Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Comparison of Pelvic Landmarks for Leg Length Discrepancy Measurement With Robotic Arm-Assisted Total Hip Arthroplasty.

Background: Leg length discrepancy (LLD) is a common complication after total hip arthroplasty (THA) leading to significant morbidity and dissatisfaction for patients. A popular system for robotic arm-assisted THA utilizes preoperative computed tomography (CT) scans for surgical planning. Accurate measurement of leg length is crucial for restoring appropriate patient anatomy during the procedure. This study investigates the interobserver and interlandmark reliability of 3 different pelvic landmarks for measuring preoperative LLD. Methods: We compiled preoperative pelvic CT scans from 99 robotic arm-assisted THAs for osteoarthritis. Radiologic leg length measurement was performed using the robotic arm-assisted THA application by 2 orthopaedic residents using reference lines bisecting the following pelvic landmarks: the anterior superior iliac spines, acetabular teardrops, and most inferior aspect of the ischial rami. Results: On multivariate analysis, there was no significant difference found (P value = .924) for leg length measurement based on the 3 different pelvic anatomical landmarks. Leg length measurements showed interobserver reliability with significant Pearson correlation coefficients (r = 1.0, 0.94, 0.96, respectively) and nonsignificant differences in LLD means between subjects on paired sample (P value = .158, .085, 0.125, respectively) as well as between landmarks on pairwise comparison. Conclusions: The 3 pelvic landmarks used in this study can be used interchangeably with the lesser trochanter as the femoral reference point to evaluate preoperative LLD on pelvic CT in patients undergoing robotic-arm assisted THA. This study is the first of its kind to evaluate the interobserver and interlandmark reliability of anatomical landmarks on pelvic CT scans and suggests interchangeability of 3 pelvic landmarks for comparing leg length differences.

Targeting disseminated estrogen-receptor-positive breast cancer cells in bone marrow.

Estrogen receptor-positive (ER+) breast cancer can recur up to 20 years after initial diagnosis. Delayed recurrences arise from disseminated tumors cells (DTCs) in sites such as bone marrow that remain quiescent during endocrine therapy and subsequently proliferate to produce clinically detectable metastases. Identifying therapies that eliminate DTCs and/or effectively target cells transitioning to proliferation promises to reduce risk of recurrence. To tackle this problem, we utilized a 3D co-culture model incorporating ER+ breast cancer cells and bone marrow mesenchymal stem cells to represent DTCs in a bone marrow niche. 3D co-cultures maintained cancer cells in a quiescent, viable state as measured by both single-cell and population-scale imaging. Single-cell imaging methods for metabolism by fluorescence lifetime (FLIM) of NADH and signaling by kinases Akt and ERK revealed that breast cancer cells utilized oxidative phosphorylation and signaling by Akt to a greater extent both in 3D co-cultures and a mouse model of ER+ breast cancer cells in bone marrow. Using our 3D co-culture model, we discovered that combination therapies targeting oxidative phosphorylation via the thioredoxin reductase (TrxR) inhibitor, D9, and the Akt inhibitor, MK-2206, preferentially eliminated breast cancer cells without altering viability of bone marrow stromal cells. Treatment of mice with disseminated ER+ human breast cancer showed that D9 plus MK-2206 blocked formation of new metastases more effectively than tamoxifen. These data establish an integrated experimental system to investigate DTCs in bone marrow and identify combination therapy against metabolic and kinase targets as a promising approach to effectively target these cells and reduce risk of recurrence in breast cancer.

Plasminogen Activator Inhibitor 1 (PAI1) Promotes Actin Cytoskeleton Reorganization and Glycolytic Metabolism in Triple-Negative Breast Cancer.

Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression. IMPLICATIONS: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

Enhanced Bone Metastases in Skeletally Immature Mice.

Bone constitutes the most common site of breast cancer metastases either at time of presentation or recurrent disease years after seemingly successful therapy. Bone metastases cause substantial morbidity, including life-threatening spinal cord compression and hypercalcemia. Given the high prevalence of patients with breast cancer, health-care costs of bone metastases (>$20,000 per episode) impose a tremendous economic burden on society. To investigate mechanisms of bone metastasis, we developed femoral artery injection of cancer cells as a physiologically relevant model of bone metastasis. Comparing young (~6 weeks), skeletally immature mice to old (~6 months) female mice with closed physes (growth plates), we showed significantly greater progression of osteolytic metastases in young animals. Bone destruction increased in the old mice following ovariectomy, emphasizing the pathologic consequences of greater bone turnover and net loss. Despite uniform initial distribution of breast cancer cells throughout the hind limb after femoral artery injection, we observed preferential formation of osteolytic bone metastases in the proximal tibia. Tropism for the proximal tibia arises in part because of TGF-ß, a cytokine abundant in both physes of skeletally immature mice and matrix of bone in mice of all ages. We also showed that age-dependent effects on osteolytic bone metastases did not occur in male mice with disseminated breast cancer cells in bone. These studies establish a model system to specifically focus on pathophysiology and treatment of bone metastases and underscore the need to match biologic variables in the model to relevant subsets of patients with breast cancer.

3D Jet Writing: Functional Microtissues Based on Tessellated Scaffold Architectures.

The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology, and stem cell biotechnology.

Functional Isolation of Tumor-Initiating Cells using Microfluidic-Based Migration Identifies Phosphatidylserine Decarboxylase as a Key Regulator.

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.