A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34 f/f mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34 f/f mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34 f/f mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.LC3 | SQSTM1/p62 | 3-MA | epidermal growth factor receptor | transferrin M acroautophagy (referred to as autophagy hereafter) is a dynamic membrane trafficking process that involves the delivery of intracellular content to lysosomes for degradation. A fully executed autophagy includes the formation of doublemembraned autophagosomes, the fusion of autophagosomes to late endosomes/lysosomes, and the digestion of the enclosed content by lysosomal hydrolases. Autophagy is constantly maintained at the basal level and is up-regulated in response to stress conditions, such as nutrient and energy limitation, hypoxia, and DNA damage. Autophagy is necessary for cellular and tissue homeostasis, by eliminating damaged organelles and misfolded proteins, and its dysregulation is implicated in developmental defects and numerous diseases (1-5).

IntroductionThe irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs.MethodsTwo metastatic human breast cancer cell lines (MDA-MB 231 and BCM2) were used to optimize the conditions of hypoxia and reoxygenation cycles. The percentage of CSCs in the cycling hypoxia selected subpopulation was analyzed based on the CD44, CD24, ESA, and E-cadherin expression by three-color flow cytometry. Colony formation assays were used to assess the ability of this subpopulation to self-renew. Limiting dilution assays were performed to evaluate the tumor-initiating and metastatic ability of this subpopulation. Induction of EMT was examined by the expression of EMT-associated markers and EMT-associated microRNAs.ResultsUsing an optimized hypoxia and reoxygenation regimen, we identified a novel cycling hypoxia-selected subpopulation from human breast cancer cell lines and demonstrated that a stem-like breast cancer cell subpopulation could be expanded through repetitive hypoxia/reoxygenation cycles without genetic manipulation. We also found that cells derived from this novel subpopulation form colonies readily, are highly tumorigenic in immune-deficient mice, and exhibit both stem-like and EMT phenotypes.ConclusionsThese results provide the validity to the newly developed hypoxia/reoxygenation culture system for examining the regulation of CSCs in breast cancer cell lines by niche factors in the tumor microenvironment and developing differential targeting strategies to eradicate breast CSCs.

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Jun NH2-terminal kinases (JNKs) regulate convergent extension movements in Xenopus embryos through the noncanonical Wnt͞ planar cell polarity pathway. In addition, there is a high level of maternal JNK activity spanning from oocyte maturation until the onset of gastrulation that has no defined functions. Here, we show that maternal JNK activation requires Dishevelled and JNK is enriched in the nucleus of Xenopus embryos. Although JNK activity is not required for the glycogen synthase kinase-3-mediated degradation of ␤-catenin, inhibition of the maternal JNK signaling by morpholino-antisense oligos causes hyperdorsalization of Xenopus embryos and ectopic expression of the Wnt͞␤-catenin target genes. These effects are associated with an increased level of nuclear and nonmembrane-bound ␤-catenin. Moreover, ventral injection of the constitutive-active Jnk mRNA blocks ␤-catenininduced axis duplication, and dorsal injection of active Jnk mRNA into Xenopus embryos decreases the dorsal marker gene expression. In mammalian cells, activation of JNK signaling reduces Wnt3A-induced and ␤-catenin-mediated gene expression. Furthermore, activation of JNK signaling rapidly induces the nuclear export of ␤-catenin. Taken together, these results suggest that JNK antagonizes the canonical Wnt pathway by regulating the nucleocytoplasmic transport of ␤-catenin rather than its cytoplasmic stability. Thus, the high level of sustained maternal JNK activity in early Xenopus embryos may provide a timing mechanism for controlling the dorsal axis formation. nucelocytoplasmic transport ͉ Wnt