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Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in P53-Mutant Triple-Negative Breast Cancer Natalie A

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Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in P53-Mutant Triple-Negative Breast Cancer Natalie A Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0519 Small Molecule Therapeutics Molecular Cancer Therapeutics Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in p53-Mutant Triple-Negative Breast Cancer Natalie A. Jabbour-Leung1, Xian Chen1, Tuyen Bui1, Yufeng Jiang1, Dong Yang1, Smruthi Vijayaraghavan1, Mark J. McArthur2, Kelly K. Hunt3, and Khandan Keyomarsi1 Abstract Triple-negative breast cancer (TNBC) is an aggressive malig- Roscovitine treatment arrests TNBC cells in the G2–M cell-cycle nancy in which the tumors lack expression of estrogen receptor, phase, priming them for DNA damage. Combination treatment progesterone receptor, and HER2. Hence, TNBC patients cannot increased frequency of DNA double-strand breaks, while simul- benefit from clinically available targeted therapies and rely on taneously reducing recruitment of homologous recombination chemotherapy and surgery for treatment. While initially respond- proteins compared with doxorubicin treatment alone. Further- ing to chemotherapy, TNBC patients are at increased risk of more, this combination therapy significantly reduced tumor developing distant metastasis and have decreased overall survival volume and increased overall survival compared with single drug compared with non-TNBC patients. A majority of TNBC tumors or concomitant treatment in xenograft studies. Examination of carry p53 mutations, enabling them to bypass the G1 checkpoint isogenic immortalized human mammary epithelial cells and and complete the cell cycle even in the presence of DNA damage. isogenic tumor cell lines found that abolishment of the p53 Therefore, we hypothesized that TNBC cells are sensitive to cell- pathway is required for combination-induced cytotoxicity, mak- cycle–targeted combination therapy, which leaves nontrans- ing p53 a putative predictor of response to therapy. By exploiting formed cells unharmed. Our findings demonstrate that sequential thespecificbiologicandmolecularcharacteristicsofTNBCtumors, administration of the pan-CDK inhibitor roscovitine before doxo- thisinnovativetherapycangreatlyimpactthetreatmentandcareof rubicin treatment is synthetically lethal explicitly in TNBC cells. TNBC patients. Mol Cancer Ther; 15(4); 593–607. Ó2016 AACR. Introduction resulting from BRCA1 mutations, may contribute to TNBC patients initially responding well to chemotherapy; however, Triple-negative breast cancer (TNBC) is a clinical diagnosis many patients' tumors recur (6). While there are several targeted that afflicts 10% to 20% of breast cancer patients (1). TNBC therapies being developed in clinical trials, including PARP and tumors are characterized by lacking expression of estrogen EGFR inhibitors, there are currently no clinically available and receptor (ER), progesterone receptor (PR) and the growth factor effective targeted therapies for TNBC patients (6–8). receptor HER2. TNBC patients are more likely to be premeno- The majority (54%–82%) of TNBC tumors harbor p53 muta- pausal, of African descent, have a poor prognosis and have tions, enabling them to bypass the G checkpoint and complete greater risk of recurrence within the first 5 years of diagnosis (2). 1 the cell cycle even with unrepaired DNA damage (6, 9, 10). In Less than one-third of metastatic TNBC patients survive 5 years comparison, only 13% of hormone receptor-positive luminal A (3). About 70% of TNBC are classified as basal-like breast cancer tumors have p53 mutations (11). Moreover, 50% of these breast (BLBC), and share many of the same characteristics, including cancers overexpress cyclin D1, inhibiting retinoblastoma (Rb) more likely occurring in patients with BRCA1/BRCA2 gene regulation of E2F (12). Notably, overexpression of cyclin E serves mutations (4, 5). Dysfunction in the DNA repair pathway, as a poor prognostic marker in breast cancer and correlates to negative ER and PR status (13, 14). Owing to deregulation of the cell cycle in cancer cells, cyclin-dependent kinase (CDK) inhibi- 1Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2Veterinary Med- tors were developed to inhibit tumor cell proliferation and icine and Surgery,The University of Texas MDAnderson Cancer Center, induce apoptosis (15). However, CDK inhibitors have not been 3 Houston,Texas. Breast Surgical Oncology,The University of Texas MD effective clinically, despite having promising results both in vitro Anderson Cancer Center, Houston, Texas. and in vivo (16, 17). Roscovitine, a pan CDK inhibitor with activity Note: Supplementary data for this article are available at Molecular Cancer against CDK1, 2, 5, 7, and 9 (18, 19), became the first orally Therapeutics Online (http://mct.aacrjournals.org/). bioavailable drug from this class to go into clinical trials based on N.A. Jabbour-Leung and X. Chen contributed equally to this article. the preclinical data showing induction of apoptosis in tumor cells. Corresponding Author: Khandan Keyomarsi, Department of Experimental However, of the 77 solid tumor patients treated with single-agent Radiation Oncology, The University of Texas MD Anderson Cancer Center, roscovitine, one partial response was seen in hepatocellular 6565 MD Anderson Blvd, Box 1052, Houston, TX 77030. Phone: 713-792- carcinoma, 2 prolonged stable disease observed in non–small 4845; Fax: 713-794-5369; E-mail: [email protected] cell lung cancer (14 and >18 months), while stable disease was the doi: 10.1158/1535-7163.MCT-15-0519 best response seen in the remaining solid tumors (20–22). One of Ó2016 American Association for Cancer Research. the reasons that these CDK inhibitors have not been more effective www.aacrjournals.org 593 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0519 Jabbour-Leung et al. 594 Mol Cancer Ther; 15(4) April 2016 Molecular Cancer Therapeutics Downloaded from mct.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0519 CDK Inhibition Sensitizes p53-Mutated Cells to Doxorubicin clinically is that they are either being used as single agents or when 10A, 76NE6, and 76NF2V, the breast cancer cell lines MDA-MB- they are used in combination therapy, both agents were delivered 157, MDA-MB-231, HCC1806, MCF-7, ZR75-1, T47D, MDA- concomitantly to the patient (23). In addition, there was no MB-468 (Supplementary Table S1A), and the isogenic colorectal þ þ attempt to identify those patients most likely to respond to these cancer cells HCT116 p53 wild-type (p53 / ) and p53 knockout À À agents based on their biology. In fact, very few patients with breast (p53 / ) were previously described (29–31). HEK-293T cells cancer of any subtype were accrued to these trials. for lentiviral packaging were maintained in DMEM supplemen- CDK1 participates in the DNA double-strand break (DSB) ted with 10% FCS. All cells were free of mycoplasma contam- repair pathway homologous recombination (HR). HR faithful- ination, and their identities were authenticated by karyotype ly repairs DNA DSBs that occur in late S, G2,andM(24).CDK and short tandem repeat analysis using the MD Anderson's activity is required for the recruitment of the endonucleases Characterized Cell Line Core Facility (http://www.mdander- Sae2 or CtIP that excise the DNA DSB to generate single strands son.org/characterized-cell-line-core-facility/index.html) on a during HR in both yeast and mammalian cells, respectively (25, routine basis (every 6 months). 26). Moreover, CDK activity is required for the recruitment and association of BRCA1 to the MRN (Mre11-Rad50-Nbs1) com- Immunofluorescence to detect DNA repair foci plex during HR (27). Concordantly, CDK inhibition with Following treatment of cells with roscovitine (IC50) for 24 roscovitine reduced the recruitment of HR downstream protein hours, doxorubicin (IC50) for 48 hours, or both drugs sequentially RPA34 in irradiated sarcoma cells due to an inability to gen- as indicated, they were fixed in 4% paraformaldehyde for 20 min- erate single strands (28). Thus, compromising HR via CDK utes followed by permeabilization with a 0.3% triton solution (20 inhibition may provide a strategy to augment TNBC cell sen- mol/L Hepes, 50 mmol/L NaCl, 3 mmol/L MgCl2, 300 mmol/L sitivity to chemotherapy. sucrose, and TritonX-100) for 20 minutes. Cells were then blocked No clinically available treatment strategies target the TNBC- with 10% BSA and 2% horse serum PBS for one hour. Antibodies deregulated cell cycle to exploit TNBC-cell sensitivity to DNA- were diluted 1:500 and 1:1000 for anti-g-H2AX (EMD Millipore) damaging agents (e.g., chemotherapeutics). Because TNBC cells and anti-Rad51 (32), respectively, and incubated at 4C over- have a deregulated G1 checkpoint, enabling them to re-enter the night. Secondary goat anti-mouse or goat anti-rabbit antibodies cell cycle while harboring DNA damage, we hypothesized that (Alexa Fluor 594 and 488, respectively, EMD Millipore) were – TNBC cells are sensitive to cell-cycle targeted combination diluted at 1:750 and incubated at room temperature at 1 hour. therapy, which leaves nontransformed cells unharmed. Ideally, Nuclei were stained with DAPI (Life Technology) at 1mg/mL for 5 this therapeutic strategy will be synthetically lethal against minutes room temperature. Cells were mounted with Dako TNBC cells by inhibiting pathways
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