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mTORC1 plays a crucial role in protein synthesis and cell proliferation and growth. It is activated by growth factors and amino acids, including essential amino acids (EAAs), such as leucine; Leu enters cells via the Leu transporter LAT1-4F2hc (also known as SLC7A5-SLC3A2) and potentially via endocytosis. Here, we investigated the contribution of the different routes of Leu entry into cells to mTORC1 activation using pharmacological inhibitors and cells that lack LAT1 or dynamin-1, -2 and -3. Our results show that LAT1 is the major route of Leu entry into cells and mTORC1 activation (~70%), whereas dynamin-dependent endocytosis and macropinocytosis contribute minimally to both (5-15%). However, macropinocytosis contributes significantly (~40%) to activation of mTORC1 by other EAAs. Surprisingly, the dynamin inhibitors dynasore and Dyngo 4A, which minimally inhibited Leu uptake, abolished mTORC1 activation independently of dynamin. Instead, dynasore inhibited RagA binding to Raptor, reducedmTORC1 recruitment to the lysosome, and inhibited Akt activation and TSC2- S939 phosphorylation; this resulted in inhibition of Rheb and mTORC1 activity. Our results suggest that these commonly used inhibitors of dynamin and endocytosis are potent suppressors of mTORC1 activation via off-target effects and not via dynamin inhibition. [ABSTRACT FROM AUTHOR]

Simple Summary: The xCT transporter of oxidized form of cysteine has been recognized as fundamental for cellular amino acid and redox homeostasis. Increasing number of data suggests that xCT inhibition-induced ferroptosis has great potential for development of novel anti-cancer therapeutics for pancreatic cancer patients. The aim of this study was to investigate potential resistance mechanisms that cancer cells with genetically disrupted xCT (xCTKO) may exploit in order to develop resistance to ferroptosis. Our data clearly showed that shuttle of reduced cysteine between cancer xCTKO and neighboring cells provide protection of the former. Importantly, this shuttle seems to be fueled by the import and reduction of oxidized cysteine by xCT-proficient feeder layer. In summary, two important findings are: (1) supply of the reduced cysteine has to be taken in consideration when xCT-based ferroptosis inducers are used, and (2) systemic inhibition of xCT could be potential approach in overcoming this resistant mechanism. In our previous study, we showed that a cystine transporter (xCT) plays a pivotal role in ferroptosis of pancreatic ductal adenocarcinoma (PDAC) cells in vitro. However, in vivo xCTKO cells grew normally indicating that a mechanism exists to drastically suppress the ferroptotic phenotype. We hypothesized that plasma and neighboring cells within the tumor mass provide a source of cysteine to confer full ferroptosis resistance to xCTKO PDAC cells. To evaluate this hypothesis, we (co-) cultured xCTKO PDAC cells with different xCT-proficient cells or with their conditioned media. Our data unequivocally showed that the presence of a cysteine/cystine shuttle between neighboring cells is the mechanism that provides redox and nutrient balance, and thus ferroptotic resistance in xCTKO cells. Interestingly, although a glutathione shuttle between cells represents a good alternative hypothesis as a "rescue-mechanism", our data clearly demonstrated that the xCTKO phenotype is suppressed even with conditioned media from cells lacking the glutathione biosynthesis enzyme. Furthermore, we demonstrated that prevention of lipid hydroperoxide accumulation in vivo is mediated by import of cysteine into xCTKO cells via several genetically and pharmacologically identified transporters (ASCT1, ASCT2, LAT1, SNATs). Collectively, these data highlight the importance of the tumor environment in the ferroptosis sensitivity of cancer cells. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1., Competing Interests: Declaration of interests M.S. reports grant support from Biogen, Astellas, Bridgebio, and Aucta, and he has served on Scientific Advisory Boards (SAB) for Roche, SpringWorks Therapeutics, and Alkermes and is currently on the SABs for Neurogene, Jaguar Gene Therapy, and Noema. All other authors declare no competing financial interests.

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.