Abstract 888: Inactivation of the DLC1 RhoGAP tumor suppressor by point mutation occurs commonly in human cancer and can result from Rho-dependent or Rho-independent mechanisms

The Rho GTPases, which are frequently activated in advanced cancer, stimulate their downstream effectors to regulate a variety of fundamental cellular process, e.g., cell cycle, cell migration, cell-cell adhesion, and cytoskeleton organization. The down-regulation of RhoGAPs is one mechanism of increased Rho activity, and members of the tumor suppressor DLC Rho-GAP family (DLC1-3) are frequently down-regulated by gene deletion or DNA methylation in human cancers. Here we report that, in addition, point mutations in the DLC1-3 coding regions occur frequently in certain cancers, most of the mutants we have analyzed have reduced tumor suppressor activity, at least two distinct mechanisms can account for this reduction, and one mechanism appears to be Rho-independent. In the TCGA database, DLC1 was mutated in 11% of gastric cancer (GAC), 10% colorectal cancer (CRC), 7% of melanomas (MEL), and 6% of lung adenocarcinoma (LAD). Most alterations were point mutations encoding a single amino acid change. Mutation of DLC2 and DLC3 also occurred in these tumor types, but were less frequent. The mutation frequency of p190-A, which is a Rho-GAP from a different gene family, was even less frequent, ranging from 4% in LAD to zero in GAC. Taken together, mutation of at least one DLC gene or p190-A was found in about 20% of LAD, CRC, and MEL, and 15% of GAC. In LAD, the DLC1 expression levels from the patients with DLC1 mutations were lower than the patients with DLC1 wild type, suggesting selection for decreased DLC1 expression in tumors harboring mutant DLC1. We constructed expression vectors for many of the DLC1 mutants and analyzed their phenotypes in human tumor cell lines. Most were found to be loss of tumor suppressor function mutants, as they were deficient for reducing growth in soft agar, cell migration, and focal adhesion turnover. One class of deficient mutants had lesions in the RhoGAP domain, which directly inactivated its RhoGAP activity and increased the activity of Rho downstream effectors. A second class had lesions in the DLC1 START (StAR-related lipid-transfer) domain, which is known to bind caveolin. although the RhoGAP activity was normal, complex formation with caveolin was much lower than with wild type DLC1. We conclude that, in addition to the frequent down-regulation of DLC1 expression via gene deletion or DNA methylation in tumors, point mutation of its coding sequence commonly inactivates the tumor suppressor activity of DLC1 at the protein level by at least two alternate mechanisms: attenuation of it binding to caveolin or of its RhoGAP activity, which up-regulates RhoA and its downstream effectors. (# Contributed equally) Citation Format: Xiaolan Qian, Dunrui Wang, Beatriz Sanchez-Solana, Brajendra K. Tripathi, Marian E. Durkin, Alex Papageorge, Douglas R. Lowy. Inactivation of the DLC1 RhoGAP tumor suppressor by point mutation occurs commonly in human cancer and can result from Rho-dependent or Rho-independent mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 888.