Glutaminase 2 Is a Novel Negative Regulator of Small Gtpase Rac1 and Mediates

1 Glutaminase 2 is a novel negative regulator of small GTPase Rac1 and mediates

2 p53 function in suppressing metastasis

4 Cen Zhang,1,4 Juan Liu,1,4 Yuhan Zhao,1 Xuetian Yue,1 Yu Zhu,1 Xiaolong Wang,1 Hao Wu1,

5 Felix Blanco,1 Shaohua Li, 2 Gyan Bhanot,3 Bruce G Haffty, 1 Wenwei Hu,1,5 Zhaohui Feng,1,5

7 1 Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers, The State

8 University of New Jersey, New Brunswick, NJ 08903, USA.

9 2 Department of Surgery, Robert Wood Johnson Medical School, Rutgers, The State University of

10 New Jersey, New Brunswick, NJ 08903, USA.

11 3 Department of Molecular Biology, Biochemistry & Physics, Rutgers, The State University of New

12 Jersey, Piscataway, NJ 08854, USA.

13 4 Co-first authors

14 5 Correspondence: W.H. (email: [email protected]) or Z.F. (email: [email protected])

15 Running Title: GLS2 inhibits Rac1 to suppress metastasis

17 Abstract

18 Glutaminase (GLS) isoenzymes GLS1 and GLS2 are key enzymes for glutamine metabolism.

19 Interestingly, GLS1 and GLS2 display contrasting functions in tumorigenesis with elusive

20 mechanism; GLS1 promotes tumorigenesis, whereas GLS2 exhibits a tumor suppressive function.

21 In this study, we found that GLS2 but not GLS1 binds to small GTPase Rac1 and inhibits its

22 interaction with Rac1 activators guanine-nucleotide exchange factors (GEFs), which in turn inhibits

23 Rac1 to suppress cancer metastasis. This function of GLS2 is independent of GLS2 glutaminase

24 activity. Furthermore, decreased GLS2 expression is associated with enhanced metastasis in human

25 cancer. As a p53 target, GLS2 mediates p53’s function in metastasis suppression through inhibiting

26 Rac1. In summary, our results reveal that GLS2 is a novel negative regulator of Rac1, and

27 uncover a novel function and mechanism whereby GLS2 suppresses metastasis. Our results also

28 elucidate a novel mechanism that contributes to the contrasting functions of GLS1 and GLS2 in

29 tumorigenesis.

31 Introduction

32 Metabolic changes are a hallmark of cancer cells (Berkers et al., 2013; Cairns et al., 2011; Ward and

33 Thompson, 2012). Increased glutamine metabolism (glutaminolysis) has been recognized as a key

34 metabolic change in cancer cells, along with increased aerobic glycolysis (the Warburg effect)

35 (Berkers et al., 2013; Cairns et al., 2011; DeBerardinis et al., 2007; Hensley et al., 2013; Ward and

36 Thompson, 2012). Glutamine is the most abundant amino acid in human plasma (Hensley et al.,

37 2013). Glutamine catabolism starts with the conversion of glutamine to glutamate, which is

38 converted to α-ketoglutarate for further metabolism in the tricarboxylic acid (TCA) cycle. Recent

39 studies have shown that increased glutamine metabolism plays a critical role in supporting the high

40 proliferation and survival of cancer cells by providing pools of the TCA cycle intermediates, as well

41 as the biosynthesis of proteins, lipids, and nucleotides (Berkers et al., 2013; Cairns et al., 2011;

42 DeBerardinis et al., 2007; Hensley et al., 2013; Ward and Thompson, 2012).

43 Glutaminase (GLS) is the initial enzyme in glutamine metabolism, which catalyzes the

44 hydrolysis of glutamine to glutamate in cells. Two genes encode glutaminases in human cells: GLS1

45 (also known as kidney-type glutaminase), and GLS2 (also known as liver-type glutaminase). GLS1

46 and GLS2 proteins exhibit a high degree of amino acid sequence similarity, particularly in their

47 glutaminase core domains. While GLS1 and GLS2 both function as glutaminase enzymes in

48 glutamine metabolism, recent studies show that they have very different functions in tumorigenesis.

49 GLS1 is ubiquitously expressed in various tissues, and its expression can be induced by the oncogene

50 MYC (Gao et al., 2009). GLS1 is frequently activated and/or overexpressed in various types of

51 cancer, including hepatocellular carcinoma (HCC) (Gao et al., 2009; Thangavelu et al., 2012; Wang

52 et al., 2010; Xiang et al., 2015). GLS1 has been reported to promote tumorigenesis in different types

53 of cancer, including HCC, which is mainly attributable to its glutaminase activity and its role in

54 promoting glutamine metabolism (Gao et al., 2009; Thangavelu et al., 2012; Wang et al., 2010;

55 Xiang et al., 2015). In contrast, GLS2 is specifically expressed in only a few tissues, including the

56 liver tissue. Recent studies including ours have shown that GLS2 is a novel target gene of the tumor

57 suppressor p53. GLS2 is transcriptionally up-regulated by p53 and mediates p53’s regulation of

58 mitochondrial function and anti-oxidant defense in cells (Hu et al., 2010; Suzuki et al.,

59 2010). Considering the critical role of p53 and its pathway in tumor suppression, the identification

60 of GLS2 as a p53 target gene strongly suggests a potential important role of GLS2 in tumor

61 suppression. Recent studies have shown that, in contrast to the tumorigenic effect of GLS1, GLS2

62 displays a tumor suppressive function (Hu et al., 2010; Liu et al., 2014a; Suzuki et al., 2010). GLS2

63 expression is frequently reduced in HCC (Hu et al., 2010; Liu et al., 2014a; Suzuki et al., 2010; Xiang

64 et al., 2015). Ectopic expression of GLS2 greatly inhibited the growth and colony formation of HCC

65 cells in vitro and the growth of HCC xenograft tumors in vivo (Hu et al., 2010; Liu et al., 2014a;

66 Suzuki et al., 2010). Given that GLS1 and GLS2 both function as glutaminase enzymes, the

67 mechanisms underlying their contrasting roles in tumorigenesis remain unclear.

68 In this study, immunoprecipitation (IP) followed by liquid chromatography-tandem mass

69 spectrometry (LC/MC-MS) analysis was employed to screen for potential proteins interacting with

70 GLS2. The small GTPase Rac1 was identified as a novel binding protein for GLS2. Rac1 cycles

71 between inactive GDP-bound and active GTP-bound forms in cells, and regulates a diverse array of

72 cellular events, including actin dynamics. The Rac1 signaling is frequently activated in various

73 types of cancer, which plays a critical role in promoting migration, invasion and metastasis of

74 cancer cells (Bid et al., 2013; Heasman and Ridley, 2008). We found that GLS2 binds to Rac1, and

75 inhibits the interaction of Rac1 with its guanine-nucleotide exchange factors (GEFs) such as Tiam1

76 and VAV1, which would normally activate Rac1. Thus, GLS2 inhibits Rac1 activity, which in turn

77 inhibits migration, invasion and metastasis of cancer cells. This function of GLS2 requires the

78 C-terminus of GLS2 and is independent of its glutaminase activity. In contrast, GLS1 does not

79 interact with Rac1 to inhibit Rac1 activity, and consequently, cannot inhibit cancer metastasis via

80 this pathway. p53 plays a pivotal role in suppressing cancer metastasis, but its underlying

81 mechanism is not fully understood (Muller et al., 2011; Vousden and Prives, 2009). Our results

82 further show that as a direct downstream target of p53, GLS2 mediates p53’s function in metastasis

83 suppression through inhibiting the Rac1 signaling. Taken together, our results demonstrated that

84 GLS2 is a novel negative regulator of Rac1, and plays a critical role in suppression of metastasis

85 through its negative regulation of Rac1 activity. Our results also revealed that GLS2 plays an

86 important role in mediating the function of p53 in suppression of cancer metastasis.

87 Results

88 Rac1 is a novel GLS2 interacting protein

89 GLS2 was reported to interact with several proteins although the biological functions of these

90 interactions remain unclear (Boisguerin et al., 2004; Olalla et al., 2001). These findings raised the

91 possibility that GLS2 may exert its function in tumor suppression through its interactions with other

92 proteins. Herein, we screened for potential GLS2-interacting proteins in human HCC Huh-1 cells

93 stably transduced with pLPCX-GLS2-Flag retroviral vectors to express GLS2-Flag and control

94 cells transduced with control vectors. Co-IP assays using an anti-Flag antibody followed by

95 LC-MS/MS assays were employed. These assays identified the small GTPase Rac1 as a potential

96 GLS2 interacting protein (Figure 1A). Rac1 is frequently activated or overexpressed in various

97 types of cancer, including HCC, and has been reported to play a critical role in promoting cancer

98 cell migration, invasion and metastasis mainly through its regulation of actin dynamics (Bid et al.,

99 2013; Heasman and Ridley, 2008).

100 The interaction between GLS2 and Rac1 was confirmed by co-IP followed by western-blot

101 assays in Huh-1 cells co-transduced with the vectors expressing GLS2-Flag and Myc-Rac1,

102 respectively. GLS2-Flag was co-precipitated by the anit-Myc antibody, and Myc-Rac1 was

103 co-precipitated by the anti-Flag antibody, indicating that GLS2-Flag interacted with Myc-Rac1 in

104 cells (Figure 1B). In contrast, no interaction was observed between GLS1-Flag and Myc-Rac1 in

105 Huh-1 cells (Figure 1C). The interaction between endogenous Rac1 with endogenous GLS2 but not

106 GLS1 was also observed in Huh-1 and HepG2 cells (Figure 1D).

107 To identify the domain of GLS2 that interacts with Rac1, three Flag-tagged deletion mutants of

108 GLS2 were constructed, including ΔN163 (deletion of N-terminal amino acids (aa) 1-163), ΔC139

109 (deletion of C-terminal aa 464-602), and C139 (C-terminal aa 464-602 only) (Figure 1E). Co-IP

110 assays in Huh-1 cells showed that the GLS2-ΔN163 and GLS2-C139, but not GLS2-ΔC139,

111 interacted with Myc-Rac1, indicating that the C-terminus of GLS2 is necessary and sufficient for

112 the interaction between GLS2 and Rac1 (Figure 1F). Since the C-terminus of GLS2 (GLS2-C139)

113 does not contain the glutaminase core domain which encodes the glutaminase catalytic domain

114 (Figure 1E) and hence lacks glutaminase activity (Figure 1G), these results demonstrate that the

115 binding of GLS2 to Rac1 is independent of its glutaminase activity.

116

117 GLS2 interacts with Rac1-GDP and inhibits the Rac1 activity

118 As a molecular Switch, Rac1 cycles between inactive GDP-bound and active GTP-bound forms in

119 cells (Bid et al., 2013; Heasman and Ridley, 2008; Raftopoulou and Hall, 2004). It is well-known

120 that constitutively active mutant Rac1 (CA Rac1-G12V) exists constitutively in the GTP-bound

121 form in cells, whereas the dominant negative Rac1 mutant (DN Rac1-T17N) exists constitutively in

122 the GDP-bound form in cells (Feig, 1999; Ridley et al., 1992). Rac1-GDP and Rac1-GTP display

123 different conformations and interact with different proteins. For instance, GEFs specifically bind to

124 Rac1-GDP to catalyze the exchange of GDP to GTP and thereby activate Rac1, whereas GAPs

125 (GTPase-activating proteins) specifically bind to Rac1-GTP to hydrolyze GTP and thereby

126 inactivate Rac1 (Cherfils and Zeghouf, 2013; Vetter and Wittinghofer, 2001).

127 To investigate the biological function of the interaction between GLS2 and Rac1, Huh-1 cells

128 were co-transfected with GLS2-Flag vectors and CA Myc-Rac1-G12V or DN Myc-Rac1-T17N

129 vectors for co-IP assays. We found that GLS2-Flag preferentially bound to Myc-Rac1-T17N but not

130 Myc-Rac1-G12V (Figure 2A). To confirm this result, lysates from Huh-1 cells co-transduced with

131 GLS2-Flag and Rac1-Myc were pretreated with GDP or GTPγS (a non-hydrolyzable analog of

132 GTP) to convert Rac1 in cell lysates into Rac1-GDP or Rac1-GTP form as previously described

133 (Castillo-Lluva et al., 2010; Fukata et al., 2002). Co-IP assays showed that GLS2-Flag

134 preferentially bound to Myc-Rac1 in cell lysates pretreated with GDP but not GTPγS (Figure 2B).

135 These results showed that GLS2 preferentially binds to Rac1-GDP, suggesting that GLS2 is

136 involved in regulating Rac1 activity.

137 We further investigated whether GLS2 inhibits the Rac1 activity in different human HCC cell

138 lines, including Huh-1 and HepG2 (p53 wild-type; WT), Hep3B (p53-null) and Huh-7 cells (p53

139 mutant). PAK1 (p21-activated kinase 1) is a critical Rac1 effector protein. It has been

140 well-established that the p21-binding domain of PAK1 binds specifically to the Rac1-GTP but not

141 Rac1-GDP in cells (Galic et al., 2014; Hayashi-Takagi et al., 2010; Palacios et al., 2002). Based on

142 this fact, the GST-p21-binding domain of PAK1 pull-down assays have been widely used to

143 measure the levels of Rac1-GTP in cells as a standard method to analyze the Rac1 activity in cells

144 (Galic et al., 2014; Hayashi-Takagi et al., 2010; Palacios et al., 2002). We found that ectopic

145 expression of GLS2-Flag greatly decreased the levels of Rac1-GTP measured by p21-binding

146 domain of PAK1 pull-down assays in Huh-1 and HepG2 cells (Figure 2C), as well as Hep3B and

147 Huh-7 cells (Figure 2C and Figure 2-figure supplement 1A). In contrast, the expression of

148 GLS2-Flag did not affect the levels of total Rac1 protein in these cells measured by western-blot

149 assays (Figure 2C and Figure 2-figure supplement 1A), indicating that GLS2 inhibits Rac1

150 activity. Consistently, knockdown of GLS2 greatly increased the levels of Rac1-GTP but not total

151 Rac1 in Huh-1 and HepG2 cells (Figure 2D) as well as in Hep3B and Huh-7 cells (Figure 2D and

152 Figure 2-figure supplement 1B, C). The endogenous levels of GLS2 in these HCC cells and the

153 levels of GLS2-Flag in HCC cells transduced with GLS2-Flag expression vectors were shown in

154 Figure 2-figure supplement 2A-D. It has been known that Rac1 binds to PAK1 and results in the

155 auto-phosphorylation of PAK1 at multiple sites, including Ser199/204, leading to PAK1 activation

156 (Chong et al., 2001; Heasman and Ridley, 2008). Therefore, we further investigated the effect of

157 GLS2 on Rac1 activity by detecting the Ser199/204 phosphorylation of PAK1 in cells. Ectopic

158 expression of GLS2-Flag in Huh-1 and HepG2 cells greatly reduced Ser199/204 phosphorylation of

159 PAK1 (Figure 2E), whereas GLS2 knockdown enhanced Ser199/204 phosphorylation of PAK1

160 (Figure 2F), which further indicates that GLS2 inhibits the Rac1 activity in HCC cells. In contrast,

161 ectopic GLS1 expression or GLS1 knockdown did not affect the Rac1 activity in HCC cells (Figure

162 2-figure supplement 1D, E).

163 Consistent with WT GLS2, the C-terminus of GLS2, GLS2-C139, specifically bound to the

164 DN Rac1-T17N but not CA Rac1-G12V in Huh-1 cells (Figure 2G). Furthermore, ectopic

165 expression of GLS2-C139 greatly inhibited the Rac1 activity in Huh-1 cells (Figure 2H). In

166 contrast, GLS2-ΔC139, which did not bind to Rac1 (Figure 1F), failed to inhibit the Rac1 activity

167 (Figure 2H). This result indicates that the interaction between GLS2 and Rac1 is critical for

168 GLS2 to inhibit the Rac1 activity. Furthermore, the function of GLS2 in binding to and inhibiting

169 Rac1 is independent of its glutaminase activity since the C-terminus of GLS2 (GLS2-C139) lacks

170 the glutaminase activity (Figure 1G). Collectively, these results revealed that GLS2 is a novel

171 negative regulator of the Rac1 signaling; GLS2 inhibits the Rac1 activity through its interaction

172 with Rac1-GDP, and furthermore, this function of GLS2 requires the C-terminus of GLS2 and is

173 independent of GLS2 glutaminase activity.

174

175 GLS2 inhibits the interaction of Rac1-GDP with Rac1 GEFs to negatively regulate Rac1

176 We further investigated the mechanism by which GLS2 inhibits Rac1. Rac1 was reported to interact

177 with other proteins through its Switch I & II regions or its C-terminus which contains the protein

178 transduction domain and GTPase C-termini (van Hennik et al., 2003; Vetter and Wittinghofer, 2001)

179 (Figure 3A). To examine the domain involved in the interaction of Rac1 with GLS2, different

180 Myc-tagged deletion mutants of Rac1 were constructed, including the ΔC33 (deletion of aa

181 160-192), the ΔN29 (deletion of aa 1-29), and the ΔSwitch (deletion of aa 30-74) (Figure 3A).

182 Co-IP assays showed that the Rac1-ΔC33 and Rac1-ΔN29, but not Rac1-ΔSwitch, interacted with

183 GLS2-Flag (Figure 3B), indicating that the Switch I & II regions are necessary for Rac1 to interact

184 with GLS2.

185 It has been well-established that Rac1 GEFs can specifically bind to Rac1-GDP through the

186 Switch I & II regions to catalyze the exchange of GDP to GTP to activate Rac1 (Rossman et al.,

187 2005; Vetter and Wittinghofer, 2001). Tiam1 and VAV1 are two most common and critical GEFs of

188 Rac1 (Heo et al., 2005; Worthylake et al., 2000). Consistent with previous reports, ectopically

189 expressed Tiam1-HA and VAV1-HA specifically bound to Rac1-GDP (shown by their preferential

190 interactions with Rac1-T17N but not Rac1-G12V; Figure 3-figure supplement 1A, B) through the

191 Switch I & II regions (Figure 3C), leading to the activation of Rac1 in Huh-1 cells (Figure 3D).

192 Since both GLS2 and Rac1 GEFs, such as Tiam1 and VAV1, bind to Rac1-GDP through the Switch

193 I & II regions, this raised the possibility that GLS2 may inhibit Rac1 activity through competing

194 with Rac1 GEFs for the Switch I & II regions of Rac1-GDP. To test this possibility, co-IP assays

195 were performed in Huh-1 cells co-transduced with DN Myc-Rac1-T17N vectors and Tiam1-HA or

196 VAV1-HA vectors, as well as increasing amount of vectors expressing GLS2-Flag. Increasing

197 amount of GLS2-Flag resulted in a progressive reduction of Tiam1-HA or VAV1-HA bound to

198 Myc-Rac1-T17N in cells (Figure 3E, F). Consistently, transducing Huh-1 and HepG2 cells with

199 increasing amount of GLS2-Flag vectors resulted in a progressive reduction of endogenous Tiam1

200 and VAV1 bound to endogenous Rac1 (Figure 3G). Furthermore, knockdown of endogenous GLS2

201 greatly promoted the interaction of endogenous Tiam1 and VAV-1 with endogenous Rac1 in Huh-1

202 and HepG2 cells (Figure 3H). These results suggest that GLS2 inhibits the Rac1 activation by

203 interacting with Rac1-GDP to block its interaction with Rac1 GEFs, such as Tiam1 and VAV1.

204

205 GLS2 inhibits migration and invasion of HCC cells through negative regulation of Rac1

206 GLS2 expression is frequently diminished in human HCC (Hu et al., 2010; Liu et al., 2014a;

207 Suzuki et al., 2010; Xiang et al., 2015). However, its role in HCC, especially HCC metastasis, is

208 poorly understood. The malignancy and poor prognosis of HCC has been related to the high

209 metastatic characteristic of HCC (El-Serag and Rudolph, 2007; Tang, 2001). Currently, the

210 mechanism underlying HCC metastasis is not well-understood. Rac1 is frequently activated or

211 overexpressed in various types of cancer, including HCC, which plays a critical role in promoting

212 cancer cell migration, invasion and metastasis (Bid et al., 2013; Heasman and Ridley, 2008). As

213 shown in Figure 4-figure supplement 1A-C, ectopic expression of CA Myc-Rac1-G12V greatly

214 promoted migration and invasion of Huh-1 and HepG2 cells as determined by trans-well assays,

215 whereas expression of DN Myc-Rac1-T17N greatly inhibited migration and invasion of these cells.

216 Therefore, our findings that GLS2 binds to and inhibits Rac1 raised the possibility that GLS2 may

217 play an important role in suppressing cancer metastasis.

218 Here, we investigated the effects of GLS2 on the abilities of migration and invasion of different

219 HCC cells, including Huh-1, HepG2, Hep3B and Huh7 cells, by using chamber trans-well assays.

220 Cells were seeded into the upper chamber containing serum-free medium without or with matrigel

221 for migration and invasion assays, respectively. Compared with cells transduced with control

222 vectors, ectopic expression of GLS2 by GLS2-Flag retroviral vectors greatly reduced the migration

223 and invasion of above-mentioned different HCC cells (Figure 4A, B). Furthermore, knockdown of

224 GLS2 by shRNA vectors greatly promoted the migration and invasion of these cells (Figure 4C, D).

225 Serum-free medium was used in the upper chamber to minimize the effect of GLS2 on cell

226 proliferation in the trans-well assays. As shown in Figure 4-figure supplement 2, no significant

227 difference in the viability and number of these cells among different groups was observed after

228 being cultured in serum-free medium for 36 h at the end of trans-well assays. Contrary to the role of

229 GLS2 in suppressing migration and invasion, ectopic expression of GLS1-Flag significantly

230 promoted the migration and invasion of Huh-1 and HepG2 cells (Figure 4E), whereas knockdown of

231 endogenous GLS1 significantly reduced the migration and invasion of these cells (Figure 4F).

232 We further investigated whether GLS2 inhibits migration and invasion of HCC cells through its

233 negative regulation of Rac1. Ectopic expression of the DN Myc-Rac1-T17N significantly reduced

234 the migration and invasion of the above-mentioned four different HCC cells (Figure 4G, H).

235 Notably, DN Myc-Rac1-T17N largely abolished the promoting effects of GLS2 knockdown on

236 migration and invasion of these cells (Figure 4G, H). Consistently, knockdown of endogenous Rac1

237 significantly reduced the migration and invasion of Huh-1 and HepG2 cells, and furthermore,

238 largely abolished the promoting effects of GLS2 knockdown on migration and invasion (Figure

239 4-figure supplement 3A-C). No significant difference in the viability and number of these cells

240 among different groups was observed after being cultured in serum-free medium for 36 h at the end

241 of trans-well assays (Figure 4-figure supplement 3D).

242 Consistent with WT GLS2, ectopic expression of the C-terminus of GLS2, GLS2-C139, which

243 interacted with Rac1-GDP and inhibited the Rac1 activity (Figure 2G, H), greatly inhibited the

244 migration and invasion of Huh-1 and HepG2 cells (Figure 4I, J). In contrast, deletion of the

245 C-terminus of GLS2 (GLS2-ΔC139), which resulted in the loss of GLS2’s ability to interact with

246 Rac1 and inhibit the Rac1 activity (Figure 2G, H), largely abolished the ability of GLS2 to inhibit

247 the migration and invasion of Huh-1 and HepG2 cells (Figure 4I, J). Taken together, these results

248 demonstrate that the negative regulation of the Rac1 activity by GLS2 is critical for GLS2 to inhibit

249 the migration and invasion of cancer cells, and furthermore, this function of GLS2 requires the

250 C-terminus of GLS2 and is independent of the glutaminase activity of GLS2.

251

252 GLS2 inhibits lung metastasis of HCC cells in vivo through negative regulation of Rac1

253 Lung metastasis is the most frequently observed distant metastasis in HCC patients (Kitano et al.,

254 2012; Uka et al., 2007). We investigated the effect of GLS2 on metastasis in vivo by employing the

255 lung metastasis model in mice. Huh-1 and HepG2 cells with ectopic GLS2-Flag expression or

256 GLS2 knockdown and their control cells were transduced with luciferase-expressing lentiviral

257 vectors and injected into BALB/c athymic nude mice via the tail vein. The metastasis of HCC cells

258 to lung was monitored by in vivo bioluminescence imaging. Bioluminescence imaging results

259 showed that ectopic expression of GLS2-Flag in both Huh-1 and HepG2 cells significantly

260 inhibited lung metastasis (Figure 5A). Histological analysis confirmed that mice injected with cells

261 with ectopic GLS2-Flag expression had fewer and smaller metastatic tumors in the lung (Figure

262 5B). Furthermore, knockdown of endogenous GLS2 led to significantly increased lung metastasis

263 of both Huh-1 and HepG2 cells analyzed by in vivo imaging and histological analysis, respectively

264 (Figure 5C, D).

265 We further investigated whether inhibition of Rac1 mediates GLS2’s function in suppression of

266 lung metastasis of HCC cells in vivo. As shown in Figure 5E, F, ectopic expression of the DN

267 Rac1-T17N greatly reduced lung metastasis of Huh-1 and HepG2 cells in nude mice. Notably, The

268 DN Rac1-T17N largely abolished the promoting effects of GLS2 knockdown on lung metastasis of

269 Huh-1 and HepG2 cells. These results together suggest that GLS2 inhibits cancer metastasis

270 through its down-regulation of the Rac1 activity.

271

272 The decreased GLS2 expression is associated with enhanced human HCC metastasis

273 Our results from cancer cell migration and invasion assays as well as lung metastasis models

274 clearly showed that GLS2 inhibited metastasis of different human HCC cells, which strongly

275 suggests that decreased expression of GLS2 in human HCC could be an important mechanism

276 contributing to the high metastasis of human HCC. To this end, we investigated the association of

277 decreased GLS2 expression with cancer metastasis in human HCC samples. We first queried the

278 The Cancer Genome Atlas (TCGA) database to compare GLS2 expression between HCC samples

279 with or without vascular invasion of HCC cells. As shown in Figure 5G, GLS2 expression was

280 significantly lower in HCCs with vascular invasion (n=57), compared with HCCs without vascular

281 invasion (n=110) (decreased by 3.03-fold; p=0.0066). Consistent results were also observed in

282 another cohort from Gene Expression Omnibus (GEO, GSE6764) by using Oncomine, a human

283 genetic dataset analysis tool. GLS2 expression was significantly lower in HCCs with vascular

284 invasion (n=18), compared with HCCs without vascular invasion (n=15) (decreased by 4.62-fold;

285 p=0.0198) (Figure 5H). These results indicated that the decreased GLS2 expression is significantly

286 associated with enhanced metastasis in human HCC.

287

288 GLS2 mediates p53’s function in suppressing HCC metastasis

289 p53 plays a critical role in inhibiting cancer metastasis. However, while extensive work has been

290 done on the mechanisms underlying p53-mediated apoptosis, cell cycle arrest and senescence, the

291 mechanism underlying p53’s function in suppressing cancer metastasis is much less

292 well-understood (Muller et al., 2011; Vousden and Prives, 2009). Previous reports including ours

293 have shown that as a direct p53 target, GLS2 is up-regulated by p53 in cells under both

294 non-stressed and stressed conditions (Hu et al., 2010; Suzuki et al., 2010). Consistently, p53

295 knockdown by shRNA greatly reduced the mRNA and protein levels of GLS2 in Huh-1 and HepG2

296 cells which express WT p53 (Figure 6A). Considering the potent activity of GLS2 in inhibiting

297 cancer cell metastasis, our findings raised the possibility that GLS2 may be an important mediator

298 of p53’s function in suppressing cancer metastasis.

299 Here, we tested this hypothesis. Knockdown of p53 significantly promoted the migration and

300 invasion of both Huh-1 and HepG2 cells measured by trans-well assays (Figure 6B, C). As shown

301 in Figure 6-figure supplement 1, no significant difference in the viability and number of these cells

302 among different groups was observed after being cultured in serum-free medium for 36 h at the end

303 of trans-well assays. Notably, while individual knockdown of GLS2 or p53 dramatically promoted

304 the migration and invasion of Huh-1 and HepG2 cells, simultaneous knockdown of GLS2 and p53

305 did not display a clear additive effect on the migration and invasion of these cells (Figure 6D, E).

306 Consistently, while individual knockdown of GLS2 or p53 dramatically promoted lung metastasis

307 of Huh-1 and HepG2 cells in mice, simultaneous knockdown of GLS2 and p53 did not display an

308 additive effect on lung metastasis of these cells (Figure 6F, G). These results demonstrate that

309 GLS2 is a novel and important mediator of p53 in suppressing cancer metastasis.

310

311 GLS2 mediates p53’s function in metastasis suppression through Rac1 inhibition

312 It has been reported that p53 inhibits Rac1 activity, but its mechanism remains unclear (Bosco et al.,

313 2010; Guo and Zheng, 2004; Muller et al., 2011). As shown in Figure 7A, B, expression of DN

314 Rac1-T17N greatly abolished the promoting effects of p53 knockdown on migration and invasion

315 of Huh-1 and HepG2 cells. Consistently, Rac1 knockdown greatly abolished the promoting effects

316 of p53 knockdown on migration and invasion of Huh-1 and HepG2 cells (Figure 7-figure

317 supplement 1A, B). These results suggest that Rac1 inhibition is an important mechanism for p53

318 to inhibit metastasis.

319 Our finding that GLS2 interacts with Rac1-GDP to inhibit Rac1 activity suggests that as a

320 direct p53 target, GLS2 could mediate p53’s function in inhibiting Rac1 activity. Notably, while

321 individual knockdown of GLS2 or p53 greatly activated Rac1, simultaneous knockdown of GLS2

322 and p53 did not display an additive effect on the Rac1 activity in Huh-1 or HepG2 cells (Figure 7C).

323 Furthermore, GLS2-Flag overexpression largely abolished the promoting effect of p53 knockdown

324 on the Rac1 activity in Huh-1 or HepG2 cells (Figure 7D). These results indicate that GLS2

325 mediates p53’s function in inhibiting Rac1 activity.

326 Our results have shown that GLS2 inhibited the interaction between Rac1 and its GEFs Tiam1

327 and VAV1 to down-regulate the Rac1 activity (Figure 3E-H). Here, we investigated whether

328 inhibition of the interaction of Tiam1 and VAV1 with Rac1 is an important mechanism for p53 to

329 down-regulate the Rac1 activity. Knockdown of WT p53 in Huh-1 and HepG2 cells, which greatly

330 decreased GLS2 protein levels (Figure 7C), clearly promoted the interaction of Tiam1 and VAV1

331 with Rac1 (Figure 7E). Notably, GLS2 knockdown in cells with p53 knockdown did not further

332 promote the interaction of Tiam1 and VAV1 with Rac1 (Figure 7E). Furthermore, GLS2

333 overexpression largely abolished the promoting effect of p53 knockdown on the interaction of

334 Tiam1 and VAV1 with Rac1 (Figure 7F). These results suggest that blocking the interaction of

335 Tiam1 and VAV1 with Rac1-GDP by GLS2 contributes greatly to p53’s function in inhibiting the

336 Rac1 activity. Collectively, our results strongly suggest that GLS2 mediates p53’s function in

337 suppression of HCC metastasis by inhibiting the interaction of Rac1 GEFs, such as Tiam1 and

338 VAV1, with Rac1-GDP to down-regulate the Rac1 activity (Figure 7G).

339

340 Discussion

341 In this study, GLS2 was identified as a novel binding protein and negative regulator for Rac1. GLS2

342 bound to Rac1-GDP through its Switch I & II regions, which is also the binding domain for Rac1

343 GEFs Tiam1 and VAV1. Thus, GLS2 blocked the binding of Tiam1 and VAV1 to Rac1-GDP, and

344 inhibited the Rac1 activation by Tiam1 and VAV1. We found that GLS2 inhibited migration and

345 invasion of HCC cells in vitro and lung metastasis of HCC cells in vivo. Blocking the Rac1 signaling

346 by expression of Rac1-T17N or knockdown of Rac1 largely abolished GLS2’s function in

347 inhibiting metastasis. These results demonstrate a novel and important role of GLS2 in suppressing

348 cancer metastasis, and also reveal that GLS2 binding to Rac1-GDP to inhibit Rac1 activity is a

349 critical underlying mechanism (Figure 7G).

350 GLS1 plays a critical role in promoting tumorigenesis through enhancing glutamine metabolism

351 (Gao et al., 2009; Thangavelu et al., 2012; Wang et al., 2010). It is unclear why GLS1 and GLS2 have

352 contrasting roles in tumorigenesis although they both function as the glutaminase enzymes. While

353 the glutaminase core domains of GLS1 and GLS2 show high homology, their C-termini show

354 relatively low homology. In this study, we found that GLS2 bound to Rac1 through its C-terminus

355 and inhibited the Rac1 activity to suppress migration and invasion of HCC cells. This effect requires

356 the C-terminus of GLS2 and is independent of its glutaminase activity. In contrast, GLS1 does not

357 bind to Rac1 or inhibit Rac1 activity. Considering the critical role of Rac1 in cancer, our results

358 provide a novel mechanism for the different roles of GLS1 and GLS2 in tumorigenesis, particularly

359 with respect to cancer metastasis. In addition to Rac1, GLS2 may interact with other proteins to

360 regulate their functions, which in turn contributes to GLS2’s function in tumor suppression. Future

361 studies should shed light on the further mechanisms of GLS2 in tumor suppression.

362 p53 plays a critical role in suppressing cancer metastasis. While extensive work has been done

363 on the mechanisms underlying p53-mediated apoptosis, cell cycle arrest and senescence, the

364 mechanism underlying p53’s function in suppressing cancer metastasis is much less

365 well-understood (Muller et al., 2011; Vousden and Prives, 2009). p53 has been reported to inhibit

366 Rac1 activity, however, the detailed mechanism is unclear (Bosco et al., 2010; Guo and Zheng,

367 2004; Muller et al., 2011). Our results show that p53 knockdown down-regulated GLS2 levels and

368 promoted the interaction of Rac1-GDP with its GEFs Tiam1 and VAV1, and thereby enhanced the

369 Rac1 activity and promoted cancer metastasis. These results demonstrate that GLS2 is a novel and

370 critical mediator for p53 in suppressing metastasis, and also reveal a novel mechanism by which p53

371 inhibits Rac1.

372 HCC is one of the most common types of cancer and the third leading cause of cancer death

373 worldwide (El-Serag and Rudolph, 2007; Jemal et al., 2011). The high malignancy and poor

374 prognosis of HCC has been related with the high metastatic characteristic of HCC, however, the

375 mechanism underlying HCC metastasis is not well-understood (El-Serag and Rudolph, 2007; Tang,

376 2001). Considering that GLS2 is frequently down-regulated in HCC (Hu et al., 2010; Liu et al.,

377 2014a; Suzuki et al., 2010; Xiang et al., 2015), our findings that GLS2 inhibits HCC metastasis and

378 loss of GLS2 promotes HCC metastasis provide a novel mechanism contributing to high metastatic

379 characteristic of HCC. These results strongly suggest that the GLS2/Rac1 signaling could be a

380 potential target for therapy in cancer, particularly in HCC.

381 In summary, our results demonstrat that GLS2 is a novel negative regulator of Rac1, and plays a

382 novel and critical role in suppression of metastasis through its negative regulation of the Rac1

383 activity. Furthermore, our results also reveal that GLS2 is a critical mediator for p53 in suppression

384 of cancer metastasis.

385

386 Materials and Methods

387 Cell lines, vectors and shRNA

388 HepG2 (p53-WT) and Hep3B (p53-null) cells were obtained from American Type Culture

389 Collection (ATCC, Manassas, VA). Huh-1 (p53-WT) and Huh-7 (p53 mutant) were obtained from

390 the Japanese Culture Collection (RIKEN BioResource Center, Saitama, Japan). All cell lines were

391 authenticated by STR profiling. Cells were regularly tested for mycoplasma using Lookout

392 Mycoplasma PCR detection kit (Sigma, MP0035) and only used when negative. The WT p53 were

393 knocked down in HepG2 and Huh-1 cells by 2 different shRNA vectors as previously described

394 (Zhang et al., 2013). The pLPCX vectors expressing Flag-tagged WT or deletion mutants of GLS2

395 were constructed by PCR amplification as we previously described (Hu et al., 2010). The

396 pLPCX-Myc-Rac1 and pLPCX-Myc-Rac1-G12V vectors were constructed by using Myc-Rac1

397 DNA fragment from pcDNA3.1-Myc-Rac1 WT and pcDNA3.1-Myc-Rac1 G12V vectors,

398 respectively (He et al., 2010). The pLPCX-Myc-Rac1-T17N vector was constructed by using a

399 Quikchange II XL Site-Directed Mutagenesis Kit (Stratagene/Agilent Technologies). The pLPCX

400 vectors expressing deletion mutants of Myc-Rac1 were constructed by PCR amplification. The

401 pLPCX-GLS1-Flag, pLPCX-Tiam1-HA and pLPCX-VAV1-HA vectors were cloned using PCR

402 amplification. Two lentiviral shRNA vectors against GLS2 (ID: V3LHS_307701 and

403 V2LHS_71048), two lentiviral shRNA vectors against Rac1 (ID: V3LHS_317664 and

404 V3LHS_317668) and control shRNA vectors were obtained from Open Biosystems (Huntsville,

405 AL).

406

407 Cell migration and invasion assays

408 The trans-well system (24 wells, 8 μM pore size, BD Biosciences) was employed for cell migration

409 and invasion assays as we previously described (Zheng et al., 2013; Zhao et al., 2015). In brief,

410 cells (2x104 for Huh-1, and 6x104 for HepG2) in serum-free medium were seeded into upper

411 chambers for migration assays. Cells on the lower surface were fixed, stained and counted at 24 h

412 after seeding. For invasion assays, cells (4x104 for Huh-1, and 1x105 for HepG2) were seeded into

413 upper chambers coated with matrigel (BD Biosciences). Cells on the lower surface were fixed,

414 stained and counted at 36 h after seeding.

415

416 In vivo lung metastasis assays

417 In vivo lung metastasis assays were performed as previously described (Li et al., 2014; Zheng et al.,

418 2013). In brief, Huh-1 and HepG2 cells (2 × 106 in 0.1mL PBS) transduced with lentiviral vectors

419 expressing luciferase were injected into 2-month-old male BALB/c nude mice via the tail vein (n=8

420 mice/group). Lung metastatic colonization was monitored and quantified at different weeks using

421 non-invasion bioluminescence imaging by IVIS Spectrum in vivo imaging system (PerkinElmer),

422 and was validated at the endpoint by routine histopathological analysis. All mouse experiments

423 were approved by the University Institutional Animal Care and Use Committee.

424

425 Western-blot assays

426 Standard western-blot assays were used to analyze protein expression in cells. The following

427 antibodies were used for assays: anti-Flag-M2 (F1804, Sigma; 1:20000 dilution), anti-β-Actin

428 (A5441, Sigma; 1:10000 dilution), anti-Myc (9E10, Roche; 1:1000 dilution), anti-HA (3F10, Roche;

429 1:1000 dilution), anti-Rac1 (23A8, Millipore; 1:5000 dilution), anti-p-PAK (Ser199/204) (09-258,

430 Millipore; 1:1000 dilution), anti-PAK (07-1451, Millipore; 1: 1000 dilution), anti-p53 (FL393, 22

431 Santa Cruz; 1:2000 dilution), anti-Tiam1 (sc-872, Santa Cruz; 1:2000 dilution), anti-VAV1 (sc-8039,

432 Santa Cruz; 1:1000 dilution). The anti-GLS2 antibody (1: 1000 dilution) was prepared as

433 previously described (Hu et al., 2010). To increase the sensitivity of the GLS2 antibody,

434 endogenous GLS2 in cells was pulled down by IP and detected by western-blot assays. The band

435 intensity was quantified by digitalization of the X-ray film and analyzed with the Image J software.

436

437 Co-IP assays

438 Co-IP assays were performed as we previously described (Liu et al., 2014b). For co-IP of

439 GLS2-Flag and Myc-Rac1 proteins, anti-Flag (M2, Sigma) and anti-Myc (9E10, Roche) agarose

440 beads were used to pull down GLS2-Flag and Myc-Rac1, respectively. For Co-IP of endogenous

441 GLS2 and Rac1, the anti-GLS2 antibody and the anti-Rac1 (23A8, Millipore) antibody were used

442 for IP, respectively. The mouse or rabbit purified IgGs were used as negative controls.

443

444 LC-MS/MS analysis

445 To determine potential GLS2 binding proteins, GLS2-Flag protein in Huh-1 cells with stable

446 expression of GLS2-Flag was pulled down by co-IP using anti-Flag (M2) beads and eluted with

447 Flag peptide. Huh-1 cells transduced with control vectors were used as a control for co-IP assays.

448 Eluted materials were separated in a 4-16% SDS-PAGE gel and visualized by silver staining using

449 the silver staining kit (Invitrogen). LC-MS/MS analysis was performed at the Biological Mass

450 Spectrometry facility of Rutgers University as previously described (Yue et al., 2015; Zhao et al.,

451 2015).

452

453 Rac1 activity analysis

454 For Rac1 activity analysis, the GST-p21-binding domain of PAK1 pull-down assays were

455 performed using a Rac1 activation assay kit (Millipore) to measure the levels of GTP-bound Rac1

456 (Rac1-GTP) in cells as previously described (Galic et al., 2014; Hayashi-Takagi et al., 2010;

457 Palacios et al., 2002). The p21-binding domain of the Rac1 effector protein PAK1 binds specifically

458 to the Rac1-GTP (Hayashi-Takagi et al., 2010; Palacios et al., 2002). The levels of precipitated

459 Rac1-GTP were measured by western-blot assays using a Rac1 antibody (23A8, Millipore) and

460 normalized to total Rac1 levels in cells measured by western-blot assays.

461

462 Quantitative Real-Time PCR assays

463 Total RNA was prepared with the RNeasy Kit (Qiagen). cDNA was prepared using a TaqMan

464 reverse transcription kit, and real-time PCR was performed with TaqMan PCR mixture (Applied

465 Biosystems, Foster City, CA) as we previously described (Hu et al., 2010; Zhang et al., 2013). The

466 expression of genes in cells was normalized to the expression of the Actin gene.

467

468 Glutaminase activity assays

469 Glutaminase activity was measured as previously described (Gao et al., 2009; Wang et al., 2010).

470 Briefly, cell lysates were incubated at 37ºC for 10 min with the assay mix consisting of 20 mM

471 glutamine, 50 mM Tris-acetate (pH 8.6), 100 mM phosphate, and 0.2 mM EDTA. The reaction was

472 quenched with the addition of 2 mL of 3 M HCl. Subsequently, the reaction mixture was incubated

473 for 30 min at room temperature with the second assay mix (2.2 U glutamate dehydrogenase, 80 mM

474 Tris-acetate (pH 9.4), 200 mM hydrazine, 0.25 mM ADP, and 2 mM nicotinamide adenine

475 dinucleotide). The absorbance was read at 340 nm using a spectrophotometer.

476

477

478 Statistical analysis

479 The differences in tumor growth among groups were analyzed for statistical significance by

480 analysis of variance, followed by Student’s t-tests using GraphPad Prism software. All other

481 P-values were obtained using two-tailed Student t-tests. **: p<0.001; *: p<0.01; #: p<0.05.

482

483

484 Author Contributions

485 Z.F. and W.H. designed experiments, analyzed the data, and wrote the manuscript. C.Z., J.L., Y.Z.,

486 X.W., Y. Z. X.Y. H.W. and F.B. designed experiments, carried out the experiments, analyzed the

487 data. G.B, S.L. and B.G.H. contributed reagents, analyzed the data and revised the manuscript..

488 489 Acknowledgements

490 We thank Dr. Arnold Levine for helpful discussion and comments. This work was supported by

491 grants from the NIH (1R01CA143204), CINJ Foundation and New Jersey Health Foundation (to

492 Z.F.), by grants from NIH (1R01CA160558-01) and Ellison Medical Foundation (to W.H.), by

493 grants from NIH (R01CA169182-01 to G.B.), and BCRF (to B.G.H.). J. L. was supported by

494 NJCCR postdoctoral fellowship.

495

496 Competing Financial Interests 497 498 The authors declare no competing financial interests. 499

500

501 502

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681

682 Figure Legends

683 Figure 1. Rac1 is a novel interacting protein for GLS2.

684 (A) The potential GLS2-interacting proteins identified by co-IP followed by LC-MS/MS analysis.

685 Huh-1 cells expressing GLS2-Flag or cells transduced with control vectors were used for co-IP with

686 the anti-Flag antibody followed by LC-MS/MS analysis. The potential GLS2 interacting proteins

687 are listed with the number of peptides identified by LC-MS/MS analysis. (B) GLS2-Flag interacted

688 with Myc-Rac1 in cells. Huh-1 cells were transduced with Myc-Rac1, GLS2-Flag and control

689 vectors as indicated for co-IP assays using the anti-Myc (left panels) and anti-Flag antibodies (right

690 panels), respectively. IP: immunoprecipitation; IB: immunoblot. (C) GLS1-Flag did not interact

691 with Myc-Rac1 in cells. Huh-1 cells were transduced with Myc-Rac1 and GLS1-Flag vectors for

692 co-IP assays using the anti-Myc (left panels) and anti-Flag antibodies (right panels), respectively.

693 (D) Endogenous GLS2 but not GLS1 interacted with endogenous Rac1 in Huh-1 and HepG2 cells

694 detected by co-IP assays. (E) Schematic representation of vectors expressing Flag-tagged wild-type

695 (WT) or serial deletion mutants of GLS2. (F) The C-terminus of GLS2, GLS2-C139, is necessary

696 and sufficient for GLS2 to interact with Rac1. Huh-1 cells were transduced with WT or different

697 mutant GLS2-Flag vectors listed in Figure 1E together with Myc-Rac1 vectors for co-IP assays. (G)

698 The relative glutaminase activities of WT and different mutant GLS2. Huh-1 and HepG2 cells were

699 transduced with WT and different mutant GLS2 vectors. The relative glutaminase activities in cells

700 transduced with WT GLS2 vectors were designated as 100. **: p<0.001. Student’s t-test.

701

702

703

704 Figure 2. GLS2 interacts with Rac1-GDP and negatively regulates the Rac1 activity.

705 (A) GLS2-Flag preferentially interacted with the DN Myc-Rac1-T17N but not the CA

706 Myc-Rac1-G12V in Huh-1 cells. Cells were transduced with GLS2-Flag vectors together with

707 Rac1-T17N or Rac1-G12V vectors for co-IP assays. (B) GLS2-Flag preferentially bound to

708 Rac1-GDP but not Rac1-GTP in cell lysates. Cell lysates from Huh-1 cells transduced with vectors

709 expressing Myc-Rac1 and GLS2-Flag were pretreated with GDP or GTPγS to convert Rac1 into

710 Rac1-GDP or Rac1-GTP form before co-IP assays. (C) Ectopic expression of GLS2 inhibited Rac1

711 activities represented by decreased levels of Rac1-GTP in HCC cells measured by the

712 GST-p21-binding domain of PAK1 pull-down assays. Left panels: Represented results of Rac1

713 activity analysis in Huh-1 and HepG2 cells. Right panels: relative Rac1-GTP/total Rac1/Actin levels

714 in Huh-1, HepG2, Hep3B and Huh-7 cells. Data present mean ± SD (n=3). *: p<0.01; Student’s

715 t-test. (D) Knockdown of GLS2 by shRNA vectors increased Rac1 activities in HCC cells. Left

716 panels: Represented results of Rac1 activity analysis in Huh-1 and HepG2 cells. Right panels:

717 relative Rac1-GTP/total Rac1 /Actin levels in Huh-1, HepG2, Hep3B and Huh-7 cells. Data present

718 mean ± SD (n=3). *: p<0.01; #: p<0.05; Student’s t-test. (E) Ectopic expression of GLS2-Flag

719 decreased the levels of p-PAK1 at Ser199/204 in Huh-1 and HepG2 cells. (F) Knockdown of GLS2

720 by shRNA vectors increased the levels of p-PAK1 at Ser199/204 in Huh-1 and HepG2 cells. (G)

721 The C-terminus of GLS2, GLS2-C139, interacted with DN Myc-Rac1-T17N but not CA

722 Myc-Rac1-G12V in Huh-1 cells detected by co-IP assays. (H) The C-terminus of GLS2,

723 GLS2-C139, inhibited the Rac1 activity in Huh-1 and HepG2 cells. Left panels: Represented results

724 of Rac1 activity analysis in Huh-1 cells transduced with different GLS2-Flag vectors. Right panels:

725 relative Rac1-GTP/total Rac1 /Actin levels in Huh-1 and HepG2. Data present mean ± SD (n=3). *:

726 p<0.01; #: p<0.05; Student’s t-test.

727 Figure 2-figure supplement 1. GLS2 inhibits Rac1 activity in HCC cells.

728 (A) Ectopic expression of GLS2-Flag inhibited Rac1 activities represented by decreased Rac1-GTP

729 levels in Hep3B and Huh-7 cells. (B) Knockdown of GLS2 by 2 different shRNA vectors in

730 different HCC cells detected by Taqman real-time PCR assays. The mRNA expression of GLS2 was

731 normalized with Actin. Data are presented as mean ± SD (n=3). **: p<0.001; Student’s t-test. (C)

732 Knockdown of endogenous GLS2 by shRNA enhanced Rac1 activities represented by increased

733 Rac1-GTP levels in Hep3B and Huh-7 cells. GLS2 knockdown was presented in Figure 2-figure

734 supplement 1B. (D) Ectopic expression of GLS1 did not clearly affect the Rac1 activity in Huh-1 or

735 HepG2 cells. (E) Knockdown of endogenous GLS1 by shRNA vectors did not clearly affect the

736 Rac1 activity in Huh-1 or HepG2 cells. In D & E: data present mean ± SD (n=3).

737

738 Figure 2-figure supplement 2. The expression of endogenous GLS2 and exogenous GLS2-Flag

739 in HCC cells.

740 (A) The expression of endogenous GLS2 protein in HCC cells. To enhance the sensitivity of the

741 anti-GLS2 antibody, endogenous GLS2 in cells was pulled down by IP assays with the anti-GLS2

742 antibody and detected by western-blot assays. (B) The expression of exogenous GLS2-Flag protein

743 in HCC cells. The levels of exogenous GLS2-Flag protein in HCC cells transduced with the

744 GLS2-Flag retrovival vector were measured by western-blot assays. (C) The relative mRNA levels

745 of GLS2 in HCC cells with or without transduction of the GLS2-Flag vector. (D) The relative

746 mRNA levels of GLS1 in HCC cells. In C and D, the mRNA levels of GLS2 and GLS1 in cells were

747 measured by real-time PCR assays and normalized with Actin. Two normal liver tissues were used

748 as controls. The relative mRNA levels of GLS2 (C) and GLS1 (D) in Huh-1 cells were designated as

749 1, respectively.

750

751 Figure 3. GLS2 inhibits the interaction of Rac1-GDP with Tiam1 and VAV1 .

752 (A) Schematic representation of Rac1 deletion mutants. Myc-tagged vectors expressing WT Rac1 or

753 serial deletion mutants were constructed. (B) GLS2 bound to Rac1 through its Switch I & II regions.

754 Huh-1 cells were transduced with different Myc-Rac1 vectors listed in Figure 3A together with

755 GLS2-Flag vectors for co-IP assays. (C) Tiam1 (upper panels) and VAV1 (lower panels) bound to

756 Rac1 through its Switch I & II regions. Huh-1 cells were transduced with different Myc-Rac1

757 vectors listed in Figure 3A together with vectors expressing Tiam1-HA or VAV1-HA for co-IP

758 assays. (D) Ectopic expression of Tiam1 and VAV1 activated Rac1 in cells. Huh-1 cells were

759 transduced with vectors expressing Tiam1-HA and VAV1-HA, respectively, and Rac1 activity was

760 analyzed. Data are presented as mean ± SD (n=3). *: p<0.01; Student’s t-test. (E, F) Ectopic

761 expression of GLS2 inhibited the interaction of DN Myc-Rac1-T17N with ectopic Tiam1-HA (E)

762 and VAV1-HA (F) in cells. Huh-1 cells were transduced with Myc-Rac1-T17N vectors and

763 Tiam1-HA (E) or VAV1-HA vectors (F) (2 μg), together with increasing amount of GLS2-Flag

764 vectors (1, 3, 6 μg) for co-IP assays. (G) Ectopic expression of GLS2 inhibited the interaction of

765 endogenous Rac1 with endogenous Tiam1 and VAV1 in cells. Huh-1 and HepG2 cells were

766 transduced with increasing amount of GLS2-Flag expression vectors (1, 3, 6 μg) for co-IP assays.

767 (H) Knockdown of endogenous GLS2 by shRNA vectors in Huh-1 and HepG2 cells promoted the

768 interaction of endogenous Rac1 with endogenous Tiam1 and VAV1 as measured by co-IP assays.

769 Figure 3-figure supplement 1: Tiam1 and VAV1 preferentially bind to Rac1-GDP.

770 (A) Tiam1-HA preferentially bound to the DN Myc-Rac1-T17N but not CA Myc-Rac1-G12V.

771 Huh-1 cells were transduced with DN Rac1-T17N or CA Rac1-G12V vectors together with vectors

772 expressing Tiam1-HA for co-IP assays. (B) VAV1-HA preferentially bound to the DN

773 Myc-Rac1-T17N but not CA Myc-Rac1-G12V. Huh-1 cells were transduced with DN

774 Myc-Rac1-T17N or CA Myc-Rac1-G12V vectors together with vectors expressing VAV1-HA for

775 co-IP assays.

776

777 Figure 4. GLS2 inhibits migration and invasion of HCC cells through negative regulation of

778 Rac1.

779 (A, B) Ectopic GLS2 expression inhibited the migration (A) and invasion (B) of different HCC cells

780 as determined by trans-well assays. Upper panels: representative images of migrating (A) and

781 invading (B) Huh-1 cells transduced with control (con) or GLS2-Flag vectors. Scale bars: 200 μm.

782 Lower panels: quantification of average number of migrating (A) and invading (B) cells/field in

783 different HCC cells transduced with control (con) or GLS2-Flag vectors. Data present mean ± SD

784 (n=6). **: p<0.001; Student’s t-test. (C, D) Knockdown of GLS2 by shRNA vectors promoted the

785 migration (C) and invasion (D) of different HCC cells. Upper panels: representative images of

786 migrating (C) and invading (D) Huh-1 cells with or without GLS2 knockdown. Scale bars, 200 μM.

787 Data present mean ± SD (n=6). **: p<0.001; Student’s t-test. (E) Ectopic GLS1 expression

788 promoted the migration (left) and invasion (right) of Huh-1 and HepG2 cells as determined by

789 trans-well assays. (F) Knockdown of GLS1 decreased the migration (left) and invasion (right) of

790 Huh-1 and HepG2 cells. In E, F: data are presented as mean ± SD (n=6). **: p<0.001; Student’s

791 t-test. (G, H) Ectopic expression of DN Rac1-T17N largely abolished the promoting effects of GLS2

792 knockdown on migration (G) and invasion (H) of different HCC cells as measured by trans-well

793 assays. Cells with knockdown of GLS2 by shRNA vectors were transduced with Rac1-T17N

794 expression vectors for trans-well assays. Data present mean ± SD (n=6). **: p<0.001; Student’s

795 t-test. (I, J) Ectopic expression of the C-terminus of GLS2, GLS2-C139, inhibited migration (I) and

796 invasion (J) of Huh-1 and HepG2 cells as measured by trans-well assays. Cells were transduced

797 with different GLS2 expression vectors described in Figure 1E for assays. Upper panels in I:

798 representative images of migrating Huh-1 cells. Data present mean ± SD (n=6). **: p<0.001;

799 Student’s t-test.

800 Figure 4-figure supplement 1: Rac1 promotes the migration and invasion of Huh-1 and HepG2

801 cells.

802 (A) Ectopic expression of CA Myc-Rac1-G12V and DN Myc-Rac1-T17N in Huh-1 and HepG2

803 cells detected by western-blot assays. Cells were transduced with Myc-Rac1-G12V and

804 Myc-Rac1-T17N vectors for assays. (B) Ectopic expression of CA Myc-Rac1-G12V promoted the

805 migration of Huh-1 and HepG2 cells, whereas ectopic expression of DN Myc-Rac1-T17N inhibited

806 the migration of Huh-1 and HepG2 cells. Upper panels: representative images of migrating Huh-1

807 cells. Scale bars: 200 μm. Lower panel: quantification of average number of migrating cells/field.

808 Data are presented as mean ± SD (n=6). **: p<0.001; Student’s t-test. (C) Ectopic expression of CA

809 Myc-Rac1-G12V promoted the invasion of Huh-1 and HepG2 cells, whereas ectopic expression of

810 DN Myc-Rac1-T17N inhibited the invasion of Huh-1 and HepG2 cells. Data are presented as mean

811 ± SD (n=6). **: p<0.001; Student’s t-test.

812 Figure 4-figure supplement 2: The viability and number of HCC cells with GLS2

813 overexpression or knockdown after being cultured in serum-free medium for 36 h.

814 (A, B) The viability of HCC cells with GLS2 overexpression (A) or knockdown (B) cultured in

815 serum-free medium for 36 h. (C, D) The relative number of HCC cells with GLS2 overexpression

816 (C) or knockdown (D) cultured in serum-free medium for 36 h. Same number of cells with GLS2

817 overexpression or knockdown and their control cells were cultured in serum-free medium for 36 h

818 before the the viability and number of cells was measured in a Vi-CELL Cell Viability Analyzer

819 (Beckman Coulter). The viability of cells was analyzed by the trypan blue cell exclusion method.

820 Figure 4-figure supplement 3: Knockdown of endogenous Rac1 largely abolishes the effect of

821 GLS2 on migration and invasion of HCC cells.

822 (A) The knockdown of Rac1 by 2 different shRNA vectors in Huh-1 and HepG2 cells measured by

823 western-blot assays. (B) Knockdown of endogenous Rac1 largely abolished the promoting effects

824 of GLS2 knockdown on migration of Huh-1 and HepG2 cells as measured by trans-well assays.

825 The endogenous Rac1 was knocked down by shRNA vectors in Huh-1 and HepG2 cells with GLS2

826 knockdown.Left panels: representative images of migrating Huh-1 cells. Scale bars, 200 μm. Right

827 panels: quantification of number of migrating cells/field. Data are presented as mean ± SD (n=6).

828 **: p<0.001; Student’s t-test. (C) Knockdown of endogenous Rac1 largely abolished the promoting

829 effects of GLS2 knockdown on invasion of Huh-1 and HepG2 cells as measured by trans-well

830 assays. Data are presented as mean ± SD (n=4). **: p<0.001; Student’s t-test. (D) The viability (left

831 panel) and number (right panel) of HCC cells with GLS2 and/or Rac1 knockdown cultured in

832 serum-free medium for 36 h. Same number of cells were cultured in serum-free medium for 36 h

833 before the the viability and number of cells was measured in a Vi-CELL Cell Viability Analyzer

834 (Beckman Coulter). The viability of cells was analyzed by the trypan blue cell exclusion method.

835

836 Figure 5. GLS2 inhibits lung metastasis of HCC cells in mice, and GLS2 expression is

837 associated with metastasis in human HCC.

838 (A, B) Ectopic expression of GLS2 inhibited lung metastasis of Huh-1 and HepG2 cells in nude

839 mice. Huh-1 and HepG2 cells with GLS2 ectopic expression were transduced with lentiviral

840 vectors expressing luciferase for lung metastasis assays. The lung metastasis was analyzed by in

841 vivo bioluminescence imaging (A) and histological analysis (B) at 7 weeks after inoculation of cells.

842 Left panels in A: representative images of lung metastasis of Huh-1 cells analyzed by in vivo

843 imaging. Right panels in A: quantification of lung photon flux (photons per second). Left panels in

844 B: representative images of H&E staining of lung metastasis of Huh-1 cells. Right panels in B: The

845 average number of tumors/lung. (C, D) Knockdown of endogenous GLS2 by shRNA promoted

846 lung metastasis of Huh-1 and HepG2 cells in nude mice. (E, F) Ectopic expression of DN

847 Rac1-T17N largely abolished the promoting effects of GLS2 knockdown on lung metastasis of

848 HCC cells in vivo. Huh-1 and HepG2 cells with stable ectopic Rac1-T17N expression and GLS2

849 knockdown were used for lung metastasis assays in mice. In A-F, data represent mean ± SD (n=8

850 mice/group). *: p< 0.01; **: p<0.001; Student’s t-test. Scale bars, 200 μM. Arrows indicate

851 metastatic tumors. (G, H) GLS2 expression is significantly decreased in human HCCs with

852 metastasis compared with HCCs without metastasis. GLS2 mRNA expression in non-metastatic

853 (without vascular invasion) and metastatic (with vascular invasion) HCCs was obtained from the

854 TCGA (G) and GSE6764 (H). p=0.0066 in G; p=0.0198 in H; Student’s t-test.

855

856 Figure 6. GLS2 mediates p53’s function in inhibiting migration, invasion and lung metastasis

857 of HCC cells.

858 (A) Knockdown of endogenous WT p53 reduced GLS2 expression in Huh-1 and HepG2 cells as

859 measured by western-blot (left) and Taqman real-time PCR assays (right), respectively. (B, C)

860 Knockdown of p53 promoted the migration (B) and invasion (C) of Huh-1 and HepG2 cells

861 measured by trans-well assays. (D, E) Simultaneous knockdown of GLS2 and p53 by shRNA

862 vectors in Huh-1 and HepG2 cells did not display an addictive promoting effect on the migration

863 (D) and invasion (E) of cells. In A-E, data represent mean ± SD (n=6). **: p<0.001; Student’s t-test.

864 (F, G) Simultaneous knockdown of GLS2 and p53 in Huh-1 and HepG2 cells did not display an

865 addictive promoting effect on lung metastasis in vivo. Huh-1 and HepG2 cells with individual

866 knockdown of GLS2 or p53, or simultaneous knockdown of GLS2 and p53 were used for assays. In

867 F, lung metastasis was analyzed by in vivo bioluminescence imaging at 7 weeks after inoculation of

868 cells. Upper panels: representative images of lung metastasis of Huh-1 cells analyzed by in vivo

869 imaging. Lower panels: quantification of lung photon flux. In G, lung metastasis was analyzed by

870 histological analysis at week 7. Left panels: H&E staining of lung metastasis of Huh-1 cells.

871 Scale bars: 200 μm. Right panels: The average number of tumors/lung. Data represent mean ± SD

872 (n=10 mice/group). **: p<0.001; Student’s t-test.

873 Figure 6-figure supplement 1: The viability and number of HCC cells with p53 knockdown

874 cultured in serum-free medium for 36 h.

875 (A, B) The viability (A) and relative number (B) of HCC cells with p53 knockdown by shRNA

876 vectors cultured in serum-free medium for 36 h. Same number of cells with or without p53

877 knockdown were cultured in serum-free medium for 36 h before the the viability and number of

878 cells was measured in a Vi-CELL Cell Viability Analyzer (Beckman Coulter). The viability of cells

879 was analyzed by the trypan blue cell exclusion method.

880

881 Figure 7. GLS2 mediates p53’s function in negative regulation of the Rac1 activity.

882 (A, B) Ectopic expression of DN Rac1-T17N greatly abolished the promoting effects of p53

883 knockdown on migration (A) and invasion (B) of Huh-1 or HepG2 cells as measured by trans-well

884 assays. Data represent mean ± SD (n=6 in A, B). **: p<0.001; Student’s t-test. (C, D) GLS2

885 mediates p53’s function in negative regulation of Rac1 activity in Huh-1 and HepG2 cells. In C,

886 knockdown of p53 in cells with GLS2 knockdown did not further promote Rac1 activity. In D,

887 GLS2 overexpression largely abolished the promoting effect of p53 knockdown on the Rac1

888 activity. Left panels: represented results of Rac1 activity analysis in cells transduced with #1

889 shRNA vectors. Right panel: relative Rac1-GTP/total Rac1/Actin levels in cells transduced with 2

890 different shRNA vectors (#1 and #2). Data represent mean ± SD (n=3). *: p<0.01; **: p<0.001;

891 Student’s t-test. (E, F) p53 inhibits the interaction of Rac1 with Tiam1 and VAV1 through GLS2 in

892 Huh-1 and HepG2 cells. In E, knockdown of p53 in cells with GLS2 knockdown did not further

893 promote the interaction of Tiam1 and VAV1 with Rac1. The knockdown of p53 and GLS2 was

894 shown in Figure 7C. Two shRNA vectors against p53 and GLS2, respectively, were used, and very

895 similar results were observed. In F, GLS2 overexpression largely abolished the promoting effect of

896 p53 knockdown on the interaction of Tiam1 and VAV1 with Rac1. (G) Proposed model for the

897 negative regulation of Rac1 activity and cancer metastasis by GLS2 and p53.

898 Figure 7-figure supplement 1: Knockdown of endogenous Rac1 greatly abolished the effects of

899 p53 on migration and invasion of HCC cells

900 (A, B) Knockdown of endogenous Rac1 greatly abolished the promoting effects of p53 knockdown

901 on migration (A) and invasion (B) of Huh-1 and HepG2 cells. The endogenous Rac1 was knocked

902 down by 2 different shRNA vectors in Huh-1 and HepG2 cells with stable p53 knockdown, and the

903 abilities of migration (A) and invasion (B) of these cells were measured by trans-well assays. The

904 knockdown of Rac1 was shown in Figure 4-figure supplement 3A. Data are presented as mean ± SD

905 (n=4). **: p<0.001; Student’s t-test.

Figure 1

A Number of Potential interacting proteins for GLS2 peptides HSPA9, heat shock 70kDa protein 9 (mortalin) 84 HSPD1, heat shock 60kDa protein 1 (chaperonin) 46 MCM7, minichromosome maintenance complex component 7 43 HSP90AB1, heat shock protein 90kDa alpha (cytosolic) 30 LMNB1, lamin B1 27 C2orf47, chromosome 2 open reading frame 47 19 RAC1, ras-related C3 botulinum toxin substrate 1 15 CTPS, CTP synthase 10 DARS, aspartyl-tRNA synthetase 8 PCNA, proliferating cell nuclear antigen 5 C19orf70, chromosome 19 open reading frame 70 4 EARS2, glutamyl-tRNA synthetase 2, mitochondrial (putative) 4

B C IP IP IP IP Input IgG Myc Input IgG Flag Input IgG Myc Input IgG Flag GLS1-Flag - + + - + + + + + + GLS2-Flag - + + + - + + + + + Myc-Rac1 + + + + + - + + - + Myc-Rac1 + + + + + - + + + - Vector Vector Flag Flag (GLS1) 50 kD (GLS2) 50 kD IB: 25 kD IB: Myc 25 kD Myc (Rac1) (Rac1) 50 kD 50 kD Actin Actin

D E Amino Huh-1 HepG2 glutaminase Acids GLS2 N-terminuscore C-terminus IP: WT 1-602 IgG Rac1 IgG GLS2 IgG Rac1 IgG GLS2 25 kD Rac1 'N163 164-602 IB: GLS2 50 kD C139 464-602 GLS1 50 kD 'C139 1-463

IP: Flag F G _ Huh-1 HepG2 GLS2-Flag N163 150 150 ' C139 WT C139 ' ** ** ** ** Myc-Rac1 + + + + + 100 100 Myc IB: 25 kD (Rac1) 50 50 Flag 50 kD

(GLS2) 20 kD activity Relative glutaminase glutaminase Relative 0 0 Input Con WT C139 'C139 Con WT C139 'C139 Myc 25 kD (Rac1) 50 kD Actin Figure 2

A C IP: Input IgG Myc Huh-1 HepG2 Con vector GLS2-Flag vector _ Vector Myc-Rac1 _ 1.5 * * Con Con GLS2 * * T17N GLS2 WT G12V T17N WT G12V T17N -Flag -FlagkD Vector GLS2-Flag + + + + + + + + + 25 Rac1-GTP 1 Flag kD (GLS2) 50 Total Rac1 25 Myc 0.5 IB: 25 (Rac1) Flag (GLS2) 50 Actin 50 0

Actin Rac1/Actin Rac1-GTP/total Huh-1 HepG2 Hep3B Huh-7

B IP: Input IgG Myc D Con-shR Huh-1 HepG2 GLS2-shR #1 Myc-Rac1 - + - - + +++ * GLS2-shR #2 GLS2-Flag * * Vector + + ++ + +++ shRNA #1 6 # #1 #2 Con GLS2 #2 kD * ------Con GLS2 GLS2 Con GLS2 # * GTPȖS + 25 # GDP ------+ Rac1-GTP 4 25 Flag kD (GLS2) Total Rac1 50 2 Myc 25 GLS2 50 IB: (Rac1)

50 0 Rac1-GTP/total Rac1/Actin Rac1/Actin Rac1-GTP/total Actin Actin Huh-1 HepG2 Hep3B Huh-7

G E Huh-1 HepG2 IP: Input IgG Myc GLS2-C139 Vector -Flag + + + + + + + + + Con GLS2 Con GLS2

-Flag -Flag Vector Myc-Rac1 - - kD p-PAK1 WT G12V T17N T17N WT G12V T17N 50 kD Flag 20 Total PAK1 (GLS2-C139) 50 kD 25 IB: Myc (Rac1) Flag (GLS2) 50 kD Actin Actin 50 kD Huh-1 * H 1.5 * Huh-1 * GLS2-Flag vector F Huh-1 HepG2 1

N163 C139 0.5 shRNA Con ' ' kD #1 Con GLS2#1 #2 WT C139 Con GLS2 GLS2#2 Con GLS2 25 Rac1-GTP 0 50 kD p-PAK1 25 HepG2 Total-Rac1 * Total PAK1 50 kD 1.5 # 50 kD * Flag (GLS2)

Actin 50 1 Rac1-GTP/ total Rac1/Actin total Rac1-GTP/ 20 0.5

0 Actin 50

N163 Con WT ' C139 C139 ' Figure 3 A B D Protein C33 N29 Switch Myc-Rac1 - ' ' ' transduction WT * GLS2-Flag 8 domain GTPase + + + + + Vector kD * Switch I Con Tiam1-HA C-termini IP: Myc VAV1-HA25 kD Rac1-GTP 6 Switch II IB: Flag Rac1 Amino Acid (GLS2) 50 25 Total Rac1 4 WT 1-192 Myc 25 (Rac1) HA (Tiam1) 2 'C33 1-159 15 150 Flag 0 'N29 30-192 Input HA (VAV1) 100

(GLS2) Rac1/Actin Rac1-GTP/total 50 50 Con 'Switch 1-29,75-192 Actin 50 Actin Tiam1 VaV1 Vector

C33 N29 Switch ' ' C Myc-Rac1 - WT ' Tiam1-HA + + + + + E F Myc-Rac1-T17N + + +++ Myc-Rac1-T17N + + +++ IP: Myc kD Tiam1-HA - ++++ VAV1-HA - IB:HA (Tiam1) 150 ++++ GLS2-Flag -- GLS2-Flag -- Myc 25 HA(Tiam1) kD kD (Rac1) 150 HA (VAV1) 100 IP: Myc

Input 15 Flag (GLS2) IP: Myc Flag (GLS2) HA 50 (Tiam1) 50 150 25 Myc (Rac1) Myc (Rac1) 25

C33 N29 switch HA(Tiam1) Myc-Rac1 ' ' 150 HA (VAV1) - WT ' 100 VAV1-HA + + + + + Flag (GLS2) Flag (GLS2) IP: Myc kD Input 50 50 IB: HA (VAV1) 100 25 Input Myc (Rac1) Myc (Rac1) 25 Myc 50 50 25 Actin Actin (Rac1) 15 Input HA (VAV1) 100

Huh-1 HepG2 Huh-1 HepG2 H G IP: IgG Rac1 IgG Rac1 GLS2-Flag - - shRNA Con Con GLS2 GLS2 Con Con GLS2 GLS2 #1 #2 #1 #2 kD Tiam1 kD Tiam1 150 150 VAV1 100 IP: Rac1 VAV1 25 IP: Rac1 100 Rac1 25 Rac1 Flag (GLS2) 50 GLS2 50 Tiam1 150 Tiam1 150 VAV1 100 25 VAV1 100 Input Rac1 Input 25 Rac1 Flag (GLS2) 50 GLS2 Actin 50 50 Actin 50 Figure 4

Migration Invasion A B GLS2-Flag vector E GLS2-Flag vector Con vector Con vector Con vector Invasion Migration GLS1-Flag vector

Huh-1 400 Huh-1 400 ** **

300 300 ** Con vector Con vector 200 200 150 GLS2-Flag vector 200 ** GLS2-Flag vector ** ** 100 150 ** 100 ** 100 **

Number of invading cells/field cells/field invading of Number 0

100 ** /field cells migrating of Number 0 ** Huh-1 HepG2 Huh-1 HepG2 50 50

0 /field cells invading of Number 0 Number of migrating cells /field cells migrating of Number Huh-1 HepG2 Hep3B Huh-7 Huh-1 HepG2 Hep3B Huh-7 F Con-shR Migration Invasion GLS1-shR #1 GLS1-shR #2 200 ** C Migration ** 150 D Invasion ** ** Con-shR GLS2-shR #1 GLS2-shR #2 ** Con-shR GLS2-shR #1 GLS2-shR #2 150 ** ** 100 ** 100

Huh-1 50 Huh-1 50

Con-shR /field cells invading of Number Con-shR 0 0 500 ** GLS2-shR #1 ** /field cells migrating of Number ** 300 ** GLS2-shR #1 Huh-1 HepG2 Huh-1 HepG2 GLS2-shR #2 ** 400 ** ** GLS2-shR #2 ** ** ** 300 ** ** 200 ** ** ** 200 ** I Migration 100 Vector 100 Con WT

0 C139 'C139 Number of invading cells /field cells invading of Number Number of migrating cells /field cells migrating of Number 0 Huh-1 HepG2 Hep3B Huh-7 Huh-1 HepG2 Hep3B Huh-7

G Migration Huh-1 ** ** 500 ** ** Con-shR ** ** ** ** GLS2-shR #1 ** 400 ** ** GLS2-shR #2 200 ** 100 ** ** 300 ** ** 150 200 100 50

100 migrating cells /field cells migrating Number of Number 50

0 migrating cells /field cells migrating Vector Con Rac1 Con Rac1 Con Rac1 Con Rac1 of Number 0 -T17N -T17N -T17N -T17N 0

Huh-1 HepG2 Hep3B Huh-7 Con WT WT C139 C139 Con C139 Vector ' C139' Huh-1 HepG2

Invasion H Invasion Con-shR J ** ** ** GLS2-shR #1 ** 100 ** 100 ** ** GLS2-shR #2 300 ** ** ** ** ** 250 ** ** ** 50 200 ** 50

150 invading cells/field invading 100 of Number 0 0

invading cells /field cells invading Number of of Number 0 Con WT C139 Con WT C139 C139 ' C139' Vector Con Rac1 Con Rac1 Con Rac1 Con Rac1 Vector -T17N -T17N -T17N -T17N Huh-1 HepG2 Huh-1 HepG2 Hep3B Huh-7 Figure 5

A Huh-1 (Week 7) Con vector B Huh-1 (Week 7) 20 Vector GLS2-Flag vector ** Con vector 7 1.5 Con GLS2-Flag vector

x10 15 Con GLS2-Flag ** ** vector 1.0 1.0 10 * 0.8

0.6 5 0.5 GLS2-Flag 0.4 vector Number of tumors / lung

Relative photon flux 0 0.2 0 Huh-1 HepG2 Huh-1 HepG2

D Huh-1 (Week 7) C Huh-1 (Week 7) Con-shR

7 GLS2-shR #1 Con-shR GLS2-shR #1 Con-shR Con-shR GLS2-shR #1 GLS2-shR #2 ** GLS2-shR #1 x10 GLS2-shR #2 1.0 ** 40 ** GLS2-shR #2 12 ** ** ** ** 0.8 30 10 ** 0.6 8 20 0.4 6 4 0.2 GLS2 10 2 -shR #2 0 Number of tumors / lung Relative photon flux 0 Huh-1 HepG2 Huh-1 HepG2 E Huh-1 (Week 7) Vector F Vector Con Rac1-T17N Con Rac1-T17N 1.0 0.8 ** Con-shR 7 Con 0.6 ** GLS2-shR #1 Con-shR Con 40 ** GLS2-shR #2 0.4 x10 GLS2-shR #1 0.2 GLS2-shR #2 35 ** 30 ** ** ** 25 ** 10 ** ** ** ** 20

8 15

6 GLS2 #1 10 GLS2 #1

shRNA shRNA 5 shRNA shRNA 4 Number of tumors / lung 0 2 Vector Con Rac1 Con Rac1 Relative photon flux 0 -T17N -T17N Vector Con Rac1 Con Rac1

GLS2 #2 Huh-1 HepG2 -T17N -T17N GLS2 #2 Huh-1 HepG2

G H GSE6764 5 TCGA 8

6 0 4

-5 2

0 log2 median-centered intensity log2 median-centered intensity -2 No Yes Vascular No Yes Vascular invasion (n=110) (n=57) invasion (n=15) (n=18)

Fold change: 3.0285 Fold change: 4.6237 p=0.006660569 p=0.019804173 Figure 6

shRNA A Con-shR B C Con-shR Con p53 #1 p53 #2 p53-shR #1 Migration Invasion Con-shR p53-shR #2 p53-shR #1 p53 50 kD ** p53-shR #1 p53-shR #2 ** ** 500 ** 1.2 ** ** p53-shR #2 400 ** GLS2 50 kD **

Huh-1 400 50 kD 1.0 ** Actin ** 300 ** 0.8 300 ** 0.6 * 200 p53 50 kD 200 0.4 GLS2 100 100

50 kD 0.2

migrating cells /field cells migrating

Number of of Number

(GLS2/Actin)

Relative mRNA levels mRNA Relative invading cells /field cells invading 50 kD of Number HepG2 Actin 0 0 0 Huh-1 HepG2 Huh-1 HepG2 Huh-1 HepG2

D Migration Con-shR ** GLS2-shR #1 F Huh-1 (Week 7) 600 ** ** GLS2-shR #2 ** shRNA 500 ** ** 400 ** Con GLS2 #1 GLS2 #2 p53 #1 p53 #2 GLS2 #1 GLS2 #2 ** 300 +p53 #1 +p53 #2 1.0 200 0.8 7

100 0.6

migrating cells/field migrating Number of of Number x10 0 0.4 shRNA Con p53 #1 p53 #2 Con p53 #1 p53 #2 0.2 Huh-1 HepG2 ** ** ** ** ** ** E 18 ** ** Invasion Con-shR ** 14 16 ** GLS2-shR #1 ** 12 ** 500 GLS2-shR #2 14 ** 12 10 ** ** 400 ** ** 10 8 ** ** ** 8 6 300 6 4 4 200 2 2 Relative photon flux

100 0 0

invading cells/field invading Number of of Number 0 Con Con shRNA p53 #1 p53 #2 p53 #1 p53 #2 p53 #1 p53 #2 p53 #1 p53 #2 GLS2 #1 GLS2 #2 GLS2 #1 GLS2 #2 shRNA Con p53 #1 p53 #2 Con p53 #1 p53 #2 + GLS2 #1 + GLS2 #1 + GLS2 #2 + GLS2 #2 Huh-1 HepG2 Huh-1 HepG2

G Huh-1 (Week 7) ** shRNA ** ** ** ** ** ** Con GLS2 #1 70 ** 50 ** ** ** 60 ** 40 50 40 30 Con 30 20 20 10

shRNA 10

Number of tumors / lung 0 0 p53 #1 Con Con

shRNA p53 #1 p53 #2 p53 #1 p53 #2 p53 #1 p53 #2 p53 #1 p53 #2 GLS2 #1 GLS2 #2 GLS2 #1 GLS2 #2 + GLS2 #1 + GLS2 #1 + GLS2 #2 + GLS2 #2 Huh-1 HepG2 Figure 7

Con-shR Huh-1 HepG2 Con p53 A Migration p53-shR #1 C GLS2 GLS2+p53 ** GLS2 GLS2 p53-shR #2 + + * ** ** ** 7 ** * * ** shRNA (#1) Con GLS2 p53 p53 Con GLS2 p53 p53 * ** * 600 ** kD * * * ** 25 6 Rac1-GTP 500 ** 5 400 25 Total-Rac1 4 300 3 200 GLS2 2 50

100 1 migrating cells/field migrating Number of of Number p53 50

0 Rac1/Actin Rac1-GTP/total 0 Vector Con Rac1-T17N Con Rac1-T17N Actin 50 shRNA #1 #2 #1 #2

Huh-1 HepG2 Huh-1 HepG2 B Invasion Con-shR D Huh-1 HepG2 ** p53-shR #1 6 * * Con-shR ** p53-shR #2 Vector Con GLS2-Flag Con GLS2-Flag * * ** 5 * p53-shR #1 ** ** shRNA(#1) Con p53 Con p53 Con p53 Con p53 kD p53-shR #2 4 400 ** 25 ** Rac1-GTP 3 300 25 Total-Rac1 2 200 Flag (GLS2) 1

100 50 Rac1-GTP/total Rac1/Actin 0 p53 50 Vector Con GLS2 Con GLS2 0 Actin 50 -Flag -Flag Vector/field cells invading of Number Con Rac1-T17N Con Rac1-T17N Huh-1 HepG2 Huh-1 HepG2 E Huh-1 HepG2 F Huh-1 HepG2 GLS2 GLS2 Vector Con GLS2-Flag Con GLS2-Flag + + shRNA (#1) Con GLS2 p53 p53 Con GLS2 p53 p53 shRNA(#1) Con p53 Con p53 Con p53 Con p53

Tiam1 Tiam1 150 kD 150 kD IP:Rac1 IP:Rac1 VAV1 100 kD VAV1 100 kD 25 kD 25 kD Rac1 Rac1

Tiam1 150 kD Tiam1 200 kD VAV1 100 kD VAV1 100 kD Input Input 25 kD 25 kD Rac1 Rac1 50 kD 50 kD Actin Actin

G Tiam1/ VAV1 Tiam1/ VAV1

Rac1 GDP Rac1 GDP

Tiam1/ Rac1 GTP VAV1 p53 GLS2 GLS2 Migration / Invasion Rac1 GDP Rac1 GDP Metastasis