KIAA1324 Suppresses Gastric Cancer Progression by ... · ities and provides new insight into the...

Molecular and Cellular Pathobiology

KIAA1324 Suppresses Gastric Cancer Progressionby Inhibiting the Oncoprotein GRP78Jin Muk Kang1, Sujin Park1, Staci Jakyong Kim1,2,3, Hyojung Kim1, Bona Lee1,4, Junil Kim1,Jinah Park1,5, Shin Tae Kim1,6, Han-Kwang Yang7,8,Woo Ho Kim7,9, and Seong-Jin Kim1,10

Abstract

Recent advances in genome and transcriptome analysis havecontributed to the identification of many potential cancer-related genes. Furthermore, biological and clinical investiga-tions of the candidate genes provide us with a better under-standing of carcinogenesis and development of cancer treat-ment. Here, we report a novel role of KIAA1324 as a tumorsuppressor in gastric cancer. We observed that KIAA1324 wasdownregulated in most gastric cancers from transcriptomesequencing data and found that histone deacetylase wasinvolved in the suppression of KIAA1324. Low KIAA1324 levelswere associated with poor prognosis in gastric cancer patients.In the xenograft model, KIAA1324 significantly reduced tumor

formation of gastric cancer cells and decreased development ofpreformed tumors. KIAA1324 also suppressed proliferation,invasion, and drug resistance and induced apoptosis in gastriccancer cells. Through protein interaction analysis, we identifiedGRP78 (glucose-regulated protein 78 kDa) as a KIAA1324-binding partner. KIAA1324 blocked oncogenic activities ofGRP78 by inhibiting GRP78–caspase-7 interaction and sup-pressing GRP78-mediated AKT activation, thereby inducingapoptosis. In conclusion, our study reveals a tumor suppressiverole of KIAA1324 via inhibition of GRP78 oncoprotein activ-ities and provides new insight into the diagnosis and treatmentof gastric cancer. Cancer Res; 75(15); 3087–97. �2015 AACR.

IntroductionGastric cancer is the fourth most common type of cancer and

the second leading cause of death from cancer worldwide (1).Although clinical treatment for gastric cancer has improved,gastric cancer therapy still remains challenging due to the diffi-culty in early detection of gastric cancer and complexities of thedisease (2). Therefore, it is crucial to investigate novel genes thatgovern the development and progression of gastric cancer toelucidate the process of gastric carcinogenesis and develop effec-tive gastric cancer treatments.

Recently, with advances in next-generation sequencing (NGS)technology, analysis of genomic and transcriptomic alterations ingastric cancer patients has been used to develop innovativemethods for diagnosis and treatment of gastric cancer (3, 4).Molecular drivers of gastric cancer have been suggested throughNGS-based genomic and transcriptomic analyses of mutation,deletion, amplification, fusion, and expression level. However,because gastric cancer is regarded as a heterogeneous and complexdisease, the biological functions and clinical relevance of thecandidate driver genes should be scrutinized for their applicationsin gastric cancer therapy.

GRP78 (glucose-regulated protein 78 kDa) is a well-knowntherapeutic target gene that is highly activated in variouscancers, including gastric cancer (5–8). GRP78, also known asHSPA5 (heat-shock protein alpha 5), is a chaperone involved inprotein folding in the endoplasmic reticulum (ER) andincreases cell survival against apoptotic stresses, includinganticancer drugs, by interacting with proapoptotic factors suchas caspase-7 and BIK (9–11). In cancer, GRP78 is often relo-calized to the plasma membrane (12, 13). Cell surface GRP78activates AKT signaling through interaction with PI3K, therebypromoting tumorigenesis (14, 15). GRP78 inhibitors, such assmall molecules and specific binding peptides, cause growthinhibition and apoptosis of cancer cells. Therefore, more effec-tive drugs targeting GRP78 have been developed for cancertherapy (12, 16, 17). GRP78 has also been investigated andsuggested as a biomarker and therapeutic target for gastriccancer therapy (18–20).

KIAA1324 gene, also known as EIG121 (estrogen-inducedgene 121), encodes a 1,013 amino acid (a.a.) transmembraneprotein that is highly conserved among species (21). Thecorrelation between KIAA1324 expression and prognosisin endometrial, ovarian, and pancreatic cancer patients was

1CHACancer Institute, College of Life Science, CHAUniversity, Seong-nam City, Republic of Korea. 2International Institute for IntegrativeSleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.3School of Integrative and Global Majors, University of Tsukuba, Tsu-kuba, Japan. 4College of Pharmacy, Sookmyung Women's University,Seoul, Republic of Korea. 5College of Pharmacy, CHA University,Seongnam City, Republic of Korea. 6Division of Pulmonary Medicine,Department of Internal Medicine, CHA Gangnam Medical Center,College of Medicine, CHA University, Republic of Korea. 7CancerResearch Institute, SeoulNationalUniversity, Seoul, RepublicofKorea.8Department of Surgery, Seoul National University College of Medi-cine, Seoul, Republic of Korea. 9Department of Pathology, SeoulNational University College of Medicine, Seoul, Republic of Korea.10Department of Biomedical Science, College of Life Science, CHAUniversity, Seongnam City, Republic of Korea.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Seong-Jin Kim, CHA Cancer Institute, CHA University,335 Pangyo-ro, Bundang-ku, Seongnam City, Kyunggi-do 463-400, Republic ofKorea. Phone: 82-31-881-7131; Fax: 82-31-881-7185; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3751

�2015 American Association for Cancer Research.

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previously reported (21–23), but the role of KIAA1324 ingastric cancer has not been investigated yet. Deng and collea-gues demonstrated that KIAA1324 localizes at the plasmamembrane and endomembranes and is involved in autophagy(24). However, the biological functions of KIAA1324 are stillpoorly understood.

Here, we identified KIAA1324 as a candidate gastric tumorsuppressor based on our transcriptome sequencing data from thetissues of gastric cancer patients. To investigate the potentialtumor suppressive role of KIAA1324 in gastric cancer, we analyzedthe correlation between KIAA1324 expression and gastric cancerpatient prognosis and evaluated the tumorigenic abilities ofgastric cancer cells expressing exogenous KIAA1324 using in vitroand in vivo assays. We observed that low KIAA1324 levels wereassociated with poor prognosis in gastric cancer patients.KIAA1324 inhibited growth, invasiveness, and tumorigenic activ-ity of gastric cancer cells and induced apoptosis by blocking theoncogenic activities of GRP78. Taken together, our data suggest anovel role of KIAA1324 as a tumor suppressor and a prognosticindicator in gastric cancer.

Materials and MethodsPrimary gastric cancer tissues

Fifty pairs of gastric cancer and normal matched control tissueswere obtained from the gastric cancer depository of the Gastro-intestinal Division in Department of Surgery at Seoul NationalUniversity Hospital (Seoul, Republic of Korea). The InstitutionalReview Board of the SeoulNational University Hospital approvedmanagement of the tissue depository and use of the tissues (IRBno. H-0806-072-248).

Cell culture and transfectionHuman gastric cancer cell lines,MKN28, AGS, and SNU16were

obtained from Korean Cell Line Bank, authenticated by shorttandem repeat profiling. These cell lines were expended and usedwithin 10 passages. The cells were maintained in RPMI1640containing 25 mmol/L HEPES (WelGENE) supplemented with10% FBS (WelGENE), 100 U/mL of penicillin, and 100 mg/mL ofstreptomycin (WelGENE). 293T cells were maintained in DMEM(WelGENE) supplemented with 10% FBS, 100 U/mL of penicil-lin, and 100 mg/mL of streptomycin. FuGENE6 (Roche AppliedScience) was used as a transfection reagent.

Establishment of gastric cancer cells expressing the KIAA1324gene or KIAA1324 shRNA

We established MKN28 and AGS cell lines expressingKIAA1324 in a doxycycline-dependent manner (tet-on) using aretroviral system. SNU16 cell lines stably expressing control orKIAA1324 shRNAwere generated as described previously (25). Toestablish MKN28 and AGS cell lines expressing KIAA1324 in adoxycycline-dependent manner (tet-on), the human KIAA1324gene was cloned into pCMV-3HA vector (Clontech). For virusgeneration, 3HA-tagged KIAA1324 gene was inserted into a ret-roviral vector, pRetroX (Clontech). Viruses containing KIAA1324gene or tetracycline response activator gene were producedaccording to the manufacturer's protocol (Cell Biolabs, Inc.).MKN28 and AGS tet-on KIAA1324 cells were generated by infec-tion with these viruses and selected by 2 mg/mL G418 and2 mg/mL puromycin. For KIAA1324 knockdown, lentiviral con-structs containing KIAA1324 shRNA (TRCN0000263309 and

TRCN0000263310) were purchased from Sigma. SNU16 celllines stably expressing control or KIAA1324 shRNA were gener-ated by lentiviral infection and selected with 2 mg/mL puromycin.

Annexin V–positive cell population analysisAnnexin V staining was performed with BD Pharmingen

Annexin V Apoptosis Detection Kit (BD Biosciences) accordingto themanufacturer's protocol. Cells were trypsinized andwashedtwice with PBS. The cells were incubated in binding buffer con-taining Annexin V-FITC and propidium iodide (PI). Stained cellswere analyzed by flow cytometry using CELLQUEST program(Becton Dickinson).

Statistical analysesAll quantitative data are expressed as the mean � SD. Kaplan–

Meier analysis and Pearson c2 tests were performed using SPSSversion 21.0 statistical software (IBM SPSS). Student t tests andone-way ANOVA were conducted using GraphPad Prism version5 (GraphPad Software Inc.). P < 0.05 was considered statisticallysignificant.

ResultsKIAA1324 expression is suppressed in most gastric cancers

To identify novel gastric cancer–related genes, we analyzedtranscriptome sequencing data acquired from 16 paired normaland tumor tissues of gastric cancer patients and 18 gastric cancercell lines (26) and sorted differentially expressed genes (DEG)between gastric cancer and normal tissues (P < 0.01 with >2-foldchange; Fig. 1A). Among the DEGs, we focused on KIAA genes,which were initially identified through the Kazusa cDNA project,because unknown genes often provide new insight into under-standing cancer, and most of KIAA genes have remained func-tionally uncharacterized (27). We confirmed that expressionpatterns of the selected KIAA genes in gastric cancer cell lineswere similar to those in primary tumor tissues (Fig. 1A). Inparticular, our data showed upregulation of KIAA1524, which isalso known asCIP2A (cancerous inhibitor of protein phosphatase2A) and was reported as an oncogene in gastric cancer (28).However, other KIAA genes have been poorly investigated, espe-cially in gastric cancer. In this study, we focused our attention onthe role of KIAA1324, which is the most significant downregu-lated KIAA gene in this set of gastric tissues and cancer cell lines.Suppressed KIAA1324 expression in most tumor tissues andgastric cancer cell lines was further validated using quantitativereverse transcription PCR (qRT-PCR; Supplementary Fig. S1). Wealso examined KIAA1324 expression in paired tissues of addi-tional gastric cancer patients using qRT-PCR. As expected,KIAA1324 expression was significantly suppressed in cancer tis-sues (P < 0.0005; Fig. 1B). Paired comparison analysis of thetissues showed that 78%of patients had low levels ofKIAA1324 atthe tumor site compared with the normal region (Fig. 1C). Theseresults were further supported by public microarray data(GSE13861; ref. 29), which showed low KIAA1324 expression ingastric tumor tissues (P ¼ 0.0002; Fig. 1D).

The loss of gene expression is mainly caused by genetic alter-ation or epigenetic modification. Therefore, to investigate themechanism by which KIAA1324 is regulated in gastric cancer celllines, we first examined the correlation between gene copy num-ber variation (CNV) and transcription level using CCLE databaseanalysis (Supplementary Fig. S2). Copy number of KIAA1324

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gene in 29 gastric cancer cell lines was barely changed andKIAA1324 expression was low in most gastric cancer cell lines.This result suggested that suppressed KIAA1324 expression isindependent of copy number variation (CNV). Next, weexplored whether KIAA1324 expression was regulated epige-netically using decitabine, a DNA methylation inhibitor, andMS-275, a synthetic histone deacetylase inhibitor. While MS-275 treatment restored KIAA1324 transcription in these gastriccancer cell lines with negligible expression, decitabine did notinfluence KIAA1324 expression (Fig. 1E). Combination treat-ment with MS-275 and decitabine in MKN1 cells enhancedrestoration of KIAA1324 transcription compared with MS-275treatment alone, but not in MKN28 and SNU638 cells. It hasbeen known that densely methylated DNA associates withtranscriptionally repressive chromatin characterized by thepresence of underacetylated histones, and these two epigeneticprocesses have been dynamically linked (30). Our data suggestthat the density of CpG island methylation and the level ofhistone deacetylation of the KIAA1324 gene vary among thethree cell lines used in our study. Taken together, these resultssuggest that epigenetic inhibition of KIAA1324 may be favored

during carcinogenesis, indicating a possible role of KIAA1324as a tumor suppressor in gastric cancer.

Low levels of KIAA1324 are associated with poor prognosis ingastric cancer patients

To evaluate the clinical impact of KIAA1324, we performed agastric tumor tissuemicroarray using the anti-KIAA1324 antibodyand tumor tissues from 428 patients. As shown in Fig. 2A, thetumor tissues were classified into four groups (negative, weak,moderate, or strong) according to KIAA1324 expression. On thebasis of this classification, we analyzed the cumulative survivalrate of gastric cancer patients who provided the tumor tissuesusing the Kaplan–Meier test (Fig. 2B). We found that lowerKIAA1324 expression was correlated with reduced survival ratesof patients (P<0.001).We also analyzed the relationship betweenKIAA1324 expression and clinicopathologic features (Fig. 2C).Patients aged 65 years or younger had lower KIAA1324 expressioncompared with older patients (P ¼ 0.038). However, KIAA1324expressionwas not associatedwith gender (P¼ 0.389). KIAA1324expression was negatively correlated with tumor invasion(P ¼ 0.001), pTNM stage (P < 0.001), lymph node metastasis

Figure 1.KIAA1324 was downregulated in gastric cancer. A, transcriptome sequencing analysis of mRNA level of KIAA genes in paired tissue samples from 16 gastriccancer patients and 18 gastric cancer cell lines. B and C, KIAA1324mRNA expression levels in paired tissue samples from 50 gastric cancer patients were examinedusing qRT-PCR (B) and comparative analysis (C) of the paired tissues was performed. D, KIAA1324 expression in gastric cancer patients was examined frompublic microarray data (GSE13861; P ¼ 0.0002). E, epigenetic modulation assay was conducted using the inhibitors, decitabine (Dec) and MS-275. KIAA1324mRNA levels in MKN1, MKN28, and SNU638 cells were assessed using RT-PCR two days after treatment with decitabine and MS-275. Student t tests wereperformed for statistical analysis (��, P < 0.0005). Error bars indicate SD.

KIAA1324, a Novel Gastric Tumor Suppressor

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(P ¼ 0.005), and distant metastasis (P ¼ 0.001). In particular,patients who had KIAA1324-deficient gastric tumors tended todevelop more advanced and invasive gastric cancer. These resultsindicate that low levels of KIAA1324 expression are significantlycorrelated with poor prognosis of gastric cancer patients.

KIAA1324 inhibits in vivo tumor formation of gastric cancercells

To investigate a possible role of KIAA1324 as a tumor suppres-sor in gastric cancer, we examined the effect of KIAA1324on in vivotumorigenesis of gastric cancer cells. For the xenograft assay, wegenerated stableMKN28 gastric cancer cell lines with tetracycline-inducible (tet-on) luciferase (Luc) or KIAA1324 expression. Lucif-erase was used as a control. We injected the MKN28 cells subcu-taneously into mice after inducing KIAA1324 expression withdoxycycline, a tetracycline analog, and observed tumor formation(Fig. 3A). Induction of KIAA1324 dramatically decreased thetumor formation ability of MKN28 cells as demonstrated bysignificantly reduced tumor sizes and weights at the time ofharvest (P < 0.0005; Fig. 3B–D). Next, we investigated whetherKIAA1324 affects the development of preformed tumors. Threeweeks after subcutaneous injection of MKN28 cells with tet-onLuc or KIAA1324, themice were fed water containing doxycyclineevery other day for a month (up to 7 weeks) to induce KIAA1324expression (Fig. 3E). Tumor formation was observed within 3weeks after injection. We measured tumor sizes weekly andobtained tumor weights at the time of harvest (Fig. 3E–H). Theresults showed that KIAA1324 induction significantly reduced thesize (P < 0.0005) and weight (P < 0.001) of preformed tumors.

KIAA1324 expression in tumors was verified by RT-PCR (Fig. 3I).These data demonstrate that KIAA1324 inhibits the tumorigenicactivity of gastric cancer cells in vivo and the development ofpreformed tumors.

KIAA1324 suppresses growth, invasion, and drug resistance ofgastric cancer cells

We further investigated whether KIAA1324 influences the char-acteristic features of gastric cancer cells, including proliferation,invasiveness, and drug resistance. In addition to MKN28 cells, weestablished stable AGS gastric cancer cell lines with tet-on Luc orKIAA1324. The proliferation assay showed that KIAA1324remarkably inhibited growth of MKN28 and AGS cells (Fig.4A). KIAA1324 also suppressed anchorage-dependent and -inde-pendent colony forming activities of gastric cancer cells (Fig. 4Band C). To examine the effect of KIAA1324 on the invasiveness ofgastric cancer cells, we assessed themigration and invasion abilityof MKN28 and AGS cells expressing KIAA1324. As shown in Fig.4D and E, KIAA1324 significantly reduced the migration andinvasion of gastric cancer cells. We next explored whetherKIAA1324 regulates the drug resistance of gastric cancer cells bytreating MKN28 and AGS cells expressing KIAA1324 with anti-cancer drugs such as cisplatin and etoposide (Fig. 4F). Cellsexpressing KIAA1324 showed decreased cell viability in presenceof cisplatin or etoposide compared with control cells. In addition,cisplatin- and etoposide-mediated apoptosis were increased inKIAA1324-expressing MKN28 cells (Supplementary Fig. S3).These results suggested that KIAA1324 caused the cells to becomemore sensitive to anticancer drugs. To further investigate whether

Figure 2.Decreased KIAA1324 expression was correlated with poor prognosis in 428 gastric cancer patients. A, representative immunostaining images of gastric cancertissues classified according to KIAA1324 expression (negative, weak, moderate, or strong). B and C, cumulative survival rate (P < 0.001; B) and clincopathologicfeatures (C) of 428 patients who were categorized into the four groups were investigated using tissue microarray and patient information. The Kaplan–Meiermethod and Pearson c2 tests were used for survival analysis and statistical analyses, respectively.

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the loss of KIAA1324 influences proliferation and drug resistancein SNU16 gastric cancer cells with KIAA1324 expression, wemadean SNU16 cell line stably expressing KIAA1324 shRNA using alentiviral system. KIAA1324 knockdown was confirmed by qRT-PCR (Fig. 4G). Although KIAA1324 knockdown did not dramat-ically affect SNU16 cell proliferation, it markedly enhancedcisplatin resistance (Fig. 4H and I). Moreover, loss of KIAA1324decreased cisplatin- and staurosporine-induced apoptosis (Fig. 4Jand Supplementary Fig. S4). Taken together, our data demon-strate that KIAA1324 inhibits the growth, invasiveness, and drugresistance of gastric cancer cells.

Ectopic expression of KIAA1324 induces apoptosis of gastriccancer cells

To examine whether KIAA1324 induces apoptosis of gastriccancer cells, we performed annexin V staining and flow cytometryanalysis (Fig. 5A). Induction of KIAA1324 expression increasedthe annexin V–positive cell population, indicating that KIAA1324induced apoptosis in MKN28 and AGS cells. We also confirmedKIAA1324-mediated apoptosis using a TUNEL assay (Fig. 5B).Next, we examined the expression of apoptosis markers usingimmunoblotting and RT-PCR.We verified activation of caspase-3,an apoptosis effector caspase protein, in gastric cancer cells

expressing KIAA1324 by detecting cleavage of caspase-3 (Fig.5C). Interestingly, expression of proapoptotic genes, such as BAXand BIM, were also increased by KIAA1324 (Fig. 5D). In addition,we investigated whether KIAA1324 affects cell-cycle distribution.KIAA1324 did not have a considerable effect on cell cycle, whileinducing apoptosis (Supplementary Fig. S5). These results suggestthat KIAA1324 exerts main effect on apoptosis. Because it hasbeen previously mentioned that KIAA1324-mediated apoptosismight occur through excessive autophagy (24), we examined theexpression of LC3B, an autophagy marker (Supplementary Fig.S6). However, we observed no significant difference betweencontrol and KIAA1324-expressing cells. In summary, our datasuggest that KIAA1324 induces apoptosis through activation of acaspase cascade rather than autophagy.

The transmembrane domain of KIAA1324 is important forKIAA1324-mediated apoptosis

It has been reported that KIAA1324 is mainly localized in themembrane fraction, and deletion of its transmembrane domain(TM) limits its localization to the cytosol (24). To explore the roleof TM in KIAA1324-induced apoptosis, we generatedMKN28 andAGS cells with a tet-on TM-deleted mutant of KIAA1324(KIAA1324 DTM). We confirmed expression of KIAA1324 DTM

Figure 3.KIAA1324 decreased the in vivo tumorigenic activity of gastric cancer cells. A, MKN28 cells harboring tetracycline-inducible (tet-on) luciferase (Luc) orKIAA1324 were treated with 1 mg/mL doxycycline for 24 hours before subcutaneous injection into 6 mice per group. After injection, mice were fed2 mg/mL doxycycline (dox) in 10% sucrose water every two days until the time of harvest. Tumor size was measured at indicated time. B, representativeimages of harvested tumors. Size (C) and weight (D) analyses of the harvested tumors. E, MKN28 cells harboring tet-on Luc or KIAA1324 were injectedsubcutaneously in 10mice per group. Three weeks after injection, mice were fed 2mg/mL doxycycline in 10% sucrose water every two days until the time of harvest.F, representative images of harvested tumors. Size (G) and weight (H) of the harvested tumors were analyzed. I, KIAA1324 expression in tumors wasexamined using RT-PCR. � , P < 0.005; ��, P < 0.001; �� , P < 0.0005; n.s., not significant.






using immunoblotting (Fig. 6A and B). As shown in Fig. 6C, TMdeletion resulted in defective cellular localization of KIAA1324.KIAA1324 DTM neither increased the annexin V–positive cellpopulation nor activated caspase-3 (Fig. 6D and E). These resultsindicate that the TM of KIAA1324 is responsible for its ability toinduce apoptosis as well as its cellular localization.

To further examine which region of KIAA1324, besides TM, isrequired for KIAA1324-mediated apoptosis, we analyzed apopto-sis in cells expressing KIAA1324 mutants with deletion of the N-terminal side (DN) or C-terminal side (DC) of the TM. KIAA1324DCexhibited similar cellular localization and apoptosis induction

as wild-type KIAA1324 (Supplementary Fig. S7), whereasKIAA1324 DN did not induce apoptosis even though its locali-zation was similar to wild-type KIAA1324 (Supplementary Fig.S8). Tofindmore specific domain that is important for KIAA1324-mediated apoptosis, we examined the effects of a.a. 304–931(DC1) and a.a. 657–931 (DC2) of KIAA1324 on apoptosis induc-tion. While KIAA1324 DC induced apoptosis, DC1 and DC2 didnot have any effect in KIAA1324-mediated apoptosis (Supple-mentary Fig. S9). These results demonstrate that KIAA1324induces apoptosis of gastric cancer cells through the TM anda.a. 1–303 region, suggesting that its cellular localization and the

Figure 4.KIAA1324 inhibited proliferation, invasiveness, and drug resistance of gastric cancer cells. A, doxycycline-induced KIAA1324 expression in MKN28 and AGS cellsharboring tet-on KIAA1324 was verified by immunoblotting. Proliferation of MKN28 and AGS cells harboring tet-on Luc or KIAA1324 was evaluateddaily in the presence or absence of 1 mg/mL doxycycline. B, representative images of methylene blue–stained colonies of MKN28 and AGS cells expressing Luc orKIAA1324. Relative colony forming unit (CFU) was calculated by dividing the colony number of doxycycline-untreated cells by that of doxycycline-treatedcells. C, soft agar colony forming assay was performed to investigate the effect of KIAA1324 on anchorage-independent colony formation of MKN28. Migration (D)and invasion (E) abilities of MKN28 and AGS cells expressing Luc or KIAA1324 were measured using Transwell migration and invasion assay, respectively.The relative migration or invasion rates were calculated by dividing cell number of doxycycline-untreated cells divided by that of doxycycline-treated cells. F, cellviability of MKN28 and AGS cells harboring tet-on KIAA1324 was measured 24 hours after treatment with 25 mmol/L cisplatin or 25 mmol/L etoposide in theabsence or presence of 1 mg/mL doxycycline. G, KIAA1324 knockdown in SNU16 cells expressing KIAA1324 shRNA was examined using qRT-PCR. H, growthof SNU16 cells expressing control orKIAA1324 shRNAwasmeasured daily. Cell viability (I) and caspase-3 activation (J) of SNU16 cells expressing control orKIAA1324shRNA were evaluated 24 hours after treatment with 25 mmol/L cisplatin by cell counts and immunoblotting, respectively. � , P < 0.005; �� , P < 0.0005.

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a.a. 1–303 region are crucial for KIAA1324-mediated apoptosis ingastric cancer cells.

GRP78 oncoprotein is identified as a KIAA1324-bindingpartner

To elucidate the regulatory mechanism of KIAA1324-inducedapoptosis, we identified KIAA1324-specific binding partnersthrough protein interaction analysis using a formaldehydecross-linking method (Fig. 7A; ref. 31). We found GRP78 as aKIAA1324-binding partner and validated the interaction betweenKIAA1324 and GRP78 (Fig. 7B). Immunofluorescence analysisalso demonstrated that KIAA1324 colocalized with GRP78 (Fig.7C). Because GRP78 is predominantly localized in the ER, weinvestigatedwhether KIAA1324 also exists in the ER by examiningcolocalization with PDI, an ER marker. As expected, KIAA1324was found in the ER (Supplementary Fig. S10). KIAA1324 wasalso located in the cell membrane in accordance with the previousreport (24).

To determine the binding region of GRP78 with KIAA1324, weexamined interactions between KIAA1324 and various domain-deleted mutants of GRP78 (Fig. 7D–F). Immunoprecipitationassays showed that a.a. 1–80 of GRP78, which contains Thr37, anATP-binding site (32), was required for the GRP78–KIAA1324interaction. In addition, analysis of the interactions betweenGRP78 and domain-deleted mutants of KIAA1324 demonstratedthat a.a. 1–303 of KIAA1324 was responsible for the interactionwith GRP78 (Fig. 7G and H and Supplementary Fig. S11).

KIAA1324 inhibits the oncogenic activity of GRP78GRP78 increases cancer cell survival by exerting antiapoptotic

activity by interacting with caspase-7 in the ER and by activatingpro-proliferative PI3K/AKT signaling in the cell membrane. Giventhat inhibition of GRP78 induces apoptosis and increases anti-cancer drug sensitivity in gastric cancer cells (16, 20), we inves-tigated the effect of siRNA-mediated GRP78 knockdown onMKN28 cells. As expected, we found that GRP78 knockdowninduced apoptosis of MKN28 cells and decreased AKT phosphor-ylation (Supplementary Fig. S12).

It has been reported that Thr37 of GRP78 is important for ATP-induced conformational change, which is critical for the interac-tion of GRP78 with its binding partners (32), and the N-terminalside of the GRP78 ATPase domain is required for AKT activation(15). Therefore, because KIAA1324 interacted with N-terminalregion of GRP78, we first investigated whether KIAA1324 affectsGRP78 binding to caspase-7. Indeed, KIAA1324wild-type andDCmutant blocked the interaction between GRP78 and caspase-7and induced cleavage of caspase-7 (Fig. 7I). However, KIAA1324DTM and N-terminal region (N) did not affect GRP78 binding tocaspase-7 despite their interaction with GRP78. This result sug-gests that the TM of KIAA1324 is required for KIAA1324 tointerferewith the interactionbetweenGRP78 and caspase-7.Next,we investigated whether KIAA1324-mediated regulation ofGRP78 affects AKT activation by examining AKT phosphorylation(Fig. 7J). The result showed reduction of AKT phosphorylation inMKN28 cells expressing KIAA1324. Taken together, these data

Figure 5.KIAA1324 induced apoptosis of gastric cancer cells. A, KIAA1324 expression was induced in MKN28 and AGS cells harboring tet-on KIAA1324 by treatmentwith 1 mg/mL doxycycline for 36 hours. Flow cytometry analysis was performed using cells stained with Annexin V-FITC and propidium iodide (PI). TheAnnexin V–positive and PI-negative population indicates the early apoptotic cells. The right panel shows quantification of early apoptotic cell population fromtriplicate samples. B, TUNEL assay was conducted using MKN28 cells harboring tet-on KIAA1324 with or without 1 mg/mL doxycycline for 48 hours. C,cleavageof caspase-3 andPARP inMKN28- andAGS-expressingKIAA1324were examinedby immunoblotting. D,mRNA levels ofBAX,BIM,KIAA1324, andGAPDH inMKN28 and AGS cells harboring tet-on KIAA1324 were evaluated by RT-PCR at the indicated times after treatment with 1 mg/mL doxycycline. � , P < 0.02;�� , P < 0.003.






demonstrate that KIAA1324 not only inhibits the interactionbetween GRP78 and caspase-7, but also may regulate GRP78-mediated AKT activation, suggesting that KIAA1324 may exertantitumor activity by suppressing the oncogenic activities ofGRP78 (Supplementary Fig. S13).

DiscussionTo date, a number of tumor suppressor genes have been

identified and investigated for their function in tumorigenesis.However, many potential tumor suppressor genes remainunknown and uncharacterized. Recently, analysis of geneticalterations and transcriptome changes in cancer tissues and celllines using NGS has become a promising method to identifycandidate tumor suppressor genes (3, 33).Moreover, studying thebiological functions of these genes improves our understanding ofthe underlying mechanisms of carcinogenesis and aids in cancerprevention and therapeutics. In the current study, KIAA1324 wasidentified as a novel tumor suppressor that is downregulated inhuman primary gastric cancer tissues and cell lines through totalmRNA sequencing analysis (Fig. 1). CNV analysis and an epige-netic modulation assay of gastric cancer cell lines showed thatsuppression of KIAA1324 expression in gastric cancer cells iscaused by epigenetic regulation rather than genetic alteration

(Fig. 1E and Supplementary Fig. S2). A study of the clinical impactof KIAA1324 demonstrated that KIAA1324 can be used as abiomarker for prognostic prediction of gastric cancer (Fig. 2).Furthermore, investigation of the effects of KIAA1324 on prolif-eration, tumorigenic activity, and apoptosis of gastric cancer cellsindicated that KIAA1324 may function as a gastric tumor sup-pressor (Figs. 3, 4, 5). In particular, induction of KIAA1324expression in preformed tumors significantly reduced tumor size(Fig. 3E–H). This result suggests that, with remarkable recentdevelopments in tumor-specific drug or gene delivery system forcancer therapy (34, 35), application of KIAA1324 gene delivery ora KIAA1324-inducible drug release system specifically targetinggastric tumors can be a feasible strategy for gastric cancer therapy.

GRP78 has been regarded as a promising therapeutic target forcancer therapy. As GRP78 enhances cancer cell survival by pro-tecting cancer cells from apoptotic stresses such as anticancerdrugs, targeting GRP78 increases efficacy of cancer treatment(17). In cancer, the role of GRP78 in the cell membrane as wellas theERhavedrawn interest because increased cell surfaceGRP78is detected in various cancers. GRP78 is principally localized in theER lumen; however, it also has been demonstrated that GRP78has putative transmembrane domains and localizes at the cellularmembrane as well as the ER membrane (9, 13). Cell surfaceGRP78 has been reported to regulate proliferation, migration,

Figure 6.The KIAA1324 transmembrane domain was required for the induction of apoptosis. A, a schematic diagram of full-length KIAA1324 (WT) and the transmembranedomain (TM) deletion constructs. B, expression of the TM deletion construct (KIAA1324DTM) in MKN28 and AGS cells harboring tet-on KIAA1324DTM wasverified by immunoblotting. C, immunofluorescence assays show cellular localization of HA-tagged KIAA1324 WT and DTM in MKN28 and AGS cells. Nucleiwere stained with DAPI. Scale bar, 20 mm. D, flow cytometry analysis was performed in MKN28 and AGS cells harboring tet-on KIAA1324DTM usingAnnexin V-FITC and propidium iodide (PI) 36 hours after treatment with 1 mg/mL doxycycline. The Annexin V-positive and PI-negative population indicates theearly apoptotic cells. The right panel shows quantification of early apoptotic cell population from triplicate samples. E, caspase-3 cleavage in MKN28 cellsexpressing KIAA1324 WT and DTM was examined by immunoblotting.

Kang et al.





and invasion of cancer cells via regulation of various cellularsignaling pathways, including the TGFb and AKT signal pathways(36–39). Peptides targeting cell surface GRP78 have suppressedtumor growth and invasion, suggesting that GRP78-targetingpeptides could be a therapeutic strategy for patients with cellsurface GRP78-positive tumors (12, 40). We demonstrated thatKIAA1324 physically interacted with the N-terminal domain ofGRP78 through its N-terminal region andmight regulate GRP78-mediated AKT activation (Fig. 7). A domain prediction programindicated that the N-terminal region of KIAA1324 may be anextracellular domain, and a previous report and our immunoflu-orescence data showed that KIAA1324 localized at cell membrane(24). Taken together, these findings suggest that KIAA1324 mayalso modulate cell surface GRP78 extracellularly through its N-terminal domain. On the basis of our findings, identification ofthe critical motif of KIAA1324, which is necessary for the inter-action with GRP78, may be a platform for the development of

GRP78-targeting KIAA1324 peptides as anticancer therapeuticagents.

In cancer, the role of GRP78 as a receptor at the cell surfacedepends on extracellular ligands. Alpha-2 macroglobulin inter-actswith theN-terminal region of cell surfaceGRP78 and activatesAKT signaling, leading to increased cell proliferation (41). How-ever, Kringle 5 induces caspase-7 activation by binding to cellsurface GRP78 (40). Par-4 also induces apoptosis via interactionwith cell surface GRP78 and activation of the FADD-caspase 8–caspase-3 pathway (42). In this study, we demonstrated thatthrough interaction with GRP78, KIAA1324 induced activationof caspase-3 and -7 and decreased AKT signaling. Consideringprevious reports, our data suggest that KIAA1324 may releasecaspase-7 from GRP78 in the ER, activate the FADD-caspase8–caspase-3 pathway in the cell membrane, and block alpha-2macroglobulin from binding to cell surface GRP78, therebyinducing apoptosis.

Figure 7.KIAA1324 inhibited antiapoptotic activity of GRP78. A, a scheme for the identification of KIAA1324-binding partners. B, twenty-four hours after treatmentwith 1 mg/mL doxycycline in MKN28 cells harboring tet-on KIAA1324, immunoprecipitation assaywas performed using the anti-HA antibody to verify the interactionbetween HA-tagged KIAA1324 and GRP78. C, immunofluorescence assays were conducted using anti-HA and anti-GRP78 antibodies to detect colocalizationof KIAA1324 and GRP78. Nuclei were stained with DAPI. D, a schematic diagram of the GRP78 domain deletion constructs. SB, substrate-binding domain.E and F, interactions of the transfected HA-tagged KIAA1324 with GFP-tagged GRP78WT and domain deletion mutants were assessed using immunoprecipitationassay in 293T cells. G, a schematic diagram of KIAA1324 domain deletion mutants. H, immunoprecipitation assay was performed by transfecting GRP78without tag and HA-tagged KIAA1324 WT and domain deletion mutants in 293T cells. I, the interaction between GRP78 and caspase-7 was evaluated in MKN28harboring tet-on KIAA1324 WT, DTM, DC, or N mutant using immunoprecipitation assay 24 hours after treatment with 1 mg/mL doxycycline. J, phosphorylationof AKT was assessed in MKN28 cells expressing Luc or KIAA1324 by immunoblotting.






Deng and colleagues reported that KIAA1324 was predomi-nantly in the membrane fraction and colocalized with autopha-gosomemarkers, suggesting that it is involved in autophagy (24).They also suggested that the observed KIAA1324-induced apo-ptosis in 293T cells occurred due to excessive autophagy. How-ever, autophagic death and apoptosis are regarded as differenttypes of cell death (43, 44). Furthermore, we could not observeKIAA1324-mediated autophagy in gastric cancer cells (Supple-mentary Fig. S6). In our study, KIAA1324 induced cell death via anapoptotic mechanism. We found that KIAA1324 blocked theinteraction between GRP78 and proapoptotic caspase-7, suggest-ing that KIAA1324 induces apoptosis of gastric cancer cells byinhibiting the antiapoptotic activity of GRP78.

It has been reported that transmembrane proteins in the ER,such as TMEM166 and TMEM214, are involved in the inductionof apoptosis (45, 46). TMEM166 contains a single transmem-brane domain and induces both autophagy and apoptosis. Com-pared with normal tissues, TMEM166 is downregulated in gastricadenocarcinoma (47). Adenovirus-mediated introduction ofTMEM166 suppressed tumor growth through autophagy andapoptosis (48). TMEM214, which contains two transmembranedomains and is localized to the outer membrane of the ER,regulates ER stress-induced apoptosis through activation of cas-pase-4 (46). Here, we observed that KIAA1324, a transmembraneprotein, induced apoptosis in the ER, and TM deletion abolishedits apoptotic activity even though the TM deletion mutant boundto GRP78. It is possible that these transmembrane proteinsinteract with each other at the ER membrane to induce apoptosisby regulating GRP78. Further investigation of the interactionamong these proteins will provide a better understanding ofKIAA1324-mediated apoptosis in cancer.

AKT signaling plays a key role in various cellular processesincluding proliferation, survival, metabolism, differentiation,and apoptosis (49). Loss of AKT inhibitors such as PTEN andSHIP or upregulation of AKT activators such as GRP78 and Srcinduces dysregulation of AKT activation and has been implicatedin carcinogenesis. In the current study, we observed thatKIAA1324 reduced AKT phosphorylation in MKN28 cells. Thisphenomenon may be attributed to KIAA1324-mediated inhibi-tion of GRP78 activity. However, we cannot exclude the possi-bilities that KIAA1324 regulates AKT signaling directly or throughinteractionwith other regulators of AKT. Therefore, further studiesto explore the possibilities will support tumor suppressive role ofKIAA1324 as a negative regulator of AKT.

The role of KIAA1324 in cancer has been evaluated only inendometrial, pancreatic, and ovarian cancer to date (21–23). Intype I endometrial cancer, KIAA1324 expression is higher at earlystage than that of benign tumors, but reduced in high grade andstage endometrial carcinoma. In addition, KIAA1324 is down-regulated in type II endometrial cancer, which is more aggressivethan type I. In pancreatic cancer, KIAA1324 is also highly

expressed in early-stage tumor, but its expression is decreased inadvanced cancer. High KIAA1324 expression in endometrial andpancreatic carcinoma is correlated with favorable prognosis incancer patients. However, in high-grade serous carcinoma of theovary/peritoneum, high expression of ERa and KIAA1324 isassociated with poor survival in cancer patients. This indicatesthat KIAA1324mayplay different roles in various types of cancers.In our study, we provide evidence that supports a tumor suppres-sive role of KIAA1324 in gastric cancer through induction ofapoptosis. Our study may lead to further investigation of thefunction of KIAA1324 in other cancers.

In conclusion, our study demonstrated that KIAA1324 wasepigenetically downregulated in gastric cancer and positivelycorrelated with prognosis of gastric cancer patients. We alsorevealed that KIAA1324 suppressed growth of gastric cancer cellsand tumors by inhibiting the oncogenic activity of GRP78. Takentogether,we suggest KIAA1324as anovel gastric tumor suppressorand provide a new insight for the application of KIAA1324 in thediagnosis and treatment of gastric cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.M. Kang, S. Park, H.-K. Yang, S.-J. KimDevelopment of methodology: J.M. Kang, S.-J. KimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.M. Kang, S. Park, S.J. Kim, H. Kim, B. Lee, W.H.Kim, S.-J. KimAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.M. Kang, S. Park, B. Lee, J. Kim, S.T. Kim, W.H. KimWriting, review, and/or revision of themanuscript: J.M. Kang, S. Park, S.J. Kim,B. Lee, J. Park, H.-K. Yang, W.H. Kim, S.-J. KimAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.M. Kang, S. Park, B. Lee, S.T. KimStudy supervision: S.-J. Kim

AcknowledgmentsThe authors thank K. Yoon, C.Y. Choi, and H.C. Yang for technical support.

Grant SupportThis work was supported by the Basic Science Research Program (2011-

0014281 to S. Park and 2014R1A6A3A01058338 to J.M. Kang) and the Bio-SynergyResearchProject (NRF-2012M3A9C4048735 to S.-J. Kim)ofMinistry ofScience, ICT, and Future Planning through the National Research Foundation,Korea.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 20, 2014; revised May 21, 2015; accepted May 26, 2015;published OnlineFirst June 4, 2015.

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2015;75:3087-3097. Published OnlineFirst June 4, 2015.Cancer Res Jin Muk Kang, Sujin Park, Staci Jakyong Kim, et al. Oncoprotein GRP78KIAA1324 Suppresses Gastric Cancer Progression by Inhibiting the

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