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. 2013 Feb 19;110(8):3029-34.
doi: 10.1073/pnas.1216141110. Epub 2013 Feb 4.

Transforming mutations of RAC guanosine triphosphatases in human cancers

Masahito Kawazu  1 , Toshihide Ueno, Kenji Kontani, Yoshitaka Ogita, Mizuo Ando, Kazutaka Fukumura, Azusa Yamato, Manabu Soda, Kengo Takeuchi, Yoshio Miki, Hiroyuki Yamaguchi, Takahiko Yasuda, Tomoki Naoe, Yoshihiro Yamashita, Toshiaki Katada, Young Lim Choi, Hiroyuki Mano
Affiliations

Transforming mutations of RAC guanosine triphosphatases in human cancers

Masahito Kawazu et al. Proc Natl Acad Sci U S A. .

Abstract

Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTP-bound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Transforming potential of RAC1 and RAC2 mutants. (A) 3T3 or MCF10A cells were infected with recombinant retroviruses encoding enhanced green fluorescent protein (EGFP) as well as wild-type or mutant forms of RAC1 or RAC2 and were then assayed for anchorage-independent growth in vitro under the presence of 10% (vol/vol) FBS. After 14 d (3T3) or 20 d (MCF10A) of culture, the cells were stained with crystal violet and examined by conventional microscopy (Left: left image of each pair), and they were monitored for EGFP expression by fluorescence microscopy (Left: right image of each pair). (Scale bars, 0.5 mm.) The numbers of cell colonies were also determined as means ± SD from three independent experiments (Right). (B) 3T3 cells expressing wild-type or mutant forms of RAC1 or RAC2 were injected s.c. into the shoulder of nude mice, and the size of the resulting tumors [(length × width)/2] was determined at the indicated times thereafter. Tumor size for 3T3 expressing NRAS(Q61K) was similarly monitored. Data are means ± SD for tumors at four injection sites. (C) HEK293T cells were transfected with expression vectors for wild-type or mutant forms of RAC1 or RAC2 together with the SRE.L reporter plasmid and pGL-TK. The activity of firefly luciferase in cell lysates was then measured and normalized by that of Renilla luciferase. Data are means ± SD from three independent experiments. (D) Lysates of 3T3 cells expressing wild-type or mutant forms of RAC1 or RAC2 were subjected to a pull-down assay with PAK1-PBD. The precipitated proteins as well as the total cell lysates were then subjected to immunoblot analysis with antibodies to RAC1 or to RAC2. The relative amounts of pulled-down RAC proteins compared with their corresponding expression levels in total cell lysate were normalized to that of wild-type RAC1 (for the RAC1 mutants) or RAC2 (for the RAC2 mutants) and are shown at the bottom.
Fig. 2.
Fig. 2.
Actin reorganization induced by the RAC1/RAC2 mutants. 3T3 cells infected with retroviruses encoding enhanced green fluorescent protein (EGFP) as well as wild-type or mutant forms of RAC1 or RAC2 were stained with Alexa Fluor 594-labeled phalloidin to visualize actin organization (Left image of each pair). The same cells were also examined for EGFP fluorescence (Right image of each pair). (Scale bars, 20 µm.)
Fig. 3.
Fig. 3.
Oncogenic RAC proteins as therapeutic targets. (A) HT1080 cells were transfected with control, RAC1, or NRAS siRNAs; lysed; and subjected to immunoblot analysis with antibodies to RAC1, NRAS, or ACTB (loading control). (B) HT1080 cells were transfected with control, RAC1, or NRAS siRNAs, as indicated, and cultured under the presence of 10% (vol/vol) FBS. Cell number was counted at the indicated times after the onset of transfection. Data are means ± SD from three independent experiments. (C) HT1080 cells were infected with a retrovirus encoding green fluorescent protein (EGFP) as well as a control or RAC1 shRNA. They were also infected with a retrovirus encoding shRNA-resistant wild-type RAC1 or RAC1(N92I), as indicated. The number of EGFP-positive cells was determined by flow cytometry after culture of the cells for the indicated times, and the size of the EGFP-positive fraction relative to that at 2 d was calculated. Data are means ± SD from three independent experiments. (D) MDA-MB-157 cells were infected with a retrovirus encoding EGFP as well as a control or RAC1 shRNA. They were also infected with a retrovirus encoding shRNA-resistant wild-type RAC1 or RAC1(P29S), as indicated. The number of EGFP-positive cells was determined by flow cytometry after culture of the cells for the indicated times, and the size of the EGFP-positive fraction relative to that at 3 d was calculated. Data are means ± SD from three independent experiments.
Fig. 4.
Fig. 4.
Biochemical properties of RAC1 mutants. (A) Bacterially expressed and purified proteins of the wild-type, P29S, N92I, or C157Y mutant of RAC1 (5 pmol each) were incubated with [35S]GTPγS in the presence of 0.8 mM Mg2+, and the amounts of [35S]GTPγS-bound proteins were determined at the indicated times. (B) [3H]GDP dissociation from [3H]GDP-bound RAC1 proteins was initiated by the addition of unlabeled GTPγS in the presence of 0.8 mM Mg2+, and the amounts of [3H]GDP-bound proteins were determined at the indicated times. (C) RAC1 proteins were preloaded with [γ32P] GTP, and then GTP hydrolysis reactions were initiated by the addition of unlabeled GTP in the presence of 0.8 mM Mg2+. Pi released from the proteins was isolated and measured at the indicated times. (D) [35S]GTPγS dissociation from [35S]GTPγS-bound RAC1 proteins was initiated by the addition of unlabeled GTPγS in the presence of 0.8 mM Mg2+, and the amounts of [35S]GTPγS-bound proteins were determined at the indicated times. (E) Schematic representation of the structure of the GTP-binding pocket of human RAC1 (ID 1mh1 in the Protein Data Bank; www.pdb.org) with α-helices and β-sheets shown in magenta and orange, respectively. The GTP analog guanosine 5′-(β,γ-imido)-triphosphate (GppNp) and Mg2+ are depicted in red and green, respectively. D11, P29, N92, and C157 amino acid residues are in orange, blue, yellow and purple, respectively. The positions of switch I and switch II regions and the P-loop are also indicated.
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