ANTICANCER RESEARCH 36: 1295-1304 (2016)

Reduced RanBPM Expression Is Associated with Distant Metastasis in Gastric Cancer and Chemoresistance

SHUAI SHAO1*, PING-HUI SUN1, LUCY K. SATHERLEY1, XIANGYU GAO1,2, KE JI1, YI FENG1, YONGNING JIA2, JIAFU JI2, WEN G. JIANG1 and LIN YE1

1Metastasis & Angiogenesis Research Group, Cardiff-China Medical Research Collaborative (CCMRC), Division of Cancer & Genetics, Cardiff University School of Medicine, Heath Park, Cardiff, U.K.; 2Key Laboratory of Carcinogenesis and Translational Research, Gastrointestinal Tumor Center, Peking University Cancer Hospital and Institute, Beijing, P.R. China

Abstract. Aim: Ran binding M (RanBPM) is a patients over the past three decades in the USA, Spain and ubiquitous, nucleocytoplasmic protein that serves as a several European countries (2, 3). Associated and scaffolding molecule. This study aimed to investigate the role such as human epidermal growth factor receptor 2, of RanBPM in gastric cancer. Materials and Methods: vascular endothelial growth factor A, fibroblast growth factor RanBPM expression in human gastric cancer tissue samples receptor and hepatocyte growth factor receptor have become was analyzed using real-time polymerase chain reaction. The targets for biological therapeutic agents (4, 5). Unfortunately, effect of RanBPM on cellular functions was examined in little progress has been made in the treatment of advanced RanBPM-knockdown gastric cells and with in vitro cell or metastatic gastric cancer and median overall survival (OS) functional assays. Results: Gastric tumors with distant remains less than 1 year (6). Therefore, there has been much metastases expressed lower levels of RanBPM transcripts interest in the identification of potential new biomarkers or compared to tumours without detectable metastases molecular targets to aid early diagnosis and facilitate the (p=0.036). RanBPM knockdown in gastric cancer cells development of novel biological therapies. reduced adhesion and promoted survival of gastric cancer Ran binding protein M (RanBPM), also as known as cells after exposure to methotrexate and fluorouracil. RanBP9, is a ubiquitously expressed nucleocytoplasmic Conclusion: RanBPM levels were reduced in gastric tumors protein (7, 8). The RanBPM is located on human with distant metastases. This suggests that loss of RanBPM 6p23 and encodes a protein of 729 amino acids expression may play an important role in gastric cancer tumor in length with a molecular weight of 90 kDa (9). RanBPM development and metastasis. Reduced RanBPM expression is contains three conserved domains, namely specific protein 1 also associated with chemoresistance of gastric cancer cells. and ryanodine receptor (SPRY) domain, platelet-activating factor acetylhydrolase IB subunit alpha (LIS1)-homology Gastric cancer remains the fourth most common cancer and motif/C-terminal to LISH (LISH/CTLH) domain and CT11- the second leading cause of cancer-related deaths worldwide RanBPM (CRA) domain (10). The SPRY domain mediates (1). Although its overall incidence and mortality rates have protein–protein interaction, for example, the interaction decreased, there has been a paradoxical increasing incidence between RanBPM with N-methyl-N’-nitro-N-nitrosoguanidine of non-cardia, intestinal-type gastric cancer in younger (MNNG)-HOS transforming gene (MET) which is the receptor of hepatocyte growth factor (11). Proteins containing LISH/CTLH domain are involved in microtubule dynamics, *Current address: Capital Medical University, 10 Xitoutiao, Feng cell migration and protein homodimerisation (12, 13). Both Tai District, Beijing 100069, China. SPRY and LISH/CTLH domains are also involved in the cytoplasmic retention of the protein (14). The CRA domain Correspondence to: Dr. Lin Ye, GF55, Henry Wellcome Building, mediates interaction between RanBPM and fragile X mental Metastasis & Angiogenesis Research Group, Cardiff-China Medical retardation protein, which may modulate RNA-binding of the Research Collaborative (CCMRC), Institute of Cancer & Genetics, latter (15). In addition to the conserved domains, there exist Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, U.K. Tel: +44 2920687861, e-mail: [email protected] six prototypical SH3-binding domains at the proline-rich N-terminus of the RanBPM protein that are predicted to bind Key Words: RanBPM, gastric cancer, invasion, growth, migration, with high affinity to proto-oncogene SRC (short for sarcoma) matrix metalloproteinase. and growth factor receptor-bound protein 2 (10). RanBPM

0250-7005/2016 $2.00+.40 1295 ANTICANCER RESEARCH 36: 1295-1304 (2016) protein contains two motifs that are important for its nuclear Materials and Methods localization. They are a proline/glutamine-rich motif at the extreme N-terminus which is predominantly responsible for Cell lines. Human gastric cancer cell lines AGS and HGC27 were the nuclear localization of RanBPM, whilst the other motif at obtained from the European Collection of Cell Cultures (Salisbury, the carboxy-terminus also contributes to a lesser extent (14). Wiltshire, UK). PC-3, DU-145, LNCaP, CAPHV10, PZHPV7, RanBPM is ubiquitously expressed in human and murine MDA-MB-231, MCF-7, BT549, BT20, ZR751, A549, SKMES1 cell lines and has been observed in the nucleus, cytoplasm, and MRC5 were purchased from the American Type Culture Collection (Manassas, VA, USA). PNT-1A and PNT-2C2 were plasma membrane and at cell junctions (8, 11, 16-18). kindly provided by Professor N Maitland, University of York. RanBPM can be part of a large protein complex that is HECV was obtained from Interlab (Naples, Italy). The cells were named the CTLH complex (19) and is also known as the maintained in Dulbecco’s modified Eagle's medium/F12 muskelin/RanBPM/CTLH complex (MRCTLH) (20). (DMEM/F12) supplemented with amphotericin B, penicillin, RanBPM has been implicated in a variety of cellular streptomycin, and 10% foetal calf serum (FCS) (Sigma-Aldrich processes, including cell adhesion, migration, microtubule Inc., Poole, Dorset, UK). The cells were incubated at 37˚C with organization and gene transcription (7, 15, 21-25). 5% CO2 and 95% humidity. RanBPM may regulate immune cell functions by Gastric tissues. Gastric adenocarcinoma or Siewert type III gastro- interacting with leukocyte- or lymphocyte-function- oesophageal junction adenocarcinoma tissues (n=245) with matched associated antigen 1 (17) and cluster of differentiation 39 adjacent normal tissues (n=158) were collected immediately after (CD39) (26). RanBPM can also regulate splicing of target surgical resection at the Beijing Cancer Hospital with informed mRNA thus contributing to normal spermatogenesis and consent from the patients. Tissue samples were stored at the Tissue male fertility (27). RanBPM is also critical for the nerve Bank of Peking University Oncology School. Clinical and system due to its interaction with neural cell adhesion histopathological information was recorded. All protocols were reviewed and approved by the Beijing Cancer Hospital Research molecule L1 and plexin A (28, 29). RanBPM is able to Ethics Committee (MTA10062009). simultaneously inhibit cell adhesion and promote amyloid β-peptide production thus contributing to the pathogenesis RNA preparation and reversetranscription–polymerase chain of Alzheimer’s disease (30, 31). Targeting RanBPM has reaction (RT-PCR). Total RNA was isolated from gastric tissues and shown certain therapeutic potential by rescuing the cells using TRI® Reagent (Sigma-Aldrich Inc.). RNA was converted neurodegenerative changes (32). to cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad RanBPM promotes pro-apoptotic pathways in response to Laboratories Ltd., Hercules, CA, USA). Following reverse transcription, PCR was performed using REDTaq® ReadyMix™ DNA damage (33). P73l promotes cell-cycle arrest and PCR Reaction Mix (Sigma-Aldrich Inc.). Primer sequences are apoptosis, while NH2-terminally truncated forms of p73 are listed in Table I. Cycling conditions were preheating at 94˚C for 5 known to have oncogenic potential (34). Binding of min, followed by 30 cycles of 30 sec at 94˚C for denaturation, 30 RanBPM with p73 significantly enhances the pro-apoptotic sec at 55˚C for annealing and 40 sec at 72˚C for elongation, activity of p73 by inhibiting its ubiquitination and extending followed by a final 10 min extension at 72˚C. The PCR products its half-life (34, 35). RanBPM is an active binding partner of were separated on a 1% agarose gel stained with Sybr Safe DNA macrophage galactose (Gal)-type C-type lectin gene 1 gel stain (Invitrogen Ltd., Paisley, Scotland, UK) and then visualized under ultraviolet light. The quality of DNA was verified and (MGL-1) and promotes MGL-1 tumor suppressor activity by normalized using glyceraldehyde 3-phosphate dehydrogenase stabilizing MGL-1 and prolonging its half-life through the (GAPDH) as a house-keeping gene. ubiquitin-proteasomal pathway (36). Previous studies have also shown that RanBPM prevents deregulated expression of Quantitative PCR (QPCR). Real-time quantitative PCR was used to some genes such as RON (Recepteur d’ Origine Nantais) determine the level of RanBPM mRNA expression using the tyrosine kinase, the adhesion molecule L1 cell adhesion prepared cDNA samples. The reaction was carried out in molecule (L1CAM), and transcription factor E74-Like Factor StepOnePlus Real-Time PCR system (Applied Biosystems, Waltham, MA, USA), with the following cycling conditions: 95˚C 3 (ELF3) (37). In short, these findings suggest that RanBPM for 10 min, followed by 100 cycles of 95˚C for 10 seco, 55˚C for functions as a putative tumor suppressor to modulate cellular 35 sec, and 72˚C for 10 sec. GAPDH was used as an internal control events, implicating that it might be involved in cancer to normalize RanBPM . An internal standard, development. simultaneously amplified with the samples, was used to generate the However, little is known about the role of RanBPM in levels of the transcripts. malignant diseases. The aim of the current study was to examine the expression of RanBPM in human gastric cancer Construction of ribozyme transgenes and knockdown of RanBPM in gastric cancer cells. Hammerhead ribozymes targeted against and determine any association with disease progression and human RanBPM were designed based on the secondary structure of patient survival. Moreover, we also examined the influence the mRNA generated using Zuker’s RNA Mfold program (38). The of RanBPM on gastric cancer cell functions using in vitro ribozymes were synthesized and cloned into pEF6/V5-His-TOPO functional assays. plasmid vector (Invitrogen Ltd.). The ribozymes were then

1296 Shao et al: RanBPM in Gastric Cancer

Table I. Primer sequences used in the current study.

Gene Sense primers (5’-) Antisense primers (5’-)

RanBPM atcggcctctctcagaacaa aatttggcggtaggtcagtg RanBPM (Q-PCR) ttaccatggggatgatggac actgaacctgaccgtacaaacagccaatgacatcacca RanBPM ribozyme ctgcaggttcgtccacggccgggtagactgatgagtccgtgagga actagttgcagcggcggctgaagcgtttcgtcctcacggact GAPDH ggctgcttttaactctggta gactgtggtcatgagtcctt GAPDH (Q-PCR) ctgagtacgtcgtggagtc actgaacctgaccgtacagagatgatgacccttttg

RanBPM: Ran binding protein M; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Q-PCR: quantitative polymerase chain reaction.

transfected into AGS and HGC27 cells using a Gene Pulser Xcell™ Table II. Ran binding protein M (RanBPM) expression in a gastric electroporation system (Bio-Rad). This was followed by selection cancer cohort. of successfully transfected cells using selection medium containing 5 μg/ml blasticidin (Sigma-Aldrich Inc.) for up to 2 weeks. TNM No. of RanBPM transcripts p-Value Following verification, stable transfectants were cultured in staging samples (copies), mean±SE maintenance medium containing 0.5 μg/ml blasticidin. The primer sequences used for the RanBPM ribozymes are shown in Table I. Normal 158 4.14×1015±3.14×1015 15 15 Wounding/migration assay. The migration assay was carried out as Tumour 245 8.98×10 ±3.90×10 0.33 T stage previously described (39). A total of 3×105 cells were seeded into each T1 12 7.83×1015±7.83×1015 well of a 24-well plate. Once the cells reached confluence, the T2 20 9.67×1014±9.51×1014 monolayer of cells was scraped to produce a linear wound using a fine- T3 29 4.24×1013±2.62×1013 0.058 gauge needle. The medium was changed twice to discard any floating T4 177 1.14×1016±5.36×1015 0.036 cells, and the plate was then returned to the incubator to maintain a N stage constant temperature of 37˚C. Cells were photographed every hour for N0 52 5.50×1015±4.24×1015 8 h using a CCD camera attached to a microscope at ×20 N1 34 7.82×1015±5.59×1015 0.74 magnification. The distance between the edges of the wounding gap N2 48 2.15×1016±1.80×1015 0.39 was measured using ImageJ software (http://imagej.net/index.html, N3 105 5.30×1015±2.77×1015 0.97 National Institutes of Health, Bethesda, MD, USA). M stage M0 215 1.02×1016±4.44×1015 Cell-matrix adhesion assay. The cell-matrix adhesion assay was M1 29 1.86×1014±1.34×1014 0.025 performed as previously described (40). A total of 2×104 cells were added to each well of a 96-well plate which had been pre-coated with 5 μg of Matrigel™ (BD Biosciences, Oxford, UK), air-dried and rehydrated with serum-free medium. After 40 minutes of incubation, non-attached cells were removed by washing with incubated for 3 days. The cells that had migrated to the other side of balanced salt solution three times, and adhered cells were fixed with the insert through the matrix were fixed with 4% formaldehyde 4% formaldehyde (v/v) for 30 min, prior to staining with 0.5% (v/v), and stained with 0.5% crystal violet (w/v). The absorbance of crystal violet (w/v). The number of adhered cells was counted under the colorimetric products was determined at a wavelength of 540 a microscope from five random fields per well, and the absorbance nm using a spectrophotometer (ELx800™; Bio-Tek) after of colorimetric products was analysed after solubilisation with 10% solubilization with 10% acetic acid. acetic acid using a spectrophotometer (ELx800™; Bio-Tek, Winooski, VT, USA) at a wavelength of 540 nm. Statistical analysis. Data were analysed using SigmaPlot software (version 12.5; SPSS, Inc., Chicago, IL, USA). The relationship In vitro cell-growth assay under normal culture conditions and after between RanBPM expression and tumor grade, TNM staging and exposure to methotrexate (MTX) and fluorouracil (5-FU). Cells nodal status was assessed using the Mann–Whitney test for non- (2,500/200 μl) were seeded into each well of a 96-well plate, and parametric data. The statistical comparisons between the RanBPM ΔRanBPM ΔRanBPM treated with MTX or 5-FU at 2.5 μg/ml and 100 μg/ml respectively. knockdown cell lines (HGC27 and AGS ) and the Plates were fixed with 4% formaldehyde (v/v) after 1, 3 and 5 days. control cell line, using cells containing a closed pEF plasmid vector pEF pEF After staining the cells with 0.5% crystal violet (w/v) and solubilizing (HGC27 and AGS ), were made using Student’s two sample t- with 10% acetic acid, the absorbance was measured using a test and two-way ANOVA. spectrophotometer (ELx800™; Bio-Tek) at a wavelength of 540 nm. Results In vitro invasion assay. Trans-well inserts with 8 μm size pores were coated with 50 μg of Matrigel™ (BD Biosciences) and air-dried. Expression of RanBPM in gastric cancer. We first Following rehydration, 2×104 cells were seeded into each insert and determined the expression of RanBPM transcript levels in

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Figure 1. Expression of Ran binding protein M (RanBPM) in gastric (A), prostate (B), breast (C), and lung (D) cancer cell lines were detected using reverse transcription polymerase chain reaction.

gastric cancer tissues using real-time PCR. RanBPM selection with blasticidin (5 μg/ml) for up to two weeks, expression was detected in both gastric cancer tissues and RanBPM expression was determined using real-time PCR adjacent normal tissues. Slightly increased levels of RanBPM (Figure 2). Reduced RanBPM transcripts were seen in both were found in the tumour samples, in comparison with HGC27ΔRanBPM and AGSΔRanBPM cells that were transfected matched background tissues, although this did not reach with anti-RanBPM ribozymes in comparison with the statistical significance (p=0.33) (Table II). In an examination corresponding control cells. The RanBPM-knockdown cells of cDNA libraries of different cancer cell lines using were then used in the subsequent experiments. conventional PCR, the expression of RanBPM was detected in several cancer cell lines, including two gastric cancer cell Knockdown of RanBPM reduced gastric cancer cell adhesion. lines, HGC27 and AGS, seven prostatic cell lines and five The adhesive ability of HGC27ΔRanBPM and AGSΔRanBPM breast cancer cell lines, although there were slight cells was examined using an in vitro cell-matrix adhesion differences in their expressions (Figure 1). RanBPM was also assay, that assessed the capacity of cancer cells to adhere to expressed by HECV (vascular endothelia) and MRC5 an artificial basement membrane. A significant decrease in (fibroblast) cell lines, which also exist in the tumours. cell adhesion was seen in both HGC27ΔRanBPM and The level of RanBPM transcript expressed in tumors with AGSΔRanBPM cells compared to HGC27pEF cells (p<0.001), distant metastases (M1) was significantly lower than that seen in and AGSpEF cells (p<0.05) (Figure 3). tumors without distant metastasis (p=0.025) (Table II). In contrast to the reduced expression in tumors with distant RanBPM knockdown promoted cell migration. We metastases, differential expression was seen in tumours with investigated the influence of RanBPM on cell migration by different levels of local invasion. The lowest expression of measuring the movement of cells after wounding. RanBPM was seen in tumors with invasion confined to the Knockdown of RanBPM resulted in a significant increase in lamina propria, muscularis mucosa or submucosa (T1) cell motility in HGC27ΔRanBPM cells compared to compared to its expression in T4 tumors where disease invaded HGC27pEF control cells, particularly in the first 6 h after the serosa (p=0.036). Moderate expression levels were seen in wounding (Figure 4A). RanBPM knockdown in AGS cells tumours invading the muscularis mucosa (Table II). did not exhibit such a strong effect on cell migration, although AGSΔRanBPM cells tended to move slightly faster Creation of gastric cancer cell sub-lines with RanBPM than the AGSpEF control cells (Figure 4B). knockdown. The two RanBPM-knockdown gastric cancer cell lines (HGC27ΔRanBPM and AGSΔRanBPM) were created by Effect of RanBPM knockdown on invasive capacity of transfection with anti-RanBPM ribozyme transgenes. After gastric cancer cell. We examined the influence of RanBPM

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Figure 2. Knockdown of Ran binding protein M (RanBPM) in gastric cancer cell lines. A: Knockdown of RanBPM in HGC27 cells was verified using real-time quantitative polymerase chain reaction. B: Figure 3. Ran binding protein M (RanBPM) promotes cell-matrix Reduced expression of RanBPM was seen in AGSΔRanBPM cells adhesion. A: Knockdown of RanBPM (AGSΔRanBPM) reduced adhesion compared to AGS WT and AGSpEF. HGC27 WT and HGC27 pEF: of HGC27 cells. B: A decrease was also seen in AGS with RanBPM HGC27 wild-type and HGC27 cells transfected with empty plasmids, knockdown in comparison with cells transfected with empty plasmids respectively. HGC27ΔRanBPM: HGC27 cells with RanBPM knockdown. (AGSpEF). Blue-dyed cells were AGSpEF adhered to the matrix Data are the mean±SD/SEM; n=3; *p<0.05. membrane (C), Blue-dyed cells were AGSΔRanBPM adhered to matrix membrane (D). Data are the mean±SD/SEM; n=6; **p<0.01.

on the invasive capacity of both HGC27 and AGS cell lines. microarray study, analysing differential gene expression in Interestingly, knockdown of RanBPM exerted a differential HT29 colon cancer cells sensitive, or resistant, to MTX (41) effect on cancer cell invasion between the two gastric (data accessible at NCBI GEO database, accession cancer cell lines. Reduced cancer cell invasion was seen in GDS3330). In our analysis of the GEO profile of RanBPM in HGC27ΔRanBPM cells compared to HGC27pEF (Figure 5A), this database, reduced expression of RanBPM was seen in whilst increased cancer cell invasion was observed in HT29 MTX-resistant cells compared with MTX-sensitive AGSΔRanBPM cells compared to AGSpEF control cells HT29 cells (p=0.0026) (Figure 6). In order to inquire into the (Figure 5B). role of RanBPM during chemotherapy, optimization experiments using different concentrations of the two drugs Involvement of RanBPM in resistance of gastric cancer cells were performed. Appropriate dosages of 5-FU for HGC27 to chemotherapeutic agents. We investigated the influence of and AGS were identified as 200 μg/ml and 100 μg/ml RanBPM on cell proliferation in vitro over a period up to 5 respectively, and the appropriate concentrations of MTX were days using the colorimetric method described in the Materials 10 μg/ml and 2.5 μg/ml respectively. After exposure (for up and Methods section. Both HGC27ΔRanBPM and to 5 days) to MTX and 5-FU, the survival rates of AGSΔRanBPM cells exhibited an increase in in vitro cell HGC27ΔRanBPM and AGSΔRanBPM were higher than those of proliferation compared to respective control cell lines, but not HGC27pEF and AGSpEF control cells. In comparison to MTX, to a significant level. Selga et al. recently performed a 5-FU was more effective, as reflected in the survival rates of

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Figure 4. Ran binding protein M (RanBPM) reduces cell migration. A: HGC27ΔRanBPM knockdown cells moved farther in comparison with HGC27 cells transfected with empty plasmid (HGC27pEF) as control after 8 h. B: Increased migration was also seen in AGS cells with RanBPM knockdown (AGSΔRanBPM) compared to AGSpEF. **p<0.01 vs. pEF control. Data are the mean±SD/SEM; n=3.

Figure 6. Expression of Ran binding protein M (RanBPM) in HT29 methotrexate (MTX)-resistant cells. Selga et al., recently performed a Figure 5. Ran binding protein M (RanBPM) affects invasion of gastric microarray analysing differential gene expression in HT29 colon cancer cancer cells. Knockdown of RanBPM (ΔRanBPM) results in reduced cells sensitive, or resistant, to MTX (data accessible at NCBI GEO invasiveness of HGC27 cells, whereas an increase was seen in AGS database, accession GDS3330). cells. Data are the mean±SD/SEM; n=3; *p<0.05.

cells. AGSΔRanBPM exhibited significantly higher survival Discussion rates compared to the AGSpEF control cells on exposure to MTX (Figure 7D). In contrast, HGC27 cells tended to be less In the present study, we examined the expression of RanBPM responsive to MTX treatment (Figure 7B). A significant in both gastric cancer tissues and gastric cancer cell lines. In increase in chemoresistance was evident in both HGC27 and the tissue samples obtained from patients, we identified AGS RanBPM-knockdown cells when they were treated with elevated expression of RanBPM in gastric tumor tissue though 5-FU for 5 days (Figure 7A and C). this was not statistically different to the levels of adjacent

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Figure 7. Ran binding protein M (RanBPM) is involved in chemotherapy resistance in gastric cancer cells. A: HGC27 cells with RanBPM knockdown (ΔRanBPM) showed stronger tolerance to fluorouracil (5-FU) (200 μg/ml) when treated for 3 days and 5 days. B: Treatment with 10 μg/ml methotrexate (MTX) for 3 days and 5 days revealed higher survival of HGC27ΔRanBPM cells in comparison to HGC27pEF as control. C, D: Higher survival rate was also seen in AGSΔRanBPM cells when treated with 100 μg/ml 5-FU and 2.5 μg/ml MTX for 3 and 5 days respectively. *p<0.05 vs. Day 1 control; **p<0.01 vs. Day 1 control; #p<0.05 vs. pEF control; ##p<0.01 vs. pEF control. Data are the mean±SEM; n=6.

background gastric tissues. Expression of RanBPM was also local invasion, the expression of RanBPM in tumors with detected in gastric cancer cell lines (HGC27 and AGS). distant metastases, namely M1 tumors, was significantly lower Screening of RanBPM mRNA expression in other cell lines compared to levels seen in tumours with no distant metastases. showed that RanBPM was also ubiquitously expressed by other This suggests that a different role may be played by RanBPM cancer cell lines and non-cancer cell lines (HECV and MRC5). in distant metastases of gastric cancer, that is in line with These results suggest that the expression level of RanBPM in findings by Jun-Dong Wei et al. showing that RanBPM acts as tissues may be heterogeneous. Further investigations using a negative regulator of cell motility (42). However, its exact fluorescence in situ hybridization or immunohistochemical impact on cellular functions and cell–cell interactions during staining may help clarify the expression of RanBPM and the metastatic process is yet to be investigated. determine its distribution in tissues and cells. RanBPM has been shown to act as a scaffolding protein, Aberrant expression of RanBPM was seen in tumours with assisting signal transduction and protein–protein interactions. different levels of local invasion. For example, the most locally For example, the role played by RanBPM in the invasive tumours (T4) expressed higher levels of RanBPM semaphorin3A/plexinA1 pathway and the interaction between compared to T1 tumours. This suggests that RanBPM may play amyloid precursor protein and lipoprotein receptor-related a promotory role during local invasive growth of gastric cancer. protein (29, 43). In the current study, we examined the impact In contrast to its expression in tumors with different degrees of of RanBPM knockdown on the cellular functions of gastric

1301 ANTICANCER RESEARCH 36: 1295-1304 (2016) cancer cells. RanBPM knockdown exerted differential effects References on adhesion, motility, invasion and growth of HGC27 and AGS cells. Knockdown of RanBPM impaired cell-matrix adhesion 1 Ferro A, Peleteiro B, Malvezzi M, Bosetti C, Bertuccio P, Levi F, of gastric cancer cells. It has been reported that intercellular Negri E, La Vecchia C and Lunet N: Worldwide trends in gastric adhesion molecule-1, integrins, and other adhesion-related cancer mortality (1980-2011), with predictions to 2015, and incidence by subtype. Eur J Cancer 50: 1330-1344, 2014. proteins are essential in cell adhesion, which mediates the 2 Anderson WF, Camargo MC, Fraumeni JF, Jr., Correa P, critical response of cells to the surrounding environment (44). Rosenberg PS and Rabkin CS: Age-specific trends in incidence of Therefore, further investigation is required to elucidate how noncardia gastric cancer in us adults. JAMA 303: 1723-1728, 2010. RanBPM is involved in the coordination of cell adhesion 3 Sonnenberg A: Time trends of mortality from gastric cancer in through interactions with these adhesion molecules. RanBPM europe. Dig Dis Sci 56: 1112-1118, 2011. can regulate cell migration by coordinating relevant signalling 4 Yoshikawa T, Tsuburaya A, Kobayashi O, Sairenji M, Motohashi via transmembrane receptors (36). On the other hand, the H, Yanoma S and Noguchi Y: Plasma concentrations of VEGF and BFGF in patients with gastric carcinoma. Cancer Lett 153: results of our wounding assay show that knockdown of 7-12, 2000. RanBPM in gastric cancer cells enhanced their motility. This 5 Gravalos C and Jimeno A: HER2 in gastric cancer: A new indicates that RanBPM may be a mediator involved in prognostic factor and a novel therapeutic target. Ann Oncol 19: inhibiting gastric cancer cell migration. A number of 1523-1529, 2008. molecules, such as 3-phosphoinositide-dependent protein 6 Cervantes A, Roda D, Tarazona N, Rosello S and Perez-Fidalgo kinase-1, have been demonstrated to participate in signal JA: Current questions for the treatment of advanced gastric transductions that regulate cell migration (45, 46). In addition, cancer. Cancer Treat Rev 39: 60-67, 2013. RanBPM may play an important role in the signal transduction 7 Nakamura M, Masuda H, Horii J, Kuma K, Yokoyama N, Ohba T, Nishitani H, Miyata T, Tanaka M and Nishimoto T: When of different pathways, including extracellular signal-regulated overexpressed, a novel centrosomal protein, RANBPM, causes kinase1/2, wingless-related integration site, NOTCH, and ectopic microtubule nucleation similar to gamma-tubulin. J Cell phosphatidylinositol-4,5-bisphosphate 3-kinase/ Protein kinase Biol 143: 1041-1052, 1998. B (Akt) pathways (37). The RanBPM knockdown cells not 8 Nishitani H, Hirose E, Uchimura Y, Nakamura M, Umeda M, only became less adhesive, but also displayed enhanced Nishii K, Mori N and Nishimoto T: Full-sized RANBPM cDNA motility to a certain degree. However, the involvement of the encodes a protein possessing a long stretch of proline and aforementioned pathways is yet to be clarified. glutamine within the N-terminal region, comprising a large protein complex. Gene 272: 25-33, 2001. We further focused on cancer cell invasion of RanBPM- 9 Wennerberg K, Rossman KL and Der CJ: The RAS superfamily knockdown cells. Interestingly, with knockdown of RanBPM, at a glance. J Cell Sci 118: 843-846, 2005. HGC27 cells appeared to be less invasive whilst AGS 10 Murrin LC and Talbot JN: RanBPM, a scaffolding protein in the knockdown cells exhibited enhanced invasiveness. This immune and nervous systems. J Neuroimmune Pharmacol 2: suggests that different interacting partners of RanBPM may 290-295, 2007. exist in the two gastric cancer cell lines, leading to differential 11 Wang D, Li Z, Messing EM and Wu G: Activation of RAS/ERK responses in their invasiveness. Further investigation into the pathway by a novel MET-interacting protein RanBPM. J Biol 277 underlying mechanism is yet to be carried out. Chem : 36216-36222, 2002. 12 Gerlitz G, Darhin E, Giorgio G, Franco B and Reiner O: Novel Although knockdown of RanBPM in gastric cancer cells did functional features of the LISH domain: Role in protein not remarkably affect their in vitro growth, enhanced survival dimerization, half-life and cellular localization. Cell Cycle 4: was seen in both HGC27 and AGS RanBPM knockdown cells 1632-1640, 2005. when they were exposed to chemotherapeutic drugs. RanBPM 13 Mateja A, Cierpicki T, Paduch M, Derewenda ZS and Otlewski J: has been demonstrated to be an activator of apoptotic The dimerization mechanism of LIS1 and its implication for pathways induced by DNA damage (33). Together with the proteins containing the LISH motif. J Mol Biol 357: 621-631, 2006. findings from the current study, it is suggested that 14 Salemi LM, Loureiro SO and Schild-Poulter C: Characterization of RanBPM molecular determinants that control its subcellular RanBPM plays a role in differential responses to certain localization. PLoS One 10: e0117655, 2015. chemotherapeutic agents and consequently influences tumour 15 Menon RP, Gibson TJ and Pastore A: The C terminus of fragile chemoresistance. Further investigation will help highlight the X mental retardation protein interacts with the multi-domain potential application of RanBPM in personalized disease Ran-binding protein in the microtubule-organising centre. J Mol management, for example in the selection of appropriate Biol 343: 43-53, 2004. chemotherapeutic agents. 16 Umeda M, Nishitani H and Nishimoto T: A novel nuclear protein, TWA1, and muskelin comprise a complex with Acknowledgements RanBPM. Gene 303: 47-54, 2003. 17 Denti S, Sirri A, Cheli A, Rogge L, Innamorati G, Putignano S, The Authors wish to thank Cancer Research Wales and Albert Hung Fabbri M, Pardi R and Bianchi E: RanBPM is a phosphoprotein Foundation for their kind support. Miss Shuai Shao is a recipient of that associates with the plasma membrane and interacts with the the Chinese Medical Research Studentship of Cardiff University. integrin LFA-1. J Biol Chem 279: 13027-13034, 2004.

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FASEB J 26: 1672-1681, 2012. 32 Woo JA, Boggess T, Uhlar C, Wang X, Khan H, Cappos G, Joly- Amado A, De Narvaez E, Majid S, Minamide LS, Bamburg JR, Morgan D, Weeber E and Kang DE: RanBP9 at the intersection between cofilin and abeta pathologies: Rescue of neurodege- nerative changes by RanBP9 reduction. Cell Death Dis 6: 1676- 1690, 2015. 33 Atabakhsh E, Bryce DM, Lefebvre KJ and Schild-Poulter C: RanBPM has proapoptotic activities that regulate cell death Received January 18, 2016 pathways in response to DNA damage. Mol Cancer Res 7: 1962- Revised February 19, 2016 1972, 2009. Accepted February 22, 2016

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