Abstract
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 γH2AX 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.
Similar content being viewed by others
References
Brenton JD, Carey LA, Ahmed AA, Caldas C (2005) Molecular classification and molecular forecasting of breast cancer: ready for clinical application? J Clin Oncol 23(29):7350–7360
Irvin WJ Jr, Carey LA (2008) What is triple-negative breast cancer? Eur J Cancer 44(18):2799–2805
Sorlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98(19):10869–10874
Bertucci F, Finetti P, Cervera N, Esterni B, Hermitte F, Viens P, Birnbaum D (2008) How basal are triple-negative breast cancers? Int J Cancer 123(1):236–240
Charafe-Jauffret E, Ginestier C, Monville F et al (2006) Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene 25(15):2273–2284
Neve RM, Chin K, Fridlyand J et al (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10(6):515–527
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298(5600):1912–1934
Hunter T (1987) A thousand and one protein kinases. Cell 50(6):823–829
Krause DS, Van Etten RA (2005) Tyrosine kinases as targets for cancer therapy. N Engl J Med 353(2):172–187
MacKeigan JP, Murphy LO, Blenis J (2005) Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat Cell Biol 7(6):591–600
Bettencourt-Dias M, Giet R, Sinka R et al (2004) Genome-wide survey of protein kinases required for cell cycle progression. Nature 432(7020):980–987
Giroux V, Iovanna J, Dagorn JC (2006) Probing the human kinome for kinases involved in pancreatic cancer cell survival and gemcitabine resistance. FASEB J 20(12):1982–1991
Rahman M, Davis SR, Pumphrey JG, Bao J, Nau MM, Meltzer PS, Lipkowitz S (2009) TRAIL induces apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype. Breast Cancer Res Treat 113(2):217–230
http://www.stanford.edu/group/gozani/Histone%20extraction%20protocol.pdf [cited]
McGowan CH, Russell P (1993) Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J 12(1):75–85
Palmer BD, Thompson AM, Booth RJ et al (2006) 4-Phenylpyrrolo[3, 4-c]carbazole-1, 3(2H, 6H)-dione inhibitors of the checkpoint kinase Wee1. Structure–activity relationships for chromophore modification and phenyl ring substitution. J Med Chem 49(16):4896–4911
Iorns E, Lord CJ, Grigoriadis A et al (2009) Integrated functional, gene expression and genomic analysis for the identification of cancer targets. PLoS ONE 4(4):e5120
Niida H, Nakanishi M (2006) DNA damage checkpoints in mammals. Mutagenesis 21(1):3–9
Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146(5):905–916
Tyner JW, Walters DK, Willis SG et al (2008) RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia. Blood 111(4):2238–2245
O’Connell MJ, Raleigh JM, Verkade HM, Nurse P (1997) Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J 16(3):545–554
Yuli C, Shao N, Rao R et al (2007) BRCA1a has antitumor activity in TN breast, ovarian and prostate cancers. Oncogene 26(41):6031–6037
Wang Y, Li J, Booher RN, Kraker A, Lawrence T, Leopold WR, Sun Y (2001) Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator. Cancer Res 61(22):8211–8217
Li J, Wang Y, Sun Y, Lawrence TS (2002) Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1 kinase inhibitor. Radiat Res 157(3):322–330
Wang Y, Decker SJ, Sebolt-Leopold J (2004) Knockdown of Chk1, Wee1 and Myt1 by RNA interference abrogates G2 checkpoint and induces apoptosis. Cancer Biol Ther 3(3):305–313
Hashimoto O, Shinkawa M, Torimura T, Nakamura T, Selvendiran K, Sakamoto M, Koga H, Ueno T, Sata M (2006) Cell cycle regulation by the Wee1 inhibitor PD0166285, pyrido [2, 3-d] pyrimidine, in the B16 mouse melanoma cell line. BMC Cancer 6:292
Syljuasen RG, Sorensen CS, Hansen LT et al (2005) Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25(9):3553–3562
Niida H, Tsuge S, Katsuno Y, Konishi A, Takeda N, Nakanishi M (2005) Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe. J Biol Chem 280(47):39246–39252
Sidi S, Sanda T, Kennedy RD et al (2008) Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. Cell 133(5):864–877
Turner N, Tutt A, Ashworth A (2004) Hallmarks of ‘BRCAness’ in sporadic cancers. Nat Rev Cancer 4(10):814–819
Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat 28(6):622–629
Chauvier D, Lecoeur H, Langonne A, Borgne-Sanchez A, Mariani J, Martinou JC, Rebouillat D, Jacotot E (2005) Upstream control of apoptosis by caspase-2 in serum-deprived primary neurons. Apoptosis 10(6):1243–1259
Gregoli PA, Bondurant MC (1999) Function of caspases in regulating apoptosis caused by erythropoietin deprivation in erythroid progenitors. J Cell Physiol 178(2):133–143
Pereira NA, Song Z (2008) Some commonly used caspase substrates and inhibitors lack the specificity required to monitor individual caspase activity. Biochem Biophys Res Commun 377(3):873–877
Schellens JH, Leijen S, Shaprio GI et al. (2009) A phase I and pharmacological study of MK-1775, a Wee1 tyrosine kinase inhibitor, in both monotherapy and in combination with gemcitabine, cisplatin, or carboplatin in patients with advanced solid tumors. J Clin Oncol 27(15s):148s
Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, Pommier Y (2008) GammaH2AX and cancer. Nat Rev Cancer 8(12):957–967
Sedelnikova OA, Bonner WM (2006) GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence. Cell Cycle 5(24):2909–2913
Hong Y, Cervantes RB, Tichy E, Tischfield JA, Stambrook PJ (2007) Protecting genomic integrity in somatic cells and embryonic stem cells. Mutat Res 614(1–2):48–55
Opar A (2009) Novel anticancer strategy targets DNA repair. Nat Rev Drug Discov 8(6):437–438
O'Shaughnessy J, Osborne C, Pippen J et al (2009) Efficacy of BSI-201, a poly(ADP-ribose) polymerase-1 (PARP1) inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): results of a randomised phase II trial. J Clin Oncol 27(15s):6s
Ashwell S, Zabludoff S (2008) DNA damage detection and repair pathways—recent advances with inhibitors of checkpoint kinases in cancer therapy. Clin Cancer Res 14(13):4032–4037
Acknowledgments
We thank Marion Nau for critical reading of this manuscript. Financial Support This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Author information
Authors and Affiliations
Corresponding author
Additional information
Lyndsay M. Murrow and Sireesha V. Garimella contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Murrow, L.M., Garimella, S.V., Jones, T.L. et al. Identification of WEE1 as a potential molecular target in cancer cells by RNAi screening of the human tyrosine kinome. Breast Cancer Res Treat 122, 347–357 (2010). https://doi.org/10.1007/s10549-009-0571-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10549-009-0571-2