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.

Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.