CHP2 Promotes Cell Proliferation in Breast Cancer Via Suppression Of

Published OnlineFirst July 2, 2018; DOI: 10.1158/1541-7786.MCR-18-0157

Oncogenes and Tumor Suppressors Molecular Cancer Research CHP2 Promotes Cell Proliferation in Breast Cancer via Suppression of FOXO3a Xiaohui Zhao1, Tian Xie2, Ting Dai1, Wenhui Zhao3, Jing Li1, Rui Xu4, Chao Jiang5, Peiqiong Li1, Junyao Deng6, Xiaobo Su1, and Ningfang Ma2

Abstract

Calcineurin B homologous protein isoform 2 (CHP2), an endogenous CHP2 inhibited, the proliferation and tumori- þ þ essential cofactor for Na /H exchanger isoform 1 (NHE1), genicity of breast cancer cells in vitro and in vivo. In addition, is identified to be expressed in various malignant cell lines. overexpression of CHP2 accelerated, whereas inhibition of However, the clinical significance and biological role of CHP2 retarded, G1–S phase cell-cycle transition in breast CHP2 in breast cancer remain to be established. Here, CHP2 cancer cells. Mechanistically, overexpression of CHP2 acti- was markedly overexpressed in breast cancer cells and clinical vated AKT signaling and suppressed the transactivation of the tumor specimens. Immunohistochemical analysis revealed forkhead box O3 (FOXO3/FOXO3a) transcription factor. that the expression of CHP2 was significantly correlated with patients' clinicopathologic characteristics like clinical stage, Implications: This study discovers a previously unrecog- and breast cancer patients with high CHP2 expression had nizedroleofCHP2intheprogressionofbreastcancerand shorter overall survival compared with patients with low supports the significance of this gene as a novel prognostic CHP2 expression. Moreover, it was demonstrated that over- biomarker and a potential therapeutic target for breast cancer. expressing CHP2 significantly enhanced, whereas silencing Mol Cancer Res; 16(10); 1512–22. 2018 AACR.

Introduction The transcription factor FOXO3a, a member of the forkhead box-containing O subfamily (3), is considered to be a tumor Breast cancer is the second most common cancer in the world, suppressor due to its antiproliferative and proapoptotic actions and the incidence of female breast cancer has continuously (4–6). Loss of the function of FOXO3a in cancer cells can result in increased (1). Despite the progress in early detection and use of the overexpression of genes that regulate cell-cycle progression, new therapeutic targets, breast cancer remains a major problem in such as cyclin-dependent kinase (CDK) inhibitors p21Cip1 (7) and public health (2). The discovery of new molecular actors involved p27Kip1 (8) and retinoblastoma-like protein p130Rb2 (9), and in the regulation of breast cancer development is essential to thereby lead to G phase cell-cycle arrest. When FOXO3a is improve the management of this disease. Therefore, understand- 1 overexpressed and translocated into the nucleus, its target genes, ing the roles and molecular mechanisms of these molecular actors including FasL (10), GADD45 (11), and Bim (12), are activated in may provide new insights into the physiology and pathology of response to ionizing radiation or UV irradiation, suggesting that breast cancer and enable the development of novel and effective FOXO3a may play an important role in apoptotic response to anticancer therapeutics. genotoxic stress. Accordingly, the negative regulation of FOXO3a may have a pivotal role in controlling cell cycling or cell survival. Better understanding the mechanisms that regulate FOXO3a 1GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, China. 2Key Laboratory of Protein Modification and Degradation activity may provide clues of novel targets for therapeutic School of Basic Medical Sciences, Affiliated Cancer Hospital and Institute of intervention. Guangzhou Medical University, Guangzhou, China. 3Juancheng People's Hos- Calcineurin B homologous protein (CHP2) belongs to the þ pital, Juancheng, China. 4Affiliated Cancer Hospital and Institute of Guangzhou super family of N-myristolated, EF-hand Ca2 -binding protein 5 Medical University, Guangzhou, China. Department of Cancer Center, People's CHPs (13–15). The biological function of CHP2 remains largely 6 þ þ Hospital of Baoan District, Shenzhen, China. Department of Histology and unknown except for a potential role in transmembrane Na /H Embryology, Southern Medical University, Guangzhou, China. exchange (15, 16) which can protect cells from serum deprivation- Note: Supplementary data for this article are available at Molecular Cancer induced death. Jin and colleagues showed that overexpression of Research Online (http://mcr.aacrjournals.org/). CHP2 in human OVCAR3 ovarian carcinoma cells contributed to X. Zhao, T. Xie, and T. Dai contributed equally to this article. an increased binding ability of the tumor cells to fibronectin (17). Corresponding Authors: Ningfang Ma, Affiliated Cancer Hospital and Institute of Li and colleagues reported that ectopic expression of CHP2 Guangzhou Medical University, Guangzhou 510095, China. Phone: 86-20-3710- promoted proliferation of HEK293 cells, whereas knockdown of 3201; Fax: 86-20-3710-3204; E-mail: [email protected]; and Xiaobo Su, endogenous CHP2 expression in HepG2 inhibited cell prolifer- GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, ation (18). In accordance with this, CHP2-transfected cells Guangzhou, 511436, China. E-mail: [email protected] showed an increased nuclear presence of NFATc3 and enhanced doi: 10.1158/1541-7786.MCR-18-0157 NFAT activity (19). In addition, Hammam and colleagues found 2018 American Association for Cancer Research. that the expression of CHP2 in leukemia primary cells was

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significantly increased, which might play an important role in to generate pSuper-retro- CHP2-RNAi(s). The targeting sequence leukemia cell growth (20). These results highlighted a potential is 50-GGGCACTGGACAGGAATAAGA-30 (synthesized by Invitro- role of CHP2 in tumorigenesis and revealed a novel function of gen). We established doxycycline-inducible CHP2-overexpressing CHP2 as an activator of the calcineurin/NFAT signaling pathway ZR-75-30 cells with Lenti-X-Tet-On-3G plasmid, and CHP2- (21). However, the clinical significance and biological role of silencing ZR-75-30 cells with pLKO-Tet-On plasmid. Lentivirus CHP2 in human breast cancer remain ambiguous. production and infection were performed as previously described In our study, we found that CHP2 was markedly overexpressed (23). Stable cell lines expressing CHP2 or CHP2 shRNAs and in a large cohort of human breast cancer samples, and the high Tet-On-CHP2 or Tet-On-CHP2-RNAi were selected for 10 days expression level of CHP2 correlated with clinical stages and poor with 0.5 mg/mL puromycin. The reporter plasmid for detecting the survival of breast cancer patients. Ectopic expression of CHP2 transcriptional activity of FOXO3a was generated as described dramatically promoted the proliferation and tumorigenicity of previously (24). breast cancer cells both in vitro and in vivo, whereas silencing CHP2 led to the reverse effects. Further, we demonstrated that over- Western blotting expression of CHP2 enhanced G1–S phase transition mediated by Western blotting was performed according to standard meth- AKT activation, which subsequently induced phosphorylation ods as described previously (25) using anti-CHP2 antibody and inactivation of FOXO3a in breast cancer cells. These findings (Signalway Antibody), anti-p21Cip1, anti-p27Kip1, anti-cyclin suggest that CHP2 may function as an oncoprotein in breast D1, anti-Rb, anti–phosphorylated-Rb (Ser780), anti-AKT, anti– cancer progression and provide new insights into the regulatory phosphorylated-AKT (Ser473), anti–GSK-3b, anti–phospho- mechanism of AKT/FOXO3a signaling. GSK-3b (Ser9), anti-FOXO3a, anti–phosphorylated-FOXO3a (Ser253; Cell Signaling Technology). Anti-GAPDH and anti– a Materials and Methods -tubulin antibodies (Sigma) were used as a loading control. Cell lines MTT cell viability assay Human mammary epithelial cells (HMEC) were established as Cells were seeded in 96-well plates at a density of 2 103 cells/ previously described (22). Breast cancer cell lines, including well. At each time point, cells were stained with 100 mL sterile MTT ZR-75-30, MDA-MB-435, T47D, BT549, MDA-MB-231, MCF-7, dye (0.5 mg/mL; Sigma) for 4 hours at 37C, followed by removal and HCC1954, were cultured in DMEM medium (Gibco) sup- of the culture medium and addition of 100 mL of dimethyl plemented with 10% FBS (HyClone). sulfoxide (Sigma). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. Each experiment was Patient information and tissue specimens performed in triplicates. This study was conducted on a total of 212 cases of paraffin- embedded, archived breast cancer samples, which had been Colony formation assay histopathologically and clinically diagnosed at the Sun Yat-sen Cells were plated in 6-well plates (5 102 cells) and cultured University Cancer Center from 2002 to 2007. Clinical stage and for 10 days as previously described (26). The colonies were clinicopathologic classification were determined according to the stained with 1% crystal violet for 30 seconds after fixation with American Joint Committee on Cancer criteria. The clinical infor- 4% formaldehyde for 5 minutes. Colonies were counted, and the mation for the patient samples is summarized in Supplementary results were shown as the fold change compared with vector Tables S1 and S2. Ethics approval and prior patient consent had control cells. been obtained from the Institutional Research Ethics Committee for the use of the clinical specimens for research purposes. Anchorage-independent growth ability assay The experiment was performed as previously described (27). Immunohistochemistry Briefly, 500 cells were trypsinized and suspended in 2 mL com- The IHC procedure and the scores of CHP2 expression were plete medium plus 0.3% agar (Sigma). The agar-cell mixture was performed as previously reported (22). Quantitative analysis of plated on top of a bottom layer with 1% agar completed medium IHC staining was performed using an Axio Vision Rel. 4.6 com- mixture. About 10 days, visible colonies that were larger than 0.1 puter image analysis system assisted by an automated measure- mm were counted. The experiment was carried out for each cell ment program (Carl Zeiss). Briefly, stained sections were evalu- line in triplicates. ated at 200 magnification and 10 representative stained fields of each section. Each stained section contained more than 95% of Bromodeoxyuridine labeling and immunofluorescence tumor cells analyzed to verify the mean optical density (MOD), Cells (5 104) were plated on coverslips (Fisher Scientific). which represented the intensity of the stained signal as measured After 24 hours, cells were incubated with bromodeoxyuridine per positive pixels. Statistical analysis of MOD data was per- (BrdUrd) for 1 hour and stained with anti-BrdUrd antibody formed using the t test to compare mean MOD differences (Upstate) according to the manufacturer's instruction. BrdUrd- between different tissue groups; P < 0.05 was considered positive cells were counted under a laser scanning microscope significant. (Axioskop 2 plus; Carl Zeiss Co. Ltd.) in ten randomly chosen fields from three independent samples, and the results were Vectors and retroviral infection presented as the mean SD. CHP2 expression construct was generated by subcloning PCR- amplified full-length human CHP2 cDNA into the pSin-EF2 Flow cytometry lentiviral vector, and a short hairpin RNA (shRNA) oligonucleo- Cells were harvested and fixed in 75% ethanol and processed tides targeting human CHP2 were cloned into pSuper-retro-puro for cell-cycle analysis by using flow cytometry. Twenty thousand

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cells were analyzed using a CytoFlex instrument (Beckman Coul- higher than those in normal breast tissues (P <0.01; Fig. 1F). ter) equipped with CytExpert software. Modfit LT 3.1 trial cell- Moreover, univariate and multivariate analyses indicated that cycle analysis software was used to determine the percentage of clinical stage, T classification, and CHP2 expression were inde- cells in the different phases of the cell cycle. pendent prognostic factors for the patients (Supplementary Table S2), suggesting that CHP2 might be a predictive biomarker for Xenografted tumor model disease outcome in patients with breast cancer. Female BALB/c nude mice (5–6 weeks of age, 18–20 g) were purchased from the Slac-Jingda Laboratory Animal and were CHP2 promotes the proliferation of breast cancer cells housed in barrier facilities on a 12-hour light/dark cycle. All Strikingly, we found that in the breast cancer specimens, the experimental procedures were approved by the Institutional Ani- areas that displayed high levels of CHP2 staining also showed mal Care and Use Committee of Guangzhou Medical University. strong Ki67 staining, whereas areas with low CHP2 expression BALB/c nude mice were randomly divided into four groups (n ¼ also displayed weakly detectable Ki67 expression (Fig. 2A). Sta- 5/group). For implantation of tumor cells, ZR-75-30 cells stably tistical analyses indicated that the expression level of CHP2 was expressing Tet-On-CHP2 and Tet-On-CHP2-RNAi (2 106) were strongly associated with the expression level of Ki67 (P < 0.01; injected into mammary fat pads of randomly two groups. To Supplementary Table S2), suggesting that CHP2 might be a induce the expression of CHP2, one group of nude mice which proproliferative factor in breast cancer cells. were injected with Tet-On-CHP2 ZR-75-30 cells was fed with We further investigated the effect of CHP2 on the proliferation 2 mg/mL doxycycline in water for 28 days (water changed every of breast cancer cells using gain and loss of function models (Fig. 2 days), and the other group was not fed. To induce the expression 2B). As shown in Fig. 2C and D, MTT and colony formation assays of CHP2-RNAi, one group of nude mice which were injected with indicated that the proliferation rate of CHP2-overexpressing cells Tet-On-CHP2-RNAi ZR-75-30 cells was fed with 2 mg/mL doxy- was significantly increased compared with the vector-control cells, cycline in water for 35 days (water was changed every 2 days), and whereas silencing CHP2 inhibited cell proliferation. Moreover, the other group was not fed. Tumors were examined twice weekly; ectopic expression of CHP2 in HMEC also exerted an accelerating length, width, and thickness measurements were obtained with role in cell proliferation (Supplementary Fig. S3A and 3B). Col- calipers, and tumor volumes were calculated. lectively, our results provided strong evidence that CHP2 played a critical role in the proliferation of breast cancer cells. Statistical analysis Statistical analyses were performed using the SPSS version 16.0 CHP2 promotes the tumorigenicity of breast cancer cells in vitro statistical software package. Statistical tests for data analysis and in vivo included log-rank test, x2 test, Spearman-rank correlation test, The anchorage-independent growth assay revealed that over- and Student two-tailed t test. Multivariate statistical analysis was expressing CHP2 significantly increased, but silencing CHP2 performed using a Cox regression model. Data represented mean decreased the anchorage-independent growth ability of ZR-75- SD. P value < 0.05 was considered statistically significant. 30 and MDA-MB-231 cells in soft agar (Fig. 3A), indicating that CHP2 played a significant role in the tumorigenicity of breast cancer cells in vitro. Results We then examined whether CHP2 could promote the tumor- CHP2 overexpression correlates with tumor progression and igenicity of breast cancer cells in vivo. As shown in Supplementary poor prognosis of breast cancer patients Fig. S4A, CHP2-transduced tumors grew significantly faster than Real-time PCR and Western blotting analyses were performed, the control tumors at each different time point (P < 0.01), whereas and the results showed that CHP2 mRNA and protein expression the tumors formed by CHP2-silenced cells grew at a much slower were markedly upregulated in all the tested breast cancer cell lines pace than the control tumors (P < 0.01; Supplementary Fig. S4A, compared with human mammary epithelial cells (HMEC; Fig. 1A left plots). Moreover, the tumors formed by CHP2-transduced and B). Consistently, we found that CHP2 expression was higher breast cancer cells were larger and had higher tumor weights than in six human breast cancer tissues than in their paired adjacent the vector-control tumors. Conversely, the tumors formed by nontumor tissues (Fig. 1C; Supplementary Fig. S1). These results CHP2-silenced cells were smaller, in both size and weight, than indicated that CHP2 expression was upregulated in breast cancer. the tumors formed from shRNA-vector control cells (Supplemen- To investigate the relationship between CHP2 expression and the tary Fig. S4A, middle and right plots). Importantly, IHC assay clinicopathologic features in breast cancer, the tissue samples of showed that areas with a strong CHP2 signals also displayed 212 cases of human breast cancer were analyzed by IHC analysis. intense cyclin D1 and Ki67 staining, whereas areas with low levels The results indicated that CHP2 was markedly upregulated in the of CHP2 expression exhibited lower cyclin D1 and Ki67 staining breast cancer samples (Fig. 1D). Statistical analysis further (Supplementary Fig. S4B). revealed that CHP2 expression was strongly associated with the We further investigated whether inducible CHP2 expression clinical stage (P < 0.01), T classification (P < 0.01), N classification alteration had effects on tumorigenicity. Dramatically, our results (P < 0.01), and M classification (P < 0.01; Supplementary Table showed that Dox-induced CHP2 tumors grew significantly faster, S1). Kaplan–Meier survival curves and the log-rank test showed whereas the tumors formed by Dox-induced CHP2-RNAi cells that CHP2 expression was significantly negatively correlated with grew at a much slower pace than controls (Fig. 3B). Tumors the overall survival (OS) of breast cancer patients (P < 0.01; Fig. formed by Dox-induced CHP2 breast cancer cells were larger and 1E). Similar results were obtained between patients in clinical had higher tumor weights. Conversely, the tumors formed by stage I–II and III–IV subgroups (Supplementary Fig. S2). Further- Dox-induced CHP2-RNAi cells were smaller, in both size and more, quantitative analysis indicated that the MODs of CHP2 weight than controls (Fig. 3B). Consistently, IHC staining staining in clinical stage I–IV primary tumors were significantly revealed that induced overexpression of CHP2 increased p-AKT,

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Figure 1. CHP2 expression is upregulated in breast cancer. A and B, Real-time PCR and Western blotting analysis of CHP2 expression in HMEC and 7 breast cancer cell lines. C, IHC analysis of CHP2 protein expression in 6 pairs matched breast cancer tissues (T) and adjacent nontumor tissues (ANT) from the same patients, and in normal breast tissues and breast cancer tissues at different clinical stages (D). E, Kaplan–Meier OS curves and univariate analyses (log-rank) comparing breast cancer patients with low (n ¼ 111) and high (n ¼ 101) CHP2- expressing tumors (P < 0.01). F, Statistical quantiﬁcation of the average MODs of CHP2 staining between normal breast tissues and breast cancer specimens at different clinical stages. , P < 0.01.

p-FOXO3a, Cyclin D1, and Ki67 expression, whereas silencing of the percentage of S phase cells. The expression of CDK inhibitors CHP2 had opposite effects (Fig. 3C). These ﬁndings indicate that p21Cip1 and p27Kip1 was drastically reduced in CHP2-overexpres- CHP2 promoted the tumorigenicity of breast cancer cells. sing cells compared with control cells at both mRNA and protein levels (Fig. 5A and 5C). This was accompanied by a concurrent

CHP2 regulates the G1–S phase transition in breast cancer increase in the levels of cell-cycle regulators cyclin D1 and p-Rb cells (Fig. 5C). Conversely, in CHP2-silenced cells, the expression of To further investigate the role of CHP2 in promoting breast p21Cip1 and p27Kip1 significantly increased, whereas the expres- cancer cells' proliferation, BrdUrd incorporation and flow cyto- sion of cyclin D1 and p-Rb decreased (Fig. 5B and C). Taken – metry assays were performed. As shown in Fig. 4A–D, overexpres- together, our results suggested that CHP2 promoted the G1 S cell- sing CHP2 significantly increased, but silencing CHP2 reduced, cycle transition in breast cancer cells.

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Figure 2. CHP2 plays a key role in breast cancer cell proliferation and tumorigenicity. A, CHP2 expression levels significantly correlated with Ki67 expression in breast cancer tissues (n ¼ 212; P < 0.01). Two representative cases are shown (left) and percentage of specimens with low or high CHP2 expression, relative to the levels of Ki67 staining (right). B, Confirmation of the overexpression or knockdown of CHP2 in ZR-75-30 and MDA-MB-231 cells by Western blotting; a-tubulin was used as a loading control. MTT assays (C) and colony formation assay (D) indicate that the growth rates increased in CHP2-overexpressing cells and decreased in CHP2-silenced cells. The number of colonies was quantified in the colony formation assay. Each bar represents the mean SD of three independent experiments. , P < 0.01.

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Figure 3. CHP2 promotes the tumorigenicity of breast cancer cells in vitro and in vivo. A, Anchorage-independent growth assay in indicated breast cancer cells. Soft-agar colony formation (colonies > 0.1 mm) was quantified after 10 days of culture. Values are mean SD of three independent experiments; , P < 0.01. B, Confirmation of the Dox-induced CHP2 and Dox-induced CHP2-RNAi in ZR-75-30 cells by Western blotting; a-tubulin was used as a loading control. Xenograft model in nude mice. Tet-On-CHP2– and Tet-On-CHP2-RNAi–overexpressing ZR-75-30 cells were inoculated into the fat pad of randomly two groups (n ¼ 5/group). Representative images of the tumors from all mice in each group (left plot). Tumor volume growth curves (middle) and mean tumor weights (right) for the tumors formed by the indicated cells. Each bar represents the mean SD of three independent experiments. , P < 0.01. C, IHC analysis and quantification of CHP2, Ki67, p-AKT(Ser473), and p-FOXO3a(Ser253) and cyclin D1 expression and H&E staining in tumor xenograft tissues. , P < 0.01.

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Figure 4.

CHP2 regulates the G1–S phase transition and alters the expression of cell-cycle regulators in breast cancer cells. A, Representative micrographs (left) and quantiﬁcation (right) of BrdUrd-incorporated cells in vector control and CHP2-overexpressing cells. B, Flow cytometric analysis of vector control and CHP2- overexpressing cells. C, Representative micrographs (left) and quantiﬁcation (right) of BrdUrd incorporation by vector and CHP2-silenced cells. D, Flow cytometric analysis of vector and CHP2-silenced cells. All values are mean SD of three independent experiments; P < 0.01.

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Figure 5.

CHP2 alters expression in G1–S phase cell-cycle regulators. A and B, Real-time PCR analysis of P21Cip1, P27Kip1, and cyclin D1 mRNA expression in breast cancer cells. Gene expression levels were normalized to GAPDH. C, Western blotting analysis of p21Cip1,p27Kip1, cyclin D1, p-Rb, and total Rb protein expression in breast cancer cells; GAPDH was used as a loading control. Each bar represents the mean SD of three independent experiments. , P < 0.01.

CHP2 downregulates FOXO3a transactivity and activates the contributed to the modulation of AKT/FOXO3a and AKT/GSK- AKT signaling pathway 3b signaling. We further examined the role of FOXO3a in Previous reports have demonstrated that FOXO3a transcription- CHP2-mediated cell proliferation. As shown in Fig. 6D, knock- ally regulates p21Cip1,p27Kip1, and cyclin D1 (28, 29), which ing down FOXO3a in CHP2-silenced cells decreased p21Cip1 prompted us to investigate whether CHP2 targets these genes by and p27Kip1 expression but increased cyclin D1 expression. modulating the transactivity of FOXO3a. As shown in Fig. 6A and B, Furthermore, MTT and colony formation assays showed that the transactivity and expression level of FOXO3a were signiﬁcantly silencing FOXO3a restored the growth rate of CHP2-silenced decreased in CHP2-overexpressing cells and increased in CHP2- cells (Fig. 6E and F). Taken together, our results suggested that silenced cells. We also found that CHP2 expression was inversely FOXO3a played a critical role in the proproliferative effect of correlated with FOXO3a in the eight freshly collected clinical breast CHP2 in breast cancer cells (Fig. 6G). cancer samples (r ¼0.7308, P < 0.05; Fig. 6C). AKT kinase is known to play a key role in phosphorylating and repressing FOXO3a transcriptional activity (30, 31). As predicted, overexpres- Discussion sing CHP2 drastically increased the levels of p-AKT and p-GSK-3b, The main ﬁndings of our study showed that CHP2 was marked- and silencing CHP2 reduced them (Fig. 6B), suggesting that CHP2 ly overexpressed in breast cancer cells and tumor tissues and was

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Figure 6. CHP2 downregulates FOXO3a transactivity and activates AKT signaling pathway. A, Related FOXO3a reporter activity in ZR-75-30 and MDA-MB-231 breast cancer cells. Values are mean SD of three independent experiments; , P < 0.01. B, Western blotting analysis of phosphorylated-AKT (Ser473), total AKT, phosphorylated-GSK-3b (Ser9), total GSK-3b, phosphorylated-FOXO3a (Ser253), and total FOXO3a proteins in breast cancer cells. a-Tubulin was used as a loading control. C, Expression (top) and correlation analysis (bottom) of CHP2 and FOXO3a expression in 8 freshly isolated human breast cancer samples. D, Western blotting analysis of p21Cip1, p27Kip1, cyclin D1, and FOXO3a proteins in CHP2 shRNA(s)–infected ZR-75-30 and MDA-MB-231 cells after transfection with FOXO3a siRNA(s). GAPDH was used as a loading control. E and F, The MTT assay shows that silencing FOXO3a increased the proliferation of CHP2-silenced cells. Error bars represent SD from three independent experiments. , P < 0.01. G, Model of the CHP2 mechanism in breast cancer cells.

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correlated with an advanced clinical stage and poor survival of and the transcription factors of FOXO3a (39). Phosphorylation of breast cancer patients. Its ectopic expression promoted the pro- FOXO3a promotes an interaction with the 14-3-3 protein and liferation and tumorigenicity of breast cancer cells in vitro and in promotes the export of FOXO3a from the nucleus to the cytoplasm, vivo. The mechanisms for promoting breast cancer cells' prolifer- which results in its ubiquitination and proteasomal degradation, ation and tumorigenicity might be activation of AKT signaling and blocks its cell-cycle arrest activity of FOXO3a (40, 41), and pathway and subsequent inhibition of FOXO3a transcriptional phosphorylation-mediated suppression of GSK3b promotes breast activity, which would decrease the expression of CDK inhibitors cancer proliferation, metastasis, and the ability to self-renew. In our p21Cip1 and p27Kip1 and upregulate cyclin D1. By providing new study, we observed similar effects, and the levels of phospho-AKT, insights into the role of CHP2 in breast cancer, our results phospho-FOXO3a, and phospho-GSK-3b were elevated in CHP2- indicated that CHP2 might function as a target for a novel tranduced cells and reduced in CHP2-silenced cells. This suggested therapeutic strategy in the treatment of patients with breast cancer. that the mechanism underlying the CHP2-mediated downregula- It has been established that CHP2 is probably a signaling tion of FOXO3a and GSK-3b might be through the activation of þ molecule that, like the other EF-hand Ca2 -binding proteins, AKT, which is a major downstream effector of EGFR (42). Inter- þ þ þ couples local Ca2 concentration changes to a variety of specific estingly, it has been reported that CHP2 is a cofactor for Na /H signal-response cascades (32, 33). However, its clinical significance exchanger isoform 1 (NHE1), which forms a complex with EGFR and biological role in human breast cancer remain unclear. In this through scaffolding regulator 1 and forms a positive regulatory study, we found that CHP2 expression was elevated in 212 cases of loop to regulate the intensity and duration of EGFR, further human breast cancer tissues and was positively correlated with adjusting the concentration of NHE1 (43). Our study showed that clinical stage and tumor–node–metastasis classification. Kaplan– overexpression of CHP2 enhanced AKT phosphorylation, likely Meier survival curves demonstrated that CHP2 overexpression was due to the formation of a complex of CHP2, NHE1, and EGFR that negatively correlated with OS of breast cancer patients (P < 0.01). further activates the AKT signaling pathway and thereby promotes Univariate and multivariate analyses indicated that CHP2 over- the development of breast cancer cells. expression might function as an independent prognostic factor for In summary, our results showed that CHP2 plays a crucial role breast cancer patients. Previous reports have provided evidence that in the development of human breast cancer and provided a more CHP2 is involved in cell growth in acute leukemia and ovarian in-depth theoretical study of the mechanisms. Studying the role cancer (20, 22). Consistent with these reports, we found that CHP2 played by CHP2 in the development of breast cancer will not only was strongly expressed in highly proliferative lesions of human promote our understanding of the pathogenesis of breast cancer, breast cancer, as indicated by a significant correlation between but also may provide a new strategy for the treatment of breast CHP2 and Ki67 expressions (P < 0.01). Therefore, our results cancer via targeting CHP2 in breast cancer cells. In addition, our supported the proposal that CHP2 is an oncogenic protein and results suggest a potential role for CHP2 as a clinically indepen- has a proliferation-promoting effect in breast cancer. dent risk prognostic factor of disease progression, prognosis, and As a tumor suppressor, FOXO3a is downregulated in various survival in breast cancer patients. Therefore, it is worthwhile to types of cancer, including breast cancer, prostate cancer, and hepa- assess the molecular diagnostic ability of CHP2 in breast cancer. tocellular carcinoma, and it may play an important and versatile role in regulating critical cellular functions such as cell-cycle arrest, Disclosure of Potential Conflicts of Interest apoptosis, and irreversible senescence (34, 35). Furthermore, No potential conflicts of interest were disclosed. FOXO3a acts as a member of transcription factors that works by Authors' Contributions regulating the expression of its downstream genes, including CDK Cip1 Kip1 – Conception and design: X. Zhao, X. Su, N. Ma inhibitors (p21 ,p27 )andcell-cycle related genes (cyclin Development of methodology: X. Zhao, X. Su, N. Ma – D1/D2; refs. 36 38). In our study, we found that CHP2 over- Acquisition of data (provided animals, acquired and managed patients, expression reduced the transcriptive activity of FOXO3a and the provided facilities, etc.): X. Zhao, T. Xie, T. Dai, W. Zhao, P. Li, J. Deng, expression of its downstream targets. Moreover, depleting of X. Su, N. Ma FOXO3a could restore the growth rate of CHP2-silenced breast Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cancer cells. Thus, we demonstrated that FOXO3a played a critical computational analysis): X. Zhao, T. Xie, T. Dai, J. Li, R. Xu, C. Jiang, X. Su, N. Ma Writing, review, and/or revision of the manuscript: X. Zhao, T. Xie, T. Dai, role in the CHP2-induced proliferation of breast cancer cells. X. Su, N. Ma Previous studies have found that CHP2 contains a nuclear export Administrative, technical, or material support (i.e., reporting or organizing sequence (NES) at the C-terminal region and mainly expresses in data, constructing databases): X. Zhao, T. Xie, X. Su, N. Ma cytoplasm and plasma membrane (13, 17, 44, 45). Interestingly, Li Study supervision: X. Zhao, X. Su, N. Ma and colleague reported that CHP2 mutation of the NES predom- inantly localized to the nucleus and resulted in enhanced prolif- Acknowledgments This work was supported by the National Natural Science Foundation of eration and tumorigenicity of HeLa cells (44). Actually, our data China (grant nos. 81502580 and 81502103), the Science and Technology also observed that a very rare number of CHP2 was stained at Department of Guangdong Province (grant nos. 201607010014 and nuclear in patient specimens by IHC. Thus, CHP2 mutation might 201607010034), Guangzhou Medical University's Scientific Research Project be speculated as a mechanism for the nuclear staining of CHP2, (grant no. 2016C40), and Guangzhou Municipal University Research Project which further exerts an oncogenic role of CHP2. However, this (grant no. 1201620284). hypothesis remains to be further investigated in future. The costs of publication of this article were defrayed in part by the payment of By showing that CHP2 overexpression decreased the transactiva- page charges. This article must therefore be hereby marked advertisement in tion of FOXO3a and its downstream targets, we hypothesized that accordance with 18 U.S.C. Section 1734 solely to indicate this fact. FOXO3a was involved in CHP2-mediated proliferative mechanisms. It has been reported that activation of AKT can trigger the Received February 13, 2018; revised May 10, 2018; accepted June 13, 2018; phosphorylation of various downstream targets, including GSK-3b published first July 2, 2018.

www.aacrjournals.org Mol Cancer Res; 16(10) October 2018 1521

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1522 Mol Cancer Res; 16(10) October 2018 Molecular Cancer Research

CHP2 Promotes Cell Proliferation in Breast Cancer via Suppression of FOXO3a

Xiaohui Zhao, Tian Xie, Ting Dai, et al.

Mol Cancer Res 2018;16:1512-1522. Published OnlineFirst July 2, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-0157

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