Intratumoral Societies - Subpopulations of Tumor Cells Expressing Different Levels of ADAM23 Seems Responsible for Different Aspects of Malignancy

Abstract

Background: Cancer evolve through the accumulation of genomic and epigenomic alterations that enable tumor progression towards malignancy. Epigenetic silencing of ADAM23 (A Desintegrin A nd Metalloprotease domain 23) gene is a recurrent event in a wide range of tumors, including pancreatic, breast, gastric and colorectal tumors. Hypermethylation of ADAM23 gene promoter has been associated with metastatic disease and poor overall survival in breast cancer patients (Verbisck et al. 2009). However the precise biological effects of ADAM23 down regulation during tumor progression is unknown. Here we sought to further clarify the role of ADAM23 in tumor progression through functional characterization of ADAM23 in vivo and in vitro. Material and Methods: We stably knocked down ADAM23 expression in MDA-MB-435 tumor cells using shRNA and ectopically expressed human ADAM23 in CMS5a, a mouse fibrosarcoma cell line. Cells transfected with empty vectors were used as controls in all comparisons. Invasion assays were performed embedding cell spheroids in a tri-dimensional collagen-type I matrix. Soft-agar and tumorigenesis assays were performed according to standardprotocols. ADAM23 expression in primary tumors displaying ADAM23 promoter hypermethylation was analyzed by in situ hybridization (approved ethics committee). Results: We demonstrated that, in both the human and murine models, reduced levels of ADAM23 correlate with a 3-fold increase in the average speed of cellular invasion through collagen matrix. By contrast, in soft-agar assays these fast invasive cells displayed a decrease of 50−70% in the colony sizes, with no changes in colony numbers. In vivo, ADAM23-positive human cells efficiently formed tumors (93%), while the tumorigenesis rate of ADAM23-negative cells was only 33%. In situ hybridization of ADAM23 hypermethylated tumors revealed the co-existence of ADAM23 negative cell clusters and clusters expressing high levels of ADAM23. Conclusions: Our results demonstrate that ADAM23 can act as a pleiotropic molecule by stimulating growth and tumorigenesis, but inhibiting invasion and suggest that the invasive phenotype is more likely to evolve after ADAM23 silencing in primary tumors. Finally, we hypothesize that the co-existence of tumor cell subpopulations expressing different level sof ADAM23 with in primary tumors may contribute to malignancy given that association of both proliferative and invasive phenotypes facilitate accomplishment of metastatic cascade.