Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca 2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H 2 O 2 ). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H 2 O 2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H 2 O 2 -responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H 2 O 2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H 2 O 2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H 2 O 2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H 2 O 2 or PRX2 oxidation but rather lead to increased nuclear H 2 O 2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1 +/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H 2 O 2 levels and local cellular responses dependent on mitochondrial ROS.

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Regulation of cell signaling cascades is critical in making sure the response is activated spatially and for a desired duration. Cell signaling cascades are spatially and temporally controlled through local protein phosphorylation events which are determined by the activation of specific kinases and/or inactivation of phosphatases to elicit a complete and thorough response. For example, A-kinase-anchoring proteins (AKAPs) contribute to the local regulated activity protein kinase A (PKA). The activity of kinases and phosphatases can also be regulated through redox-dependent cysteine modifications that mediate the activity of these proteins. A primary example of this is the activation of the epidermal growth factor receptor (EGFR) and the inactivation of the phosphatase and tensin homologue (PTEN) phosphatase by reactive oxygen species (ROS). Therefore, the local redox environment must play a critical role in the timing and magnitude of these events. Mitochondria are a primary source of ROS and energy (ATP) that contributes to redox-dependent signaling and ATP-dependent phosphorylation events, respectively. The strategic positioning of mitochondria within cells contributes to intracellular gradients of ROS and ATP, which have been shown to correlate with changes to protein redox and phosphorylation status driving downstream cellular processes. In this review, we will discuss the relationship between subcellular mitochondrial positioning and intracellular ROS and ATP gradients that support dynamic oxidation and phosphorylation signaling and resulting cellular effects, specifically associated with cell migration signaling.

Miro1 is an outer mitochondrial membrane protein that links mitochondria to motor protein complexes to support subcellular mitochondrial trafficking. Preliminary observations and published work has established that metastatic breast cancer cells have increased expression of Miro1 in comparison to breast cancer cells with low metastatic potential. Furthermore, tumors from breast cancer patients with high Miro1 expression have poorer survival rates compared to patients harboring tumors with low Miro1 expression. This data suggests that Miro1 has a functional role in aggressive breast cancers although this has not been widely studied or confirmed in advanced preclinical models. The objective of our research is to investigate the role of Miro1 in breast cancer tumorigenesis and metastasis. We hypothesize that that Miro1-mediated mitochondrial dynamics support subcellular signaling events driving cell migration, invasion, and tumorigenesis. To examine the function of Miro1 in promoting metastatic phenotypes in vitro, we generated human breast cancer cell lines (MDA-MB-231, MDA-MB-468, T-47d, and MCF7) with Miro1 knockdown using adenoviral shRNA. Knockdown of Miro1 to levels greater than 50% of that in control cells resulted in mitochondria sequestered around the nucleus and reduced cell migration. Additionally, MDA-MB-231 cells with ≥50% Miro1 knockdown resulted in a significant decrease in cell invasion through Matrigel coated membranes in transwell invasion assays. Our research has also uncovered altered phosphorylation of key cell migration and stress-response proteins including vinculin, focal adhesion kinase and ERK ½ when Miro1 is knocked down. To investigate the role of Miro1 in vivo, we generated a novel transgenic mouse model with inducible, tissue specific Miro1 deletion in mammary epithelial cells with concurrent polyomavirus middle T-antigen oncogene activation. We found that when breast cancer formation is induced utilizing the transgenic middle-T antigen system, our control mice form tumors at all mammary gland sites and have metastasis to the lungs. Conversely, mice with concurrent Miro1 deletion and middle T- antigen activation fail to develop tumors. This data suggests that Miro1 has a functional role in both mammary tumor onset and tumor cell migration and invasion, providing a possible new biomarker of tumor status and pathway for therapeutic intervention. Citation Format: Randi Gravelle, Margaret McCoy, Nathaniel Shannon, Martin Chang, Brian Cunniff. The role of miro1-mediated mitochondrial positioning in the development and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 87.

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