1. NF-κB activation is required for EMT and metastasis.
NF-κB activation is required for EMT and metastasis. A, NF-κB activity, reported as relative light units +/− SD, from MCF10A cells infected with shRNAs targeting RASAL2 and DAB2IP and coinfected with either vector control or the IKBα-SR. Coinfection with the IKBα-SR completely ablates NF-κB activity. B, Transwell invasion of MCF10A cells infected with control shRNAs or shRNAs targeting RASAL2 and DAB2IP. Invasion was measured after 24 hours and reported as average +/− SD. Suppression of NF-κB activity (coinfection of the IKBα-SR) had no effect on invasion. C, Western blot showing the expression of the EMT markers E-cadherin and N-cadherin in MCF10A cells that were infected with control shRNAs or shRNAs targeting RASAL2 and/or DAB2IP in the absence or presence of the IKBα-SR. Lysates were run in duplicate. RASAL2, N-cadherin, and SLUG were probed for on the same gel with the loading control GAPDH (1), whereas DAB2IP and E-cadherin were probed for separately with the loading control GAPDH (2). D, Real-time PCR quantification of FN1, VIM, SLUG, SNAIL, and ZEB1 in MCF10A cells in response to RASAL2 and DAB2IP suppression with and without the IKBα-SR. Data show average relative amount of mRNA +/− SD. E, Representative H&E images of lungs, with McNeu metastases marked by red circles. McNeu cells were infected with nontargeting control shRNAs or shRNAs targeting RASAL2 and DAB2IP with and without the IKBα-SR. Cells were injected into tail veins of syngeneic mice, lungs were harvested 3 weeks after injection, and the number of metastases per mouse was quantified. Scale bars correspond to 500 μm. F, Coinfection of the IKBα-SR significantly suppressed metastasis formation (P = 0.037, Mann–Whitney), indicated by an asterisk. Horizontal bars indicate the median number of metastatic lesions per mouse. Note: The data indicated in gray are a duplicate from Fig. 3G.
Sarah Naomi Olsen et al. Cancer Discov 2017;7:
©2017 by American Association for Cancer Research