www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 19 A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability Maria Carmela Cantisani1, Alessia Parascandolo2, Merja Perälä3,4, Chiara Allocca2, Vidal Fey3,4, Niko Sahlberg3,4, Francesco Merolla5, Fulvio Basolo6, Mikko O. Laukkanen1, Olli Pekka Kallioniemi7, Massimo Santoro2,8, Maria Domenica Castellone8 1IRCCS SDN, Naples, Italy 2Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita’ Federico II, Naples, Italy 3Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland 4Center for Biotechnology, University of Turku, Turku, Finland 5Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, Italy 6Division of Pathology, Department of Surgery, University of Pisa, Pisa, Italy 7FIMM-Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland 8Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” (IEOS), C.N.R., Naples, Italy Correspondence to: Maria Domenica Castellone, e-mail: [email protected] Keywords: kinases, screening, siRNA, thyroid carcinoma Received: October 01, 2015 Accepted: March 02, 2016 Published: April 4, 2016 ABSTRACT RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6- RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. INTRODUCTION occurs in thyroid cancer [1–4]. Accordingly, point mutations of the BRAF serine/threonine kinase as well Thyroid cancer is the most common endocrine as rearrangements (RET/PTC) of RET receptor tyrosine malignancy, whose incidence has been steadily increasing kinase (RTK) or of other RTKs are found in the majority over the past 30 years [1–3]. Most common thyroid cancer of PTC cases [1–6]. Mutations of RAS, an upstream types arise from follicular epithelial cells and are classified activator of both MAPK and PI3K, are common in FTC in well-differentiated papillary (PTC) and follicular and in follicular-variant PTC [5–7]. Mutations or copy (FTC), poorly differentiated (PDC), and anaplastic number alterations of PI3K and AKT are found in ATC (undifferentiated) (ATC) carcinoma; PTC accounts for the and radioiodine-refractory thyroid cancer [3, 8–10]. vast majority of thyroid cancer cases [1–3]. Unbiased RNA interference (RNAi) screenings, Oncogenic conversion of kinases involved in the based on the use of siRNAs collections targeting the entire MAPK (mitogen activated protein kinases) and PI3K genome or relevant gene families, are being exploited to (phosphatidyl-inositol-3-kinase)/AKT signaling cascades identify novel molecular mediators of tumorigenesis and www.impactjournals.com/oncotarget 28510 Oncotarget therapeutic targets [11]. Here, through a siRNA-based Supplemental Information, Figure S1 and raw data are kinome screen, we identified a set of kinases and related available in Supplemental Information, Table S1. Overall, proteins as novel drivers of thyroid cancer cells viability. silencing of 50 genes reduced TPC1 cell viability. This list included a pseudogene LOC392265 and a Ser/Thr-like RESULTS kinase MGC44796 (later identified as STK40) that were excluded from further studies (Supplemental Information, High-throughput siRNA-based screening in Table S2). TPC1 thyroid cancer cells Though TPC1 cells depend on RET/PTC1 for their viability [12], RET was not present among the hits because We designed a functional screening using the the two RET targeting siRNAs included in our library did human papillary carcinoma cell line, TPC1, harboring not efficiently knock-down the RET/PTC1 expression at the endogenous level the RET/PTC1 (CCDC6-RET) (data not shown). rearrangement [12] and a commercially available siRNA library containing synthetic siRNAs targeting the 518 Validation of antiproliferative hits in TPC1 cells human protein kinases as well as kinase-related and -associated proteins. The library included two independent To confirm the results of the screening, we measured siRNAs targeting each transcript and negative controls TPC1 cell viability upon transfection with the same 2 consisting of siRNAs targeting green fluorescent protein siRNAs from the library (siRNA1 and 2) as well as with 2 (GFP), eight non targeting scrambled sequences, as well independent siRNAs (siRNA3 and 4) targeting a distinct as buffer alone. The screening was performed in duplicate. mRNA region of the 48 hits (Supplemental Information, Seventy-two hours after transfection, viable cells were Table S2); average results are reported in Figure 1 and stained and antiproliferative hits were identified as genes raw data are shown in Supplemental Information, Table whose knock-down, by at least one of the two siRNAs, S3. Nine out of 48 genes were not confirmed to reduce cell reduced (by at least 30%; loess-log ≤-0.5) cell viability viability and were therefore excluded from further tests. in both replicates upon normalization to the median value Among the remaining ones, silencing of 27 genes reduced of negative controls. A plot of the results is shown in dye incorporation by at least 30% (loess-log ≤-0.5) Figure 1: Identification of genes essential for TPC1 cell viability. TPC1 cells were transfected in duplicate with 4 independent siRNAs (Supplemental Information, Table S2) targeting different regions of the 48 antiproliferative hits. Seventy-two hours after transfection, cell viability was measured by CellTiter-Blue assay and expressed as log2 (loess-log normalization). The reported values are the average results of the transfection of 4 siRNAs for each gene in duplicate normalized by negative controls (average results of Non specific siRNA Control and AllStars Negative siRNA Control). The bars represent 95% confidence intervals. Dotted line represents an arbitrary cut-off value (loess-log ≤-0.5) applied to select the down hits. www.impactjournals.com/oncotarget 28511 Oncotarget Table 1: List of the 14 antiproliferative hits identified in TPC1 cells Symbol Gene name Genbank ID AGC: cAMP-dependent, cGMP-dependent and protein kinase C AKT2 v-akt murine thymoma viral oncogene homolog 2 NM_001626 PKN1 protein kinase N1 NM_002741 PRKACB protein kinase cAMP dependent, catalitic, beta NM_182948 CMGC group AKAP9 A kinase (PRKA) anchor protein (yotiao) 9 NM_005751 mitogen-activated protein kinase kinase kinase 7 interacting MAP3K7IP1 NM_006116 protein 1 GPCR coupled kinases GRK4 G protein-coupled receptor kinase 4 NM_182982 STE: serine/threonine kinase family MAPKAPK2 mitogen-activated protein kinase-activated protein kinase 2 NM_004759 TK: tyrosine kinases EPHA2 EPH receptor A2 NM_004431 EPHA7 EPH receptor A7 NM_004440 EPHB2 EPH receptor B2 NM_017449 EPHB6 EPH receptor B6 NM_004445 FYN FYN oncogene NM_002037 HCK hemopoietic cell kinase NM_002110 TKL: tyrosine kinase like LIMK1 LIM domain kinase 1 NM_002314 (average value of the 4 different siRNAs) as compared different from RET. To this aim, we selected 3 additional to the negative controls (siRNAs with no homology thyroid cancer cell lines (BCPAP, 8505C and CAL62) of to any known mammalian gene: Non specific siRNA different histotypes and featuring different complements Control, catalogue no. 1022079 and AllStars Negative of genetic lesions. BCPAP and 8505C cells bear a BRAF siRNA Control, catalogue no. SI03650318, Qiagen); V600E and CAL62 bears a KRAS G12R mutation, in particular, in the case of 20 genes the reduction was respectively (Supplemental Information, Table S4) [13]. significant (p <0.05) with each one of the four different The 14 hits were individually silenced by siRNA1 and 2 used siRNAs. We therefore restricted our analysis to these in the three cancer cell lines, compared to NTHY cells. 20 hits and tested effects of their silencing in a control Silencing of most of them (14/14 genes for BCPAP, cell line (NTHY) obtained from normal thyroid tissue and 11/14 for CAL62 and 14/14 for 8505C cells) significantly immortalized by SV40 Large T. Silencing of six (CIB3, (p <0.01) reduced viability of cancer (Figure 3) but not ITPKA, MAPK7, MARCKS, PAK2, STK33) genes NTHY (Figure 4) cells. Exceptions were represented by affected the viability also of control cells (data not shown) FYN and PRKACB that reduced CAL62 cell viability and therefore they were excluded from further tests, thus only when silenced with siRNA1 and LIMK1 that did not finally resulting in 14 selected hits (Table 1 and Figure 2). reduce dye incorporation with either siRNA. To confirm gene knock-down, we performed Validation of antiproliferative hits in a panel of quantitative