BTG2 is a tumor suppressor gene upregulated by p53 and PTEN in human bladder carcinoma cells

Abstract Although widely deemed as a tumor suppressor gene, the role of B‐cell translocation gene 2 (BTG2) in bladder cancer is still inconclusive. We investigated the role and regulatory mechanism of BTG2 in bladder cancer. BTG2 expression in human bladder tissues was determined by RT‐qPCR and immunoblotting assays. Expressions of BTG2 and PTEN in bladder carcinoma cells were determined by immunoblotting, RT‐qPCR, or reporter assays. The 3H‐thymidine incorporation assay, flow cytometry, and the xenograft animal model were used to determine the cell growth. BTG2 expression was lower in human bladder cancer tissues than normal bladder tissues. Highly differentiated bladder cancer cells, RT4, expressed higher BTG2 than the less‐differentiated bladder cancer cells, HT1376 and T24. Overexpression of BTG2 in T24 cells inhibited cell growth in vitro and in vivo. Camptothecin and doxorubicin treatments in RT‐4 cells or transient overexpression of p53 into p53‐mutant HT1376 cells induced p53 and BTG2 expression. Further reporter assays with site‐mutation of p53 response element from GGGAAAGTCC to GGAGTCC within BTG2 promoter area showed that p53‐induced BTG2 gene expression was dependent on the p53 response element. Ectopic PTEN overexpression in T24 cells blocked the Akt signal pathway which attenuated cell growth via upregualtion of BTG2 gene expression, while reverse effect was found in PTEN‐knockdown RT‐4 cells. PTEN activity inhibitor (VO‐OHpic) treatment decreased BTG2 expression in RT‐4 and PTEN‐overexpressed T24 cells. Our results suggested that BTG2 functioned as a bladder cancer tumor suppressor gene, and was induced by p53 and PTEN. Modulation of BTG2 expression seems a promising way to treat human bladder cancer.


Introduction
BTG3, and Tob genes. TIS21, the homologous of BTG2, was first isolated from 3T3 fibroblasts [4]. Then, the human BTG2 was cloned from chromosomal segment 1q32 [5]. The function of BTG2 in cancer growth inhibition has been previoulsy explored in several reports [6][7][8]. Mao et al. further indicated that BTG2 caused G1 or G2/M cell cycle arrest dependent on the cell types [9]. Our group has proved that BTG2 inhibited cell growth and induced either p53 dependently or independently in human prostate cancer cells [10]. BTG2 has further identified as one of the prostate-derived ets factor (PDEF) downstream genes in prostate cancer and bladder cancer cells [11,12]. However, the exact role of BTG2 in bladder cancer is still inconclusive. Hoffman et al. showed the raloxifene inhibitory effect on the RT4 cell growth via enhancement of BTG2 expression, suggesting BTG2 may play as a tumor suppressor gene in the bladder cancer [13]. On the contrary, one report indicated that endogenous expression of BTG2 stimulated the migration of bladder cancer cell and higher BTG2 expression correlated with poor survival of patients with bladder cancer [14]. Therefore, it is suggested further study to clarify the BTG2 role in bladder cancer.
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been widely known as a tumor suppressor gene and PTEN mutation or deletion is frequently noted in a lot of cancers [15]. The most known function of PTEN is the negative regulator of PI3K/Akt/ mTOR pathway, which is a crucial signal transduction pathway for cancer cell growth [16]. For bladder cancer, loss of PTEN expression has been correlated with the disease invasiveness [17]. However, the details regarding PTEN influences on the cell growth of bladder cancer and the PTEN downstream genes have not studied yet.
In this study, we investigated the roles of BTG2 and PTEN as well as the regulatory mechanisms of BTG2 in human bladder cancer. We aimed to provide new targets for bladder cancer therapy to improve the survival rate for the patients.

Cell cultures and chemicals
The bladder carcinoma cell lines RT4, HT1376, and T24 were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan, ROC) and maintained as described before [12]. Fetal calf serum (FCS) was purchased from HyClone (Logan, UT), RPMI 1640 media was obtained from Invitrogen (Carlsbad, CA), Matrigel was purchased from BD Biosciences (Bedford, MA), and PTEN inhibitor, VO-OHpic trihydrate, from Sigma (St. Louis, MO).

Tissue collection and analysis
Tissues of human bladder comprised biopsy specimens obtained from patients admitted to the Department of Urology, Chang Gung University Hospital (Tao-Yuan, Taiwan) and the protocol for tissue collection and analysis was approved by the Institutional Review Board of the Chang Gung Memorial Hospital (Approval: IRB 102-3721B). Bladder tissues were classified based on the pathological examinations of the parallel preparations from respective samples by attending pathologists.

Expression vector constructs and stable transfection
The amplified cDNA fragment containing the human BTG2 coding region was cloned into the eukaryotic expression vector pcDNA3 (Invitrogen) as described in detail previously [18]. The human PTEN natural ORF mammalian expression plasmid (HG10421-UT; pCMV3-PTEN) was purchased from Sino Biological Inc. (Bejing, PR China). Electroporation was used to introduce expression vectors into the T24 cells, and the cells were selected with G418 (for T24-BTG2) or hygomycin (for T24-PTEN) as described in detail previously [12]. The mock-transfected T24 (T24-DNA) cells were transfected with an empty expression vector (pcDNA3) and then clonally selected as the same as overexpressed cells.

H-thymidine incorporation assay
The 3 H-thymidine incorporation assay was used to measure cell proliferation as previously described [18].

Flow cytometry
Cells were serum starved for 24 h and then cultured in RPMI 1640 medium with 10% FCS for another 24 h. Cell cycle analysis was performed using the FACS-Calibur Cytometer and CellQuestPro Software (BD Biosciences); the data were analyzed using ModFit LT Mac 3.0 Software.

Real-time reverse transcription-polymerase chain reaction (RT-qPCR)
Total RNA was isolated using TRIzol ® reagent, and cDNA was synthesized using the Superscript III pre-amplification system (Invitrogen). FAM ™ dye-labeled TaqMan ® MGB probes as well as PCR primers for human BTG2 (Hs0019 8887_m1), 18S (Hs03003631_g1), PTEN (Hs99999905_ m1), and GAPDH (Hs99999905_m1) were purchased from Applied Biosystems (Foster City, CA). GAPDH (glyceraldehyde 3-phosphate dehydrogenase; for the study of cells) and S18 (for the study of tissues) were used as internal positive probes. Real-time reverse transcriptionpolymerase chain reaction (RT-qPCR) was performed and the mean cycle threshold (C t ) values were calculated for internal control and target genes as described in detail previously [12].

Reporter vector constructs and reporter assay
The human BTG2 (−297 to −1), reporter vectors were constructed as described in detail previously [11,12]. The mutant p53 response elements in BTG2 reporter vectors were constructed as described previously [18]. Cells were seeded at a density of 10 4 cells/well in a 24-well plate and allowed to grow for 24 h. Cells were then transiently transfected with luciferase reporter vector for additional 48 h and relative luciferase activities were then measured and reported in relative light units (RLU) as previously described [19].

Tumor xenograft study
The animal study has obtained approval from the Institutional Animal Care and Use Committee of the College of Medicine, Chang Gung University (IACUC Approval No.: CGU15092). Animal studies were performed in accordance with Laboratory Animal Facilities and Care guidelines (Council of Agriculture, Executive Yuan, Taiwan). Eighteen 4-week-old male BALB/cAnN-Foxn1 NU mice were used in this study. Animals were purchased from the National Laboratory Animal Center, Taipei, Taiwan. Each mouse was anesthetized with a 100 μL intraperitoneal injection of a mixture of 2.5% tribromoethanol and 2.5% tert-amyl alcohol in Tris buffer solution. Prior prepared cancer cells (3 × 10 6 cells/100 μL) were mixed (1:1) with Matrigel and subcutaneously injected into one side of the back near the shoulder of each mouse. Mice were kept in a barrier facility under HEPA filtration and animal health was monitored twice per week during experiment. Xenograft growth was measured by Vernier calipers at intervals as indicated, and tumor volume was calculated using a previously described formula, namely Volume = [π/6 × largest diameter × (smallest diameter) 2 ] [20].

Statistical analysis
Results are expressed as means ± SE of at least three independent experiments. Significant differences between groups were determined by one-way ANOVA and the Student t-test. All statistical analyses were carried out using the statistical package SigmaStat for Windows (Version 2.03, SPSS, Chicago, IL).

Evaluation of BTG2 expression in human bladder cancer tissues and cell lines
The mRNA expression of BTG2 was evaluated by RT-qPCR. As shown in Figure 1A, BTG2 mRNA expression was higher in normal bladder tissues than cancerous tissues with the ∆∆CT of 2.85. Further measurement from paired normal and cancerous bladder tissues revealed that bladder cancer tissues presented with lower BTG2 mRNA expression (∆∆CT = 1.85, Fig. 1B) in comparison with bladder normal tissues. Results of immunoblotting assays also indicated that expression of BTG2 was lower in the cancer part than paired normal tissues (Fig. 1C). As compared to the highly differentiated bladder cancer cells, RT4, with other two less differentiated bladder cancer cells, HT1376 and T24, RT4 cells has higher BTG2 expression than HT1376 and T24 cells as determined by immunoblotting (Fig. 1D, top) and RT-qPCR (Fig. 1D, bottom) assays.

Evaluation of BTG2 role in human bladder cancer cell in vitro and in vivo
To evaluate BTG2 role in human bladder cancer, we stably transfected BTG2 into T24 cells, and obtained T24-BTG2-1 and T24-BTG2-2 cells. As shown in Figure 2A, T24-BTG2-1 and T24-BTG2-2 cells presented higher BTG2 mRNA and protein expressions than T24-DNA cells (T24 cells with mock BTG2 transfection). The cell proliferation of T24-BTG2-1 and T24-BTG2-2 cells were attenuated as compared with T24-DNA cells as determined by 3 H-thymidie incorporation assay (Fig. 2B). In vivo animal study also revealed that xenografted T24-BTG2-2 cells grew much slowly than T24-DNA cells (Fig. 2C). To investigate further the influences of BTG2 on T24 cell growth, the cell cycle distribution of T24-DNA, T24-BTG2-1, and T24-BTG2-2 cells were evaluated by flow cytometry. Figure 2D demonstrated higher S and G2/M phase cells in both T24-BTG2-1 and T24-BTG2-2 cells. Collectively, our results suggested that BTG2 played as a tumor suppressor gene in human bladder cancer because BTG2 expressed lower in human bladder cancer tissues, and forced expression of BTG2 in human bladder cancer cells decreased cell growth in vitro and in vivo, which was partly attributed to cell cycle arrest induction at G2/M phase.
Evaluation of PTEN effect on cell growth and BTG2 mRNA expressions in human bladder cancer cells PTEN expression was evaluated in RT4, HT1376, and T24 cells with highest and lowest PTEN expressions in RT4 and T24 cells, respectively (Fig. 4A). To investigate PTEN effect on human bladder cancer, PTEN was knocked down or overexpressed in RT4 (Fig. 4B) or T24 (Fig. 4C) cells, respectively. The BTG2 expressions were decreased by PTEN knockdown in bladder cancer cells as RT4_ shPTEN cells (RT4 cells with PTEN knockdown) exhibited lower BTG2 mRNA expression than RT4_shCtrl cells (RT4 cells with mock knockdown) (Fig. 4B); while T24-PTEN cells (T24 cells with PTEN overexpression) presented higher BTG2 mRNA expression than T24-DNA cells (T24 cells with mock overexpression) (Fig. 4C). The result was further supported by the reporter assays, which showed that BTG2 reporter activities were increased in a dose-dependent manner as treated by PTEN expression vectors (Fig. 4D). Figure 4E shows that T24-PTEN cells had lower cellular proliferation rate than T24-DNA cells; while RT4_shPTEN cells exhibited higher cell proliferation rate than RT4_shCtrl cells (Fig. 4F). Collectively, our results indicated that PTEN repressed cell growth of the bladder cancer in vitro, and negatively modulated BTG2 mRNA expression in bladder cancer cell.

Evaluation of PTEN downstream signals and genes in human bladder cancer cells
We further evaluated PTEN downstream signals expressions in bladder cancer cells. T24-PTEN cells showed lower pAKTs473, pAKTt308, pGSK3b, pmTOR, and pP70S6K expressions than T24-DNA cells; while RT4_shPTEN cells presented higher pAKTs473, pAKTt308, pGSK3b, pmTOR, and pP70S6K expressions than RT4_shCtrl cells (Fig. 5A). Figure 5A demonstrated that PTEN increased BTG2 protein expression in human bladder cancer cells as T24-PTEN cells exhibited higher BTG2 expression than T24-DNA cells; while RT4_shPTEN cells revealed lower BTG2 expression than RT4_shCtrl cells. Then, we treated RT4 cells with VO-OHpic trihydrate, one kind of PTEN activitiy inhibitor, and the expression of p-Akt (t308 and s473) was increased, but BTG2 was decreased while PTEN and Akt expressions remained the same (Fig. 5B). The BTG2 mRNA expression was inhibited by VO-OHpic trihydrate in RT4 cells (Fig. 5C) and T24-PTEN (Fig. 5D) cells. The reporter assay for BTG2 reporter vector-transfected T24-PTEN cells treated by varied concentrations of VO-OHpic trihydrate revealed that the BTG2 reporter activity was decreased by VO-OHpic trihydrate (Fig. 5E). Collectivley, our results indicated that BTG2 expression in human bladder cancer cells was stimulated by PTEN.

Discussion
In this study, we demonstrated that BTG2 served as a tumor suppressor gene in human bladder cancer in vitro and in vivo and lower BTG2 expression was found in human bladder cancer tissues as compared to normal bladder tissues. The expressions of BTG2 were stimulated by p53 and PTEN in human bladder cancer cells. PTEN deficiency also enhanced cell growth of the human bladder cancer. Our results suggested that modulation of BTG2 expression is a new therapeutic direction for human bladder cancer.
BTG2 belongs to the BTG/TOB anti-proliferative proteins family, besides BTG2, which also comprises BTG1, BTG3, BTG4, TOB1, and TOB2 featuring the conserved N-terminal BTG domain [21,22]. Although widely deemed as a tumor suppressor gene, the role of BTG2 in human bladder cancer has not disclosed fully with higher BTG2 expression associated with reported poor prognosis of human bladder cancer patients [14]. Our results indicate that human bladder cancer exhibited lower BTG2 mRNA and protein expression as compared to normal bladder tissues (Fig. 1A, B, and C). The higher differentiated human bladder cancer cells, RT4, possessed higher BTG2 protein expression than other two less differentiated human bladder cancer cells, HT1376 and T24 (Fig. 1D). To understand BTG2 role in bladder cancer, BTG2 was then transfected into T24 cells. Figure 2B demonstrated that both T24-BTG2-1 and T24-BTG2-2 cells presented with slower proliferative rate as compared with T24-DNA cells. The xenografted T24-BTG2-2 tumor exhibited smaller tumor volume than T24-DNA cell group (Fig. 2C). Collectively, since BTG2 presented with higher expression in normal bladder tissues than bladder cancer tissues and forced expression of BTG2 in human bladder cancer cells inhibited cancer cell growth in vitro and in vivo, we thus concluded that BTG2 played as a tumor suppressor gene in human bladder cancer.
Cell cycle progression is the necessity for cell to proliferate and is under well orchestration and strict control to maintain human tissue homeostasis. The uncontrolled cell proliferation of cancer mainly can be attributed to the cell cycle deregulation [23]. Thus, cell cycle progression emerges as a good target for cancer treatment. BTG2 was proposed as a pan-cell cycle regulator before [24], which could induce G1/S or G2/M arrest in a tissue-or cell-specific manner. Previous studies indicated that overexpression of BTG2 induces growth inhibition of 293 and OSCC cells by modulation of cyclin A, cyclin B, cyclin D1, or cyclin E [25]. As we analyzed cell cycle distribution of T24-DNA, T24-BTG2-1, and T24-BTG2-2 cells by flow cytometry, higher G2/M and S phase cell percentages were found in T24-BTG2-1 and T24-BTG2-2 cells (Fig. 2D), indicating BTG2 transfection could induce G2/M arrest in human bladder cancer cells, leading to the growth inhibition found in Figure 2B and C.
p53 is a well-known tumor suppressor gene and p53 mutations have been identified in a variety of human cancers [26]. Previously, p53 response element has found to exist within BTG2 promoter area, indicating BTG2 expression is modulated by p53 [5,10,27]. To investigate whether BTG2 expression is modulated by p53 in human bladder cancer cells, camptothecin (0.25-1 μmol/L) and doxorubicin (0.05-0.2 μg/mL) were applied to treat p53 wild-type RT4 cells. Figure 3A, B and C demonstrated that both drugs could induce p53 and BTG2 expressions in RT4 cells dose-dependently. Transient overexpression of p53 in p53-null HT1376 cells increased both p53 and BTG2 expressions (Fig. 3D), while knockdown p53 in p53-wild-type RT4 cells decreased both p53 and BTG2 expressions (Fig. 3E), which was also supported by the increased BTG2 reporter activity as treated by varied doses of p53 expression vectors in HT1376 cells (Fig. 3F). The reporter assays with 5′-deletion and site-mutation of p53 response elements within BTG2 promoter area further confirmed that p53 induced BTG2 gene expression through interacting with the p53 response element located at BTG2 promoter area (Fig. 3G). Taken together, our results indicated that BTG2 was stimulated by p53 in human bladder cancer cells. These results are consistent with our previous studies in the prostate carcinoma cells [18,27]. Besides p53, several reports has indicated that JNK, ERK, p38, NFκB, WNT/β-catenin, AKt/sp1/ NOx4, and Src/FAK pathways also modulate BTG2 expressions in different cancer cells [28][29][30][31][32]. PTEN, identified in 1997 in chromosome 10q23, is a well-known tumor suppressor gene. The finding of the late stage cancer usually has inactivated PTEN renders PTEN a hot issue for cancer treatment research in the past decades [33,34]. The main function of PTEN lies in the negative regulation of PI3K/Akt/mTOR pathway, which plays a vital role in regulating many important signaling pathways, which mainly induce cell growth and metastasis [15]. As we evaluated PTEN expressions in three kinds of human bladder cancer cells, RT4, the mostly differentiated cells among these three kinds of cancer cells, presented with the highest PTEN expression (Fig. 4A). As we knocked down or overexpressed PTEN, the phosphorylation of downstream signal proteins were changed, including pAKTs473, pAKTt308, pGSK3b, pmTOR, and  (Fig. 5A). The findings of that T24-PTEN cells had lower cell proliferative rate than T24-DNA cells and RT4_shCtrl had lower cell proliferative rate as compared to RT4_shPTEN cells demonstrated the tumor suppressor gene role of PTEN in human bladder cancer cells (Fig. 4E  and 4F). Since early study indicated that PTEN and    Fig. 4 and 5). Thus, we concluded that PTEN insufficiency would increase cell growth of the human bladder cancer with BTG2 positively regulated by PTEN. Previous study has indicated that PTEN induced p53 acetylation which regulated p53 protein stability in osteosarcoma U2OS cells [35]. Whether PTEN induced p53 protein stability to upregulate BTG2 gene expression in bladder carcinoma cells needs further investigation. However, based on our study showing ectopic overexpression of PTEN in the p53-null T24 cells induced BTG2 expression, PTEN upregulated BTG2 expression is, in part, not via the p53 signal pathway in bladder cancer cells (Fig. 4C).

Conclusion
Although most bladder cancer patients diagnosed in the early stage, still 70% of patients have cancer recurrence and 10% of the recurrent patients have bladder cancer with muscle involvement, which has the high possibility of concurrent distant metastasis, leading to the poor prognosis [36]. Thus, to find out more targets for bladder cancer treatment is warranted. Our current work demonstrated that BTG2 functioned as a tumor suppressor gene in human bladder cancer and induced by p53 and PTEN. PTEN served as a tumor suppressor gene as well in human bladder cancer. Our results suggest that modulation of BTG2 expression is a promising direction for bladder cancer treatment.