Amyloid-β precursor protein promotes cell proliferation and motility of advanced breast cancer
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Amyloid-β precursor protein (APP) is a highly conserved single transmembrane protein that has been linked to Alzheimer disease. Recently, the increased expression of APP in multiple types of cancers has been reported where it has significant correlation with the cancer cell proliferation. However, the function of APP in the pathogenesis of breast cancer has not previously been determined. In this study, we studied the pathological role of APP in breast cancer and revealed its potential mechanism.
The expression level of APP in multiple breast cancer cell lines was measured by Western blot analysis and the breast cancer tissue microarray was utilized to analyze the expression pattern of APP in human patient specimens. To interrogate the functional role of APP in cell growth and apoptosis, the effect of APP knockdown in MDA-MB-231 cells were analyzed. Specifically, multiple signal transduction pathways and functional alterations linked to cell survival and motility were examined in in vivo animal model as well as in vitro cell culture with the manipulation of APP expression.
We found that the expression of APP is increased in mouse and human breast cancer cell lines, especially in the cell line possessing higher metastatic potential. Moreover, the analysis of human breast cancer tissues revealed a significant correlation between the level of APP and tumor development. Knockdown of APP (APP-kd) in breast cancer cells caused the retardation of cell growth in vitro and in vivo, with both the induction of p27kip1 and caspase-3-mediated apoptosis. APP-kd cells also had higher sensitivity to treatment of chemotherapeutic agents, TRAIL and 5-FU. Such anti-tumorigenic effects shown in the APP-kd cells partially came from reduced pro-survival AKT activation in response to IGF-1, leading to activation of key signaling regulators for cell growth, survival, and pro-apoptotic events such as GSK3-β and FOXO1. Notably, knock-down of APP in metastatic breast cancer cells limited cell migration and invasion ability upon stimulation of IGF-1.
The present data strongly suggest that the increase of APP expression is causally linked to tumorigenicity as well as invasion of aggressive breast cancer and, therefore, the targeting of APP may be an effective therapy for breast cancer.
KeywordsAKT Amyloid-β precursor protein Apoptosis Breast cancer Invasion p27kip1
Amyloid-β precursor protein
Insulin-like growth factor-1
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand
Amyloid-β precursor protein (APP) is a highly conserved single transmembrane protein with a receptor-like structure and has been linked with Alzheimer disease [1, 2] while its normal physiological function is unclear. Several APP isoforms derived from alternative splicing processes have been reported and diverse products including soluble APP (sAPP) or abnormal amyloid-β peptide through α-, β-, or γ-secretase-mediated cleavage(s) are post-translationally generated [3, 4]. APP is ubiquitously expressed in a broad spectrum of cell types including non-neuronal cells, while the nature of APP has been mainly studied in neuronal cells due to its pathological significance in Alzheimer disease. Several pathophysiological functions of APP have been proposed such as its potential role in cell growth and cell adherence [5, 6, 7]. It has been demonstrated that APP is engaged in neuronal growth cone adhesion and plays a role as an independently operating cell adhesion molecule for binding to extracellular matrices such as laminin . Specifically, it has been reported that APP is linked to proliferation of thyroid epithelial cells and epidermal basal cell proliferation [8, 9, 10, 11] and, interestingly, the increased expression of APP in several types of cancers including pancreatic, lung, colon and breast cancer has been reported [10, 11, 12, 13, 14, 15]. These studies suggested that APP has growth-promoting effect as an autocrine growth factor while the underlying mechanism in the regulation of cellular signaling and gene expression has not been fully explored. The potential role of APP in cancer cell motility is also supported by studies which show APP plays a role in migration of neuronal precursor cells and neurite outgrowth [16, 17].
In this study, we explored the pathological role of APP in malignancy of breast cancer and its potential molecular mechanism related with cell proliferation and metastasis. Breast cancer is the most common cancer diagnosed among women worldwide  and metastatic breast cancer is significantly correlated with poor prognosis and a main cause of death while the underlying molecular pathogenic mechanism still remains to be delineated. We found that the expression level of APP is mechanistically linked with tumorigenicity and malignancy of breast cancer. APP knockdown (APP-kd) in breast cancer cells reduced cell growth via p27kip1 induction, promoting apoptosis, increasing sensitivity to therapeutic treatments, and delayed cell migration and invasion ability upon stimulation. These results suggest that targeting APP may effectively suppress the growth and invasion of malignant breast cancer cells.
Cell culture and reagents
MDA-MB-231 cells were grown in DMEM, and 67NR, 4T07, and 4T1 breast cancer cell lines were grown in RPMI supplemented with 10% (vol/vol) FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml; Invitrogen, Rockville, MD). The four human breast cancer cell lines MCF10A1 (M-I), MCF10AT1k.cl2 (M-II), MCF10CA1h (M-III), and MCF10CA1a.cl1 (M-IV) were obtained from Dr. Anita Roberts (NCI/NIH, Bethesda, MD). M-I, M-II, M-III, and M-IV cells were grown in DMEM/F12 (Invitrogen, Carlsbad, CA) containing 5% horse serum (Invitrogen) at 37°C with 5% CO2. M-I and M-II cells were supplemented additionally with 10 μg/ml insulin (Sigma, St. Louis, MO), 20 ng/ml epidermal growth factor (Sigma), 0.5 μg/ml hydrocortisone (Sigma), and 100 ng/ml cholera toxin (Sigma). Antibodies specific for APP (22C11) were purchased from EMD Millipore; APP (4G8) from Covance. Specific antibodies for p27(C-19) and p21 (F-5) were from Santacruz and anti-β-actin (AC-15) was from Sigma. Antibodies purchased from Cell Signaling were AKT (#9772), pAKT Thr308 (#4056), pAKT Ser473 (#9271), pFOXO1 Thr24 (#9464), pGSK3 Ser9 (#9336), pp65 Ser536 (#3033), pERK1/2 (#9101), β-Catenin (#9562), PARP (#9542), and cleaved Caspase-3 (#9661). Anti-survivin antibody (AB8228) was purchased from Abcam. The anti-CD44 antibody (#15675-1-AP) was from Proteintech group and anti-GSK3b (KAP-ST002E) antibody was from Stressgen.
Knockdown of human APP using lentiviral infection system
Knockdown of human endogenous APP gene expression was carried out using the lentivirus shRNA expression system and experimental method as previously described . The target sequence of human APP (shAPP-5: 5’-CCCTGTTCATTGTAAGCACTT, shAPP-7: 5’-GCAGACACAGACTATGCAGAT) or control luciferase was used. In order to produce viral particles, the shRNA constructs and virus packaging plasmids were transfected into fresh 293T cells and then harvested the viral supernatant and filtered through 0.45 μm syringe filter prior to infection. Target cells were infected with virus by spinning at 2000 rpm for 30 min. Semi-quantitative RT-PCR and immunoblotting were carried out to measure knock-down efficiency.
Western blotting and RT-PCR
The cells were harvested and lysed in RIPA buffer. Equal amounts of protein were loaded and separated in SDS-PAGE gel and then transferred to PVDF membrane. The blot was incubated in blocking solution (5% milk/TBST) and then incubated with primary antibody followed by incubation with secondary HRP conjugated antibody for 1 or 2 hours. The blot was washed 3 times for 5 minutes with TBST between the incubations. Eventually, the change of target protein expression was detected by conducting reaction with Chemiluminescent Substrate (Thermo Scientific), exposing, and developing the film. RT-PCR for measuring the level of APP mRNA expression was performed with the primers specific to human APP .
Detection of apoptotic cell population
MDA-MB-231 cells (5×104) freshly infected with shLuc, shAPP-5, or shAPP-7 lentiviral particles were immediately seeded in 6-well plates. In order to detect early apoptotic events, we employed Annexin V staining method (eBioscience) which can detect phosphatidylserine on the outer plasma membrane upon initiation of apoptosis. Cell viability staining was carried out using propidium iodide (PI) to identify early-stage apoptotic cells. The FACS analysis was immediately followed after staining the cells.
Cell growth assay
The control and APP-kd of MDA-MB-231cells (2×103) were seeded in 6-well plate in triplicate and maintained in normal growth medium. The sub-confluently growing cells were counted using coulter counter (Beckman) at day 2 and 4.
Wound-healing assay and cell invasion assay
To compare the cell motility, the MDA-MB-231 control (shluc) or APP knockdown (shAPP-7) MDA-MB-231 cells were examined in wound healing assay. The confluently grown cells were wounded with 200 μl tips and followed by either no treatment or treated with IGF-1 (25 ng/ml) for 18 hours in 0.1% serum containing medium. Subsequently, cells were fixed with 2% paraformaldehyde and then stained with rapid 3 step staining set (Richard-Allen Scientific) for clear visualization of migrated cells. The initial wounded edges were marked with dotted lines. Representative results from at least three independent experiments are shown. Cell invasion assays were performed by seeding cells in Boyden chamber (BD Bioscience) coated with matrigel in serum-free medium with or without IGF-1 (50 ng/ml) in the bottom of each wells for 18 hours. The migrated cells were visualized by staining and photographing under the microscope.
Xenograft mouse model
The breast cancer cells were seeded freshly prior to injection. The control and shAPP MDA-MB-231 (1×106) cells were prepared in the solution (1:1) of PBS and growth factor-reduced matrigel and followed by injection into athymic nude mice subcutaneously. Primary tumor outgrowth was monitored every 4 days by taking measurements of the tumor length (L) and width (W). Tumor volume was calculated as πLW2/6 . The mice were maintained up to 6 weeks and sacrificed for tumor excision. The tumor growth was compared to the counterpart and imaged. All animal housing and procedures were performed in compliance with guidelines established by the Institutional Animal Care and Use Committee at Case Western Reserve University.
The breast cancer tissue array was purchased from US Biomax (Cat# BRC961). For immunohistochemistry for the APP detection, the tissue microarrays were hydrated through two changes of xylene and descending ethanol solutions for 10 min each, followed by a 30 min submersion in 3% H2O2 and finally Tris-buffered saline (TBS). The slides were incubated in 10% normal goat serum (NGS) in TBS for 30 min and the primary antibody was applied overnight. A monoclonal antibody specific to APP, 22C11 (recognizing the N-terminal domain of full length amyloid-β precursor protein; EMD Millipore, 1:250), was applied to the microarrays and then the peroxidase-anti-peroxidase technique was employed and developed with 3′-3’-diaminobenzidine (Dako).
Data are presented as means ± standard deviation. Differences between the experimental groups were compared with Student’s paired two tailed t-test. A p-value less than 0.05 was considered statistically significant.
The level of APP expression is linked to malignancy of breast cancer cells
Reduction of the expression of APP prevents cell growth in MDA-MB-231 cells
We investigated the pathophysiological function of APP by knocking it down using the shRNA targeting APP in MDA-MB-231 malignant human breast cancer cells (Figure 1A). Both mRNA and protein expression of APP were markedly reduced in APP-kd cells compared to control cells (Figure 1C). APP protein expression of MDA-MB-231 was comparable to that of M-IV cells while MDA-MB-231, but not M-IV cells, showed fair amount of soluble APP secretion that is known to enhance cell growth and survival [25, 26]. Next, we examined cell proliferation in normal growth medium with 10% FBS in the control (shluc) and APP-kd (shAPP) cells. Consistent with our hypothesis, reduction of APP expression significantly affected cell proliferation and viability (Figure 1D,E). To confirm the effect of APP on cell growth further, we performed FACS analysis to determine cell cycle phase. The cell cycle analysis showed that APP-kd cells were arrested largely in G1 phase (45.2%) compared to control (31.4%), but low percentage of APP-kd cells (19.4%) was in S phase as compared to that of control cells (25.5%) (Figure 1F). Retarded cell growth and G1 arrest of APP-kd cells suggest that APP is likely engaged in expression of cell cycle inhibitors working on G1 phase such as p27kip1 and p21cip1[27, 28].
APP enhances cell proliferation via regulation of p27kip1
It has been established that p27kip1 has dual function as either a tumor suppressor or promoter because nuclear p27Kip1 works as an anti-proliferative protein, while cytoplasmic p27kip1 promotes cytoskeleton remodeling that is important for tumor cell motility and dissemination. In particular, subcellular location of p27Kip1 is significantly correlated with survival of breast cancer patients [29, 30]. In order to verify functional competency of p27kip1 as a cell cycle inhibitor, we analyzed cellular localization of p27kip1 with immunocytochemistry. A substantial amount of p27kip1 is still located in nuclear compartment of APP-kd cells even after one hour in serum-containing medium (Figure 2D). Conversely, in control cells, p27kip1 located in nuclei required much longer exposure time to be displayed owing to the substantial decrease of total protein with 10% serum stimulation, and potentially the redistribution of p27kip1 to cytoplasmic compartment. These results indicate that serum-sensitive signaling pathways regulating p27kip1 expression and cytoplasmic translocation were skewed by APP knockdown. These data also suggest that APP plays a crucial role for cell proliferation of malignant breast cancers by modulating the expression of cyclin-dependent kinase inhibitor, p27kip1.
APP modulates breast cancer cell survival
APP affects cell growth in 3D culture and in xenografted mouse model
APP is engaged in IGF1-induced AKT activation
APP reduction reduces cell motility in MDA-MB-231 cells
Increased expression of APP in human breast cancer tissues
In order to examine the clinical relevance of APP expression in breast cancer, a tissue microarray (TMA) containing various grades of breast cancer tissues and normal breast tissues was analyzed with an anti-APP antibody (22C11). In the normal breast tissues, there was minimal to no staining of the breast epithelium. However, the vast majority of the invasive breast carcinomas showed some degree of APP expression. In total, there were 40 invasive breast carcinomas that could be evaluated on the TMA sections stained with 22C11 antibody. No staining was observed in 3 (7.5%) of the cases. Weak staining was observed in 10 (25%) of the cases, moderate staining in 18 (45%), and strong staining in 9 (22.5%). Though the number of cases in this series is small, there was a trend seen where the higher grade tumors showed more intense staining than the lower grade tumors overall (Figure 6D-F). These results strongly support our hypothesis that elevated APP expression has close correlation with tumor cell growth and progression.
Our data strongly indicate the pathological role of APP in breast cancer. First, we demonstrated increased expression APP in breast cancer cells and its correlation with malignancy. Second, the inhibition of APP expression in breast cancer cells effectively prevents cell growth and motility in vitro and in vivo models. Third, we also demonstrated that APP is mechanistically linked to the AKT/FOXO and AKT/GSK3-β pathways which are known to modulate cell growth, survival, and invasion of breast cancer cells through the regulation of target genes including p27kip1 and survivin. Importantly, knocking down of APP expression resulted in retarded cell growth in vitro and in vivo xenografted mouse model. We found that the slower cell proliferation was, in part, caused by the upregulated cell cycle inhibitor p27kip1 expression in APP-kd cells. Thus, increased APP expression is inversely correlated with p27kip1 expression in malignant breast cancers. Since the reduced p27kip1 expression is correlated with tumor aggressiveness and poor patient survival , this finding suggests that APP plays a significant role in regulation of p27kip1 in a malignant human breast cancer. In addition, knockdown of APP in breast cancers augmented apoptotic markers and it is likely that advanced breast cancers (M-II, M-III, and M-IV) with knockdown of APP are more prone to enter into apoptosis. Similarly, in addition to the result of MCF-10A cells, APP knockdown in MDA-MB-231 promotes sensitivity to therapeutic treatments of TRAIL or 5-FU, implying that targeting APP in malignant breast cancers may promote the sensitivity to therapeutic drugs. Since homozygous APP-deficient mice are viable and normal in development , it seems that normal breast epithelial cell growth is not affected by knockdown of APP expression. However, advanced breast cancers may struggle to survive in the absence of APP, presumably because they have evolved to survive better, at least in part, in an APP-dependent manner. After the submission of this manuscript, Goodarzi et al.  published an article demonstrating the biological effect of APP in the regulation of breast cancer progression. Their results suggest that APP might suppress aggressiveness of breast cancer cells. While those results are not overlapped with the phenotype of our APP knockdown experiments, both reports strongly suggest the pathological role of APP in breast cancer pathogenesis. The discrepancy between two studies might be explained by different cellular conditions used in the studies. While they examined the role of APP under the condition of TARBP2 knockdown, our study examined a direct function of APP in the parental MDA-MB-231 cells without any other combinatorial genetic modifications. These results strongly suggest that the pathological role of APP in breast cancer pathogenesis works diversely upon the cellular context and this needs to be addressed in the future study.
Our data also suggest that APP is involved in IGF-1/AKT signaling pathways, which are key regulatory pathways for cell growth and survival of breast cancer. APP-kd cells displayed mitigated AKT activation which leads to decreased inhibitory phosphorylation of GSK3β (Ser9) and FOXO1 (T24). GSK3β is known to suppress β-catenin-dependent oncogenic signaling pathway by phosphorylating β-Catenin [44, 45]. Activation of β-catenin is reported in subgroup of triple negative breast cancers (i.e., aggressive breast cancers possessing lack of estrogen receptor, progesterone receptor, and Her2 receptor expression) and is associated with poor clinical outcomes . On the other hand, FOXO family including FOXO1 can induce cell cycle inhibitors (e.g., p27kip1, p21cip1) and pro-apoptotic molecules (e.g., BIM, BNIP3, FASL, TRAIL, and survivin) . The anti-apoptotic protein, survivin, is a family member of inhibitors of apoptosis (IAP) which embodies diverse cellular function, encompassing mitosis, metabolism, and survival by promoting adaptation to stresses . As such, FOXO-survivin and β-catenin-survivin regulatory pathways are considered to play an essential role for the expression of survivin in breast cancer [38, 44]. Thus, our results strongly suggest that APP-mediated regulation of AKT/FOXO and AKT/GSK3β pathways are playing a significant role for breast cancer development. Supporting this hypothesis, a previous study demonstrated that sAPPα stimulates AKT/GSK3β pathway in neuronal cells and consequently resulted in its neuroprotective effect .
Interestingly, APP is also known to promote cell migration in neuronal progenitor cells  and engage in neuronal growth cone adhesion where it plays a role as an independently operating cell adhesion molecule for binding to extracellular matrices such as laminin . Acquiring cell motility is a key aspect enabling cancer cells to invade into adjacent tissue and disseminate into the secondary organs. We therefore examined the cell motility and invasion ability of MDA-MB-231 after knocking down of APP expression. Upon stimulation with IGF-1 that promotes cell migration and cancer metastasis, APP-kd cells migrate slowly in response to IGF-1 partly due to limited activation of AKT. It is well known that AKT plays an important role in the process of EMT via repression of E-cadhrin . In addition, β-catenin is also closely engaged in EMT and cell migration [50, 51]. Our findings that APP is functionally linked with AKT activation and GSK-3β/β-catenin pathways warrant the future study that elevated APP in malignant breast cancers is associated with dissemination of breast cancer into other target organs by promoting EMT process.
In summary, we found that the expression of APP is increased both in mouse and human malignant breast cancer cell lines and similarly in human breast cancer tissues. The APP expression is important to regulate cell growth, apoptosis, and motility of breast cancer, possibly through engagement of AKT-mediated signaling pathways. Overall, our findings provide substantial groundwork for the pathophysiological function of APP and its underlying mechanism that promotes breast cancer malignancy.
We thank Sandra Siedlak for the technical assistance. This study was supported by the National Institutes of Health (AG028679) to HGL.
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