686. CAR-Expression Is Essential for Tumor Inititation in Lung Cancer Xenografts

Abstract

Top of pageAbstract Rationale: CAR, the Coxsackievirus Adenovirus Receptor, has primarily been studied in its role as the initial cell surface attachment receptor for Coxsackie and group C-Adenoviruses (Ad). Recent studies indicate that CAR serves to mediate homotypic cell-cell adhesion as a component of the tight junction and/or adherens junction. CAR's role in tumorigenesis has been considered, but no mechanistic studies probing this role have been reported. Experimental Design: Non-small cell lung cancer (NSCLC) cells with exuberant CAR expression (FACS: 99.9% positive w/ MCF 55.2) were stably transfected with control and AS-CAR vectors, and CAR(−)clones isolated (FACS: 1.9% positive w/ MCF 9.6). Cells were phenotypically characterized and tumorigenesis was compared in xenograft models. In separate experiments, CAR was functionally blocked using a monoclonal antibody and a specific ligand (Ad fiber knob), and the impact on tumor formation assessed. Results: Expectedly, CAR (−) cells were refractory to Ad-transduction and demonstrated marked reductions in CAR-mRNA by RT-PCR. Silencing CAR expression in cells that exhibited relatively “high” surface expression of this molecule profoundly inhibited their ability to develop xenografts in host mice (Day 40 tumor volume: CAR (+) cells 574 + 304 mm3, CAR (−) cells 0 mm3; n = 10 scid/scid mice, p<0.001). In survival studies, whereas 5/5 mice sufficed to tumor by day 53 post implantation by CAR (+) cells, all 5/5 mice injected with CAR (−) cells survived (p<0.001). Indeed, 4/5 mice injected with CAR(−)cells were euthanized without tumor on day 120. However, one of 5 mice injected with CAR (−) cells displayed delayed tumor growth beginning at 3 months post-injection, and when this tumor was extirpated and analyzed, CAR-expression had re-emerged. In the second series of experiments, co-injection of NSCLC-cells with high and intermediate surface expression of CAR with a blocking antibody inhibited tumorigenesis in nude mice by these cells (p<0.05). The impact of Ad-fiber knob and siRNA-inhibition of CAR-expression in situ is currently underway. Conclusion: Results to date indicate that CAR subserves a novel biological function in tumor-initiation by lung cancer xenografts. The molecular and cellular mechanisms for this function are being explored.