Abstract A25: Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F

Abstract

Abstract Rationale: Cancers of the lung and bronchus are responsible for nearly 30% of all cancer-related deaths in the US, and mutations in the tumor-suppressor gene TP53 occur in 54% of lung adenocarcinomas and 86% of squamous cell carcinomas. Compared with other cancers, TP53 mutational hotspots in lung cancer more often include missense mutations at codons V157, R158, and A159, with frequencies approaching that of the canonical mutational hotspot R273. The resulting mutant p53 (mutp53) protein often exhibits not only loss of tumor suppressor capability but also gain of oncogenic function (GOF). Our previous work identified a novel, p53-dependent transcriptome including alterations in expression of urokinase plasminogen activator (uPA), a key regulator of the plasminogen activation system. When activated, uPA converts plasminogen to plasmin, which cleaves extracellular matrix components and promotes cell migration. We evaluated uPA expression and cell migration in human lung cancer cell lines expressing wild-type or mutant p53. Methods: Human lung cancer cell lines with wild-type p53 (A549) or with somatic mutations at the endogenous p53 locus (NCI-H1781 and NCI-H2087, V157F; NCI-H441, R158L; NCI-H2110, R158P; NCI-H2009, R273L) were obtained from ATCC. Using RNAi (shRNA and siRNA) cells were depleted of p53 as confirmed by puromycin selection of cells infected with lentivirus, in the case of shRNA. Whole-cell lysates were harvested for protein and RNA purification, and conditioned medium was harvested for measurement of secreted proteins. Immunoblotting and qRT-PCR were used to assess expression of protein and mRNA, respectively. A wound healing assay was performed following siRNA transfection to determine cell migration in cells expressing vs. depleted of wild-type or mutant p53. Results: Silencing of p53 by shRNA and siRNA targeted to TP53 was confirmed by Western blot and qRT-PCR. With p53 depletion, protein and mRNA expression of the uPA pro-enzyme pro-uPA and its activated B- and A-chains increased in the H2087 (V157F mutp53) cell line. No p53-dependent change was seen in uPA expression in H2009 cells (R273L mutp53) despite a similar relative quantity of uPA protein. Secreted uPA also increased as measured in conditioned media from H2087 cells (V157F mutp53). A wound healing assay showed increased migration by mutp53-depleted H2087 cells compared with mutp53-expressing H2087 cells, while A549 cells (wt p53) showed no p53-dependent difference in migration rate. Conclusions: To our knowledge, GOF phenotypes in lung cancer cells carrying V157F mutp53 have not been characterized. We have shown here that mutp53 represses uPA expression in human lung cancer cells with endogenous V157F mutp53, and depletion of p53 in these cells leads to an increase in migration. These findings prompt a reexamination of the signaling pathways associated with uPA. Further study is needed to elucidate the mechanism of the p53-dependent alterations in uPA expression and to establish additional biologic effects of V157F mutp53. Citation Format: Julie A. Barta, Steven B. McMahon. Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A25.