Deconstructing the Intracellular Events Leading to Enhanced Melanoma Cell Proliferation by CEACAM1 Protein

CEACAM1 protein expression in primary cutaneous melanoma lesions predicts poor survival. We have previously found that CEACAM1 facilitates cell cycle and net proliferation of melanoma cells. CEACAM1 is a transmembrane glycoprotein contains extracellular, transmembrane and intracellular domain. Previously we showed that the entire protein is crucial for proliferation. In order to deconstruct the intracellular events that lead to enhanced proliferation, we created a series of point mutations in CEACAM1. The constructs were stably transfected into CEACAM1-negative melanoma cells and tested for proliferation. Remarkably, mutation analysis identifies the ITIM residues (tyrosine 493 and 520) as well as serine508 to hold a critical role in CEACAM1-mediated proliferation. Indeed, mutation in these amino acids entirely abrogates the proliferative effect of CEACAM1 on melanoma cells both in vitro and in a xenograft animal model. Accordingly, the role of SHP phosohatases. that known to interact with CEACAM1 in lymphocytes. was studied. Co-IP assays showed more interactions of SHP-1 with CEACAM1 in melanoma cells transfected with wild type compare to mutated CEACAM1, unlike SHP-2 that showed no difference. The results of the Co-IP assays were supported by knockdown assays with siRNA that showed only the effect of SHP-1 on proliferation but not SHP-2. This implies that the effect is mediated by SHP-1.

Finally, we found that CEACAM1 affect important cell cycle inhibitors: mir-34 and p21. Cells transfected with CEACAM1 showed lower levels of p21 (RNA and protein) and mir-34 (RNA) compare to cells with mock transfection.

In conclusion, we propose a new pathway that facilitating melanoma cell proliferation, mediated by recruitment of SHP-1 to key residues in CEACAM1 protein that leads to down regulation of cell cycle inhibitors.