For Triple Negative BCa (TN BCa), there is a current lack of understanding of driver pathways and hence are often treated using more generic therapies. Currently there are no clinically accepted targets for the treatment for TN BCa and to predict its potential to metastasize. Metabolites are the end products of protein activity and are less complex, more stable, and can be easily measured in a clinical setting. Most of the metabolism in an actively growing cell (tumor) occurs in the cytoplasm and mitochondria. The latter is considered the energy house of the cell and has been strongly implicated in tumor development and progression and thus serving as a potential target for chemotherapy. With the advent of Transmitochondrial cybrid (cybrid) technology, it is now possible to examine the specific contribution of tumor-associated mitochondria to neoplastic growth and development under a defined nuclear background. Cybrids are constructed by fusing enucleated cells harboring mitochondria of interest with rho0 recipient cells (cells harboring ablated mitochondrial DNA). Using this technology in both an in vitro and in vivo setting, Dr. Kaipparettu has demonstrated a key role for mitochondrial retrograde regulation (MRR) progression of TN BCa. Here we propose to combine Dr. Kaipparettu's (PI) expertise in BCa and cybrid technology with Dr. Sreekumar's (Co-I) expertise on BCa metabolomics to evaluate a clinically challenging question, "How can we better distinguish metastatic TN BCa?" Strategically, we will use cybrid system to analyze the mitochondria specific alterations in metastatic BCa. rho0 cells from different TN breast-derived non-cancerous and cancerous nuclear background are already available in Kaipparettu lab. Into these recipient cells, we will transplant the mitochondria from TN benign breast epithelium non-to-moderately metastatic and highly metastatic BCa. The cybrids thus generated will be confirmed for their mitochondrial function, nuclear origin by next-gen sequencing and evaluated for phenotypic changes using in vitro and in vivo tumor forming/invasion assays. Following this, the cybrids and tumors will be profiled for their metabolome using a combination of gas/liquid chromatography-coupled mass spectrometry and metabolic phenotyping. The metabolic profiles will be examined using an established biostatistics and bioinformatics pipelines together with the biostatistician Dr. Creighton to generate metabolic signatures and pathways associated with TN BCa metastasis. The data will be compared with existing patient-derived metabolic profiles from patient-derived xenografts and independent patient specimens having long term clinical follow up to nominate clinically relevant metabolites. The nominated pathways will be evaluated for their role in TN BCa progression using cell line and xenograft models of the disease. Overall, we expect to develop the first-of-its-kind mitochondria-driven metabolome for TN breast cancer progression and metastasis. Clinically, we expect these to be translated to identify new drug targets for TN breast cancer.
Though mitochondria play an important role in promoting cancer progression, by altering the metabolic profile of the tumor, to date, the unique metabolic profile of the tumor, regulated by mitochondria has never been completely examined. Using a combination of cybrid technology together with unbiased mass spectrometry-based metabolomic profiling and metabolomics phenotyping, this proposal aims to identify the mitochondria-regulated metabolomic signature for triple negative breast cancer. The metabolites in this signature will have clinical significance as the metabolic pathways defined by this study could reveal novel mechanism for the aggressiveness of triple negative breast cancer and help to identify targets for future drug development.
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