- Email: [email protected]
AAG8 promotes carcinogenesis by activating STAT3
CLS-08153; No of Pages 7 Cellular Signalling xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Cellular Signalling
1
AAG8 promotes carcinogenesis by activating STAT3
2Q1
Bing Sun a, Masahiro Kawahara b, Shogo Ehata c, Teruyuki Nagamune a,b,⁎
3 4 5
a
6
a r t i c l e
7 8 9 10 11
Article history: Received 4 February 2014 Received in revised form 2 April 2014 Accepted 2 April 2014 Available online xxxx
12 13 14 15
Keywords: AAG8 STAT3 Cancer
O
R O
i n f o
a b s t r a c t
P
Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is involved in ER (endoplasmic reticulum)-associated degradation and has been intensively elaborated in neuroscience. However, its rationale in carcinogenesis has rarely been noticed. In this study, we explored the intrinsic oncogenetic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and elucidated that it alternatively activated STAT3 in addition to IL6/JAK pathway. Based on these findings and a drug screening study, we demonstrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. Taken together, our findings shed light on the fundamental evidences for identification of AAG8 as an oncoprotein and potential target for cancer prevention, as well as highlight the importance of ER proteins in contributing to JAK/STAT signaling and carcinogenesis. © 2014 Published by Elsevier Inc.
T
E
c
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
D
b
F
journal homepage: www.elsevier.com/locate/cellsig
28
C
32 30 29 31
1. Introduction
34
Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is predominantly expressed at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and distributes dynamically. It modulates both MAM-specific and plasma membrane proteins and mitochondrial metabolism [1]. In particular, AAG8 is involved in ER-associated degradation [2] and has been elaborated in neuroscience [3]. Mutations of AAG8 cause neurodegenerative diseases such as amyotrophic lateral sclerosis [4]. However, its roles in cancer have just recently been noticed. We previously discovered that AAG8 antagonists potentially inhibit melanoma cell growth and proposed AAG8 as a promising target for melanoma therapy [5]. However, the lack of gainor loss-of-function studies has precluded a clear understanding of the rationale of AAG8 in carcinogenesis. The STAT (signal transducer and activator of transcription) family consists of seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. STATs are pivotal in modulating cellular functions in response to cytokines, interferons, and various growth factors,
41 42 43 44 45 46 47 48 49 50 51 52 53
R
R
39 40
N C O
37 38
U
35 36
E
33
⁎ Corresponding author at: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1138656, Japan. Tel.: +81 3 5841 7328; fax: +81 3 5841 8657. E-mail address: [email protected] (T. Nagamune).
16 17 18 19 20 21 22 23 24 25 26 27
which activate JAKs (Janus kinases), leading to key tyrosine phosphorylation on their receptors. JAKs activation allows the binding of STATs via their SH2 domains to these phosphotyrosine docking sites. STATs are in turn tyrosine phosphorylated, thus allowing their dimerization and activation. STATs have been shown to be controlled by several negative regulatory mechanisms. Notably, the SOCS (suppressor of cytokine signalling) family negatively regulates STAT activation [6]. STAT3 is a wellknown transcription factor that has been intensively investigated in cancer and immunity [7,8]. Upon phosphorylation at Y705, activated STAT3 translocates into the nucleus to initiate transcription. STAT3 hyperactivation is a feature of the majority of solid cancers. However, the regulation of STAT3 activation is not fully understood. In this study, we explored the intrinsic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and demonstrated that it alternatively activated STAT3 in addition to IL6/JAK pathway.
54
2. Materials and methods
71
2.1. Cell lines and reagents
72
DLD-1, HCT116, PANC1, AGS, MKN7, MSTO211H and B16 cell lines were obtained from American Type Culture Collection (USA). COLO205 cell line was purchased from RIKEN Cell Bank (Japan). Cell culture was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Carlsbad,
73 74
http://dx.doi.org/10.1016/j.cellsig.2014.04.001 0898-6568/© 2014 Published by Elsevier Inc.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
75 76 77
96
2.3. Establishing stable cell lines
97 98
For AAG8 overexpression, the SIGMAR1 gene was cloned from the cDNA of DLD-1 cells with the forward primer 5′-ACCCAAGCTGGCTA GAATGCAGTGGGCCGTG-3′ and reverse primer 5′-GTGGATCCGAGCTC GTCAAGGGTCCTGGCCAAAG-3′ and subcloned into pcDNA3.1 vector (Life Technologies) between NheI and KpnI sites. DLD-1 and AGS cells were transfected with either the empty vector or the plasmid expressing AAG8 using Lipofectamine LTX with Plus reagent (Life Technologies) according to the manufacturer's instructions. 48 h after transfection, cells were selected by 700 μg/ml G418 (WAKO, Wako, Japan). For gene knockdown, we employed the RNA polymerase II promoter U6 in pLKO.1 vector to express shRNA targeting the 5′-CCTCAACCCAGCAG CAATTTG-3′ sequence of SIGMAR1 gene. Lentivirus incorporated with shRNA was generated in HEK293T cells by combining packing plasmid pCMV-dR8.91, envelope plasmid VSV-G, (gifts from Dr. Kenneth Rock, University of Massachusetts Medical School, Worcester, MA) and the pLKO.1 plasmids. DLD-1 cells were infected with the lentivirus and selected by 3 μg/ml puromycin (SIGMA). HCT116 and AGS cells were transfected directly with the shRNA plasmids using Lipofectamine LTX with Plus reagent (Invitrogen) according to the manufacturer's protocol. 48 h after transfection, cells were selected by 1 μg/ml puromycin (SIGMA). A scramble shRNA plasmid (kindly provided by Dr. David Sabatini, Addgene plasmid 1864) was used as the control [10].
105 106 107 108 109 110 111 112 113 114 115 116 117 118
C
103 104
E
101 102
R
99 100
R
92 93
O
90 91
C
89
2.4. Transient API4 knockdown
120 121
126
DLD-1 cells were transfected with the pLKO.1 shRNAs targeting the 5′-CGTCCGGTTGCGCTTTCCTTT-3′ sequence (shAPI4-1) and the 5′CCGCATCTCTACATTCAAGAA-3′ sequence (shAPI4-2) of BIRC5 (API4) gene using Lipofectamine LTX with Plus reagent (Invitrogen) according to the manufacture's protocol. A scramble shRNA plasmid was used as the control. Seventy-two hours after transfection, cells were treated as indicated.
127
2.5. Growth assay and apoptosis assay
128
For the growth assay, dead cells were stained with trypan blue and the total cell number was evaluated with Countess™ (Life Technologies). For the apoptosis assay, cells were treated with chemicals as indicated in 3D Matrigel culture and then stained with 1 nM ethidium bromide (EtBr) for 5 min. The stained DNA were observed and photographed under a fluorescence microscope (OLYMPUS).
129 130 131 132 133
U
124 125
N
119
122 123
2.7. Western blot
141
Cells were lysed with Laemmli buffer, and each lysate sample was loaded into two adjacent lanes, if indicated, of a 10% polyacrylamide gel for minimizing loading differences. For PKM2 detection, cytoplasmic and nuclear fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, RL, USA) according to the manual's instructions. Proteins were separated at 30 mA and transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Membranes were blocked for 1 h at room temperature using 5% skim milk or 5% BSA (for phosphorylation detection) in TBS-Tween (TBS-T). Western blot analysis was performed according to the antibody manufacturer's specifications. The membranes were incubated with primary antibodies overnight in either 5% BSA or 5% skim milk in TBS-T at 4 °C. The membranes were washed thrice in TBS-T. The appropriate HRPconjugated secondary antibody was added into 5% skim milk in TBS-T, followed by three washes in TBS-T. The membranes were developed using a Luminata Crescendo Western HRP substrate (Millipore). Antibodies used in this work are as follows: pSTAT3 (#9145) antibodies were from Cell Signalling Technology (Danvers, MA, USA). STAT3 (sc-482) antibodies were from Santa Cruz Biotechnology. AAG8 (HPA018002) and GAPDH (G9295) antibodies were from SIGMA. API4 (NB500-201H) and LMNB (NBP1-19804) antibodies were from NOVUS (Littleton, CO, USA). PKM2 (ab150377) antibody was from abcam (Cambridge, MA, USA). The secondary HRPconjugated anti-rabbit IgG antibody (G21234) was from Life Technologies.
142 143
2.8. Statistical analysis
167
All statistical analyses were performed using Origin 8 Software. Error bars indicate standard errors of the mean (S.E.M.). Time courses or dose dependence was analyzed by two-tailed unpaired t-test or one-way ANOVA followed by appropriate post hoc test.
168
3. Results
172
3.1. Oncogenetic AAG8
173
We firstly observed that specific AAG8 antagonist BD1047 induced growth-suppressive phenotype of colorectal COLO205 cancer cells, pancreatic PANC1 cancer cells, and gastric AGS cancer cells in 3D Matrigel culture (Fig. 1A and Fig. S1A, B). Moreover, BD1047 potently suppressed mesothelioma MSTO211H cell growth in 3D culture; in contrast, AAG8 agonist PRE084 failed to suppress growth and tube formation, although a different phenotype was observed (Fig. S1C). In addition, BD1047 induced apoptosis of 3D-cultured COLO205 cells (Fig. S2A). BD1047 also dose-dependently suppressed the growth of colorectal COLO205 and DLD-1 cancer cells, as well as gastric MKN7 cancer cells (Fig. S2B–D). Gain- and loss-of-function approaches were employed for further confirmation. AAG8 overexpression promoted proliferation of both DLD-1 and gastric AGS cancer cells (Fig. 1B, left and Fig. S3). In line with this, AAG8 knockdown with short hairpin RNA (shRNA) delayed DLD-1 cell proliferation (Fig. 1B, right) and suppressed AGS cell growth in 3D culture (Fig. S4A, B), which closely mimicked the phenotype changes observed with AAG8 antagonist (Fig. S1A). Interestingly,
174
F
94 95
3D on-top culture of cancer cells was as previously described with some modifications [9]. Briefly, surface of 6-well plates was coated with pre-thawed Matrigel (500 μl/well) with a pipette tip. For each well, 3 × 105 or 106 cells were resuspended in 3 ml of complete Dulbecco's Modified Eagle's Medium containing 5% Matrigel and pipetted onto the pre-coated surface. Chemicals were added into the medium as indicated. Cells were then cultured for the indicated days before further assays. Cells were observed and photographed under a phase contrast microscope (OLYMPUS, Tokyo, Japan).
135 136
O
87 88
84
Animal experiments were performed in accordance with the policies of the Animal Ethics Committee of the University of Tokyo. Female BALB/c nu/nu mice (4 weeks of age) were purchased from the Sankyo Labo Service Corporation. Three million cells with Matrigel in 100 μl were injected into the flank of each mouse subcutaneously, and tumors were measured as described previously [11].
R O
2.2. 3D culture
82 83
134
P
86
80 81
2.6. Xenografts
T
85
CA, USA) in a standard incubator at 37 °C with 5% CO2. AAG8 antagonists BD1047 and BD1063, and AAG8 agonist PRE084 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Matrigel™ basement membrane matrix was from BD Bioscience (Bedford, MA, USA). Recombinant human IL-6 was from Genzyme-Techne (Minneapolis, MN, USA). Gemcitabine was from SIGMA (St Louis, MO, USA). JSI124, YM155, Ruxolitinib, JAK Inhibitor I, and JAK Inhibitor VI were included in the SCADS Inhibitor Kits.
D
78 79
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
E
2
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
137 138 139 140
144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
169 170 171
175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
A
3
R O
O
F
COLO205
E
C
T
E
D
P
B
U
N C O
R
R
C
Fig. 1. Oncogenetic effects of AAG8. (A) Phase contrast images showing COLO205 cells cultured in 3D Matrigel and treated with or without 100 μM BD1047 (AAG8 antagonist) for 48 h. (B) Immunoblot of AAG8 and proliferation assay of DLD-1 cells with stable overexpression (left) or AAG8 knockdown (right). Total lysates from control and stable knockdown cell lines were immunoblotted with AAG8 antibody; GAPDH served as the loading control. Cell number was counted every 3 days. Overexpression: initial cell number = 5 × 103. Knockdown: initial cell number = 5 × 104. n = 3. Error bars (s.e.m.) are indicated. *p b 0.05, ***p b 0.001 (two-tailed unpaired t-test). (C) Three million DLD-1 cells were injected subcutaneously into athymic nude mice. Representative pictures of mice are shown in left panel and quantitative measurements are shown in right. n = 7 (shCtrl), n = 8 (shAAG8). Error bars (s.e.m) are indicated. **p b 0.01 (one way repeated ANOVA followed by Tukey's test).
192 193 194 195
although AAG8 knockdown resulted in few to no alternations of the morphogenesis of colorectal HCT116 cancer cells in 3D culture, it increased their sensitivity to gemcitabine, a clinical cancer drug (Fig. S4C, D). In agreement with the data in vitro, AAG8 knockdown
slowed xenograft tumor formation of DLD-1 cells in vivo (Fig. 1C). These results collectively illustrate the tumor-promoting roles of AAG8, implying that AAG8 serves as an oncoprotein, and strongly indicate AAG8 as a potential target for tumor chemotherapy.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
196 197 198 199
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
201 202
We unanticipatedly discovered STAT3 inactivation in both PANC1 (Fig. 2A) and AGS cells treated with BD1047 in a dose-dependent manner (Fig. 2B), which might explain the underlying molecular mechanisms of AAG8 in promoting carcinogenesis. To confirm this, a timedependent assay revealed that STAT3 activity began to decrease 3 h after BD1047 treatment and was largely suppressed after 6 h in mouse melanoma B16 cells (Fig. 2C). We supposed that AAG8 may act as a STAT3 activator to enhance cancer cell proliferation. Supporting this hypothesis, we found that STAT3 Y705 phosphorylation level was increased by ectopic AAG8 expression in DLD-1 cells (Fig. 3A, left). Consistently, AAG8 knockdown decreased STAT3 activation in both DLD-1 cells (Fig. 3A, right), as well as in AGS cells (Fig. 3B). These findings indicate AAG8 as an upstream STAT3 activator.
208 209 210 211 212 213
3.3. Dual STAT3 activation by AAG8 and JAK signalling
215
We next performed a SCADS (screening committee of anticancer drugs) screening using DLD-1 cells stably expressing both AAG8 and a luciferase STAT3 reporter, such that STAT3 activity could be monitored after drug treatment. Among 364 chemicals with 232 targets, an API4 (apoptosis inhibitor 4) inhibitor YM155, was found to dramatically (fold change N 20) decrease STAT3 activity in these cells (data not shown). This is consistent with the published data that YM155 reduced STAT3 phosphorylation in PANC1 cells [12] and suggests that YM155 might block STAT3 activation either dependent on or independent of AAG8-related signalling. To further disentangle this event, DLD-1 cells with stable AAG8 knockdown were treated with or without YM155 for 12 h, followed by IL6 stimulation. As a result, IL6-induced robust STAT3 activation was largely abolished by YM155 treatment (Fig. 4A), confirming the STAT3 inhibitory effect of YM155 in SCADS screening and suggesting that YM155 disturbs the signalling activities, which are indispensable for IL6-induced STAT3 activation. In contrast, AAG8 knockdown contributes no change to IL6-induced STAT3 phosphorylation level (Fig. 4A), meaning that AAG8 activates STAT3 beyond IL6dependency. Substantiating this conjecture, specific AAG8 antagonists BD1047 or its analog BD1063 failed to decrease IL6-induced STAT3 activation in DLD-1 cells (Fig. 4B). Surprisingly, AAG8 knockdown further diminished the remaining pSTAT3 in YM155-treated cells (Fig. 4A). Based on the above findings, we hypothesized that AAG8 knockdown could also decrease STAT3 activity in cells with inhibition of IL6/JAK pathway. As expected, similar results to Fig. 4A were obtained with two JAK inhibitors JAK inhibitor I and Ruxolitinib (Fig. 4C). To analyze the temporal regulation of STAT3 by YM155 in AAG8knockdown cells, we decreased the time of YM155 treatment to 6 h or increased it to 18 h (Fig. 5A). YM155 treatment for 6 h hardly affected API4; however, AAG8 knockdown led to decreased STAT3 activity in
231 232 233 234 235 236 237 238 239 240 241 242 243 244
C
229 230
E
227 228
R
225 226
R
223 224
O
221 222
C
220
3.4. Combined inhibition of AAG8 and JAK signalling
282
We then supposed that combining YM155 and AAG8 knockdown could synergistically suppress STAT3 activation and cancer cell growth. Accordingly, we observed that YM155 treatment significantly limited DLD-1 cell growth in 3D culture, which was enhanced by AAG8 knockdown (Fig. 7A), suggesting the synergistic antitumor effects by combined inhibition of these two proteins. Similarly, AAG8 knockdown significantly slowed the proliferation of DLD-1 cells treated with the JAK inhibitor JSI-124 (Fig. 7B) and JAK3 Inhibitor VI (Fig. 7C).
283 284
N
218 219
U
216 217
T
214
R O
206 207
P
205
245 246
D
203 204
YM155-treated cells. In contrast, YM155 treatment for 18 h led to decreased API4 expression, dramatically lowered STAT3 activity, and even reduced total STAT3 level. Notably, AAG8 knockdown further abolished STAT3 activation and reduced API4 in YM155-treated cells. These data exclude the possibility that YM155 inhibits STAT3 activation dependent on AAG8-related signalings. These results together give rise to the conclusion that AAG8 alternatively activates STAT3 in addition to IL6/JAK pathway. API4 is an anti-apoptotic protein, and its transcription can be concurrently modulated by several transcription factors such as STAT3 [13], β-catenin, and YAP1 [14]. We could not establish the stable API4 knockdown DLD-1 cell line, perhaps due to its cytotoxicity, as reported elsewhere [15]. To investigate whether API4 is required for IL6-induced STAT3 activation, we transiently knocked down API4 in DLD-1 cells with two specific shRNAs (Fig. 5B). To our surprise, API4 depletion was dispensable for IL6-induced STAT3 phosphorylation in these cells (Fig. 5B). Although YM155 has been principally regarded as an API4 inhibitor, its specificity remains uncertain. Since YM155 dramatically decreased IL6-induced STAT3 activation while API4 knockdown did not, we supposed that YM155 might employ other inhibition mechanisms for this inactivation. There are at least two pieces of evidence supporting this argument. Firstly, in Fig. 5A, while YM155 treatment for 6 h did not decrease API4 expression, it had already cooperated with AAG8 knockdown to synergistically decrease STAT3 activation. Secondly, YM155 directly targets ILF3 (interleukin enhancer-binding factor 3), a transcription factor, to suppress API4 expression [16]. Conclusively, some proteins besides API4 could be suppressed by ILF3dependent YM155 treatment. In summary, YM155 appears to be a potent STAT3 inhibitor independent of its suppression on API4. Furthermore, although PKM2 (pyruvate kinase M2) was reported to translocate into the nucleus and function as a direct kinase for STAT3 phosphorylation at Y705 [17], we could not detect the changes in both expression level and cellular distribution of PKM2 upon AAG8 overexpression or knockdown (Fig. 6), suggesting that PKM2 might not be involved in AAG8-induced STAT3 activation. Taken together, our data identified AAG8 as an alternative STAT3 activator in addition to JAKs and PKM2 kinases.
F
3.2. Identification of AAG8 as a STAT3 activator
O
200
E
4
Fig. 2. STAT3 inactivation by AAG8 antagonism. (A) Immunoblot of pSTAT3 and STAT3 in PANC1 cells in 3D culture treated with 100 μM BD1047 for 48 h. (B) Immunoblot of pSTAT3 and STAT3 in AGS cells in 2D culture treated with BD1047 of indicated concentrations for 24 h. (C) Immunoblot of pSTAT3 and STAT3 in B16 cells in 2D culture treated with 100 μM BD1047 for different periods of time (0, 1, 3, 6, 9 and 12 h). Total lysates from control and treated cells were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281
285 286 287 288 289 290
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
5
Fig. 3. STAT3 activation by AAG8. (A) Immunoblot of pSTAT3 and STAT3 in DLD-1 cells with stable AAG8 overexpression (left panel) or stable AAG8 knockdown (right panel). Mean values of pSTAT3 versus STAT3 levels were labeled with control cells as standard. (B) Immunoblot of pSTAT3 and STAT3 in stable AAG8-knockdown AGS cells. Mean values of pSTAT3 versus STAT3 levels were labeled with control cells as standard. Total lysates from control and treated cells were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control.
T
E
D
A
E
C
B
R
299 300
R
297 298
C
_
_
shCtrl
shAAG8
N C O
295 296
IL6
Ruxolitinib
+
+
shCtrl
shAAG8
+ shCtrl
+ shAAG8
pSTAT3 STAT3
U
293 294
F
In this study, we show that AAG8, a chaperon protein, promotes carcinogenesis by activating STAT3. Inhibition of AAG8 by antagonists or shRNA efficiently suppressed cancer cell growth in vitro and tumor formation in vivo. Furthermore, by drug screening analysis, we pinpoint AAG8 as an alternative STAT3 activator in addition to IL6/JAK signalling. This study elucidates, for the first time, the critical roles of AAG8 in regulating JAK/STAT3 signalling pathway, although previous studies demonstrated that AAG8 associated with several ion channels and/or receptors to regulate cellular ion signalling [3,18].
O
292
The hallucinogen N,N-dimethyltryptamine (DMT) has been identified as an endogenous agonist of AAG8 [19]. Exogenously, a plethora of ligands of AAG8 have been synthesized [20,21]. Although an AAG8 antagonist has been assessed for pain treatment in phase I studies [22], few have been tested for their anti-cancer property. Our evaluation of the anti-tumor effects of AAG8 antagonists suggests the novel use of classical neurological drugs on cancer treatment. Promisingly, some synthesized AAG8 ligands have been reported to specifically bind to AAG8 in the nanomolar range [1]. Further efforts are needed to determine whether the anti-cancer ability of AAG8 antagonists could be translated in vivo.
R O
4. Discussion
P
291
IL6
_
_
shCtrl
shAAG8
JAK inhibitor I
+
+
+
+
shCtrl
shAAG8
shCtrl
shAAG8
pSTAT3 STAT3
Fig. 4. STAT3 activation by AAG8 beyond JAK signalling. (A) AAG8-knockdown DLD-1 cells were treated with or without 100 nM YM155 for 12 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates from control and stable knockdown cell lines were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control. (B) Immunoblot of pSTAT3 and STAT3 in DLD-1 cells treated with BD1047 or BD1063 for 12 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control. (C) Upper: AAG8-knockdown DLD-1 cells were treated with or without 1 μM Ruxolitinib for 20 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Juxtaposed lanes that were non-adjacent in the gel are indicated by a vertical black line. Lower: AAG8-knockdown DLD-1 cells were treated with or without 100 nM JAK inhibitor I for 24 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates from control and stable knockdown cells were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control. Mean values of pSTAT3 versus STAT3 levels in Ruxolitinib- or JAK inhibitor I-treated cells were labeled with shCtrl cells as the standard.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
301 302 303 304 305 306 307 308 309 310 311
6
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
B
R O
O
F
A
IL6
P
PBS
D
pSTAT3
E
STAT3
T
API4
R
R
O C N
314 315
At present, the exact mechanisms by which AAG8 activates STAT3 is uncertain. STAT3 phosphorylation of Y705 is concurrently and tightly controlled by multiple kinases and protein tyrosine phosphatases, which are a large and structurally diverse family of enzymes that
U
312 313
E
C
Fig. 5. STAT3 regulation by YM155 and API4 knockdown. (A) Temporal STAT3 regulation by YM155. Immunoblot of pSTAT3, STAT3 and API4 in AAG8-knockdown DLD-1 cells treated with or without YM155 for the indicated time, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates were immunoblotted with pSTAT3, STAT3 and API4 antibodies; GAPDH served as the loading control. (B) Effect of API4 knockdown on IL6-induced STAT3 activation. Immunoblots of pSTAT3, STAT3 and API4 in transient API4-knockdown DLD-1 cells treated with PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates were immunoblotted with pSTAT3, STAT3 and API4 antibodies; STAT3 served as the loading control.
Fig. 6. Dispensable effects of AAG8 on PKM2. (A) Immunoblot of PKM2 in DLD-1 cells with either AAG8 overexpression (left) or knockdown (right). Total lysates were immunoblotted with AAG8 antibody; GAPDH served as the loading control. (B) Immunoblot of PKM2 of total, cytoplasmic, and nuclear fractions in AAG8-knockdown HCT116 cells. Lysates of indicated fractions were immunoblotted with PKM2 antibody. LMNB served as the loading control for the nuclear fraction. GAPDH served as the loading control for total and cytoplasmic fractions.
catalyze the dephosphorylation. We demonstrated that STAT3 kinases are dispensable in AAG8-induced STAT3 activation. However, there is a possibility that dephosphorylation might account for AAG8-related regulation. For instance, PTPMeg2 is a physiologic STAT3 phosphatase that can directly dephosphorylate STAT3 at the Tyr705 residue [23]. This possibility merits future detailed evaluation. Despite great efforts focusing on STAT3 for cancer therapy, few efficient strategies have been developed [24]. Single usage of chemical drugs targeting upstream kinases of STAT3, such as JAK2 inhibitors, often results in drug insensitivity due to acquired resistance [25]. In the anticancer drug screening, we identified YM155 as a potent STAT3 suppressant, suggesting that YM155 blocks the signalling activities which are required for IL6-induced STAT3 activation. We further illustrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. As single use of both JAK inhibitors and API4 inhibitor YM155 has clinical limitations [25,26], our drug combination strategy provides a promising therapeutic approach for increasing the antitumor efficacy and decreasing drug resistance.
316 317
5. Conclusions
334
The present study characterized AAG8 as an oncoprotein in multiple types of cancers through investigating its cancer-promoting effects and the underlying mechanisms. We uncovered the molecular clues that AAG8 is an alternative upstream STAT3 activator in addition to IL6/JAK signalling pathway. Tandem inhibition of AAG8 and JAK signalling synergistically suppresses cancer cell growth. Taken together, our findings
335
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333
336 337 338 339 340
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
7
Acknowledgments
345
We thank Dr. Daizo Koinuma and Dr. Kohei Miyazono (The University of Tokyo) for their helpful suggestions and discussions; and Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan for the generous gift of The SCADS Inhibitor Kits I-IV. B. S. was supported by China Scholarship Council (CSC) and Center for Medical System Innovation (CMSI) of The University of Tokyo.
346 347
Appendix A. Supplementary data
355
348 349 350 351 352
F
353 354
O
Supplementary data to this article can be found online at http://dx. 356 doi.org/10.1016/j.cellsig.2014.04.001. 357
R O
References
N C O
R
R
E
C
T
E
D
P
[1] K.S.C. Marriott, M. Prasad, V. Thapliyal, H.S. Bose, J. Pharmacol. Exp. Ther. 343 (2012) 578–586. [2] T. Hayashi, E. Hayashi, M. Fujimoto, H. Sprong, T.P. Su, J. Biol. Chem. 287 (2012) 43156–43169. [3] S. Kourrich, T. Hayashi, J.Y. Chuang, S.Y. Tsai, T.P. Su, A. Bonci, Cell 152 (2013) 236–247. [4] A. Al-Saif, F. Al-Mohanna, S. Bohlega, Ann. Neurol. 70 (2011) 913–919. [5] B. Sun, M. Kawahara, T. Nagamune, Cancer Med. (2014). [6] X.X.O. Yang, H.Y. Zhang, B.S. Kim, X.Y. Niu, J. Peng, Y.H. Chen, R. Kerketta, Y.H. Lee, S. H. Chang, D.B. Corry, D.M. Wang, S.S. Watowich, C. Dong, Nat. Immunol. 14 (2013) (732 − +). [7] M. Hatziapostolou, C. Polytarchou, E. Aggelidou, A. Drakaki, G.A. Poultsides, S.A. Jaeger, H. Ogata, M. Karin, K. Struhl, M. Hadzopoulou-Cladaras, D. Iliopoulos, Cell 147 (2011) 1233–1247. [8] J.W. Shui, A. Larange, G. Kim, J.L. Vela, S. Zahner, H. Cheroutre, M. Kronenberg, Nature 488 (2012) (222 − +). [9] G.Y. Lee, P.A. Kenny, E.H. Lee, M.J. Bissell, Nat. Methods 4 (2007) 359–365. [10] D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Science 307 (2005) 1098–1101. [11] Y. Shirai, S. Ehata, M. Yashiro, K. Yanagihara, K. Hirakawa, K. Miyazono, Am. J. Pathol. 179 (2011) 2920–2930. [12] Y.S. Na, S.J. Yang, S.M. Kim, K.A. Jung, J.H. Moon, J.S. Shin, D.H. Yoon, Y.S. Hong, M.H. Ryu, J.L. Lee, J.S. Lee, T.W. Kim, PLoS ONE 7 (2012). [13] N. Sen, X.B. Che, J. Rajamani, L. Zerboni, P. Sung, J. Ptacek, A.M. Arvin, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 600–605. [14] J. Rosenbluh, D. Nijhawan, A.G. Cox, X.N. Li, J.T. Neal, E.J. Schafer, T.I. Zack, X.X. Wang, A. Tsherniak, A.C. Schinzel, D.D. Shao, S.E. Schumacher, B.A. Weir, F. Vazquez, G.S. Cowley, D.E. Root, J.P. Mesirov, R. Beroukhim, C.J. Kuo, W. Goessling, W.C. Hahn, Cell 151 (2012) 1457–1473. [15] A.V. Sarthy, S.E. Morgan-Lappe, D. Zakula, L. Vernetti, M. Schurdak, J.C.L. Packer, M.G. Anderson, S. Shirasawa, T. Sasazuki, S.W. Fesik, Mol. Cancer Ther. 6 (2007) 269–276. [16] N. Nakamura, T. Yamauchi, M. Hiramoto, M. Yuri, M. Naito, M. Takeuchi, K. Yamanaka, A. Kita, T. Nakahara, I. Kinoyama, A. Matsuhisa, N. Kaneko, H. Koutoku, M. Sasamata, H. Yokota, S. Kawabata, K. Furuichi, Mol. Cell. Proteomics 11 (2012). [17] X.L. Gao, H.Z. Wang, J.J. Yang, X.W. Liu, Z.R. Liu, Mol. Cell 45 (2012) 598–609. [18] D. Balasuriya, A.P. Stewart, D. Crottes, F. Borgese, O. Soriani, J.M. Edwardson, J. Biol. Chem. 287 (2012) 37021–37029. [19] D. Fontanilla, M. Johannessen, A.R. Hajipour, N.V. Cozzi, M.B. Jackson, A.E. Ruoho, Science 323 (2009) 934–937. [20] L.X. Wang, J.A. Eldred, P. Sidaway, J. Sanderson, A.J.O. Smith, R.P. Bowater, J.R. Reddan, I.M. Wormstone, Mech. Ageing Dev. 133 (2012) 665–674. [21] A. Hyrskyluoto, I. Pulli, K. Tornqvist, T.H. Ho, L. Korhonen, D. Lindholm, Cell Death Dis. 4 (2013). [22] M. Abadias, M. Escriche, A. Vaque, M. Sust, G. Encina, Br. J. Clin. Pharmacol. 75 (2013) 103–117. [23] R. Li, Z.L. Hu, S.Y. Sun, Z.G. Chen, T.K. Owonikoko, G.L. Sica, S.S. Ramalingam, W.J. Curran, F.R. Khuri, X.M. Deng, Mol. Cancer Ther. 12 (2013) 2200–2212. [24] M. Sen, S.M. Thomas, S. Kim, J.I. Yeh, R.L. Ferris, J.T. Johnson, U. Duvvuri, J. Lee, N. Sahu, S. Joyce, M.L. Freilino, H.B. Shi, C.Y. Li, D. Ly, S. Rapireddy, J.P. Etter, P.K. Li, L. Wang, S. Chiosea, R.R. Seethala, W.E. Gooding, X.M. Chen, N. Kaminski, K. Pandit, D.E. Johnson, J.R. Grandis, Cancer Discov. 2 (2012) 694–705. [25] P. Koppikar, N. Bhagwat, O. Kilpivaara, T. Manshouri, M. Adli, T. Hricik, F. Liu, L.M. Saunders, A. Mullally, O. Abdel-Wahab, L. Leung, A. Weinstein, S. Marubayashi, A. Goel, M. Gonen, Z. Estrov, B.L. Ebert, G. Chiosis, S.D. Nimer, B.E. Bernstein, S. Verstovsek, R.L. Levine, Nature 489 (2012) 155-U222. [26] R.J. Kelly, A. Thomas, A. Rajan, G. Chun, A. Lopez-Chavez, E. Szabo, S. Spencer, C.A. Carter, U. Guha, S. Khozin, S. Poondru, C. Van Sant, A. Keating, S.M. Steinberg, W. Figg, G. Giaccone, Ann. Oncol. 24 (2013) 2601–2606.
U
Fig. 7. Tandem AAG8–JAK inhibition. (A) Phase contrast images showing acinar morphology of the indicated DLD-1 cells in 3D culture on day 5 after treatment with or without 100 nM YM155. (B) Growth assay of AAG8-knockdown DLD-1 cells treated with 1 μM JSI-124 (JAK inhibitor) for 24 h. Initial cell number = 6 × 105. n = 3. Error bars (s.e.m.) are indicated. ***p b 0.001 (one way ANOVA followed by Tukey's test). (C) Phase contrast images showing acinar morphology of the indicated DLD-1 cells in 3D culture on day 10 after treatment with or without 100 nM JAK3 inhibitor VI.
341 342 343 344
shed light on the fundamental evidences for identification of AAG8 as a potential target for cancer prevention and highlight the importance of ER chaperon proteins in contributing to JAK/STAT signalling and carcinogenesis.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
358 359 360 361 362 363 364 365 366 Q3 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414
Contents lists available at ScienceDirect
Cellular Signalling
1
AAG8 promotes carcinogenesis by activating STAT3
2Q1
Bing Sun a, Masahiro Kawahara b, Shogo Ehata c, Teruyuki Nagamune a,b,⁎
3 4 5
a
6
a r t i c l e
7 8 9 10 11
Article history: Received 4 February 2014 Received in revised form 2 April 2014 Accepted 2 April 2014 Available online xxxx
12 13 14 15
Keywords: AAG8 STAT3 Cancer
O
R O
i n f o
a b s t r a c t
P
Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is involved in ER (endoplasmic reticulum)-associated degradation and has been intensively elaborated in neuroscience. However, its rationale in carcinogenesis has rarely been noticed. In this study, we explored the intrinsic oncogenetic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and elucidated that it alternatively activated STAT3 in addition to IL6/JAK pathway. Based on these findings and a drug screening study, we demonstrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. Taken together, our findings shed light on the fundamental evidences for identification of AAG8 as an oncoprotein and potential target for cancer prevention, as well as highlight the importance of ER proteins in contributing to JAK/STAT signaling and carcinogenesis. © 2014 Published by Elsevier Inc.
T
E
c
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
D
b
F
journal homepage: www.elsevier.com/locate/cellsig
28
C
32 30 29 31
1. Introduction
34
Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is predominantly expressed at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and distributes dynamically. It modulates both MAM-specific and plasma membrane proteins and mitochondrial metabolism [1]. In particular, AAG8 is involved in ER-associated degradation [2] and has been elaborated in neuroscience [3]. Mutations of AAG8 cause neurodegenerative diseases such as amyotrophic lateral sclerosis [4]. However, its roles in cancer have just recently been noticed. We previously discovered that AAG8 antagonists potentially inhibit melanoma cell growth and proposed AAG8 as a promising target for melanoma therapy [5]. However, the lack of gainor loss-of-function studies has precluded a clear understanding of the rationale of AAG8 in carcinogenesis. The STAT (signal transducer and activator of transcription) family consists of seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. STATs are pivotal in modulating cellular functions in response to cytokines, interferons, and various growth factors,
41 42 43 44 45 46 47 48 49 50 51 52 53
R
R
39 40
N C O
37 38
U
35 36
E
33
⁎ Corresponding author at: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1138656, Japan. Tel.: +81 3 5841 7328; fax: +81 3 5841 8657. E-mail address: [email protected] (T. Nagamune).
16 17 18 19 20 21 22 23 24 25 26 27
which activate JAKs (Janus kinases), leading to key tyrosine phosphorylation on their receptors. JAKs activation allows the binding of STATs via their SH2 domains to these phosphotyrosine docking sites. STATs are in turn tyrosine phosphorylated, thus allowing their dimerization and activation. STATs have been shown to be controlled by several negative regulatory mechanisms. Notably, the SOCS (suppressor of cytokine signalling) family negatively regulates STAT activation [6]. STAT3 is a wellknown transcription factor that has been intensively investigated in cancer and immunity [7,8]. Upon phosphorylation at Y705, activated STAT3 translocates into the nucleus to initiate transcription. STAT3 hyperactivation is a feature of the majority of solid cancers. However, the regulation of STAT3 activation is not fully understood. In this study, we explored the intrinsic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and demonstrated that it alternatively activated STAT3 in addition to IL6/JAK pathway.
54
2. Materials and methods
71
2.1. Cell lines and reagents
72
DLD-1, HCT116, PANC1, AGS, MKN7, MSTO211H and B16 cell lines were obtained from American Type Culture Collection (USA). COLO205 cell line was purchased from RIKEN Cell Bank (Japan). Cell culture was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Carlsbad,
73 74
http://dx.doi.org/10.1016/j.cellsig.2014.04.001 0898-6568/© 2014 Published by Elsevier Inc.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
75 76 77
96
2.3. Establishing stable cell lines
97 98
For AAG8 overexpression, the SIGMAR1 gene was cloned from the cDNA of DLD-1 cells with the forward primer 5′-ACCCAAGCTGGCTA GAATGCAGTGGGCCGTG-3′ and reverse primer 5′-GTGGATCCGAGCTC GTCAAGGGTCCTGGCCAAAG-3′ and subcloned into pcDNA3.1 vector (Life Technologies) between NheI and KpnI sites. DLD-1 and AGS cells were transfected with either the empty vector or the plasmid expressing AAG8 using Lipofectamine LTX with Plus reagent (Life Technologies) according to the manufacturer's instructions. 48 h after transfection, cells were selected by 700 μg/ml G418 (WAKO, Wako, Japan). For gene knockdown, we employed the RNA polymerase II promoter U6 in pLKO.1 vector to express shRNA targeting the 5′-CCTCAACCCAGCAG CAATTTG-3′ sequence of SIGMAR1 gene. Lentivirus incorporated with shRNA was generated in HEK293T cells by combining packing plasmid pCMV-dR8.91, envelope plasmid VSV-G, (gifts from Dr. Kenneth Rock, University of Massachusetts Medical School, Worcester, MA) and the pLKO.1 plasmids. DLD-1 cells were infected with the lentivirus and selected by 3 μg/ml puromycin (SIGMA). HCT116 and AGS cells were transfected directly with the shRNA plasmids using Lipofectamine LTX with Plus reagent (Invitrogen) according to the manufacturer's protocol. 48 h after transfection, cells were selected by 1 μg/ml puromycin (SIGMA). A scramble shRNA plasmid (kindly provided by Dr. David Sabatini, Addgene plasmid 1864) was used as the control [10].
105 106 107 108 109 110 111 112 113 114 115 116 117 118
C
103 104
E
101 102
R
99 100
R
92 93
O
90 91
C
89
2.4. Transient API4 knockdown
120 121
126
DLD-1 cells were transfected with the pLKO.1 shRNAs targeting the 5′-CGTCCGGTTGCGCTTTCCTTT-3′ sequence (shAPI4-1) and the 5′CCGCATCTCTACATTCAAGAA-3′ sequence (shAPI4-2) of BIRC5 (API4) gene using Lipofectamine LTX with Plus reagent (Invitrogen) according to the manufacture's protocol. A scramble shRNA plasmid was used as the control. Seventy-two hours after transfection, cells were treated as indicated.
127
2.5. Growth assay and apoptosis assay
128
For the growth assay, dead cells were stained with trypan blue and the total cell number was evaluated with Countess™ (Life Technologies). For the apoptosis assay, cells were treated with chemicals as indicated in 3D Matrigel culture and then stained with 1 nM ethidium bromide (EtBr) for 5 min. The stained DNA were observed and photographed under a fluorescence microscope (OLYMPUS).
129 130 131 132 133
U
124 125
N
119
122 123
2.7. Western blot
141
Cells were lysed with Laemmli buffer, and each lysate sample was loaded into two adjacent lanes, if indicated, of a 10% polyacrylamide gel for minimizing loading differences. For PKM2 detection, cytoplasmic and nuclear fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, RL, USA) according to the manual's instructions. Proteins were separated at 30 mA and transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Membranes were blocked for 1 h at room temperature using 5% skim milk or 5% BSA (for phosphorylation detection) in TBS-Tween (TBS-T). Western blot analysis was performed according to the antibody manufacturer's specifications. The membranes were incubated with primary antibodies overnight in either 5% BSA or 5% skim milk in TBS-T at 4 °C. The membranes were washed thrice in TBS-T. The appropriate HRPconjugated secondary antibody was added into 5% skim milk in TBS-T, followed by three washes in TBS-T. The membranes were developed using a Luminata Crescendo Western HRP substrate (Millipore). Antibodies used in this work are as follows: pSTAT3 (#9145) antibodies were from Cell Signalling Technology (Danvers, MA, USA). STAT3 (sc-482) antibodies were from Santa Cruz Biotechnology. AAG8 (HPA018002) and GAPDH (G9295) antibodies were from SIGMA. API4 (NB500-201H) and LMNB (NBP1-19804) antibodies were from NOVUS (Littleton, CO, USA). PKM2 (ab150377) antibody was from abcam (Cambridge, MA, USA). The secondary HRPconjugated anti-rabbit IgG antibody (G21234) was from Life Technologies.
142 143
2.8. Statistical analysis
167
All statistical analyses were performed using Origin 8 Software. Error bars indicate standard errors of the mean (S.E.M.). Time courses or dose dependence was analyzed by two-tailed unpaired t-test or one-way ANOVA followed by appropriate post hoc test.
168
3. Results
172
3.1. Oncogenetic AAG8
173
We firstly observed that specific AAG8 antagonist BD1047 induced growth-suppressive phenotype of colorectal COLO205 cancer cells, pancreatic PANC1 cancer cells, and gastric AGS cancer cells in 3D Matrigel culture (Fig. 1A and Fig. S1A, B). Moreover, BD1047 potently suppressed mesothelioma MSTO211H cell growth in 3D culture; in contrast, AAG8 agonist PRE084 failed to suppress growth and tube formation, although a different phenotype was observed (Fig. S1C). In addition, BD1047 induced apoptosis of 3D-cultured COLO205 cells (Fig. S2A). BD1047 also dose-dependently suppressed the growth of colorectal COLO205 and DLD-1 cancer cells, as well as gastric MKN7 cancer cells (Fig. S2B–D). Gain- and loss-of-function approaches were employed for further confirmation. AAG8 overexpression promoted proliferation of both DLD-1 and gastric AGS cancer cells (Fig. 1B, left and Fig. S3). In line with this, AAG8 knockdown with short hairpin RNA (shRNA) delayed DLD-1 cell proliferation (Fig. 1B, right) and suppressed AGS cell growth in 3D culture (Fig. S4A, B), which closely mimicked the phenotype changes observed with AAG8 antagonist (Fig. S1A). Interestingly,
174
F
94 95
3D on-top culture of cancer cells was as previously described with some modifications [9]. Briefly, surface of 6-well plates was coated with pre-thawed Matrigel (500 μl/well) with a pipette tip. For each well, 3 × 105 or 106 cells were resuspended in 3 ml of complete Dulbecco's Modified Eagle's Medium containing 5% Matrigel and pipetted onto the pre-coated surface. Chemicals were added into the medium as indicated. Cells were then cultured for the indicated days before further assays. Cells were observed and photographed under a phase contrast microscope (OLYMPUS, Tokyo, Japan).
135 136
O
87 88
84
Animal experiments were performed in accordance with the policies of the Animal Ethics Committee of the University of Tokyo. Female BALB/c nu/nu mice (4 weeks of age) were purchased from the Sankyo Labo Service Corporation. Three million cells with Matrigel in 100 μl were injected into the flank of each mouse subcutaneously, and tumors were measured as described previously [11].
R O
2.2. 3D culture
82 83
134
P
86
80 81
2.6. Xenografts
T
85
CA, USA) in a standard incubator at 37 °C with 5% CO2. AAG8 antagonists BD1047 and BD1063, and AAG8 agonist PRE084 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Matrigel™ basement membrane matrix was from BD Bioscience (Bedford, MA, USA). Recombinant human IL-6 was from Genzyme-Techne (Minneapolis, MN, USA). Gemcitabine was from SIGMA (St Louis, MO, USA). JSI124, YM155, Ruxolitinib, JAK Inhibitor I, and JAK Inhibitor VI were included in the SCADS Inhibitor Kits.
D
78 79
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
E
2
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
137 138 139 140
144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
169 170 171
175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
A
3
R O
O
F
COLO205
E
C
T
E
D
P
B
U
N C O
R
R
C
Fig. 1. Oncogenetic effects of AAG8. (A) Phase contrast images showing COLO205 cells cultured in 3D Matrigel and treated with or without 100 μM BD1047 (AAG8 antagonist) for 48 h. (B) Immunoblot of AAG8 and proliferation assay of DLD-1 cells with stable overexpression (left) or AAG8 knockdown (right). Total lysates from control and stable knockdown cell lines were immunoblotted with AAG8 antibody; GAPDH served as the loading control. Cell number was counted every 3 days. Overexpression: initial cell number = 5 × 103. Knockdown: initial cell number = 5 × 104. n = 3. Error bars (s.e.m.) are indicated. *p b 0.05, ***p b 0.001 (two-tailed unpaired t-test). (C) Three million DLD-1 cells were injected subcutaneously into athymic nude mice. Representative pictures of mice are shown in left panel and quantitative measurements are shown in right. n = 7 (shCtrl), n = 8 (shAAG8). Error bars (s.e.m) are indicated. **p b 0.01 (one way repeated ANOVA followed by Tukey's test).
192 193 194 195
although AAG8 knockdown resulted in few to no alternations of the morphogenesis of colorectal HCT116 cancer cells in 3D culture, it increased their sensitivity to gemcitabine, a clinical cancer drug (Fig. S4C, D). In agreement with the data in vitro, AAG8 knockdown
slowed xenograft tumor formation of DLD-1 cells in vivo (Fig. 1C). These results collectively illustrate the tumor-promoting roles of AAG8, implying that AAG8 serves as an oncoprotein, and strongly indicate AAG8 as a potential target for tumor chemotherapy.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
196 197 198 199
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
201 202
We unanticipatedly discovered STAT3 inactivation in both PANC1 (Fig. 2A) and AGS cells treated with BD1047 in a dose-dependent manner (Fig. 2B), which might explain the underlying molecular mechanisms of AAG8 in promoting carcinogenesis. To confirm this, a timedependent assay revealed that STAT3 activity began to decrease 3 h after BD1047 treatment and was largely suppressed after 6 h in mouse melanoma B16 cells (Fig. 2C). We supposed that AAG8 may act as a STAT3 activator to enhance cancer cell proliferation. Supporting this hypothesis, we found that STAT3 Y705 phosphorylation level was increased by ectopic AAG8 expression in DLD-1 cells (Fig. 3A, left). Consistently, AAG8 knockdown decreased STAT3 activation in both DLD-1 cells (Fig. 3A, right), as well as in AGS cells (Fig. 3B). These findings indicate AAG8 as an upstream STAT3 activator.
208 209 210 211 212 213
3.3. Dual STAT3 activation by AAG8 and JAK signalling
215
We next performed a SCADS (screening committee of anticancer drugs) screening using DLD-1 cells stably expressing both AAG8 and a luciferase STAT3 reporter, such that STAT3 activity could be monitored after drug treatment. Among 364 chemicals with 232 targets, an API4 (apoptosis inhibitor 4) inhibitor YM155, was found to dramatically (fold change N 20) decrease STAT3 activity in these cells (data not shown). This is consistent with the published data that YM155 reduced STAT3 phosphorylation in PANC1 cells [12] and suggests that YM155 might block STAT3 activation either dependent on or independent of AAG8-related signalling. To further disentangle this event, DLD-1 cells with stable AAG8 knockdown were treated with or without YM155 for 12 h, followed by IL6 stimulation. As a result, IL6-induced robust STAT3 activation was largely abolished by YM155 treatment (Fig. 4A), confirming the STAT3 inhibitory effect of YM155 in SCADS screening and suggesting that YM155 disturbs the signalling activities, which are indispensable for IL6-induced STAT3 activation. In contrast, AAG8 knockdown contributes no change to IL6-induced STAT3 phosphorylation level (Fig. 4A), meaning that AAG8 activates STAT3 beyond IL6dependency. Substantiating this conjecture, specific AAG8 antagonists BD1047 or its analog BD1063 failed to decrease IL6-induced STAT3 activation in DLD-1 cells (Fig. 4B). Surprisingly, AAG8 knockdown further diminished the remaining pSTAT3 in YM155-treated cells (Fig. 4A). Based on the above findings, we hypothesized that AAG8 knockdown could also decrease STAT3 activity in cells with inhibition of IL6/JAK pathway. As expected, similar results to Fig. 4A were obtained with two JAK inhibitors JAK inhibitor I and Ruxolitinib (Fig. 4C). To analyze the temporal regulation of STAT3 by YM155 in AAG8knockdown cells, we decreased the time of YM155 treatment to 6 h or increased it to 18 h (Fig. 5A). YM155 treatment for 6 h hardly affected API4; however, AAG8 knockdown led to decreased STAT3 activity in
231 232 233 234 235 236 237 238 239 240 241 242 243 244
C
229 230
E
227 228
R
225 226
R
223 224
O
221 222
C
220
3.4. Combined inhibition of AAG8 and JAK signalling
282
We then supposed that combining YM155 and AAG8 knockdown could synergistically suppress STAT3 activation and cancer cell growth. Accordingly, we observed that YM155 treatment significantly limited DLD-1 cell growth in 3D culture, which was enhanced by AAG8 knockdown (Fig. 7A), suggesting the synergistic antitumor effects by combined inhibition of these two proteins. Similarly, AAG8 knockdown significantly slowed the proliferation of DLD-1 cells treated with the JAK inhibitor JSI-124 (Fig. 7B) and JAK3 Inhibitor VI (Fig. 7C).
283 284
N
218 219
U
216 217
T
214
R O
206 207
P
205
245 246
D
203 204
YM155-treated cells. In contrast, YM155 treatment for 18 h led to decreased API4 expression, dramatically lowered STAT3 activity, and even reduced total STAT3 level. Notably, AAG8 knockdown further abolished STAT3 activation and reduced API4 in YM155-treated cells. These data exclude the possibility that YM155 inhibits STAT3 activation dependent on AAG8-related signalings. These results together give rise to the conclusion that AAG8 alternatively activates STAT3 in addition to IL6/JAK pathway. API4 is an anti-apoptotic protein, and its transcription can be concurrently modulated by several transcription factors such as STAT3 [13], β-catenin, and YAP1 [14]. We could not establish the stable API4 knockdown DLD-1 cell line, perhaps due to its cytotoxicity, as reported elsewhere [15]. To investigate whether API4 is required for IL6-induced STAT3 activation, we transiently knocked down API4 in DLD-1 cells with two specific shRNAs (Fig. 5B). To our surprise, API4 depletion was dispensable for IL6-induced STAT3 phosphorylation in these cells (Fig. 5B). Although YM155 has been principally regarded as an API4 inhibitor, its specificity remains uncertain. Since YM155 dramatically decreased IL6-induced STAT3 activation while API4 knockdown did not, we supposed that YM155 might employ other inhibition mechanisms for this inactivation. There are at least two pieces of evidence supporting this argument. Firstly, in Fig. 5A, while YM155 treatment for 6 h did not decrease API4 expression, it had already cooperated with AAG8 knockdown to synergistically decrease STAT3 activation. Secondly, YM155 directly targets ILF3 (interleukin enhancer-binding factor 3), a transcription factor, to suppress API4 expression [16]. Conclusively, some proteins besides API4 could be suppressed by ILF3dependent YM155 treatment. In summary, YM155 appears to be a potent STAT3 inhibitor independent of its suppression on API4. Furthermore, although PKM2 (pyruvate kinase M2) was reported to translocate into the nucleus and function as a direct kinase for STAT3 phosphorylation at Y705 [17], we could not detect the changes in both expression level and cellular distribution of PKM2 upon AAG8 overexpression or knockdown (Fig. 6), suggesting that PKM2 might not be involved in AAG8-induced STAT3 activation. Taken together, our data identified AAG8 as an alternative STAT3 activator in addition to JAKs and PKM2 kinases.
F
3.2. Identification of AAG8 as a STAT3 activator
O
200
E
4
Fig. 2. STAT3 inactivation by AAG8 antagonism. (A) Immunoblot of pSTAT3 and STAT3 in PANC1 cells in 3D culture treated with 100 μM BD1047 for 48 h. (B) Immunoblot of pSTAT3 and STAT3 in AGS cells in 2D culture treated with BD1047 of indicated concentrations for 24 h. (C) Immunoblot of pSTAT3 and STAT3 in B16 cells in 2D culture treated with 100 μM BD1047 for different periods of time (0, 1, 3, 6, 9 and 12 h). Total lysates from control and treated cells were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281
285 286 287 288 289 290
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
5
Fig. 3. STAT3 activation by AAG8. (A) Immunoblot of pSTAT3 and STAT3 in DLD-1 cells with stable AAG8 overexpression (left panel) or stable AAG8 knockdown (right panel). Mean values of pSTAT3 versus STAT3 levels were labeled with control cells as standard. (B) Immunoblot of pSTAT3 and STAT3 in stable AAG8-knockdown AGS cells. Mean values of pSTAT3 versus STAT3 levels were labeled with control cells as standard. Total lysates from control and treated cells were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control.
T
E
D
A
E
C
B
R
299 300
R
297 298
C
_
_
shCtrl
shAAG8
N C O
295 296
IL6
Ruxolitinib
+
+
shCtrl
shAAG8
+ shCtrl
+ shAAG8
pSTAT3 STAT3
U
293 294
F
In this study, we show that AAG8, a chaperon protein, promotes carcinogenesis by activating STAT3. Inhibition of AAG8 by antagonists or shRNA efficiently suppressed cancer cell growth in vitro and tumor formation in vivo. Furthermore, by drug screening analysis, we pinpoint AAG8 as an alternative STAT3 activator in addition to IL6/JAK signalling. This study elucidates, for the first time, the critical roles of AAG8 in regulating JAK/STAT3 signalling pathway, although previous studies demonstrated that AAG8 associated with several ion channels and/or receptors to regulate cellular ion signalling [3,18].
O
292
The hallucinogen N,N-dimethyltryptamine (DMT) has been identified as an endogenous agonist of AAG8 [19]. Exogenously, a plethora of ligands of AAG8 have been synthesized [20,21]. Although an AAG8 antagonist has been assessed for pain treatment in phase I studies [22], few have been tested for their anti-cancer property. Our evaluation of the anti-tumor effects of AAG8 antagonists suggests the novel use of classical neurological drugs on cancer treatment. Promisingly, some synthesized AAG8 ligands have been reported to specifically bind to AAG8 in the nanomolar range [1]. Further efforts are needed to determine whether the anti-cancer ability of AAG8 antagonists could be translated in vivo.
R O
4. Discussion
P
291
IL6
_
_
shCtrl
shAAG8
JAK inhibitor I
+
+
+
+
shCtrl
shAAG8
shCtrl
shAAG8
pSTAT3 STAT3
Fig. 4. STAT3 activation by AAG8 beyond JAK signalling. (A) AAG8-knockdown DLD-1 cells were treated with or without 100 nM YM155 for 12 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates from control and stable knockdown cell lines were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control. (B) Immunoblot of pSTAT3 and STAT3 in DLD-1 cells treated with BD1047 or BD1063 for 12 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control. (C) Upper: AAG8-knockdown DLD-1 cells were treated with or without 1 μM Ruxolitinib for 20 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Juxtaposed lanes that were non-adjacent in the gel are indicated by a vertical black line. Lower: AAG8-knockdown DLD-1 cells were treated with or without 100 nM JAK inhibitor I for 24 h, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates from control and stable knockdown cells were immunoblotted with pSTAT3 antibody; STAT3 served as the loading control. Mean values of pSTAT3 versus STAT3 levels in Ruxolitinib- or JAK inhibitor I-treated cells were labeled with shCtrl cells as the standard.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
301 302 303 304 305 306 307 308 309 310 311
6
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
B
R O
O
F
A
IL6
P
PBS
D
pSTAT3
E
STAT3
T
API4
R
R
O C N
314 315
At present, the exact mechanisms by which AAG8 activates STAT3 is uncertain. STAT3 phosphorylation of Y705 is concurrently and tightly controlled by multiple kinases and protein tyrosine phosphatases, which are a large and structurally diverse family of enzymes that
U
312 313
E
C
Fig. 5. STAT3 regulation by YM155 and API4 knockdown. (A) Temporal STAT3 regulation by YM155. Immunoblot of pSTAT3, STAT3 and API4 in AAG8-knockdown DLD-1 cells treated with or without YM155 for the indicated time, followed by PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates were immunoblotted with pSTAT3, STAT3 and API4 antibodies; GAPDH served as the loading control. (B) Effect of API4 knockdown on IL6-induced STAT3 activation. Immunoblots of pSTAT3, STAT3 and API4 in transient API4-knockdown DLD-1 cells treated with PBS or 10 ng/ml IL6 treatment for 1 h. Total lysates were immunoblotted with pSTAT3, STAT3 and API4 antibodies; STAT3 served as the loading control.
Fig. 6. Dispensable effects of AAG8 on PKM2. (A) Immunoblot of PKM2 in DLD-1 cells with either AAG8 overexpression (left) or knockdown (right). Total lysates were immunoblotted with AAG8 antibody; GAPDH served as the loading control. (B) Immunoblot of PKM2 of total, cytoplasmic, and nuclear fractions in AAG8-knockdown HCT116 cells. Lysates of indicated fractions were immunoblotted with PKM2 antibody. LMNB served as the loading control for the nuclear fraction. GAPDH served as the loading control for total and cytoplasmic fractions.
catalyze the dephosphorylation. We demonstrated that STAT3 kinases are dispensable in AAG8-induced STAT3 activation. However, there is a possibility that dephosphorylation might account for AAG8-related regulation. For instance, PTPMeg2 is a physiologic STAT3 phosphatase that can directly dephosphorylate STAT3 at the Tyr705 residue [23]. This possibility merits future detailed evaluation. Despite great efforts focusing on STAT3 for cancer therapy, few efficient strategies have been developed [24]. Single usage of chemical drugs targeting upstream kinases of STAT3, such as JAK2 inhibitors, often results in drug insensitivity due to acquired resistance [25]. In the anticancer drug screening, we identified YM155 as a potent STAT3 suppressant, suggesting that YM155 blocks the signalling activities which are required for IL6-induced STAT3 activation. We further illustrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. As single use of both JAK inhibitors and API4 inhibitor YM155 has clinical limitations [25,26], our drug combination strategy provides a promising therapeutic approach for increasing the antitumor efficacy and decreasing drug resistance.
316 317
5. Conclusions
334
The present study characterized AAG8 as an oncoprotein in multiple types of cancers through investigating its cancer-promoting effects and the underlying mechanisms. We uncovered the molecular clues that AAG8 is an alternative upstream STAT3 activator in addition to IL6/JAK signalling pathway. Tandem inhibition of AAG8 and JAK signalling synergistically suppresses cancer cell growth. Taken together, our findings
335
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333
336 337 338 339 340
B. Sun et al. / Cellular Signalling xxx (2014) xxx–xxx
7
Acknowledgments
345
We thank Dr. Daizo Koinuma and Dr. Kohei Miyazono (The University of Tokyo) for their helpful suggestions and discussions; and Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan for the generous gift of The SCADS Inhibitor Kits I-IV. B. S. was supported by China Scholarship Council (CSC) and Center for Medical System Innovation (CMSI) of The University of Tokyo.
346 347
Appendix A. Supplementary data
355
348 349 350 351 352
F
353 354
O
Supplementary data to this article can be found online at http://dx. 356 doi.org/10.1016/j.cellsig.2014.04.001. 357
R O
References
N C O
R
R
E
C
T
E
D
P
[1] K.S.C. Marriott, M. Prasad, V. Thapliyal, H.S. Bose, J. Pharmacol. Exp. Ther. 343 (2012) 578–586. [2] T. Hayashi, E. Hayashi, M. Fujimoto, H. Sprong, T.P. Su, J. Biol. Chem. 287 (2012) 43156–43169. [3] S. Kourrich, T. Hayashi, J.Y. Chuang, S.Y. Tsai, T.P. Su, A. Bonci, Cell 152 (2013) 236–247. [4] A. Al-Saif, F. Al-Mohanna, S. Bohlega, Ann. Neurol. 70 (2011) 913–919. [5] B. Sun, M. Kawahara, T. Nagamune, Cancer Med. (2014). [6] X.X.O. Yang, H.Y. Zhang, B.S. Kim, X.Y. Niu, J. Peng, Y.H. Chen, R. Kerketta, Y.H. Lee, S. H. Chang, D.B. Corry, D.M. Wang, S.S. Watowich, C. Dong, Nat. Immunol. 14 (2013) (732 − +). [7] M. Hatziapostolou, C. Polytarchou, E. Aggelidou, A. Drakaki, G.A. Poultsides, S.A. Jaeger, H. Ogata, M. Karin, K. Struhl, M. Hadzopoulou-Cladaras, D. Iliopoulos, Cell 147 (2011) 1233–1247. [8] J.W. Shui, A. Larange, G. Kim, J.L. Vela, S. Zahner, H. Cheroutre, M. Kronenberg, Nature 488 (2012) (222 − +). [9] G.Y. Lee, P.A. Kenny, E.H. Lee, M.J. Bissell, Nat. Methods 4 (2007) 359–365. [10] D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Science 307 (2005) 1098–1101. [11] Y. Shirai, S. Ehata, M. Yashiro, K. Yanagihara, K. Hirakawa, K. Miyazono, Am. J. Pathol. 179 (2011) 2920–2930. [12] Y.S. Na, S.J. Yang, S.M. Kim, K.A. Jung, J.H. Moon, J.S. Shin, D.H. Yoon, Y.S. Hong, M.H. Ryu, J.L. Lee, J.S. Lee, T.W. Kim, PLoS ONE 7 (2012). [13] N. Sen, X.B. Che, J. Rajamani, L. Zerboni, P. Sung, J. Ptacek, A.M. Arvin, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 600–605. [14] J. Rosenbluh, D. Nijhawan, A.G. Cox, X.N. Li, J.T. Neal, E.J. Schafer, T.I. Zack, X.X. Wang, A. Tsherniak, A.C. Schinzel, D.D. Shao, S.E. Schumacher, B.A. Weir, F. Vazquez, G.S. Cowley, D.E. Root, J.P. Mesirov, R. Beroukhim, C.J. Kuo, W. Goessling, W.C. Hahn, Cell 151 (2012) 1457–1473. [15] A.V. Sarthy, S.E. Morgan-Lappe, D. Zakula, L. Vernetti, M. Schurdak, J.C.L. Packer, M.G. Anderson, S. Shirasawa, T. Sasazuki, S.W. Fesik, Mol. Cancer Ther. 6 (2007) 269–276. [16] N. Nakamura, T. Yamauchi, M. Hiramoto, M. Yuri, M. Naito, M. Takeuchi, K. Yamanaka, A. Kita, T. Nakahara, I. Kinoyama, A. Matsuhisa, N. Kaneko, H. Koutoku, M. Sasamata, H. Yokota, S. Kawabata, K. Furuichi, Mol. Cell. Proteomics 11 (2012). [17] X.L. Gao, H.Z. Wang, J.J. Yang, X.W. Liu, Z.R. Liu, Mol. Cell 45 (2012) 598–609. [18] D. Balasuriya, A.P. Stewart, D. Crottes, F. Borgese, O. Soriani, J.M. Edwardson, J. Biol. Chem. 287 (2012) 37021–37029. [19] D. Fontanilla, M. Johannessen, A.R. Hajipour, N.V. Cozzi, M.B. Jackson, A.E. Ruoho, Science 323 (2009) 934–937. [20] L.X. Wang, J.A. Eldred, P. Sidaway, J. Sanderson, A.J.O. Smith, R.P. Bowater, J.R. Reddan, I.M. Wormstone, Mech. Ageing Dev. 133 (2012) 665–674. [21] A. Hyrskyluoto, I. Pulli, K. Tornqvist, T.H. Ho, L. Korhonen, D. Lindholm, Cell Death Dis. 4 (2013). [22] M. Abadias, M. Escriche, A. Vaque, M. Sust, G. Encina, Br. J. Clin. Pharmacol. 75 (2013) 103–117. [23] R. Li, Z.L. Hu, S.Y. Sun, Z.G. Chen, T.K. Owonikoko, G.L. Sica, S.S. Ramalingam, W.J. Curran, F.R. Khuri, X.M. Deng, Mol. Cancer Ther. 12 (2013) 2200–2212. [24] M. Sen, S.M. Thomas, S. Kim, J.I. Yeh, R.L. Ferris, J.T. Johnson, U. Duvvuri, J. Lee, N. Sahu, S. Joyce, M.L. Freilino, H.B. Shi, C.Y. Li, D. Ly, S. Rapireddy, J.P. Etter, P.K. Li, L. Wang, S. Chiosea, R.R. Seethala, W.E. Gooding, X.M. Chen, N. Kaminski, K. Pandit, D.E. Johnson, J.R. Grandis, Cancer Discov. 2 (2012) 694–705. [25] P. Koppikar, N. Bhagwat, O. Kilpivaara, T. Manshouri, M. Adli, T. Hricik, F. Liu, L.M. Saunders, A. Mullally, O. Abdel-Wahab, L. Leung, A. Weinstein, S. Marubayashi, A. Goel, M. Gonen, Z. Estrov, B.L. Ebert, G. Chiosis, S.D. Nimer, B.E. Bernstein, S. Verstovsek, R.L. Levine, Nature 489 (2012) 155-U222. [26] R.J. Kelly, A. Thomas, A. Rajan, G. Chun, A. Lopez-Chavez, E. Szabo, S. Spencer, C.A. Carter, U. Guha, S. Khozin, S. Poondru, C. Van Sant, A. Keating, S.M. Steinberg, W. Figg, G. Giaccone, Ann. Oncol. 24 (2013) 2601–2606.
U
Fig. 7. Tandem AAG8–JAK inhibition. (A) Phase contrast images showing acinar morphology of the indicated DLD-1 cells in 3D culture on day 5 after treatment with or without 100 nM YM155. (B) Growth assay of AAG8-knockdown DLD-1 cells treated with 1 μM JSI-124 (JAK inhibitor) for 24 h. Initial cell number = 6 × 105. n = 3. Error bars (s.e.m.) are indicated. ***p b 0.001 (one way ANOVA followed by Tukey's test). (C) Phase contrast images showing acinar morphology of the indicated DLD-1 cells in 3D culture on day 10 after treatment with or without 100 nM JAK3 inhibitor VI.
341 342 343 344
shed light on the fundamental evidences for identification of AAG8 as a potential target for cancer prevention and highlight the importance of ER chaperon proteins in contributing to JAK/STAT signalling and carcinogenesis.
Please cite this article as: B. Sun, et al., Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.04.001
358 359 360 361 362 363 364 365 366 Q3 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414