Int J Clin Exp Pathol 2013;6(4):660-668 www.ijcep.com /ISSN:1936-2625/IJCEP1301062 Original Article A RTK-based functional RNAi screen reveals determinants of PTX-3 expression Hua Liu*, Xin-Kai Qu*, Fang Yuan, Min Zhang, Wei-Yi Fang Department of Cardiology, Shanghai Chest Hospital affiliated to Shanghai JiaoTong University, Shanghai, China. *These authors contributed equally to this work. Received January 30, 2013; Accepted February 15, 2013; Epub March 15, 2013; Published April 1, 2013 Abstract: Aim: The aim of the present study was to explore the role of receptor tyrosine kinases (RTKs) in the regu- lation of expression of PTX-3, a protector in atherosclerosis. Methods: Human monocytic U937 cells were infected with a shRNA lentiviral vector library targeting human RTKs upon LPS stimuli and PTX-3 expression was determined by ELISA analysis. The involvement of downstream signaling in the regulation of PTX-3 expression was analyzed by both Western blotting and ELISA assay. Results: We found that knocking down of ERBB2/3, EPHA7, FGFR3 and RET impaired PTX-3 expression without effects on cell growth or viability. Moreover, inhibition of AKT, the downstream effector of ERBB2/3, also reduced PTX-3 expression. Furthermore, we showed that FGFR3 inhibition by anti-cancer drugs attenuated p38 activity, in turn induced a reduction of PTX-3 expression. Conclusion: Altogether, our study demonstrates the role of RTKs in the regulation of PTX-3 expression and uncovers a potential cardiotoxicity effect of RTK inhibitor treatments in cancer patients who have symptoms of atherosclerosis or are at the risk of athero- sclerosis. Keywords: PTX-3, RNAi screening, RTKs, target therapy, cardiotoxicity, atherosclerosis Introduction ties [5-7]. However, in many cases, adverse car- diac events in the clinic were not anticipated Treatment of patients with cancer has changed based on the fact that predefined cardiac end- radically over the last several years with the points was not included in early clinical trials advent of “targeted therapeutics.” This target- and the difficulties to diagnose heart failure in ed approach, predominantly via inhibition of patients with cancer. Since kinase inhibition tyrosine kinase activity, has markedly improved has revolutionized the treatment of cancer, the management of cancers. Given the initial which can be managed effectively for years and success of this approach, the number of tar- could be eventually regarded as a chronic dis- geted therapy drugs entering into development ease, one could except that the rate of cardio- in the last 5 years has increased dramatically. toxicity in cancer patients treated with kinase And so far, there are hundreds of kinase inhibi- inhibitors would increase in future. tors somewhere in between discovery and mar- ket, with 80% of drug development being in Dysfunctions of cardiovascular system induced cancer [1]. by kinase inhibitor therapy are varied and have included heart failure, LV dysfunction, conduc- Although target therapy has an improve antitu- tion abnormalities, QT prolongation, acute coro- mor activity with fewer toxic side effects than nary syndromes, myocardial injury, arterial traditional anticancer therapies including radia- thromboses and hypertension [8]. Since athero- tion therapies and chemotherapies, many sclerosis is considered as the significant under- approved RTK inhibitors have been found to lying cause of cardiovascular disease (CVD) [9, evoke cardiac dysfunction in some cancer 10], inhibition of driver kinases might therefore patients [2-4] . Furthermore, there is a strong potentially compromise the cardiovascular sys- link between kinase pathway inhibition and car- tem function by accelerating atherosclerosis. diotoxicity as compared with other organ toxici- However, the side effects of treatment with Function of RTKs in PTX-3 expression kinase inhibitors on atherosclerosis in cancer RPMI 1640 medium supplemented with 10% patients have not been fully investigated due to FBS (Hyclone). All media were supplemented lack of appropriate preclinical model. with penicillin (100 IU·ml-1) and Streptomycin (100 μg·ml-1) (Life Technologies). Given the critical role of inflammation in athero- sclerosis, circulating factors related to inflam- Induction of PTX-3 expression by LPS stimuli mation and atherosclerosis therefore attracted attention [11]. Of interest, pentraxin protein The cells were washed and incubated in endo- family is highly associated with CVD [11]. toxin-free RPMI 1640 (10% FBS) at 5 X 104 cells Pentraxin protein family consists of C-reactive each well in 24-well plate, with or without LPS protein (CRP), serum amyloid P component (lipopolysaccharide, Escherichia coli 0127:B8, (SAP) and pentraxin-3 (PTX-3) [11-13], all of Sigma, L4516 ) stimuli for 6 hrs at 37°C in the which have the features of pattern recognition presence of 5% CO2. The optimized concentra- receptors and are involved in human humoral tion of LPS to induce PTX-3 expression was -1 immune response [14, 15]. In contrast to CRP, tested with 0, 20, 50 and 100 ng· ml . PTX-3 demonstrates to be more specifically U937 cells induced by series of concentration associated with advanced atherosclerosis [15- of LPS were homogenized with 0.5 ml Trizol (life 17]. PTX-3 is highly expressed in advanced ath- technologies) and total RNA was isolated as erosclerosis tissues [18, 19], including macro- instruction manual (life technologies). Reverse phages, surviving endothelial cells, activated transcription was carried out at 42°C for 1 hour monocytes and infiltrating neutrophils [19]. with oligo dT. Real-time PCR primers were Amounts of evidence suggest the possibility designed as follows: forward primer: tgtatgt- that the increased levels of PTX-3 in subjects gaatttggacaacgaa; reverse primer: cattccgagt- with CVD may reflect a protective physiologic gctcctgac. Reaction program was 2 min at response that correlates with the severity of 95°C, 40 cycles of 95°C for 30 s, 60°C for 30 s the disease [20-22]. More importantly, defi- and 72°C for 30 s. The concentration of LPS ciency of the long pentraxin PTX-3 promotes induced the maximal expression of PTX-3 was vascular inflammation and atherosclerosis selected to carry out RNAi screening. [23]. RNAi screening To systematically investigate the function of receptor tyrosine kinases (RTKs), the main tar- U937 cells were incubated in endotoxin-free gets in target cancer therapies, on atheroscle- RPMI 1640 (10% FBS) at 1 × 104 cells each well rosis, we set out to screen a short hairpin RNA in 96-well plates, and then infected respective- (shRNA) library representing the full comple- ly with 55 RTK lentiviral shRNAs in the pres- ment of 56 human RTKs (Table 1) for genes ence of polybrene to the final concentration of whose inhibition could impair PTX-3 expression 8 μg· ml-1, each gene in the library was duplicate in U937 cells. Our results revealed multiple in two plates. LPS was added to cell culture to functionally important pathways, particularly final concentration 100 ng· ml-1 120 h after the FGFR/p38 signaling in the regulation of infection for 6 hrs. After the treatment, cells PTX-3 expression. Our study suggests that clini- were pelleted and divided into two parts: the cally used kinase inhibitor might contribute to supernate was collected to measure PTX-3 lev- atherosclerosis, in turn inducing cardiotoxicity, els by ELISA, and the pellets were used to by interfering with PTX-3 expression. These detect cell viability by MTS assay (Promega, findings could provide a preclinical model for G3580) according to the operation manual. studying the side effects of kinase inhibitors on atherosclerosis. Determination of PTX-3 expression by ELISA assay Materials and methods PTX-3 levels were measured by the Sandwich Cell culture ELISA as previously described. Briefly, ELISA plates were coated with 100 ng each well of 293T cells (ATCC) were cultured in DMEM medi- mouse anti-human PTX-3 antibody diluted in um supplemented with 10% FBS (Hyclone). PBS by overnight incubation at 4°C. Washing U937 cells (ATCC, CRL-1593.2) were grown in buffer (PBS containing 0.05% Tween 20) was 661 Int J Clin Exp Pathol 2013;6(4):660-668 Function of RTKs in PTX-3 expression Table 1. RTKs genes for screen Gene symbol Official Full Name Inhibitor Associated with cardiotoxicity (Ref) ERBB4 v-erb-a erythroblastic leukemia viral oncogene homolog 4 NA NA MST1R macrophage stimulating 1 receptor NA NA RYK receptor-like tyrosine kinase NA NA KDR kinase insert domain receptor sorafenib/sunitinib [8, 32] NTRK1 neurotrophic tyrosine kinase, receptor, type 1 NA NA STYK1 serine/threonine/tyrosine kinase 1 NA NA EPHA3 EPH receptor A3 NA NA FLT1 fms-related tyrosine kinase 1 sorafenib/sunitinib [8, 32] FGFR1 fibroblast growth factor receptor 1 PD-173074 NA AXL AXL receptor tyrosine kinase NA NA NTRK2 neurotrophic tyrosine kinase, receptor, type 2 NA NA TEK TEK tyrosine kinase, endothelial NA NA EGFR epidermal growth factor receptor erlotinib/gefitinib/lapatinib [1, 8] ROR2 receptor tyrosine kinase-like orphan receptor 2 NA NA FLT3 fms-related tyrosine kinase 3 sorafenib/sunitinib [8, 32] ALK anaplastic lymphoma receptor tyrosine kinase crizotinib NA ERBB3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 AZD8931 NA (avian) ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 Lapatinib [1, 32] INSRR insulin receptor-related receptor NA NA EPHA6 EPH receptor A6 NA NA EPHA5 EPH receptor A5 NA NA EPHA7 EPH receptor A7 NA NA FGFR3 fibroblast growth factor receptor 3 Linifanib/AZD4547