Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.
Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combinedclustered,regularlyinterspacedshortpalindromicrepeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unalteredin vivosettings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.
From the ‡Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Xu
X
Li
From the ‡Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Min
M
Gao
‖Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030.
Jong Min
JM
Choi
‖Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030.
Beom-Jun
BJ
Kim
From the ‡Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Mao-Tian
MT
Zhou
From the ‡Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Zhen
Z
Chen
‖Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030.
Antrix N
AN
Jain
‖Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030.
Sung Yun
SY
Jung
**Department of Radiation Oncology, Center for Radiological Research, Columbia University, New York, New York 10032.
Jingsong
J
Yuan
‡‡Department of Developmental and Cell Biology, University of California at Irvine, Irvine, California 92697 jchen8@mdanderson.org yiw@bcm.edu wenqiw6@uci.edu.
Wenqi
W
Wang
‖Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030; jchen8@mdanderson.org yiw@bcm.edu wenqiw6@uci.edu.
Yi
Y
Wang
From the ‡Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030; jchen8@mdanderson.org yiw@bcm.edu wenqiw6@uci.edu.
Junjie
J
Chen
D064113:CRISPR-Cas Systems; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002470:Cell Survival; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D025542:Neurofibromin 1 / Q000235:genetics / Q000378:metabolism*; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C544374:LAMTOR1 protein, human; D025542:Neurofibromin 1; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases