The cancer-testis antigen a-kinase anchor protein 3 facilitates breast cancer progression via activation of the PTEN/PI3K/AKT/mTOR signaling

ABSTRACT The cancer-testis antigen A-kinase anchor protein 3 (AKAP3) has been shown to have a strong association with breast cancer (BC). However, its role in BC progression received scant attention. We aimed to explore the prognostic implication of aberrant AKAP3 expression for a better knowledge of BC progression and improved treatment. AKAP3 expression was quantitated using tissue microarrays and immunohistochemistry (IHC). Cell viability, invasion, migration, apoptosis, and expressions of PTEN/PI3K/AKT/mTOR signaling components were assessed in AKAP3-overexpressed or si-AKAP3-transfected BC cells. Finally, elevated AKAP3 expression was observed in BC versus paracancerous tissues. BC patients with high AKAP3 expression showed a worse prognosis than low expression patients (P < 0.0001). AKAP3 overexpressions fueled cell growth, proliferation, migration, and invasion in HCC1937 and MDA-MB-468 BC cell lines, alongside increased expressions of PI3K/AKT/mTOR signaling components and PTEN suppression. These effects were pronouncedly reversed, together with elevated apoptosis, in cells transfected with si-AKAP3. Therefore, AKAP3 is upregulated in BC and promotes BC cell growth, invasion, and migration via PTEN/PI3K/AKT/mTOR signaling activation. It may serve as a prognosis indicator for BC survival.


Introduction
Breast cancer (BC) is a malignant tumor derived from the glandular epithelium of the breast. It shows region-specific prevalence in North America, northern Europe, and Oceania [1] and is the leading malignant tumor among women worldwide, with the rate of new cases growing at a higher-than-average speed of 3% per year [2]. It is also the fourth leading cause of death from cancer in China, according to the 2015 cancer statistics [3]. Except for known tumorigenic factors, such as serum hormone, unhealthy diet, a history of familial BC, and environment [2,4], proto-oncogenes, inactivation of tumor suppressor genes, mutations, and other genetic abnormalities also contribute to the occurrence of BC [5,6]. Currently, significant progress has been made in the identification of more biomarkers [7], thanks to the known biomarkers of BC survival or treatment and the application of new technologies in BC molecular research [8], which underlies robust diagnostic algorithms based on biomarker combinations. In this respect, comprehensive expression profile analysis of genes for identification of new biomarkers is still a feasible way to improve treatment decision-making and patient prognosis.
Cancer-testis antigens (CTAs) are frequently detectable in normal testicular tissues, spermatozoa, and various types of tumor cells in humans and other mammals and lowly expressed in normal somatic cells [9]. Current knowledge on CTA genes reveals that the majority of these genes belong to distinct multigene families and are located on the X chromosome [10]. They are primarily expressed in germ cells of the normal testis (instead of other normal tissues) and most malignant tumor cells derived from different types of tissues and elicit immune responses to antigenic stimuli in tumors [11]. Since the melanoma antigen gene (MAGE) family was discovered by Vander Bruggen in 1991 using T cell epitope cloning [12], more than 200 CTA genes have been identified and shown to have close associations with mutations in tumorigenic genes or tumor suppressor genes during gametogenesis [13]. Therefore, CTAs are considered as one of the most promising targets for cancer vaccine therapy, of which MAGE-A, IL-13Rα, and NYESO-1 have been studied in patients to verify whether they can cause tumor regressions [9,14,15].
Human A-kinase anchoring protein 3 (AKAP3) is a CTA family member essential for tumorigenesis. The human AKAP3 gene that maps on chr12:4,615,508-4,649,047 and encodes AKAP3 is a member of the AKAP family of functionally related proteins that target protein kinase A (PKA) to discrete locations within the cell. It has been shown to interact with the R-subunit of PKA and sperm-associated proteins to regulate motility, capacitation, and the acrosome reaction [16]. Its roles in the occurrence and development of ovarian cancer have been reported [17]. However, there are limited studies on AKAP3's roles in distinct tumors.
The PI3K/AKT/mTOR signaling has been shown to involve in cell differentiation, proliferation, anti-apoptosis in breast cancer cells, and oxidative stress and angiogenesis [18]. PETN is an inhibitor of the PI3K/AKT/mTOR signaling. PTEN methylation, alongside PI3K mutations and AKT activation, has been reported to be closely associated with response and resistance to anticancer therapy, particularly hormonal therapy resistance, and prognosis in BC [19]. Currently, direct evidence of the correlation between AKAP3 and the PI3K/AKT/mTOR signaling is unavailable, but CTA research in lung cancers has demonstrated the relationship of another cancer-testis antigen LDHC with this signaling, which may promote GSK-3β-dependent phosphorylation of AKT [20]. AKAP3 is a dual-specificity molecule that modulates PKA isotypes. The latter can trigger the PI3K/AKT signaling in MCF-7 breast cancer cells [21,22]. Thus, it is intriguing to hypothesize that AKAP3 restoration or downregulation may alter the activity of the PI3K/AKT/ mTOR signaling. In the present study, we aimed to characterize the expression pattern of AKAP3 in BC using high-throughput tissue microarrays and IHC and explore correlations between aberrant AKAP3 expression and clinicopathological features and prognosis of BC patients. We used transfections to block or overexpress AKAP3 in human BC cell lines. We hypothesized that AKAP3 could promote BC malignant behavior via PI3K/AKT/mTOR signaling activation. , and intraductal carcinoma (1 case). Two of them were triple-negative breast cancer (TNBC) cases. All cases were confirmed to have primary cancer without distant organ metastasis. Sixty-nine cases had lymph node metastasis, and 70 showed the absence of lymph node metastasis. In six cases, the presence of metastasis was not recorded. The sixth edition of AJCC Staging System was utilized to stage BC patients as follows: stage 0 (n = 1), stage 1 (n = 22), stage 2 (n = 78), and stage 3 (n = 39). Five participants had missing stage records (n = 5). A tissue microarray (HBreD050Bc01, SHANGHAI OUTDO BIOTECH) comprises 24 TNBC cases were also used for the analysis. All patients were followed up until July 2014. Approval for tissue microarray analysis was given by the research ethics committee.

Cell culture, vector construction, and construction of AKAP3-overexpressed and silenced BC cell lines
Human MCF-7, HCC1937, MDA-MB-231, and MDA-MB-468 BC cell lines, and Hs578Bst breast cell line and HEK293a cell line (Shanghai Cell Bank of Chinese Academy of Sciences, Shanghai, China) were selectively cultured in DMEM high-glucose medium or RPMI-1640 medium containing 10% FBS in a 37°C, 5% CO 2 incubator. MDA-MB-231 cells were maintained in Leibovitz's L-15 medium supplemented with 10% FBS in a CO 2 -free incubator at 37°C. The culture medium was replaced with a fresh medium every 2-3 days, and cells were passaged every 5-7 days. GV358-EGFP lentivirus vectors overexpressing AKAP3 (Oe-AKAP3) and GV115-EGFP vectors expressing RNA interference (RNAi) against AKAP3 (si-AKAP3) were successfully constructed and packaged in cells. AKAP3overexpressed or silenced cell lines were screened using 0.4 μg/mL puromycin.

Real-time qRT-PCR
Total RNA was extracted from cell lysates using a cell/tissue RNA extraction kit (Promega) and reversely transcribed into cDNA using the Transcriptor First-strand cDNA Synthesis Kit (ROX). The cDNA was amplified using the ABI7500 fluorescence quantitative PCR system and SYBR Green Master (ROX) with AKAP3specific forward (5'-ACAAGGCTGAGAGTTATTCCCT-3') and reverse primers (5'-CTCACCCAGAGTTTTCGCACA-3', product length: 158 bp). The primers of the housekeeping gene GAPDH included forward 5'-GACAGTCAGCCGCATCTTCT-3' and reverse 5'-GCGCCCAATACGACCAAATC-3' (product length: 104bp). The PCR cycling conditions were as follows: one cycle of pre-denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15s, and one cycle of annealing at 60°C for 1 min. Relative AKAP3 mRNA expression was calculated as a fold change of 2 −ΔΔCT value. The Hs578Bst breast cell line as normal control, and HEK293a cell were treated as AKAP3-negative control.

Cell viability assay
Cells in the logarithmic growth phase were adjusted to 5 × 10 3 cells/well and cell suspension was prepared with DMEM high-glucose medium or RPMI-1640 medium. 100 μL per well was inoculated in a 96-well culture plate. After culture for 0, 24, 48, 72, and 96 h, 10 μL of CCK-8 reagent was added to each well and cultured for 3 h. The optical density (OD) values were measured at dual wavelengths (450 and 630 nm) using a microplate reader (Thermo), which was proportional to the number of cells. The percent viability of each sample was calculated depending on the OD values of four replicate wells. Growth curve analysis was performed to monitor cell proliferation [24].

Wound healing migration assay
BC cells (5 to 8 × 10 5 cells/well) were seeded into a 6-well plate and incubated to a confluence of 95% or more. A vertical scratch wound was made by scraping a line down the center of each well with a disposable 200-μL micropipette tip. Nonadherent cells were removed with two washes of PBS, and remnant cells were photographed at 0, 48, and 72 h. The number of migrated cells was counted and compared to baseline values (0 h) to reflect cell migration ability [25].

Transwell invasion and migration assay
Cell concentration was adjusted to 1.5 × 10 5 cells/mL. To perform a Transwell assay, Transwell chambers containing Matrigel-coated filters with an 8-µm pore size were utilized. Matrigel-coated filters were not employed in migration assays. BC cell suspension (200 μL per well) was added to the upper chamber, and 600 μl of culture medium containing 30% fetal bovine serum was added to the lower chamber. Cells were allowed to invade or migrate for 48 or 72 h. The cells in the lower chamber were fixed with methanol for 30 min and stained with 0.1% crystal violet dye solution for 20 min. After washes with PBS, cells were photographed and counted under a microscope. Five fields were randomly selected, and the number of cells migrating across a filter was counted to assess cell invasion or migration ability [25].

TUNEL assay
MCF-7 and MDA-MB-231 cells were collected, washed, and fixed with 4% paraformaldehyde for 30 min and then incubated with PBS containing 0.3% Triton X-100 for 5 min at room temperature. Cells were incubated in 0.3% H 2 O 2 in PBS for 20 min at room temperature to suppress endogenous peroxidase activity. Subsequently, a biotin-conjugated reagent (50 μL/well) was added to each well for 60 min at 37°C. After one wash with PBS, cells were incubated with stop solution (0.1-0.3 mL/well) for 10 min at room temperature and with 50 μL of streptavidin solution (Beyotime, China) for 30 min at room temperature. The reaction was visualized with DAB (0.2-0.5 mL/well). The nucleus was counterstained with hematoxylin. Cells were assessed as apoptotic when the nucleus was stained brown or dark brown. For each group, apoptotic cells were counted in five fields randomly selected and the data were analyzed [26].

DNA ladder assay
DNA laddering is a significant indicator of cell apoptosis. Cell apoptosis can be confirmed while observing DNA ladder formation. Therefore, DNA ladder assays were performed. Trypsin-digested cells were washed once with PBS or saline and centrifuged at 1,000-2,000 × g for 1-2 min. Cell lysates were mixed with protease K (5 μL/1 mL), vortexed, and placed in a tube in the water bath at 50°C overnight to digest. Tris-equilibrated phenol solution (500 μL, pH 8.0) was added to the cell lysate and thoroughly vortexed. The supernatant was extracted, mixed with 60 μL of 10 M ammonium acetate and 600 μL of anhydrous ethanol, and turned upside down several times until DNA precipitation occurred. The mixture was centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was removed. DNAs were washed once with 70% ethanol, dried, and dissolved in TE buffer. DNA fragmentation was measured using 1% agarose gel electrophoresis [27].

Statistical analysis
SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Measurement data were presented as the mean ± standard deviation (SD). The two-sample t-test was applied for normally distributed continuous variables. Differences in categorical variables (e.g., clinicopathological features and AKAP3 expression scores) between groups were compared using the Chi-square test. Kaplan-Meier survival analysis was performed to assess potential correlations between aberrant AKAP3 expression and overall survival (OS). A P-value of < 0.05 was considered statistically significant.

AKAP3 serves as a prognostic marker in BC
AKAP3 expression patterns in BC tissue and cell lines and the prognostic significance of aberrant AKAP3 expression were assessed. AKAP3 was primarily expressed in the cytoplasm of tumor cells (Figure 1(a)) and was detectable in 136/145 (93.8%) BC tissue samples, including 79 cases with AKAP3 high expression patterns (++/+++) and 57 with AKAP3 low expression patterns (±/+). Moreover, the positive expression rate of AKAP3 was estimated to be of 65.38% (17/26) in TNBC cases. In paratumorous tissues (n = 90), AKAP3 was lowly expressed (±/+) in 28 cases and undetectable (−) in 62 cases. AKAP3 expression IHC score significantly increased in stage 3A-3C versus 0-2B BC patients (P = 0.0027, Figure 1(b)). However, there were no significant correlations of AKAP3 upregulation with clinicopathological prognostic parameters (Table 1). BC patients with low AKAP3 expression could enjoy longer OS than those with high AKAP3 (Figure 1(c)) (P < 0.0001). Similarly, AKAP3 mRNA expression was significantly elevated in MDA-MB-231 and MCF-7 BC cells compared to that achieved in MDA-MB-468 and HCC1937 cells. HEK293a cell line failed to express AKAP3 mRNA and was regarded as negative control (Figure 1(d)). Further, AKAP3 expression had a positive correlation with EGFR and Ki67 expressions, a negative correlation with p53, but no associations with HER2, ER and PR (Figure 1(e-j)).

AKAP3 overexpression promotes BC cell proliferation, invasion, and migration
To confirm whether AKAP3 overexpression could affect cell viability, we performed CCK-8, wound healing migration, and Transwell invasion and migration assays in HCC1937 and MDA-MB-468 cell lines whose AKAP3 was endogenously downregulated. HCC1937 and MDA-MB-468 cells overexpressed AKAP3 at mRNA and protein levels after transfection with Oe-AKAP3 (Figure 2(a,b)). Compared to sh-NC, AKAP3-overexpressing cells had greater proliferation (Figure 2(c)), migration,  and invasion abilities in both cell lines ( Figure 2(d-g)).

AKAP3 knockdown suppresses BC cell growth, proliferation, invasion, and migration
We chose MCF-7 and MDA-MB-231 cell lines that had endogenous high AKAP3 for knockdown experiments. In qRT-PCR and immunoblotting assays, AKAP3 mRNA and protein levels sharply decreased in si-AKAP3transfected MCF-7 and MDA-MB-231 cells (Figure 3(a)). As shown in Figure 3(b), the number of MCF-7 and MDA-MB-231 cells pronouncedly decreased after si-AKAP3 transfection, together with suppressed cell growth and proliferation (Figure 3(c)), compared to empty vector (sh-NC) transfected cells. In Transwell invasion and migration assays, si-AKAP3transfected cells exhibited suppressed invasion and migration abilities compared to sh-NC cells (Figure 3(d-g)). These findings suggest that AKAP3 knockdown inhibits the malignant behavior of MCF-7 and MDA-MB-231 cells.

AKAP3 knockdown triggers BC cell apoptosis
Apoptosis analyses were conducted to investigate whether the suppressive effects of AKAP3 downregulation on cell viability were due to apoptosis. MCF-7 and MDA-MB-231 cell lines were selected. We found that compared to sh-NC nuclear DNA degradation was enhanced in apoptotic cells, together with a more drastic decrease in total DNA at 96 h, after AKAP3 knockdown (Figure 4(a)). An increase in the number of apoptotic cells was detectable in the si-AKAP3 group (Figure 4(b,c)), which was consistent with downregulated anti-apoptotic Bcl-2 and elevated proapoptotic caspase-3 in transfected cells (Figure 4(d,e)).

Discussion
Evaluation of biomarker state or endpoints remains a valid instrument designated to screen the target population and monitor the efficacy of BC treatment. Since the immunogenicity of BC cells was previously reported [29], CTA has been extensively explored and proven an effective biomarker for immunotherapy in BC [30,31]. Two ongoing clinical trials focus on immunotherapy with MAGE-A-and NY-ESO -1-based polyvalent vaccines in patients with primary or metastatic BC [32]. Better knowledge of mechanisms linking roles of CTA genes aids in the development of anti-CTA targeted immunotherapy for BC, particularly advanced BC. AKAP3 is a member of the CTA family and is specifically expressed in the testis of humans and other mammals [16,17,33]. The efficacy of AKAP3 as a prognostic biomarker and an immunotherapy target has been demonstrated in ovarian cancer [33] and hepatocellular carcinoma [34]. However, its efficacy in BC has not been reported.  advanced-(stage 3A-3C) versus early-stage [stage 0-2B) BC patients. Further, AKAP3 expression was positively correlated with EGFR and Ki67 expressions and inversely related to p53 expression. These findings indicate that AKAP3 upregulation participates in the occurrence and development of BC. AKAP3 upregulation also had a significant correlation with poor OS of BC patients, suggesting that AKAP3 may serve as a prognostic indicator for survival in BC. However, the AKAP3 expression pattern differs among BC subtypes. Esmaeili et al. reportedthat AKAP3 was downregulated in triple-negative BC (TNBC] and high AKAP3 expression had a significant association with a better 5-year disease-free survival [35]. However, our subtype analyses showed that a high AKAP3 expression rate (65.38%, 17/26) was observed TNBC patients. A possible reason for these inconsistent correlation findings can be prognostic curves based on AKAP3 mRNA expression in Esmaeili's study versus those based on AKAP3 protein expression in the present study. It was reported that the percentage agreement between gene and protein expression was only 40%-50%, which underlies the difference between prognosis analysis results based on gene and protein expression data [36]. Future studies systematical assessment of aberrant AKAP3 expression in each BC subtype are needed.
We observed significantly enhanced cell growth, proliferation, invasion, and migration in HCC1937 and MDA-MB-468 cells after Oe-AKAP3 transfection. These effects were robustly inhibited by si-AKAP3, alongside increased apoptosis, in MCF-7 and MDA-MB-231 cell lines. This oncogenic effect of AKAP3 has been reported in other cancer types. [37] demonstrated that AKAP-3 was highly expressed in patients with epithelial ovarian cancer and confirmed its role as an oncogene in epithelial ovarian cancer.
The PI3K/AKT/mTOR signaling is critical in cellcycle regulation, which is directly involved in cell dormancy, proliferation, carcinogenesis, and lifespan of BC cells [33,38]. PTEN selectively antagonizes the PI3K/AKT/mTOR pathway [39]. PI3K activation converts phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to the second messenger PIP3. The latter binds to the PH domain of the intracellular signal transduction proteins AKT and PDK1, thus phosphorylating AKT at Ser308 via PDK1 activation [40]. Activated AKT regulates cell behavior by phosphorylating downstream enzymes, kinases, and transcription factors [40]. In this study, we found that AKAP3 overexpression downregulated PTEN expression and activated the PI3K/ AKT/mTOR signaling, promoting cell growth, proliferation, invasion, and metastasis in BC cells. These effects were potently suppressed by AKAP3 knockdown. Our findings indicate that AKAP3 regulates the malignant behavior of BC cells via activation of the PTEN/PI3K/AKT/mTOR signaling.

Conclusion
AKAP3 is upregulated in BC cancerous tissues. AKAP3 elevation triggers the malignant behavior of BC via PTEN/PI3K/AKT/mTOR activation. It is associated with poor survival of patients with advanced BC, suggesting the potential of high AKAP3 to serve as a biomarker for prognosis prediction or a potential target for the immunotherapy for BC. We plan to carry out transcriptome and proteomic analysis in cell lines stably transfected with the AKAP3 gene in the future to screen regulators associated with AKAP3's effects on BC and related mechanisms.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.