ORIGINAL ARTICLE Src-Homology 2 Domain-Containing Phosphatase 2 in Resected EGFR Mutation-Positive Lung Adenocarcinoma Masaoki Ito, MD,a,b,c Jordi Codony-Servat, PhD,a Ana Giménez-Capitán, BSc,a Mireia Serra-Mitjans, MD,d Francisco Pérez-Ochoa, MD,e,f David Llige, BSc,b Imane Chaib, PhD,b Ramón Rami-Porta, MD,d,g Carme Obiols, MD,d Sergi Call, MD,d,h Manuela Iglesias, MD,d José Belda-Sanchis, MD,d Xavier Tarroch-Sarasa, MD,e Niki Karachaliou, MD,a,** Miguel Angel Molina-Vila, PhD,a Morihito Okada, MD,c Rafael Rosell, MDb,i,* a Pangaea Oncology, Laboratory of Molecular Biology, Quirón-Dexeus University Institute, Barcelona, Spain b Laboratory of Cellular and Molecular Biology, Institute for Health Science Research Germans Trias i Pujol (IGTP), Badalona, Spain c Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan d Thoracic Surgery Service, University Hospital Mutua de Terrassa, Terrassa, Spain e Department of Pathology, University Hospital Mutua de Terrassa, Terrassa, Spain f Department of Pathology, Atryshealth - Centro Medico Teknon, Barcelona, Spain g Network of Centers for Biomedical Research in Respiratory Diseases (CIBERES), Lung Cancer Group, Terrassa, Spain h Department of Morphological Sciences, School of Medicine, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain i Institute of Oncology Rosell (IOR), Quirón-Dexeus University Institute, Barcelona, Spain Received 14 May 2020; revised 28 July 2020; accepted 13 August 2020 Available online - 21 August 2020 ABSTRACT EGFR-mutant cell lines, the combination of SHP099 or RMC- 4550 (SHP2 inhibitors) with erlotinib revealed synergism Introduction: EGFR mutation-positive lung adenocarci- via abrogation of phosphorylated AKT (S473) and ERK1/2 noma (LUAD) displays impaired phosphorylation of ERK (T202/Y204). Although erlotinib translocates phosphory- and Src-homology 2 domain-containing phosphatase 2 lated SHP2 (Y542) into the nucleus, either RMC-4550 alone, (SHP2) in comparison with EGFR wild-type LUADs. We or in combination with erlotinib, relocates SHP2 into the hypothesize that SHP2 expression could be predictive in cytoplasm membrane, limiting AKT and ERK1/2 activation. patients positive with resected EGFR mutation versus pa- tients with EGFR wild-type LUAD. Methods: We examined resected LUAD cases from Japan *Corresponding author. and Spain. mRNA expression levels of AXL, MET, CDCP1, Disclosure: The authors declare no conflict of interest. STAT3, YAP1, and SHP2 were analyzed by quantitative Address for correspondence: Rafael Rosell, MD, Institut d’Investigació reverse transcriptase polymerase chain reaction. The ac- en Ciències de la Salut Germans Trias i Pujol Campus Can Ruti (Edifici Muntanya), Ctra. de Can Ruti, Cami de les Escoles s/n, 08916 Badalona, tivity of SHP2 inhibitors plus erlotinib were tested in EGFR- Barcelona, Spain. E-mail: rrosell@iconcologia.net mutant cell lines and analyzed by cell viability assay, **Current address: Merck Healthcare KGaA Darmstadt, Germany. Western blot, and immunofluorescence. Cite this article as: Ito M, et al. Src-Homology 2 Domain-Containing Results: A total of 50 of 100 EGFR mutation-positive LUADs Phosphatase 2 in Resected EGFR Mutation-Positive Lung Adenocarcinoma. JTO Clin Res Rep 1:100084 relapsed, among them, patients with higher SHP2 mRNA ª 2020 The Authors. Published by Elsevier Inc. on behalf of the expression revealed shorter progression-free survival, in International Association for the Study of Lung Cancer. This is an comparison with those having low SHP2 mRNA (hazard open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). ratio: 1.83; 95% confidence interval: 1.05–3.23; p ¼ ISSN: 2666-3643 0.0329). However, SHP2 was not associated with prognosis https://doi.org/10.1016/j.jtocrr.2020.100084 in the remaining 167 patients with wild-type EGFR. In JTO Clinical and Research Reports Vol. 1 No. 4: 1–11 2 Ito et al JTO Clinical and Research Reports Vol. 1 No. 4 Conclusions: Elevated SHP2 mRNA levels are associated Src-homology-2 domain containing phosphatase-2 with recurrence in resected EGFR mutation-positive LUADs, (SHP2, encoded by the PTPN11 gene) is a promi- but not in EGFR wild-type. EGFR tyrosine kinase inhibitors gratory signal from numerous growth-factors, cytokines can enhance SHP2 activation, hindering adjuvant therapy. and receptor tyrosine kinases (RTKs). Phosphorylated SHP2 inhibitors could improve the benefit of adjuvant SHP2 (pSHP2)-Y542 is a readout of resistance to TKIs in therapy in EGFR mutation-positive LUADs. melanoma.18 SHP2 activates Src family kinases, mainly  2020 The Authors. Published by Elsevier Inc. on behalf of Src, YES, and FYN, and downstream targets, inducing the International Association for the Study of Lung Cancer. migration and invasion.19 In a previous study, we re- This is an open access article under the CC BY-NC-ND li- ported that high levels of SHP2 mRNA expression cense (http://creativecommons.org/licenses/by-nc-nd/ correlate with poor progression-free survival and OS in 4.0/). advanced EGFR mutation-positive LUADs treated with EGFR TKIs.20 Moreover, combinatory therapy with SHP2 Keywords: Lung adenocarcinoma; EGFR; SHP2; Surgery; inhibitors was noted to be effective in BRAF-mutant lung Recurrence cancer cells.21 Early studies indicated that EGFR-mutant cells have limited ERK signaling and SHP2, which partly explains the sensitivity to EGFR TKIs.22 We assessed the mRNA levels of several RTKs and non-RTKs, including Introduction MET, AXL, CDCP1, STAT3, YAP1, and SHP2, in 267 Accurate prediction of recurrence in early-stage lung resected LUADs, mostly stage IA. In this study, we adenocarcinomas (LUADs) is attained by gene expression investigated whether membranous SHP2 translocated to profiling,1 multiregion sequencing, and bespoke multiplex- the nucleus as a mechanism of resistance to first- polymerase chain reaction (PCR) assays.2,3 However, the generation TKI (erlotinib) in EGFR-mutant cells and benefit of adjuvant therapy in patients with high risk of whether mRNA levels of SHP2 could be prognostic in recurrence has not been improved, neither with cisplatin- EGFR mutation-positive surgically resected LUADs based chemotherapy (4% of survival benefit at 5 y), nor with bevacizumab.4 The 5-year survival rate in early, Methods completely resected lung cancer varies from 67% in pa- tients with T1-N0 (stage IA) to 23% for T1-3N2 (stage Patients IIIA) disease.5 Moreover, adjuvant cisplatin-based chemo- Surgical specimens of primary invasive resected therapy in stage IA could have a detrimental effect on LUADs between February 2007 and December 2015 at survival,6 which was not observed with adjuvant oral the Hiroshima University Hospital and between uracil-tegafur.7 In early resected EGFR mutation-positive November 2017 and September 2018 at the University LUADs, gefitinib and erlotinib were tested in the BR19 Hospital Mutua de Terrassa were collected. Patients, af- and RADIANT trials, respectively. The BR19 study ter preoperative chemotherapy, chemoradiotherapy or included a small number of EGFR mutation-positive incomplete resection, or with unavailable specimens or LUADs with better overall survival (OS) for the placebo- clinicopathologic data, were excluded from the study. treated group (hazard ratio of death was 3.16, p ¼ Patients with adenocarcinoma in situ and minimally 0.15).8 Nor was any survival benefit seen with erlotinib invasive adenocarcinoma, were also excluded, owing to versus placebo in stage IB to IIIA EGFR mutation-positive no recurrence after complete resection. All cases were LUADs (RADIANT).9 The benefit of gefitinib versus diagnosed according to the 2015 WHO pathologic clas- vinorelbine plus cisplatin was seen in patients with stage sification23 and the International Association for the II to IIIA EGFR mutation-positive LUAD.10 Several ongoing Study of Lung Cancer eighth TNM staging system was studies of adjuvant EGFR tyrosine kinase inhibitors (TKIs) utilized for staging.24 The Institutional Review Boards are ongoing in resected lung cancer.11 Caution with adju- approved the design of this study for (IRB number: E- vant EGFR TKIs should be taken because EGFR-mutant 908 in Hiroshima University, EO/1745 in University lung cancer cells treated with erlotinib or afatinib acti- Hospital Mutua de Terrassa), and all study participants vate Notch signaling, increasing aldehyde dehydrogenase provided informed written consent. The research was stem-like cells.12,13 EGFR TKIs in combination with STAT3 conducted in accordance with the 1964 Declaration of inhibitors,13 AKT inhibitors,14 Src inhibitors,15 STAT3, and Helsinki and its later amendments. Src inhibitors16 or TPX-0005 (repotrectinib)17 can revert the resistance to EGFR TKIs in EGFR-mutant cells. Previ- Tumor Tissue Specimens Evaluation ously, we found that elevated mRNA expression of AXL Formalin-fixed paraffin-embedded (FFPE) surgically and CDCP1 negatively influences progression-free survival resected tissues were cut into four slices of 4 mm for and OS in advanced EGFR mutation-positive LUADs.17 RNA extraction. Sliced samples were deparaffinized November 2020 SHP2 in Resected EGFR Mutation-Positive LUAD 3 using xylene. After the removal of xylene using ethanol, purchased by the American Type Culture Collection. tissues were lysed in buffer containing tris-chloride, HCC827 cells, harboring EGFR exon 19 deletion (E746- ethylenediaminetetraacetic acid, sodium dodecyl sul- A750), and H1666 EGFR wild-type cells were pur- fate, and proteinase K. Next, RNA was extracted using a chased by American Type Culture Collection. Cells mixture of phenol and chloroform followed by precipi- were cultured with Roswell Park Memorial Institute tation using isopropanol, glycogen (10901393001, medium supplemented with 10% fetal bovine serum Darmstadt, Merck), and sodium acetate. The RNA was and 50 mg/mL penicillin-streptomycin. Cells were resolved in nuclease-free water and purified using a DNA maintained in a humidified atmosphere with 5% car- removal kit (AM1906, Thermo Fisher Scientific, Wal- bon dioxide at 37 C. Erlotinib and the SHP2 inhibitor tham, MA). Complementary DNA was synthesized using SHP099 were obtained from Selleckchem, Houston, reverse transcriptase-PCR with M-MLV (28025013, Texas, whereas the SHP2 inhibitor RMC-4550 was Thermo Fisher Scientific), dNTP Mix (110002, BIORON obtained from CHEMIETEK, Indianapolis, Indiana. GmbH, Römerberg, Germany), Random primer (48190- Erlotinib was used at concentrations from 0.5 mM to 011, Thermo Fisher Scientific), and RNase OUT 2.5 mM, corresponding to the plasma concentrations of (10777019, Thermo Fisher Scientific). The complemen- less than 50 mg/day and around 150 mg/day in tary DNA was mixed with Taqman Universal Master Mix clinical settings, respectively.5 Each reagent was used (4318157, Thermo Fisher Scientific) containing specific in a single-reagent treatment or in combination in the primers and probes for each gene. The sequences of three cell lines. In the combination treatment, the primers and probes are shown in Supplementary synergistic effect of erlotinib plus SHP099 or RMC- Table 1. Real-time PCR was performed using the ABI 4550 was evaluated. Details regarding the methods Prism 7900HT Sequence Detection System (Thermo of the preclinical experiments, such as, cell viability Fisher Scientific), and mRNA expression was quantified assay, colony formation assay, Western blotting anal- using the following formula; 2(DCt sampleDCt calibrator), in ysis, and immunofluorescence experiments are pro- which DCt values of the calibrator and sample were vided in the Methods section of the Supplementary determined by subtracting the cycle threshold (Ct) value Appendix. of the house-keeping gene (b-actin) from the Ct value of the target gene. Commercial MVP Total RNA Human liver Statistical Analysis and lung controls were used as calibrators (540017 and To estimate the prognostic impact of the biomarkers 540019, Agilent Technologies, Santa Clara, CA). In the explored in this study, the mRNA expression levels be- quantitative process, the SD of the Ct values was kept tween recurrent and nonrecurrent cases were compared less than 0.30 in two or three independent analyses. The considering the EGFR mutation status, and the heatmap was made using SHINYHEATMAP.COM (http:// recurrence-free survival (RFS) and OS. RFS was defined shinyheatmap.com/) after the quantified mRNA was from the day of operation to the day when recurrence converted to logarithm and standardized using the was detected radiologically or the death owing to any following formula: [log(mRNA) – average value]/SD. cause. OS was defined from the day of operation to the DNA was extracted from five slices of 5 mm FFPE day of death from any cause. RFS and OS were calculated tissues using the QIAamp DNA FFPE Tissue Kit (51304, by the Kaplan-Meier method. Qiagen GmbH, Hilden, Germany) in accordance with the The significance of difference between RFS and OS manufacturer’s protocol. The EGFR mutation status curves was determined by the log-rank test unless sur- (G719X in exon 18, deletions in exon 19, and L858R/ vival curves cross the point of less than 80% of survival L861Q in exon 21) was detected by a peptide nucleic probability. The significance of frequencies for the data acid-locked nucleic acid PCR clamp-based detection test of the patients was compared using chi–square test or using a 7900HT Fast Real-Time PCR System (Thermo Yates-square test. Age was compared as continuous Fisher Scientific Inc.) as previously described.1,2 variables using Mann-Whitney U tests. For the preclinical studies, the strength of interaction Cell Culture Experiments between reagents was determined by the combination Cell Lines and Reagents. Four EGFR-mutant lung index according to the method of the isobologram- cancer cell lines were used. The human LUAD PC9 combination index (Chou-Talalay method) as previ- cells, harboring EGFR exon 19 deletion (E746-A750) ously described.3 The expression levels of protein and were provided by F. Hoffmann-La Roche Ltd. with the mRNA were compared using the Wilcoxon t-test or authorization of Dr. Mayumi Ono (Kyushu University, Mann-Whitney U-test. The in vitro examinations were Fukuoka, Japan) H1975 cells, harboring both sensi- repeated at least three times. Two-sided statistics were tizing L858R and resistant T790M mutation, were employed and a p-value of less than 0.05 was regarded 4 Ito et al JTO Clinical and Research Reports Vol. 1 No. 4 Table 1. Clinicopathologic Characteristics of Enrolled Patients With pN0-2 Adenocarcinoma (N ¼ 267) EGFR Status Clinicopathologic Characteristic Mutant (N ¼ 100) Wild Type (N ¼ 167) p Value Positive EGFR mutation status, N (%) G719X in exon 18 6 (6.0) — — Deletion in exon19 38 (38.0) — — Mutation in exon 21 52 (52.0) — — L858R 49 (49.0) — — L861Q 3 (3.0) — — Double mutation 4 (4.0) — — Age, y Median (interquartile range) 70 (13.0) 69 (14.0) 0.635 Range 41–89 40–91 — Sex, N (%) Male 35 (35.0) 109 (65.3) <0.001 Female 65 (65.0) 58 (34.7) — Smoking status, N (%) Ex- or current 37 (37.0) 114 (68.3) <0.001 Never 63 (63.0) 52 (31.1) <0.001 Unknown 0 (0.0) 1 (0.6) 0.795 Surgical procedure, N (%) Pneumonectomy 0 (0.0) 1 (0.6) 0.795 Lobectomy 74 (74.0) 117 (70.1) 0.490 Segmentectomy 23 (23.0) 35 (21.0) 0.695 Wedge resection 3 (3.0) 14 (8.4) 0.138 Predominant subtype, N (%) Lepidic 24 (24.0) 34 (20.4) 0.485 Papillary 58 (58.0) 70 (41.9) 0.0109 Acinar 8 (8.0) 31 (18.6) 0.0288 Solid 5 (5.0) 20 (12.0) 0.0936 Micropapillary 5 (5.0) 7 (4.2) 0.997 Invasive mucinous 0 (0.0) 5 (3.0) 0.200 Pleural invasion, N (%) Negative 72 (72.0) 125 (74.9) 0.608 Positive 28 (28.0) 42 (25.1) — Lymphatic invasion, N (%) Negative 71 (71.0) 133 (79.6) 0.108 Positive 29 (29.0) 34 (20.4) — Vascular invasion, N (%) Negative 73 (73.0) 122 (73.1) 0.992 Positive 27 (27.0) 45 (26.9) — Intrapulmonary metastasis, N (%) Negative 96 (96.0) 161 (96.4) 0.870 Positive 4 (4.0) 6 (3.6) — Pathologic N descriptor, N (%) N0 71 (71.0) 140 (83.8) 0.0127 N1 8 (8.0) 13 (7.8) 0.864 N2 21 (21.0) 14 (8.4) 0.00311 Pathologic stage, N (%) IA1–IA3 45 (45.0) 86 (51.5) 0.304 IB 20 (20.0) 42 (25.1) 0.335 IIA–IIB 11 (11.0) 21 (12.6) 0.701 IIIA–IIIB 24 (24.0) 18 (10.8) 0.00408 Recurrence, N (%) No 53 (53.0) 125 (74.9) <0.001 Yes 47 (47.0) 42 (25.1) — Locoregional 35 (35.0) 34 (20.4) 0.633 Distant 8 (8.0) 7 (4.2) 0.811 Locoregional þ distant 4 (4.0) 1 (0.6) 0.365 November 2020 SHP2 in Resected EGFR Mutation-Positive LUAD 5 Figure 1. Comparison of mRNA expression of six RTKs and non-RTKs according to EGFR mutation, recurrent status, and prognostic impact of SHP2 expression. (A) Heatmap depicting mRNA expression of six RTKs and non-RTKs. The recurrent cases and nonrecurrent cases were gathered to upper and lower location, respectively. The expression patterns of SHP2, CDCP1, and MET are comparable. In contrast, YAP, STAT3, and AXL revealed similarity in expression patterns. (B) Comparison of mRNA level according to EGFR mutation and recurrent status. SHP2 expression is significantly higher in recurrent cases than recurrence-free cases with EGFR mutation. As the heatmap suggested, CDCP1 and MET reveal similarity with SHP2; CDCP1 and MET reveal a tendency for higher expression in recurrent cases than recurrence-free cases with EGFR mutation (the difference did not attain significance). YAP1, STAT3, and AXL are higher expressed in recurrence-free cases, regardless of EGFR mutation status. (C) Recurrence-free and OS curves according to EGFR mutation or SHP2 expression level. The cutoff level of mRNA expression was determined at the point where RFS was divided in the most efficient manner around the median expression level (from third to fifth octile of mRNA expression level). Wild-type cases revealed higher RFS compared with EGFR-mutant cases. In EGFR-mutant cases, higher expression of SHP2 reveals a significantly worse prognosis than cases with lower SHP2 expression. The 5-year RFS in EGFR mutation-positive LUADs was 31.5% with high SHP2 expression, versus 50.6% for those with low SHP2 (hazard ratio, 1.83; 95% CI: 1.05–3.23; p ¼ 0.0329). The 5-year OS in wild-type LUADs was 79.9% with no influence of SHP2 mRNA. In EGFR mutation-positive LUADs with low SHP2 mRNA, the 5-year OS was 86.8% versus 60.7% for those with high SHP2 mRNA (hazard ratio, 2.28; 95% CI: 1.03–4.58; p ¼ 0.0414). CI, confidence interval; LUAD, lung adenocarcinoma; OS, overall survival; RFS, recurrence-free survival; RTK, receptor tyrosine kinase; SHP2, Src-homology 2 domain-containing phosphatase 2. as significant. Statistical analyses were performed using illustrated in Table 1. The median age of the patients was SPSS version 20.0 (IBM Corp., Armonk, NY) and StatMate 69 years (range, 40–91) and 53.9% were male. Among V (ATMS Co., Ltd., Tokyo, Japan). them, 232 patients (86.9%) had pathologically confirmed N0-1 LUAD and 100 (37.7%) were EGFR Results mutation-positive LUADs. The median follow-up period Effect of SHP2 Expression on the Outcome of was 45 months (range, 1.4–150 mo). Among the patients Patients With Resected EGFR Mutation-Positive positive with EGFR mutation, 65 (65.0%) had patholog- LUAD ically verified stage I (IA1–IB), and half of them had a Firstly, mRNA expression of the RTKs, AXL and MET, recurrence within a median period of 47 months (range, CDCP1, and the non-RTKs, YAP1, STAT3, and SHP2 were 5–150). As illustrated in Figure 1A and B, there was a examined in EGFR mutation-positive and EGFR mutation- trend for higher SHP2, MET, and CDCP1 mRNA expres- negative resected LUADs sion for patients with tumor recurrence compared with The mRNA expression of the six biomarkers was those who did not have tumor recurrence. In contrast, evaluated in tumor samples from 267 surgically resected the mRNA expression of YAP1, STAT3, and AXL was LUADs. The clinical characteristics of the patients are higher in patients without tumor recurrence. 6 Ito et al JTO Clinical and Research Reports Vol. 1 No. 4 Figure 2. SHP2 inhibitor RMC-4550 decreases activation of protein involved in erlotinib resistance. (A) Erlotinib or RMC-4550 monotherapy or combination of these two compounds was tested in PC9 cell line for 18 hours and molecular effects were analyzed by Western blot technique. The expression level is shown as a fold change compared with control (¼ 1.0). Actin was used as a housekeeping protein. The experiment was performed at least three times. (B) Western blotting reveals the expression level of pSHP2 (Y542) after erlotinib treatment according to concentration (0.5 mM, 1.0 mM, and 2.5 mM) and treatment time (24, 48, and 72 hours) in PC9 cell lines. The expression level of pSHP2 (Y542) significantly increases through the reduced dose (0.5 mM, 1.0 mM) to clinically normal dose (2.5 mM), and assay period employed in this study (24–72 h). The quantification of phosphorylation level is reported as a fold change compared with control (¼ 1.0). Actin was used as a housekeeping protein. The experiment was performed at least three times. pAKT, phosphorylated AKT; pEGFR, phosphory- lated EGFR; pERK, phosphorylated ERK; pSHP2, phosphorylated SHP2; SHP2, Src-homology 2 domain-containing phosphatase 2. The second part of the study focused on resected Effect of EGFR and SHP2 Inhibition in EGFR- EGFR mutation-positive LUADs. The 5-year RFS in wild- Mutant Cell Lines type LUADs was 68.6%, without differences according to On the basis of our findings, we further explored the SHP2 mRNA expression. However, for EGFR mutation- biological role of SHP2 in EGFR-mutant LUAD cell lines. positive LUADs with low SHP2 mRNA, the 5-year RFS In the PC9 EGFR-mutant cell line, SHP2 phosphorylation was 50.6% versus 31.5% in those with high SHP2 mRNA was increased upon erlotinib therapy, compared with (hazard ratio, 1.83; 95% CI: 1.05–3.23; p ¼ 0.0329) the baseline levels, as illustrated by Western blotting in (Fig. 1C). The 5-year OS in wild-type LUADs was 79.9% Figure 2A. The increased SHP2 phosphorylation upon with no influence of SHP2 mRNA. In EGFR mutation- erlotinib therapy in PC9 cells was confirmed with positive LUADs with low SHP2 mRNA, the 5-year OS different doses of erlotinib (0.5–2.5 mM) for 24, 48, and was 86.8% versus 60.7% for those with high SHP2 72 hours (Fig. 2B). When erlotinib was combined with mRNA (hazard ratio, 2.28; 95% CI: 1.03–4.58; p ¼ the SHP2 inhibitor, RMC-4550, the phosphorylation of 0.0414) (Fig. 1C). No significant differences in RFS or OS SHP2 (Y542), as well as, EGFR (Y1068) and downstream of patients with EGFR mutation-positive LUAD were components, such as, AKT (S473) and ERK1/2 (T202/ observed according to CDCP1 and MET mRNA Y204), were significantly suppressed, and the suppres- expression. sion was more profound with the combination, November 2020 SHP2 in Resected EGFR Mutation-Positive LUAD 7 Figure 3. Combination of erlotinib and SHP2 inhibitor potentiate cell viability and colony formation inhibition. (A) The combination treatment using erlotinib plus SHP2 inhibitor (SHP099 or RMC-4550) was assayed. Cells were exposed to increasing concentrations, on the basis of IC50 concentrations of erlotinib, SHP90 inhibitor (SHP099 or RMC-4550) or com- bination of erlotinib and SHP2 inhibitor, in the three EGFR-mutant cell lines, HCC827, PC9, and H1975 for 72 hours and cell viability was analyzed by MTT assay. The combination treatment indicates synergism in the three EGFR-mutant cell lines. The colored graph represents cell viability according to drug concentration (from 26 to 22 times of IC50 of each inhibitor). The dashed lines in x axis and y axis represent the IC50 concentration and the point of 50% cell viability, respectively. Black-white graphs reveal CIns at each drug concentration (from 26 to 21 times of IC50 of each inhibitor). Total CIn (calculated as the 8 Ito et al JTO Clinical and Research Reports Vol. 1 No. 4 compared with each of the inhibitors alone (Fig. 2A). In Discussion three EGFR-mutant cell lines (PC9, HCC827, and H1975) Patients from Hiroshima, Japan and Terrassa, Spain, the combination of erlotinib with SHP2 inhibitors (RMC- mostly early-stage LUADs, were examined, including 4550 and SHP099) was synergistic, as revealed in a 100 EGFR mutation-positive LUADs. The frequency of 3-day monolayer culture and in a long-term 10-day EGFR mutations is geographically distinct by regions, colony formation assay (Fig. 3A and B and with a high frequency in Asians, in comparison with Supplementary Table 1 in Supplementary Appendix). Caucasians.26 The recent Nederlands-Leuvens Long- It was further examined whether the synergistic ef- kanker Screenings Onderzoek study has revealed a fect of erlotinib with SHP2 inhibitors is related to the reduction in lung cancer mortality with volume-based increased SHP2 phosphorylation observed with erlotinib low-dose computed tomography screening among monotherapy. Intriguingly, in the PC9 cell line, the former and current male smokers. Screening-detected sensitivity to RMC-4550 decreased after exposure to lung cancers are more often stage IA or IB (58.6%).27 erlotinib (Supplementary Fig. 1), suggesting that the Most patients included in our study, with or without erlotinib-induced increment of SHP2 phosphorylation harboring EGFR mutations, were stage IA (IA1, IA2, and does not augment the effectiveness of the SHP2 inhibitor IA3) or IB (T2a > 3–4 cm).28 SHP2 surfaced as the only in the EGFR-mutant PC9 cell line. The protein tyrosine significant marker of RFS and OS, but exclusively in phosphatase SHP2 is required for complete ERK activa- EGFR mutation-positive NSCLC. This finding was partly tion downstream of EGFR and other RTKs. SHP2 is expected. Activation of SHP2 by phosphorylation at distributed to both the cytoplasm and nucleus. The tyrosine 542 is required for ERK activation in response localization of SHP2 in the nucleus potentially promotes to growth factors. It was previously reported that SHP2 tumor invasion, migration and metastases.25 The cellular Y542 phosphorylation was induced in H1666 cells in localization of pSHP2 in the PC9 cell line, and upon response to EGF by 5 minutes, but its phosphorylation treatment with erlotinib, RMC-4550 (SHP2 inhibitor), was not induced in H3255 cells with EGFR mutations at and the combination, were, then, analyzed by immuno- any time point. The results shed light on the seminal fluorescence analyses. As illustrated in Figure 4 and finding that SHP2 function is impaired in NSCLC cells Supplementary Figure 2, pSHP2 was in the nucleus and expressing mutant versus wild-type EGFR.25 In EGFR- cytoplasm of the PC9 cell line. Upon erlotinib treatment, mutant cells, SHP2 is sequestered at the plasma mem- pSHP2 translocated partially to the nucleus, whereas brane, impeding the ability of SHP2 to promote ERK upon RMC-4550 treatment, it was assembled on the cell activity. Weak ERK activity can favor initial response to membrane. Erlotinib combined with RMC-4550 pre- EGFR TKIs. In this study, fluorescently labeled SHP2 vented the nuclear import of pSHP2 (Fig. 4). Intensive was translocated to the nucleus and only faintly visible staining in the nucleus and membrane was confirmed as in the cell membrane after erlotinib treatment (Fig. 4). a spike of pixel intensity after erlotinib or RMC-4550 However, after RMC-4550 treatment (SHP2 inhibitor), treatment, respectively (Supplementary Fig. 2). The SHP2 was repositioned in the cell membrane (Fig. 4). SHP2 phosphorylation in the wild-type cell line, H1666, Furthermore, the combination of erlotinib and RMC- was widely diffused in the nuclei (Supplementary 4550 prevented nuclear accumulation of pSHP2 Fig. 3A). In patients with EGFR wild type, no differ- (Fig. 4). It was observed that the combination of erlo- ences in RFS and OS were seen according to SHP2 levels tinib and RMC-4550 reduced the nuclear expression of (Supplementary Fig. 3B). pEGFR in PC9 cells (Supplementary Fig. 3). In this Phosphorylated EGFR (pEGFR) on the tyrosine res- study, we were unable to reveal that YAP1 mRNA levels idue 1068 was expressed in the nucleus and cytoplasm predicted RFS and OS. In a previous study, we reported in the PC9 cell line. Upon RMC-4550 treatment, pEGFR that either gefitinib or osimertinib (first- and third- remained in the perinuclear region, whereas upon erlo- generation EGFR TKIs, respectively) induced YAP tinib monotherapy and combination with RMC-4550, the phosphorylation and elevated YAP mRNA levels were pEGFR expression was reduced (Supplementary Fig. 4). associated with shorter progression-free survival and average of each CIn) ranged 0.63 to 0.76. The experiment was performed at least three times. (B) The three cell lines, HCC827, PC9, and H1975, were exposed to increasing doses of RMC-4550 in combination with increasing doses of erlotinib for x days. The colonies were then fixed and stained with crystal violet and a representative colony formation assay is showed. Colored chart representing the ratio of crystal violet concentration (control is fixed as 1.00). The graphs display CIns at each drug concentration. Erlotinib plus RMC-4550 combination revealed synergism in all three cell lines. Each CIn ranged synergism level: 0.559 to 0.088. The experiment was performed at least three times and a representative colony formation assay is reported. CI, confidence interval; CIn, combination index; IC50, concentration that inhibits 50%; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; SHP2, Src-homology 2 domain-containing phosphatase 2. November 2020 SHP2 in Resected EGFR Mutation-Positive LUAD 9 Figure 4. Immunostaining experiments estimating pSHP2 (Y542) expression after monotherapy or combination therapy. Representative immunofluorescence images of location of pSHP2 in PC-9 cells after erlotinib or RMC-4550 treatment. Fixed cells were stained using DAPI (blue) for nuclear counterstaining, phalloidin (red) for actin, and pSHP2 (Y542) (green) anti- body. The scale bar corresponds to 50 mm. The shape of PC9 cells changes from spindle to round after erlotinib or RMC-45500 treatment. DAPI, 40 ,6-diamidino-2-phenylindole; pSHP2, phosphorylated SHP2; SHP2, Src-homology 2 domain-containing phosphatase 2. OS in patients with EGFR-mutant NSCLC treated with expression in this patient subgroup for further selection EGFR TKIs.16 In this study, the YAP mRNA levels were of optimal adjuvant combinatory therapy where SHP2 not related to prediction of RFS or OS, neither in EGFR inhibitors could be an essential part of the treatment.33 mutant LUADS nor wild-type LUADs. This apparent Nevertheless, our study has some limitations. Firstly, discrepancy can be explained by the fact that resected although most patients were stage I, a few patients EGFR mutation-positive LUADs did not receive neo- with stage III were also included, and no homogenous adjuvant therapy and, therefore, neither SHP2, YAP, nor adjuvant therapy was administered. The Japanese pa- other transcription factors, could be activated. The tients received adjuvant oral uracil-tegafur, but there is combination of erlotinib and RMC-4550 was highly no reliable data on the adherence to adjuvant treat- synergistic in three EGFR-mutant cell lines (Fig. 2). In ment. Notwithstanding, the study highlights the func- other studies, SHP2 depletion also leads to substantial tion of SHP2 in EGFR mutation-positive LUAD patients, inhibition of colony formation in RTK-dependent cancer and the data in EGFR-mutant cells advocates adjuvant cells.29-31 Moreover, SHP2 inhibition prevents resis- treatment with TKIs plus SHP2 inhibitors. In addition, tance to MEK inhibitors in several tumors.32 This study we recently reviewed 1155 stage IAI to IIIA resected reveals that SHP2 plays a crucial role in EGFR-mutant LUADs and confirmed that RFS was significantly lower cells and erlotinib causes mislocalization of SHP2 in in the subgroup of EGFR mutation-positive cases the nucleus, potentially promoting tumor invasion, (excluding minimally invasive carcinomas, adenocarci- migration, and metastases. Therefore, studies in EGFR noma in situ, and lepidic variants), in comparison with mutation-positive LUADs with the combination of EGFR EGFR wild-type LUADs.34 “We validated the synergism TKI and SHP2 inhibitors should be promoted. More- in MTT by colony forming assay. However, the range of over, this study was able to include patients with stage reagent concentrations was not wide and high syner- IA, which reinforces the need to determine SHP2 gism might be partly due to repeated treatments every 10 Ito et al JTO Clinical and Research Reports Vol. 1 No. 4 3 days in colony forming assay. Further validation 7. Kato H, Ichinose Y, Ohta M, et al. A randomized trial of including the toxicity should be performed in other cell adjuvant chemotherapy with uracil-tegafur for adeno- lines, with a wider range of concentration, or in vivo.” carcinoma of the lung. N Engl J Med. 2004;350:1713– 1721. In conclusion, SHP2 is an important mechanism of 8. Goss GD, O’Callaghan C, Lorimer I, et al. Gefitinib versus recurrence in EGFR mutation-positive NSCLC and the placebo in completely resected non-small-cell lung combination with SHP2 inhibitors can improve adju- cancer: results of the NCIC CTG BR19 study. J Clin Oncol. vant therapy with EGFR TKIs. Further studies can also 2013;31:3320–3326. define whether SHP2 can prevent metastasis by abro- 9. Kelly K, Altorki NK, Eberhardt WE, et al. Adjuvant gating myeloid-derived suppressor cells.35 Lewis lung erlotinib versus placebo in patients with stage IB-IIIA cancer mice treated with adjuvant epigenetic therapy non-small-cell lung cancer (RADIANT): a randomized, have longer survival than those treated with double-blind, phase III trial. J Clin Oncol. 2015;33:4007–4014. chemotherapy.36 10. Zhong WZ, Wang Q, Mao WM, et al. Gefitinib versus vinorelbine plus cisplatin as adjuvant treatment for Acknowledgments stage II-IIIA (N1-N2) EGFR-mutant NSCLC (ADJUVANT/ CTONG1104): a randomised, open-label, phase 3 study. This study was conducted in part at the Analysis Center Lancet Oncol. 2018;19:139–148. of Life Science, Natural Science Center for Basic Research 11. Sandler JE, D’Aiello A, Halmos B. Changes in store for and Development, Hiroshima University, Hiroshima, Japan, early-stage non-small cell lung cancer. J Thorac Dis. and supported by a La Caixa Foundation grant and the 2019;11:2117–2125. Spanish Association Against Cancer (PROYE18012ROSE). 12. Arasada RR, Amann JM, Rahman MA, Huppert SS, Carbone DP. EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling. Supplementary Data Cancer Res. 2014;74:5572–5584. Note: To access the supplementary material accompa- 13. Codony-Servat C, Codony-Servat J, Karachaliou N, nying this article, visit the online version of the JTO et al. Activation of signal transducer and activator of Clinical and Research Reports at www.jtocrr.org and at transcription 3 (STAT3) signaling in EGFR mutant non- small-cell lung cancer (NSCLC). Oncotarget. 2017;8: https://doi.org/10.1016/j.jtocrr.2020.100084. 47305–47316. 14. Jacobsen K, Bertran-Alamillo J, Molina MA, et al. 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