CITED2 Modulates Breast Cancer Metastatic Ability through Effects on IKKα

Abstract

Previously, we identified the transcriptional co-activator CITED2 as a potential facilitator of bone metastasis using a murine mammary cancer model. Extending these studies to human breast cancer, it was observed that CITED2 mRNA expression was significantly elevated in patient specimens of metastatic breast cancer relative to primary tumors, with highest levels in metastasis to bone relative to non-bone sites. To further evaluate CITED2 functions in breast cancer metastasis, CITED2 expression was stably reduced in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, cell migration and invasion were significantly reduced, as was the establishment of metastasis following intra-cardiac administration in athymic nude mice. To explore the mechanism behind these effects, gene expression following CITED2 knockdown in MDA-MB-231 cells by cDNA microarray was performed. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced along with several NF-κB targets with known roles in metastasis (OPN, MMP9, uPA, SPARC, IL-11 and IL-1β). Further, ChIP assay revealed recruitment of CITED2 to the promoter of IKKα, indicating a direct role in regulating its expression. Consistent with reduced IKKα expression, CITED2 knockdown inhibited both canonical and non-canonical NF-κB signaling. Finally, restoration of IKKα expression following CITED2 knockdown in MDA-MB-231 and MDA-MB-468 cells rescued their invasive ability. Collectively, these data demonstrate that CITED2 modulates metastatic ability in human breast cancer cells, at least in part, through the regulation of IKKα. Implications The current study highlights the role of CITED2 in facilitating breast cancer metastasis, partly via regulation of IKKα.

CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells

“…The human breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultured as previously described ( 25 ). These cell lines were authenticated by the ATCC using DNA profiling and cytogenetic analysis and used for experiments within 6 months from the time of resuscitation.…”

“…These cell lines were authenticated by the ATCC using DNA profiling and cytogenetic analysis and used for experiments within 6 months from the time of resuscitation. Scramble and short hairpin (sh)CITED2-expressing MDA-MB-231 and MDA-MB-468 cells were generated following a previously described protocol ( 24 , 25 ), using the lentiviral shRNA expression vector pLKO.1-puro (Addgene plasmid 8453; Addgene, Inc., Cambridge, MA, USA) containing a shRNA sequence specific for scramble or CITED2, as previously described ( 15 , 28 ).…”

“…Total RNA from the cell lines (three biological replicates per experimental condition) and tumor tissues (6 tumors per experimental group for MDA-MB-231; 4 tumors per experimental group for MDA-MB-468) were obtained as previously described ( 24 ). Amplification of 36B4 served as the internal control in the RT-qPCR reaction ( 25 ). Relative expression between samples was calculated using the comparative 2 −ΔΔCq method ( 30 ).…”

“…Furthermore, it has been reported that CITED2 serves a role in promoting skin, colon, lung and breast cancer ( 22 – 27 ). Beyond potentially facilitating breast cancer metastasis ( 25 ), it has been suggested that CITED2 may promote primary tumor growth ( 24 ). This effect may be mediated, at least partly, by modulating tumor vasculature via regulation of VEGFA; however, the mechanisms by which CITED2 regulates tumor growth remain to be fully elucidated.…”

CDK7 Inhibition Is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition

“…Transcriptional co-regulator CITED2 has recently been reported as a prognostic factor in breast cancer patients with important roles in cancer progression, chemoresistance and metastasis [22,23]. Therefore, we examined in more detail the association of CITED2 with response to CDK7 inhibition.…”