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BRAFV600E is the most prevalent mutation in thyroid cancer and correlates with poor prognosis and therapy resistance. Although selective inhibitors of BRAFV600E have been developed, more advanced tumors such as anaplastic thyroid carcinomas show a poor response in clinical trials. Therefore, the study of alternative survival mechanisms is needed. Since metabolic changes have been related to malignant progression, in this work we explore metabolic dependencies of thyroid tumor cells to exploit them therapeutically. Our results show that respiration of thyroid carcinoma cells is highly dependent on fatty acid oxidation and, in turn, fatty acid mitochondrial availability is regulated through macroautophagy/autophagy. Furthermore, we show that both lysosomal inhibition and the knockout of the essential autophagy gene, ATG7, lead to enhanced lipolysis; although this effect is not essential for survival of thyroid carcinoma cells. We also demonstrate that following inhibition of either autophagy or fatty acid oxidation, thyroid tumor cells compensate oxidative phosphorylation deficiency with an increase in glycolysis. In contrast to lipolysis induction, upon autophagy inhibition, glycolytic boost in autophagy-deficient cells is essential for survival and, importantly, correlates with a higher sensitivity to the BRAFV600E selective inhibitor, vemurafenib. In agreement, downregulation of the glycolytic pathway results in enhanced mitochondrial respiration and vemurafenib resistance. Our work provides new insights into the role of autophagy in thyroid cancer metabolism and supports mitochondrial targeting in combination with vemurafenib to eliminate BRAFV600E-positive thyroid carcinoma cells. Abbreviations: AMP: adenosine monophosphate; ATC: anaplastic thyroid carcinoma; ATG: autophagy related; ATP: adenosine triphosphate; BRAF: B-Raf proto-oncogene, serine/threonine kinase; Cas9: CRISPR-associated protein; CREB: cAMP responsive element binding protein; CRISPR: clustered regularly interspaced short palindromic repeats; 2DG: 2-deoxyglucose; FA: fatty acid; FAO: fatty acid oxidation; FASN: fatty acid synthase; FCCP: trifluoromethoxy carbonyl cyanide phenylhydrazone; LAMP1: lysosomal associated membrane protein 1; LIPE/HSL: lipase E, hormone sensitive type; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; PRKA/PKA: protein kinase cAMP-activated; PTC: papillary thyroid carcinoma; SREBF1/SREBP1: sterol regulatory element binding transcription factor 1. [ABSTRACT FROM AUTHOR]

Previous results from our laboratory showed that GH4C1 cells with low-cholesterol cell content had increased adenylyl cyclase (AC) activity with a parallel increase in G protein α subunits associated to the plasma membrane. This effect was directly related to mevalonate availability. In the present report, we characterized the high-affinity GTPase activity present in GH4C1 cell membranes and studied its regulation by cholesterol cell content. The high-affinity GTPase activity, measured as the [γ32P]GTP hydrolysis rate, was both time-dependent and protein concentration-dependent. Cultured cells with lipoprotein-deficient serum (LPDS) showed decreased cholesterol cell content and decreased GTPase activity. The kinetic analysis, as interpreted by Lineweaver–Burk plots, indicated that low-cholesterol cell content had no effect on the apparent affinity for GTP, but resulted in a 47% decrease in the maximal velocity of the reaction. Addition of 25-hydroxycholesterol (25-HC), an inhibitor of the expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and synthetase to cells in LPDS, further decreased GTPase activity in a dose-dependent manner. This effect was reverted by exogenous cholesterol, but not by mevalonate. Studies with bacterial toxins revealed that neither cholera toxin (CTX) nor pertussis toxins (PTX) were able to revert the inhibition produced by low-cholesterol cell content. These results allowed us to postulate that cholesterol modulates GTPase activity in both Gs and Gi protein families. To analyse further the mechanism of modulation of GTPase activity by cholesterol cell content, [35S]GTPγS binding in membranes of GH4C1 cells was studied. Changes in cholesterol cell content did not have any effect on GTP binding. Data demonstrated that high-affinity GTPase activity in plasma membrane of GH4C1 cells is direct stimulated by cholesterol cell content and not by mevalonate availability. This example provides a mechanism by which cholesterol cell content can modulate signal transduction mediating by G proteins. [Copyright &y& Elsevier]

Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a `knockout' of the raf-1 gene in mice as welt as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1-/- mice die in embryogenesis and show vascular defects in the yolk sac and placenta as welt as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1FF/FF mice has no detectable activity towards MEK in vitro, and yet raf-1FF/FF mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1-/- and raf-1FF/FF mice, ERIC activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis. [ABSTRACT FROM AUTHOR]

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours. [ABSTRACT FROM AUTHOR]

Abstract: BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic V600EBRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of V600EBRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel V600EBRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target V600EBRAF signalling to effectively control progression of thyroid cancer. [Copyright &y& Elsevier]

Abstract: BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic V600EBRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease. [Copyright &y& Elsevier]