Papel de V600EBRAF en la reprogramación del metabolismo de glucosa y glutamina de células tumorales tiroideasimplicaciones terapéuticas

Laburpena

Cancer is one of the main causes of death in the world and the study of new therapy strategies for the treatment of this disease has made great progress. Notably, we have better knowledge of the genetic and molecular alterations that regulate the acquisition of certain capabilities by tumor cells, that allow them to survive and proliferate under conditions that would normally be deleterious. One of the leading signaling pathways altered in cancer and directly related to many of these hallmarks is the RAS-RAF-MEK-ERK pathway, which regulates many cellular processes. The BRAF protein is mutated in different types of cancer. The most frequent BRAF mutation consists of the substitution of a valine for a glutamate (V600EBRAF), that renders this kinase constitutive active leading to hyperactivation of ERK, and supposes more than 95% of the mutations found in this oncogene. The expression of this mutant is especially common in melanoma and thyroid tumors, where its presence increases the proliferative, invasive and metastatic capacity of these cells. For all these reasons, large selective inhibitors of this mutant have been developed. However, although some of these inhibitors are approved by the treatment of metastatic melanoma and produce an improvement in patient survival, the results obtained in thyroid cancer are not as expected. It has been observed that a significant percentage of thyroid cancers progress to a more advanced stage of the disease and/or develop resistance to BRAF inhibitors, but the associated mechanisms are barely known. Recently, new emphasis has been given to the altered cellular metabolism of proliferating cancer cells, which require high amount of glucose and glutamine for energy production and macromolecules biosynthesis. This metabolic reprogramming is orchestrated by oncogenes activation leading to abnormal proliferation and tumor development. Moreover, this hallmark of malignancy has been associated with chemotherapy resistance of thyroid cancer cells. Because of this, many metabolic enzymes inhibitors are currently been investigated to improve current therapies. For all these reasons, we studied the relationship between the mutant V600EBRAF and metabolic reprogramming, as well as the molecular alterations and signaling pathways involved in this process. In addition, we analyzed the role of glucose and glutamine metabolism in the proliferation and survival of thyroid tumor cells and the effect of their modulation on the efficacy of therapeutic strategies by using the inhibition of V600EBRAF in vitro and in vivo models. For this purpose, we used three thyroid cancer cell lines harboring V600EBRAF mutation, in which we modulated its activity or expression by using inhibitors or siRNA. Moreover, to address the role of of glucose and glutamine metabolism, the cells were treated with inhibitors of their catabolism or incubated in deprived cultured mediums. Our results show that V600EBRAF thyroid tumor cells depend on the availability and metabolism of glucose and glutamine for their survival, and inhibition of their catabolic processes leads to increased cell death. Furthermore, we demonstrate that V600EBRAF is involved in the maintenance of cellular respiration, ATP production and glucose and glutamine oxidation dependence to maintain cellular respiration. Additionally, V600EBRAF increases glucose uptake and glycolytic flux through increased expression of GLUT-1 and HK-II by increasing, at least in part, the activity of the transcription factor HIF-1α. We also demonstrate that inhibition of the V600EBRAF oncogene triggers an increase of activity of energy cellular sensor AMPK and inhibition of mTORC1, which can contribute to to the effects of V600EBRAF inhibition on glucose metabolism. Regarding glutamine metabolism, we show that glutamine uptake and glutaminolysis are essential for proliferation and survival of thyroid cancer cells. Moreover, we also demonstrated that V600EBRAF inhibition does not modify the intracellular levels of this amino acid, but increases asparagine synthesis, by enhancing ASNS levels through the eIF2α–ATF4 reticulum stress pathway. On the other hand, the combination therapy of glucose (2DG) or asparagine (LAsparaginase) metabolism inhibitors and the V600EBRAF inhibitor (PLX4720) potentiates the cytotoxic effects that individual treatments exert on cell death. Importantly, 2DG treatment improve antitumor efficiency of PLX4720 in a xenograft model in vivo. Together, the results reported here contribute to a better understanding of the molecular mechanisms underlying the survival of thyroid cancer cells. Indeed, all these data demonstrate that oncogenic V600EBRAF plays an important role in the metabolic reprogramming of glucose and glutamine metabolism. Moreover, we provide evidences that support the therapeutic intervention of inhibit the glucose and glutamine metabolism in thyroid cancer, and our data support the idea that the inhibition of glucose and glutamine/asparagine metabolism are good strategies that can improve therapies against thyroid tumors harboring the mutation V600EBRAF.