SNAT7 regulates mTORC1 via macropinocytosis.

Mammalian target of rapamycin complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Some amino acids signal to mTORC1 through the Rag GTPase, whereas glutamine and asparagine activate mTORC1 through a Rag GTPase-independent pathway. Here, we show that the lysosomal glutamine and asparagine transporter SNAT7 activates mTORC1 after extracellular protein, such as albumin, is macropinocytosed. The N terminus of SNAT7 forms nutrient-sensitive interaction with mTORC1 and regulates mTORC1 activation independently of the Rag GTPases. Depletion of SNAT7 inhibits albumin-induced mTORC1 lysosomal localization and subsequent activation. Moreover, SNAT7 is essential to sustain KRAS-driven pancreatic cancer cell growth through mTORC1. Thus, SNAT7 links glutamine and asparagine signaling from extracellular protein to mTORC1 independently of the Rag GTPases and is required for macropinocytosis-mediated mTORC1 activation and pancreatic cancer cell growth.

AKAP13 couples GPCR signaling to mTORC1 inhibition.

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.

GRP75 mediates endoplasmic reticulum-mitochondria coupling during palmitate-induced pancreatic ß-cell apoptosis.

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER-mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca2+ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca2+ levels and ROS generation, augment ER-mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER-mitochondrial interaction leading to apoptosis in pancreatic islet cells.

YTHDF3 Induces the Translation of m6A-Enriched Gene Transcripts to Promote Breast Cancer Brain Metastasis.

Brain metastasis is a major cause of cancer mortality, but its molecular mechanisms are severely understudied. In addition, little is known regarding the role of m6A reader YTHDF3 in human diseases. Here, we show that YTHDF3 overexpression clinically correlates with brain metastases in breast cancer patients. YTHDF3 promotes cancer cell interactions with brain endothelial cells and astrocytes, blood-brain barrier extravasation, angiogenesis, and outgrow. Mechanistically, YTHDF3 enhances the translation of m6A-enriched transcripts for ST6GALNAC5, GJA1, and EGFR, all associated with brain metastasis. Furthermore, overexpression of YTHDF3 in brain metastases is attributed to increased gene copy number and the autoregulation of YTHDF3 cap-independent translation by binding to m6A residues within its own 5' UTR. Our work uncovers an essential role of YTHDF3 in controlling the interaction between cancer cells and brain microenvironment, thereby inducing brain metastatic competence.

Altered Lipid Tumor Environment and Its Potential Effects on NKT Cell Function in Tumor Immunity.

Natural killer T (NKT) cells are CD1d restricted T cells that mostly recognize lipid antigens. These cells share characteristics with both adaptive and innate immune cells and have multiple immunoregulatory roles. In a manner similar to innate immune cells, they respond quickly to stimuli and secrete large amounts of cytokines, amplifying and modulating the immune response. As T cells, they express T cell receptors (TCRs) and respond in an antigen-specific manner like conventional T cells. There are at least two subtypes of NKT cells, type I and type II, that differ in the nature of their TCR, either semi-invariant (type I) or diverse (type II). The two sub-types generally have opposing functions in tumor immunity, with type I promoting and type II suppressing tumor immunity, and they cross-regulate each other, forming an immunoregulatory axis. The tumor has multiple mechanisms by which it can evade immune-surveillance. One such mechanism involves alteration in tumor lipid repertoire and accumulation of lipids and fatty acids that favor tumor growth and evade anti-tumor immunity. Since NKT cells mostly recognize lipid antigens, an altered tumor lipid metabolic profile will also alter the repertoire of lipid antigens that can potentially affect their immune-modulatory function. In this review, we will explore the effects of alterations in the lipid metabolites on tumor growth, antigen cross-presentation, and overall effect on anti-tumor immunity, especially in the context of NKT cells.

Long non-coding RNA H19 inhibition promotes hyperglycemia in mice by upregulating hepatic FoxO1 levels and promoting gluconeogenesis.

In a previous report from our laboratory, it was reported that hepatic levels of the long non-coding RNA (lncRNA), H19 are decreased in diabetic mice which elevates hepatic gluconeogenesis and glucose output. But, the mechanisms of H19 inhibition in elevating gluconeogenic genes' transcription and promoting hepatic glucose output were not known. In this study, we aimed to decipher this regulatory role of H19 on glucose metabolism and on FoxO1, an important transcriptional regulator of gluconeogenesis. While H19 inhibition in HepG2 cells increased the levels of FoxO1, its overexpression led to significant inhibition in FoxO1 levels, thereby identifying H19 as an important regulator of FoxO1. Our data also demonstrates that in the absence of H19, there is increased occupancy of p53 on the FoxO1 promoter that possibly is responsible for increased FoxO1 transcription. In vivo silencing of H19 in normal mice caused hyperglycemia, hyperinsulinemia and impaired glucose, insulin, and pyruvate tolerance. Serum triglyceride and cholesterol levels, however, did not show any change. H19 inhibition significantly elevated the hepatic levels of FoxO1 and all the gluconeogenic genes. While fasting increased gluconeogenic genes' transcription, the levels of H19 were decreased and these patterns reversed upon refeeding the mice. Thus, gluconeogenic genes and H19 levels show inverse patterns of expression, and these results indicate towards an important regulatory role of the lncRNA, H19. It acts as an upstream regulator of gluconeogenesis by regulating the transcription of FoxO1, an important transcription factor of gluconeogenic genes, and hence, regulates hepatic glucose metabolism. KEY MESSAGES: H19 regulates FoxO1 transcript and protein levels. H19 inhibition increases p53 occupancy on the FoxO1 promoter that promotes FoxO1 transcription. H19 inhibition in vivo induces hyperglycemia and impairs glucose, insulin, and pyruvate tolerance. In vivo H19 inhibition increases the hepatic transcript levels of gluconeogenic genes and FoxO1. Transcript levels of H19 and gluconeogenic genes are inversely regulated during fed and fasted states.

Intratumorally delivered formulation, INT230-6, containing potent anticancer agents induces protective T cell immunity and memory.

The benefits of anti-cancer agents extend beyond direct tumor killing. One aspect of cell death is the potential to release antigens that initiate adaptive immune responses. Here, a diffusion enhanced formulation, INT230-6, containing potent anti-cancer cytotoxic agents, was administered intratumorally into large (approx. 300mm3) subcutaneous murine Colon26 tumors. Treatment resulted in regression from baseline in 100% of the tumors and complete response in up to 90%. CD8+ or CD8+/CD4+ T cell double-depletion at treatment onset prevented complete responses, indicating a critical role of T cells in promoting complete tumor regression. Mice with complete response were protected from subcutaneous and intravenous re-challenge of Colon26 cells in a CD4+/CD8+ dependent manner. Thus, immunological T cell memory was induced by INT230-6. Colon26 tumors express the endogenous retroviral protein gp70 containing the CD8+ T-cell AH-1 epitope. AH-1-specific CD8+ T cells were detected in peripheral blood of tumor-bearing mice and their frequency increased 14 days after treatment onset. AH-1-specific CD8+ T cells were also significantly enriched in tumors of untreated mice. These cells had an activated phenotype and highly expressed Programmed cell-death protein-1 (PD-1) but did not lead to tumor regression. CD8+ T cell tumor infiltrate also increased 11 days after treatment. INT230-6 synergized with checkpoint blockade, inducing a complete remission of the primary tumors and shrinking of untreated contralateral tumors, which demonstrates not only a local but also systemic immunological effect of the combined therapy. Similar T-cell dependent inhibition of tumor growth was also found in an orthotopic 4T1 breast cancer model.

Metastatic Brain Tumors Disrupt the Blood-Brain Barrier and Alter Lipid Metabolism by Inhibiting Expression of the Endothelial Cell Fatty Acid Transporter Mfsd2a.

Disruption of the blood-brain barrier (BBB) by cancer cells is linked to metastatic tumor initiation and progression; however, the pathways that drive these events remain poorly understood. Here, we have developed novel patient-derived xenograft (PDX) models of brain metastases that recapitulate pathological growth features found in original patient samples, thus allowing for analysis of BBB disruption by tumor cells. We report that the BBB is selectively disrupted in brain metastases, in part, via inhibition of the endothelial cell-expressed docosahexaenoic acid (DHA) transporter, major facilitator superfamily domain 2a (Mfsd2a). Loss of Mfsd2a expression in the tumor endothelium results in enhanced BBB leakage, but reduced DHA transport and altered lipid metabolism within metastases. Mfsd2a expression in normal cerebral endothelial cells is cooperatively regulated by TGFß and bFGF signaling pathways, and these pathways are pathologically diminished in the brain metastasis endothelium. These results not only reveal a fundamental pathway underlying BBB disruption by metastatic cancer cells, but also suggest that restoring DHA metabolism in the brain tumor microenvironment may be a novel therapeutic strategy to block metastatic cell growth and survival.

Correction: FRIZZLED7 Is Required for Tumor Inititation and Metastatic Growth of Melanoma Cells.

[This corrects the article DOI: 10.1371/journal.pone.0147638.].

FRIZZLED7 Is Required for Tumor Initiation and Metastatic Growth of Melanoma Cells.

Metastases are thought to arise from cancer stem cells and their tumor initiating abilities are required for the establishment of metastases. Nevertheless, in metastatic melanoma, the nature of cancer stem cells is under debate and their contribution to metastasis formation remains unknown. Using an experimental metastasis model, we discovered that high levels of the WNT receptor, FZD7, correlated with enhanced metastatic potentials of melanoma cell lines. Knocking down of FZD7 in a panel of four melanoma cell lines led to a significant reduction in lung metastases in animal models, arguing that FZD7 plays a causal role during metastasis formation. Notably, limiting dilution analyses revealed that FZD7 is essential for the tumor initiation of melanoma cells and FZD7 knockdown impeded the early expansion of metastatic melanoma cells shortly after seeding, in accordance with the view that tumor initiating ability of cancer cells is required for metastasis formation. FZD7 activated JNK in melanoma cell lines in vitro and the expression of a dominant negative JNK suppressed metastasis formation in vivo, suggesting that FZD7 may promote metastatic growth of melanoma cells via activation of JNK. Taken together, our findings uncovered a signaling pathway that regulates the tumor initiation of melanoma cells and contributes to metastasis formation in melanoma.

Suppression of induced but not developmental apoptosis in Drosophila by Ayurvedic Amalaki Rasayana and Rasa-Sindoor.

Earlier we showed formulation-specific beneficial effects of dietary supplement of Ayurvedic Amalaki Rasayana (AR, a herbal formulation) and Rasa-Sindoor (RS, a mercury-based organo-metallic formulation) on various biological parameters in Drosophila, parallel to traditional Ayurvedic literature. These formulations also suppressed cell death and pathology in fly models of neurodegeneration. To understand basis of inhibition of apoptosis, we examined effects of AR and RS on induced and developmental apoptosis in Drosophila. Dietary AR or RS significantly reduced apoptosis induced by GMR-GAL4-, sev-GAL4- or hs-GAL4-directed expression of Rpr, Hid or Grim (RHG) proapoptotic proteins or by GMR-GAL4-directed DIAP1-RNAi, resulting in significant restoration of organism's viability and eye morphology. AR or RS supplement enhanced levels of inhibitor of apoptosis proteins, DIAP1 and DIAP2, and of Bancal/Hrb57A, while the levels of RHG proteins and of initiator Dronc and effecter Drice caspases were reduced in non-apoptotic wild type as well as in RHG over-expressing tissues. Levels of Dronc or Drice remained unaffected in cells developmentally destined to die so that developmental apoptosis occurred normally. Elevated levels of DIAPs and reduced levels of RHG proteins and caspases reflect a more robust physiological state of AR or RS fed organisms allowing them to tolerate greater insults without triggering the cell-death response. Such homeostatic effects of these Rasayanas seem to contribute to 'healthy ageing', one of their effects suggested in traditional Ayurvedic practices.