Transient &quotkiss-and-run&quot endosome-mitochondria interactions can mediate mitochondrial iron translocation (MIT) but the associated mechanisms are still elusive. We show that Divalent Metal Transporter 1 (DMT1) modulates MIT via endosome-mitochondria interactions in invasive MDA-MB-231, but not in non-invasive T47D breast cancer cells. CRISPR/Cas9-based DMT1 knockout (KO) stable cells were used to demonstrate that DMT1 regulates MIT, endosomal speed, and labile iron pool (LIP) levels only in MDA-MB-231. DMT1 silencing increases PINK1/Parkin mitophagy markers, the autophagy marker LC3B, as well as mitochondrial ferritin in MDA-MB-231, but not in T47D. Strikingly, re-expression of DMT1 in MDA-MB-231 DMT KO cells rescues all protein levels evaluated. DMT1 silencing decreases Tom20 colocalization with PMPCB, a DMT1 interactor that regulates mitophagy hyperactivation. In MDA-MB-231 both mitochondrial metabolism and invasion were impaired by DMT1 silencing and rescued by DMT1 re-expression. DMT1 acts as a bridge between endosomes and mitochondria to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cancer cell migration.

Mitochondria‐early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE‐mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti‐import) and high ferroportin (FPN) (pro‐export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple‐negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome‐mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE‐mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re‐expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro‐Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE‐mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE‐mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1‐mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin‐dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re‐expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re‐expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE‐mitochondria interac...

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 processe...

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.