Περίληψη:
Background: ERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways. Methods: We used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-κB pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA. Results: Cell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors. Conclusions: The results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients. © 2019 The Author(s).
Συγγραφείς:
Zoi, I.
Karamouzis, M.V.
Xingi, E.
Sarantis, P.
Thomaidou, D.
Lembessis, P.
Theocharis, S.
Papavassiliou, A.G.
Λέξεις-κλειδιά:
denosumab; epidermal growth factor receptor 2; immunoglobulin enhancer binding protein; osteoclast differentiation factor; pertuzumab; receptor activator of nuclear factor kappa B; trastuzumab; epidermal growth factor receptor; immunological antineoplastic agent; osteoclast differentiation factor; receptor activator of nuclear factor kappa B; TNFSF11 protein, human, antineoplastic activity; antiproliferative activity; apoptosis; Article; BT-474 cell line; cancer growth; cancer patient; cancer tissue; cell growth; cell migration; cell specificity; cell viability; clonogenic assay; cohort analysis; controlled study; dimerization; drug potentiation; drug targeting; flow cytometry; human; human cell; human epidermal growth factor receptor 2 positive breast cancer; human tissue; immunofluorescence test; immunohistochemistry; in vitro study; MCF-7 cell line; MDA-MB-453 cell line; metastasis potential; protein analysis; protein expression; protein protein interaction; proximity ligation assay; reverse transcription polymerase chain reaction; signal transduction; SK-BR-3 cell line; theoretical model; Western blotting; wound healing assay; XTT assay; breast tumor; cell culture; drug effect; female; gene expression; genetics; metabolism; molecularly targeted therapy; pathology; tumor cell line, Antineoplastic Agents, Immunological; Breast Neoplasms; Cell Line, Tumor; Cells, Cultured; ErbB Receptors; Female; Gene Expression; Humans; Immunohistochemistry; Molecular Targeted Therapy; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction
