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PPP1R1B-STARD3 chimeric fusion transcript in human gastric cancer promotes tumorigenesis through activation of PI3K/AKT signaling


Fusion genes act as potent oncogenes, resulting from chromosomal rearrangements or abnormal transcription in many human cancers. Although multiple gastric cancer genomes have been sequenced, the driving recurrent gene fusions have not been well characterized. Here, we used paired-end transcriptome sequencing to identify novel gene fusions in 18 human gastric cancer cell lines and 18 pairs of primary human gastric cancer tissues and their adjacent normal tissues. Multiple samples revealed expression of PPP1R1B-STARD3 fusion transcript. The presence of PPP1R1B-STARD3 correlated with elevated levels of PPP1R1B mRNA. PPP1R1B-STARD3 fusion transcript was detected in 21.3% of primary human gastric cancers but not in adjacent matched normal gastric tissues. Based on reverse transcription PCR analysis of DNA, unlike other fusions described in gastric cancer, the PPP1R1B-STARD3 appears to be generated by RNA processing without chromosomal rearrangement. Overexpression of PPP1R1B-STARD3 in MKN-28 significantly increased cell proliferation and colony formation. This increased proliferation was mediated by activation of phosphatidylinositol-3-kinase (PI3K)/AKT signaling. Furthermore, expression of PPP1R1B-STARD3 fusion transcript enhanced the tumor growth of MKN-28 cells in athymic nude mice. These findings show that PPP1R1B-STARD3 fusion transcript has a key role in subsets of gastric cancers through the activation of PI3K/AKT signaling.


Gastric cancer was the fourth most common cancer diagnosis worldwide in men and fifth most common in women in 2011.1 It is a leading cause of global cancer mortality, with a 5-year survival rate of 27%. Approximately, 70% of new cases occur in developing countries, including Eastern Asia and many parts of South America.2 The main risk factors of gastric cancer are Helicobacter pylori infection, dietary factors and smoking. In addition, the result of multistep conversion of normal cell to malignant cell is due to genetic and epigenetic alterations of cell adhesion molecules, oncogenes, cytokines and tumor-suppressor genes such as CDH1,3 ERBB2 and KRAS,4 TLR4,5 p536 and RUNX3,7 respectively.

Fusion genes, caused by genomic aberrations, such as translocation, amplification and rearrangement, are known to trigger human cancers.8, 9, 10 Fusion transcript is a chimeric RNA from two different genes. The fusion transcript unrelated to chromosomal rearrangements often occurs by transcription-induced chimeras. Transcription-induced chimeras can be generated from two genes, which are located up to 50 kb from each other, and are more likely to occur between genes of a short distance.11 The two possible mechanisms of transcription-induced chimeras in the formation of the gene fusions contribute to their biological significance. One mechanism is read-through, which is a chimeric transcript that starts in the first exon of the upstream gene and stops at the termination of the downstream gene with the splicing region between two genes. This mechanism can be explained as a ‘run-off’ transcription of the upstream gene or ‘leakage’ accidence of the transcriptional machinery. And these events create a frameshift, causing a premature stop of the fusion gene.12, 13 The other mechanism is trans-splicing, which appears between the separate pre-mRNA of tandem genes. Chimeric transcripts give rise to a diverse range of functional protein by generating chimeric protein and altered regulation of mRNA in normal human tissues.14 However, in carcinogenesis, the accumulation of aberrant expression of chimeric transcript causes dysfunction of protein by dysregulation of RNA processing.15, 16

PPP1R1B was initially identified in the brain and has been implicated in physiologic processes, including neuronal disease and dopaminergic abnormality.17, 18, 19 PPP1R1B has also been involved in common carcinomas,20 including gastric cancer.21, 22 PPP1R1B-STARD3 fusion transcript was first reported in breast cancer.23 However, the function of PPP1R1B-STARD3 fusion in cancers has not been investigated.

Here, we identified PPP1R1B-STARD3 fusion in gastric cancer using next-generation sequencing. The PPP1R1B-STARD3 expression was validated in gastric cell lines and human gastric patient tissues by using reverse transcription PCR and quantitative real-time PCR (Q-PCR). PPP1R1B-STARD3 increased cell proliferation through the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in vitro. And PPP1R1B-STARD3 affected the tumorigenic capacity of gastric cancer cells in vivo.


Discovery of the PPP1R1B-STARD3 fusion transcript in gastric cancer

To investigate fusion genes in gastric cancer, we conducted paired-end transcriptome sequencing with 18 gastric cancer cell lines and 18 pairs of primary human gastric cancer tissues and their adjacent normal tissues. The deFuse algorithm (version 0.4.1) was used with default parameters to identify fusion transcripts. PPP1R1B-STARD3 fusion gene was identified in NCI-N87 gastric cancer cell line. The sequence reads were aligned with the reference transcriptome to determine the fusion junction (Figures 1a and b, Supplementary Figures S2a and b). PPP1R1B and STARD3 lay adjacent to each other on chromosome 17, separated by 455 bp. PPP1R1B-STARD3 fusion transcript is made of PPP1R1B exons 1–6 and STARD3 exons 2–15. A fusion of the complete exon 6 of PPP1R1B with the beginning of exon 2 of STARD3 resulted in a read-through fusion protein (Figure 1c). It is predicted that translation initiation from an ATG site in PPP1R1B exon 1 could produce a 245-amino-acid protein, comprising of 188 amino acids of PPP1R1B and 57 amino acids from STARD3. The latter resulted from an out-of-frame of STARD3. Translation from this fusion apparently produced loss of c-terminal sequences from the full 204 amino acid of PPP1R1B protein but retained all functionally relevant domains (Supplementary Figure S1c). We estimated the RNA expressions of PPP1R1B and STARD3 by read distributions. Read sequences were annotated to mRNAs (Figure 1d) and exons of the genomes (Supplementary Figure S1d). PPP1R1B gene was highly expressed without relation to fusion junction, whereas STARD3 expression increased from exon 2 in comparison to exon 1, which was breakpoint junction of the fusion transcript. This suggested that abnormal overexpression of STARD3 was rendered by PPP1RB-STARD3 fusion transcript.

Figure 1

Discovery of PPP1R1B-STARD3 fusion transcript in gastric cancer. (a) Identification of PPP1R1B-STARD3 fusion transcript by RNA sequencing. The sequences of spanning reads are displayed across the fusion junction. Black and blue indicate PPP1R1B exon 6–7 and STARD3 exon 2, respectively. (b) The number of paired-end reads whose 5′ and 3′ end sequences are successfully aligned to each of fused genes across the exon junction of PPP1R1B and STARD3 and spanning reads are shown. (c) Schematic representations of the wild-type PPP1R1B and STARD3 genes with breakpoints of respective exon numbers and mRNA positions for each gene. The fusion occurred between the sixth exon of PPP1R1B and the second exon of STARD3. Red line indicates the position of start and stop codon of the predicted fusion protein. The chromatogram by Sanger sequencing presents the fusion junction. (d) RNA expression levels of PPP1R1B and STARD3 are estimated through read distributions, which were annotated to their mRNAs.

Expression of PPP1R1B-STARD3 fusion gene in primary gastric cancer tissues and gastric cancer cell lines

To confirm whether PPP1R1B-STARD3 was expressed in other gastric cell lines and clinical specimens, we performed reverse transcription PCR and Q-PCR screening of PPP1R1B-STARD3 with 18 gastric cancer cell lines and 47 pairs of primary human gastric cancer tissues and their adjacent normal tissues. PPP1R1B-STARD3 was expressed in about 33% of gastric cancer cell lines (6/18), which correlated to fragments per kilobase per million read (FPKM) values estimated by RNA sequencing (Figures 2a and b, Supplementary Figures S2a and d, and Table 1). This fusion transcript was generated by RNA processing without DNA-level rearrangement (Supplementary Figure S2b). Interestingly, the PPP1R1B-STARD3 fusion transcript was differentially expressed in tumors compared with normal samples. PPP1R1B-STARD3 was expressed in about 21.3% of gastric tumor tissues (10/47), whereas expression was not detected in their adjacent normal tissues (Figure 2d, Supplementary Figures S2c–e, and Table 1).

Figure 2

PPP1R1B-STARD3 expression in gastric cancer cell lines and primary gastric cancer tissues. (a) RNA transcriptional levels of PPP1R1B-STARD3, PPP1R1B and STARD3 in 18 gastric cancer cell lines were measured by Q-PCR. (b) The levels of RNA expression of PPP1R1B and STARD3 were estimated by RNA sequencing. (c and e) Comparison of PPP1R1B and STARD3 expressions in 18 gastric cancer cell lines (c) and 47 gastric cancer patient tumor tissues (e). *P<0.001 and **P<0.0006, which is expression of fusion transcript versus PPP1R1B in gastric cancer cell lines and gastric tumor tissues, respectively. NS indicates not significant. (d) The representative Q-PCR result of PPP1R1B-STARD3 and each parental gene expressions in 47 pairs of primary human gastric cancer tissues and their adjacent normal tissues was shown.

Table 1 The frequency of fusion gene in 18 gastric cancer cell lines and in 47 pairs of primary human gastric cancer tissues and their adjacent normal tissues

PPP1R1B and STARD3 genes are located within PPP1R1B-STARD3-ERBB2-GRB7 amplicon on human chromosome 17q 12. It was reported that PPP1R1B, STARD3, ERBB2 and GRB7 are frequently co-amplified in human gastric cancer.24 To confirm if PPP1R1B-STARD3 transcript was to be generated by this amplicon, we analyzed expression of these genes in gastric cancer cell lines. Cell lines with high expression of PPP1R1B-STARD3, excluding NCI-N87, did not show high expression of the amplicon (Supplementary Figures S3a and b). We also analyzed copy number variations of PPP1R1B-STARD3-ERBB2-GRB7 amplicon by whole-genome sequencing.25 However, copy number gain or loss was not observed in the PPP1R1B-STARD3-ERBB2-GRB7 amplicon (Supplementary Figure S3c). Therefore, it was not possible that fusion transcripts arise from cryptic genomic rearrangement by the amplicon.

Moreover, cells expressing high levels of PPP1R1B wild-type transcript had a tendency to be PPP1R1B-STARD3 fusion positive. As shown in Figures 2c and e, a highly significant correlation was found between expression of PPP1R1B parental gene and fusion transcript. This fusion transcript was identified in several different cancer cell lines such as breast cancer, endometrial cancer and ovarian cancer (Supplementary Figure S4). We observed that a positive correlation between PPP1R1B-STARD3 chimeric and PPP1R1B expressions in several cancer cell lines was consistent with that in gastric cancer. The expression of PPP1R1B-STARD3 was specific not only in gastric cancers but also in other cancers, implying that this fusion transcript may be involved in cancer development.

Induction of cancer cell proliferation and colony formation by PPP1R1B-STARD3 fusion gene

As demonstrated in Figure 2a, we observed PPP1R1B-STARD3 in six human gastric cancer cell lines. We confirmed endogenous protein expression of PPP1R1B-STARD3 fusion gene in the cell lines, which showed a high level of fusion expression in NCI-N87, KATOIII and MKN-45 (Figure 3a). Given that the dysregulation of proliferation is an important step for cancer cell development, we next investigated whether the ectopic expression of the fusion gene influenced cell proliferation. Using retroviral constructs, we stably expressed empty vector, PPP1R1B or PPP1R1B-STARD3 in MKN-28, a fusion negative cell line. As shown in Figure 3b, the expression of the fusion gene at the protein level was confirmed by immunoblotting. MKN-28 expressing PPP1R1B-STARD3 had significantly increased in proliferation rate when compared with vector controls (Figure 3c). In addition, PPP1R1B-STARD3-expressing MKN-28 cells grew at a higher density and formed more foci during the focus-forming assay (Figure 3d). We obtained similar results from AGS cells expressing PPP1R1B-STARD3 (Supplementary Figure S5). Overall, these results suggested that expression of exogenous PPP1R1B-STARD3 fusion gene conferred a growth and a proliferative advantage in gastric cancer.

Figure 3

Characterization of PPP1R1B-STARD3 fusion gene. (a) Endogenous expressions of PPP1R1B wild-type (WT; lower bands) and PPP1R1B-STARD3 (upper bands) in fusion-positive and -negative gastric cancer cell lines. (b) Western blot analysis showing the levels of PPP1R1B and PPP1R1B-STARD3 in empty vector, PPP1R1B- or PPP1R1B-STARD3-expressing MKN-28 stable cells. (c) Proliferation assay of three stable cell lines was performed using Cell Titer-Glo Luminescent assay kit (*P<0.05; **P<0.005). (d) Focus-forming assay demonstrates significant increase in relative cell survival in MKN-28 cells stably expressing PPP1R1B-STARD3 (top). Quantitative result of focus formation assays was displayed (bottom). (e) MKN-28 cells stably expressing empty vector, PPP1R1B or PPP1R1B-STARD3 were injected subcutaneously in to nude mice. (f) Tumor weights were significantly higher in the PPP1R1B-STARD3 group. *P<0.05, which is fusion versus PPP1R1B WT expressing tumors. **P<0.0001, which is PPP1R1B WT versus empty vector expressing tumors. (g) Tumor volume was measured at the indicated times. Each data point represents the mean (±) standard deviation for six xenografts (*P=0.028; **P=0.0044; ***P=0.0001).

Promotion of tumor growth in vivo by PPP1R1B-STARD3 fusion protein

Overexpression of PPP1R1B showed an oncogenic potential in NIH 3T3 cells and induced tumorigenesis in gastric cancer.22 To determine if overexpression of the fusion gene increased growth in vivo, we used the xenograft mouse model. MKN-28 cells stably expressing empty vector, PPP1R1B or PPP1R1B-STARD3 were injected subcutaneously (5 × 106 cells per site) in to nude mice, and the growth of the tumors was monitored (Figure 3e). These recurrent tumors displayed high-level expression of the PPP1R1B and PPP1R1B-STARD3 (Supplementary Figure S6). As shown in Figures 3f and g, MKN-28 cells with PPP1R1B-STARD3 expression generated significantly larger tumors, further confirming that PPP1R1B-STARD3 reinforced tumorigenicity in vivo.

Regulation of the PI3K/AKT survival pathway by PPP1R1B-STARD3 fusion gene in gastric cancer cells

It has been previously demonstrated that PPP1R1B activates AKT phosphorylation.26 Therefore, we examined whether PPP1R1B-STARD3 also activates AKT phosphorylation. The activation of the PI3K/AKT signaling cascades regulates cell proliferation, survival, angiogenesis, invasion and metastasis.27, 28 To test whether PPP1R1B-STARD3 is involved in the PI3K/AKT signaling pathway, we assessed the activation of AKT in three cell lines. Intriguingly, AKT phosphorlyation was induced in PPP1R1B and even more significantly so in PPP1R1B-STARD3 stable cells (Figure 4a and Supplementary Figure S7a). To clarify if the enhanced AKT activation is due to a shorter PPP1R1B protein, or if the additional amino acids derived from STARD3 sequences have any role, we constructed truncated form of PPP1R1B (PPP1R1B truncated), which deleted the STARD3 part in the PPP1R1B-STARD3 fusion sequences. Activation of AKT phosphorylation by truncated form of PPP1R1B was similar to that of PPP1R1B wild type (Supplementary Figure S7b). PPP1R1B wild type and truncated form of PPP1R1B induced proliferation of MKN-28 similarly compared with vector control, whereas PPP1R1B-STARD3 further increased proliferation (Supplementary Figure S7c). This correlated well with AKT activation levels induced by these proteins. As AKT activation level induced by PPP1R1B-STARD3 fusion protein is greater than those by PPP1R1B wild type and truncated form of PPP1R1B, it is likely that the sequences derived from STARD3 part of PPP1R1B-STARD3 fusion sequences may potentiate the ability of PPP1R1B to activate AKT. Because elevated levels of PPP1R1B-STARD3 transcript were associated with elevated levels of PPP1R1B wild-type transcript, we compared AKT activation in MKN-28 cells stably expressing PPP1R1B-STARD3 alone or PPP1R1B wild type and PPP1R1B-STARD3 together to test whether expression of both PPP1R1B wild type and PPP1R1B-STARD3 further enhances AKT phosphorylation. Both wild-type and fusion overexpressing cells showed increased AKT activation greater than wild type or fusion alone overexpressing cells (Supplementary Figure S7d).

Figure 4

Induction of the PI3K-AKT activation by PPP1R1B-STARD3 in gastric cancer cells. (a) Western blot analysis of AKT and phospho-AKT (S473) in empty vector, PPP1R1B- or PPP1R1B-STARD3-expressing MKN-28 stable cells. (b) AKT activations with or without PI3K inhibitor, LY294002, treatment (50 μM) in three stable cell lines were examined by western blotting. (c) Overexpression of PPP1R1B-STARD3 in MKN-28 cells significantly reduced cell proliferation with (red lines) or without (black lines) LY294002 (10 μM) treatment for 4 days (*P=0.0036; **P=0.0022; ***P<0.0001). (d) Focus-forming assays demonstrate significant reduction in relative cell survival in the MKN-28 cells stably expressing PPP1R1B-STARD3 after LY294002 (25 μM) treatment for 14 days (left panel). Quantitative result of focus formation assays was shown (right panel; *P=0.0023; **P=0.0001; ***P<0.0001). DMSO, dimethyl sulfoxide; WT, wild type.

To further test whether induction of AKT phosphorlyation was involved in the PI3K/AKT pathway, we treated cells with PI3K inhibitor, LY294002. The result showed that AKT activation was completely suppressed following LY294002 treatment (Figure 4b and Supplementary Figure S7e). To confirm that the repression of AKT activation was due to PIK3CA inhibition and not an off-target effect, we treated the stable cells with another AKT inhibitor, AKT-IV. The result also demonstrated that AKT activation was completely suppressed by AKT-IV treatment (Supplementary Figure S7f). We next examined proliferation rate of three stable cell lines with or without LY294002 stimulation to explore a growth advantage of PPP1R1B-STARD3 through the PI3K/AKT pathway. Even though proliferation of empty vector and PPP1R1B wild-type cells slightly increased, the proliferation reduction in the fusion was observed at a concentration of 10 μM of LY294002 after 96 h (Figure 4c). To further confirm the enhanced proliferative response of PPP1R1B-STARD3 in MKN-28, we performed focus-forming assay after LY294002 treatment for 14 days. Consistently, inhibition of AKT signaling significantly decreased cell foci formation (Figure 4d), indicating that PPP1R1B-STARD3 has an important role in gastric cancer development through the PI3K/AKT pathway.


This study provides a new insight into chimeric RNAs expressed in human gastric cancer. We identified PPP1R1B-STARD3 as a novel chimeric transcript in gastric cancer cell lines and tumors using paired-end transcriptome sequencing. PPP1R1B was found to be overexpressed in several human cancers including gastric cancer,26, 29 which implied the involvement of PPP1R1B-STARD3 in carcinogenesis. Even though PPP1R1B-STARD3 fusion gene was first identified in HCC1569 breast cancer cell line,23 the biological function of PPP1R1B-STARD3 has not yet been examined in cancer development.

A fusion of the complete exon 6 of PPP1R1B with the beginning of exon 2 of STARD3 resulted in a read-through fusion protein. PPP1R1B-STARD3 chimeric transcript was not generated by the PPP1R1B-STARD3-ERBB2-GRB7 amplicon (Supplementary Figure S3). Moreover, RNA processing not DNA rearrangement produced this transcript (Supplementary Figure S2b). Chimeric RNAs were similar to gene fusions in the sense that the mechanisms were generated by two genes. The biological significance of chimeric RNAs would also be similar to that of gene fusions.15 And, chimeric RNAs, like gene fusions, are expected to increase the diversity of proteins in normal cells.14 On the other hand, abnormal expression of chimeric transcripts provided cell growth and survival in human cancer.30 PPP1R1B-STARD3 chimeric fusion transcripts were expressed far higher rate than a few gene fusion in gastric cancer cell lines and human gastric patient cancer tissues at a relatively high percentage of 33.3% and 21.3%, respectively.31, 32 Therefore, a high occurrence rate of PPP1R1B-STARD3 may be a more useful target in cancer diagnosis and therapy compared with that of low occurrence fusion genes.

Our experiments investigated the mechanism and function of PPP1R1B-STARD3 in gastric cancer. Intriguingly, the Q-PCR results showed that PPP1R1B parental gene involved in the expression of chimeric transcript was significantly overexpressed in gastric cancers, whereas the expression level of STARD3 did not show any correlation with the fusion transcript (Figures 2c and e). This expression pattern in other cancer cell lines was consistent with that of gastric cancer cell lines (Supplementary Figure S4). Random transcriptional leakage is likely to generate more events in highly expressed genes. Furthermore, given that PPP1R1B parental gene was differentially expressed in gastric cancers in comparison to matched normal samples, it was speculated that PPP1R1B regulatory factors modulated both parental and fusion genes. The tumor-associated aberrant regulation of cis- or trans-elements in transcriptional regulation level potentially affects their gene expression and biological functions.30 These elements such as suppressors or regulators may influence the promoter or termination of PPP1R1B in transcription and RNA splicing.

Expression of PPP1R1B-STARD3 markedly enhanced proliferation as well as tumorigenicity of gastric cancer cells compared with PPP1R1B wild type. In this study, we found that PPP1R1B-STARD3 activates the PI3K/AKT signaling pathway (Figure 4). As PPP1R1B wild type was known to activate AKT signaling by promoting interaction between EGFR and ERBB3,26 it is likely that the function of PPP1R1B-STARD3 retained all functionally relevant domain of PPP1R1B is similar to PPP1R1B. Interestingly, the effect of PPP1R1B-STARD3 on cell proliferation and tumorigenesis was slightly greater than that of PPP1R1B. Therefore, we cannot exclude the possibility that 57 amino acids of novel sequences from STARD3 may also contribute to gastric tumorigenesis.

Taken together, PPP1R1B-STARD3 fusion transcript promotes tumorigenesis in gastric cancer. Moreover, the fusion gene increased cell proliferation through activation of the PI3K/AKT pathway. We suggest that PPP1R1B-STARD3 may be a candidate for a new genetic and epigenetic marker in gastric cancer.

Materials and methods

Primary tissues and cell lines

Eighteen pairs of gastric cancer and normal matched control tissues for RNA sequencing were obtained from the Gastrointestinal Division in Department of Surgery at Seoul National University Hospital. The usage of the patient tissues and related clinicopathologic data, and the protocols and informed consent documents for this study were approved by the institutional review board of the Seoul National University Hospital (IRB no. H-0806-072-248). The clinical information of the gastric cancer tissues is provided in Supplementary Table S1. Eighteen human gastric cancer cell lines, purchased from Korean Cell Line Bank, were used in this study (SNU-1, SNU-5, SNU-16, SNU-216, SNU-484, SNU-520, SNU-601, SNU-620, SNU-638, SNU-668, SNU-719, MKN-1, MKN-28, MKN-25, MKN-74, KATOIII, AGS and NCI-N87). These gastric cancer cell lines were maintained with RPMI (WelGENE, Daegu, Republic of Korea) containing 25 mM HEPES, 10% fetal bovine serum (WelGENE) and 1% penicillin/streptomycin (WelGENE).

RNA sequencing

Total RNA for RNA sequencing was isolated by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturing instructions. And then, miRNeasy (Qiagen, Hilden, Germany) were used for the purification of total RNA after DNase I treatment following their specific protocol.

Illumina platform for analyzing fusion genes of gastric cancer transcriptomes with 90 bp paired-end library were used according to the manufacturer’s instructions (Illumina, San Diego, CA, USA).

Reads alignment and fusion gene detection from RNA sequencing data

Paired-end sequence reads were aligned to hg19 human reference genome (NCBI build 37) with a TOPHAT algorithm (ver. 2.0.4).33 Transcript assembly and gene expression level estimation were conducted by using Cufflinks34 ver. 2.0.2, which calculates the fragments per kilobase per million reads. Gene model information from Ensembl release 62 ( was used in both Tophat and Cufflinks algorithms.

Fusion genes were detected using RNA-seq data by the deFuse algorithm35 ver.0.4.1 with a human genome sequence and gene model from Ensembl release 62, UCSC hg19 repeat data, and NCBI unigene data. Supporting reads, that is, split reads and spanning reads, of a gene fusion point were selected by a script ‘’ of the defuse package.

Validation of fusion gene expression

Expression levels of PPP1R1B, STARD3 and PPP1R1B-STARD3 were determined by reverse transcription PCR, and Q-PCR using SYBR Green assay. All oligonucleotide primers, listed in Supplementary Table S2, were synthesized by Bioneer Company. Samples were normalized against 18S rRNA, and each target was measured in triplicates. Total RNA was extracted from human gastric patient tissues with Tri reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. SuperScript II (Invitrogen) was used for first-strand cDNA synthesis following the manufacturing instructions. Genomic DNAs of 18 gastric cancer cell lines were isolated by QIAmp DNA mini kit (Qiagen) according to the manufacturer’s instructions.

Reagents and antibodies

LY294002, PI3K/AKT inhibitor was purchased from SelleckBio (Houston, TX, USA). Antibodies were obtained from Abcam (Cambridge, MA, USA) (anti-PPP1R1B), Cell Signaling Technology (Cell signaling, Inc, Danvers, MA, USA) (anti-pAKT Ser473 and anti-AKT) and Sigma-Aldrich (Milwaukee, WI, USA) (anti-α-tubulin).

Retroviral production and infection

The Platinum-GP (Plat-GP) cells were transfected with pMX-puro empty vector, pMX-puro-PPP1R1B wild-type or pMX-puro-PPP1R1B-STARD3-expressing vector using FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA). After 48 h, viral supernatants were collected for infection of MKN-28 cells with polybrene (10 μg/ml; Sigma-Aldrich). Puromycin-resistant transfectants were selected and maintained in RPMI with puromycin (4 μg/ml).

Cell proliferation assay

Proliferation studies were performed using cell counts and Cell Titer-Glo Luminescent Cell Viability Assay (G7572; Promega, Madison, WI, USA). For the cell count experiment, MKN-28 cells stably expressing empty vector, PPP1R1B or PPP1R1B-STARD3 were plated with 1 × 104 cells per well in a 12-well plate containing RPMI medium with 10% fetal bovine serum and incubated overnight. Subsequently, cells were incubated with or without LY294002 for an additional 72 h. After trypsinization, cells were stained with trypan blue and counted using a Neubauer chamber (Celeromics, Valencia, Spain). The mean±s.d. of at least three independent experiments was displayed.

For Cell Titer-Glo Luminescent Assay, MKN-28 cells stably expressing empty vector, PPP1R1B or PPP1R1B-STARD3 were plated with 3 × 103 cells per well in a 96-well plate in RPMI medium with 10% fetal bovine serum and incubated overnight. And then, cell proliferation was measured every day according to the manufacturer’s instructions. All experiments were carried out in triplicates.

Focus-forming assay

Five hundred cells were seeded per well in six-well plates. After incubation for 2 weeks, colonies were rinsed with PBS and stained with 2% methylene blue. All experiments are carried out in triplicates.

In vivo experiments

MKN-28 cell lines stably expressing empty vector, PPP1R1B or PPP1R1B-STARD3 fusion gene were injected subcutaneously (5 × 106 cells per site) in to nude mice. Mice were killed 4 weeks after injection, and tumors were isolated. Tumor volume (V) was calculated by using the formula (LXSXS) × 0.5, where L and S were the long and short dimensions, respectively. All animals were maintained according to the CHA Hospital Animal Care and Use Committee guidelines under protocol number IACUC120026.


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This work was supported in part by the National Research Foundation of Korea (NRF) grants (2009-0081756 and 2012M3A9C4048736 to S-JK). JB and SL were supported by TheragenEtex and Genome Research Foundation internal funds. JB was supported by the Industrial Strategic Technology Development Program (10040231) funded by the Ministry of Knowledge Economy (MKE, Korea).

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Correspondence to S-J Kim.

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The authors declare competing financial interests. Some authors are present employees of TheragenEtex, and some of them have personal financial interests as shareholders in TheragenEtex.

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Author Contributions

SMY and KY performed research, analyzed data and wrote the manuscript; SL analyzed and interpreted sequencing data; EK and PK performed research and collected data; JMK performed an animal study; JC, YC and HY contributed to the manuscript; T-SH, S-HK and H-KY collected clinical information and prepared clinical samples; SJ and JB analyzed and collected sequencing data; S-JK designed research, interpreted data and drafted the manuscript.

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Yun, S., Yoon, K., Lee, S. et al. PPP1R1B-STARD3 chimeric fusion transcript in human gastric cancer promotes tumorigenesis through activation of PI3K/AKT signaling. Oncogene 33, 5341–5347 (2014).

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  • fusion transcript
  • gastric cancer
  • transcriptome sequencing

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