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Amplification of TLK2 Induces Genomic Instability Via Impairing the G 2−M Checkpoint

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Amplification of TLK2 Induces Genomic Instability Via Impairing the G 2−M Checkpoint Published OnlineFirst August 3, 2016; DOI: 10.1158/1541-7786.MCR-16-0161 DNA Damage and Repair Molecular Cancer Research Amplification of TLK2 Induces Genomic Instability via Impairing the G2–M Checkpoint Jin-Ah Kim1,2,3, Meenakshi Anurag1,2,3, Jamunarani Veeraraghavan1,2,3, Rachel Schiff1,2,3, Kaiyi Li1,2,4, and Xiao-Song Wang1,2,3,5,6,7,8 Abstract Managing aggressive breast cancers with enhanced chromosomal irradiation or doxorubicin. To our knowledge, this is the first report instability (CIN) is a significant challenge in clinics. Previously, we linking TLK2 function to CIN, in contrast to the function of its described that a cell cycle–associated kinase called Tousled-like paralog TLK1 as a guardian of genome stability. This finding yields kinase 2 (TLK2) is frequently deregulated by genomic amplifications new insight into the deregulated DNA damage pathway and þ þ in aggressive estrogen receptor–positive (ER ) breast cancers. In this increased genomic instability in aggressive ER breast cancers. study, it was discovered that TLK2 amplification and overexpression mechanistically impair Chk1/2-induced DNA damage checkpoint Implications: Targeting TLK2 presents an attractive thera- TLK2 fi signaling, leading to a G2–M checkpoint defect, delayed DNA repair peutic strategy for the -ampli ed breast cancers that process, and increased CIN. In addition, TLK2 overexpression mod- possess enhanced genomic instability and aggressiveness. estly sensitizes breast cancer cells to DNA-damaging agents, such as Mol Cancer Res; 14(10); 920–7. Ó2016 AACR. Introduction genome-wide copy number aberrations are often used as a surro- þ gate marker to evaluate the level of CIN in cancer (4). Estrogen receptor–positive (ER ) breast cancers (also known as In the presence of DNA damage during S–G phase, cell cycle is luminal breast cancers) account for a vast majority of all breast 2 arrested at G –M checkpoint to ensure the cells to repair their DNA cancers and can be classified into A and B intrinsic subtypes. In 2 before enter into mitosis. The key regulatory step of mitotic entry contrast to the slow-growing and endocrine-sensitive luminal A þ is the activation of Cdk1 via dephosphorylation of Cdk1 at Thr14 tumors, the luminal B tumors are more aggressive form of ER and Tyr15, which is carried out by the Cdc25 phosphatase (5). breast cancers characterized by higher proliferation index and worse G –M checkpoint signaling in response to DNA damage activates clinical outcome after endocrine therapy. Recent large-scale geno- 2 Chk1 and Chk2, which in turn repress Cdc25 phosphatases, mic profiling studies suggest that the markedly enhanced accumu- resulting in the inactivation of CDK1 and cell-cycle arrest at lation of chromosomal aberrations is characteristic of luminal B G –M checkpoint (6). After DNA repair is completed, the mitotic breast tumors (1). Chromosomal instability (CIN) is the major 2 kinases, such as AURKA and PLK1, have a key role in G –M form of genomic instability in human cancers and is characterized 2 checkpoint recovery. In multiple tumors, the amplification and by an increased rate of numerical and structural alterations in the overexpression of AURKA or PLK1 are known causes of G –M chromosomes. CINs have been linked to disease progression, 2 checkpoint defect and enhanced CIN, which is often related to distant metastasis, and therapeutic resistance in breast cancer (1, increased tumor aggressiveness (7). Thus, targeted agents are 2), which pose a great challenge to clinical management. Because of being actively developed against these mitotic kinases, and the mechanistic connection, it is increasingly accepted that numerical Aurora A kinase inhibitors are now in advanced clinical devel- and structural CINs cannot be considered in isolation (3); thus, opments for treating solid tumors (8, 9). It is therefore critical to discover additional genetic aberrations of cell-cycle kinases inde- pendent of AURKA or PLK1 that promote G –M checkpoint 1 2 Lester & Sue Smith Breast Center, Baylor College of Medicine, Hous- defects and CIN in the luminal B breast cancers so as to develop ton,Texas. 2Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston,Texas. 3Department of Medicine, Baylor College of Medicine, new targeted therapies. Houston, Texas. 4Department of Surgery, Baylor College of Medicine, In our previous study, we have identified a cell-cycle kinase Houston,Texas. 5Department of Molecular and Cellular Biology, Baylor 6 called "Tousled-like kinase 2" (TLK2) that is targeted for ampli- College of Medicine, Houston, Texas. University of Pittsburgh Cancer fi þ Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. 7Depart- cation in approximately 10.5% of ER breast tumors, and ment of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania. amplification of TLK2 appears to be enriched in the luminal B 8 Women's Cancer Research Center, University of Pittsburgh, Pitts- breast cancers. The resulting overexpression of TLK2 endows burgh, Pennsylvania. increased invasiveness of luminal breast cancer cells and correlates þ Note: Supplementary data for this article are available at Molecular Cancer with a poorer outcome of ER breast cancer patients. In the current Research Online (http://mcr.aacrjournals.org/). study, we discovered that TLK2 overexpression correlates with Corresponding Author: Xiaosong Wang, University of Pittsburgh, 5117 Centre increased CIN of breast cancers measured by genome-wide copy Avenue, Room G.5a, Pittsburgh, PA 15213. Phone: 412-623-1587; Fax: 412-623- number aberrations, which is independent of the known CIN 1010; E-mail: [email protected] causal factors, such as AURKA and PLK1. TLKs are nuclear- doi: 10.1158/1541-7786.MCR-16-0161 enriched cell-cycle kinases that have maximal activity during S Ó2016 American Association for Cancer Research. phase and are rapidly inactivated in response to the DNA damage 920 Mol Cancer Res; 14(10) October 2016 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 3, 2016; DOI: 10.1158/1541-7786.MCR-16-0161 Role of TLK2 in Breast Cancer Genomic Instability induced by ionizing radiation (IR; refs. 10, 11). As the role of temperature and then incubated with Alexa 488 goat anti-rabbit TLK2 in DNA damage response (DDR) was largely based on the antibody (Invitrogen) for 1 hour at room temperature. Cells were studies focusing on TLK1 (11, 12), the role of TLK2 in DDR is analyzed using FACSCanto II Cell Analyzer (BD Biosciences). poorly understood, and there is no report about its function in CIN in breast cancer. Our further experimental studies revealed Neutral comet assay the crucial role of TLK2 overexpression in impairing G2–M check- Comet assay kit was purchased from TREVIGEN and assay was point signaling, delayed DNA repair, and increased CIN. These followed as per the manufacturer's protocols (https://www.trevi- data further support the rationale to target the cell-cycle kinase gen.com/cat/1/3/0/CometAssay/). Briefly, 1 Â 105 cells were col- TLK2 in the management of more aggressive luminal breast lected and suspended in 500 mL of cold PBS. A total of 20 mL of cell cancers. suspension was mixed with 200 mL of LM agarose, and then 50 mL of the cell mixture was placed to the sample area of slide. After Materials and Methods incubation at 4C in the dark for 30 minutes, slides were immersed in chilled lysis solution overnight at 4C and incubated in the Genomic instability index calculation chilled neutral electrophoresis buffer for 30 minutes. Following the Affymetrix SNP 6.0 array–based CNV data (level 3) were electrophoresis at 21 V for 75 minutes at 4 C, slides were immersed retrieved for 1,083 The Cancer Genome Atlas (TCGA) breast- in DNA precipitation solution for 30 minutes at room temperature invasive carcinoma samples. To extract a set of high-confidence and then in 70% EtOH for 30 minutes at room temperature. After copy number alternations (CNA) we used the segment mean drying the samples, slides were incubated with 2.5 mg/mL of threshold of 0.3 for copy number gain and À0.3 for copy number propidium iodide in the dark for 30 minutes, and then slides loss, as previously reported (13). For a given sample, we calculated were dried at 37 C. Tail moment of each cell was analyzed by the genomic instability index from these CNAs using the follow- Comet assay IV software (Perceptive Instruments Ltd.). ing equation: Total CNA segment lengthðÞ bps GII ¼ Â no: of CNAs Double thymidine block HG19 chromosome sizeðÞ bps T47D cells were treated with 10 mmol/L thymidine (Thy) for 18 hours, released for 9 hours after washing three times with PBS, and The copy number break point index for each breast tumor was then blocked again with 10 mmol/L Thy for 22 hours. calculated as the sum of the copy number break points of each ¼ chromosome ( total number of copy number segments of each Western blot analysis À chromosome 1). The cutoff of TLK2 overexpression was cal- Cells were extracted in RIPA lysis buffer (Sigma-Aldrich), sup- þ Â culated on the basis of median 1 MAD (median absolute plemented with complete protease inhibitor cocktail tablet deviation). MAD is calculated using the R with default constant (Roche). Following primary antibodies were used for Western ¼ ( 1.4826). PAM50-based clinical subtypes of breast cancer for blot analysis: anti-TLK2 (Bethyl Laboratories), anti-GAPDH TCGA samples were derived from the UCSC Cancer Genome (Santa Cruz Biotechnology). Anti-pChk2 (T68), Chk2, pChk1 Browser (https://genome-cancer.ucsc.edu/). (S317), Chk1, pATM (S1981), ATM, pATR (S428), ATR, cyclin B1, pCdk1 (Y15), pH3 (S10), and pAurora kinases antibodies were Cell culture purchased from Cell Signaling Technology. T47D and MCF10A cells were obtained by Dr. Dean P. Edwards (Department of Molecular and Cellular Biology, Baylor College of Engineering doxycycline-inducible plasmids and stable cell Medicine, Houston, TX 77030, USA) from ATCC included in the lines NCI-ATCC ICBP 45 cell line kit.
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