Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening
| Autor: | Kristie Gehlhaus, Natasha J. Caplen, Jason J. Pitt, Jennifer L Dine, Magdalena Grandin, Sirisha Chakka, Stanley Lipkowitz, Sireesha V. Garimella, Marion M. Nau |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Cancer Research
Small interfering RNA Cell Survival DNA repair Immunoblotting Cell bcl-X Protein Apoptosis Breast Neoplasms Cysteine Proteinase Inhibitors Biology Piperazines Nitrophenols TNF-Related Apoptosis-Inducing Ligand Breast cancer Cell Line Tumor medicine Humans skin and connective tissue diseases Caspase 7 Medicine(all) Caspase 8 Sulfonamides Caspase 3 Kinase Biphenyl Compounds Transfection medicine.disease Gene Expression Regulation Neoplastic Pyrimidines src-Family Kinases medicine.anatomical_structure Oncology Drug Resistance Neoplasm SKBR3 Cancer research RNA Interference Oligopeptides Research Article |
| Zdroj: | Breast Cancer Research : BCR |
| ISSN: | 1465-542X 1465-5411 |
| DOI: | 10.1186/bcr3645 |
| Popis: | Introduction Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells. Methods We conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors. Results The primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity. Conclusions These data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink