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 ad-BTG2 virus. Note no significant reduction of Bcl-XL and cleavage of PRAP in ad-LacZ transduced A549 cells. (B) Immunoblot analysis for level of Bcl-XL after cisplatin treatment. After indicated amount of cisplatin treatment in A549 cell, Bcl-XL protein was decreased in dose and time dependent manner. (C) Changes of BTG2 and Bcl-XL mRNA after cisplatin treatment. Note reciprocal relationship between BTG2 and Bcl-XL mRNA level after cisplatin treatment. (D) Comparison of Ad-LacZ or Ad-TIS21 transduced Hela Cell death after cisplatin treatment. Left, phase contrast microscopic findings of HeLa cells treated with cisplatin after 24 hours of ad-LacZ or ad-BTG2 virus transduction. Right, cell viability measured by tryptophan blue exclusion assay. More cell death was observed in ad-BTG2 transduced HeLa cells treated with indicated concentration of cisplatin. (E) Comparison of BTG2 WT or BTG2 Y65A transfected Hela Cell death after cisplatin treatment. Left, phase contrast microscopic findings of HeLa cells treated with cisplatin after 24 hours of BTG2 WT or BTG2 Y65A plasmid transfection. Right, cell viability measured by tryptophan blue exclusion assay. Increased cell death observed in BTG2 WT transfected HeLa cells was not shown in BTG2 Y65A plasmid transfection.

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Figure S4. BTG2 mRNA level according to chemotherapy sensitivity from gene expression data of 30 cancer cell lines [58].

Figure S5. BTG2 and Bcl-XL expression after oxaliplatin treatment in several gastric cancer cell lines.

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Figure S6. Expression pattern of BTG2 and Bcl-XL mRNA in TCGA database

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3.7. High BTG2 expression was associated with favorable platinum-based chemotherapy