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

Abstract

The transcriptional co-regulator Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) may promote breast tumor growth; however, the mechanisms by which its effects are mediated remain to be fully elucidated. Tumor-associated macrophages serve an important function in tumor development and progression and are recruited by chemotactic factors produced by cells within the tumor microenvironment. The present study assessed the effects of CITED2 silencing on macrophage recruitment in two xenograft mouse models of human breast cancer, one in which tumor growth was sensitive to CITED2 silencing (MDA-MB-231) and one in which it was insensitive (MDA-MB-468). The present study identified that silencing CITED2 significantly attenuated macrophage infiltration in MDA-MB-231 but not MDA-MB-468 orthotopic tumors, concordant with its effect on tumor growth. Correspondingly, conditioned media obtained from CITED2-silenced MDA-MB-231 cells exhibited a significantly decreased ability to induce macrophage recruitment by Transwell migration assay, whereas the chemotactic effect of MDA-MB-468 conditioned media was unaffected. Examining the expression of macrophage chemoattractants within orthotopic tumors and tumor cell-conditioned media revealed a significant decrease in C-C motif chemokine ligand (CCL)20 mRNA and protein expression following CITED2-silencing in MDA-MB-231 cells, compared with that in cells transfected with scramble shRNA. However, mRNA and protein expression was unaffected by CITED2-silencing in MDA-MB-468 cells. Furthermore, chromatin immunoprecipitation analysis revealed that CITED2 was localized to the CCL20 promoter in MDA-MB-231 cells, suggesting that it serves a direct function in its regulation, which is consistent with the effect of CITED2 silencing on CCL20 expression. Lastly, neutralizing CCL20 in the conditioned media of MDA-MB-231 cells significantly inhibited macrophage recruitment. Collectively, these results suggest that CITED2 is involved in modulating macrophage recruitment, representing a novel mechanism through which it may influence tumor growth. This may be partly mediated by regulating tumor cell production of the chemokine CCL20.

IRGM promotes glioma M2 macrophage polarization through p62/TRAF6/NF‐κB pathway mediated IL‐8 production

“…Interestingly, IRGM also promotes MIP‐3α release and inhibited IL‐6 release (Figure D and 4E). The chemokine MIP‐3α has not reported a role in M2 macrophage polarization, while MIP‐3α could influence tumor growth by inducing macrophage recruitment as a chemokine (Jayaraman et al, ), which suggested IRGM could recruit macrophage into glioma by releasing MIP‐3α. We speculate both IL‐8 and MIP‐3α play key roles in glioma immune suppressive microenvironment formation.…”

“…IL‐8 promotes a pro‐oncogenic inflammatory microenvironment by inducing M2‐type tumor‐associated macrophages (TAMs) (Xiao et al, ; Li et al, ). The chemokine MIP‐3α (also called CCL20) could influence tumor growth through mediating macrophage recruitment (Jayaraman et al, ). IL‐6 is a multifunctional cytokine released by M1 macrophages as proinflammatory cytokine, and could inhibit M2 macrophages polarization (Sansone et al, ; Chishti et al, ).…”

Targeting Macrophage-Recruiting Chemokines as a Novel Therapeutic Strategy to Prevent the Progression of Solid Tumors

“…For example, it is reported that CCL20, a ligand for CCR6, is abundant in PyMT mammary tumors and genetic deletion of Ccr6 gene in PyMT mice significantly reduces the number of TAMs in mammary tumors ( 68 ). Although the cell type that secretes CCL20 is unknown in the PyMT model, a recent study demonstrates that MDA-MB-231 human breast cancer cells express high level of CCL20 and that inhibition of CCL20 expression in cancer cells reduces TAM accumulation in xenografts ( 69 ). It is also reported that a highly metastatic derivative of MDA-MB-231 cells (named BM1) expresses high levels of CCL5 (a ligand for CCR5) and treatment of BM1 tumor-bearing mice with a CCR5 antagonist significantly reduces the number of TAMs in tumors ( 70 ).…”

“…These results suggest that breast cancer cells can utilize CCL20-CCR6 and CCL5-CCR5 signaling in order to recruit TAMs. In the MDA-MB-231 breast cancer model ( 69 ), expression of CCL20 and TAM accumulation in xenografted tumors are suppressed by knock down of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2), a transcriptional co-regulator whose expression is increased in human invasive ductal carcinoma compared to normal mammary tissues and further enhanced in metastatic breast cancer ( 71 , 72 ). In the BM1 as well as 4T1 mammary tumor models, forced expression of Raf kinase inhibitory protein (RKIP) suppresses CCL5 secretion from cancer cells and reduces TAM accumulation in the xenograft ( 70 , 73 ).…”

CITED2 and the modulation of the hypoxic response in cancer

“…Alternatively, CITED2 may in this case act as co-activator of other transcription factors, such as SMAD2/3[ 18 ], which are mediators of TGF-β signaling pathway, or TFAP2A, showed to cooperate with CITED2 for normal vascularization of the myocardium during heart development[ 70 ]. Other studies also reported a reduction in primary tumor growth due to the attenuation of tumor-associated macrophage (promoting tumor development and progression) recruitment in response to CITED2 depletion in cancer cells and the consequent downregulation of the macrophage chemoattractant CCL20[ 71 ]. This suggests that CITED2 promotes tumor-associated macrophage recruitment and infiltration in breast tumors.…”