Angiotensin II Regulates Mitochondrial mTOR Pathway Activity Dependent on Acyl-CoA Synthetase 4 in Adrenocortical Cells.

Two well-known protein complexes in mammalian cells, mTOR type 1 and type 2 (mTORC1/2) are involved in several cellular processes such as protein synthesis, cell proliferation, and commonly dysregulated in cancer. An acyl-CoA synthetase type 4 (ACSL4) is one of the most recently mTORC1/2 regulators described, in breast cancer cells. The expression of ACSL4 is hormone-regulated in adrenocortical cells and required for steroid biosynthesis. mTORC1/2 have been reported to be crucial in the proliferation of human adrenocortical tumor cells H295R and interestingly reported at several subcellular locations, which has brought cell biology to the vanguard of the mTOR signaling field. In the present work, we study the regulation of mTORC1/2 activation by angiotensin II (Ang II)-the trophic hormone for adrenocortical cells-the subcellular localization of mTORC1/2 signaling proteins and the role of ACSL4 in the regulation of this pathway, in H295R cells. Ang II promotes activation by phosphorylation of mTORC1/2 pathway proteins in a time-dependent manner. Mitochondrial pools of ribosomal protein S6, protein kinase B (Akt) in threonine 308, and serine 473 and Rictor are phosphorylated and activated. Glycogen synthase kinase type 3 (GSK3) is phosphorylated and inactivated in mitochondria, favoring mTORC1 activation. Epidermal growth factor, a classic mTORC1/2 activator, promoted unique activation kinetics of mTORC1/2 pathway, except for Akt phosphorylation. Here, we demonstrate that ACSL4 is necessary for mTORC1/2 effectors phosphorylation and H295R proliferation, triggered by Ang II. Ang II promotes activation of mitochondrial mTORC1/2 signaling proteins, through ACSL4, with a direct effect on adrenocortical cellular proliferation.

Regulation and role of Acyl-CoA synthetase 4 in glial cells.

Acyl-CoA synthetase 4 (Acsl4), an enzyme involved in arachidonic acid (AA) metabolism, participates in physiological and pathological processes such as steroidogenesis and cancer. The role of Acsl4 in neurons and in nervous system development has also been documented but little is known regarding its functionality in glial cells. In turn, several processes in glial cells, including neurosteroidogenesis, stellation and AA uptake, are regulated by cyclic adenosine monophosphate (cAMP) signal. In this context, the aim of this work was to analyze the expression and functional role of Acsl4 in primary rat astrocyte cultures and in the C6 glioma cell line by chemical inhibition and stable silencing, respectively. Results show that Acsl4 expression was regulated by cAMP in both models and that cAMP stimulation of steroidogenic acute regulatory protein mRNA levels was reduced by Acsl4 inhibition or silencing. Also, Acsl4 inhibition reduced progesterone synthesis stimulated by cAMP and also affected cAMP-induced astrocyte stellation, decreasing process elongation and increasing branching complexity. Similar effects were observed for Acsl4 silencing on cAMP-induced C6 cell morphological shift. Moreover, Acsl4 inhibition and silencing reduced proliferation and migration of both cell types. Acsl4 silencing in C6 cells reduced the capacity for colony proliferation and neurosphere formation, the latter ability also being abolished by Acsl4 inhibition. In sum, this work presents novel evidence of Acsl4 involvement in neurosteroidogenesis and the morphological changes of glial cells promoted by cAMP. Furthermore, Acsl4 participates in migration and proliferation, also affecting cell self-renewal. Altogether, these findings provide insights into Acsl4 functions in glial cells.

Regulatory mechanisms leading to differential Acyl-CoA synthetase 4 expression in breast cancer cells.

Acyl-CoA synthetase 4 (ACSL4) overexpression plays a causal role in the aggressiveness of triple negative breast cancer. In turn, a negative correlation has been established between ACSL4 and estrogen receptor alpha (ERα) expression. However, the upstream regulatory mechanisms leading to differential ACSL4 expression between triple negative breast cancer and ERα-positive cells remained unknown. We performed the characterization of the human ACSL4 promoter and the identification of transcription factors involved. Deletional analysis demonstrated the proximal 43 base pairs of the promoter are involved in overexpression. By site directed mutagenesis we describe that retinoid-related orphan receptor alpha (RORα), Sp1 and E2F elements are involved in the promoter activity. We established for the first time that estrogen-related receptor alpha (ERRα) is a transcription factor involved in the higher activation of the human ACSL4 promoter in breast cancer cells. Furthermore, a combination of inhibitors of ACSL4 and ERRα produced a synergistic decrease in MDA-MB-231 cell proliferation. We also demonstrated that ERα restoration in triple negative breast cancer cells downregulates ACSL4 expression. The results presented in this manuscript demonstrated transcriptional mechanism is involved in the different expression of ACSL4 in human breast cancer cell lines of different aggressiveness.