β-Catenin nuclear localization positively feeds back on EGF/EGFR-attenuated AJAP1 expression in breast cancer

283 cases of paraffin-embedded breast cancer patients’ specimen and 25 pairs of fresh tumor tissues were randomly selected at Cancer Hospital of Tianjin Medical University. The patients received treatments from January 1, 2006 to December 31, 2006. None of the patients underwent chemotherapy or radiotherapy before surgery. The patient clinical pathologic features are showed in Additional file 1: Table S1. All cases had decent follow-up and reliable clinical data. Besides, this study followed the Declaration of Helsinki, and the patients provided written informed consents.

All paraffinized tissue blocks were cut at 4 μm thicknesses and detected by the SP immunochemistry kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). IHC assay was conducted as previously described [27]. The rabbit monoclonal anti-human AJAP1 antibody (Bioss, China) at 1:100 dilution or the mouse monoclonal anti-human β-catenin antibody (CTNNB1, Boster) at 1:200 dilution was used for IHC. Two senior pathologists (Yun Niu and Shuhua Lv) evaluated the score without any knowledge of the clinicopathological outcomes of the patients. The percentage of positivity of the tumor was scored as “0” (no tumor cells), “1” (1–25%), “2” (26–50%), “3” (51–75%), and “4” (> 75%). The staining intensity of the positive tumor cells was scored as “0” (no staining), “1” (weak staining), “2” (moderate staining), and “3” (strong staining). As for AJAP1, the multiplier of the scoring of (0–3) for low expression and (4–12) for high expression were used. The IHC staining results of β-catenin were evaluated independently according to the subcellular localization of the nucleus and membrane. A positive/abnormal nuclear expression was defined as over 10% of the nuclear-stained tumor cells. An abnormal membranous expression demonstrated either no immunoreactivity in the membrane or less than 10% of the cancer cells with a positive membranous staining [28, 29].

All cell lines were purchased from the American Type Culture Collection (ATCC, USA). T47D, SK-BR-3, MCF-7 and MDA-MB-231 were cultured with the 1640 (Gibco, USA), MCF10A was cultured in DMEM-F12 (Gibco, USA) media. MDA-MB-453 was cultured in L-15 media. All media contained 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and cell culture carried out at 37 °C in a 5% CO₂ incubator.

The cells were grown in 24 -well plates and then stimulated with 50 ng/ml EGF for 24 h. After 1 day, the cells were fixed, rehydrated, and incubated with β-catenin (Santa Cruz Biotech, China), AJAP1 (Bioss, China), and fluorescein-labeled secondary antibodies. The nuclei were stained with DAPI for 10 min. The slides were examined through a fluorescent confocal microscope.

Cellular protein was extracted by RIPA buffer containing PMSF. The Nuclear and Cytoplasmic Isolation Kit (KeyGEN, China) was used for the cytoplasmic and nuclear protein extraction according to the manufacturer’s instructions. The proteins were separated on 10% SDS-PAGE gels, transferred onto PVDF membranes, blocked using 5% skim milk, and incubated with primary antibodies overnight at 4 °C. The protein bands were detected by the ECL detection Kit (Solarbio, China).

The related siRNAs and control plasmids were showed in Additional file 2: Table S2. The viruses were packaged in 293 T cells according to the manufacturer’s instructions. The infected cells were selected with 2 μg/mL puromycin.

Total RNAs were extracted using Trizol reagent (Takara, Japan). Reverse transcription was conducted by the SuperScript RT kit (Takara, Japan). The qRT-PCR assay was performed with SYBR Green PCR kit (Takara, Japan). Primers used are listed in Additional file 2: Table S2. GAPDH was used as the internal control. The relative expression levels were calculated by the 2^-ΔΔCt method. All experiments were assayed in triplicate.

The co-IP of AJAP1 and β-catenin was performed using the Pierce Co-IP kit (Thermo Scientific, USA) according to the manufacturer’s protocol as described previously [30]. The protein quantity was then detected by western blotting.

5 × 10⁴ cells were seeded in 24-well plates and cultured for 24 h at a density of 70–80%. The cells were transferred with 2 μg of β-catenin-responsive firefly luciferase reporter plasmid TOP-FLASH or the negative control FOP-FLASH (Merck-Millipore) using fugene6 (Invitrogen, USA) according to the manufacturer’s instructions. The cells were subjected to luciferase reporter assay 24 h after transfection according the Dual Luciferase Reporter Assay System (Promega, Madison, WI). The relative Renilla luciferase activity was normalized to that of firefly luciferase activity. Each experiment was measured in triplicate.

2 × 10⁴ cells were seeded in the upper chamber with RMPI 1640 medium without FBS and a medium containing 10% FBS at 37 °C in the lower chamber. After culturing at 37 °C for 24 h, the cells in the upper chamber were harvested and then stained according to the three-step set (Thermo Scientific, USA) protocol. Scratches were made when the cells reached a 95% confluence in each well using a 10 μL plastic pipette tip. The scratches were measured and photographed. Each experiment was conducted at least three times.

2 × 10³ cells were seeded in 96-well plates and the activities of the viable cells were measured at different time points using a microplate reader. In the colony formation assay, 8 × 10² cells were cultured in 6-well plates for 2 weeks. The colonies were stained with crystal violet and counted.

Female BALB/c nude mice (4–6 week) were injected with 5 × 10⁶ treated cells and kept in the Tianjin Medical University Cancer Institute and Hospital SPF’s animal feeding center. The tumors were measured as described previously [31]. All animals were sacrificed after 60 days. All experimental procedures were approved by the International Animal Care and Use Committee of Tianjin Medical University Cancer Institute and Hospital.

To determine the expression profile of AJAP1 in breast cancer tissues, breast cancer datasets from Oncomine and GOBO datasets were analyzed in this study. The dataset from Finak (GOBO)and Oncomine showed that AJAP1 was downregulated in the breast cancer tissues compared with the normal tissues (Fig. 1a left panel). In addition, GOBO dataset demonstrated that the expression of AJAP1 was related with cancer subtypes (Fig. 1a middle panel, p = 0.00402) and grades (Fig. 1a right panel, p = 0.00275). These conclusions were confirmed by Western blotting and qRT-PCR of 25 pairs of breast cancer and corresponding normal tissues (Fig. 1b).

Furthermore, 283 breast cancer formalin-fixed, paraffin-embedded tumor slides with complete clinicopathological characteristics and follow-up data were utilized to analyze the expression of AJAP1. In this work, AJAP1 was mainly located in the cytoplasm and was downregulated in breast cancer. Moreover, its expression was decreased in the progression of breast cancer (Fig. 1c). As shown in Table 1, low AJAP1 expression was significantly associated with advanced histological grade and higher lymph node involvement. In addition, Cox proportional hazards regression analyses (Additional file 3: Table S3) demonstrated that AJAP1 was an independent prognostic marker for overall survival (p = 0.024). Meanwhile, Kaplan-Meier analysis showed that patients with low expression of AJAP1 exhibited poor survival (Fig. 1d).

To better explore the role of AJAP1 in breast cancer, AJAP1 expression in different wild types of breast cancer cell lines were detected. T47D and MDA-MB-231 cells showed typical high and low expression of AJAP1, respectively (Fig. 1e). T47D cells with knocking down of AJAP1 expression and MDA-MB-231 cells with overexpression of AJAP1 were then generated (Fig. 1f) to evaluate the proliferation, migration and invasion of breast cancer by MTT, Transwell, colony-formation and wound-healing assays. These results showed that AJAP1-depletion in T47D cells promoted the capacity of proliferation (Fig. 1g, Fig. 1i), migration (Fig. 1h) and invasion (Fig. 1j). In contrast, AJAP1 overexpressed in MDA-MB-231 cells showed the opposite results. Furthermore, flow cytometry (Fig. 1k) results showed that downregulation of AJAP1 in T47D cells significantly reduced the population of G0/G1 cells but increased S/G2/M cells. On the contrary, AJAP1-overexpressed MDA-MB-231 cells showed the opposite results. In conclusion, AJAP1 was a putative tumor suppressor in breast cancer.

The 283 breast cancer cases were then used to explore the relationship between β-catenin expression and clinicopathological parameters. Figure 2a represents the typical progression of breast cancer. It was observed that β-catenin presented a strong uniform membrane in the luminal cells of para-carcinoma normal tissues (Fig. 2a, left). In the high-grade ductal carcinoma in situ and invasive cancer, β-catenin demonstrated different expression patterns of cytoplasmic (Fig. 2a, middle) and nuclear expression (Fig. 2a, right). The different expression patterns of AJAP1 and β-catenin in different cases were illustrated in Fig. 2b–e. The abnormal membranous expression of β-catenin and the positive nuclear/cytoplasmic expression of β-catenin were detected in 43.5% (123/283) and 78.1% (221/283) of the breast cancer tissues, respectively. As shown in Table 2, the β-catenin expression in membrane staining was correlated with histological grade (p = 0.005) and lymph node (LN) involvement (p = 0.018) in breast cancer patients. Likewise, the expression of β-catenin in the nucleus and cytoplasm was related to histological grade (p < 0.0001) and LN involvement (p = 0.010) in breast cancer patients.

There were different expression forms of AJAP1 and β-catenin observed in Fig. 2b–e, thus the correlation between the expressions of β-catenin and AJAP1 was analyzed. Table 3 showed that the low expression of AJAP1 was linked to abnormal membrane expression (p < 0.0001) and cytoplasmic/nuclear staining of β-catenin (p = 0.001). Moreover, the membrane staining of β-catenin had a significant difference with the cytoplasmic/nuclear expression (Table 4, p < 0.001).

As shown in Fig. 3a, abnormal membrane expression, cytoplasm and nuclear staining of β-catenin characterized a poor prognosis, while univariate and multivariate analyses showed that the location of β-catenin was not an independent prognostic factor for OS (Additional file 3: Table S3).

Many studies hold different perspectives on the prognosis value of β-catenin. The membrane expression of β-catenin seemed to predict OS better in the I/II histological grade (p = 0.0039) and no LN involvement (p = 0.0394) as shown in Fig. 3b. However, the cytoplasmic or nuclear expression of β-catenin did not show a significant difference in the different risks of subgroups in breast cancer patients (Fig. 3c).

Patients with a low expression of AJAP1 predicted poor OS in all the subgroups except the patients without involvement of LN (Fig. 3d, p = 0.8234). In summary, these results indicated that AJAP1 expression could be a prognostic marker in the different risks of subgroups of breast cancer patients, while β-catenin didn’t have the same function.

To further explore how AJAP1 mediated β-catenin, the relationship and potential mechanism between AJAP1 and the different locations of β-catenin in breast cancer were investigated. Firstly, it was found that AJAP1 and β-catenin interacted in T47D and MDA-MB-231 cells (Fig. 4a). Co-IP assay was also conducted to confirm this theory (Fig. 4b). Loss of AJAP1 reduced the β-catenin cytoplasm expression but increased its nuclear location in T47D. In contrast, upregulated AJAP1 in MDA-MB-231 cells showed opposite results (Fig. 4c). To determine the exact path of β-catenin degradation mediated by AJAP1, the ubiquitination level of β-catenin in AJAP1-deficient T47D cells was examined. As shown in Fig. 4d, the ubiquitination of β-catenin was reduced in the AJAP1 knockdown cells, demonstrating that AJAP1-depletion activated Wnt signaling which caused β-catenin degradation.

Above experiments showed that the knockdown of AJAP1 influenced the location of β-catenin, however it is unclear whether the transcriptional activity of β-catenin was also changed. TCF/LEF-responsive luciferase (TOP-FlASH) and non-responsive (FOP-FLASH) reporters were used to evaluate the effect of AJAP1 on the β-catenin transcriptional activity. As shown in Fig. 4e, upregulation of AJAP1 led to a lower transcription activity from the β-catenin/TCF/LEF-dependent promoter (TOP-FLASH) but had little effect on FOP-FLASH. Conversely, downregulation of AJAP1 obviously increased the transcription activity of the β-catenin/TCF/LEF-dependent promoter (Fig. 4f). To further examine the influence of knockdown or overexpression of AJAP1 on β-catenin’s downstream gene expression, the expressions of C-myc and CyclinD1 were analyzed by Western blotting and qRT-PCR. Overexpression of AJAP1 in MDA-MB-231 and T47D cells decreased C-myc and CyclinD1 expression both in protein and mRNA levels (Fig. 4g and h). On the contrary, knockdown of AJAP1 showed the opposite results (Fig. 4i and j). These findings showed that AJAP1 depletion increased β-catenin nuclear expression, activated the transcriptional activity of β-catenin and promoted its expressions of downstream genes (C-myc and CyclinD1).

The above results indicated that AJAP1 inhibited cell invasion, proliferation and migration ability in breast cancer. Then we explored whether the function of AJAP1 in breast cancer progression relied on mediation of β-catenin expression. For this purpose, three groups of T47D cells were constructed: depletion of AJAP1 (AJAP1 KD), AJAP1 knockdown and β-catenin knockdown (AJAP1/β-catenin KD), and the control. Figure 5a shows the different protein levels of AJAP1 and β-catenin in the three groups. Transwell assay and wound healing were used to examine the migratory and invasion abilities of these cells. As shown in Fig. 5b and Fig. 5d, AJAP1/β-catenin KD group suppressed the migratory and invasion abilities in comparison with the AJAP1 KD and control groups. Colony formation and MTT assays were performed on the three groups and the results showed that the proliferation ability of the AJAP1/β-catenin KD group was also lower than AJAP1 KD and control groups (Fig. 5c and Fig. 5e). The cell cycle results demonstrated that the AJAP1/β-catenin KD group had a higher number of G0/G1 cells but a lower number of S/G2/M cells than the other two groups (Fig. 5f). This phenomenon indicated that inhibited β-catenin expression reversed the effect caused by the depletion of AJAP1.To better analyze the significance of β-catenin in the tumorigenicity of AJAP1-mediated tumor progression, stable T47D cells were successfully established in the AJAP1 KD, AJAP1/β-catenin KD, and control groups. The AJAP1/β-catenin KD group had the lowest tumor capacity and tumor growth rate among the three groups (Fig. 5g).

The expression levels of ki67, C-myc, CyclinD1, lung and liver metastasis situation were evaluated in the mouse xenografts (Fig. 5h and i). It is clear that AJAP1/β-catenin KD group shows the lower expression of Ki67, less metastasis in lung and liver compared with the other two groups. This group also shows the lowest C-myc and CyclinD1 expressions (Fig. 5h and i). Collectively, AJAP1 inhibited breast cancer growth by regulating β-catenin transcriptional activity through suppressing C-myc and CyclinD1 expression both in vivo and in vitro.

The above experiments verified β-catenin as the downstream target of AJAP1 in controlling breast cancer tumorigenesis and metastasis. However, it is necessary to further investigate which molecule controls the AJAP1 expression or mediate the conjunction between AJAP1 and β-catenin. Using the String online prediction software and the reported literature (Additional file 4: Figure S1), it was presumed that EGFR or EGF could participate in the AJAP1-β-catenin axis modulation process. To verify this assumption, EGF was added to T47D and MDA-MB-231 cells to observe the locational changes of β-catenin and AJAP1 through immunofluorescence assay. Consistently, Fig. 6a-c showed that EGF led the β-catenin expression transfer from the membrane/cytoplasm to the nucleus. Besides, the expression of AJAP1 was reduced but didn’t show locational changes. Western blot validated the same results in the MDA-MB-231 and T47D cell lines (Fig. 6d). In the subsequent co-IP assay, Fig. 6e presented that interaction between AJAP1 and β-catenin was obviously deceased by EGF. However, the total β-catenin of the cells did not change significantly. These experiments confirmed that β-catenin transferred from the cytoplasm to the nucleus. Subsequently, transcriptional activity of β-catenin and its downstream genes, such as C-myc and CyclinD1, were explored via luciferase assay and qRT-PCR. Figure 6f shows that the overexpression of AJAP1 with EGF reduced the relative TOP/FOP activity and the C-myc 、CyclinD1 mRNA levels in comparison with the group treated EGF alone. Taken together, EGF promoted the transfer of β-catenin from the membrane/cytoplasm to the nucleus and activated its transcriptional activity by inhibiting the expression of AJAP1.

EGF is considered as a ligand part of EGFR, and previous studies have demonstrated that its activation could stabilize and enhance the β-catenin nuclear accumulation in cancer [32‐34]. Thus, investigations were carried out on whether EGFR had the same function as EGF and whether it participated in the process of AJAP1-mediated β-catenin expression. Cells without EGF stimulation were used to observe above changes. As shown in the first and second panels of Fig. 6g, EGFR depletion obviously inhibited the expression of AJAP1, reduced the expression of β-catenin and its downstream genes (C-myc and CyclinD1). However, knockdown AJAP1 and EGFR group also enhanced these effects and increased the relative β-catenin TOP/FOP activity.

Overall, these results showed that EGFR and EGF could inhibit the expression of AJAP1 and promot the β-catenin nuclear localization in the breast cancer cell lines.

Numerous reports showed that overexpression of EGFR decreased OS and DFS in women with early breast cancer [35, 36]. Therefore, it also seemed to be a major driving factor of malignancy in breast cancer [37‐39]. Experiments were carried out to study whether AJAP1 had the same effect on EGFR in vivo. As shown in Fig. 6h, AJAP1/EGFR KD group improved the tumor growth and weight, β-catenin nuclear expression and the expressions of C-myc and CyclinD1 compared with the other two groups isolated from the breast cancer cell-inoculated nude mice.

Figure 6i showed that AJAP1 could interact with β-catenin and the loss of AJAP1 could promote breast cancer progression and metastasis by stimulating β-catenin nuclear translocation and TCF/LEF1 transcriptional activity, increasing C-myc and CyclinD1 expression. Furthermore, EGF/EGFR could inhibit the expression of AJAP1 and negatively control the location and the activity of β-catenin. EGF reduced the AJAP1–β-catenin complex joint efficiency by reducing AJAP1 expression and dissociating β-catenin from the junctions. Further, dissociative β-catenin could accumulate in the cytoplasm and then move to the nucleus, which aggravates the breast cancer malignancy. Collectively, EGF/EGFR/AJAP1 axis controled breast cancer tumor progression and metastasis by mediating the β-catenin nuclear transactivation.