Biology of Human Tumors Mitochondrial Reprogramming Regulates Breast Cancer Progression Anbarasu Kannan 1 , Robert B. Wells 2 , Subramaniam Sivakumar 3 , Satoshi Komatsu 1 , Karan P. Singh 4 , Buka Samten 5 , Julie V. Philley 6 , Edward R. Sauter 7 , Mitsuo Ikebe 1 , Steven Idell 1,8 , Sudeep Gupta 9 , and Santanu Dasgupta 1 Abstract Purpose: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers. Experimental Design: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development. Results: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis–associated protein and MFN2, an impor- tant regulator of mitochondrial fusion. Following its overexpres- sion in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cyto- chrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors. Conclusions: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348–60. Ó2016 AACR. Introduction Breast cancer represents 14.0% of all new cancer cases and is the second most common cause of cancer-associated morbidity among the U.S. women (1, 2). In 2015, there will be an estimated 231,840 cases and 40,290 deaths (2). Being highly heterogeneous and metastatic, breast cancer poses significant challenges to clinical management (1, 3). Early breast cancer detection has a better chance of cure or prolonged disease-free survival compared with the metastatic disease (4). Although at least one progression model of normal tissue to invasive cancer has been proposed using cell morphology (5), the molecular drivers behind the initiation and stage-wise progression of breast cancer are not well characterized. Continuous proliferation and apoptosis resistance are hall- marks of cancer cells (6). Abnormal mitochondrial function and reprogramming contribute to these hallmarks at least in part and hence are implicated in biomarker development (6, 7). Mito- chondrial fusion is a process of fusion of damaged mitochondria to healthy ones (6, 8). Studies suggest that production in tumors of normal mitochondria could be tumor suppressive by promot- ing oxidative metabolism and enhanced reactive oxygen species (ROS) production (8). On the other hand, mitochondrial bio- genesis is a process involving replication of the mitochondrial genome and coordinated expression of both nuclear and mito- chondria-encoded molecules and assembly of the oxidative phos- phorylation complexes (6, 8, 9). Many factors, including MFN2, PINK1, PGC-1a, and mitochondrial transcription factor A (MT- TFA) play critical role in regulating mitochondrial fusion, bio- genesis, and maintaining mitochondrial integrity (6, 8, 9). The role of mitochondrial fusion and biogenesis in breast cancer development and progression remains largely unknown. Exosomes are 50 to 200 nm, small secreted endocytic vesicles present in all cell types and body fluids (10–12). Cancer exosomes (CE) carry survival information in the form of nucleic acids and proteins, shuttle constantly between the cancer cells through the circulation, and influence growth and progression (10–12). Characterizing the CEs and deciphering the cancer-promoting 1 Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, Texas. 2 Department of Pathology,The University of Texas Health Science Center at Tyler, Tyler, Texas. 3 Department of Biochemistry, Sri Sankara Arts and Science College, Kanchipuram, Tamil Nadu, India. 4 University of Alabama at Birmingham Comprehensive Cancer Center's Biosta- tistics and Bioinformatics Shared Facility, University of Alabama at Birmingham, Birmingham, Alabama. 5 Department of Microbiology and Immunology, The University of Texas Health Science Center, Tyler, Texas. 6 Department of Medicine, The University of Texas Health Science Center, Tyler, Texas. 7 Department of Surgery, The University of Texas Health Science Center, Tyler, Texas. 8 The Texas Lung Injury Institute, The University of Texas Health Science Center, Tyler, Texas. 9 Department of Medical Oncology,Tata Memorial Cen- ter, Mumbai, Maharashtra, India. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Santanu Dasgupta, The University of Texas Health Science Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708. Phone: 903-877- 7007; Fax: 903-877-7558; E-mail: santanu.dasgupta@uthct.edu doi: 10.1158/1078-0432.CCR-15-2456 Ó2016 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 22(13) July 1, 2016 3348 on June 11, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst February 17, 2016; DOI: 10.1158/1078-0432.CCR-15-2456