Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May;47(5):326-37.
doi: 10.1002/mc.20389.

DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms

Kevin D Healy  1 , Louis Hodgson, Tai-Young Kim, Adam Shutes, Savitri Maddileti, Rudolph L Juliano, Klaus M Hahn, T Kendall Harden, Yung-Jue Bang, Channing J Der
Affiliations

DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms

Kevin D Healy et al. Mol Carcinog. 2008 May.

Abstract

Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multi-domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full-length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-delta1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.

PubMed Disclaimer

Figures

Figure 1
Figure 1
DLC-1 is a RhoGAP. (A) Schematic of the structure of wild type and mutant human DLC-1 proteins used in this study. (B) DLC-1 is a RhoGAP for RhoA, (C) RhoB, and (D) RhoC. The GTP hydrolysis rate of recombinant GST fusion proteins of RhoA, RhoB, and RhoC was measured in the presence and absence of 0.30 μM DLC-1. GAP activity of a missense mutant of DLC-1, with substitution of a conserved arginine residue in the RhoGAP catalytic domain (DLC-1(R718E)), was also determined. The small GTPases were preloaded with GTP, and incubated with a fluorescently-labeled phosphate binding protein (PBP) which undergoes a dramatic increase in fluorescence upon binding inorganic phosphate released from GTP hydrolysis. Real time GTP hydrolysis was monitored by measuring the increases in fluorescence, which directly correlated with GTP hydrolysis. Data shown are representative of two independent experiments.
Figure 1
Figure 1
DLC-1 is a RhoGAP. (A) Schematic of the structure of wild type and mutant human DLC-1 proteins used in this study. (B) DLC-1 is a RhoGAP for RhoA, (C) RhoB, and (D) RhoC. The GTP hydrolysis rate of recombinant GST fusion proteins of RhoA, RhoB, and RhoC was measured in the presence and absence of 0.30 μM DLC-1. GAP activity of a missense mutant of DLC-1, with substitution of a conserved arginine residue in the RhoGAP catalytic domain (DLC-1(R718E)), was also determined. The small GTPases were preloaded with GTP, and incubated with a fluorescently-labeled phosphate binding protein (PBP) which undergoes a dramatic increase in fluorescence upon binding inorganic phosphate released from GTP hydrolysis. Real time GTP hydrolysis was monitored by measuring the increases in fluorescence, which directly correlated with GTP hydrolysis. Data shown are representative of two independent experiments.
Figure 2
Figure 2
The isolated RhoGAP domain shows greatly enhanced catalytic activity for RhoA, RhoB, RhoC and Cdc42. Recombinant full length and an isolated RhoGAP catalytic domain fragment (amino acids 609-878) of DLC-1 were expressed and purified for analysis of in vitro GAP activity. Real time GTP hydrolysis was measured with a fluorescently labeled PBP, as described in Fig. 1, for GST fusion proteins of wild type (A, B) RhoA, (C, D) RhoB, (E, F) RhoC, (G, H) Cdc42, and (I, J) Rac1. Data shown are representative of three independent experiments.
Figure 2
Figure 2
The isolated RhoGAP domain shows greatly enhanced catalytic activity for RhoA, RhoB, RhoC and Cdc42. Recombinant full length and an isolated RhoGAP catalytic domain fragment (amino acids 609-878) of DLC-1 were expressed and purified for analysis of in vitro GAP activity. Real time GTP hydrolysis was measured with a fluorescently labeled PBP, as described in Fig. 1, for GST fusion proteins of wild type (A, B) RhoA, (C, D) RhoB, (E, F) RhoC, (G, H) Cdc42, and (I, J) Rac1. Data shown are representative of three independent experiments.
Figure 2
Figure 2
The isolated RhoGAP domain shows greatly enhanced catalytic activity for RhoA, RhoB, RhoC and Cdc42. Recombinant full length and an isolated RhoGAP catalytic domain fragment (amino acids 609-878) of DLC-1 were expressed and purified for analysis of in vitro GAP activity. Real time GTP hydrolysis was measured with a fluorescently labeled PBP, as described in Fig. 1, for GST fusion proteins of wild type (A, B) RhoA, (C, D) RhoB, (E, F) RhoC, (G, H) Cdc42, and (I, J) Rac1. Data shown are representative of three independent experiments.
Figure 3
Figure 3
DLC-1 protein expression is lost in NSCLC cell lines and associated with increased RhoA-GTP formation. (A) DLC-1 protein expression is lost in some NSCLCs. Lysates from nine NSCLC cell lines were resolved by SDS-PAGE and transferred to a PVDF membrane. A monoclonal antibody was used for western blot analysis of DLC-1 expression (612020; BD Biosciences). A parallel blot for β-actin was done to ensure equivalent loading of total cell lysate protein. (B) Ectopic expression of wild type but not mutant DLC-1 is associated with a reduction of RhoA-GTP activity in NSCLCs. Mass populations of two NSCLC cell lines, deficient in endogenous DLC-1 protein expression, were established via infection with the pBabe-puro retrovirus vector encoding wild type full length DLC-1 or the GAP-dead mutant, DLC-1(R718E). RhoA-GTP levels were assessed by incubating lysates with GST-Rhotekin-RBD that was precoupled to glutathione-sepharose beads. Precipitated and total lysates (4% loading control) were analyzed by western blotting with anti-RhoA and -DLC-1 antibodies. Data shown are representative of two independent experiments.
Figure 4
Figure 4
DLC-1 expression reduces RhoA activity at the leading edge of cellular protrusions. MEFs stably expressing a RhoA biosensor were transiently-transfected with mCherry-tagged DLC-1 and plated on fibronectin. (A) Time-lapse images of RhoA activity during cellular protrusion. Regions of intense RhoA activity are shown in red. Scale bar, 10 μm. (B) Quantification of RhoA activity at varying distances from the edge of the cell. RhoA activity was quantified along 120 line-scans drawn perpendicular to the edge of 8 mCherry control cells and 103 line-scans perpendicular to the edge of 8 mCherry DLC-1 cells. Data points represent the average ± SD (C) mCherry DLC-1 is expressed at focal adhesions in the MEFs. Expression of the mCherry fluorescence is shown for representative control and DLC-1 cells.
Figure 5
Figure 5
Ectopic re-expression of DLC-1 impairs NSCLC anchorage-independent growth. NCI-H23 and A549 NSCLC cells stably-infected with the pBabepuro vector encoding DLC-1 or DLC-1(R718E) were evaluated for colony formation in soft agar and the number of proliferating viable colonies was quantitated after 21 days. (A) Viable colonies were stained with MTT and photographed. (B) The total number of colonies (> 10 cells) within five representative fields of view was quantified. Data shown are the average ± SD of triplicate wells and are representative of three independent experiments. *, significant at P ≤ 0.005 vs. vector; †, significant at P < 0.01 vs. vector and vs. wild type DLC-1.
Figure 6
Figure 6
DLC-1 reduces NSCLC invasion in vitro. NCI-H23 cells stably-infected with the pBabe-puro vector encoding DLC-1 or DLC-1(R718E) were assessed for invasion through Matrigel. Cells were dissociated, resuspended in serum-free growth media containing 1% BSA, and incubated for 22 h at 37°C in a Matrigel invasion chamber; the lower well contained growth medium supplemented with 3% FCS. Non-invaders were removed, and the chambers were fixed and stained. (A) Invading cells were photographed under 10X magnification. (B) Quantification of invading cells. Data shown are the mean ± SD of triplicate wells and are representative of two independent experiments. *, significant at P ≤; 0.01 vs. vector.
Figure 7
Figure 7
DLC-1 does not stimulate phosphoinositide hydrolysis activity of PLC-δ1. Cos-7 cells were transiently transfected with vectors encoding various PLC isoforms and known or putative activators. Intracellular inositol phosphate accumulation was quantified as described in Methods. (A) PLC-β2 phospholipid hydrolysis activity is activated by constitutively activated Rac3. To verify the assay conditions, we coexpressed PLC-β2 with Rac3 G12V, an established activator of PLC-β2, and quantified [3H]inositol phosphate accumulation. (B) PLC-δ1 activity is not stimulated by DLC-1. Zero toδ100 ng of expression vector encoding PLC-δ1 was co-transfected with 0, 25, 50, 100, or 200 ng of DNA encoding human DLC-1, and the accumulation of [3H]inositol phosphates was quantified. Data shown are the mean ± SD of triplicate determinations and are representative of results obtained in two independent experiments.
See this image and copyright information in PMC

References

    1. Kim TY, Jong HS, Song SH, et al. Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanism in gastric cancer cells. Oncogene. 2003;22:3943–3951. - PubMed
    1. Seng TJ, Low JS, Li H, et al. The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation. Oncogene. 2007;26:934–944. - PubMed
    1. Ullmannova V, Popescu NC. Expression profile of the tumor suppressor genes DLC-1 and DLC-2 in solid tumors. Int J Oncol. 2006;29:1127–1132. - PubMed
    1. Yuan BZ, Durkin ME, Popescu NC. Promoter hypermethylation of DLC-1, a candidate tumor suppressor gene, in several common human cancers. Cancer Genet Cytogenet. 2003;140:113–117. - PubMed
    1. Yuan BZ, Miller MJ, Keck CL, Zimonjic DB, Thorgeirsson SS, Popescu NC. Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP. Cancer Res. 1998;58:2196–2199. - PubMed

Publication types

MeSH terms