Arl13b Promotes Gastric Tumorigenesis by Regulating Smo Trafficking and Activation of the Hedgehog Signaling Pathway

Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-16-2461

Cancer Molecular and Cellular Pathobiology Research

Arl13b Promotes Gastric Tumorigenesis by Regulating Smo Trafﬁcking and Activation of the Hedgehog Signaling Pathway Jia Shao1, Linlin Xu1, Limin Chen1, Quqin Lu2, Xinsheng Xie1, Wei Shi1, Huanting Xiong1, Chao Shi1, Xuan Huang1, Jinhong Mei3, Hai Rao4, Hua Lu5, Nonghua Lu6, and Shiwen Luo1

Abstract

Inhibitors of the Hedgehog (Hh) pathway transducer cells, Arl13b stimulated proliferation, migration, and invasion Smoothened (Smo) have been approved for cancer treatment, in vitro and in vivo. In clinical specimens of gastric cancer, Arl13b but Smo mutations often lead to tumor resistance and it expression correlated strongly with tumor size and depth remains unclear how Smo is regulated. In this study, we iden- of invasion; patients with high levels of Arl13b had a poor tiﬁed the small GTPase Arl13b as a novel partner and regulator prognosis. Our results show how Arl13b participates in Hh of Smo. Arl13b regulated Smo stability, trafﬁcking, and local- pathway activation in gastric cancer. Cancer Res; 77(15); 4000–13. ization, which are each crucial for Hh signaling. In gastric cancer 2017 AACR.

Introduction the Hh signaling pathway is mostly quiescent in adult tissues (5). Aberrantly activated Hh signaling could lead to oncogenesis in The Hedgehog (Hh) signaling pathway was initially identified various tissues or organs, including basal cell carcinoma, medul- in Drosophila melanogaster (1), but its core components and loblastoma, pancreatic cancer, colon cancer, gastric cancer, and regulatory mechanism are evolutionarily conserved from inverte- glioblastoma (6–10). Smo plays an important signal transduction brates to vertebrates, with few exceptions (2). In vertebrates, Hh role in these physiologic and oncopathologic events by trans- signaling is initiated by the binding of Hh ligands (Shh, Ihh, and ducing Hh signaling. Dhh) to a 12-transmembrane receptor Patched (Ptch) present in Smo is a membrane frizzled family G-protein–coupled receptor receiver cells. The binding of an Hh ligand to Ptch relieves the (GPCR), and its trafficking to and enrichment in the PC are key inhibition of Smoothened (Smo) by Ptch, resulting in the trans- steps in the activation of the Hh signaling pathway. Smo contains location of Smo to the primary cilium (PC). Upon entering in the an extracellular N-terminal domain, a central seven-transmem- cilia, derepressed Smo triggers the activation of zinc-finger tran- brane domain, and an intracellular C-terminal domain (C-tail; scription factors Gli1, Gli2, and Gli3, which then translocate to ref. 11). The N-terminal domain and seven-transmembrane heli- the nucleus and modulate the expression of their downstream cal domain primarily function as physiologic ligand-binding sites target genes (3). Hh signaling plays an important role in body that are critical for Smo activity (12), while the C-tail mediates the patterning and organ development during embryogenesis by activation of Hh signaling (13–15). Posttranslational modifica- regulating the proliferation, migration, and differentiation of tions, such as phosphorylation or ubiquitination, and protein– target cells in a concentration-dependent manner (4). However, protein interactions occurring on the C-tail have profound effects on Smo activity (12, 15–17). In response to Hh ligands, the C-tail of Smo becomes hyperphosphorylated, which triggers a confor- 1Center for Experimental Medicine, The First Affiliated Hospital of Nanchang mational change associated with the enrichment of Smo in the PC University, Nanchang, Jiangxi, China. 2Department of Epidemiology & Biosta- tistics, School of Public Health, Nanchang University, Nanchang, Jiangxi, China. and activates downstream signals in vertebrate, whereas its ubi- 3Department of Pathology, The First Affiliated Hospital of Nanchang University, quitination promotes the endocytosis and degradation of Smo Nanchang, Jiangxi, China. 4Department of Molecular Medicine, University of (18). Regulatory or effector proteins, such as b-arrestin, Costal 2, Texas Health, San Antonio, Texas. 5Department of Biochemistry & Molecular and Gprasp2, facilitate Smo activation by directly binding to its C- Biology and Tulane Cancer Center, Tulane University School of Medicine, New tail (12, 15, 19). Although several molecules have been identified Orleans, Louisiana. 6Institute of Digestive Disease, The First Affiliated Hospital of to regulate Smo via its C-tail in response to Hh signaling, it Nanchang University, Nanchang, Jiangxi, China. remains unclear whether there are other yet unknown Smo Note: Supplementary data for this article are available at Cancer Research regulators that are important for the Hh signaling pathway. Online (http://cancerres.aacrjournals.org/). In an attempt to address this issue, we identified Arl13b as a J. Shao, L. Xu, and L. Chen contributed equally as first authors of this article. novel regulator of Smo in the Hh pathway. Arl13b is an ADP- Corresponding Author: Shiwen Luo, The First Affiliated Hospital of Nanchang ribosylation factor (Arf)-like small GTPase that is involved in University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006, the formation and function of cilia as well as vesicle trafficking, China. Phone: 86-791-88692139; Fax: 86-791-88623153; E-mail: cellular differentiation, cell movement, and cytoskeletal pro- [email protected] cesses (20). Arl13b is specifically enriched in the cilia of many doi: 10.1158/0008-5472.CAN-16-2461 organisms. In the absence of Arl13b, the cilia are short and 2017 American Association for Cancer Research. display a specific structural defect in the ciliary axoneme (21).

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Mutations in the human Arl13b gene result in Joubert syn- To determine the direct interaction between Smo and Arl13b, drome, an autosomal-recessive disorder that is known to have MBP, MBP-Smo (550–787) and His6-Arl13bD19 were produced perturbed ciliary function (22, 23). Arl13b-null mutant mice in E. coli and purified. The target proteins that purity was above display the phenotypes of Joubert syndrome patients and 90% were subjected to further pull-down assay. Then, amylose show coupled defects in cilia structure and the Hh signaling resin contain MBP or MBP-Smo (550–787) was incubated with pathway (24). Although one study showed that Arl13b could His6-Arl13bD19 at 4C for 3 hours, and were washed three times affect the localization of some Hh signaling components, such with ice-cold PBS. Finally, the precipitated proteins were used to as the distribution of Smo within the cilium (25), it remains SDS-PAGE analysis and Coomassie brilliant blue staining. completely unknown how exactly Arl13b functions in response to Hh signaling, including whether it works directly with Smo Immunofluorescence and IHC in this signaling pathway and whether it plays a role in tumor To detect colocallization of Arl13b and Smo, AGS cells were formation associated with this signaling pathway. treated with N-Shh for 12 hours. To detect localization of Smo in Our study presented here was designed to address these ques- the PC, NIH3T3 cells with or without Arl13b knockdown were tions. As detailed below, we found that Arl13b can promote the treated with FBS-free DMEM for 24 hours and stimulated with N- tumor growth of gastric cancer by directly regulating Smo traf- Shh for 2 hours. Cells cultured on coverslips were fixed in 4% PFA ficking and subsequent activation of the Hh signaling pathway. in PBS and incubated at 4C for 30 minutes with 0.1% TritonX- Our results also suggest that Arl13b could serve as a novel 100. For immunostaining, fixed slides and cells were incubated at molecular target for developing antigastric cancer therapy. 4C overnight with primary antibodies in PBS containing 0.5% goat serum, 1% BSA, and 0.1% TritonX-100. After the samples Materials and Methods were washed 3 times with PBS, they were incubated at room Cell line temperature for 1 hour with Alexa 488-, 594-, or 647-conjugated Cell lines AGS and 293T were purchased from the ATCC goat anti-rabbit/mouse secondary antibodies, and mounted with between 2010 and 2015. NIH3T3 was purchased from ATCC in Vectashield mounting medium (Vector). Representative images March 2016. MKN45 and MKN28 cells were purchased from the were acquired using an LSM700 confocal microscope (Zeiss). China Centre for Typical Culture Collection at Wuhan University IHC staining was performed as described previously (27). between 2010 and 2013. AGS, MKN28, MKN45, and 293T cells Tissue sections were incubated in 10 mmol/L sodium citrate were authenticated using short tandem repeat (STR) profiling and buffer (pH 6.0) at sub-boiling temperatures for 10 minutes, rinsed were negative for mycoplasma contamination detecting via PCR- in PBS, and incubated with 10% normal goat serum to block based assay in December 2016. These cells used in the study were nonspecific staining. Sections were incubated with a primary cultured not more than 3 months after resuscitation and cultured antibody (1:200) at 4 C in a humidified chamber overnight and as recommended by the manufacturers in a humidified incubator immunoreactivity was visualized using a Polink-2 HRP DAB with 5% CO2 at 37 C. Detection Kit following the manufacturer's procedure. Images were captured using an FSX100 microscope equipped with a Western blotting and real-time PCR digital camera system (Olympus). Samples were examined by 3 Protein extracts were obtained using extraction buffer and were individual researchers to independently obtain pathologic infor- analyzed via Western blotting. The band intensity was analyzed mation using the German semiquantitative scoring method. Each with ImageJ software. Real-time PCR was performed with an ABI specimen was scored for the intensity of nucleic, cytoplasmic, and Sep Plus One sequence detection system (Applied Biosystems). membrane staining (no staining ¼ 0, weak staining ¼ 1, moderate staining ¼ 2, strong staining ¼ 3) and for the extent of stained cells Protein–protein interaction (0% ¼ 0, 1%–24% ¼ 1, 25%–49% ¼ 2, 50%–74% ¼ 3, 75%– To detect the interaction between Smo and Arl13b, Arl13bD19 100% ¼ 4). The final immunoreactive score was the product of the GST-fusion proteins were produced in BL21, purified, and immo- intensity score multiplied by the extent score. Consecutive sec- bilized on glutathione Sepharose 4B beads (Amersham Pharma- tions were stained by H&E to distinguish cancer tissues from cia). The beads were then incubated with lysates from 293T cells adjacent normal epithelium. transfected with Flag-Smo. Bead-associated proteins were subjected to SDS-PAGE and Western blot analysis. Furthermore, Cell proliferation, migration, and invasion assay MBP-Smo (550–787) that prepurified on amylose resin was To analyze cell proliferation, an approximately equal number incubated with lysates from 293T cells transfected with Flag- of MKN45, AGS, or MKN28 cells (3 103/well) were equiva- Arl13b, the precipitated proteins were used to analysis via Western lently plated in 6-well plates (with triplicate wells for each cell blot assays. type), in DMEM supplemented with 10% FBS. The cells were To detect the interaction of Smo and Arl13b in mammalian cultured for up to 12–15 days and then stained with crystal violet. cells, cells transfected with indicated vectors were solubilized in Dishes were graphed, and positive colonies containing more than the lysis buffer containing 0.5% Lubrol-PX, 50 mmol/L KCl, 2 50 cells were counted under a microscope. mmol/L CaCl2, 20% glycerol, 50 mmol/L Tris-HCl, and proteases Cell migration was measured using a scratch (wounding heal- and phosphatases inhibitors (pH 7.4). Precleared cell lysates were ing) assay. Cells were plated in 6-well plates to create a confluent incubated with 0.5 mg of indicated antibodies at 4C overnight. monolayer. Then, the monolayer was scraped in a straight line to The reaction was incubated with 50 mL of 1:1 slurry beads create a scratch or wound with a p200 pipet tip. The cells were conjugated with protein G or A (Roche) for 3 hours at 4C. Beads washed once with growth medium and incubated in DMEM were washed 4 times with lysis buffer before the addition of SDS containing 2% FBS. The remaining cells were treated with N-Shh sample buffer and subjected to Western blot analysis as described conditional medium and imaged using a phase-contrast micro- previously (26). scope. The wound area was quantified using NIH Image-Pro Plus

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software. The data are expressed as the means of four independent Hospital of Nanchang University (permit number: 2011-021). All experiments SD. surgeries were performed under sodium pentobarbital anesthesia, Cell invasion assays were performed in Transwell plates (8-mm with minimal suffering. pore size, 6.5-mm diameter; Corning Life Sciences) precoated with Matrigel Basement Membrane Matrix (1 mg/mL; BD Bios- Human tissue specimens ciences) according to the manufacturer's protocol. Briefly, 3 104 Human specimens were retrieved via surgical intervention cells in 200 mL of FBS-free medium were seeded into top cham- without prior radiotherapy or chemotherapy. All samples were bers. Bottom wells in the system were filled with 800 mL of N-Shh collected at the First Affiliated Hospital of Nanchang University conditional medium supplemented with 1%–2% FBS. After the between January 2009 and September 2013. The inclusion criteria assays had been run for 24 hours at 37 C, nonmigrated or are described in the Supplementary Information. The study pro- noninvaded cells were removed from the top surface of the filter. tocol was approved by the Institutional Review Board of the Frist Cells on the bottom surface of the membrane were fixed with ice- Affiliated Hospital of Nanchang University. Detailed clinical and cold methanol and stained with crystal violet. Cell numbers were pathologic information for the patients is summarized in Table 1. counted under an optical microscope. Each experiment was repeated at least three times. Statistical analysis Unless otherwise indicated, the data were expressed as the Flow cytometric analysis mean SD from experiments performed at least three times. 293T cells were transfected with the shRNA-control or shRNA- Differences between two groups were assessed with Student t test Arl13b vector. Three days after transfection, cells were detached or one-way ANOVA. Differences were considered significant if P < with enzyme-free cell dissociation buffer (Gibco) and stained 0.05. All analyses were carried out using SPSS v.13.0 software with an anti-Smo-N (extracellular domain) antibody and fol- (SPSS Inc.). lowed by staining with Alexa Fluor 488–conjugated goat anti- þ rabbit IgG (H L) in PBS. The cells were then subjected to analysis Results of Smo cell surface level using a Beckman cytometer (MOFLO XDP). Identification of Arl13b as a Smo-interacting partner Although Smo is well established to play a central role in Luciferase assay transducing Hh signaling (12), the mechanism(s) underlying its AGS or MKN28 cells were seeded into a 24-well plate. After the signal transduction function still remains poorly understood. To cells were cultured overnight, they were cotransfected with the address this issue, we searched for Smo-interacting partners by pGL4.7-8 GBS reporter plasmid and the pGL4.2-TK plasmid performing a yeast two-hybrid screen using the functionally with miRNAi-Arl13b or miRNAi control, and Arl13b-myc or myc important C-tail of Smo (15) as a bait. From this screen, we vector, respectively. Luciferase assays were performed 48 hours found three positive clones that encode two different N-terminal after transfection using a Dual Luciferase Reporter Assay System fragments of Arl13b, indicating that Arl13b could be a potential (Promega). Smo-interacting partner. To confirm this hypothesis, we performed an in vitro GST pull-down assay using Flag-Smo expressed Lentivirus infection and xenografts in 293T cells and purified GST-Arl13b lacking its N-terminal 19 The Lenti-X-shRNA Tet-On construct (pGV307-RFP) for amino acids (GST-Arl13bD19). We used GST-Arl13bD19 because shRNA-Arl13b knockdown was generated, packed, and purified its full-length form is often difficult to be purified from bacteria by GeneChem. The shRNA target sequence was 50-CAGATA- due to this N-terminal hydrophobic region (28). As shown GAACCATGT-30. Lentivirus infection was done according to the in Fig. 1A, recombinant Smo was pulled down with GST- protocol provided by the manufacturer. Briefly, 5 104 Arl13bD19. This Smo–Arl13b interaction was direct, as it was MKN45, SGC7901 or MKN28 cells were incubated with 1 confirmed by using another pull-down assay with purified MBP- 108 IU of virus and 8 mg/mL polybrene (Sigma-Aldrich) for 12 Smo [residues 550-787; MBP-Smo (550-787)] and His6- hours. Cells were induced in 2 mg/mL doxycycline for 48 hours Arl13bD19 (Fig. 1B). andfollowedby5mg/mL puromycin 14 days after infection to To further validate the Arl13b–Smo interaction in mammalian select stably infected cells. cells, we performed coimmunoprecipitation (IP) experiments For in vivo experiments, 2 107 stably infected MKN45, and found that Flag-Smo and GFP-Arl13b were coimmunopre- SGC7901 [Lenti-control (Vector) and Lenti-shRNA-Arl13b cipitated with each other in 293T cells (Fig. 1C and D). Consistent (Arl13b-KD)] or MKN28 [Lenti-control (Vector) and Lenti-Arl13b with these results, endogenous Smo protein could be immuno- (Arl13b)] cells were resuspended in sterile PBS (200 mL) and precipitated with the Arl13b antibody (Fig. 1E), indicating that injected subcutaneously into both flanks of 5-week-old female Smo and Arl13b can form a protein complex in cells. This BALB/c-nu mice (SLAC Laboratory Animal Co. Ltd). One week interaction between endogenous Smo and Arl13b was markedly after injection, the mice were administered 2 mg/ml doxycycline induced by Hh ligands (Fig. 1F). Finally, immunofluorescence and 5% sucrose in sterile drinking water. The doxycycline-contain- staining showed that Arl13b and Smo colocalized in the cyto- ing water was replenished every 3 days. Tumor sizes in both flanks plasm of the cells (Fig. 1G), indicating that binding of these of the mice were measured using Vernier calipers thrice weekly, proteins occurs in the cytoplasm. and the tumor volume was calculated using the formula volume, Next, we tried to map their binding domains using different V ¼ (L W2)/2. After 4 weeks, the xenografts were harvested for fragments of these two proteins. Smo contains three major seg- IHC and Western blot analysis. Eight female nude mice (4–5 ments (Fig. 2A). In agreement with the results from the yeast two- weeks old) were included in each group. All animal experiments hybrid screening, MBP-Smo (550–787), but not MBP alone, was were approved by the Ethical Committee of the First Affiliated able to pull down full-length Arl13b (Fig. 2B), suggesting that the

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Table 1. Association of Arl13b and Smo expression levels with different clinicopathologic characteristics in gastric cancers Arl13b Expression Smo Expression Clinicopathologic Low High Low High Measurement data n Mean SD Mean SD P Mean SD Mean SD P Age (y) 154 58.25 12.14 57.98 15.35 0.898 58.32 10.510 58.08 13.421 0.924 Tumor size (cm) 154 3.75 2.05 4.49 1.99 0.026a 3.25 1.869 4.30 2.053 0.006b Enumeration data n Count n% Count n% P Count n% Count n% P Gender Male 58 37.70% 42 27.30% 25 16.23% 75 48.70% Female 29 18.80% 25 16.20% 9 5.84% 45 29.22% Total 154 87 56.50% 67 43.50% 0.608 34 22.08% 120 77.92% 0.234 Degree of differentiation Well 2 1.30% 1 0.60% 1 0.65% 2 1.30% Moderately 36 23.40% 18 11.70% 11 7.14% 43 27.92% Poor 49 31.80% 48 31.20% 22 14.29% 75 48.70% Total 154 87 56.50% 67 43.50% 0.125 34 22.08% 120 77.92% 0.719 T factor T1 18 11.70% 6 3.90% 12 7.79% 12 7.79% T2 13 8.40% 2 1.30% 7 4.55% 8 5.19% T3 53 34.40% 56 36.40% 14 9.09% 95 61.69% T4 3 1.90% 3 1.90% 1 0.65% 5 3.25% Total 154 87 56.50% 67 43.50% 0.005b 34 22.08% 120 77.92% 0.000b Lymph node metastasis(N factor) N0 29 18.80% 16 10.40% 14 9.09% 31 20.13% N1 32 20.80% 19 12.30% 14 9.09% 37 24.03% N2 15 9.70% 23 14.90% 5 3.25% 33 21.43% N3 11 7.10% 9 5.80% 1 0.65% 19 12.34% Total 154 87 56.50% 67 43.50% 0.091 34 22.08% 120 77.92% 0.04a Neural invasion Yes 34 22.10% 34 22.10% 8 5.19% 60 38.96% No 53 34.40% 33 21.40% 26 16.88% 60 38.96% Total 154 87 56.50% 67 43.50% 0.148 34 22.08% 120 77.92% 0.006b Vascular invasion Yes 27 17.50% 30 19.50% 7 4.55% 50 32.47% No 60 39.00% 37 24.00% 27 17.53% 70 45.45% Total 154 87 56.50% 67 43.50% 0.08 34 22.08% 120 77.92% 0.025a Distant invasion Yes 5 3.20% 5 3.20% 1 0.65% 9 5.84% No 82 53.20% 62 40.30% 33 21.43% 111 72.08% Total 154 87 56.50% 67 43.50% 0.748 34 22.08% 120 77.92% 0.461 Clinical stage Stage I 24 15.60% 7 4.50% 14 9.09% 17 11.04% Stage II 11 7.10% 12 7.80% 7 4.55% 16 10.39% Stage III 35 22.70% 30 19.50% 10 6.49% 55 35.71% Stage IV 17 11.00% 18 11.70% 3 1.95% 32 20.78% Total 154 87 56.50% 67 43.50% 0.063 34 22.08% 120 77.92% 0.001b aP < 0.05, statistical difference. bP < 0.01, statistical difference.

C-tail of Smo is sufficient for Arl13b-binding. This result was R79Q showed a reduced interaction with Smo, whereas G28V verified in mammalian cells transfected with the plasmids encod- increased the Arl13b-Smo binding (Fig. 2G and H). Correlated ing the C-tail of Smo and full-length Arl13b (Fig. 2C). Arl13b is with this result, mycophenolic acid (MPA), an inhibitor of inosine comprised of an N-terminal GTP-binding domain, a central coil- monophosphate dehydrogenase (IMPDH), which can reduce coiled domain, and a C-terminal proline-rich domain (28) (Fig. intracellular GTP levels (30), was found to inhibit the interaction 2D). Using several truncated Arl13b proteins (Fig. 2D), we per- between Arl13b and Smo (Supplementary Fig. S1). Taken togeth- formed a set of co-IP Western blot experiments and found that er, these results identify Arl13b as a novel Smo-binding protein while the coil-coiled domain and proline-rich region of Arl13b and demonstrate that the C-tail of Smo directly interacts with the were dispensable for its interaction with Smo, the GTP-binding GTP-binding domain of Arl13b. domain was essential for Arl13b to form a complex with Smo, indicating that Arl13b interacts with Smo through its GTP-bind- Arl13b promotes Hh signaling through Smo stabilization ing domain (Fig. 2D and E). An in vitro GST pull-down assay Next, we determined the functional outcome(s) of the Arl13b– validated the binding of this GTP-binding domain (20–150) to Smo interaction. As the Arl13b–Smo interaction is enhanced the C-terminus of Smo (Fig. 2F). upon N-Shh stimulation (Fig. 1F), we first tested whether Arl13b As the N-terminal Arl13b is responsible for its GTPase activity, could affect the level of Smo. Interestingly, titrating the amount of we generated the active G28V mutant (23) and inactive R79Q ectopic Flag-Arl13b led to an increase of endogenous Smo protein mutant (29) of Arl13b to evaluate whether the GTP-binding site is levels in a dose-dependent manner (Fig. 3A), but alteration of critical for the interaction between Arl13b and Smo. Indeed, Arl13b did not affect Smo transcription (Supplementary Fig. S2A

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Figure 1. The interaction between Arl13b and Smo. A, Arl13b interacts with Smo. B, Arl13b binds Smo directly. Puriﬁed MBP-Smo(550-787) immobilized on amylose resin was incubated with prepuriﬁed His6-Arl13bD19. C and D, Interaction of Arl13b with Smo in mammalian cells. E, Endogenous Arl13b binds to Smo in gastric cancer cells. F, N-Shh stimulation increases the interaction of endogenous Arl13b with Smo in gastric cancer cells. G, Colocalization of Arl13b and Smo. Cells were costained with antibodies against Arl13b (Alexa Fluor 594, red) and Smo (Alexa Fluor 488, green). Arrows, colocalization.

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Figure 2. Mapping the domains responsible for the Smo–Arl13b interaction. A, Schematic illustration of the domains of Smo and its association with Arl13b. B and C, C-terminal tail of Smo interacts with Arl13b. D, Diagrammatic representation of Arl13b and various deletions used to determine the Smo-binding domain. E, Identiﬁcation of the Arl13b domains responsible for Smo interaction. Lysates from 293T cells transfected with Flag-Smo and various GFP-tagged Arl13b derivatives were subjected to immunoprecipitation (IP) with anti-Flag antibody. Asterisks indicate the domains interacted with Smo. F, N-terminal region of Arl13b interacts with C-terminal tail of Smo. G and H, The Smo–Arl13b interaction was inﬂuenced by the GTPase activity of Arl13b. 293T cells lysates cotransfected with wild type (WT), G28V mutant, or R79Q mutant of GFP-Arl13b and Flag-Smo (550–787) or empty vector were immunoprecipitated with anti-Flag antibody. Data are shown as mean SD (n ¼ 3). , P < 0.01.

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Figure 3. Arl13b enhances Smo stability, trafﬁcking, and activity. A, Overexpression of Arl13b increases the Smo protein level. 293T cells were transfected with indicated doses of Flag-Ar13b. B and C, Arl13b knockdown accelerates the degradation of Smo protein. 293T cells expressing miR-control or miR-Arl13b-478 were treated with cycloheximide (CHX) for indicated times. KD, knockdown. Data are shown as mean SD (n ¼ 3). , P < 0.01. D, Ubiquitylation- and proteasome- dependent degradation of Smo. AGS cells cotransfected with Flag-Smo and miR-control or miR-Arl13b-478 were treated with or without 50 mmol/L MG-132. E, Arl13b (1–150) functions as a dominant-negative modulator on the stabilization of Smo. 293T cells were transfected with indicated doses of GFP-Arl13b(1-150). F and G, Arl13b (1–150) signiﬁcantly reduces the colony formation of gastric cancer cells. AGS cells transfected with GFP-Arl13b, GFP-Arl13b(1-150), or GFP were sorted and used for colony formation assays. Data are shown as mean SD (n ¼ 3). , P < 0.01. H, Knockdown of Arl13b reduces Smo surface expression. I and J, Arl13b regulates localization of Smo in the PC. Cells were costained with antibodies against Ac-tubulin (Ac-Tub; Alexa Fluor 594, red) and Smo (Alexa Fluor 647, green). Arrow, PC. Data are shown as mean SD (n ¼ 40). , P < 0.01.

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and S2B), suggesting that Arl13b may regulate Smo stability by these cancer cells. Indeed, knockdown of Arl13b dramatically directly binding to this protein. To test this conjecture, we per- blocked the N-Shh–induced wound healing of MKN45 cells, and formed an Arl13b knockdown experiment followed by analysis of also inhibited N-Shh–mediated invasion of the cells (Fig. 4B–E). the half-life of Smo. Knockdown of Arl13b led to a marked decrease These results were also reproduced using AGS cells (Supplemen- of the Smo's half-life from approximately 6 hours to less than 2 tary Fig. S4B–S4E). In addition, Arl13b knockdown significantly hours (Fig. 3B and C; Supplementary Fig. S2C). In addition, retarded the N-Shh–induced colony formation and growth of proteasome inhibitor MG-132, but not lysosomal inhibitor Mon- MKN45 cells (Fig. 4F; Supplementary Fig. S4F and S4G). These ensin, increased the level of Smo protein (Supplementary Fig. S2D). results indicate that Arl13b is critical for the proliferation, migra- Consistently, knockdown of Arl13b led to an increase in the tion, and invasion of gastric cancer cells. ubiquitylation of Flag-Smo, which was more apparent in the Conversely, overexpression of Arl13b using the lentivirus sys- presence of MG-132 (Fig. 3D). These results indicate that Arl13b tem in MKN28 cells (Supplementary Fig. S5A) drastically protects Smo from ubiquitination and ubiquitination-dependent increased the percentage of wound repair with N-Shh treatment proteolysis. Intriguingly, The N-terminal Smo-binding domain of of the cells (Fig. 4G and H). Also, Arl13b overexpression signif- Arl13b (residues 1–150) reduced the Smo protein levels in a dose- icantly enhanced N-Shh–induced cell invasion, colony forma- dependent manner (Fig. 3E) and inhibited the proliferation of tion, and growth (Fig. 4I–K; Supplementary Fig. S5B and S5C). gastric cancer cells (Fig. 3F and G), suggesting that binding to Smo Altogether, these results demonstrate that Arl13b plays a crucial itself is not sufficient to protect Smo from ubiquitination-mediated role in the Hh signaling–mediated proliferation, migration, and degradation; instead, this binding might prevent the protective invasion of gastric cancer cells. effect of full-length Arl13b on Smo in a dominant-negative fashion. We then determined whether Arl13b might affect Smo traffick- Arl13b accelerates gastric carcinogenesis in vivo ing. Immunofluorescence staining revealed that Smo was To determine whether the cellular phenotypes described above enriched in the cytoplasmic membrane of Arl13b-expressed cells, could be reproduced in a more biologically significant context, but reduced on the surface of Arl13b-depleted cells (Supplemen- we generated xenograft tumor models using gastric cancer cell tary Fig. S2E), which was confirmed by flow cytometric analysis lines MKN45, SGC7901, and MKN28. These cell lines expressing (Fig. 3H). Also, knockdown of Arl13b reduced the localization of scrambled shRNA, Arl13b-shRNA(Arl13b-KD), or ectopic Arl13b Smo to the PC (Fig. 3I and J; Supplementary Fig. S2F). We further were subcutaneously injected into the flanks of nude mice. Knock- evaluated whether Arl13b regulates the effects of Smo inhibitors, down of Arl13b led to dramatic decreases of the average tumor the expression of the Hh signaling target genes Gli1 and Bcl2 were volume and the average tumor weight compared with the control detected, and we found that Smo inhibitors were ineffective in the group (Fig. 5A–C). In line with these results, the expression of presence of overexpression of Arl13b (Supplementary Fig. S2G Gli2, Gli1, Smo, snail, N-cadherin, vimentin, and p-FAK was dra- and S2H). These results indicate that Arl13b can mediate the matically reduced in the tumor tissues of the Arl13b-KD group trafficking of Smo and its activity. (Fig. 5D, left), similar to the results obtained in vitro (Fig. 4A). The It was reported that mice lacking Arl13b exhibit abnormal Hh IHC results indicated that Ki-67 strictly associated with cell pro- signaling (21), and Arl13b is required for the entry of Smo into the liferation, and MMP9 involved in cancer invasion and metastasis cilium via unknown mechanisms (25). We found Arl13b regu- were significantly decreased in Arl13b knockdown xenograft tu- lates the expression of Gli1 in NIH3T3 cells, which is Hh respon- mors (Fig. 5E). Furthermore, we observed similar results in tumor sive (Supplementary Fig. S3A and S3B). We further determined xenograft of SGC7901 cells (Supplementary Fig. S6). Consistent whether Arl13b affects the Hh signaling response in gastric cancer with these results, the overexpression of Arl13b resulted in a cells. Indeed, knockdown of Arl13b reduced, but overexpression dramatic increase in both tumor volume (by 3-fold) and tumor of Arl13b enhanced luciferase expression driven by a Gli2-binding weight (by 2-fold) compared with that of the control group site–containing promoter (Supplementary Fig. S3C and S3D). In (Fig. 5F–H). Consistently, overexpression of Arl13b significantly line with these results, the expression of several Hh signaling increased the level of Gli2, Gli1, Smo, snail, N-cadherin, vimentin, components, its downstream targets, and the epithelial mesen- and p-FAK in Arl13b xenograft tumors (Fig. 5D, right). The levels chymal transition (EMT)-related proteins was reduced in Arl13b- of Smo, Ki67 and MMP9 were also markedly enhanced in ectopic depleted cells, but upregulated in Arl13b-overexpressed cells with Arl13b-expressed xenograft tumors (Fig. 5E). These results demon- or without Hh treatment (Supplementary Fig. S3E and S3F). These strate that Arl13b plays a critical role in driving the growth of results indicate that Arl13b not only regulates the activity of Smo, xenograft gastric tumors by mediating the Hh signaling pathway. but also influences the Hh signaling response, and suggest that Arl13b might play a role in the proliferation, migration, and Arl13b expression levels correlate with the clinical invasion of cancer cells. pathogenesis of gastric cancer To translate the above described oncogenic role of Arl13b in Arl13b facilitates Shh-induced malignancy of gastric gastric cancer development and progression to clinical signifi- cancer cells cance, we recruited 154 eligible gastric cancer patients and ana- Although Hh signaling has been shown to be crucial for gastric lyzed the expression of Arl13b and Smo, as well as the correlation cancer development and metastasis (31), the role of Arl13b in between their expression and the stage or progression of gastric these oncogenic processes remains unknown. To test this, we cancers in pairs of tumor specimens. First, we evaluated the employed two cell lines AGS and MKN45, which contain higher specificity of the antibodies used in IHC assays, and found that levels of Arl13b (Supplementary Fig. S4A). Knockdown of Arl13b anti-Arl13b and anti-Smo antibodies could effectively and spe- markedly reduced the expression of Gli2, Gli1, and Smo as well as cifically recognize the indicated proteins (Supplementary Fig. S7). that of EMT-related proteins (Fig. 4A), suggesting that Arl13b Then, we found Arl13b and Smo protein levels were significantly might be required for the proliferation, migration, and invasion of increased in gastric tumors compared with the paired adjacent

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Figure 4. Arl13b increases Shh-induced gastric cancer migration and invasion. A, Arl13b regulates the levels of Hh pathway components and EMT-related proteins in gastric cancer cells. Lysates of MKN45 contorl (Vector) or Arl13b stable knockdown (Arl13b-KD) cells were subjected to Western blotting. B and C, Arl13b knockdown reduces N-Shh–induced migration of MKN45 cells. Images were acquired at indicated time. Data are shown as mean SD (n ¼ 3). , P < 0.01. D and E, Knockdown of Arl13b decreases N-Shh–stimulated invasion of MKN45 cells. Data are shown as mean SD (n ¼ 4). , P < 0.01. F, Arl13b knockdown inhibits the N-Shh–stimulated colony formation of MKN45 cells. The data are shown as mean SD (n ¼ 3). , P < 0.01. G and H, Arl13b overexpression enhances the N-Shh–induced migration of MKN28 cells. Data are shown as mean SD (n ¼ 3). , P < 0.01. I and J, Arl13b overexpression signiﬁcantly increases the N-Shh-stimulated invasion of MKN28 cells. Data are shown as mean SD (n ¼ 4). , P < 0.01. K, Arl13b overexpression increases N-Shh–stimulated colony formation in MKN28 cells. Data are shown as mean SD (n ¼ 3). , P < 0.01.

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Figure 5. Arl13b promotes gastric carcinogenesis in vivo. A–C, Arl13b knockdown inhibits tumor growth. Eight nude mice were injected subcutaneously with 2 107 cells/ mouse for each of the indicated stable cell lines of MKN45-Arl13b-KD. Results are presented as tumor volume (A), isolated tumors (B), and tumor weights (C). Data are shown as mean SD (n ¼ 8). , P < 0.05; , P < 0.01. D, Arl13b regulates levels of Hh pathway components and EMT related proteins in vivo. E, Arl13b regulates metastasis and proliferation of tumor cells. Tumors were isolated, ﬁxed, and employed to IHC assays. F–H, Overexpression of Arl13b promotes tumor growth. Eight nude mice were injected subcutaneously with 2 107 cells/mouse for each of the indicated stable cell lines of MKN28-Arl13b. Results are represented as tumor volume (F), isolated tumors (G), and tumor weights (H). Data are shown as mean SD (n ¼ 8). , P < 0.05; , P < 0.01.

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tissues via IHC analysis (Fig. 6A–C), consistent with the results Our study further demonstrated that the GTP-binding motif is from the xenograft experiments described above (Fig. 5). In 8 pairs critical for the interaction between Arl13b and Smo using two of representative tumor and adjacent tissues, the levels of Arl13b point mutations in Arl13b. The small GTPases of the Ras super- and Smo were revealed to be much higher than that in adjacent family contain a P loop (GXXXXGKS/T) that is required for gastric tissues (Fig. 6D). These results indicate that both Arl13b guanine nucleotide binding (33). A previous study showed that and Smo are highly expressed in human gastric cancers. substitution of the first G with V frequently results in decreased Next, we analyzed the correlation of the expression of Arl13b GTP hydrolysis and generates a dominant-active form (23). and Smo with the survival rates of patients with gastric cancers Similarly, we introduced a mutation at the corresponding site of using the Kaplan–Meier method. As shown in Fig. 6E–H, univar- Arl13b and generated a G28V mutation. We also made an R79Q iate analysis of tumor samples for disease-free survival and overall substitution in the N-terminus of Arl13b, which was previously survival revealed that the group of patients exhibiting lower identified in Joubert syndrome patients to interfere with GTP expression of Arl13b or Smo displayed a better prognosis (Fig. binding in a dominant-negative fashion (29). Using these 6E–H). We then analyzed the correlation between the expression mutants of Arl13b, we showed that the Arl13b–Smo interaction of Arl13b or Smo and the clinical and pathologic features of is dependent on its GTPase activity, as the G28V mutation patients with gastric cancers. We used scores of 0 to 12 to estimate enhanced the Arl13b–Smo interaction, while the R79Q mutation the expression of Arl13b or Smo, defining a score of less than 6 as suppressed this interaction (Fig. 2G and H). Also, an IMPDH low expression, and a score equal to or greater than 6 as high inhibitor MPA that can decrease cellular GTP levels reduced this expression. Similar to the case for Smo (Table 1), the higher interaction. These results indicate that the GTPase domain is expression of Arl13b in gastric cancer samples was strongly pivotal for the interaction of Arl13b with Smo, and suggest that correlated with both tumor size (P ¼ 0.026) and the depth of this interaction may serves as a target site for screening inhibitors invasion (P ¼ 0.005). But, in contrast to the case for Smo, little that could be developed into a potential anticancer therapy as correlation was observed between the expression of Arl13b and further discussed below. tumor TNM stage (Table 1). In line with the above IHC results, analysis of the GEO database Arl13b regulates Smo trafficking and Hh signaling activity also showed much higher expression of Arl13b at the RNA level in Our functional studies of the Arl13b–Smo interaction revealed gastric cancer tissues than that in paired normal tissues (Supple- that Arl13b can markedly enhance the stability of Smo (Fig. 3A). mentary Fig. S8A and S8B). On the basis of gene expression By binding to Smo, Arl13b blocked its ubiquitination and thus patterns, gastric cancers are classified into three subtypes: meta- prevented its degradation (Fig. 3D). Arl13b appeared to inhibit bolic, proliferative, and mesenchymal, which have features of 5- Smo degradation through a proteasome-dependent mechanism, fluorouracil sensitivity, genomic instability and cancer stem cells, but not via the lysosome-dependent mechanism, in mammalian respectively (32). Arl13b was found to be highly expressed in the cells. As activated Smo is often stabilized on the plasma mem- proliferative and mesenchymal subtypes of gastric cancers (Sup- brane in Drosophila or on the PC in vertebrates (34), our results plementary Fig. S8C). Hence, taken together, these results dem- suggest that Arl13b might affect the trafficking of Smo in mam- onstrate that Arl13b is highly associated with the progression of malian cells. human gastric cancer and could be a biomarker for the prediction Indeed, this was the case, as Arl13b promoted the plasma of prognosis. membrane localization of Smo and inhibited Smo ubiquitination that mediates the endocytic trafficking of Smo (Fig. 3H; fi Discussion Supplementary Fig. S2E). In vertebrates, traf cking of Smo to the PC is essential for Smo signal transduction (13). In the The seven-transmembrane receptor Smo is a key player in the presence of Hh ligand, Smo is transported to and enriched in Hh signaling pathway. However, it remains poorly understood cilia, triggering the activation of Hh signaling. There are three how Smo is regulated in response to Hh signaling. The current models proposed for Hh ligand–inducedSmotransporttothe study identified Arl13b as a novel regulator and partner of Smo via PC, (i) direct pathway: direct trafficking of Smo from the Golgi directly binding to this protein. As further discussed below, to the base of the cilium; (ii) lateral transport pathway: trans- Arl13b can regulate the trafficking and subsequent activation of port to the cell surface followed by lateral transport to the Smo in response to Hh signaling, and play a crucial role in cilium; (iii) recycling pathway: surface localization followed by promoting the progression of gastric cancer (Fig. 6I). internalization into a recycling pathway (35). Previously, Smo was shown to move from the plasma membrane to the ciliary Arl13b physically interacts with Smo membrane through the lateral transport pathway (35). A later Although previous studies have shown that both Smo and study showed that Arl13b regulates Smo trafficking to the PC Arl13b are required for Hh signaling, and Arl13b regulates the via the endocytic recycling pathway (20). Together with our distribution of Smo within the PC (25), it remains completely results as presented here (Fig. 3I and J), these studies suggest unknown how Arl13b regulates the localization of Smo and that Arl13b may mediate lateral transport of Smo from the whether these proteins directly interact with each other. Using plasma membrane to the PC by directly binding to Smo and the C-terminus of human Smo as bait in yeast two-hybrid screen- inhibiting its ubiquitination and degradation. ing, we identified Arl13b as a potential novel Smo-interacting Our study establishes Arl13b as a positive regulator of Hh protein (Fig. 1). Our further biochemical and cellular analyses not signaling, which is consistent with the previous finding that mice only validated the physical interaction between Smo and Arl13b lacking Arl13b display aberrant Hh signaling (21). Interestingly, (Fig. 1B), but also mapped their binding domains to the C-tail of our study also showed that the N-terminal domain of Arl13b Smo and the N-terminal GTP-binding motif of Arl13b (Fig. 2). (residues 1–150) acts as a dominant-negative modulator as it Remarkably, this interaction was responsive to Hh signaling. competed with full-length Arl13b for Smo-binding, leading to the

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Figure 6. High levels of Arl13b and Smo are closely related to poor survival in gastric cancer patients. A–C, Arl13b and Smo are overexpressed in gastric cancer tissues compared with adjacent tissues as examined by IHC. A, The representative images with magnified local images reflecting detailed information are shown on the right. Plotting of the Arl13b and Smo scores in each gastric carcinoma and adjacent tissues (B) and box plots of the scores of Arl13b and Smo expression (C) are shown. Statistical significance was analyzed using the Mann–Whitney U test, n ¼ 154. D, Arl13b and Smo are highly expressed in gastric cancer tissues. Proteins isolated from gastric cancer and adjacent nontumorous tissues from 8 patients were subjected to Western blotting. A, adjacent; C, tumor. E–H, The high levels of Arl13b and Smo are correlated with poor survival of patients with gastric cancer. Univariate analysis was carried out to analyze disease-free survival and overall survival of patients with gastric cancer. I, Hypothetical model of Arl13b promotes tumorigenesis via mediating Smo trafficking and activating of Hh signaling. In the absence of Shh (top), Ptch enriches in and around the PC and inhibits Smo translocation to PC. The repressed form of Gli (GliR) cleaved by proteasome accumulates and enters the nucleus to block expression of target genes. In the presence of Shh ligand (bottom), Arl13b strongly interacts with Smo, facilitating trafficking of Smo to the PC. PC-localized Smo induces accumulation of Gli activated form (GliFL) in the cytoplasm and nucleus, results in activation of target genes, and then promotes the proliferation of cancer cells.

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degradation of Smo (Fig. 3E). Hence, our results suggest that in vivo (Figs. 4 and 5). Remarkably, by analyzing clinical gastric targeting the Arl13b–Smo interaction with peptides or small cancer specimens, we found that Arl13b levels are highly corre- compounds may be a good approach to inactivate the Hh lated with tumor size and the late stage of invasion in gastric signaling. cancers. More remarkably, patients with a higher level of Arl13b Aberrant Hh signaling has been implicated in various cancers, display a poor prognosis (Fig. 6E–H and Table 1). including gastric cancer, basal cell carcinoma, and pancreatic Our further analysis showed that the RNA level of Arl13b is cancer. Activating mutations of Smo (e.g., V321M, L412F, increased in the proliferative and mesenchymal subtypes of gastric F460L, W535L, and R562Q) have strong effects on its activity cancers, compared with the metabolic subtype. Correspondingly, and can drive the formation of basal cell cancer and medullo- genes critical for cell cycle and DNA replication were upregulated blastoma (36–38). Several Smo inhibitors, such as cyclopamine, in the proliferative subtype, and the mesenchymal subtype has IPI-926, GDC-0449, and LDE-225, are currently in clinical trials the features of cancer stem cells with aberrantly activated Hh for various cancer treatments (39–42). GDC-0449 has been signaling (32). Together, our results suggest that Arl13b could approved for the treatment of locally advanced and metastatic serve as a biomarker for the late stage of gastric cancers, especially basal cell cancer and medulloblastoma (43). Cyclopamine Hh signaling associated ones. Although additional studies are potently inhibits the proliferation of gastric cancer cells, which needed to understand how the level and/or activity of Arl13b is express high levels of Smo (9). However, a de novo mutation in regulated in these gastric cancers, our current study offers useful Smo (W535L, SmoM2) results in tumor resistance against GDC- information for future precision oncology with a new biomarker 0449 or other Smo inhibitors (37). Upregulation of Arl13b for this type of cancer. significantly inhibited the activity of cyclopamine (Supplemen- tary Fig. S2H). Identification of peptides or small compounds that Disclosure of Potential Conflicts of Interest interfere with the Arl13b–Smo interaction might offer a way to No potential conflicts of interest were disclosed. overcome the resistance caused by Smo mutation, and to develop a new molecule-targeted therapy against Smo-related cancers by Authors' Contributions synergizing the effect of Smo inhibitors. Conception and design: S. Luo Development of methodology: L. Chen Acquisition of data (provided animals, acquired and managed patients, Arl13b serves as a biomarker of cancer provided facilities, etc.): J. Shao, L. Xu, L. Chen, X. Xie, W. Shi, H. Xiong, Although the PC, a slender microtubule-based subcellular X. Huang, N. Lu organelle, projects on the surface of most mammalian cells and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, plays key roles in vertebrate development and tissue homeostasis, computational analysis): J. Shao, L. Xu, Q. Lu, C. Shi, H. Rao, N. Lu this organelle is also highly involved in cancers and inherited Writing, review, and/or revision of the manuscript: J. Shao, L. Chen, H. Rao, human diseases, such as cystic kidney disease (44). Genetic H. Lu, S. Luo Administrative, technical, or material support (i.e., reporting or organizing studies in mice have shown that the PC is essential for Hh data, constructing databases): L. Xu, J. Mei signaling transduction (45). Arl13b is a marker of the PC. Recent Study supervision: S. Luo studies have demonstrated that Arl13b regulates cell migration Other (helped in some experimental design and instructions for some invitro and cell-cycle progression (46, 47). However, it remains unclear mechanistic experiments): H. Lu how exactly Arl13b functions in cilia and whether Arl13b plays a role in tumorigenesis. Our studies as presented here not only Acknowledgments illustrate the biochemical function of Arl13b by directly interact- We are grateful to Dr. Cheng Luo, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for technical assistance of protein purification. ing with Smo, stabilizing this Hh signaling protein, and mediating Hh signaling as discussed above, but also unveil the oncogenic Grant Support role of this GTPase protein in gastric cancer. This work was supported in part by grants from the National Natural Science Gastric cancer is one of the most frequent and fatal malignan- Foundation of China (31171359 and 31460305 to S. Luo; 81460376 to L. Xu). cies worldwide (48). It was reported that abnormal activation of H. Lu was supported in part by NIH-NCI grants R01CA095441 and Hh signaling plays a role in the progression of gastric cancer (31). R01CA172468. H. Rao was supported by grant R01GM118350 from the NIH. In this study, by investigating the effects of Ar13b on the pro- The costs of publication of this article were defrayed in part by the gression of gastric cancer cells in vitro and in vivo, we found that payment of page charges. This article must therefore be hereby marked knockdown of Arl13b leads to a significant reduction in the advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. proliferation, migration, and invasion of gastric cancer cells, but overexpression of Arl13b causes a marked increase in the prolif- Received September 14, 2016; revised March 21, 2017; accepted June 5, 2017; eration, migration, and invasion of gastric cancer cells in vitro and published OnlineFirst June 13, 2017.

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Arl13b Promotes Gastric Tumorigenesis by Regulating Smo Trafficking and Activation of the Hedgehog Signaling Pathway

Jia Shao, Linlin Xu, Limin Chen, et al.

Cancer Res 2017;77:4000-4013. Published OnlineFirst June 13, 2017.

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