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Pancreatic ductal adenocarcinoma (PDA) is aggressive cancer characterized by rapid progression, metastatic recurrence, and highly resistant to treatment. PDA cells exhibit aerobic glycolysis, or the Warburg effect, which reduces the flux of pyruvate into mitochondria. As a result, more glycolytic metabolites are shunted to pathways for the production of building blocks (e.g., ribose) and reducing agents (e.g., NADPH) for biosynthesis that are necessary for cell proliferation. In addition, PDA cells are highly addicted to glutamine for both maintaining biosynthetic pathways and achieving redox balance. Mitochondrial uncoupling facilitates proton influx across the mitochondrial inner membrane without generating ATP, leading to a futile cycle that consumes glucose metabolites and glutamine. We synthesized a new mitochondrial uncoupler MB1-47 and tested its effect on cancer cell metabolism and the anticancer activity in pancreatic cancer cell models and murine tumor transplantation models. MB1-47 uncouples mitochondria in the pancreatic cancer cells, resulting in: (1) the acceleration of pyruvate oxidation and TCA turnover; (2) increases in AMP/ATP and ADP/AMP ratios; and (3) a decrease in the synthesis rate of nucleotides and sugar nucleotides. Moreover, MB1-47 arrests cell cycle at G0-G1 phase, reduces clonogenicity, and inhibits cell growth of murine and human pancreatic cancer cells. In vivo studies showed that MB1-47 inhibits tumor growth in murine tumor transplantation models, and inhibits the hepatic metastasis when tumor cells were transplanted intrasplenically. Our results provide proof of concept for a potentially new strategy of treating PDA, and a novel prototype experimental drug for future studies and development.

Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point(TM) that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

Metabolism of cancer cells is characterized by aerobic glycolysis, or the Warburg effect. Aerobic glycolysis reduces pyruvate flux into mitochondria, preventing a complete oxidation of glucose and shunting glucose to anabolic pathways essential for cell proliferation. Here we tested a new strategy, mitochondrial uncoupling, for its potential of antagonizing the anabolic effect of aerobic glycolysis and for its potential anticancer activities. Mitochondrial uncoupling is a process that facilitates proton influx across the mitochondrial inner membrane without generating ATP, stimulating a futile cycle of acetyl- CoA oxidation. We tested two safe mitochondrial uncouplers, NEN (niclosamide ethanolamine) and oxyclozanide, on their metabolic effects and anti-cancer activities. We used metabolomic NMR to examine the effect of mitochondrial uncoupling on glucose metabolism in colon cancer MC38 cells. We further tested the anti-cancer effect of NEN and oxyclozanide in cultured cell models, APCmin/+ mouse model, and a metastatic colon cancer mouse model. Using a metabolomic NMR approach, we demonstrated that mitochondrial uncoupling promotes pyruvate influx to mitochondria and reduces various anabolic pathway activities. Moreover, mitochondrial uncoupling inhibits cell proliferation and reduces clonogenicity of cultured colon cancer cells. Furthermore, oral treatment with mitochondrial uncouplers reduces intestinal polyp formation in APCmin/+ mice, and diminishes hepatic metastasis of colon cancer cells transplanted intrasplenically. Our data highlight a unique approach for targeting cancer cell metabolism for cancer prevention and treatment, identified two prototype compounds, and shed light on the anti-cancer mechanism of niclosamide.

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

Type 2 diabetes (T2D) has reached an epidemic level globally. Most current treatments ameliorate the hyperglycemic symptom of the disease but are not effective in correcting its underlying cause. One important causal factor of T2D is ectopic accumulation of lipids in metabolically sensitive organs such as liver and muscle. Mitochondrial uncoupling, which reduces cellular energy efficiency and increases lipid oxidation, is an appealing therapeutic strategy. The challenge, however, is to discover safe mitochondrial uncouplers for practical use. Niclosamide is an anthelmintic drug approved by the US Food and Drug Administration that uncouples the mitochondria of parasitic worms. Here we show that niclosamide ethanolamine salt (NEN) uncouples mammalian mitochondria at upper nanomolar concentrations. Oral NEN increases energy expenditure and lipid metabolism in mice. It is also efficacious in preventing and treating hepatic steatosis and insulin resistance induced by a high-fat diet. Moreover, it improves glycemic control and delays disease progression in db/db mice. Given the well-documented safety profile of NEN, our study provides a potentially new and practical pharmacological approach for treating T2D.

Autophagy is a mechanism for intracellular degradation of cytoplasmic components including macromolecules and organelles. It has been shown to be involved in a large number of physiological and pathological processes. Studies have recently uncovered an important role of autophagy in the process of adipogenesis. In addition, some interesting correlations between autophagy activity and adipose functions have been made. This chapter explores the roles autophagy plays in adipocytes, detailing the core machinery of autophagy and highlighting what is currently understood about the functions of autophagy in differentiating and mature white adipocytes.

Autophagy is an evolutionarily conserved cytoplasmic degradation pathway. A fundamental function of autophagy at the cellular level is to recycle cytoplasmic components, including macromolecules and organelles. Autophagy is essential for cell survival under nutrient deprived conditions. In addition, working in concert with other proteins in cells, the regulated and cargo-specific autophagic degradation is crucial for cellular maintenance and reducing cellular damage. Since the initial reports that autophagy is implicated in tumor suppression more than a decade ago, great advancements have been made in research on both autophagy and on its role in tumorigenesis. Here we summarize some of the important results from literature, mostly mouse genetics and human cancer genetics data. These data support a working model that autophagy suppresses tumorigenesis primarily in a cell-­intrinsic and indirect manner, most likely through reducing cellular damage by degrading some tissue-specific cell-damaging factors. In addition, non-cell autonomous processes triggered by autophagy defect may also contribute to tumor development. The current information on the unequivocal role of autophagy in suppressing tumorigenesis strongly favors future exploration in area of developing practical autophagy activating approaches for tumor chemoprevention.

One fundamental change in cancer cells in oncogenesis is the alteration of glucose metabolism. With few exceptions, cancer cells exhibit aerobic glycolysis. A majority of pyruvate derived from glycolysis is converted to lactate instead of entering mitochondria for oxidative phosphorylation. This feature of cancer cells is also referred as “the Warburg effect”. The functional significance of the inefficient metabolic mode to cancer cells is becoming clear. In essence, reduction in the flux of pyruvate into mitochondria prevents complete oxidation of glucose. This increases production of NADPH and metabolic intermediates required for biosynthesis of macromolecules essential for cell proliferation. Pyruvate flux into mitochondria can be dramatically enhanced through mitochondrial uncoupling, a process that allows protons cross the mitochondrial inner membrane without producing ATP. As a result, mitochondrial uncoupler promotes “futile” oxidation of acetyl-CoA, leading to complete glucose oxidation. Therefore, safe mitochondrial uncouplers could be potent anti-cancer agents by antagonizing the function of the Warburg effect. Here we tested this novel cancer chemotherapeutic strategy with niclosamide ethanolamine (NEN), a well-characterized mitochondrial uncoupler with an excellent safety profile, and one of its derivative (OXY-1) in culture cell models and a mouse model. Mouse colon cancer cells (MC38) and mouse pancreatic cells (Panc02) were treated with NEN and OXY-1. At effective mitochondrial uncoupling concentrations, NEN and OXY-1 reduced cells viability, induced cell cycle arrest, and reduced clonegenicity. Moreover, NEN and OXY-1 also inhibited cell migration. These effects were associated with the activation of AMPK and reduction of NADPH/NADP ratio. We further examined the anti- cancer effect of NEN and OXY-1 on a metastatic cancer mouse model. First, we tested if the uncouplers are effective in inhibiting cancer growth. The MC38 or Panc02 cells were injected into the liver of NOD mice. Oral treatment of OXY-1 (800 ppm in food) significantly reduced tumor size and tumor incidence in mouse liver. We then tested if these drugs could inhibit tumor metastasis. MC38 or Panc02 cells were intrasplenically injected into the mice and the tumor metastasis to liver was analyzed. Oral NEN (2000 ppm in food) or OXY-1 (800 ppm in food) either totally prevented tumor metastasis to liver or drastically reduced metastatic tumor numbers and tumor volume. Taken together, our results strongly suggest that the safe and mild mitochondrial uncouplers NEN and OXY-1 could be effective in preventing and treating hepatic metastasis of colon and pancreatic cancers. Our results may provide a new and attractive way for preventing and treating these incurable tumors. Citation Format: AMER H. ALASADI, Jingjing Guo, Hanlin Tao, Shengkan Jin. Preventing and treating hepatic metastatic colon and pancreatic cancers by targeting cell metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 45.