Iron transporter DMT1 regulates endosome‐mitochondria dynamics, mitochondrial metabolism and invasive migration in breast cancer cells

Abstract

Iron is essential to support tumor initiation, growth and metastasis. 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. Recently, mitochondrial morphology, dynamics and function have been shown to be regulated by interaction with other organelles, such as ER and endosomes. We have shown that interaction between early endosomes (EE) and mitochondria regulates iron translocation into the mitochondria in epithelial cells. Furthermore, blocking iron release has been shown to prolong EE-mitochondria interactions and increase endosomal dynamics. Divalent metal transporter 1 (DMT1) modulates iron transport from endosomes into mitochondria and has been suggested as a regulator of endosome-mitochondria interactions. We have evaluated the role of DMT1 in EE-mitochondria interactions, EE dynamics and its relationship with cancer-related processes. To evaluate organelle morphology and dynamics, mammary epithelial MCF10A and human breast cancer MDA-MB-231 and T47D cells, representative of triple negative and estrogen receptor positive breast cancer types, respectively, were subjected to time-lapse live-cell and immunofluorescence imaging assays. Z-stack images were collected using high-resolution microscopy and subjected to 3D rendering using IMARIS software. The Agilent Seahorse Cell Mito Stress assay was used to assess mitochondrial function and an inverted invasion assay to evaluate invasive migration. Comparison of non-cancerous epithelial cells MCF10A and MDA-MB-231 and T47D cells showed heterogeneity in expression of proteins related to iron transport and signaling, e.g. DMT1, mitochondrial-ferritin, transferrin receptor and EGFR. Interestingly, EE dynamics, as shown by the mean track speed (MTS) of EE, are elevated in MDA-MB-231 and T47D in comparison to MCF10A. We are currently analyzing how alterations in EE dynamics affect the frequency of “kiss and run” EE-mitochondria interactions events in non-cancerous vs. breast cancer cells. CRISPR/Cas9 was used for silencing of DMT1 in MDA-MB-231 and T47D cells. DMT1 depletion decreases EE MTS in MDA-MB-231 but not in T47D. Moreover, DMT1 silencing in MDA-MB-231 and T47D increases EE-mitochondria distance separation while increasing proximity between late endosomes (LE) and mitochondria. These results are consistent with elevated EE-mitochondria and reduced LE-mitochondria colocalization levels. Moreover, ERK and AKT activation is inhibited upon DMT1 silencing, concurrently with decreases in invasive migration. Mitochondrial metabolism was severely impaired upon DMT1 silencing in both MDA-MB-231 and T47D cells. Overall, our results suggest that in breast cancer cells, DMT1 regulates endosomal dynamics to maintain adequate EE-mitochondria interactions and support higher levels of iron translocation into mitochondria. Furthermore, DMT1 is necessary for maintaining higher levels of mitochondrial metabolism required for invasive migration and other cancer-related processes, indicating that DMT1 function may be at the basis of more aggressive phenotypes in breast cancer.