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Enhanced fatty acid oxidation confers resistance to glutaminase inhibition in triple-negative breast cancer cells

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Larissa Menezes dos Reis
Total Authors: 1
Document type: Doctoral Thesis
Institution: Universidade Estadual de Campinas, Instituto de Biologia
Defense date:
Advisor: Sandra Martha Gomes Dias

Triple-negative breast cancer accounts for about 15% of all breast cancer, characterized by a lack of estrogen and progesterone receptor expression (ESR and PGR, respectively) and the absence of human epithelial growth factor receptor (ERBB2) amplification. Therefore, this subtype does not respond to conventional treatments for breast cancer. Recent studies have shown a relationship between triple-negative breast cancer and altered metabolism, suggesting that metabolic reprogramming (with dependence on the amino acid glutamine) may be key to disease progression and promising in the development of new therapies. Inhibition of Glutaminase enzyme (GLS), first in the glutamine metabolism pathway, has been evaluated in the treatment of triple-negative breast tumors with compound CB-839 (Clinical Phase II) and, therefore, we evaluated which alternative metabolic mechanisms could be triggered with the treatment, causing tumor resistance to the compound. Initially, we confirmed literature data showing a varyable dependence on glutamine and sensitivity to CB-839 in TN cell lines and, thus, identified resistant (proliferation inhibition less than 50% compared to vehicle tretatment) and sensitive (death or proliferation inhibition more than 50% of that of the vehicle-treated cells) cells lines. Then, from the analysis of transcriptomic data from cells lines and tumor tissues, we observed that the genes related to lipid metabolism are differentially expressed between resistant and sensitive cells and between tumors with low or high GLS expression. Specifically, several genes related to the beta-oxidation process have increased expression in tumor tissues with low GLS expression. In addition, patients with tumors with lower GLS associated to higher CPT1 expression show worse prognosis. In order to validate whether beta-oxidation is an important metabolic pathway in resistant cells lines, we evaluated CPT1 and CPT2 protein expression, CPT1 activity, and etomoxir sensibility (beta-oxidation inhibitor) levels on those cells. Cells resistant to CB-839 have higher CPT2 protein level, higher CPT1 activity and are more sensitive to etomoxir (in relation to proliferation) than sensitive cells. We also observed that CB-839 treatment impacted less the mitochondrial morphology and ATP levels of resistant compared to sensitive cells. Combined treatment of CB-839 with etomoxir in resistant cells promoted a greater impact on cellular respiration of fatty acids, intracellular ATP level, cell proliferation, invasion and migration phenotypes. The levels of Tyr172 phosphorylation in AMPK and Ser72 in ACC are higher in tumors with lower GLS expression, providing the molecular bases for the activation of beta-oxidation in CB-839-resistant cells. In summary, we conclude that glutamine and fatty acid metabolism provide metabolic plasticity for triple-negative breast tumors and that the protein levels of CPT1 and CPT2 should be used as markers of response to CB-839 treatmen (AU)

FAPESP's process: 14/18061-9 - Study of alternative anaplerotic sources of the TCA as new targets against triple-negative breast cancer
Grantee:Larissa Menezes dos Reis
Support type: Scholarships in Brazil - Doctorate (Direct)