) PDX tumors. H, DAB2IP expression in a panel of ER+ human breast cancer cell lines and normal immortalized mammary epithelial cells (MCF10A). Cell lines with minimal or no DAB2IP are indicated in bold. I, Western blot showing an increase in HRAS-GTP, NRAS-GTP, and pERK levels in CAMA1 cells following shRNA-mediated inactivation of DAB2IP or control shRNA. Samples were run on the same gels, but a lane was cut out as indicated by the line. Lysates were run in duplicate. DAB2IP, HRAS/HRAS-GTP, and pERK were probed for on the same gel, whereas NRAS/NRAS-GTP were probed for on a separate gel. J, Soft-agar colony formation of CAMA1 cells infected with a 3′-UTR shRNA targeting DAB2IP or nontargeting control, followed by expression of EGFP control vector, wild-type DAB2IP, or DAB2IP GAP point mutant (R413L). Data show relative number of colonies +/− SD. Data show relative number of colonies +/− SD, and all statistically significant differences are indicated by an asterisk. There was a significant increase in soft-agar colony formation upon DAB2IP suppression [P < , t test comparing EGFP/shCNT vs. EGFP/shDAB2IP(1)]. Reconstitution with wild-type DAB2IP rescued colony formation [not statistically significantly (ns) compared to the control]; however, the R413L GAP mutant failed to rescue [P < , t test comparing EGFP/shCNT vs. R413L/shDAB2IP(1)]. K, Western blot showing a reduction of HRAS-GTP, KRAS-GTP, and pERK levels in MCF7 cells upon expression of DAB2IP compared with EGFP. Samples were run on the same gel, but a lane was cut out as indicated by the line. Lysates were run in duplicate. DAB2IP, HRAS/HRAS-GTP, KRAS/KRAS-GTP, and pERK were probed for on the same gel, whereas NRAS/NRAS-GTP were probed for on a separate gel. L, Soft-agar colony formation of MCF7 cells expressing DAB2IP or EGFP. Data show relative number of colonies +/− SD. There was a statistically significant decrease in soft-agar colony formation upon DAB2IP reconstitution (P = , t test). M, Western blot showing that expression of the R413L DAB2IP-GAP mutant fails to suppress HRAS-GTP, KRAS-GTP, NRAS-GTP, and pERK levels in MCF7 cells. Lysates were run in duplicate. DAB2IP, HRAS/HRAS-GTP, KRAS/KRAS-GTP, and pERK were probed for on the same gel, whereas NRAS/NRAS-GTP were probed for on a separate gel. N, Soft-agar colony formation of MCF7 cells expressing the R413L GAP mutant or EGFP. Data show relative number of colonies +/− SD. There was no statistically significant decrease in anchorage-independent growth upon ectopic expression of the R413L mutant. O, Xenograft tumor formation of CAMA1 cells infected with DAB2IP-targeting CRISPR gRNA or empty control vector. Cells were injected subcutaneously into female NOD/SCID mice. Horizontal bars indicate median tumor volume, and the error bars indicate +/− interquartile range. There was a statistically significant increase in tumor volume upon CRISPR-mediated DAB2IP knockout as indicated by the asterisks [P = for sgDAB2IP(1); P = for sgDAB2IP(2), Mann–Whitney].
Sarah Naomi Olsen et al. Cancer Discov 2017;7:
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