eIF4A/PDCD4 Pathway, a Factor for Doxorubicin Chemoresistance in a Triple-Negative Breast Cancer Cell Model

Abstract

Cells employ several adaptive mechanisms under conditions of accelerated cell division, such as the unfolded protein response (UPR). The UPR is composed of a tripartite signaling system that involves ATF6, PERK, and IRE1, which maintain protein homeostasis (proteostasis). However, deregulation of protein translation initiation could be associated with breast cancer (BC) chemoresistance. Specifically, eukaryotic initiation factor-4A (eIF4A) is involved in the unfolding of the secondary structures of several mRNAs at the 5′ untranslated region (5′-UTR), as well as in the regulation of targets involved in chemoresistance. Importantly, the tumor suppressor gene PDCD4 could modulate this process. This regulation might be disrupted in chemoresistant triple negative-BC (TNBC) cells. Therefore, we characterized the effect of doxorubicin (Dox), a commonly used anthracycline medication, on human breast carcinoma MDA-MB-231 cells. Here, we generated and characterized models of Dox chemoresistance, and chemoresistant cells exhibited lower Dox internalization levels followed by alteration of the IRE1 and PERK arms of the UPR and triggering of the antioxidant Nrf2 axis. Critically, chemoresistant cells exhibited PDCD4 downregulation, which coincided with a reduction in eIF4A interaction, suggesting a sophisticated regulation of protein translation. Likewise, Dox-induced chemoresistance was associated with alterations in cellular migration and invasion, which are key cancer hallmarks, coupled with changes in focal adhesion kinase (FAK) activation and secretion of matrix metalloproteinase-9 (MMP-9). Moreover, eIF4A knockdown via siRNA and its overexpression in chemoresistant cells suggested that eIF4A regulates FAK. Pro-atherogenic low-density lipoproteins (LDL) promoted cellular invasion in parental and chemoresistant cells in an MMP-9-dependent manner. Moreover, Dox only inhibited parental cell invasion. Significantly, chemoresistance was modulated by cryptotanshinone (Cry), a natural terpene purified from the roots of Salvia brandegeei. Cry and Dox co-exposure induced chemosensitization, connected with the Cry effect on eIF4A interaction. We further demonstrated the Cry binding capability on eIF4A and in silico assays suggest Cry inhibition on the RNA-processing domain. Therefore, strategic disruption of protein translation initiation is a druggable pathway by natural compounds during chemoresistance in TNBC. However, plasmatic LDL levels should be closely monitored throughout treatment.

Low-Density Lipoproteins Increase Proliferation, Invasion, and Chemoresistance via an Exosome Autocrine Mechanism in MDA-MB-231 Chemoresistant Cells

“…An increment in cellular invasion coupled to MMP-9 activity in the extracellular medium was registered (Figure 2), although Dox treatment induced a reduction in this process only in parental cells. Therefore, our evidence suggests a potential role of LDL cholesterol overload in oncogenic processes [17]. This proposal was supported by the influence of LDL stimuli in the spheroid growth assay mainly in variant B cells, and the extended stability registered in variant B regarding the parental spheroids under Dox treatment (Figure 2).…”

“…Previous results suggest that PDCD4 down-regulation is a condition in doxorubicin chemoresistance in TNBC cells, modulating responses connected with the PERK pathway of UPR [17]. In this work, after treatment with LDL, exosomes derived from chemoresistant cells exert an autocrine phenomenon that induces cellular proliferation, migration, and invasion.…”

“…Cell cultures were treated with a lethal Dox dose (500 nM) for 24 h, followed by a gradual decreasing dose for 60 days until a dose of 12.5 nM was reached and a recovery period of 30 days. This led to the generation of a variant that will from now on be referred to as variant B [17]. Chemoresistance was coupled with higher concentrations of Dox in the extracellular medium in variant B cells, a phenomenon analyzed later.…”

“…Furthermore, the secretion of UPR targets such as chaperones may drive cell communication pathways [17]. Hsp90 and Hsp70 chaperones must modulate cellular signaling mechanisms and contribute in a non-cell-autonomous maintenance of proteostasis [28]; therefore, their secretion may be contained in exosomes under eIF4F activation [48,49].…”

“…A condition that suppresses the assembly of the eIF4F complex is the binding of eIF4A with programmed cell death protein 4 (PDCD4) [16]. PDCD4 activity has been associated with the inhibition of tumor cell proliferation and chemoresistance [17]. A PDCD4 regulatory mechanism is triggered by its degradation induced by the mTOR/p70 ribosomal protein kinase S6 (p70S6kα) pathway, a central modulator of cell growth, proliferation, survival, and differentiation in response to extracellular stimuli [18], possibly involving vesicles such as exosomes (Exs).…”