High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven macropinocytosis.
Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of macropinocytosis. To measure oncogene-dependent macropinocytosis, we used HeLa cells expressing oncogenic HRAS(G12D) driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of macropinocytosis. We detected macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70 kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of macropinocytosis as well as novel targets.
1 RNAi and Gene Editing Core Facility, Memorial Sloan-Kettering Cancer Center , New York, New York.
Myles
M
Fennell
2 NCI-Designated Cancer Center Cell Death and Survival Networks Program, Sanford-Burnham Medical Research Institute , La Jolla, California.
Cosimo
C
Commisso
3 Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York.
Craig
C
Ramirez
1 RNAi and Gene Editing Core Facility, Memorial Sloan-Kettering Cancer Center , New York, New York.
Ralph
R
Garippa
3 Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York.
Dafna
D
Bar-Sagi
D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009857:Oncogenes; D010873:Pinocytosis; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
D034741:RNA, Small Interfering; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)