KSR1 Is Coordinately Regulated with Notch Signaling and Oxidative Phosphorylation in Thyroid Cancer

J LEE and others The role of KSR1 in 54:2 115–124 Research thyroid cancer

KSR1 is coordinately regulated with Notch signaling and oxidative phosphorylation in thyroid cancer

Jandee Lee1, Mi-Youn Seol1, Seonhyang Jeong2, Hyeong Ju Kwon3, Cho Rok Lee1, 2 1 1 2 Cheol Ryong Ku , Sang-Wook Kang , Jong Ju Jeong , Dong Yeob Shin , Correspondence Kee-Hyun Nam1, Eun Jig Lee2, Woong Youn Chung1 and Young Suk Jo2 should be addressed to Y S Jo Departments of 1Surgery, 2Internal Medicine, and 3Pathology, Yonsei Cancer Center, Severance Hospital, Email Yonsei University College of Medicine, 120-752 Seoul, Korea [email protected]

Abstract

Kinase suppressor of RAS1 (KSR1) is a scaffold protein implicated in RAS-mediated RAF Key Words activation. However, the molecular function of KSR in papillary thyroid cancer (PTC) is " kinase suppressor of RAS unknown. Thus, this study aimed to characterize the role of KSR1 in patients with PTC. (KSR) qRT-PCR and immunohistochemistry (IHC) revealed inter-tumor heterogeneities in the " connector enhancer of KSR (CNKSR) expression of KSR1 in PTC tissues. Interestingly, BRAFV600E-positive PTC showed higher " thyroid cancer KSR1 mRNA expression than BRAFV600E-negative PTC (P!0.001). Gene Set Enrichment " notch signaling Analysis (GSEA) using public repositories showed that high KSR1 expression coordinately " oxidative phosphorylation upregulated Notch signaling (nominal PZ0.019, false discovery rate (FDR) q-valueZ0.165); this ﬁnding was supported by GeneNetwork analysis, indicating that KSR1 expression is positively correlated with NOTCH1 expression (rZ0.677, PZ6.15!10K9). siRNA against KSR1

Journal of Molecular Endocrinology (siKSR1) signiﬁcantly decreased ERK phosphorylation induced by BRAFV600E, resulting in reduced expression of NOTCH1 and HES1, targets of Notch signaling. GSEA revealed that high KSR1 expression was also associated with downregulation of genes related to oxidative phosphorylation (OxPhos). Consistent with this, electron microscopy showed that PTCs with high KSR1 expression exhibited structural defects of the mitochondrial cristae. Furthermore, siKSR1-transfected BCPAP and 8505C cells generated fewer colonies in colony-forming assays. In addition, GSEA showed that high expression of KSR2 and connector enhancer of KSR1 (CNKSR1) also coordinately upregulated Notch signaling (KSR2: nominal PZ0.0097, FDR q-valueZ0.154 and CNKSR1: nominal P!0.0001, FDR q-valueZ0.00554), and high CNKSR2 was associated with downregulation of the OxPhos gene set (nominal P!0.0001, FDR q-value !0.0001). In conclusion, KSR1 is coordinately regulated with Notch signaling and OxPhos in PTC, because its scaffold function might be required to sustain the Journal of Molecular proliferative signaling and metabolic remodeling associated with this type of cancer. Endocrinology (2015) 54, 115–124

Introduction

Papillary thyroid cancer (PTC) is the most common Pacini et al. 2006, Kweon et al.2013); however, a signiﬁcant endocrine malignancy, and generally has favorable proportion of patients with PTC exhibit poor clinical outcomes (DeGroot et al. 1990, Wada et al. 2003, outcomes such as lymph node (LN) metastasis, distant

http://jme.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JME-14-0270 Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 11:21:27AM via free access Research J LEE and others The role of KSR1 in 54:2 116 thyroid cancer

metastasis, and even fatality (Mirallie et al.1999, Pereira et al. ERK signal propagation (Berman et al. 2003, Liu et al. 2009, 2005, Bilimoria et al.2007). Surgery followed by radioactive Lito et al. 2013). iodine therapy has been the gold-standard treatment regimen In this study, we investigated the expression status of for differentiated thyroid cancer (DTC; Mazzaferri & Jhiang KSR1 in patients with PTC before treatment with targeted 1994, Pacini et al. 2006, American Thyroid Association agents, with the goal of gaining insights into the role of Guidelines Taskforce on Thyroid N et al. 2009, Alzahrani & KSR1 in RAS–RAF–MAPK signal propagation in PTC and Xing 2013); however, recurrent or metastasized lesions identifying potential targets for drugs that would be sometimes cannot be cured by surgical resection or radio- effective against PTC, regardless of the presence or absence active iodine therapy (Riesco-Eizaguirre et al.2009, Xing et al. of de novo or acquired drug resistance to novel targeted 2013). Recently, sorafenib (Nexavar) has been approved by agents. To this end, we examined the expression levels the FDA to treat patients with locally advanced or metastatic of KSR1 by qRT-PCR and immunohistochemistry (IHC), DTC that is refractory to radioactive iodine; however, the which revealed KSR1 inter-tumor heterogeneity. Bioinfor- Phase 3 DECISION trial did notdemonstrateasignificant matic analysis showed that KSR and CNKSR are associated improvement in overall survival (Brose et al.2014). with Notch signaling and mitochondrial oxidative The RAS–RAF–MAPK pathway is an intracellular chain phosphorylation (OxPhos). Furthermore, the results of of proteins that transduces a signal from a membrane cell-based assays using siRNA against KSR1 (siKSR1) and receptor to the nucleus. In many cancers, including electron microscopy (EM) of human PTC samples BRAFV600E-driven tumors, the sustained activation of indicated that KSR1 participates in the propagation of this pathway causes uncontrolled growth and cell survival, the Notch signal, mitochondrial respiration, and colony promoting carcinogenesis (Hanahan & Weinberg 2011). formation in vitro under BRAFV600E-activated conditions. The signaling pathway comprises many proteins, including various adaptor and scaffold proteins. Recent studies have shown that two of these scaffold proteins, kinase Materials and methods suppressor of RAS (KSR) and connector enhancer of KSR (CNKSR), regulate RAS-mediated RAF activation via novel Subjects mechanisms (Anselmo et al. 2002, Lanigan et al. 2003, We enrolled 165 patients who underwent thyroidectomy Claperon & Therrien 2007, Brennan et al. 2011). for the management of classical PTC between January Several compounds, including sorafenib, dabrafenib, 2013 and January 2014 at Yonsei Cancer Center, Severance and vemurafenib, have inhibitory effects on the RAS–RAF– Hospital, Seoul, South Korea. All samples were micro- Journal of Molecular Endocrinology MAPK pathways (Bollag et al. 2010, 2012, Chapman et al. scopically dissected at the time of surgery and validated by 2011, Hauschild et al. 2012); however, tumors acquire hematoxylin–eosin staining after the operation. The resistance to RAF inhibitors by several mechanisms (Lito samples were taken from the central part of the cancer et al. 2013). In BRAFV600E-driven tumors, ERK-dependent and from contralateral histologically normal tissue. All negative feedback suppresses rzeceptor tyrosine kinase protocols were approved by the Institutional Review Board (RTK)-mediated signaling, resulting in low levels of of Severance Hospital. GTP-bound RAS (Villanueva et al. 2011, Lito et al. 2013); however, inhibition of ERK by RAF inhibitors substantially DNA isolation and sequencing decreases ERK-dependent negative feedback, resulting in Genomic DNA from formalin-fixed paraffin-embedded the restoration of RTK signaling and activation of RAS specimens was prepared (QIAamp DNA FFPE Tissue Kit; (Corcoran et al. 2012, Montero-Conde et al.2013). Qiagen). To examine for the presence of the BRAFT1799A Regarding the mechanism of drug resistance related to mutation, the BRAF gene was amplified by PCR using the the loss of ERK-dependent negative feedback, we postulate following primers: forward 50-ATG CTT GCT CTG ATA GGA that KSR and CNKSR play a role in the restoration of the AA-30 and reverse 50-ATT TTT GTG AAT ACT GGG GAA-30. ERK proliferative signal induced by RTK signaling The PCR products were sequenced on an ABI PRISM 3100 (Therrien et al. 1998, Anselmo et al. 2002, Ohmachi et al. automated capillary DNA Sequencer (Applied Biosystems). 2002, Lanigan et al. 2003, Claperon & Therrien 2007). In support of this idea, ERK activation induced by RTK RNA isolation and qRT-PCR signaling is mediated by RAF dimers that are insensitive to RAF inhibitors, indicating that formation of a signalosome Total RNA was extracted using TRIzol (Invitrogen). cDNA complex tethered by scaffold proteins is a pivotal step in was prepared from total RNA using the Super Script

First-Strand Synthesis System (Invitrogen). Real-time PCR http://www.genenetwork.org/). Statistical analysis was measurement of individual cDNAs was performed using carried out using SPSS version 18.0 for Windows (IBM SYBR Green dye to measure duplex DNA formation on Corporation, Armonk, NY, USA) or GraphPad Prism a Light Cycler System (Roche Diagnostics). cDNA were (GraphPad Software, Inc., San Diego, CA, USA). ampliﬁed using the following primers: KSR1,50-GGG GAG Comparisons of means were performed by paired t-test CAC AAG GAG GAC T-30 and 50-GCG TGC AGG GGA ATA or Mann–Whitney U test. Data are expressed as means 0 0 CAG G-3 ; Notch 1 (NOTCH1), 5 -GAG GCG TGG CAG ACT GS.E.M., *P!0.05, **P!0.01, and ***P!0.001. All reported ATG C-30 and 50-CTT GTA CTC CGT CAG CGT GA-30; P values are two sided. Notch 4 (NOTCH4), 50-GAT GGG CTG GAC ACC TAC AC-30 and 50-CAC ACG CAG TGA AAG CTA CCA-30; NADH Cells, transfection, and immunoblot analysis dehydrogenase (ubiquinone) 1 alpha subcomplex, 4(NDUFA4), 50-ATG CTC CGC CAG ATC ATC G-30 and BCPAP and 8505C cells were cultured in RPMImedia (Sigma– 50-TGC CAG ACG CAA GAG ATA CAG-30; cytochrome c Aldrich) and HeLa cells were grown in DMEM (Sigma– oxidase assembly protein (COX11), 50-AGA GGG TAG AGC Aldrich). The cells were transiently transfected with pEFm- CGT TTC TTA G-30 and 50-GCG TGT GAA AGG GTT CGA G- BRAFV600E or SiKSR1 (sc-35762, Santa Cruz Biotechnology) 30; KSR2,50-GGA AGG GCT TAG GAC CAA ATG-30 and using Lipofectamine PLUS (Invitrogen). The cells were lysed 50-CAT CGA CGA TTC GGA ACC AGT-30; hairy and in lysis buffer, and the lysates were separated by SDS–PAGE. enhancer of split 1 (HES1), 50-TCA ACA CGA CAC CGG After proteins were transferred to a nitrocellulose membrane ATA AAC-30 and 50-GCC GCG AGC TAT CTT TCT TCA-30; (Amersham Biosciences), the membranes were blocked with CNKSR1,50-AAG CCT GAC AGA GGG ACT TCT-30 and 5% skimmed milk and incubated with the indicated primary 50-AGG ACA TCA ATA GGG GTC TTG G-30; CNKSR2, antibodies overnight at 4 8C; after washing, the membranes 50-TCA GTT GTG GAT CTG ATT GGA GC-30 and 50-CGA were incubated with secondary antibodies for 1 h at room CAG GGA TAT GAT GTG GTC AC-30; and glyceraldehyde- temperature. The immunoreactive bands were developed 3-phosphate dehydrogenase (GAPDH), 50-GGA GCG AGA using peroxidase-conjugated secondary antibodies (WEST- TCC CTC CAA AAT-30 and 50-GGC TGT TGT CAT ACT TCT ZOL, iNtRON Biotechnology, Kyungki-Do, South Korea). CAT GG-30. QRT-PCR experiments were repeated three The primary antibodies used in this study were anti-BRAF times, and each experiment was performed in triplicate. (F-7) (sc-5284, Santa Cruz Biotechnology), anti-KSR1 Relative mRNA expression levels were calculated using the (sc-25416, Santa Cruz Biotechnology), anti-Notch1 (#4380, K equation 2DCtnZ2CtGAPDH Cttarget, and then expressed as a Cell Signaling, Danvers, MA, USA), anti-phospho-p44/42

Journal of Molecular Endocrinology ratio (level in PTC/level in matched normal thyroid tissue). MAPK (Thr202/Tyr204) (#4376, Cell Signaling), and anti-actin (sc-1616, Santa Cruz Biotechnology).

IHC analysis EM analysis Paraffin-embedded tissue sections (4 mmthick)were deparaffinized in xylene and stained with anti-KSR1 Tissues were fixed in 2% paraformaldehyde (Sigma–Aldrich) antibody (sc-25416, Santa Cruz Biotechnology). The and 2% glutaraldehyde (Sigma–Aldrich) overnight. After avidin–biotin complex/HRP Kit (DAKO, Copenhagen, rinsing in 0.1 M cacodylate, samples were postfixed in 1%

Denmark) was used according to a standard three-step OsO4 in cacodylate buffer (Hampton Research, Aliso Viejo, technique. Negative controls were incubated with PBS CA, USA). The samples were dehydrated through graded instead of primary antibody, and normal adrenal gland ethanol (10–100%, Merck KgaA) for 10 min each, and then tissues were used as positive controls. embedded in Epon (EMS, Hatfield, PA, USA). The prepared samples were analyzed by an EM (Tecnai G2 Spirit Twin; FEI company; Korea Basic Science Institute). The morphology of Public data and statistical analysis mitochondria cristae was quantified by measuring the Data from the Gene Expression Omnibus (GEO) of NCBI lengths of cristae in a mitochondrion and normalizing the (http://www.ncbi.nlm.nih.gov/projects/geo; accession lengths by the area of the mitochondrion using ImageJ no. GSE33630) were subjected to Gene Set Enrichment Software (NIH, Bethesda, MD, USA; Schneider et al.2012). Analysis (GSEA) (Subramanian et al. 2005). Analysis of the For comparison, the average ratio of cristae lengths to correlation between KSR1 and NOTCH1 was performed mitochondrial area in PTC samples with low KSR1 mRNA using GeneNetwork (a free scientific web resource, expression was set to 100% (Costa et al. 2010).

Colony formation assays inter-tumor heterogeneity of KSR1 expression (Fig. 1A); however, 108 (65.5%) out of 165 cases expressed higher The cells (5.0!102 cells/well) were seeded into six-well cell levels of KSR1 mRNA in tumors than that in control culture plates and allowed to grow for 14 days. Every tissue, and the difference was statistically significant 2 days, the cells were transfected with siKSR1 (sc-35762, (paired t-test, P!0.001). Interestingly, the mean relative Santa Cruz Biotechnology) using JetPei (Polyplus- KSR1 mRNA level of BRAFV600E-positive PTC samples Transfection SA, Illkirch, France). The colonies were fixed in methanol and stained with crystal violet (Sigma– was higher than that of BRAFV600E-negative PTC ! Aldrich). Visible colonies containing w50 or more cells samples (P 0.001, Fig. 1B). To validate our qRT-PCR were counted. Three independent experiments were data, we conducted IHC analysis using anti-KSR1 Z performed in triplicate (Franken et al. 2006). antibody (n 165). Tumor cells in PTCs with high levels of KSR1 mRNA consistently exhibited abundant protein expression relative to adjacent normal tissues (Fig. 1C and Results D). Furthermore, metastatic LNs from PTCs with high levels of KSR1 mRNA also exhibited markedly elevated Expression of KSR1 in normal human thyroid tissues KSR1 expression (Fig. 1E). Taken together, these data and PTCs indicate that a subset of PTCs express higher levels of To evaluate the expression pattern of KSR1/2, we first KSR1 mRNA than that of normal thyroid tissues, performed qRT-PCR using cDNA from PTCs and matched suggesting that PTC can be classified according to KSR1 contralateral normal thyroid tissues. The results revealed expression status.

C A KSR1

1.2 ***

1.1

1.0

Journal of Molecular Endocrinology 0.9 D

Relative mRNA expression ratio mRNA expression Relative 0.8 Normal PTC

B KSR1 1.3 *** 1.2 E 1.1

1.0

0.9

0.8

Relative mRNA expression ratio mRNA expression Relative 0.7 Negative Positive BRAFV600E mutation

Figure 1 Expression of kinase suppressor of RAS1 (KSR1) in normal human thyroid the relative mRNA level in the PTC/relative mRNA level in matched tissues and papillary thyroid cancers (PTCs). (A) Result of qRT-PCR to normal thyroid tissue. Mean ratios were compared by paired t-test. compare KSR1 mRNA expression in PTCs vs matched normal thyroid tissues. ***P!0.001. (C, D and E) Representative immunohistochemical staining (B) Comparison of KSR1 mRNA expression in PTCs according to BRAFV600E for KSR1 in tissue sections of PTC or metastatic lymph nodes (original mutation status. Relative mRNA expression ratio in PTCs was calculated as magniﬁcation, !200).

Analysis of enriched gene sets in PTCs expressing As summarized in Supplementary Table 1, see section on high levels of KSR1 mRNA supplementary data given at the end of this article, the top 20 gene sets enriched in high-KSR1 PTCs included Next, we sought to identify enriched gene sets in PTCs the wingless-type MMTV integration site family (WNT) expressing high levels of KSR1 mRNA.Tothisend,we signaling pathway (nominal PZ0.0014, false discovery used public repository GEO dataset GSE33630, which rate (FDR) q-valueZ0.131), colorectal cancer (nominal includes data from 49 PTCs obtained from Ukraine via PZ0.0138, FDR q-valueZ0.1475), and the Notch signal- the Chernobyl Tissue Bank. Among these, we selected the ing pathway (nominal PZ0.019, FDR q-valueZ0.1647) 16 PTCs with the highest KSR1 expression (high-KSR1 PTCs) and 16 PTCs with the lowest KSR1 expression (Fig. 2A). Compatible with the results of GSEA, Gene- (low-KSR1 PTCs) and subjected these samples to GSEA. Network analysis using repository data (Record ID:

Low KSR1 High KSR1 A DLL4 JAG1 DVL2 DVL3 MAML1 CREBBP Nominal P value=0.019 MAML2 NUMB FDR q-value=0.165 CTBP2 JAG2 CTBP1 ADAM17 EP300 NCOR2 DLL1 DTX3 NOTCH4 NOTCH3 LFNG NUMBL MFNG NOTCH1

BC n = 53 Spearman’s rank correlation

Journal of Molecular Endocrinology 60

60 77 64 8 6 50 43 49 66 14 102 39 62 ** 83 42 C57BL/6J 6 ** 40 70 D2B6F1 69 48 4 100 32 B6D2F1 29 68 101 30 74 4 84 85 11 75 31 NOTCH1 40 27 2 56 20 DBA/2J 45 79 2 103 ** 67 89 ** 90 87 65 34 mRNA expression Relative mRNA expression Relative 80 10 95 63 44 0 0 01 ρ 73 =0.677 97 NOTCH1 NOTCH4 NDFUA4 COX11 50 –9 12 99 P=6.15×10 0 24 10 20 30 40 50 60 KSR1

Figure 2 Bioinformatic analysis indicating the relationship between kinase exon level: 10388917). (C) QRT-PCR data indicated mRNA expression of suppressor of RAS1 (KSR1) and Notch signaling. (A) Gene Set Enrichment NOTCH1, NOTCH4, NDUFA4,andCOX11 in PTC samples with low KSR1 Analysis revealed enrichment of genes involved in Notch signaling in expression (white bars, nZ7 per group) and in PTC samples with high KSR1 papillary thyroid cancers (PTCs) with high KSR1 expression. (B) Gene- expression (blue bars, nZ7 per group). Mean comparisons were performed Network Analysis revealed a strong positive correlation between KSR1 and by the Mann–Whitney U test. Data are meanGS.E.M. and P values are NOTCH1 expression levels in repository data (Spearman’s rank correlation, two-sided, **P!0.01. rZ0.677, PZ6.15!10K9; INIA Adrenal Affy MoGene 1.0ST (Jun 12) RMA

10388917) also revealed a strong positive correlation for Notch activation induced by BRAFV600E oncogenic between KSR1 and NOTCH1 expression levels (Supple- kinase. mentary Fig. 1 and Fig. 2B). As shown in Supplementary Table 2, the top 20 gene sets enriched in low-KSR1 PTCs Defects in cristae shape in PTCs with high KSR1 included OxPhos (nominal P!0.001, FDR q-valueZ Because our GSEA analysis indicated the reduction 0.0069) (Supplementary Fig. 2). In accord with the results in OxPhos in high-KSR1 PTCs, as veriﬁed by qRT-PCR in of GSEA, qPCR data obtained using cDNA from the study our study subjects, we observed the mitochondrial subjects indicated that PTC samples with high KSR1 morphology of PTC using EM. This analysis revealed that expression had higher NOTCH1 and NOTCH4 mRNA human PTCs expressing higher levels of KSR1 contained levels and lower NDUFA4 and COX11 mRNA levels than more fragmented mitochondria and exhibited more PTC samples with low KSR1 expression (Fig. 2C). Taken profound defects in cristae structure than PTCs expressing together, the results of GSEA, GeneNetwork analyses, and low levels of KSR1 (Fig. 4A and Supplementary Fig. 3, see qPCR suggest that high KSR1 expression is associated section on supplementary data given at the end of this with upregulation of the Notch signaling pathway and article). In addition, mitochondrial cristae length in PTC downregulation of OxPhos. samples with high KSR1 expression was shorter than that in PTC samples with low KSR1 expression (P!0.01, Fig. 4B). KSR1 is required for BRAFV600E-induced NOTCH1 activation The effect of siKSR1 on cell proliferation

Based on our observation of a relationship between KSR1 Our GSEA, cell-based assays, and EM consistently indi- and Notch signaling, we asked whether KSR1 is required cated that KSR1 is related to NOTCH1 and OxPhos, all of for BRAFV600E-induced Notch activation. As shown in which can affect cell proliferation during carcinogenesis. Fig. 3A, NOTCH1 expression was markedly increased in To conﬁrm the impact of KSR1 on tumor behavior, HeLa cells by BRAFV600E, accompanied by robust ERK we performed a colony-forming assay using BCPAP phosphorylation. Remarkably, when we treated HeLa cells cells (BRAFV600E-positive PTC cells) and 8505C cells with siKSR1, ERK phosphorylation by BRAFV600E was (BRAFV600E-positive anaplastic thyroid cancer cells) barely detectable, and NOTCH1 expression was also treated with siKSR1. After 14 days of growth with siKSR1 signiﬁcantly affected (Fig. 3B). QRT-PCR analysis revealed treatment for every 2 days, the number of colonies was

Journal of Molecular Endocrinology that siKSR1 decreased expression of NOTCH1 and HES1,a signiﬁcantly decreased (Fig. 4C and D), suggesting that representative of NOTCH1 target (Fig. 3C and D). Taken KSR1 plays a pivotal role in cell proliferation in together, cell-based assays conﬁrmed that KSR1 is required BRAFV600E-driven carcinogenesis.

AB CD NOTCH1 HES1 BRAF BRAF 1.5 1.5

NOTCH1 NOTCH1 (NTM) (NTM) 1.0 1.0

pERK pERK 0.5 *** 0.5 *** Actin KSR1 0.0 0.0 Relative mRNA expression Relative Control BRAFV600E Actin Control siKSR1 mRNA expression Relative Control siKSR1 BRAFV600E BRAFV600E Control siKSR1 BRAFV600E

Figure 3 Effects of kinase suppressor of RAS1 (KSR1) knockdown on activation of cells with BRAFV600E and siKSR1, western blotting analyses were NOTCH1 signaling by oncogenic BRAFV600E kinase. (A) Forty-eight hours performed to detect the indicated proteins. (C and D) Under the same after transfection of HeLa cells with BRAFV600E, western blotting analyses experimental condition as in B, qRT-PCR was performed. All data are were performed to detect the indicated proteins. NTM, transmembrane/ meansGS.E.M., ***P!0.001. intracellular region. (B) Forty-eight hours after co-transfection of HeLa

ABCD

BCPAP 8505C 120 BCPAP 80 600 100 60 80 400 60 ** 40 40 *** 200

No. of colonies No. 20 of colonies No. ** 20 8505C area (% of low KSR1) area (% of low 0 0 0 Cristae length/mitochondrial Low High Low KSR1 High KSR1 Control siKSR1 Control siKSR1 KSR1 KSR1 Control siKSR1

Figure 4 Electron microscopy and colony-forming assays. (A) Mitochondrial nZ7 per group). See the detailed description in ‘Materials and methods’ morphology in human papillary thyroid cancers (PTCs) with low or high section. (C) Representative ﬁgures of colony-forming assays. (D) Compari- kinase suppressor of RAS1 (KSR1) expression (original magniﬁcation, son of number of colonies between control and siKSR1-treated HeLa cells. !30 000). (B) Morphometric analysis of mitochondrial cristae. Data Means were compared by Mann–Whitney U test. All data are meansGS.E.M., represent meanGS.E.M. (50 random selected mitochondria/sample, **P!0.01 and ***P!0.001.

Analysis of the enriched gene sets in PTCs expressing Discussion high levels of KSR2, CNKSR1,orCNKSR2 The discovery of oncogenic BRAFV600E led to intensive As our data suggested that KSR1 expression correlates with investigation of the RAS–RAF–MAPK signaling pathway increased Notch1 signaling and decreased OxPhos, we (Davies et al. 2002, Namba et al. 2003), but the exact decided to investigate whether KSR2 and CNKSR1/2, mechanism of signal propagation through this pathway which are necessary for the regulation of RAF in the remains elusive. As the key roles played by protein–protein MAPK pathway, are also involved in these processes. In the interactions (e.g. RAF homo- and heterodimers) in RAF case of KSR2, 85 (51.5%) PTC samples exhibited high activation have been revealed, the complexity of the RAS– mRNA levels (Fig. 5A), but the difference in expression RAF–MAPK pathway has taken central stage (Montagut between the tumor tissue and the control tissue was not et al. 2008, Bollag et al. 2010, Hatzivassiliou et al. 2010, statistically signiﬁcant. The top 20 gene sets enriched in Johannessen et al. 2010). In addition to RAF dimerization, PTC samples expressing high levels of KSR2 included those additional regulatory interactions are important: for

Journal of Molecular Endocrinology of WNT (nominal P!0.001, FDR q-valueZ0.163) and example, heat shock protein 90 stabilizes the RAF protein Notch pathways (nominal PZ0.009, FDR q-valueZ0.154) itself, whereas the adaptor protein 14-3-3 stabilizes the (Supplementary Table 3, see section on supplementary active conformation of RAF (Tzivion & Avruch 2002, data given at the end of this article, Fig. 5D,and Grbovic et al. 2006). KSR1/2, a pseudokinase scaffold Supplementary Fig. 4). As shown in Fig. 5B and C, qRT- protein, regulates the MAPK cascade (including MEK and PCR revealed heterogeneous expression of CNKSR1/2 in ERK) by interacting with RAF proteins (Claperon & PTC samples. As shown in Supplementary Table 4, the top Therrien 2007). In addition, CNKSR1/2 are also necessary 20 gene sets enriched in the high CNKSR1 mRNA components in the regulation of the RAS–RAF–MAPK expression group included genes of the Notch signaling signaling pathway (Therrien et al. 1998). pathway (nominal P!0.001, FDR q-valueZ0.006, Fig. 5E In our study, expression levels of KSR1/2 and and Supplementary Fig. 5A). In GSEA of CNKSR2, low CNKSR1/2 were somewhat heterogeneous. Nonetheless, CNKSR2 expression was associated with genes related to a signiﬁcant proportion of PTCs exhibited elevated OxPhos (nominal P!0.001, FDR q-value !0.001) (Supple- expression of these scaffold proteins relative to matched mentary Table 5, Fig. 5F, and Supplementary Fig. 5B). normal tissues, indicating that PTC can be classiﬁed by Taken together, these data indicate that, similar to the KSR1/2 and CNKSR1/2 expression status. Moreover, expression of KSR1, the expression of KSR2, CNKSR1, and BRAFV600E positive PTC samples showed higher KSR1 CNKSR2 is also associated with the regulation of the Notch mRNA expression, indicating that KSR1 scaffold function signaling pathway and OxPhos. Supporting this idea, KSR1 might be essential for BRAFV600E signaling. In GSEA, high mRNA levels showed a strong positive correlation with expression of KSR1 and KSR2 was associated with coordi- KSR2 and CNKSR1 expression, and tended to correlate nated upregulation of Notch signaling. Previous studies with CNKSR2 expression (Supplementary Fig. 6). using pancreatic cancer cells suggested that the MAPK

A KSR2BC CNKSR1 CNKSR2

1.4 1.25 *** 2.0 NS *** 1.15 1.2 1.5

1.05

1.0 1.0 0.95

0.8 0.85 0.5 Relative mRNA expression ratio mRNA expression Relative ratio mRNA expression Relative ratio mRNA expression Relative Normal PTC Normal PTC Normal PTC

DENotch signaling pathway Notch signaling pathwayF Oxidative phosphorylation

Nominal P value=0.0097 Nominal P value <0.0001 Nominal P value <0.0001 FDR q-value=0.154 FDR q-value=0.00554 FDR q-value <0.0001

KSR2 CNKSR1 CNKSR2

Figure 5 Expression of kinase suppressor of RAS2 (KSR2) and connector enhancer was the same as that used in Fig. 1A. (D and E) Gene Set Enrichment of kinase suppressor of RAS1/2 (CNKSR1/2) in normal human thyroid Analysis (GSEA) showing that the Notch signaling pathway was enriched in tissues and papillary thyroid cancers (PTCs). (A, B and C) QRT-PCR results PTCs with high KSR2 (D) and CNKSR1 (E) expression. (F) GSEA showing that comparing KSR2 (A), CNKSR1 (B), and CNKSR2 (C) mRNA expression in PTC gene set related to oxidative phosphorylation was enriched in PTCs with tissue with that in matched normal thyroid tissues. The analytic method low CNKSR2 expression. ***P!0.001.

pathway is able to promote Notch signaling activation by induced by RTK signaling, resulting in the expression of g-secretase complex-dependent proteolysis (Tremblay et al. dual-speciﬁcity phosphatases (DUSPs) and Sprouty (SPRY) 2013). Although we could not demonstrate g-secretase proteins (Pratilas et al. 2009, Lee et al. 2012). However, complex activity using the g-secretase peptide cleavage selective BRAFV600E inhibitors restore RTK-induced assay (Farmery et al. 2003, McLeod et al. 2009), we observed MAPK signal propagation by inhibiting this negative

Journal of Molecular Endocrinology an increase in the level of the transmembrane/intracellular feedback. In the resultant new steady state, KSR1/2 and region (NTM) of NOTCH1 upon BRAFV600E transfection, CNKSR1/2 work coordinately to propagate the MAPK which is consistent with the results of the previous study. signal. Indeed, CNKSR1 is required for RAS signaling, Remarkably, knockdown of KSR1 by siRNA abrogated linking RHO and RAS signal transduction (Jaffe et al. NOTCH1 activation by BRAFV600E, suggesting that KSR1 2004). In addition, CNKSR1 acts as a scaffold protein for is required for BRAFV600E-induced Notch activation. KSR1 the AKT–FOXO signaling axis, and also promotes invasion expression was also associated with downregulation of of cancer cells via NFkB-dependent signaling (Fritz & gene sets related to OxPhos. This was veriﬁed by perform- Radziwill 2010, 2011, Fritz et al. 2010). In our GSEA, gene ing qRT-PCR expression analysis of NOTCH1, NOTCH4, sets related to Notch signaling or OxPhos were enriched in NDUFA4, and COX11 mRNA using cDNA from PTC samples high-CNKSR1 or low-CNKSR2 PTCs, respectively, of our study subjects. In addition, EM clearly revealed suggesting that Notch signaling and OxPhos are regulat- defective mitochondrial cristae in PTC samples expressing ory targets of CNKSR1/2 through the RAS–RAF–MAPK high levels of KSR1. In light of these observations, we signaling pathway. postulated that activation of Notch signaling combined In summary, our data indicate that KSR1/2 and with downregulated OxPhos strongly promotes cancer-cell CNKSR1/2, scaffold proteins of RAS–RAF–MAPK signaling, proliferation. In support of this idea, colony-forming assays are consistently associated with upregulation of NOTCH1 demonstrated that the proliferation of BCPAP and 8505C signaling and downregulation of gene sets related to tumor cells, which harbor the BRAFV600E mutation, was OxPhos. As PTCs exhibit a linear signal cascade during signiﬁcantly affected by KSR1 knockdown. carcinogenesis regardless of the initiating oncogenic The transcription-mediated negative feedback pro- events, the enriched gene sets common to PTCs expressing voked by BRAFV600E can repress MAPK activation high levels of KSR1/2 and CNKSR1/2 represent the most

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Received in ﬁnal form 6 January 2015 Accepted 20 January 2015 Accepted Preprint published online 21 January 2015