Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for ...asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation.
Alteration of the PTEN/PI3K pathway is associated with late-stage and castrate-resistant prostate cancer (CRPC). However, how PTEN loss is involved in CRPC development is not clear. Here, we show ...that castration-resistant growth is an intrinsic property of
Pten null prostate cancer (CaP) cells, independent of cancer development stage. PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of
Ar in the epithelium promotes the proliferation of
Pten null cancer cells, at least in part, by downregulating the androgen-responsive gene
Fkbp5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy.
► Castration-resistant proliferation of Pten null prostate cancer cells ► PTEN loss suppresses AR-transcription factor activity and promotes AR independence ► AR inhibits AKT through FKBP5 scaffold protein and PHLPP phosphatase ► Cotargeting of AR and AKT/mTOR signaling leads to better therapeutic outcome
Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes ...in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation.
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•Adenovirus E4ORF1 promotes increased glycolytic flux in cultured epithelial cells•E4ORF1 augments MYC-dependent transcription of key metabolic genes•E4ORF1 activation of MYC increases nucleotide biosynthesis from glucose metabolites•E4ORF1-induced MYC activation promotes optimal adenovirus replication
Upon infection, viruses exploit the host cell’s machinery to reprogram cellular metabolism for virus progeny generation. Thai et al. show that adenovirus reprograms host cell glucose metabolism via E4ORF1-induced MYC activation, which enhances glycolytic flux and enables nucleotide biosynthesis from glucose metabolites to promote optimal viral replication.
Colony stimulating factor 1 (CSF-1) recruits tumor-infiltrating myeloid cells (TIM) that suppress tumor immunity, including M2 macrophages and myeloid-derived suppressor cells (MDSC). The CSF-1 ...receptor (CSF-1R) is a tyrosine kinase that is targetable by small molecule inhibitors such as PLX3397. In this study, we used a syngeneic mouse model of BRAF(V600E)-driven melanoma to evaluate the ability of PLX3397 to improve the efficacy of adoptive cell therapy (ACT). In this model, we found that combined treatment produced superior antitumor responses compared with single treatments. In mice receiving the combined treatment, a dramatic reduction of TIMs and a skewing of MHCII(low) to MHCII(hi) macrophages were observed. Furthermore, mice receiving the combined treatment exhibited an increase in tumor-infiltrating lymphocytes (TIL) and T cells, as revealed by real-time imaging in vivo. In support of these observations, TILs from these mice released higher levels of IFN-γ. In conclusion, CSF-1R blockade with PLX3397 improved the efficacy of ACT immunotherapy by inhibiting the intratumoral accumulation of immunosuppressive macrophages.
PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy ...based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.
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•PD-L1 is primarily regulated by interferon gamma signaling in melanoma cells•PD-L2 is regulated by both interferon beta and gamma signaling•Regulation of PD-1 ligands works mainly through the JAK1/2-STAT1/3-IRF1 axis
Garcia-Diaz et al. performed a small hairpin RNA screen and genetic and functional studies to map the signaling pathways that result in reactive PD-L1 and PD-L2 on melanoma cells upon interferon gamma exposure. The authors highlight the importance of the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis for clinical responses to PD-1 blockade therapy.
Significance MITF, a master regulator of melanocytes and a major melanoma oncogene amplified in 30-40% of melanomas, determines proliferative or invasive phenotypes. Previously unrecognized as a ...driver of lysosomal biogenesis, we found that MITF expression correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis. This accumulation of incomplete organelles expands the late endosomal compartment, enhancing Wnt signaling by entrapping the Wnt machinery in multivesicular bodies. Wnt signaling can stabilize many proteins besides β-catenin. Our study identifies MITF as an oncogenic protein stabilized by Wnt, and describes three novel glycogen synthase kinase 3-regulated phosphorylation sites in this oncogene. This study deepens our knowledge on proliferative stages of melanoma: MITF, multivesicular bodies, and Wnt may form a feedback loop that drives proliferation.
Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.
The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including ...inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.
Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated, primed, pluripotent states established by specific culture conditions. Increased intracellular ...α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate, and elevated succinate levels delay, primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead, αKG induced, and succinate impaired, global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state.
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•hPSCs produce TCA cycle metabolites despite low OXPHOS•α-ketoglutarate (αKG) promotes early differentiation of hPSCs•Accumulation of succinate or depletion of αKG delays differentiation of hPSCs•αKG/succinate alters histone methylation corresponding to differentiation kinetics
α-ketoglutarate (αKG) is an important cofactor for demethylation reactions that helps to maintain naive pluripotent stem cells. TeSlaa et al. show that at later stages of pluripotency, αKG can promote early differentiation, highlighting that the cellular context and potentially the stage of cellular maturity can alter the effect of αKG.
The use of potent therapies inhibiting critical oncogenic pathways active in epithelial cancers has led to multiple resistance mechanisms, including the development of highly aggressive, small cell ...neuroendocrine carcinoma (SCNC). SCNC patients have a dismal prognosis due in part to a limited understanding of the molecular mechanisms driving this malignancy and the lack of effective treatments. Here, we demonstrate that a common set of defined oncogenic drivers reproducibly reprograms normal human prostate and lung epithelial cells to small cell prostate cancer (SCPC) and small cell lung cancer (SCLC), respectively. We identify shared active transcription factor binding regions in the reprogrammed prostate and lung SCNCs by integrative analyses of epigenetic and transcriptional landscapes. These results suggest that neuroendocrine cancers arising from distinct epithelial tissues may share common vulnerabilities that could be exploited for the development of drugs targeting SCNCs.
Type I interferons (IFN-α and IFN-β) are important for protection against many viral infections, whereas type II interferon (IFN-γ) is essential for host defense against some bacterial and parasitic ...pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-β and IFN-γ gene expression programs. IFN-γ and its downstream vitamin D—dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-γ—induced macrophage vitamin D—dependent antimicrobial peptide response was inhibited by IFN-β and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.
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