Identification of WEE1 as a potential molecular target in cancer cells by RNAi screening of the human tyrosine kinome.
Breast cancers can be classified into those that express the estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER-2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple-negative or basal-like breast cancer). In order to identify potential molecular targets in breast cancer, we performed a synthetic siRNA-mediated RNAi screen of the human tyrosine kinome. A primary RNAi screen conducted in the triple-negative/basal-like breast cancer cell line MDA-MB231 followed by secondary RNAi screens and further studies in this cell line and two additional triple-negative/basal-like breast cancer cell lines, BT20 and HCC1937, identified the G2/M checkpoint protein, WEE1, as a potential therapeutic target. Similar sensitivity to WEE1 inhibition was observed in cell lines from all subtypes of breast cancer. RNAi-mediated silencing or small compound inhibition of WEE1 in breast cancer cell lines resulted in an increase in gammaH2AX levels, arrest in the S-phase of the cell cycle, and a significant decrease in cell proliferation. WEE1-inhibited cells underwent apoptosis as demonstrated by positive Annexin V staining, increased sub-G1 DNA content, apoptotic morphology, caspase activation, and rescue by the pan-caspase inhibitor, Z-VAD-FMK. In contrast, the non-transformed mammary epithelial cell line, MCF10A, did not exhibit any of these downstream effects following WEE1 silencing or inhibition. These results identify WEE1 as a potential molecular target in breast cancer.
Murrow
Lyndsay M
LM
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892-4256, USA.
Garimella
Sireesha V
SV
Jones
Tamara L
TL
Caplen
Natasha J
NJ
Lipkowitz
Stanley
S
D017209:Apoptosis; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis; D011980:Receptors, Progesterone / Q000032:analysis; D013997:Time Factors
D061945:Caspase Inhibitors; D020169:Caspases; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; D006657:Histones; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; C468783:H2AX protein, human; C508053:ERBB2 protein, human; C499349:WEE1 protein, human