SUMMARY Stem cell markers such as NANOG have been implicated in various cancers; however, the functional contribution of NANOG to cancer pathogenesis has remained unclear. Here, we show that Toll-like receptor 4 (TLR4) signaling phosphorylates E2F1 to transactivate NANOG. Down-regulation of Nanog reduces tumor progression. NANOG ChIP-seq identified genes associated with NANOG-dependent mitochondrial metabolic pathways to maintain tumor-initiating stem-like cells (TICs). The causal roles of NANOG in mitochondrial metabolic reprogramming occurred through the inhibition of oxidative phosphorylation (OXPHOS) with decreased production of mitochondrial ROS and activation of fatty acid oxidation (FAO), which was required for self-renewal and drug resistance. Restoration of OXPHOS activity and inhibition of FAO rendered TICs susceptible to a standard care chemotherapy drug, sorafenib. This study provides insights into the mechanisms of NANOG-mediated generation of TICs, tumorigenesis and chemo-resistance due to metabolic reprograming of mitochondrial functions.

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Abstract. The requirements for nuclear targeting of a number of U snRNAs have been studied by analyzing the behavior of in vitro-generated transcripts after microinjection into the cytoplasm of Xenopus oocytes.Like the previously studied U1 snRNA, U2 snRNA is excluded from the nucleus when it does not have the 2,2,7mGpppN cap structure typical of the RNA polymerase II (pol II)-transcribed U snRNAs. Surprisingly, two other pol H-transcribed U snRNAs, U4 and U5, have a much less stringent requirement for the trimethyl cap structure. The 3,-monomethyl triphosphate cap structure of the RNA polymerase III-transcribed U6 snRNA, on the other hand, is shown not to play a role in nuclear targeting. Wheat germ agglutinin, which is known to prevent the import of many proteins into the nucleus, inhibits nuclear uptake of U6, but not of U1 or U5 snRNAs. Conversely, a 2,2,7mGpppG dinucleotide analogue of the trimethyl cap structure inhibits transport of the pol II U snRNAs, but does not detectably affect the transport of either U6 snRNA or a karyophilic protein. From these results it can be deduced that U6 enters the nucleus by a pathway similar or identical to that used by karyophilic proteins. The composite nuclear localization signals of the trimethyl cap-containing U snRNPs, however, do not function in the same way as previously defined nuclear targeting signals.

Monoclonal antibodies directed against the lac carrier protein purified' from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several-clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture' supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. In contrast, the antibody has no effect on the generation ofthe proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-a-D-galactopyranoside. In order to achieve 50% inhibition of transport activity, a 2-to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration ofantibody required for a given degree ofinhibition is proportional to the amount of lac carrier in the membrane. Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.Transport of f-galactosides in Escherichia coli is catalyzed by the product of the lac y gene (1), the lac carrier protein, which translocates 13-galactosides with protons in a symport reaction (2). Accordingly, in the presence of a proton electrochemical gradient (ASH+), hydrogen ion moves down the electrochemical gradient and drives the uphill translocation of sugar (see ref. 3 for a review). The lac carrier was identified as a membranebound protein chemically in 1965 (4) and functionally in 1970 (5). Eight years later in rapid succession, the lac y gene was cloned in a recombinant plasmid, its product was amplified (6) and synthesized in vitro (7), and the sequence of the protein was deduced from the DNA sequence (8). Shortly thereafter, it was demonstrated (9) that lactose transport activity can be solubilized and reconstituted into proteoliposomes, and a highly specific photoaffinity label for the lac carrier was developed (10). Most recently, use of these techniques has led to the purification of a single protein, its identification as the product of the lac y gene, and the demonstration that it is the only polypeptide in the cytoplasmic membrane required for lactose/ proton symport (11, 12).The lac carrier protein is a 46.5 kilodalton (kDal) polypeptide chain of 417 amino acid residues of known sequence (8,11). A preliminary secondary structure model for the molecule has been formulated (unpublished data) based on the hydropathic nature of the protein along its sequence...