Preparation of Ad-BTG2 virus and Reconstitution of BTG2 gene Expression

Adenovirus with BTG2 gene (Ad-BTG2) was prepared by transfection of hemagglutinin-tagged BTG2(BTG2-HA) cDNA into 293A cells according to the established method in our laboratory [Park TJ et al., 2008].

- 33 - 3. mTOR Kinase Assays

Assay protocol was adopted from Ikenoue et al []. Immunoprecipitates isolated from the cell lysates transduced with Ad-LacZ and Ad-BTG2 virus were used as the in vitro kinase source.

4. In vivo Analyses and Preparation of Mouse Tissues

Mice were maintained under the specific-pathogen-free condition and all animal procedures were followed by Ajou University Institutional Review Board. Organs collected from the BTG2-wild type (C57BL/6, 16 week-old) and BTG2-KO (17 week old) female mice (n = 3/each group/experiment) were snap-frozen in liquid nitrogen before use.

Tissue homogenates were prepared in RIPA buffer and analysed by SDS-PAGE. The same protocol was applied for PHLPP2 protein expression analysis from human breast cancer tissues. All experiments used for mouse tissues were repeated more than twice. No blind experiments were performed.

5. Chromatin Immunoprecipitation Analysis (ChIP)

ChIP analysis was performed by the method described previously [Sundaramoorthy S et al., 2013]. Precipitated DNAs were analyzed for PCR amplification using Taq-DNA-polymerase (NanoHelix, Daejon, South Korea) using the primers listed in Table 1.

6. Immunohistochemistry and Survival Analysis

Immunohistochemical reactions were conducted on the 4 μm sections of formalin-fixed, paraffin-embedded tissue blocks of human breast cancers as the described method [Devanand P et al., 2014] using anti-BTG2 and anti-AKT antibodies. Clinical information about patients was collected from the Ajou University Hospital after informed consent, and tumour tissues were classified based on the degree of lymph node invasion. Tumour and the matched normal tissues were obtained according to the regulations of Institutional Review Board at the Ajou University Hospital (AJIRB-GEN-SMP-11-066). For overall survival analysis between BTG2 expression and ER:LN (estrogen receptor:lymph node) status, Kaplan-Meier analysis was performed and log-rank test was applied for the statistical significance (www.kmplot.com) [Gyorffy B et al., 2010] .

- 34 - 7. Immunoprecipitation

To explore protein-protein interaction, cells with BTG2-expressed or the control were sonicated in E1A lysis buffer (250mM NaCl, 50mM HEPES, pH7.5, 0.1% NP-40, 5mM EDTA, and protease/phosphatase inhibitors), and then subjected to immunoprecipitation as described previously [Ryu MS et al., 2004].

8. MTT assay

Proliferations of MCF-7 and MDA-MB-231 cells were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny ltetrazoliumbromide (MTT) assay. Cells infected with Ad-BTG2 or Ad-LacZ were seeded into 96-well plates (quadruplicates) and incubated for 48 h in the fresh media, and then added 20 μl of MTT (5 mg/ml; Sigma, St. Louis, MO, USA) solution at 37°C for 4 h. The supernatants were removed, and 100 μl of dimethylsulfoxide (Sigma, St. Louis, MO, USA) was added to each well. Colour formation was analysed by absorbance at 570 nm.

9. Colony Formation assay

MDA-MB-231 and MCF-7 cells were infected with Ad-BTG2 or Ad-LacZ and incubated until 48 h, and the cells (2000 cells/60mm dish) were incubated for 14 days more. The plates were washes with 1xphosphate-buffered saline (PBS) and then stained with Crystal Violet solution (0.5%) for 30 minutes at room temperature. Finally the plates were washed with 1xPBS until individual colonies were seen clearly.

10. FACS analysis

Cell cycle analysis was performed by flow cytometry (BD FACScan II, BD Biosciences, San Jose, CA) after staining the DNA with propidium iodide (Sigma) according to the manufacturer’s instruction. Complete details of the protocol have been described elsewhere [Sundaramoorthy S et al., 2013].

11. Transfections of siRNA and Plasmid DNAs

siRNAs against BTG2, Rictor, AKT1, AKT2 and PKCα were synthesized (Genotech, Daejeon, South Korea) and transfected into cells by using Lipofectamine 2000 (Invitrogen,

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Carlsbad, CA) according to the manufacturer’s instruction. siRNAs and the reagent mixture in opti-MEM medium (GIBCO, Life Technologies, Grand Island, NY) were added to the cells for 5h and then cultivated in the fresh media up to 48h. The siRNA sequences were described in Table 1.

12. Reverse transcriptional PCR and RT–qPCR

Total cellular RNAs were extracted from cells with RNAiso Plus (Takara Inc, Kyoto, Japan), and the RNAs (1 μg) were reverse-transcribed by the PCR kit (Takara Inc. Kyoto, Japan). The primers used are described in Table 1. The transcripts were analysed under optimal PCR conditions to avoid saturation phenomenon. The cDNAs were used for RT-qPCR analysis with the specific primers. 18S and glyceraldehyde 3-phosphate dehydrogenase gene expressions were used as the control. Reaction was carried out with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) under the following conditions; initial activation at 95°C for 15 min, followed by 45 cycles of 95°C for 20 s and 60°C for 20 s with the reaction mix.

13. Subcellular fractionation

The protein sequence of NFAT1 was obtained from Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/) and then the sequences were analyzed by Scansite motif software (scansite.mit.edu/).

14. Live cell imaging of GFP-AKT PH localization

MDA-MB-231 cells transduced with either Ad-BTG2 or Ad-LacZ were transfected with GFP-AKT-PH plasmid containing the pleckstrin homology domain of AKT. The cells were incubated for 48 h and the images were taken by live cell imaging. Multiple images spanning the entire cell culture dish were taken and the cells per field were counted. GFP-AKT-PH localization to the membrane was counted and presented as percentage over the total cell numbers.

15. GEO data analysis

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Datasets were downloaded from NCBI and expression data of each gene from normal and breast cancer patients were extracted. SPSS software was used to analyse Spearmen’s correlation values among the genes.

16. In-vitro Protein Purifications

Induction of GST-fusion protein was carried out in BL21 cells with isopropyl β-D-thiogalactoside (500 μM) at 30°C. The cells were lysed in PBS, pH 7.4 containing 5 mM 2-mercaptoethanol, 1 μg/ml leupeptin, and 0.5% Triton X-100, and then cell debris were removed by centrifugation (12,000 × g, 30 min, 4°C). The supernatants containing the GST-fusion proteins were purified with GSH beads (Amersham Biosciences) according to the manufacturer's instruction, and the recombinant proteins were analysed by SDS-PAGE and Coomassie blue stain. GST-S6K1 construct was transfected into HEK293A cells, and the cells were serum starved for 24h before rapamycin (30nM) treatment for 2h to obtain non-phosphorylated GST-S6K1. The cells lysates were centrifuged at 13,000 rpm for 10 mins to remove cell debris. GSH beads were added to the supernatant to purify the GST-S6K1 protein.

17. Statistical analysis

Normally distributed data was analysed by Student’s t-test for in vitro studies. Pearson’s Chi-Square tests were performed using SPSS software for analysing in vivo immunohistochemical study and followed by relative risk analysis of lymph node invasion based on the level of BTG2 expression. No statistical methods were used to predetermine sample size. Results obtained from the test samples are expressed as mean + SD versus control. P-value < 0.05 was considered as significant. Randomization and blind experiments were performed for immunohistochemistry of breast cancer.

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III. RESULTS

A. BTG2 differentially regulates the mTOR interaction with Raptor and Rictor We have previously reported that BTG2 enhances phosphorylation of AKT in normal and cancer cells [Choi JA et al., 2016; Kim BC et al., 2008; Sundaramoorthy S et al., 2013]. To investigate how BTG2 activates AKT in breast cancer cells (Figure 7A, 7B), BTG2 gene transduction was employed in triple negative breast cancer MDA-MB-231 cells, and the effects of the transduced BTG2 on pAKT-S473 were confirmed using short interfering RNAs against BTG2 (Figure 7C). Cell fractionation analyses revealed greater accumulation of pAKT-S473 in the cytoplasm compared to that in the nucleus of BTG2 expressers (Figure 7D). Exogenous BTG2 interacted with endogenous AKT protein (Figure 7E) without regulating the mRNA of AKT isoforms in both invasive and non-invasive breast cancer cells (Figure 7F). Upon further examination of the activation of AKT by analyzing downstream targets, phosphorylation of GSK3β-S9, but not vimentin expression, was observed to be increased in aBTG2-dose-dependent manner (Figure 7G).

Although the kinase responsible for phosphorylation of AKT-S473 is context-dependent [Persad S et al., 2001; Feng J et al., 2004; Partovian C et al., 2004], mTORc2 has been reported to be a kinase that is responsible for the complete activation of AKT [Sarbassov DD et al., 2005]. Therefore, the effects of BTG2 expression on the activities of mTORc1 and mTORc2 were evaluated by in vivo immunoprecipitation and immunoblot analyses in highly invasive MDA-MB-231 cells (Figures 1A and 1B) as well as in non-invasive MCF-7 breast cancer cells (Figure 9A). BTG2 reduced Raptor binding to mTOR (Figure 8A left panel), whereas it increased the interaction of Rictor with mTOR (Figure 8B, left panel).

Measurement of the level of p-mTOR-S2448 by reciprocal immunoprecipitation with anti-Raptor and anti-Rictor antibodies also revealed inhibition of mTORc1 (Figure 8A right panel), in contrast to mTORc2 activation by BTG2 expression (Figure 8B, right panel).

Figure 8C and Figure 9B show that Torin1 and PP242-dual mTOR kinase inhibitors-inhibited BTG2-induced pAKT-S473 in several breast cancer cells, substantiating the increased activity of mTOR kinase by BTG2 in both invasive and non-invasive breast cancer cells. However, rapamycin treatment failed to inhibit BTG2-induced AKT phosphorylation; instead, it enhanced the phosphorylation (Figure 8D), which strongly

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suggests that BTG2-induced pAKT-S473 are regulated by activated mTORc2 in cells.

Since BTG2 inhibited the mTOR interaction with Raptor, BTG2-regulated phosphorylation of p70S6K-a downstream effector of mTORc1-was examined, and a significant loss of p-p70S6K was observed in BTG2 expressers (Figure 8E). The data were further evaluated in MEF cells, and the results demonstrated that the basal level of p-p70S6K was much higher in BTG2-KO-MEF than that in the wild type. p-p70S6K was completely lost following rapamycin treatment in both cell types (Figure 8F), suggesting that mTORc1 activity was inhibited by BTG2 expression in normal and breast cancer cells. Considering that constitutively active p70S6K inhibits mTORc2 [Treins C et al., 2010; Liu P et al., 2013], our present data propose that BTG2-induced pAKT-S473 might be due to the de-repression of mTORc2 downstream of mTORc1, by inhibition of mTOR binding to Raptor, rather than enhancement of the mTOR-Rictor interaction.

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Figure 7. Forced expression of BTG2 induces AKT phosphorylation at Ser473 residue.

A) Concentration dependent activation of AKT at S473 residue in MDA-MB-231 cells.

Cell were infected with various doses of Ad-LacZ or Ad-BTG2-HA viruses for 48h and then harvested for immunoblot analysis to detect the level of p-AKT-S473. GAPDH was used as a loading control. B) Time dependent activation of AKT at S473 residue in MDA-MB-231 cells after infection with 100 moi of Ad-LacZ or Ad-BTG2-HA, and then harvested at the different time points. Note maximum activity of BTG2 upto 48 h after the transduction. C) Cells were infected with Ad-BTG2-HA for 4 h and then incubated overnight before transfection with 50nM of si-BTG2 or si-Control to knockdown the exogenously expressed BTG2 gene. Immunoblot analysis was performed in 24h. D) Cells infected with Ad-LacZ or Ad-BTG2-HA for 48h were subjected to cell fractionation to examine the localization of pAKT under the influence of BTG2gene expression. Note cytosolic and nuclear expressions of pAKT-S473 in response to BTG2 expression. E) Interaction of BTG2-HA with AKT in MDA-MB-231 cells, examined by immunoprecipitation-immunoblot analysis with 1.0 µg of anti-HA and anti-AKT antibodies. F) Realtime qPCR analysis revealing no significant regulations of AKT1 and AKT2 mRNA expressions by BTG2 gene in the non-invasive MCF7 and two different invasive breast cancer cell lines. G) Immunoblot analysis showing activation of GSK-3

at the downstream of the BTG2-induced pAKT-S473 in the MDA-MB-231 cells, however, vimentin expression was not changed.

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Figure 8. BTG2 differentially regulates interaction of mTOR with Raptor and Rictor.

A) In vivo immunoprecipitation revealing inhibition of mTOR binding to Raptor by the BTG2 gene. MDA-MB-231 cells were infected with 100 moi of adenovirus carrying BTG2-HA and LacZ for 5 h, and then incubated for 48 h in fresh media until harvest in E1A buffer. Cell lysates were then incubated at 4°C overnight with 1 µg of anti-mTOR (left panel) and anti-Raptor (right panel) antibodies. Immunoprecipitates were pulled down by protein G beads for 1.5 h. Whole cell lysates (40 g) were loaded as an input. B) In vivo immunoprecipitation revealing the increased interaction of mTOR with Rictor caused by the BTG2 gene. The assay process was the same as mentioned above, except incubation of whole cell lysates with anti-mTOR and anti-Rictor antibodies, at 4°C overnight. C) Invasive breast cancer cells were transduced with 100 moi of Ad-BTG2 and Ad-LacZ for 5 h, and the mTOR kinase inhibitors, Torin 1 and PP242, were applied for 2 h.

Note the changes of the pAKT-S473 levels. D) MDA-MB-231 cells infected with the virus were treated with rapamycin for 2 h before detection of pAKT. GAPDH was used as a control. E) Breast cancer cells were transduced with 100 moi of adenovirus and incubated for 48 h, and the regulation of p70S6K activation was examined by immunoblot analysis. F) Wild type and BTG2-KO MEFs were cultured in 60 mm dishes overnight and serum-starved for 24 h before rapamycin treatment for 2 h (0, 10 and 20 nM), and then subjected to immunoblot analysis.

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B. Interaction of Raptor with BTG2 inhibits mTORc1 activity in breast cancer cells To evaluate whether BTG2-induced pAKT-S473 is PIP3-dependent, membrane translocation of GFP tagged AKT-PH was examined by immunocytochemistry, and increased translocation of GFP-AKT-PH to the cancer cell membrane was observed in BTG2 expresser cells compared to that observed in controls (43.7% vs. 3.2%, Figure 9C).

This observation was in agreement with the report that AKT activation by mTORc2 is PI3K-dependent [Liu P et al., 2015]. To further study how BTG2 inhibits mTORc1 activity, we explored whether BTG2 interacts with the downstream effector of mTORc1. In vitro GST-S6K1 pulldown and in vivo immunoprecipitation analyses were performed, and no interaction of BTG2 with GST-S6K1 was observed (Figures 10A and 10B). When the kinase activity of mTORc1 was evaluated in vitro using anti-Raptor immunoprecipitates and purified GST-S6K1 (Figure 10C), the Raptor immunoprecipitates clearly phosphorylated GST-S6K1 in LacZ expresser cells, but not in BTG2 expresser cells (Figure 10D), further supporting the hypothesis of inhibition of mTORc1 activity by BTG2.

We then performed a GST pulldown assay using MDA-MB-231 cell lysates along with purified GST and GST-BTG2 proteins; GST-BTG2 interacted with Raptor and mTOR proteins, and the interaction was further demonstrated by in vivo reciprocal immunoprecipitation analyses (Figures 10E-10G). The BTG2-Raptor interaction was further confirmed by transfecting 293TN cells with the HA-RAPTOR and v5-BTG2 plasmids (Figure 9D). All of the results indicated that BTG2 -induced pAKT-S473 is dependent on PIP3 formation at the cell membrane and that the interaction of BTG2 with Raptor inhibits mTORc1 activity. We further examined the above-mentioned results in tsc1-null and tsc2-null MEFs in a p53-null background. As shown in Figure 11A, BTG2 overexpression reduced p-p70S6K-T389 in tsc2-null cells, and the interaction of RAPTOR with mTOR was inhibited by the BTG2 gene in both tsc1-null and tsc2-null cells (Figures 11B and 11C). Moreover, mRNA and protein expression of tsc1/2 were significantly lower in BTG2-/- MEFs than that in BTG2+/+ MEFs, and were recovered by transduction with the Ad-BTG2 virus (Figures 11D-11G), suggesting that the expression and activity of the tsc1 and tsc2 genes can be substituted by BTG2 expression.

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- 45 -

Figure 9. Expression of BTG2 induces AKT phosphorylation that is localized to the cell membrane

A) In vivo immunoprecipitation exhibiting the inhibition of mTOR binding to Raptor by BTG2gene. MCF-7 cells seeded in 100 mm dish were infected with 100 moi of adenovirus carrying either with BTG2-HA or LacZ gene for 5h and then incubated for 48 h in the fresh media. To perform immunoprecipitation and immunoblot analysis, the cells were lysed in E1A buffer and then followed by incubation at 4oC overnight with 1.0 µg of anti-mTOR antibody. The immunoprecipitates were pulled down by protein G beads for 1 and half h, and then separated on the SDS-PAGE before immunoblot analyses with the corresponding antibodies. The whole cell lysates (40 µg) were loaded as an input. B) Immunoblot analyses. MCF-7 and MDA-MB-453 cells were transduced with 100 moi of Ad-BTG2 aor Ad-LacZ for 5h, and the mTOR kinase inhibitor, Torin 1, was applied for 2h. Note significant loss of pAKT-S473 level by the inhibitor treatment. C) MDA-MB-231 cells were infected with Ad-LacZ and Ad-BTG2-HA, followed by transfection with GFP-AKT-PH plasmid containing pleckstrin homology domain of AKT. The cells were visualized under live microscope and differentially counted. Note the more activation of GFP-AKT-PH in the BTG2-HA transduced cells compared with that of the Ad-LacZ expresser. D) Immunoprecipitation assay: 293TN cells lysates were prepared with E1A buffer in 48 h of the transfection with HA-RAPTOR and v5-BTG2 plasmids. And then incubated with anti-HA antibody at 40C and the immune complex was pulled down through protein G beads at room temperature for 1.5 h. Immunoblot analysis was performed to check the interaction of BTG2 with HA-RAPTOR. Note HA-RAPTOR and v5-BTG2 interaction. The star indicates nonspecific band.

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Figure 10. Interaction of Raptor with BTG2 inhibits mTORc1 activity in breast cancer cells

A) 293A cells transfected with 1 µg of GST-S6K1 and v5-BTG2were lysed in E1A buffer and incubated with GSH beads at 4°C overnight to examine whether GST-S6K1 binds to v5-BTG2. B) 293A cells transfected with pcDNA3-BTG2-HA or GST-S6K1 were lysed in E1A buffer and incubated with 1 µg anti-GST antibody to examine its interaction with BTG2-HA. C) 293A cells transfected with GST-S6K1 and serum-starved for 24 h were treated with 30 nM rapamycin for 2 h to obtain GST-S6K1. Cell lysates were incubated with GSH beads, and then purified GST-S6K1 was obtained by SDS-PAGE and Coomassie blue staining. D) MDA-MB-231 cells infected with Ad-BTG2 were lysed in CHAPS buffer and incubated with 0.5 µg anti-Raptor antibody at 4°C overnight before pull down by protein G beads. The immunoprecipitates were incubated with purified GST-S6K1 and ATP at 30°C for 30 min after washing and analyzed using immunoblot analysis. E) Coomassie blue staining showing the purified GST-BTG2 and GST proteins from BL21 cells transformed with GST-BTG2 and GST constructs. BL21 cell supernatants were incubated with GSH beads at 4°C overnight, and pulled down to collect bound proteins. F) Purified GST-BTG2 and GST proteins were incubated with MDA-MB-231 cell lysates at 4°C overnight followed by examination of the interaction of GST-BTG2 with Raptor and mTOR using GSH beads. G) MDA-MB-231 cells transduced with either BTG2or Ad-LacZ were harvested in 48 h and lysed in E1A buffer before incubation with 1 µg anti-Raptor (left panel) and anti-HA (right panel) antibodies.

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Figure 11. Expression and activity of tsc1/2 can be upregulated by the BTG2 gene in MEF

A) tsc+/+, tsc1-/-, and tsc2-/- MEFs infected with either Ad-LacZ or Ad-BTG2-HA were -tubulin was used as loading control. Immunoprecipitation and immunoblot analyses in tsc1-/- (B) and tsc2-/- (C) MEFs transduced with 100 moi of Ad-LacZ or Ad-BTG2-HA. Cell lysates in E1A buffer (1.0 mg) were subjected to immunoprecipitation with 1 µg of anti-Raptor antibodies at 4°C overnight, and then pulled down by protein G beads to analyze the effect of BTG2 on mTORc1 formation. Note the significant reduction in the Raptor-mTOR interaction in the BTG2 expressers compared to that in the LacZ controls. D) Downregulation of tsc1 and tsc2 gene expression in BTG2-KO MEFs compared to that in wild-type cells, as measured by RT-qPCR. Expression was normalized by the 18S rRNA level. The results are expressed as the mean ± S.D. of two independent experiments. E) BTG2-dependent upregulation of tsc1 expression in BTG2-/- MEFs. Total cell RNAs were extracted from BTG2-KO MEFs transduced with different doses of Ad-BTG2. Expression of tsc1, tsc2 and BTG2 genes was examined by RT-qPCR. The results are expressed as the mean ± S.D. of two independent experiments. F) Immunoblot analysis demonstrating the reduced expression of the tsc1 protein in BTG2-null MEFs compared to that of the wild type cells. G) Recovery of tsc1 protein expression after transduction with the Ad-BTG2 gene in the BTG2-null MEFs.

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C. BTG2 upregulates mTORc2 activity in both normal and cancer cells

C. BTG2 upregulates mTORc2 activity in both normal and cancer cells