11 months ago

2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Monitoring cellular signaling and autophagy Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to a lysosome for degradation. Phosphatidylinositol-3-phosphate, or PI(3)P, is generated on the earliest autophagic membrane (phagophore) and recruits effector proteins needed for this process. The production of PI(3)P by the class III PI3- kinase Vps34 has been well established, but phosphatases that dephosphorylate this lipid during autophagy are unknown. To identify such enzymes, we screened human phosphatase genes by RNA interference (RNAi) and found that loss of a specific phosphatase in the human genome increases cellular PI(3)P and hyperactivates autophagy. This autophagic phenotype was confirmed in knock-out MEFs when compared with wild-type counterparts. Further, we discovered that this classically defined phosphatase harbors lipid phosphatase activity and its active site binds PI(3)P. Our findings suggest a novel role for these enzymes in cancer and provide insight into the regulation of autophagy. Mechanistic knowledge of this process is critical for understanding and targeting therapies for several human diseases, including Alzheimer disease and prostate cancer, in which abnormal autophagy may be pathological. Uncontrolled cellular survival and chemoresistance is a therapeutic problem that severely limits successful treatment of most human cancers. This is particularly true of colorectal cancer, in which the development of resistance is common: most anticancer regimens are ineffective, with the five-year survival rates for late-stage colorectal cancer being only 8%. How colorectal cancer resistance develops is largely unknown, and the response to therapy varies based on individual patient tumors. With this in mind, how can we prevent cancer emergence or progression at the level of individual tumors? Recent studies have shown that a large percentage of colorectal tumors have mutations in a key gene, for class I PI3 kinase. While mutations play an important causative role in colorectal cancer, it is currently unclear how these mutations can be exploited as drug targets and whether we can develop targeted cancer agents based on the gene. We have ongoing projects to determine the molecular pathways (and genes) that can be used to prevent the progression of precancerous lesions to colorectal cancer. Parkinson disease–associated genes in cancer Dysregulation of receptor tyrosine kinase signaling is a common oncogenic mechanism in human cancer. Abnormal activation of these receptors is associated with a loss of growth factor dependence, resulting in uncontrolled proliferation and survival of cancer cells. The receptor tyrosine kinase MET is often genetically amplified and overexpressed in human tumors. Because simple overexpression of MET is insufficient to mediate its activation, additional “hits” such as activating mutations or overexpression of its ligand, hepatocyte growth factor (HGF), often accompany MET genetic amplification. In the absence of these secondary events, it is not always clear how MET becomes activated and drives oncogenesis. We have identified a novel mechanism for MET activation that is driven by genetic co-amplification of a second protein kinase. The identification of alternative mechanisms that mediate MET activation in these instances is crucial for Figure 2 the design of rationally targeted therapies aimed at interruption of oncogenic signaling in cancer. This project is a collaboration with VARI’s Kyle Furge, Bin Teh, and George Vande Woude. Figure 2. Depletion of specific kinases using RNAi decreases receptor tyrosine kinase activation. Global analysis of RTK phosphorylation in kidney cancer cells demonstrates significant activation of only EGFR (black arrow) and MET (white arrow) under basal conditions. 32

VARI | 2009 External Collaborators Dana Farber Cancer Institute, Boston, Massachusetts McGill Cancer Center, Montreal, Canada Michigan Medical, P.C., Grand Rapids Michigan State University, East Lansing Newcastle University, Newcastle upon Tyne, U.K. Novartis Institutes for Biomedical Research, Cambridge, Massachusetts Ontario Cancer Institute, Toronto, Canada Spectrum Health, Grand Rapids, Michigan St. Jude Children’s Hospital, Memphis, Tennessee Translational Genomics Research Institute, Phoenix, Arizona University of Virginia, Charlottesville Recent Publications Nicklin, Paul, Philip Bergman, Bailin Zhang, Ellen Triantafellow, Henry Wang, Beat Nyfeler, Haidi Yang, Marc Hild, Charles Kung, Christopher Wilson, et al. 2009. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136(3): 521–534. Looyenga, Brendan D., Alyse M. DeHaan, and Jeffrey P. MacKeigan. 2008. PINK1 (PARK6). UCSD-Nature Molecule Pages. June. doi:10.1038/mp.a003826.01. From left: Sian, Wolters, MacKeigan, Nelson, Looyenga, Goodall, Karnes, Westrate 33

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