DLC1 is the principal biologically-relevant down-regulated DLC family member in several cancers

Abstract

The RHO family of RAS-related GTPases in tumors may be activated by reduced levels of RHO GTPase accelerating proteins (GAPs). One common mechanism is decreased expression of one or more members of the Deleted in Liver Cancer (DLC) family of Rho-GAPs, which comprises three closely related genes (DLC1, DLC2, and DLC3) that are down-regulated in a wide range of malignancies. Here we have studied their comparative biological activity in cultured cells and used publicly available datasets to examine their mRNA expression patterns in normal and cancer tissues, and to explore their relationship to cancer phenotypes and survival outcomes. In The Cancer Genome Atlas (TCGA) database, DLC1 expression predominated in normal lung, breast, and liver, but not in colorectum. Conversely, reduced DLC1 expression predominated in lung squamous cell carcinoma (LSC), lung adenocarcinoma (LAD), breast cancer, and hepatocellular carcinoma (HCC), but not in colorectal cancer. Reduced DLC1 expression was frequently associated with promoter methylation in LSC and LAD, while DLC1 copy number loss was frequent in HCC. DLC1 expression was higher in TCGA LAD patients who remained cancer-free, while low DLC1 had a poorer prognosis than low DLC2 or low DLC3 in a more completely annotated database. The poorest prognosis was associated with low expression of both DLC1 and DLC2 (P < 0.0001). In cultured cells, the three genes induced a similar reduction of Rho-GTP and cell migration. We conclude that DLC1 is the predominant family member expressed in several normal tissues, and its expression is preferentially reduced in common cancers at these sites.

Keywords: DLC genes; RhoGAP; TCGA; bioinformatics; tumor suppressor.

Figure 1. DLC1, DLC2 and DLC3 gene expression in control tissue adjacent to the tumors

Basal RNA expression levels of DLC1, DLC2 and DLC3 from normal tissue in lung A. and B., liver C., breast D. and colorectum E. are derived from the TCGA dataset (RNA-Seq Version 2, Level 3). The vertical axis differs for some panels. The mean and standard errors of adjacent controls from correspondent cancer have been plotted. F. DLC1 gene expression in different tissues. LSC = lung squamous cell carcinoma; LAD = lung adenocarcinoma.

Figure 2. Fold change in DLC1, DLC2, and DLC3 expression between tumor and adjacent control tissue

The fold change of DLC1, DLC2 and DLC3 RNA-Seq Version 2 values from individual paired control to tumor of the TCGA dataset are plotted for lung squamous cell carcinoma A., lung adenocarcinoma B., hepatocellular carcinoma C., breast cancer D. and colorectal adenocarcinoma E.

Figure 3. Down-regulation of DLCs is associated with poor prognosis

A-C. Comparison of DLC gene expression of patients with follow up status based on “new tumor event dx indicator” of TCGA lung adenocarcinoma clinical data as of October 2015. The most recent clinical patient status has been selected, and DLC gene expression (RNA-Seq Version 2) mean and standard errors of the mean are plotted against “new tumor” status. D-H. Kaplan-Meier survival analysis: Down-regulation of DLC1 and DLC2 is associated with poor prognosis. From the Director's Challenge Lung Study cohort of 442 lung adenocarcinomas. High and Low in the Figure legend represent the status of the mRNA expression level compared to the median of the expression for corresponding gene. D-F. Survival comparison between patients with low vs. high expression of the designated DLC gene. G. Survival comparison between patients whose DLC1 and DLC2 expressions are low vs. all others. H. Survival comparison between patients whose DLC1 and DLC3 expressions are low vs. all others.

Figure 4. DLC copy number variation and gene expression in tumors

Comparison of DLC1 and DLC2 RNA-Seq values and copy number (CN) variation in TCGA HCC A., LAD B., and LSC C. The patients are grouped based on copy number loss variation (value log2 <-0.5 and log2 > = −0.5). D. DLC1 expression in adjacent control lung tissue grouped according to the copy number status of the respective LAD and LSC tumors.

Figure 5. DLC promoter methylation and gene expression in tumors

TCGA level 3 data from JHU_USC__ HumanMethylation450 directory of each selected cancer were used for analysis. DLC means and standard errors of cancer and controls in LSC A-C., LAD D-F. and HCC G-I. were calculated using beta values from all available probes in the DLC1 variant 2 A., D., G. DLC2 alpha, variant 1 B., E., H. and DLC3 beta variant 3 C., F., I. (sequence details in Figure S2).

Figure 6. TP53 mutation and DLC expression in lung adenocarcinoma and lung squamous cell carcinoma

TCGA LAD A. and LSC B. datasets. DLC expression levels (mean + standard error) are plotted against groups of patients with or without TP53 mutations. MU = TP53 mutation. WT = TP53 wild type.

Figure 7. DLC RhoGAP and bioactivity in transfected human lung cancer cell lines

GFP-tagged DLC1, DLC2 and DLC3α constructs have been stably transfected into H1299 A. and B. and H358 C., D. and E. cells. The expression of GFP or GFP-DLCs in the established stable clones has been analyzed by IP followed by IB with anti-GFP antibody. RhoGAP activity was measured by Rhotekin pull-down assay (A and C). In the cell migration assay, the migrated H1299 and H358 transfectants in the botom chamber of 24 well inserts were stained and photographed using a light microscope. The quantitation was performed colorometrically as described in materials and methods (B and D). Equal numbers of H358 stably transfected cells were seeded in soft agar for growth and quantitation as shown to compare the effect of anchorage-independent cell growth (E).