High inorganic phosphate intake promotes tumorigenesis at early stages in a mouse model of lung cancer

© 2015 Lee et al. Inorganic phosphate (Pi) is required by all living organisms for the development of organs such as bone, muscle, brain, and lungs, regulating the expression of several critical genes as well as signal transduction. However, little is known about the effects of prolonged dietary Pi consumption on lung cancer progression. This study investigated the effects of a highphosphate diet (HPD) in a mouse model of adenocarcinoma. K-rasLA1 mice were fed a normal diet (0.3% Pi) or an HPD (1% Pi) for 1, 2, or 4 months. Mice were then sacrificed and subjected to inductively coupled plasma mass/optical emission spectrometry and laser ablation inductively coupled plasma mass-spectrometry analyses, western blot analysis, histopathological, immunohistochemical, and immunocytochemical analyses to evaluate tumor formation and progression (including cell proliferation, angiogenesis, and apoptosis), changes in ion levels and metabolism, autophagy, epithelial-to-mesenchymal transition, and protein translation in the lungs. An HPD accelerated tumorigenesis, as evidenced by increased adenoma and adenocarcinoma rates as well as tumor size. However, after 4 months of the HPD, cell proliferation was arrested, and marked increases in liver and lung ion levels and in energy production via the tricarboxylic acid cycle in the liver were observed, which were accompanied by increased autophagy and decreased angiogenesis and apoptosis. These results indicate that an HPD initially promotes but later inhibits lung cancer progression because of metabolic adaptation leading to tumor cell quiescence. Moreover, the results suggest that carefully regulated Pi consumption are effective in lung cancer prevention

Caspase involvement in autophagy

Caspases are a family of cysteine proteases widely known as the principal mediators of the apoptotic cell death response, but considerably less so as the contributors to the regulation of pathways outside cellular demise. In regards to autophagy, the modulatory roles of caspases have only recently begun to be adequately described. In contrast to apoptosis, autophagy promotes cell survival by providing energy and nutrients through the lysosomal degradation of cytoplasmic constituents. Under basal conditions autophagy and apoptosis cross-regulate each other through an elaborate network of interconnections which also includes the interplay between autophagyrelated proteins (ATGs) and caspases. In this review we focus on the effects of this crosstalk at the cellular level, as we aim to concentrate the main observations from research conducted so far on the fine-tuning of autophagy by caspases. Several members of this protease-family have been found to directly interact with key ATGs involved in different tiers across the autophagic cascade. Therefore, we firstly outline the core mechanism of macroautophagy in brief. In an effort to emphasize the importance of the intricate cross-regulation of ATGs and caspases, we also present examples drawn from Drosophila and plant models regarding the contribution of autophagy to apoptotic cell death during normal development

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Functional characterization of lysosomal interaction of Akt with VRK2

Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns (3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondria) outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy

Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).Funding agencies: he National Cancer Institute, United States CA224175 (D.L.), CA210240 (D.L.), CA232093(D.L.), CA163117 and CA207983 (D.L.), CA163120 (D.L.), CA169416 (D.L.), CA169538 (D.L.),CA218513 (D.L., H.Z.) and AI144301 (D.L., V.P.); the United States Department of Defense, UnitedStates W81XWH-13-1-0425 (D.L.), W81XWH-13-1-0427, W81XWH-13-1-0249 (D.L.), and W81XWH-14-1-0199 (D.L.); National Institutes of Health, United States/WCM CTSC, United States (NIH/NCATS (UL1TR00457) (H.Z.); NIH/NCATS (UL1TR002384) (D.L. and H.Z.); the Hartwell Foundation,United States (D.L.); the Thompson Family Foundation (D.L., D.K.); the STARR Consortium I9-A9-056(D.L., H.Z.) and I8-A8-123 (D.L.); the Pediatric Oncology Experimental Therapeutics Investigator’sConsortium (D.L., T.M.T.); Alex’s Lemonade Stand Foundation, United Sates (D.L.); the Breast Can-cer Research Foundation, United States (D.L.); the Feldstein Medical Foundation (D.L.); the TortolaniFoundation (D.L.); the Clinical &amp; Translational Science Center (D.L., H.Z.); the Mary Kay Ash Chari-table Foundation (D.L., I.M.); the Malcolm Hewitt Weiner Foundation (D.L.); the Manning Founda-tion (DL., A.H.); the Daniel P. and Nancy C. Paduano Family Foundation (D.L); the James PaduanoFoundation (D.L.); the Sohn Foundation (D.L.); the AHEPA Vth District Cancer Research Foundation(D.L., L.B., S.L.); the Daedalus Fund Selma and Lawrence Ruben Science to Industry Bridge Award(D.L.); the Children’s Cancer and Blood Foundation, United States (D.L.); Susan G. Komen Postdoc-toral Fellowship PDF15331556, JST PRESTO, Japan 30021, and JSPS KAKENHI, Japan JP19K23743(A.H.); the National Research Foundation of Korea, South Korea (NRF) grant funded by the Korea government (MSIT) (2019R1C1C1006709, 2018R1A5A2025079, and 2020M3F7A1094093); a grantof the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Insti-tute (KHIDI), funded by the Ministry of Health &amp; Welfare, South Korea (KHIDIHI19C1015010020);"The Alchemist Project" through the Ministry of Trade, Industry and Energy (MOTIE, Korea)(20012443), and Severance Hospital Research fund for Clinical excellence (SHRC) (C-2020-0032and C-2020-0025) (H.S.K.); The Swedish Cancer Society Pancreatic Cancer Fellowship, the Lions In-ternational Postdoctoral fellowship and the Sweden-America stipend (L.B.); the Sa ̃ o Paulo ResearchFoundation (FAPESP) Sa ̃ o Paulo, Brazil (2017/07117-7) (G.C.T); the Swiss National Science Founda-tion (SFNS), Switzerland Postdoc Mobility grant (P2SKP3_174785 and P400PB_186791) (F.A.V.); andthe DoD PRCRP Horizon Award, United States (W81XWH-19-PRCRP-HA) (S.L).</p