Abstract
Concomitant loss of lumen formation and cell adhesion protein CEACAM1 is a hallmark feature of breast cancer. In a three-dimensional culture model, transfection of CEACAM1 into MCF7 breast cells can restore lumen formation by an unknown mechanism. ID4, a transcriptional regulator lacking a DNA binding domain, is highly up-regulated in CEACAM1-transfected MCF7 cells, and when down-regulated with RNAi, abrogates lumen formation. Conversely, when MCF7 cells, which fail to form lumena in a three-dimensional culture, are transfected with ID4, lumen formation is restored, demonstrating that ID4 may substitute for CEACAM1. After showing the ID4 promoter is hypermethylated in MCF7 cells but hypomethylated in MCF/CEACAM1 cells, ID4 expression was induced in MCF7 cells by agents affecting chromatin remodeling and methylation. Mechanistically, CaMK2D was up-regulated in CEACAM1-transfected cells, effecting phosphorylation of HDAC4 and its sequestration in the cytoplasm by the adaptor protein 14-3-3. CaMK2D also phosphorylates CEACAM1 on its cytoplasmic domain and mutation of these phosphorylation sites abrogates lumen formation. Thus, CEACAM1 is able to maintain the active transcription of ID4 by an epigenetic mechanism involving HDAC4 and CaMK2D, and the same kinase enables lumen formation by CEACAM1. Because ID4 can replace CEACAM1 in parental MCF7 cells, it must act downstream from CEACAM1 by inhibiting the activity of other transcription factors that would otherwise prevent lumen formation. This overall mechanism may be operative in other cancers, such as colon and prostate, where the down-regulation of CEACAM1 is observed.
Highlights
Lumen formation, a central feature of mammary morphogenesis, is lost during mammary tumorigenesis, starting with filling in the lumen with cancer cells in ductal carcinoma in situ [1] and becoming more evident in invasive solid tumors [2]
Once we demonstrated that a key threonine residue (Thr-457) and a back-up serine (Ser-459) were phosphorylated during lumen formation, and null mutations in these two residues blocked lumen formation [7], we performed gene chip experiments to determine which genes changed between MCF7 cells transfected with wild type CEACAM1-SF (SW cells) and those transfected with the double alanine null mutants (DA cells)
We first validated its up-regulation by qPCR analysis of RNA isolated from MCF7 cells that were transfected with empty vector (V), CEACAM1-SF wild type cells (SW), and the phosphorylation site, double alanine mutant cells (DA) grown for 0, 2, 4, and 6 days in a three-dimensional culture
Summary
Lumen formation, a central feature of mammary morphogenesis, is lost during mammary tumorigenesis, starting with filling in the lumen with cancer cells in ductal carcinoma in situ [1] and becoming more evident in invasive solid tumors [2]. We demonstrated that MCF7 breast cancer cells that fail to express CEACAM1 or form a lumen in the three-dimensional culture, regain this property when transfected with CEACAM1 [6].
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