HER-2 is overexpressed in 20–30% of BC, indicating poor prognosis. RANKL/RANK pathway is implicated in HER-2(+) BC. The molecular mechanism and the potential dual targeting have been evaluated and showed promising results.
We used SKBR3, BT474, MCF7, MDA-MB-453 human BC cell lines. We examined the expression of RANK and RANKL using RT-PCR, Western-Blot and immunofluorescence. The interaction between RANK and HER-2 was detected using Proximity Ligation Assay (PLA) which enables the visualization of interacting proteins. NF-κB pathway activation was studied using Western-Blot. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. Cell viability and cell migration was evaluated using XTT and scratch assay, respectively.
Cell lines differentially express RANK and RANKL. RANK receptor dimerizes with HER-2. RANK/HER-2 dimer number depends on HER-2 expression (SKBR3: 5.4, BT474: 8.2, MCF7: 0.7, MDA-MB-453: 0.3). RANK/HER-2 dimers are decreased in the presence of the inhibitors D, T and P, while they are 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 BT474 (m:8.2, D:3.1, T:4.3, DT:0.7, TP:3.4, DTP:3.2, R:11.6). MCF7 and MDA-MB-453 do not correspond to inhibitors. The combination treatment of SKBR3 with the inhibitors further decreased NF-κB pathway activation compared to single targeting. SKBR3, after inhibitors treatment, also exhibit reduced cell proliferation (m:100%, D:84.7%, T:51.57%, DT:48.61%, R:111%) and lower metastatic potential (m:26.02%, D:33.56%, T:16.54%, DT:13.75%, R:32.36%) compared to mock cells (m) and increased values in RANKL presence. The combination treatment of SKBR3 with D and T has an advantage in functional proliferation assays compared to single targeting.
The results indicate a novel association between HER-2 and RANKL/RANK pathway that affects HER-2(+) BC growth. We also present data suggesting that the combination of HER-2 targeting and RANKL inhibition has therapeutic benefit and should be further tested for certain BC patients.
Michalis V. Karamouzis.
Has not received any funding.
All authors have declared no conflicts of interest.