BTG2 interaction proteins screening with protein chip array

The HuProt Human Proteome Microarray was purchased from CDI laboratories (http://www.cdi-lab.com). The Chip includes 16,368 unique full-length humanrecombinant proteins in duplicate along with several control proteins such as IgG, GST, BSA-biotin, and histones as previously described [56].

Human cDNA of BTG2 was subclonned into various GST-V5-HIS fusion vectors and purified, Purified hBTG2 proteins were applied to human protein microarray and hBTG2 interacting proteins were detected by using Alexa-Fluor 488 conjugated V5 antibody(Invitrogen, Carlsbad, CA). V5 antibody alone was used as control. Briefly, the protein chip was first incubated with blocking buffer (5% BSA in PBS with 0.05% (v/v) Tween 20) for 30 minutes at room temperature and V5-His-hBTG2 or V5 antibody were further incubated under the lifterslip (Thermo scientific, USA) for 1 hour at room temperature. After washing three times with 1x PBS containing 0.05% Tween 20 by gentle shaking for 10 min each, the microarray was incubated with Alexa-Fluor conjugated antibody.

Subsequently, the microarray was washed three times and then the values of probe signal were obtained using a GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA).

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3. RESULTS

3.1. BTG2 regulates mRNA and protein level of anti-apoptotic protein, Bcl-XL

To investigate the possibility that BTG2, as a binding partner of mRNA deadenylase, CAF1, can influence cell viability by regulating mRNA stability of anti-apoptotic proteins, the basal level of mRNA of Bcl-2, Bcl-XL and MCL1 was observed after overexpression of BTG2 in several cancer cell lines. In A549, lung carcinoma cell line, decreased basal levels of Bcl-2, Bcl-XL and MCL1 was observed after BTG2 overexpression. However, in BTG2-overexpressed other cell lines, depression of Bcl-2 and MCL1 mRNA was not consistently observed. After all, only the decrease of Bcl-XL mRNA was consistently observed in all investigated cell lines after BTG2 expression (Figure 1A). To exclude the possibility of decreased transcription of Bcl-XL as a mechanism of decreased Bcl-XL mRNA level in overexpressed cells, the level of pre-mRNA was compared between control and BTG2-overexpressed cells. As expected, the significant difference was not observed in the level of BCL-XL pre-mRNA (Figure 1B). After transcription, alternative splicing of pre-mRNA of Bcl-XL produces short or long form mRNA, so called Bcl-XS and Bcl-XL, respectively and it was already reported that the alternative splicing toward producing more Bcl-XS along with decreased Bcl-XL can induce the loss of cell viability [57]. However, by observing the decrease of both long and short form of Bcl-X (Figure 1C), we can verify that BTG2 has no significant influence on alternative splicing process in this experiment setting. Although, along with decrease of Bcl-2 or MCL1 mRNA levels, in some cell lines, the decreased protein level of Bcl-2 or MCL1 was observed, decreased protein level of Bcl-XL was consistently confirmed by western blot analysis in all those cell lines after 48 hours of BTG2 overexpression (Figure 1D). In addition, the increased level of BCL-XL mRNA was observed in liver tissue from BTG2/TIS21-knock out mouse, along with increased other anti-apoptotic genes, Bcl2, MCL1 and BCL2L10 Figure S1. Taken together, these results indicated that BTG2 can regulate mRNA level of Bcl-XL not by transcriptional but by post transcriptional level.

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Figure 1.BTG2 decreases mRNA and protein level of anti-apoptotic protein, Bcl-XL. (A) mRNA level of Bcl2, Bcl-XL, MCL1 in BTG2 overexpressed cancer cells. RNA from cells infected with Ad-BTG2 or Ad-LacZ for 48 hours was subjected to quantitative real-time PCR with their specific primers of Bcl2, Bcl-XL, MCL1 and GAPDH. mRNA expressions were normalized to that of GAPDH. Note statistically significant decrease of Bcl-Xl mRNA in BTG2 overexpressed cancer cells. (B) Comparison of Bcl-X enogenous pre-mRNA in LacZ and BTG2 overexpressed cancer cells. Note no significant difference of Bcl-X pre-mRNA level in three different cancer cell lines after BTG2 overexpression in contrast to the level of mature Bcl-XL mRNA. (C) mRNA level of Bcl-XL and Bcl-XS in BTG2 overexpressed cancer cells. Similar degree of decrease of Bcl-XL and Bcl-XS was observed in BTG2 overexpressed A549 cells. (D) Immunoblot analysis of Bcl2, Bcl-XL, MCL1 protein level in BTG2 overexpressed cancer cells. Note significant decrease of Bcl-XL protein in agreement with the decrease in Bcl-XL mRNA in BTG2 overexpressed cancer cells.

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Figure S1. Gene expression data of microarray from liver tissue of TIS21 wild type and knock out mouse.

Figure S2. Comparison of mRNA stability between LacZ and BTG2 overexpressed HeLa cells.

１６ 3.2. BTG2 regulates mRNA stability of Bcl-XL

To prove the mechanism that BTG2 can reduce mRNA of Bcl-XL, not by regulation of transcription or splicing process, but by regulation of mRNA stability, remaining mRNA of Bcl-XL was compared between control and BTG2-overexpressed cell lines after blocking further transcription by actinomycin D treatment. In three different BTG2-overexpressed cancer cell lines, statistically significant decrease of mRNA was observed after actinomycin D treatment only in Bcl-XL, not in Bcl2 or MCL1 (Figure 2A). It was reported that prolonged treatment of actinomycin D as a chemotherapeutic agent can decrease cell viability [58]. As expected, increased loss of cell viability was observed in BTG2 overexpressed cancer cells after 12hr treatment of acinomycin D (Figure 2B).

Increased apoptotic cell death is anticipated when Bcl-XL, as anti-apoptotic protein, is decreased.

Morphological feature of dead cells and increased cleaved PARP along with decreased Bcl-XL expression suggested increased apoptotic cell death in BTG2 overexpressed cell treated with acinomycin D. Although it was reported that basal BTG2 expression is relatively low in cancer tissues and cell lines compared normal tissues or cell lines, cancer cell lines still express variable level of BTG2. Therefore, endogenous BTG2 was knocked down with BTG2 siRNA in Hele cells, expressing relatively high level of BTG2 among investigated cancer cell lines (Figure 2C). When BTG2 was knocked down, basal level of Bcl-XL mRNA (Figure 2C) and mRNA stability of Bcl-XL measured by real-time PCR after acinomycin D treatment were increased (Figure 2D). Together, these data suggest that BTG2 can reduce the expression of Bcl-XL and promote cell death, when proper stimulation was applied, by down-regulating mRNA stability of Bcl-XL.

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Figure 2. BTG2 downregulates mRNA stability of Bcl-XL.

(A) mRNA stability of Bcl2, Bcl-XL, MCL1 in BTG2 overexpressed cancer cells. After actinomycin D (5ug/m) treatment, RNA from cells infected with Ad-BTG2 or Ad-LacZ was subjected to quantitative real-time PCR with their specific primers of Bcl2, Bcl-XL, MCL1 and GAPDH. mRNA expressions were normalized to that of GAPDH. Note decreased mRNA stability was observed only in Bcl-XL. (B) Cell death after actinomycin D treatment. Significant apoptotic cell death was observed along with a decrease in Bcl-XL protein in BTG2 overexpressed cells. (C) Increased Bcl-XL mRNA after BTG2 knock down. Note significant upregulation of Bcl-XL mRNA by knockdown of BTG2 in HeLa cells by transfection with siRNA-BTG2. (D) Comparison of Bcl-XL mRNA stability between siRNA-control and siRNA-BTG2 transfected HeLa cells after actinomycin D treatment. Note statistically significant increase of Bcl-XL mRNA stability in siRNA-BTG2 transfected HeLa cells.

２０ 3.3. BTG2 interacts with CNOT7 and hnRNP C

BTG2 has no known enzymatic activity itself. Thus, it was hypothesized that BTG2 might reduce mRNA stability of Bcl-XL by interacting with other proteins. PRMT1 as representative binding partner with BTG2, mediates methylation of arginine residue on several proteins including RNA binding proteins such as AUF1 (hnRNP D) and nucleolin, It is also reported that enzyme activity of PRMT1 can be regulated by BTG2 [59]. In fact, AUF1 is well known RNA binding protein as negative regulation of mRNA stability [60]. In vitro, increased methylation of GST-AUF by PRMT1 was observed when 1ug of recombinant BTG2 was co-incubated (Figure S3A.). However, when PRMT1 was efficiently knocked down by different kinds of siPRMT1s, no significant difference of Bcl-XL mRNA stability was observed (Figure S3B.) and decreased Bcl-XL mRNA after BTG2 overexpression was not recovered by siPRMT1 (Figure S3C.) In addition, the phenomenon of BTG2-mediating enhanced cell death was not reversed when PRMT1 was knocked down (data not shown).

Together, these data suggested that regulation of Bcl-XL mRNA stability was not mediated by PRMT1.

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Figure S3. Regulation of Bcl-xL mRNA stability by BTG2 is not dependent on PRMT1.

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CNOT7 (CAF1a),mRNAdeadenylase, is another well-known interacting partner of BTG/TOB family, and it was suggested that TOB can mediate recruitment of CNOT7 (CAF1a) to target mRNA directly interacting with PABP by PAM2 motifs located on C-terminal [61]. However, since BTG2 has no PAM2 motifs, it was hypothesized that unknown BTG2 interacting protein which has mRNA binding ability might mediate the interaction between BTG2 and target mRNA. According to a recent study, BTG2 can interacts with cytoplasmic poly(A) binding protein (PABPC)1 and stimulates CAF1 deadenylase activity [51]. To find out unknown BTG2 interacting proteins, in vitro binding assay using protein chip with recombinant BTG2 protein was performed and 210 BTG2 binding protein was discovered. As predicted, binding intensity score of CNOT7, known interacting protein was relatively high among other candidate proteins as expected (Figure 3A).

HnRNP C is a nuclear RNA-binding protein with roles in pre-mRNA splicing [62], mRNA stability [63], and translational modulation [64] with nascent mRNA transcripts. On literature searching, hnRNP C was identified as one of the proteins interacting to 3'UTR of Bcl-XL mRNA along with nucleolin, YB-1, and NF-AT, and only the role of nucleolin as stabilizing Bcl-XL mRNA in response to UAV irradiation was studied [65]. Thus, hnRNP C was selected for further investigation.

To confirm the in vitro interaction between BTG2 and hnRNP C or CNOT7 observed in protein array result, immunoprecipitation assay was performed in 293T cell. Since the level of endogenous hnRNP C protein was relatively abundant compared to that of BTG2 or CNOT7, BTG2 and CNOT7 but not hnRNP C was overexpressed in 293T cell by BTG2-HA and CNOT7-V5 plasmid. After immunoprecipitating with HA antibody, strong interaction between BTG2 and known interacting protein, CNOT7 was observed (Figure 3B). In this experimental condition, following immunoprecipitation was performed. After immunoprecipitating with HA antibody, interaction between BTG2 and endogenous hnRNP C was detected by western blotting (Figure 3C, D). In another cancer cell lines, interaction between BTG2-HA and hnRNP C was confirmed by immunoprecipitation assay (Figure 3E, F).

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２４ Figure 3. BTG2 interacts with CNOT7 and hnRNP C

(A) Binding intensity of recombinant BTG2 and candidate proteins by protein chip array. Note high binding intensity between CNOT7, known interacting protein, and BTG2. HnRNP C, previously discovered as one of the proteins interacting to 3'-UTR of Bcl-XL mRNA [65], was chosen for further study. (B) Immunoprecipitation with BTG2-HA by HA antibody in CNOT7-V5 and BTG3-HA plasmid transected 293T cells. Note strong interaction between BTG2 and known interacting protein, CNOT7. In this experimental condition, following immunoprecipitation was performed for exploring interaction between BTG2 and hnRNP C. (C) Immunoprecipitation with BTG2-HA by HA antibody in BTG2-HA plasmid transected 293T cells. 293T cells transfected with BTG2-HA plasmid was equally divided and was immunoprecipitated with isotype control IgG antibody and HA antibody.

Strong interaction between BTG2 and hnRNP C was found only in sample incubating HA antibody.

(D) Reciprocal immunoprecipitation. Similar results were obtained by immunoprecipitating hnRNP C.

(E) Immunoprecipitation with BTG2-HA by HA antibody in BTG2-HA plasmid transected MCF7 cells. (F) Immunoprecipitation with hnRNP C in BTG2-HA plasmid transected A549 cells.

２５ 3.4. HnRNP C interacts with 3’-UTR of Bcl-XL mRNA

RNA immunoprecipitation to pull down hnRNP C together with its potential mRNA targets was conducted to investigate the interaction between hnRNP C and 3’-UTR of Bcl-xL mRNA. This method has been successfully used to examine in vivo RNA-protein interactions [52]. By immunoprecipitation of hnRNP C protein, followed by detection of 3’-UTR of Bcl-xL mRNA specific primers by reverse transcription and PCR amplification., the 3’-UTR of Bcl-xL mRNA was significantly enriched in the hnRNP C immunoprecipitations (Figure 4A). This result indicated that Bcl-XL mRNA was bound by endogenous hnRNP C in intact cells. Next, to indentify the regions of 3’-UTR of Bcl-xL mRNA to which hnRNP C binds, biotinylated transcripts representing four segments of 3’-UTR of Bcl-xL mRNA was used for biotin pulldown analysis. After each of four biotinylated RNA fragments was incubated with cell lysates, pulldown with streptavidin bead was performed, and the level of hnRNP C was determined by western blot analysis. The strongest interaction was observed with fragment D, which contained AUUUUA regions, known RNA-binding sequence of hnRNP C (Figure 4B). Taken together, these results suggested that BTG2 can bind 3’-UTR of Bcl-xL mRNA indirectly through the hnRNP C and might mediate degradation of target mRNA by recruiting CNOT7.

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２７ Figure 4. HnRNPC interacts with Bcl-XL mRNA 3’-UTR

(A) RNA immunoprecipitation (RIP) is performed with whole cell extracts to enrich RNAs interacting with hnRNP C or control IgG. The presence of Bcl-XL mRNA 3’-UTR was determined by 3’-UTR specific RT-PCR in control IgG and anti-hnRNP C immunocomplexes. (B) Top, schematic depicting the Bcl-XL mRNA 3'-UTR tested by biotin pulldown assays. Bottom, after incubation with the indicated biotinylated RNAs fragment, the presence of hnRNP C was detected by immunoblot analysis. Biotinylated 3′ UTR of GAPDH served as negative control.

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3.5. mRNA downregulation of Bcl-XL is dependent on interaction between BTG2 and CNOT7 To determine whether the change of Bcl-XL mRNA stability in BTG2 overexpressed and knock down cells was due to the interaction between BTG2 and deadenylase, CNOT7, previously reported BTG2 mutants (BTG2 G64A, W103A, and BTG2 Y65A) defective in binding CNOT7, was produced.

After transfection of BTG2 wild-type or mutants and C-terminal V5 tagged CNOT7 in 293T cells, immunoprecipitation with HA antibody was performed. As previous reports, the binding affinity of BTG2 G64A, W103A or BTG2 Y65A to CNOT7 was significantly decreased compared to that of wild type BTG2 to CNOT7, although complete abolishment of interaction between mutant BTG2 and CNOT7, which was observed in previous report, was not shown in this experiment (Figure 5A).

However, the binding between BTG2 and hnRNP C was maintained even in BTG2 Y65A (Figure 5B).

When BTG2 Y65A was overexpressed, increased mRNA degradation of Bcl-XL after actinomycin D treatment, which was observed in wild type BTG2 overexpressed cells, was not shown (Figure 5C).

However, when baseline level of hnRNP C was decreased by sihnRNP C, the change of Bcl-XL was not revealed statistically significant difference. Taken together, these data suggested that mRNA stability of Bcl-XL was dependent on the interaction between BTG2 and deadenylase, CNOT7.

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Figure 5. mRNA downregulation of Bcl-XL is dependent on interaction between BTG2 and CNOT7

(A) Immunoprecipitation with HA antibody in 293T cell lysates transfecting either BTG2 WT, BTG2 G64A, W103A, or BTG2 Y65A and CNOT7-V5 and immunoblotting with V5 antibody for detecting CNOT7-V5. Note significant reduction of interaction between BTG2 G64A, W103A, or BTG2 Y65A and CNOT7. (B) Immunoprecipitation with HA antibody in 293T cell lysates transfecting either BTG2 WT or BTG2 Y64A and immunoblotting with hnRNP C antibody for detecting hnRNP C. Note maintenance of interaction between BTG2 Y64A and hnRNP C. (C) mRNA level of Bcl-XL after actinomycin D (5ug/m) treatment. Increased reduction of Bcl-XL mRNA level in BTG2 WT overexpressed 293T cells was not observed in BTG2 Y64A overexpressed 293T cells. (D) mRNA level of Bcl-XL after sihnRNP C transfection. Left, the efficiency of knock down by sihnRNP C was measured. Right, Bcl-XL mRNA level was compared in cells transfected with siControl and sihnRNP C. There was no significant difference in Bcl-XL mRNA level after hnRNP C knock down.

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3.6. BTG2 enhances cell death through the regulation of Bcl-XL

Previous studies suggested that BTG2, as tumor suppressor gene, can augment cancer cell death.

In normal and cancer cells, basal level of endogenous BTG2 is tightly regulated with short protein half-life about 15 min and BTG2 expression, as well-known one of the p53 downstream genes, is significantly increased in various stressful situation such as DNA damage. Therefore, when DNA damage was induced, increased BTG2 might augment cancer cell death by downregulation of Bcl-XL.

To investigate this hypothesis, at first, by using ad-BTG2 virus, BTG2 was overexpressed in A549 cancer cell lines. Overexpression of BTG2 without any additional stimulation could induce cancer cell death in dose-dependent manner (Figure 6A). Morphologic feature and increased cleaved PARP along with reduction of Bcl-XL suggested the main type of induced cell death was apoptosis (Figure 6A).

However, the level of BTG2 overexpressed by ad-BTG2 virus transduction-inducing direct cell death is far from physiologic. Since previous study showed that the cell lines demonstrating relative resistance to 70,000 cytotoxic agents in the 60 cell lines of the National Cancer Institute's in vitro anticancer drug screen were characterized by high BCL-XL expression, BCL-X was suggested to have unique role in general resistance to cytotoxic agents [66]. In addition, BTG2 was demonstrated as mediator of cisplatin induced anti-proliferation effect on prostate cancer cell [57]. Thus, chemotherapeutic agent, cisplatin, most commonly used in chemotherapy regimens for solid tumor treatment was applied for inducing cell death to mimic more relevant physiologic and clinical situation. During cell death induced by cisplatin, Bcl-XL was decreased in cisplatin dose and time dependent manner (Figure 6B). At early time points, before definitive cell death was observed, BTG2 mRNA was increased along with decrease of Bcl-XL mRNA within 8hr after cisplatin treatment (Figure 6C). When BTG2 was overexpressed up to the level without spontaneous cell death, cisplatin-induced cell deaths was augmented in BTG2 overexpressed cells (Figure 5D) and these increased cell death was not observed when BTG2 mutant (Y65A) which has defect in binding to CNOT7, was overexpressed (Figure 6E).

To validate and broaden these observations in more objective and clinically meaningful situation, gene expression data [67] of 30 cancer cell lines with information of resistance towards 11 anticancer drugs at clinically achieved concentrations was analyzed. In this database, BTG2 expression was also significantly higher among cisplatin sensitive cell lines compared that of insensitive cell lines (Figure S4). This reciprocal gene level of BTG2 and Bcl-XL after platinum chemotherapy was consistently observed in various kinds of oncogene and tumor suppressor gene mutated gastric cancer cell lines (Figure S5). In addition, this negative correlation of BTG2 and Bcl-XL gene was found with variable degrees of correlation in several cancer types of TCGA database (Figure S6). Taken together, these data revealed that BTG2 could enhance chemotherapy induced cancer cell death through the regulation of Bcl-XL mRNA stability mediated by interacting hnRNP C and CNOT7.

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Figure 6. BTG2 enhances cisplatin-induced cancer cell death.

(A) Cell death of LacZ or BTG2 overexpressed A549 cells. Indicated moi of conrol LacZ or ad-BTG2 virus was transduced in A549 cells. Top, phase contrast microscopic findings of A549 cells transduced with indicated moi of adenovirus for 48 hours. Significant the cell death was observed from A549 cells transduced 400 moi of ad-BTG2 virus. In contrast, there was no significant cell death until 1000 moi of ad-LacZ virus transduction. Bottom, immunoblot analysis for level of Bcl-XL and cleaved PRAP after BTG2 overexpression. In agreement with observed cell death, significant reduction of Bcl-XL and cleavage of PRAP was observed from A549 cells ransduced 400 moi of

(A) Cell death of LacZ or BTG2 overexpressed A549 cells. Indicated moi of conrol LacZ or ad-BTG2 virus was transduced in A549 cells. Top, phase contrast microscopic findings of A549 cells transduced with indicated moi of adenovirus for 48 hours. Significant the cell death was observed from A549 cells transduced 400 moi of ad-BTG2 virus. In contrast, there was no significant cell death until 1000 moi of ad-LacZ virus transduction. Bottom, immunoblot analysis for level of Bcl-XL and cleaved PRAP after BTG2 overexpression. In agreement with observed cell death, significant reduction of Bcl-XL and cleavage of PRAP was observed from A549 cells ransduced 400 moi of