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Cited by 103Alzheimer's disease research and treatmentsCancer Cells and MetastasisMechanisms of cancer metastasis

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Amyloid-β precursor protein promotes cell proliferation and motility of advanced breast cancer

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Lim, Seunghwan, Yoo, Byoung Kwon, Kim, Hae-Suk, Gilmore, Hannah L, Lee, Yonghun, Lee, Hyun-pil, Kim, Seong-Jin, Letterio, John, Lee, Hyoung-gon·2014·BMC Cancer
DOI 10.1186/1471-2407-14-928PMC PMC4295427
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Abstract

BACKGROUND: 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. METHODS: 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. RESULTS: 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 p27(kip1) 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. CONCLUSION: 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.

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Figure 1 The elevated expression of APP engaged in breast cancer cell proliferation. (A) APP expression is detected by 22C11 mouse monoclonal anti-APP antibody in human breast cancer cell lines and correlates with increasing malignancy. (+); a positive control of APP protein overexpressed in neuronal cells. (B) The expression of APP is compared in mouse breast cancer cells with increasing metastatic potential. (C) APP protein expression was present at a similar level in both M-IV and MDA-MB-231. Knock down of APP expression was verified in RT-PCR following lentiviral infection encoding shAPP in MDA-MB-231. APP knockdown resulted in decreased expression of APP and soluble APP. The equal volume of conditioned media was condensed by using Centricon and analyzed in Western blot. For the loading control, β-actin was uesd. (D) Cells (2x103) were seeded in 6-well plate and cell numbers counted using coulter counter at day 2 and 4. (E) MDA-MB-231 cells were seeded at two different numbers and the cell growth was compared by MTT assay. (F) MDA-MB-231 cells fixed and stained with propidium iodide (PI) were subjected to cell cycle analysis by FACS.
Figure 1 The elevated expression of APP engaged in breast cancer cell proliferation. (A) APP expression is detected by 22C11 mouse monoclonal anti-APP antibody in human breast cancer cell lines and correlates with increasing malignancy. (+); a positive control of APP protein overexpressed in neuronal cells. (B) The expression of APP is compared in mouse breast cancer cells with increasing metastatic potential. (C) APP protein expression was present at a similar level in both M-IV and MDA-MB-231. Knock down of APP expression was verified in RT-PCR following lentiviral infection encoding shAPP in MDA-MB-231. APP knockdown resulted in decreased expression of APP and soluble APP. The equal volume of conditioned media was condensed by using Centricon and analyzed in Western blot. For the loading control, β-actin was uesd. (D) Cells (2x103) were seeded in 6-well plate and cell numbers counted using coulter counter at day 2 and 4. (E) MDA-MB-231 cells were seeded at two different numbers and the cell growth was compared by MTT assay. (F) MDA-MB-231 cells fixed and stained with propidium iodide (PI) were subjected to cell cycle analysis by FACS.CC BY · PubMed Central
Figure 2 APP involved in the induction of cell cycle inhibitor p27 kip1 in breast cancer cells. (A) Knock-down of APP in MDA-MB-231 cells using two different shRNA constructs of APP (shAPP-5 and shAPP-7) resulted in marked suppression of both cellular and soluble form of APP expression. The p27kip1 expression was elevated in shAPP-5 and shAPP-7 cells. (B) The p27kip1 and p21cip1 expression was evaluated in M-I and M-IV after introduction of shluc, shAPP-5, or shAPP-7. (C) The control and shAPP-7 cells were incubated in serum deprived medium for 3 hours and then released with 10% serum for the indicated time points. The cells were harvested and subjected to assessment of p27kip1 and p21cip1 expression. (D) The cells incubated in serum-free medium for 18 hours were treated with 10% serum for 60 minutes and then the images were acquired to show subcellular localization of p27kip1. The nuclear localized p27kip1 was confirmed by merging with DAPI images. The longer image acquisition was needed to detect p27kip1 in the control (shluc) cells due to the low expression of p27kip1. Scale bar = 20 μm.
Figure 2 APP involved in the induction of cell cycle inhibitor p27 kip1 in breast cancer cells. (A) Knock-down of APP in MDA-MB-231 cells using two different shRNA constructs of APP (shAPP-5 and shAPP-7) resulted in marked suppression of both cellular and soluble form of APP expression. The p27kip1 expression was elevated in shAPP-5 and shAPP-7 cells. (B) The p27kip1 and p21cip1 expression was evaluated in M-I and M-IV after introduction of shluc, shAPP-5, or shAPP-7. (C) The control and shAPP-7 cells were incubated in serum deprived medium for 3 hours and then released with 10% serum for the indicated time points. The cells were harvested and subjected to assessment of p27kip1 and p21cip1 expression. (D) The cells incubated in serum-free medium for 18 hours were treated with 10% serum for 60 minutes and then the images were acquired to show subcellular localization of p27kip1. The nuclear localized p27kip1 was confirmed by merging with DAPI images. The longer image acquisition was needed to detect p27kip1 in the control (shluc) cells due to the low expression of p27kip1. Scale bar = 20 μm.CC BY · PubMed Central
Figure 3 Reduction of APP expression is associated with the apoptotic induction in breast cancer cells. (A) A series of MCF-10A cells were infected with lentivirus encoding control (shluc) or APP shRNA (shAPP-7) and then tested for APP expression by immunoblotting. Under this condition, alteration of apoptotic indicators such as cleaved PARP and cleaved Caspase-3 were compared. (B) MDA-MB-231 cells were infected with lentivirus encoding shluc, shAPP-5, or shAPP-7. Each cell line was treated with TRAIL (10 ng/ml) or 5-FU (200 μM) for 24 hours. (C, D) The on-going early apoptotic events were compared by staining for extracellular Annexin V and cell viability with propidium iodide (PI). The apoptotic cell populations with Annexin V high and PI low were indicated as percentage.
Figure 3 Reduction of APP expression is associated with the apoptotic induction in breast cancer cells. (A) A series of MCF-10A cells were infected with lentivirus encoding control (shluc) or APP shRNA (shAPP-7) and then tested for APP expression by immunoblotting. Under this condition, alteration of apoptotic indicators such as cleaved PARP and cleaved Caspase-3 were compared. (B) MDA-MB-231 cells were infected with lentivirus encoding shluc, shAPP-5, or shAPP-7. Each cell line was treated with TRAIL (10 ng/ml) or 5-FU (200 μM) for 24 hours. (C, D) The on-going early apoptotic events were compared by staining for extracellular Annexin V and cell viability with propidium iodide (PI). The apoptotic cell populations with Annexin V high and PI low were indicated as percentage.CC BY · PubMed Central
Figure 4 APP modulates breast cancer cell growth in 3D culture and in xenografted model. MDA-MB-231 cells were subjected to 3D Matrigel on-top assay. The cells were seeded (2x104/well) in 48-well plate coated with Matrigel in triplicate and then cultured for 7 days with medium change in every two days. The morphology of growing cells were obtained (A) and followed by MTT assay (B). (C) The control and shAPP-7 MDA-MB-231 (2x106) cells were injected into nude mice s.c. (n = 6) and allowed to grow for 6 weeks. The grown tumors were excised and the grown tumor size compared. (Scale bar = 1cm) (D) The independent xenograft study (2.5x105 cells s.c injected; n = 5, respectively) revealed that shAPP-7 MDA-MB-231 cell growth rate was largely decreased as compared to control group (p < 0.01).
Figure 4 APP modulates breast cancer cell growth in 3D culture and in xenografted model. MDA-MB-231 cells were subjected to 3D Matrigel on-top assay. The cells were seeded (2x104/well) in 48-well plate coated with Matrigel in triplicate and then cultured for 7 days with medium change in every two days. The morphology of growing cells were obtained (A) and followed by MTT assay (B). (C) The control and shAPP-7 MDA-MB-231 (2x106) cells were injected into nude mice s.c. (n = 6) and allowed to grow for 6 weeks. The grown tumors were excised and the grown tumor size compared. (Scale bar = 1cm) (D) The independent xenograft study (2.5x105 cells s.c injected; n = 5, respectively) revealed that shAPP-7 MDA-MB-231 cell growth rate was largely decreased as compared to control group (p < 0.01).CC BY · PubMed Central
Figure 5 APP significantly impacts IGF-1-mediated activation of AKT and its downstream effectors. Both MDA-MB-231 control (shluc) and APP-kd (shAPP) cells were treated with EGF (50 ng/ml), LPS (100 ng/ml), or IGF-1 (100 ng/ml) as indicated. (A) EGF-mediated Erk activation was assessed in the APP knock-down cells post stimulation with EGF. (B) LPS-mediated activation of pro-inflammatory response in the APP knockdown cells was tested by demonstrating the level of IκBα expression and NF-κB activation (phosphorylated p65 at S536). (C) IGF-1-stimulated Akt activation and phosphorylation of Akt target proteins such as GSK3β (S9) and FOXO1 (T24) were examined. (D) APP affects the expression of β-Catenin, a target of GSK3β, and its downstream targets such as Survivin and CD44, but not Cyclin D1.
Figure 5 APP significantly impacts IGF-1-mediated activation of AKT and its downstream effectors. Both MDA-MB-231 control (shluc) and APP-kd (shAPP) cells were treated with EGF (50 ng/ml), LPS (100 ng/ml), or IGF-1 (100 ng/ml) as indicated. (A) EGF-mediated Erk activation was assessed in the APP knock-down cells post stimulation with EGF. (B) LPS-mediated activation of pro-inflammatory response in the APP knockdown cells was tested by demonstrating the level of IκBα expression and NF-κB activation (phosphorylated p65 at S536). (C) IGF-1-stimulated Akt activation and phosphorylation of Akt target proteins such as GSK3β (S9) and FOXO1 (T24) were examined. (D) APP affects the expression of β-Catenin, a target of GSK3β, and its downstream targets such as Survivin and CD44, but not Cyclin D1.CC BY · PubMed Central
Figure 6 APP promotes cell migration of MDA-MB-231 and its expression is elevated in invasive breast cancer of human tissues. (A) The cell motility of APP knockdown (shAPP) MDA-MB-231 was examined in wound healing assay. Following the wounding, cells were untreated or treated with IGF-1 (25 ng/ml) for 18 hours in 0.1% serum containing medium. Cells were then fixed and stained for clear demonstration (scale bar = 200 μm). (B) The role of APP for cell migration was evaluated in Boyden chamber assay in serum-free medium with or without IGF-1 (50 ng/ml) for 18 hours. The rectangular area was further magnified for demonstration of different cell morphology. (C) The migrated cells in panel B were counted in three randomly selected areas. (D) No staining for APP (22C11) is present in this normal terminal duct lobular unit. (E) The well-differentiated grade 1 invasive ductal carcinoma shows weak staining for APP. (F) The poorly-differentiated grade 3 invasive ductal carcinoma shows strong staining for APP. Scale bar = 100 μm.
Figure 6 APP promotes cell migration of MDA-MB-231 and its expression is elevated in invasive breast cancer of human tissues. (A) The cell motility of APP knockdown (shAPP) MDA-MB-231 was examined in wound healing assay. Following the wounding, cells were untreated or treated with IGF-1 (25 ng/ml) for 18 hours in 0.1% serum containing medium. Cells were then fixed and stained for clear demonstration (scale bar = 200 μm). (B) The role of APP for cell migration was evaluated in Boyden chamber assay in serum-free medium with or without IGF-1 (50 ng/ml) for 18 hours. The rectangular area was further magnified for demonstration of different cell morphology. (C) The migrated cells in panel B were counted in three randomly selected areas. (D) No staining for APP (22C11) is present in this normal terminal duct lobular unit. (E) The well-differentiated grade 1 invasive ductal carcinoma shows weak staining for APP. (F) The poorly-differentiated grade 3 invasive ductal carcinoma shows strong staining for APP. Scale bar = 100 μm.CC BY · PubMed Central

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Metadata

Authors
Lim, S., Yoo, B. K., Kim, H.-S., Gilmore, H. L., Lee, Y., Lee, H.-P., Kim, S.-J., Letterio, J., & Lee, H.-G.
Affiliations
  • Case Comprehensive Cancer Center (US)
  • Virginia Commonwealth University (US)
  • Case Western Reserve University (US)
  • CHA University (KR)
  • Rainbow Babies & Children's Hospital (US)
Corresponding author
  • Lim, Seunghwan
  • Lee, Hyun-pil
  • Lee, Hyoung-gon
ORCID
Venue
BMC Cancer
Publisher
Springer Science and Business Media LLC
Published
2014-12
Volume
14
Issue
1
Pages
928-928
ISSN
1471-2407 (ISSN-L)
DOI
10.1186/1471-2407-14-928
PMC
PMC4295427
OpenAlex
W2086492959
Cited by
100
References
54
Citations by year
2026: 10 · 2025: 8 · 2024: 9 · 2023: 8 · 2022: 9 · 2021: 9 · 2020: 13 · 2019: 11 · 2018: 10 · 2017: 6 · 2016: 6 · 2015: 1
FWCI
2.89
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Cancer (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (AG028679)
  • National Institute on Aging (AG028679, R01AG028679)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mechanisms of cancer metastasis
Keywords
amyloid precursor protein, breast cancer, metastasis, cell proliferation, cell migration and invasion, apoptosis, p27, caspase-3, Akt signaling, insulin-like growth factor I, GSK3β, FOXO1, chemosensitivity, TRAIL, 5-fluorouracil, therapeutic target
MeSH terms
Animals, Breast Neoplasms, Cell Movement, Cell Survival, Disease Models, Animal, Female, Humans, Insulin-Like Growth Factor I, Neoplasm Staging, Gene Expression, Amyloid beta-Protein Precursor, Apoptosis, Xenograft Model Antitumor Assays, Cell Line, Tumor, Cell Proliferation, Cyclin-Dependent Kinase Inhibitor p27, Proto-Oncogene Proteins c-akt, Mice, Gene Knockdown Techniques

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