Emerging evidence reveals enrichment of glioma-initiating cells (GICs) following therapeutic intervention. One factor known to contribute to this enrichment is cellular plasticity-the ability of glioma cells to attain multiple phenotypes. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open-chromatin marks in known astrocytic enhancers for interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p = 0.001) and positively correlated with that of genes associated with the GIC phenotypes, such as KLF4, c-Myc, and HIF2α (p < 0.001). Immunohistochemical analysis of patient samples demonstrated elevated IL-8 expression in about 60% of recurrent GBM tumors relative to matched primary tumors and this expression also positively correlates with time to recurrence. Exposure to IL-8 significantly enhanced the self-renewing capacity of PDX GBM (average threefold, p < 0.0005), as well as increasing the expression of GIC markers in the CXCR2 population. Furthermore, IL-8 knockdown significantly delayed PDX GBM tumor growth in vivo (p < 0.0005). Finally, guided by in silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that autocrine IL-8 alters cellular plasticity and mediates alterations in histone status. These findings suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.

Chronic inflammation predisposes to aging-associated disease, but it is unknown whether immunity regulation might be important for extending healthy lifespan. Here we show that in C. elegans, dietary restriction (DR) extends lifespan by modulating a conserved innate immunity pathway that is regulated by p38 signaling and the transcription factor ATF-7. Longevity from DR depends upon p38-ATF-7 immunity being intact but downregulated to a basal level. p38-ATF-7 immunity accelerates aging when hyperactive, influences lifespan independently of pathogen exposure, and is activated by nutrients independently of mTORC1, a major DR mediator. Longevity from reduced insulin/IGF-1 signaling (rIIS) also involves p38-ATF-7 downregulation, with signals from DAF-16/FOXO reducing food intake. We conclude that p38-ATF-7 is an immunometabolic pathway that senses bacterial and nutrient signals, that immunity modulation is critical for DR, and that DAF-16/FOXO couples appetite to growth regulation. These conserved mechanisms may influence aging in more complex organisms.

Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation. Glutamine is catabolyzed to α-ketoglutarate (αKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. Here we demonstrate that mTORC1 positively regulates GLS and glutamine flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Heterotaxy characterized by abnormal left-right body asymmetry causes diverse congenital anomalies. Organ rotation is a crucial developmental process to establish the left-right patterning during animal development. However, the molecular basis of how organ rotation is regulated is poorly understood. Here we report that Drosophila UV-resistance associated gene (UVRAG), a tumor suppressor that regulates autophagy and endocytosis, plays unexpected roles in controlling organ rotation. Loss-of-function mutants of UVRAG show seriously impaired organ rotation phenotypes, which are associated with defects in endocytic trafficking rather than autophagy. Blunted endocytic degradation by UVRAG deficiency causes endosomal accumulation of Notch, resulting in abnormally enhanced Notch activity. Knockdown of Notch itself or expression of a dominant negative form of Notch transcriptional co-activator Mastermind is sufficient to rescue the rotation defect in UVRAG mutants. Consistently, UVRAG-mutated heterotaxy patient cells also display highly increased Notch protein levels. These results suggest evolutionarily conserved roles of UVRAG in organ rotation by regulating Notch endocytic degradation.

Neural stem cell (NSC)-based carriers have been presented as promising therapeutic tools for the treatment of infiltrative brain tumors due to their intrinsic tumor homing property. They have demonstrated the ability to migrate towards distant tumor microsatellites and effectively deliver the therapeutic payload, thus significantly improving survival in experimental animal models for brain tumor. Despite such optimistic results, the efficacy of NSC-based anti-cancer therapy has been limited due to the restricted tumor homing ability of NSCs. To examine this issue, we investigated the mechanisms of tumor-tropic migration of an FDA-approved NSC line, HB1.F3.CD, by performing a gene expression analysis. We identified vascular endothelial growth factor-A (VEGFA) and membrane-bound matrix metalloproteinase (MMP14) as molecules whose expression are significantly elevated in migratory NSCs. We observed increased expression of VEGF receptor 2 (VEGFR2) in the focal adhesion complexes of migratory NSCs, with downstream activation of VEGFR2-dependent kinases such as p-PLCγ, p-FAK, and p-Akt, a signaling cascade reported to be required for cellular migration. In an in vivo orthotopic glioma xenograft model, analysis of the migratory trail showed that NSCs maintained expression of VEGFR2 and preferentially migrated within the perivascular space. Knockdown of VEGFR2 via shRNAs led to significant downregulation of MMP14 expression, which resulted in inhibited tumor-tropic migration. Overall, our results suggest, the involvement of VEGFR2-regulated MMP14 in the tumor-tropic migratory behavior of NSCs. Our data warrant investigation of MMP14 as a target for enhancing the migratory properties of NSC carriers and optimizing the delivery of therapeutic payloads to disseminated tumor burdens.

Chronic inflammation facilitates tumor progression. We discovered that a subset of non-small cell lung cancer cells underwent a gradually progressing epithelial-to-mesenchymal (EMT) phenotype following a 21-day exposure to IL-1β, an abundant proinflammatory cytokine in the at-risk for lung cancer pulmonary and the lung tumor microenvironments. Pathway analysis of the gene expression profile and in vitro functional studies revealed that the EMT and EMT-associated phenotypes, including enhanced cell invasion, PD-L1 upregulation, and chemoresistance, were sustained in the absence of continuous IL-1β exposure. We referred to this phenomenon as EMT memory. Utilizing a doxycycline-controlled SLUG expression system, we found that high expression of the transcription factor SLUG was indispensable for the establishment of EMT memory. High SLUG expression in tumors of lung cancer patients was associated with poor survival. Chemical or genetic inhibition of SLUG upregulation prevented EMT following the acute IL-1β exposure but did not reverse EMT memory. Chromatin immunoprecipitation and methylation-specific PCR further revealed a SLUG-mediated temporal regulation of epigenetic modifications, including accumulation of H3K27, H3K9, and DNA methylation, in the CDH1 (E-cadherin) promoter following the chronic IL-1β exposure. Chemical inhibition of DNA methylation not only restored E-cadherin expression in EMT memory, but also primed cells for chemotherapy-induced apoptosis.

Noncanonical mechanistic target of rapamycin (mTOR) pathways remain poorly understood. Mutations in the tumor suppressor folliculin (FLCN) cause Birt-Hogg-Dubé syndrome, a hamartomatous disease marked by mitochondria-rich kidney tumors. FLCN functionally interacts with mTOR and is expressed in most tissues, but its role in fat has not been explored. We show here that FLCN regulates adipose tissue browning via mTOR and the transcription factor TFE3. Adipose-specific deletion of FLCN relieves mTOR-dependent cytoplasmic retention of TFE3, leading to direct induction of the PGC-1 transcriptional coactivators, drivers of mitochondrial biogenesis and the browning program. Cytoplasmic retention of TFE3 by mTOR is sensitive to ambient amino acids, is independent of growth factor and tuberous sclerosis complex (TSC) signaling, is driven by RagC/D, and is separable from canonical mTOR signaling to S6K. Codeletion of TFE3 in adipose-specific FLCN knockout animals rescues adipose tissue browning, as does codeletion of PGC-1β. Conversely, inducible expression of PGC-1β in white adipose tissue is sufficient to induce beige fat gene expression in vivo. These data thus unveil a novel FLCN-mTOR-TFE3-PGC-1β pathway-separate from the canonical TSC-mTOR-S6K pathway-that regulates browning of adipose tissue.

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.

N6-adenosine methylation (m6A) of messenger RNA (mRNA) plays key regulatory roles in gene expression. Accurate measurement of m6A levels is thus critical to understand its dynamic changes in various biological settings. Here, we provide a protocol to quantitate the levels of adenosine and m6A in cellular mRNAs. Using nuclease and phosphatase, we digest mRNA into nucleosides, which are subsequently quantified using liquid chromatography mass spectrometry. For complete details on the use and execution of this protocol, please refer to Cho et al. (2021).

N6-adenosine methylation (m6A) is the most abundant mRNA modification that controls gene expression through diverse mechanisms. Accordingly, m6A-dependent regulation of oncogenes and tumor suppressors contributes to tumor development. However, the role of m6A-mediated gene regulation upon drug treatment or resistance is poorly understood. Here, we report that m6A modification of mitogen-activated protein kinase 13 (MAPK13) mRNA determines the sensitivity of cancer cells to the mechanistic target of rapamycin complex 1 (mTORC1)-targeting agent rapamycin. mTORC1 induces m6A modification of MAPK13 mRNA at its 3' untranslated region through the methyltransferase-like 3 (METTL3)-METTL14-Wilms' tumor 1-associating protein(WTAP) methyltransferase complex, facilitating its mRNA degradation via an m6A reader protein YTH domain family protein 2. Rapamycin blunts this process and stabilizes MAPK13. On the other hand, genetic or pharmacological inhibition of MAPK13 enhances rapamycin's anticancer effects, which suggests that MAPK13 confers a progrowth signal upon rapamycin treatment, thereby limiting rapamycin efficacy. Together, our data indicate that rapamycin-mediated MAPK13 mRNA stabilization underlies drug resistance, and it should be considered as a promising therapeutic target to sensitize cancer cells to rapamycin.

Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial-mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.

Silnoon (Sln) is a monocarboxylate transporter (MCT) that mediates active transport of metabolic monocarboxylates such as butyrate and lactate. Here, we identify Sln as a novel LKB1-interacting protein using Drosophila melanogaster genetic modifier screening. Sln expression does not affect cell cycle progression or cell size but specifically enhances LKB1-dependent apoptosis and tissue size reduction. Conversely, down-regulation of Sln suppresses LKB1-dependent apoptosis, implicating Sln as a downstream mediator of LKB1. The kinase activity of LKB1 induces apical trafficking of Sln in polarized cells, and LKB1-dependent Sln trafficking is crucial for triggering apoptosis induced by extracellular butyrate. Given that LKB1 functions to control both epithelial polarity and cell death, we propose Sln is an important downstream target of LKB1.

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. Here, we have characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. Our Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, we demonstrated that ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, our genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.

As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here, we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.

Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.