Abstract 2396: Role of iron transporter DMT1 in endosome-mitochondria interactions and mitochondrial metabolism in breast cancer cells

Abstract

Iron is essential to support tumor initiation, growth and metastasis. Recently, we have shown that interaction between early endosomes and mitochondria regulates intracellular iron transport in epithelial cells. Mitochondria are the ultimate cellular iron sink, where iron can be either used for Iron-Sulfur cluster and heme synthesis or stored in mitochondrial ferritin. Further understanding how endosomes interact with mitochondria to modulate iron transport in breast cancer cells should provide novel insights into both prevention and treatment of breast cancer. Here, we sought to evaluate the role of the iron transporter Divalent Metal Transporter 1 (DMT1) in endosome-mitochondria interactions, iron transport into mitochondria and its functional consequences in invasive migration and mitochondrial metabolism in breast cancer cells. For silencing experiments, we used CRISPR/Cas9 technology validated by immunoblotting. Leica Thunder microscope and LAS software were used for imaging. High resolution imaging was performed using Airyscan LSM880. Z-stack images were subjected to 3D rendering using IMARIS software to evaluate organelle morphology and organelle-organelle interactions. The Agilent Seahorse Cell Mito Stress assay was used to assess mitochondrial function. Expression of different proteins related to iron transport and signaling, e.g. DMT1, mitochondrial-ferritin, transferrin receptor and EGFR showed substantial heterogeneity between non-cancerous epithelial cells MCF10A and breast cancer cells triple negative MDA-MB-231 and estrogen receptor positive T47D cells. Live cell imaging experiments show that transferrin-containing endosomes in MDA-MB-231 and T47D are more motile compared to MCF10A. Moreover, events of “kiss and run” endosome-mitochondria interactions are more frequent in breast cancer cells. DMT1 knock-out (KO) seems to alter both early and late endosomes distribution and its colocalization with mitochondria in breast cancer cells. Functionally, DMT1 (KO) in both MDA-MB-231 and T47D decreases ERK and AKT activation which is consistent with measurable decrease in invasive migration. Metabolically, Seahorse Mito-Stress assay indicate that basal respiration, proton leak, spare capacity and non-mitochondrial oxygen consumption were severely impaired upon DMT1 KO in MDA-MB-231 and T47D cells. Overall, our results suggest that endosome-mitochondria interactions and dynamics that are required for iron import into mitochondria may be involved in the establishment of a more aggressive and invasive tumor phenotype in breast cancer. Citation Format: Jonathan Barra, Iram Nelson, Lauren Elder, Ling Wang, Margarida M. Barroso. Role of iron transporter DMT1 in endosome-mitochondria interactions and mitochondrial metabolism in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2396.

Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites

“…4F and Fig. S4A), which suggests that iron may directly transit from PfDMT1 on the FV periphery to the mitochondrion without a cytoplasmic intermediate, analogous to what has been observed at transient endosome/lysosome-mitochondrial contact sites in mammalian cells (23, 63, 64). A similar mechanism may account for iron delivery to the apicoplast, which is also frequently observed in close proximity to the FV (65).…”

“…Loss of PfDMT1 alone in -aTC conditions did not depolarize mitochondria, which accumulated MitoTracker Red CMXRos in discrete fluorescent foci similar to +aTC conditions. (23,63,64). A similar mechanism may account for iron delivery to the apicoplast, which is also frequently observed in close proximity to the FV (65).…”