beta-catenin signaling pathway

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Overexpressed ACBD3 has prognostic value in human breast cancer and promotes the self-renewal potential of breast cancer cells by activating the Wnt/beta-catenin signaling pathway ⁎

Yun Huanga,1, Le Yangb,1, Yuan-yuan Peic,1, Jie Wangd,e, Hongmei Wuc, Jie Yuanf, , Lan Wanga,

⁎

a

Department of Pathogen Biology and Immunology, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China Department of Basic Medicine, Nanyang Medical College, Nanyang, Henan 473061, China c Shenzhen Long-gang Maternal and Child Health Hospital Centralab, Shenzhen 518172, China d Institution of Pharmaceutical Bioactive Substances, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China e Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou 510006, China f Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Breast cancer ACBD3 Cancer stem cell Tumorigenesis Wnt/beta-catenin

Acyl-CoA binding domain containing 3 (ACBD3) is involved in the maintenance of Golgi structure and function through its interaction with the integral membrane protein. However, the clinical significance and biological role of ACBD3 in breast cancer remain unclear. Herein, we found that the mRNA and protein levels of ACBD3 were markedly up-regulated in breast cancer cells and tissues. Immunohistochemical analysis of breast cancer tissues demonstrated that ACBD3 overexpression was significantly associated with advanced clinicopathological features. Univariate and multivariate analysis indicated that ACBD3 overexpression correlates with poor prognosis in breast cancer. Furthermore, overexpressing ACBD3 promoted, while silencing ACBD3 inhibited, selfrenewal and tumorigenesis in breast cancer cells in vitro and in vivo respectively. Importantly, upregulating ACBD3 promoted the self-renewal and tumorigenesis of breast cancer cells via activating the Wnt/beta-catenin signaling, and the pro-self-renewal effect of ACBD3 in breast cancer was antagonized by the Wnt signaling inhibitor TCF4-siRNA and Lef1-siRNA.These findings indicate that ACBD3 may represent candidate therapeutic targets to enable the elimination of breast cancer stem cells, providing the preclinical proof-of-concept for the prevention and treatment of breast cancer.

1. Introduction With roughly half a million deaths per year worldwide, breast cancer has become the second cause of tumor-related deaths in females [1]. The treatment and prognosis of breast cancer are complex and vary dramatically depending on the subtype of breast cancer [2]. At present, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (Her2) are the most common receptors in breast cancer cells and specifically determine therapeutic approaches and prognosis of breast cancer [3]. Quantities of investigations show that most of the patients died of tumor recurrence within 5–10 years after surgery or chemotherapy [4]. Adjuvant therapies have

significantly reduced breast cancer recurrence rates, but a substantial proportion of breast cancer patients are faced with recurrence. It is believed that breast cancer stem cells (BCSCs) are probably the major source of breast cancer recurrence [5]. Because of the abilities of exhibiting self-renewal activity and long-term cancer-propagating capacity and developing acquired drug resistance, CSCs can produce more malignant subclones over time [6]. Moreover, the efflux chemotherapy drugs function of CSC are associated with the activation of the critical signaling pathways, such as epidermal growth factor receptor (EGFR), Notch pathway and Wnt/beta-catenin pathway [7–9]. Wnt signaling influences tissue homeostasis, cell renewal, and regeneration in a continuous way [10,11]. As a key nuclear effector of canonical Wnt

Abbreviations: ACBD3, Acyl-CoA binding domain containing 3; ER, estrogen receptor; PR, progesterone receptor; Her2, human epidermal growth factor receptor-2; CSC, cancer stem cell; BCSC, breast cancer stem cell; EGFR, epidermal growth factor receptor; ACBP, acyl-CoA-binding protein; PTEN, phosphatase and tensin homolog; NBEC, Normal human breast epithelial cell; IF, Immunofluorescent; NOD/SCID, nonobese diabetes/severe combined immunodeficiency disease; TCGA, The Cancer Genome Atlas; PKARIA, Protein Kinase-A Regulatory Subunit Iα; TSPO, translocator protein ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Yuan), [email protected] (L. Wang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.yexcr.2018.01.003 Received 15 October 2017; Received in revised form 25 December 2017; Accepted 2 January 2018 0014-4827/ © 2018 Elsevier Inc. All rights reserved.

Please cite this article as: Huang, Y., Experimental Cell Research (2018), https://doi.org/10.1016/j.yexcr.2018.01.003

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Fig. 1. ACBD3 is up-regulated in breast cancer. (A) Expression of ACBD3 was up-regulated in 1099 breast cancer tissues compared with 111 normal breast tissue samples in the TCGA profile. (B) ACBD3 expression was markedly elevated in 111 paired breast cancer tissues compared with patient-matched adjacent normal tissues in the TCGA profile. (C) ACBD3 expression was markedly increased among different subtypes of breast cancer. (D) Real-time PCR analysis of ACBD3 expression in NBEC1, NBEC2 and breast cancer cell lines. (E) Western blotting of ACBD3 expression in NBEC1, NBEC2 and breast cancer cell lines. α-Tubulin was detected as a loading control in the western blotting. (F) ACBD3 mRNA expression level in nine paired breast cancer tissues. The average ACBD3 mRNA expression level was normalized to the expression of GDPDH. (G) ACBD3 protein expression level in nine paired breast cancer tissues.

and embryonic-like stem cells that are derived from placenta. It is assumed that ACBD6 functions in tumorigenesis [22]. ACBD7 is expressed in spleen, thymus and brain [23]. Considering the roles of these proteins, we speculate that ACBPs may be linked to cell differentiation and metabolism. ACBD3 was first observed to be intracellularly localized in the Golgi body, and is released from the Golgi body into the cytosol [24,25]. ACBD3 has not only been reported to be involved in iron homeostasis via its interaction with the divalent metal transporter 1 [26], but also involved in apoptosis through interaction with golgin160 caspase cleavage fragments [24,27,28]. Moreover, microarray data has mentioned that ACBD3 is involved in cell cycle control [29]. In Gefitinib-non-responders non-small cell cycle control, it is 3.8 fold higher and it also plays a role in cellular asymmetric division in neural progenitor cell-fate specification [30]. As has been reported, ACBD3 plays a role in cellular asymmetric division in neural progenitor cellfate specification [31]. Evidence showed that standard chemotherapy can be resisted by CSCs which have similar features to normal tissue stem cells. And it is those cancer stem cells that do the work of driving tumor regrowth [32]. Strong evidence supported that CSCs with similar features to normal tissue stem cells are resistant to standard chemotherapy and drive tumor regrowth. These findings indicate that ACBD3 plays an important role in tumorigenesis; however, it remains unclear whether ACBD3 can regulate tumorigenesis. Since asymmetric cell division and self-renewal is also characteristic of CSCs, it would be of interest to make a further investigation about the role of ACBD3 in

signaling, beta-catenin plays a role in triggering transcription of Wntspecific genes responsible for the control of cell fate decisions in many cells and tissues [12]. Besides, beta-catenin-mediated regulation of cMyc and p21 may help balance the cell death and proliferation in breast cancer [13]. All these observations point to the need of developing new BCSC-eliminating treatment strategies through which cure rates and survival can be improved The acyl-CoA-binding protein (ACBP), including ACBD1, ACBD2, ACBD23,ACBD4, ACBD5, ACBD6, and ACBD7, possesses a conserved ACBP domain at the N-terminal end [14]. Functionally, these proteins are released to the cytosol, interacting with other signaling molecules to regulate various cellular processes [15]. By shuttling acyl-CoA between the mitochondria and ER (microsomes), ACBD1 plays a role in protecting the long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity [16,17]. It was reported that ACBD2 has also been linked to hepatocellular carcinoma-associated antigen 64, indicating its role in human hepatocellular carcinoma [18]. ACBD3 was reported to be located in cytoplasm and membrane in eukaryotic cell, especially in the cells, tissues and systems of active metabolism, such as live and kidney [19]. ACBD4 is up-regulated in a panel of cancer cell lines treated with the histone deacetylase inhibitor valproic acid [20]. ACBD5 is up-regulated in phosphatase and tensin homolog (PTEN) positive neural stem cells in vitro, compared with PTEN null neurosphere cultures, indicating a potential function of tumor formation regulation [21]. ACBD6 is expressed in circulating CD34 + progenitors, 2

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Fig. 2. High ACBD3 expression in breast cancer tissues correlates with advanced clinicopathological features and poor patient survival. (A) Representative images of ACBD3 expression in normal breast tissues and breast cancer tissues of different clinical stages. (B) Quantification of the average mean optical density (MOD) for ACBD3 in normal breast tissue and tissues from different clinical stages of breast cancer. (C) Kaplan-Meier overall survival curves for all 253 breast cancer patients stratified by high and low expression of ACBD3. (D and E) Kaplan-Meier overall survival curves for all 253 breast cancer patients stratified by subgroups of patients with different stages (clinical stage I, II; clinical stage III, IV).

2.2. Plasmids and generation of stably-engineered cell lines

tumor stem cell. To get a better understanding of the onset of cancer, more study on the cellular function of ACBD3 in CSC and tumorigenesis should be made. In the present study, we report that expression of ACBD3 is significantly elevated in human breast cancer cells and tissues, and the expression correlates with advanced clinicopathological features and poor prognosis. Overexpressing ACBD3 promotes, while silencing ACBD3 inhibits self-renewal and tumorigenesis of breast cancer cells. Our results further reveal that ACBD3 promotes the tumorigenicity of breast cancer cells in vivo. Importantly, upregulating ACBD3 promoted the self-renewal and tumorigenesis of breast cancer cells via activating the Wnt/beta-catenin signaling. These findings uncover a novel ACBD3mediated regulatory mechanism in breast cancer and indicate that ACBD3 may be used as a valuable therapeutic target for the treatment of breast cancer.

ACBD3 (528 amino acids) construct was generated by subcloning the PCR-amplified human ACBD3 coding sequence into pMSCV-puro (Clontech Laboratories Inc.). The preparation of pure recombinant ACBD3 polypeptide was done according to a previously described protocol [33]. For depletion of ACBD3, human siRNA sequence was cloned into the pSuper-retro-puro plasmid. Retroviral production and infection were performed as described previously [33]. The reporter plasmids containing wild-type (CCTTTGATC; TOP flash) or mutated (CCTTTGGCC; FOP flash) TCF/LEF DNA binding sites were purchased from Upstate Biotechnology. 2.3. RNA extraction, reverse transcription and real-time PCR

2. Methods

RNA extraction, reverse transcription and real-time PCR were performed according to standard methods as previously described [33]. Primers were listed in the Supplemental Table 4.

2.1. Cell culture and human breast cancer specimens

2.4. Western blotting analysis

The breast cancer cell lines MDA-MB-453, MDA-MB-415, BT549, MDA-MB-231, ZR-75-30, SKBR3, T47D and MCF-7, normal human breast epithelial cells (NBECs) and breast cancer specimens were established as previously described [33]. This study was conducted with a total of 253 paraffin-embedded human breast cancer specimens that were histopathologically diagnosed at the Sun Yat-sen University Cancer Center from 1998 to 2003. Clinical information about the samples is summarized in Supplemental Table 1. The use of the clinical specimens was approved by the local Institutional Review Board, the Ethical Committee of the Sun Yat-sen University Cancer Center, and conformed to the ethical guidelines of the Helsinki Declaration.

Western blotting analysis was performed according to standard methods as previously described [34]. The following primary antibodies were used: anti-ACBD3 (sc-333, dilution 1:500; Santa Cruz Biotechnology, CA, USA), anti-beta-catenin, anti-α-tubulin, anti-TCF-4, anti-LEF1 and anti-GAPDH (1:1000, Sigma, Saint Louis, MI, USA). 2.5. Mammosphere culture 1000 cells were seeded in suspension in serum-free DMEM-F12 as described by Song et al. [34]. Cultures were fed once every three days. On day 20, the length and width measurements of the mammospheres was obtained using Zeiss Axiovision software (Carl Zeiss, Jena, Germany). 3

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Fig. 3. ACBD3 modulates the self-renewal of breast cancer cells. (A) The GSEA plot shows that ACBD3 expression positively correlates with proliferation and cell cycle-activated gene signatures. (B) Real-time PCR of the indicated breast cancer cells transfected with ACBD3-vector, ACBD3, ACBD3-RNAi-vector, ACBD3-RNAi1 or ACBD3-RNAi2. (C) Western blotting of the indicated breast cancer cells transfected with ACBD3-vector, ACBD3, ACBD3-RNAi-vector, ACBD3-RNAi1 or ACBD3-RNAi2. (D) Hoechst 33342 staining and flow cytometry revealed that overexpression of ACBD3 significantly increased the SP cells, while downregulation of endogenous ACBD3 significantly reduced the SP cells. (E) the average percentage of the SP cells in indicated cells in three independent detections.

2.6. Hoechst 33342 staining and flow cytometry

2.8. Luciferase reporter assay

To identify and isolate side population cells, the cells were dissociated and resuspended at 1 × 106 cells/mL in DMEM supplemented with 5% fetal bovine serum, pre-incubated at 37 °C for 30 min without or with 100 mM verapamil (Sigma). If verapamil was used in the subsequent steps, it was included at 50 mM. Cells were labeled with 2.5 mg/mL hoechst 33342 (Sigma) in staining media at 37 °C for 90 min, incubated on ice for 10 min, washed twice with ice-cold PBS, and then analyzed (20,000 cells per experiment with three replication) on a FACStar plus (BDIS) cell sorter which was equipped with dual Coherent I-90 lasers.

Cells were seeded in triplicate in 24-well plates and allowed to settle for 24 h, then transfected with the indicated plasmids plus 1 ng of the pRL-TK Renilla plasmid using Lipofectamine 2000 Reagent (Life Technologies). 48 h later,cells were assayed by using the DualLuciferase Reporter Assay (Promega) according to the manufacturer's instructions.

2.9. Xenograft tumor model Human-non-obese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice (4–5 weeks of age, 18–20 g) were purchased from the Center of Experimental Animal of Guangzhou University of Chinese Medicine. The Institutional Animal Care and Use Committee of Sun Yat-sen University approved all experimental procedures. The mice were randomly divided into groups (5 mice/group). Each mouse was inoculated in situ with A549-vector cells in the left breast and A549ACBD3 cells in the right mammary pad; the different groups of mice were inoculated with different number of cells: 5 × 104 cells/mouse, 1 × 104 cells/mouse, 5 × 103 cells/mouse, 1 × 103 cells/mouse, 5 × 102 cells/mouse, 1 × 102 cells/mouse and 10 cells/mouse. On day 50,

2.7. Immunofluorescent assay Immunofluorescent (IF) staining was performed on cells cultured on cover slips using an anti-human beta-catenin monoclonal antibody (Santa Cruz Biotechnology; 1:200 dilution). DAPI was used to stain the nuclei. And images were acquired using a laser scanning microscope (Axioskop 2 Plus, Carl Zeiss Co. Ltd., Jena, Germany).

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Fig. 4. ACBD3 regulates the tumorigenesis of breast cancer. (A-C) Representative micrographs, sphere sizes and numbers in the anchorage-independent growth assay. Each bar represents the mean values ± SEM of three independent experiments. *P < 0.05. (D) Images of excised tumors from five NOD/SCID mice at 49 days after injection with the indicated cells. (E) Average weight of excised tumors. (F) Representative images of sections sliced from the indicated tumors and stained with anti-ACBD3 and anti-Bmi-1, respectively. *P < 0.05.

3. Results

the animals were euthanized and the tumors were excised and weighed. All institutional and national guidelines for the care and use of laboratory animals were followed.

3.1. ACBD3 is up-regulated in breast cancer The published data from The Cancer Genome Atlas (TCGA) was used to analyze the mRNA expression of ACBD3 in breast cancer tissues. We found that mRNA of ACBD3 was up-regulated in 1099 breast cancer samples (Tumor) compared with 111 adjacent normal tissue samples (Normal, P < 0.001, Fig. 1A). As shown in Fig. 1B, ACBD3 mRNA was significantly up-regulated in the breast cancer tissues compared with the paired adjacent normal tissues (Normal, P < 0.001). By analyzing the mRNA expression of ACBD3 in breast cancer tissues with the help of different molecular subtypes, which show significant heterogeneity of breast cancer, we got a further understanding about the clinical significance and biological role of ACBD3 in breast cancer. Compared with that in normal breast tissues, ACBD3 was markedly up-regulated in patients with basal-like, her2-enriched, luminal A, luminal B and normal-like (Normal, P < 0.001, Fig. 1C). To verify these results derived from analyses of these published data, normal breast epithelial cells (NBEC1 and NBEC2) and eight

2.10. Statistical analysis All statistical analysis were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Associations between ACBD3 expression and the clinicopathological characteristics of the patients were analyzed using the Chi-squared test. Bivariate correlations between study variables were calculated using the Spearman's rank correlation coefficient. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Survival data were evaluated using uni-variate and multivariate Cox regression analysis. A two-tailed P-value of less than 0.05 was considered statistically significant in all experiments.

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Fig. 5. ACBD3 activates beta-catenin in breast cancer cells. (A) The GSEA plot shows that ACBD3 expression positively correlates with CTNNB1-activated gene signatures and TCF4activated gene signatures. (B) The indicated cells were transfected with TOP flash or FOP flash and Renilla pRL-TK, and subjected to dual luciferase assays 48 h after transfection. Firefly luciferase reporter gene activity was normalized to Renilla luciferase activity. (C) Subcellular localization of beta-catenin was assessed by immunofluorescent staining in the indicated cells. (D) Western blotting analysis (up) and TOP/FOP flash (down) of Wnt/beta-catenin-associated proteins in the indicated breast cancer cells. (E) Representative micrographs (up) and quantification (down) of mammosphere formation.

G). Taken together, these results strongly indicate that ACBD3 is upregulated in human breast cancer.

breast cancer cells were cultured and analyzed in the present study. Real-time PCR analysis and western blotting verified that the mRNA level and the protein level of ACBD3 was indeed up-regulated in cultured breast cancer cells compared with NBEC1 (Fig. 1D and E). To examine the expression of ACBD3 in clinical breast cancer specimens, nine paired breast tumor tissues (T) and the matched adjacent nontumor breast tissues (ANT) were analyzed. Real-time PCR analysis and western blotting revealed that ACBD3 were markedly overexpressed in the human primary breast tumor tissues compared to the ANT (Fig. 1F,

3.2. Upregulation of ACBD3 is associated with advanced clinicopathological features and poor prognosis in breast cancer We examined ACBD3 expression by immunohistochemical analysis of 253 paraffin-embedded, archived human breast cancer tissues (Supplemental Table 1). As shown in Fig. 2A, ACBD3 expression was 6

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Fig. 6. Analysis of ACBD3 and Wnt/beta-catenin pathway in human breast cancer samples. (A) IHC staining of breast cancer specimens. IHC analyses were performed independently twice on each sample. Two representative cases are shown. Original magnification, ×400. (B) Percentage of specimens showing ACBD3 expression based on the cytoplasmic/nuclear or membrane localization of beta-catenin. *P < .05, name test.

smaller mammospheres (Fig. 4A–C). Then, evaluation on the effect of ACBD3 on the tumorigenesis in vivo was taken. When subcutaneously implanted into NOD/SCID mice, ACBD3-overexpressing BT549 cells formed significantly higher numbers of larger tumors compared to vector-transfected BT549 cells (Fig. 4D); as few as 100 ACBD3-overexpressing cells could form tumors in NOD/SCID mice. As shown in Fig. 4E, tumor weight was increased remarkably in the ACBD3-overexpression group compared with the control group. Furthermore, IHC analysis revealed that the ACBD3 overexpressing tumor tissues displayed higher Bmi-1 stem cell indexes (Fig. 4F). Collectively, these results suggested that ACBD3 promotes the tumorigenicity of breast cancer cells in vitro and in vivo.

primarily observed within the cytoplasm and was elevated in 239/253 (94.5%) malignant specimens compared with the benign specimens. Quantitative analysis revealed that ACBD3 was expressed at higher level in advanced stages of disease (stage III–IV) and at lower level in early-stage tumors (stage I–II) (Fig. 2B). In addition, there were strong positive associations between ACBD3 expression and clinical stage (P < 0.001), T classification (P < 0.001), N classification (P < 0.001) and M classification (P = 0.037) (Supplemental Table 2). Kaplan-Meier survival analysis revealed that the patients with high ACBD3 expression exhibited shorter overall survivals compared to patients with low ACBD3 expression (Fig. 2C, P < 0.001). Furthermore, patients with high ACBD3 expression had significantly shorter overall survival compared to those with low expression of ACBD3 in the stage I + II subgroup (n = 146; P = 0.0216; Fig. 2D) and stages III + IV subgroup (n = 107; P = 0.0169; Fig. 2E). Moreover, multivariate survival analysis indicated that ACBD3 expression level was an independent prognostic factor for the assessment of patient outcomes (Supplemental Table 3).

3.5. Wnt/beta-catenin signaling is involved in ACBD3-mediated selfrenewal GSEA was further performed to identify the pathways involved in ACBD3-mediated breast cancer progression. As shown in Fig. 5A, ACBD3 expression level was positively correlated with the CTNNB1activated gene signatures and the TCF4-activated gene signatures, suggesting that the Wnt/beta-catenin pathway may mediate the effects of ACBD3 on cellular self-renewal. Overexpression of ACBD3 markedly increased the transactivation activity of beta-catenin in BT549 and T47D cells, as indicated by beta-catenin reporter gene assays based on the TCF and LEF genes (Fig. 5B). Conversely, knockdown of ACBD3 reduced TCF/LEF transcription activity in BT549 and T47D cells (Fig. 5B). These results suggested that ACBD3 might contribute to activation of the Wnt/beta-catenin signaling pathway. We therefore performed immunofluorescent staining assays to detect the location of beta-catenin. In agreement with this observation, overexpression of ACBD3 resulted in substantial nuclear accumulation of beta-catenin in A549 and T47D breast cancer cells, while knockdown of ACBD3 reduced nuclear translocation of beta-catenin (Fig. 5C). These data suggested that ACBD3 enhances the nuclear translocation of beta-catenin and consequently promotes transcription of TCF/LEF. To further validate the role of beta-catenin in the ACBD3-induced self-renewal potential of breast cancer cells, we blocked the Wnt/betacatenin pathway by knocking down TCF4 or LEF1 in ACBD3-overexpressing BT549 and T47D cells. Inhibition of beta-catenin signaling not only reduced the protein expression and transcriptional activity of TCF/LEF (Fig. 5D), but also abrogated the self-renewal ability induced by overexpression of ACBD3 (Fig. 5E). Taken together, these results indicate that Wnt/beta-catenin signaling is a functional mediator of ACBD3-induced self-renewal in breast cancer cells.

3.3. ACBD3 modulates breast cancer cells self-renewal On the basis of mRNA expression data from the TCGA, we used Gene Set Enrichment Analysis (GSEA) to confirm the biological role of ACBD3 in breast cancer. Results indicated that high levels of ACBD3 correlated significantly with stemcell-associated gene signatures (Fig. 3A). Considering the ER or PR receptor states and expression level of ACBD3, we generated ACBD3-stably expressing BT549 and T47D breast cancer cell lines by ectopically overexpressing ACBD3 and endogenously knocking down ACBD3 via retrovirus infection; real-time PCR and western blotting were performed to measure the mRNA and protein levels of ACBD3 expression (Fig. 3B and C). Consistent with the GSEA analysis, hoechst 33342 staining and flow cytometry revealed that the ectopic expression of ACBD3 significantly increased, while silencing ACBD3 reduced, the hoechst 33342-effluxed cell numbers in both BT549 and T47D breast cancer cells (Fig. 3D and E). These results indicated that ACBD3 enhances the self-renewal ability of breast cancer cells. 3.4. ACBD3 promotes the tumorigenesis of breast cancer On the purpose of investigating the effects of ACBD3 on the tumorigenic activity of breast cancer cells, we assessed mammosphere formation in suspension culture assay and found overexpression of ACBD3 enhanced the formation of a higher number of more bulky mammospheres, while silencing of ACBD3 reduced, the self-renewal ability of BT549 and T47D cells through formation of a lower number of 7

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Table 5). Moreover, in accord with the conclusion that ACBD3 protein is released from the Golgi body into the cytosol, intracellular localization of ACBD3 protein in breast cancer is cytoplasmic. According to the microarray data from published cancer related databases, ACBD3 plays a role in cell cycle control and retinoblastoma protein (pRB), a critical tumor, takes the control of ACBD3's regulation [46]. Similarly, knockout of the mouse ACBD3 gene revealed a potential link between ACBD3 and numb signaling in asymmetric cell division [47]. Moreover, ACBD3 was reported to play a role in cellular asymmetric division in neural progenitor cell [47]. Since asymmetric cell division occurs widely from embryonic stem cells to CSCs, it would be of interest to further investigate the role of ACBD3 in CSCs. For the first time, we confirmed that ACBD3 could regulate the self-renewal potential of breast cancer cells. Given the potentially important role of ACBD3 in BCSCs, we investigated how ACBD3 regualts CSC. Existing research has found that ACBD3 silencing PRKAR1A expression induced growth inhibition and apoptosis of cancer cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/beta-catenin pathway signaling [45]. On the one hand, Wnt signaling represents powerfully regulated cell fate in animal throughout their lifespan [48], on the other hand, beta-catenin is the key effector responsible for transduction of the Wnt signal to the nucleus and it triggers transcription of Wnt-specific genes [49]. Therefore, we speculated that ACBD3 could affect the Wnt/beta-catenin signaling pathway. We observed that overexpression of ACBD3 enhanced the nuclear translocation of betacatenin in both breast cancer cell lines and clinical specimens. That is, our observations may have potential implications for the critical role of beta-catenin in cell progressions.

3.6. Overexpression of ACBD3 is associated with activation of the betacatenin pathway in human breast cancer Immunohistochemical staining was performed to further explore the clinical relevance of these findings in human breast cancer. An association was observed between the ACBD3 expression level and betacatenin nuclear expression in the 136 clinical specimens tested (Fig. 6A). As shown in Fig. 6B, breast cancer samples with high ACBD3 expression showed higher levels of beta-catenin activation (34/42 samples; 81.0%) than those with low ACBD3 expression (35/94 samples; 37.3%; Fig. 5B; P < 0.05). These data indicate that overexpression of ACBD3 is associated with activation of beta-catenin in human breast cancer. 4. Discussion Stem cells, undifferentiated and pluripotent cells, can produce more new stem cells (self-renewal) and differentiate into specialized cells such as vascular cells, immune cells and mammary ductal cells [35–37]. Cancer stem cell (CSC) exhibits self-renewal activity and long-term cancer-propagating capacity, making treatment incomplete and leading to the recovery of cancer [38]. In other words, CSC have been recognized as a major‘target cell population’in oncology in recent years. Cancer treatment could be potentially improved under the identification of the effective regulatory molecules of CSC. Actually, a number of different moleculars have been described to play a role in the evolution and maintenance of CSC. For example, high Heat Shock Factor 1 (HSF1) not only correlates with CSC marker expression, but also induces the CSC phenotype [39]. By activating the Wnt signaling pathway, SRY-box 2 (Sox2) overexpression increases the proportion of BCSCs [40]. As a necessary factor to maintain self-renewal ability, Kruppel like factor 4 (KLF4) promote the migration and invasion of the tumor cells in vivo [41]. Critical and delicate as it is, the regulation of stem cell self-renewal and tumor suppression via asymmetric cell division is of value to be studied. The present study indicates that ACBD3 plays a significant role in the self-renewal of stem cells in breast cancer, as overexpression of ACBD3 increased mammosphere formation and the proportion of side population cells in BT549 and T47D cells; therefore, ACBD3 may represent a potential target for elimination of BCSCs during breast cancer therapy. ACBD3 was first identified by yeast 2-hybrid screening and mammalian cell co-precipitation studies as a translocator Protein and Protein Kinase-A Regulatory Subunit Iα(PKARIA)-binding protein [24]. The mitochondrial membrane translocator protein (TSPO), was reported to protect cells from free radical damage [42]. Classical and novel TSPO ligands for the mitochondrial TSPO can modulate nuclear gene expression, which functions included cell viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis [43,44]. The protein kinase A regulatory subunit 1 alpha (PRKAR1A) pathway is overexpressed in varieties of tumors and cancer cell lines [45]. Following the pattern of PRKAR1A expression, ACBD3 was reported to express in steroidogenic tissues, which shows the participation of ACBD3 in PRKAR1A-mediated tumorigenesis and hypercortisolism [45]. However, the expression level in breast tissue,especially in breast cancer is not clear. Herein, analyzing TCGA data, western blotting, real-time PCR and immunohistochemical analysis firstly indicate that ACBD3 protein is in the state of low expression in normal breast tissues and high expression in breast cancer tissues. Our further evaluation of ACBD3 expression in 253 paraffinembedded, archived clinical tumor tissue specimens showed that the level of ACBD3 protein was strongly correlated with clinical staging (P < 0.001), T classification (P < 0.001), N classification (P < 0.001), and M classification (P = 0.037) of breast cancer patients. As to the samples from TCGA database, 579 patients had a description of the relapse states among the total 1099 patients. Chi-Square tests showed that there is a positive correlation between ACBD3 expression and relapse state (Supplemental

5. Conclusion This study demonstrates that ACBD3 is up-regulated in breast cancer and associated with advanced clinicopathological features as well as poor prognosis in breast cancer. Meanwhile, ACBD3 regulates the enrichment of self-renewing BCSCs via activation of the Wnt/betacatenin signaling pathway. Besides, ACBD3 may represent candidate therapeutic targets to enable the elimination of BCSCs, which may allow the development of innovative therapies for breast cancer. Funding This work was supported by Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306023, 2014), Natural Science Foundation of Guangdong Province, China (No. 2017A030313549, 2017), Medical Scientific Research Foundation of Guangdong Province (No. A2016091, 2017), Shenzhen Science and Technology Project (No. JCYJ20170307144612471, 2017). Disclosure of potential conflicts of interest Conflicts of interest: none. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2018.01.003. References [1] O.M. Ginsburg, R.R. Love, Breast cancer: a neglected disease for the majority of affected women worldwide, Breast J. 17 (3) (2011) 289–295. [2] H.J. Senn, St. Gallen consensus 2013: optimizing and personalizing primary curative therapy of breast cancer worldwide, Breast Care 8 (2) (2013) 101. [3] C.A. Parise, V. Caggiano, Risk of mortality of node-negative, ER/PR/HER2 breast cancer subtypes in T1, T2, and T3 tumors, Breast Cancer Res Treat. (2017).

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