Inês Direito1, Liliana Monteiro1, Tânia Melo2, Daniela Figueira1, João Lobo3,4,5, Vera Enes1, Gabriela Moura1, Rui Henrique3,4,5, Manuel Santos1, Carmen Jerónimo3,4,5, Francisco Amado2, Margarida Fardilha1 and Luisa A. Helguero1
Protein quality control (PQC) network-autophagy, proteasome and the unfolded protein response (UPR)-is triggered by stress and is overactive in acquired antiestrogen (AE) therapy resistance. We hypothesized that AE therapy induces accumulation of aggresomes in sensitive cells hindering the activation of survival pathways. AE-sensitive (MCF-7 and T-47D) or resistant (MCF-7R and T-47DR) breast cancer (BC) cells were treated with AE for 24h. Insoluble proteins were analyzed by LC-MS/MS. RTCB expression was inhibited with siRNAs and its effects evaluated by cell counting, IF and WB. Aggresome load correlated with apoptosis and was increased in sensitive cells. LC-MS/MS analysis identified a set of proteins with essential function in PQC only in sensitive cells, among them the UPR modulator RTCB. Aggregation of RTCB induced by AE correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in resistant cells and increased the formation of aggresomes, leading to apoptosis. Analysis of primary human BC and metastases appearing in the same patient after AE therapy showed that RTCB is only localized to aggresomes in the primary tumours. Different protein aggregation patterns indicate loss of function of essential proteins that can be used to identify AE-resistant BC cells and improve the response to therapy. This supports the idea that AE-resistant cells that originate metastasis have a higher capacity to preserve RTCB function and, consequently, to successfully induce UPR and autophagy to maintain low levels of protein aggregation.