“Fatty Acid β-Oxidation Pathway in Canine Mammary Tumors: biochemical, immunohistochemical and pharmacological studies”

Metabolic plasticity describes the ability of the cells to adapt their metabolic status in response to changes in the external microenvironment to support or allow rapid proliferation, continuous growth, and survival in adverse conditions. The metabolic activities in quiescent cells are totally different with respect to those of proliferating ones, in fact, under quiescent conditions, cells have a basal rate of glycolysis, converting glucose to pyruvate, which is then oxidized in the tricarboxylic acid cycle (TCA) into acetyl-CoA and carbon dioxide (CO2) within the mitochondria. The acetyl-CoA then enters the TCA cycle (also known as the citric acid or Krebs cycle) where it is fully oxidized to CO2 and H2O, and produces huge amounts of energy during the process of oxidative phosphorylation (OXPHOS). Nevertheless, quiescent cells can also use fatty acids as building blocks to fulfill their physiological function and to maintain their structural integrity as well as redox and energy balance. On the contrary, during proliferation, metabolism turns towards an anabolic metabolism, with high glycolytic flux and lactate production. Tumor cells have a higher rate of glucose metabolism than their normal counterparts and preferentially use glycolysis instead of OXPHOS even under appropriate oxygen concentrations (Aerobic glycolysis or Warburg Effect). During aerobic glycolysis, glucose is converted to pyruvate, and the final product of this reaction is lactate, which is exported out of the cell, contributing to extracellular acidification. The resulting acidic environment promotes the degradation of the extracellular matrix by proteinases and increases angiogenesis through the release of vascular endothelial growth factor. Recently, it has been demonstrated that most cancer cells to fulfill their increased energy requests, can exploit other metabolic pathways, such as Fatty Acid β –oxidation which represents one of the most crucial mechanisms that can accompany cancer-associated metabolic reprogramming. The use of fatty acids as energy substrates requires about 25 different enzymes and transport proteins, which carry out fatty acids, import them into mitochondria, and facilitate the β-oxidation steps. In particular, the Carnitine System (CS) is considered as a gridlock to finely trigger the metabolic flexibility of cancer cells. The components of the CS play a crucial role in tuning the switch between glucose and fatty acid metabolism. Here, we report, in Canine Mammary Tumors (CMTs), the expression of the CS components in both canine tissue samples derived from bitches suffering from mammary tumors and CMT cell lines. In particular, the analysis of CMTs and mammary gland control specimens confirmed the aberrant expression of the four components involved in this transporting system (CPT1A, CACT, CRAT e CPT2) in primary tumors and especially in well-differentiated (grade 1) tumors in comparison to moderately- (grade 2) and poorly- (grade 3) differentiated tumors. The role of chemotherapy in bitches with malignant mammary tumors has not been clearly defined for all tumor types and, therefore, we have also tested the cytotoxic and proapoptotic effects of the CPT1A inhibitor ST1326 on CMT cell lines obtained from bitches with different mammary malignancies. Cell viability was evaluated in CMT cell lines (CMT-U309, P114, CMT-U27, CMT-U131 and CMT-U229) cells by using trypan blue staining. The exposure of the CMT cell lines to increasing concentrations of ST1326 [1-20 μM] for 48h has determined a significant reduced rate in cell viability. Furthermore, we have also evaluated whether the compound ST1326 is also able to induce cell death in CMT cell lines. CMT-U131 and CMT-U229 have been the cells with the greater sensitivity to ST1326 treatment in comparison to the other cell lines tested. The ability of ST1326 to induce apoptosis in CMT-U229 and CMT-U131 has been examined by employing Annexin V-FITC Assay using Flow Cytometry. The exposure of CMT-U229 cells to 10 µM ST1326 lead to a significant increase (8.40%) of cells in the early apoptotic phase and also a significant increase (8.25%) at concentration of 20 µM in the late apoptotic phase, respectively. The treatment of CMT-U131 has also displayed a significant increase (13.8%) and (40.85%) in the late apoptotic phase at concentration of 10 or 20 µM ST1326, respectively. Finally, by western blot analysis we have investigated which molecular signaling pathways were involved in ST1326-mediated cell death. Our results have shown that ST1326 reduces, at least in part, the phosphorylated levels of serine/threonine‑protein kinase AktSer473, reduces the total levels of extracellular signal-related kinase (ERK) protein as well as increases the phosphorylated levels of ERK protein. Taken all together, our results identify CPT1A as a novel target for CMTs treatment and suggest new druggable pathways for prevention and treatment of these tumors, highlighting, for the first time, a new and emerging deregulated biochemical pathway in canine tumor metabolic reprogramming