Cancer-Associated Point Mutations in the DLC1 Tumor Suppressor and Other Rho-GAPs Occur Frequently and Are Associated with Decreased Function

Abstract

In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity. SIGNIFICANCE: These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.

Figure 1.. Correlations between the frequency of DLC1 mutation and total number of mutations in 12 tumor types in TCGA.

(A) Correlation for the 12 tumor types between total number of codon mutations and number of DLC1 mutations. r = 0.79. (B) Uterine cancer: Total number of mutations vs. DLC1 mutations. The total number of all codon mutations are plotted for individual patients. Left panel: the dark vertical bars represent each of the 48 patients with one or more DLC1 mutation, while the light vertical bars represent each of the 52 patients with the highest number of total codon mutations who have wild type DLC1. Right panel: The Statistical analysis used the Mann-Whitney U test, which compared the total mutation numbers of the patients with and without DLC1 mutations. The vertical axis represents the average value plus standard error. (C) Colon cancer: Total number of mutations vs. DLC1 mutations. The data are presented and analyzed similarly to B, except that the dark vertical bars represent each of the 34 patients with DLC1 mutations, while the light vertical bars represent each of the 66 patients with the highest number of total codon mutations who have wild type DLC1.

Figure 2.. Most DLC1 mutants with missense mutation in the Rho-GAP domain are less active than WT DLC1.

(A) HCT 116 cells were transiently transfected with GFP or full-length GFP-DLC1 followed by Rhotekin pull-down assay to detect their RhoA-GTP levels. The GFP expression level is shown for the transfectants. (B) H1299 cells were transiently transfected with GFP or GFP-GAP followed by Rhotekin pull down assay. The expression level of GFP-tagged proteins is shown for the transfectants. (C) The phospho-cofilin levels in SW620 cells transiently expressing GFP or GFP-GAP were analyzed by immunoblot. The expression level of GFP, total RHOA, and Cofilin are also shown in each panel.

Figure 3.. Compared with WT DLC1, biological activity is reduced for most DLC1 mutants with mutations in the Rho-GAP domain.

(A) H1299 cells transiently expressing GFP or GFP-DLC1 (top) were analyzed for cell migration in transwell dishes. Representative micrographs of migrated cells are shown (bottom). The total number of migrated cells, in triplicate, for each mutant is shown as mean ± SD (middle). (B) SW620 stable clones expressing GFP or GFP-DLC1 mutants were analyzed by Rhotekin PD assay (top). Cells were seeded in soft agar for 3 weeks. Representative views of colonies under microscope and the stained colonies in the dish are shown (bottom). Number of colonies (≥ 0.4 mm) in triplicate dishes have been plotted as mean ± SD (middle).

Figure 4.. Interaction between DLC1 mutants and GST 14-3-3 proteins or GST Caveolin-1.

(A, B) DLC1 S327R mutant and double mutant S327R/S431A are deficient for binding to GST 14-3-3 proteins. As indicated in the panels, HEK 293T cells were cotransfected with GST or GST 14-3-3 proteins (theta, beta, or eta) and GFP, GFP-DLC1-WT, or GFP-DLC1 mutants. After 48 hours, cells were lysed and pulled-down by Glutathione sepharose-4B (Gluta) and immunoblotted (IB) using GFP or GST antibodies. (C) DLC1 E966K mutant binds poorly to GST Caveolin-1 (GST CAV1). HEK 293T cells were cotransfected with GST or GST Caveolin-1 and GFP, GFP-DLC1-WT or GFP-DLC1-E966K mutant and subjected to Glutathione pull-down as described for (A, B). A deletion mutant of DLC1 lacking amino acids 929–957 was used in the pull-down as a negative control for Caveolin-1 binding (see text). WCE: whole cell extracts.

Figure 5.. DLC1 S327R and E966K mutants have WT Rho-GAP activity, but inhibit cell migration and colony formation in soft agar less efficiently than DLC1-WT.

(A, B, C) Rho-GAP activity. Active Rho (GTP) in HCT 116 (A, B) or H1299 (C) cells expressing DLC1-WT or the indicated mutants were analyzed by Rhotekin pull-down (PD) assay followed by anti-RHOA immunoblotting (IB). GFP-DLC1 expression and total RHOA expression were also confirmed by immunoblotting. (D) Migration in transwell. H1299 cells were transfected with the indicated constructs. 24 hours after transfection, 1 × 10⁵ cells were seeded in a transwell dish and allow to migrate for 18 hours. The migrated cells were stained with crystal violet, photographed (upper panel), and quantified at OD590nm (lower panel). The total number of migrated cells, in triplicate, for each mutant is shown as mean ± SD. (E) Soft agar growth. Stable clones for control GFP, GFP-DLC1-WT or the indicated mutants were generated in SW620 cells. 1 × 10⁵ cells were seeded in soft agar and allow to form colonies for approximately 28 days, and those colonies >0.2mm were photographed (middle panel) and counted (lower panel). Number of colonies (≥ 0.2mm) in triplicate dishes have been plotted as mean ± SD. DLC1 expression was confirmed by immunoblotting (upper panel).