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The Combination of TPL2 Knockdown and Tnfα Causes Synthetic Lethality Via Caspase-8 Activation in Human Carcinoma Cell Lines

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The Combination of TPL2 Knockdown and Tnfα Causes Synthetic Lethality Via Caspase-8 Activation in Human Carcinoma Cell Lines The combination of TPL2 knockdown and TNFα causes synthetic lethality via caspase-8 activation in human carcinoma cell lines Oksana B. Serebrennikovaa, Maria D. Paraskevopouloua, Elia Aguado-Frailea,1, Vasiliki Tarasliaa, Wenying Rena,2, Geeta Thapaa, Jatin Ropera,3, Keyong Dua,2, Carlo M. Croceb,c,4, and Philip N. Tsichlisa,b,c,4 aMolecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111; bDepartment of Cancer Biology and Genetics, The Ohio State University, Columbus, OH 43210; and cThe Ohio State University Comprehensive Cancer Center, Columbus, OH 43210 Contributed by Carlo M. Croce, May 21, 2019 (sent for review January 29, 2019; reviewed by Emad S. Alnemri and Wafik El-Deiry) Most normal and tumor cells are protected from tumor necrosis subset of tumor cells and we delineate the relevant TPL2-regulated factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 pathway(s). kinase tumor progression locus-2 (TPL2) as a player contributing TPL2 is an oncoprotein that is activated by provirus insertion to the protection of a subset of tumor cell lines. The combination in Moloney murine leukemia virus-induced rodent lymphomas of TPL2 knockdown and TNFα gives rise to a synthetic lethality and mammary tumor virus-induced mammary adenocarcinomas phenotype via receptor-interacting serine/threonine-protein ki- (13, 14). Expression of constitutively active TPL2 from a thymus- nase 1 (RIPK1)-dependent and -independent mechanisms. Whereas targeted transgene confirmed its oncogenic potential (15). Sub- wild-type TPL2 rescues the phenotype, its kinase-dead mutant sequently, it was shown that TPL2 is obligatory for the trans- does not. Comparison of the molecular events initiated by small duction of signals initiated by triggering TLRs and other TPL2 TPL2 ± α interfering RNA for (si ) TNF in treatment-sensitive and receptors in different cell types and that it plays an impor- -resistant lines revealed that the activation of caspase-8, downstream tant role in innate and adaptive immunity and inflammation (16, of miR-21-5p and cFLIP, is the dominant TPL2-dependent event. More 17) (SI Appendix, SI Introduction). Although studies published important, comparison of the gene expression profiles of all of the over the last 5 y provide evidence that TPL2 has the potential to CELL BIOLOGY tested cell lines results in the clustering of sensitive and resistant lines function as a tumor suppressor for several types of tumors (18, into distinct groups, providing proof of principle for the feasibility of 19), TPL2 is primarily known to function as an oncogene. In generating a predictive tool for treatment sensitivity. human tumors, it is often expressed at high levels, and its ex- pression promotes cell proliferation and drug resistance (20). TPL2 | TNFα | synthetic lethality | caspase | apoptosis In the present study, we show that the combination of TPL2 knockdown and TNFα induces synthetic lethality in TPL2- α α umor necrosis factor (TNF ), a member of the tumor ne- expressing human tumor cell lines. Using pharmacological and Tcrosis factor cytokine superfamily, is a type II transmembrane protein that forms membrane-associated homotrimers. Pro- Significance teolytic cleavage of the membrane-bound cytokine is mediated by the TNFα converting enzyme (TACE or ADAM17), which α α releases a soluble trimeric form of the cytokine. TNFα is pro- Tumor necrosis factor (TNF ) fails to induce apoptosis in most duced primarily by activated macrophages, although it can also tumor cells. Defining the mechanism(s) responsible for the protection of these cells from TNFα-induced apoptosis may be produced by other types of cells, including neutrophils, eo- have significant therapeutic implications. Here, we show that sinophils, mast cells, natural killer cells, and cluster of differen- + tumor progression locus-2 (TPL2), a MAP3 kinase activated by tiation 4-positive (CD4 ) lymphocytes. Its expression is induced TNFα, protects from TNFα-induced apoptosis by preventing the by Toll-like receptor (TLR), Nod-like receptor, and C-type lectin activation of caspase-8, and that the combination of TPL2 receptor signals, as well as by cytokine and chemokine receptor + knockdown and TNFα treatment induces synthetic lethality. signals. In CD4 T cells, it can also be induced by T cell receptor More important, sensitivity to synthetic lethality can be pre- signals (1, 2). dicted from the gene expression profiles of the tumor cells. In α TNF signaling can be initiated by both the membrane-bound addition to their therapeutic potential, these results identify an and soluble forms of the cytokine, and it has an integral role in important node in TNFα signaling. innate immunity and inflammation (1). Although in some normal and tumor cells, it induces apoptosis, most cells are protected. In Author contributions: O.B.S. and P.N.T. designed research; O.B.S., M.D.P., E.A.-F., V.T., some of them, protection is due to the expression of the caspase- W.R., and G.T. performed research; J.R., K.D., and C.M.C. contributed new reagents/ana- 8 inhibitor cFLIP and other antiapoptotic factors and is nuclear lytic tools; O.B.S., M.D.P., and E.A.-F. analyzed data; O.B.S. and P.N.T. wrote the paper; and M.D.P. contributed the bioinformatics analyses. factor-κB (NF-κB)–dependent (3–5). In others, it depends on the Reviewers: E.S.A., Thomas Jefferson University; and W.E.-D., Brown University. ubiquitin ligases cIAP1 and cIAP2, which ubiquitinate receptor- The authors declare no conflict of interest. interacting serine/threonine-protein kinase 1 (RIPK1) and block its ability to activate caspase-8 (6). The role of these mechanisms Published under the PNAS license. 1 has been confirmed with the use of NF-κB and protein synthesis Present address: Clinical Biomarkers Department, Agios Pharmaceuticals, Cambridge, MA 02139. inhibitors (cycloheximide [CHX]), which block the NF-κB–me- 2Present address: Department of Medicine, Tufts Medical Center, Boston, MA 02111. diated protection (7), and with the use of Smac mimetics, which – α 3Present address: Division of Gastroenterology, Department of Medicine, Duke University, block cIAP1 and cIAP2 (6 8). TNF and CHX-induced death is Durham, NC 27710. α RIPK1-independent, while TNF and Smac mimetic-induced 4To whom correspondence may be addressed. Email: [email protected] or philip. death is RIPK1-dependent (7). The RIPK1-dependent cell [email protected]. κ death is inhibited by the I B kinase complex and MK2, which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inhibit RIPK1 (9–12). In this report, we identify the MAP3 ki- 1073/pnas.1901465116/-/DCSupplemental. nase tumor progression locus-2 (TPL2) as a protective player in a Published online June 25, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1901465116 PNAS | July 9, 2019 | vol. 116 | no. 28 | 14039–14048 Downloaded by guest on September 28, 2021 genetic tools, we showed that the dominant event responsible for Results TNFα-induced apoptosis in small interfering RNA for TPL2 The Knockdown of TPL2 Renders a Subset of Human Cancer Cell Lines (siTPL2)/TNFα-treated cells is the activation of caspase-8, Sensitive to the Apoptotic Action of TNFα. To determine the role of downstream of miR-21-5p and cFLIP. The mitochondrial path- TPL2 in the response of different cell lines to TNFα, we exam- way of apoptosis is also activated, but it has an auxiliary role. ined the effects of TNFα stimulation in a set of cancer cell lines Most important, sensitive and resistant lines cluster in distinct 48 h after treatment with siTPL2 or siControl RNAs. Monitoring groups when their gene expression profiles are compared, sug- cell survival with light microscopy and the CellTiter-Glo assay gesting the feasibility of a tool that predicts treatment sensitivity. 24 h later revealed that although TNFα and TPL2 knockdown A HeLa HCT 116 U-2 OS NCI-H460 MIA PaCa-2 MDA-MB-436 120 120 120 120 120 120 *** *** *** *** 80 80 80 80 80 *** 80 *** sic 40 40 40 40 40 40 siTPL2 Viability, % 0 0 0 0 0 0 TNF 100 sic TNF B C 150 75 sid1 siRNA c d1 d2 d3c d1 d2 d3 sid2 0.2 0.05 *** Dox ( g/ml) 2 P-ERK1/2 -37 100 50 sid3 *** *** ERK1/2 -37 *** Dox, 0 ug/mlg/ml siRNATT T T Dox, 0.05 ug/mlg/ml HA-TPL2 Cell death, % 25 Tpl2 50 -50 * -50 g/ml Viability, % Dox, 0.2 ug/ml -37 -37 0 GAPDH Dox, 2 ug/mlg/ml GAPDH 0 TNF TNF 90 D 120 *** 60 ** 80 sic siRNA ccTT sic kd TPL2Tpl2 30 sic HA-TPL2 40 siTPL2 -50 Cell death, % siTPL2 Viability, % -37 siTPL2 kd Tpl2TPL2 GAPDH 0 0 TNF TNF TRAIL 150 miR-21-5p E1 E2 ** F1 2 *** * ** 100 TNF % 1 sic miRNA, siRNAccT T 50 Mitochondrial siTPL2 0 procaspase-8 -50 expression relative membrane potential, 0 sic siTPL2 p43/41 -37 TNF (h) 0 3620 FLIP 2.5 L -25 F2 *** *** TNF (h) 01360136 2 -20 siRNAcccT c TTT 1.5 *** p18 cytochrome c -15 RNA, 1 sic -20 -20 SMAC/DIABLO 0.5 siTPL2 β-actin expression relative cl. caspase-3 -37 0 -15 TNF (h) 0 24 TNF (h) 036036 -100 cl. PARP E3 siRNAccT c TT TNF siRNAccT T TPL2 -50 BAX -20 -37 IP FLIP GAPDH IgG -50 L -50 6A7 Ab FLIP -25 BAX -20 S -37 GAPDH -37 GAPDH WCL Fig. 1. siTPL2 and TNFα induce synthetic lethality in human cancer cells by promoting the activation of caspase-8 and the mitochondrial pathway of apo- ptosis. (A) Viability of cancer cells transfected with control or TPL2 siRNA was measured 24 h after the exposure to TNFα, using the CellTiter-Glo assay.
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