![]() ![]() BC is a highly heterogeneous disease, which has been classified into distinct subtypes according to clinicopathologic features and molecular profile. 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.Īccording to global statistics, breast cancer (BC) is the most common cancer in the female population. ![]() The results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits 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. Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. 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). 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 receptor dimerizes with ERBB family members. RANK immunostaining was also detected in human BC tissue samples. ResultsĬell lines express RANK and RANKL. For cell migration evaluation, scratch assay was performed. ![]() Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. NF-κB pathway activation was studied using Western blot. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. We used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. 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. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. Statistically significant differences (p<0.05) are indicated by asterisks.ERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The PLA signals (in red) were counted with the Duolink ImageTool software and the average number of spots in the nucleus per cell (200 cells per sample were counted) is presented in the graph. Control and MADA fibroblasts left untreated or treated with rapamycin, were labeled with anti-Oct-1 and anti-prelamin A (SC - 6214) antibodies and probed with Duolink (Sigma) detection reagents according to the manufacturer. ( C) Proximity Ligation Assay (PLA) between prelamin A and Oct-1. The percentage of nuclei with Oct-1 mislocalized in nuclear clusters is reported in the graph as mean values +/− standard deviation. The intensity of fluorescence is represented on a pseudocolor scale (palette bar). The pseudo-coloring of Oct-1 pictures obtained by using the Photoshop 7 color mapping function reveals the accumulation of Oct-1 in nuclear foci and at the periphery and recovery by rapamycin treatment in MADA. Arrows indicate Oct-1 nuclear foci, arrowheads indicate recovered Oct-1 localization in the nucleoplasm. ( B) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for Oct-1. Levels of LAP2alpha fluorescence intensity and the percentage of nuclei with LAP2alpha mislocalized in nuclear clusters are reported in the upper and lower graph respectively as mean values +/− standard deviation. ( A) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for LAP2alpha. LAP2alpha and Oct-1 localization are affected in MADA and rescued by rapamycin.
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