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. 2008 Jun 1;22(11):1439-44.
doi: 10.1101/gad.1672608.

DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma

Wen Xue  1 , Alexander Krasnitz, Robert Lucito, Raffaella Sordella, Linda Vanaelst, Carlos Cordon-Cardo, Stephan Singer, Florian Kuehnel, Michael Wigler, Scott Powers, Lars Zender, Scott W Lowe
Affiliations

DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma

Wen Xue et al. Genes Dev. .

Abstract

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

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Figures

Figure 1.
Figure 1.
DLC1 is a candidate tumor suppressor on chromosome 8p22. (A,B) DNA copy number profiles for chromosome 8 of two representative human HCC samples reveals chromosome 8p22 deletions containing DLC1. The blue data points represent the averaged fluorescent ratio (tumor vs. normal) and the orange lines correspond to the value determined by copy number segmentation. Arrows denote the DLC1 locus. (C) Deletion counts per case for DLC1, p16INK4a, PTEN, TP53, and β-Tubulin (TUBB) in five types of human carcinomas. The counts were obtained from ROMA profiles of 257 breast, 137 colon, 86 liver, 213 lung, and 46 pancreas cancers, respectively. β-Tubulin serves as a negative control. The error bars indicate the 90% confidence intervals for each quantity. (D) Deletion counts in ROMA profiles of 257 human breast cancers. A deletion count in each profile set was obtained by finding the maximal tier number for 24,719 genes across the genome in each profile in the set and summing the result over the set. The counts are plotted against the gene ordinal number, with the genes sorted by their genomic transcription start position. Shown is chromosome 8 and 9. The points in the vicinity of DLC1 and p16INK4A are highlighted by red circles. (E) High-magnification view of 8p22 region in D. DLC1 and FLJ25402 reside at a local deletion epicenter. FLJ25402 is an uncharacterized gene. (F) qPCR analysis of DLC1 expression in mouse embryonic liver progenitor cells (LPC), immortalized liver progenitor cells (MIL53 and PHM1), mouse HCC cells, and primary mouse HCC tumors. Numbers indicate the average value of each group.
Figure 2.
Figure 2.
DLC1 loss triggered by in vivo RNAi promotes tumorigenesis in a mouse liver cancer model. (A) Immunoblots of 293T cells cotransfected with a 6xMyc-tagged murine dlc1 cDNA (lanes 2–4) and a control shRNA (lane 2) or DLC1 shRNAs (lanes 3,4). Tubulin serves as a loading control. (B) DLC1 qRT–PCR of p53-null liver progenitor cells coinfected with Myc and a control shRNA or two DLC1 shRNAs. (C) Cells as in B were plated at equal number and stained with crystal violet after 8 d. Shown are representative results from three experiments. (D) DLC1 loss cooperates with Myc and p53 loss to accelerate liver tumor formation. Kaplan-Meier survival curve of mice transplanted with p53-null liver progenitor cells coinfected with Myc and DLC1 shRNAs (n = 6 for each group). (E) Representative images of explanted livers. GFP imaging identifies shRNA transduced cells. (F) Histopathology of representative tumors. Proliferating cells were labeled by Ki67 staining.
Figure 3.
Figure 3.
Reintroduction of DLC1 reverts tumorigenesis. (A) Immunoblots of Ras-driven hepatoma cells infected with control retrovirus (v) or retrovirus expressing DLC1 cDNA. (B) Bioluminescence imaging of in situ liver tumor. Hepatoma cells as in A are transplanted into livers of NCR nu/nu mice and imaged at day 14. (C) Quantification of luciferase signal as in B. Error bars denote SD (n = 3). (D) Bioluminescence and GFP imaging of explanted livers at day 14.
Figure 4.
Figure 4.
DLC1 knockdown deregulates RhoA activity, which is sufficient to accelerate tumorigenesis. (A) RhoA-GTP pull-down assay of p53-null liver progenitor cells coinfected with Myc and a control shRNA or DLC1 shRNAs. (B) Quantification of A. Error bars denote SD (n = 3). (C) DLC1 knockdown increases actin stress fiber formation. p53−/−;Myc Liver progenitor cells infected with shDLC1 or RhoAV14 were serum starved and stained with fluorescent phalloidin. (D) Immunoblots of p53−/−-null hepatoblasts infected with Myc and a constitutively active RhoAV14 allele with 6xMyc tag at the N terminus. (E) Activated RhoA cooperates with Myc and loss of p53 to accelerate liver tumor formation. Kaplan-Meier survival curve of syngeneic mice transplanted with p53-null liver progenitor cells coinfected with Myc and RhoAV14 (n = 6 for each group). (F) Representative images of explanted livers at day 40 following cell transplantation. GFP imaging identifies retrovirally transduced cells.
Figure 5.
Figure 5.
HCC mediated by DLC1 loss requires RhoA and is sensitized to Rho inhibitors. (A) Hepatoma cell line BNL is infected by retroviruses expressing control shRNA (control) or three RhoA shRNAs and immunoblotted for RhoA protein. Tubulin serves as a loading control. Numbers denote relative protein abundance. (B) RhoA shRNAs selectively suppress tumors harboring low levels of DLC1. Relative tumor volume is the last measurement in Figure S6A-D. (*) P < 0.05; (**) P < 0.0005; (#) P > 0.05. DLC1 mRNA level is measured by qRT–PCR and normalized to control liver progenitor cells. (C,D) Loss of DLC1 sensitizes liver cells to ROCK kinase inhibitors Y27632 and Fasudil. Error bars denote SD (n = 3).
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