Cell lines and tissue samples. The 18 human NSCLC cell lines used in this. study included eight adenocarcinoma cell lines (ADCs; A427, A549, LC319,

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1 Supplementary Materials and Methods Cell lines and tissue samples. The 18 human NSCLC cell lines used in this study included eight adenocarcinoma cell lines (ADCs; A427, A549, LC319, NCI-H1373, PC-3, PC-9, PC-14, and NCI-H1781), nine squamous-cell carcinoma cell lines (SCCs; EBC-1, LU61, NCI-H520, NCI-H1703, NCI-H2170, RERF-LC-AI, SK-MES-1, NCI-H226, and NCI-H647), and one large-cell carcinoma cell line (LCC; LX1). The human esophageal carcinoma cell lines used in this study were as follows; 13 SCC cell lines (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, TE10, TE11, TE13, TE14, TE15) and one ADC cell line (TE7). All cells were grown in monolayers in appropriate media supplemented with 10% fetal calf serum (FCS) and were maintained at 37 o C in an atmosphere of humidified air with 5% CO 2. Human small airway epithelial cells (SAEC) were grown in optimized medium (SAGM) purchased from Cambrex Bio Science Inc. Primary NSCLC and ESCC samples had been obtained earlier with informed consent (15, 16, 25). A total of 406 formalin-fixed samples of primary NSCLCs (254 ADCs, 112 SCCs, 28 LCCs, 12 ASCs; 126 female and 280 male patients; median age of 65.0 ± 9.8 SD with a range of years), and adjacent normal lung tissues, had been obtained earlier along with clinicopathological data from patients undergoing surgery at Saitama Cancer Center (Saitama, Japan). These NSCLC patients received resection of their primary cancers without preoperative chemo- and/or radiotherapy, and among them only patients with positive lymph-node metastasis were treated with cisplatin-based adjuvant chemotherapies after their surgery. A total of 265 formalin-fixed primary ESCCs (26 female and 239 male patients; median age of 62.0 ± 7.9 SD with a range of 38

2 - 76 years) and adjacent normal esophageal tissue samples had also been obtained from patients undergoing curative surgery at Keiyukai Sapporo Hospital (Sapporo, Japan). These ESCC patients received resection of their primary cancers without preoperative chemo- and/or radiotherapy. NSCLC specimen and five tissues (heart, liver, lung, kidney, and testis) from post-mortem materials (2 individuals with SCC) were also obtained from Hiroshima University. The pathological stage was determined according to the classification of the Union Internationale Contre le Cancer (36). This study and the use of all clinical materials mentioned were approved by individual institutional Ethical Committees. Serum samples. Serum samples were obtained with informed consent from 74 healthy individuals as controls (14 females and 60 males; median age 48.0 ± 7.47 SD with a range of years), and from 65 non-neoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) enrolled as a part of the Japanese Project for Personalized Medicine (BioBank Japan) or admitted to Hiroshima University Hospital (8 females and 57 males; median age of 66.0 ± 5.92 SD with a range of years). All of these COPD patients were current and/or former smokers (The mean [± 1SD] of pack-year index (PYI) was 55.6 ± 50.1 SD; PYI was defined as the number of cigarette packs [20 cigarette per pack] consumed a day multiplied by years). Serum samples were also obtained with informed consent from 112 NSCLC patients admitted to Hiroshima University Hospital, as well as Kanagawa Cancer Center Hospital (40 females and 72 males; median age 66.0 ± 12.0 SD with a range of 30-84) and 81

3 esophageal-cancer patients who were admitted to Keiyukai Sapporo Hospital or who were registered in the BioBank Japan (12 females and 69 males; median age 65.0 ± 5.1 SD with a range of 37-74). These 112 NSCLC cases included 85 ADCs and 27 SCCs. Samples were selected for the study on the basis of the following criteria: (1) patients were newly diagnosed and previously untreated and (2) their tumors were pathologically diagnosed as lung or esophageal cancers (stages I - IV). Serum was obtained at the time of diagnosis and stored at -150 C. Semi-quantitative RT-PCR analysis. Total RNA was extracted from cultured cells and clinical tissues using Trizol reagent (Life Technologies, Inc. Gaithersburg, MD) according to the manufacturer s protocol. Extracted RNAs and normal human-tissue poly(a) RNAs were treated with DNase I (Roche Diagnostics, Basel, Switzerland) and then reverse-transcribed using oligo (dt) primer and SuperScript II reverse transcriptase (Life Technologies, Inc.). Semi-quantitative RT-PCR experiments were carried out with synthesized LY6K gene-specific primers (5 -ATTCGCTACTGCAATTTAGAGG-3 and 5 -GTTTAATGCAACAGGTGACAACG-3 ), or with beta-actin (ACTB)-specific primers (5 -GAGGTGATAGCATTGCTTTCG-3 and 5 -CAAGTCAGTGTACAGGTAAGC-3 ). All PCR reactions involved initial denaturation at 94 o C for 2 min followed by 22 (for ACTB) or 30 cycles (for LY6K) of 94 o C 30 s, 58 o C for 30 s, and 72 o C for 60 s on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). Northern-blot analysis. Human multiple-tissue blots (BD Biosciences, Palo

4 Alto, CA) were hybridized with 32 P-labeled PCR products. PCR product of LY6K was prepared as a probe by RT-PCR using primers 5'-AGGGTGACAATAGAGTGTGGTGT-3' and 5'-GTTTAATGCAACAGGTGACAACG-3'. Prehybridization, hybridization, and washing were performed according to the supplier s recommendations. The blots were autoradiographed with intensifying screens at -80 o C for one week. Immunohistochemistry and Tissue Microarray. Tumor-tissue microarrays were constructed using 406 formalin-fixed primary NSCLCs and 265 ESCCs, as published previously (37-39). Briefly, the tissue area for sampling was selected based on visual alignment with the corresponding HE-stained section on a slide. Three, four, or five tissue cores (diameter 0.6 mm; height 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block using a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched from each case, and 5-µm sections of the resulting microarray block were used for immunohistochemical analysis. To investigate the status of the LY6K protein in clinical lung-cancer samples that had been embedded in paraffin blocks, we stained the sections in the following manner. Briefly, a rabbit polyclonal anti-human LY6K antibody (TM38) was added after blocking of endogenous peroxidase and proteins. The sections were incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. Substrate-chromogen was added and the specimens were counterstained with hematoxylin. Three independent investigators assessed LY6K positivity

5 semi-quantitatively without prior knowledge of clinicopathological data. The intensity of LY6K staining was evaluated using following criteria: strong positive (2+), dark brown staining in more than 50% of tumor cells completely obscuring cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cells; absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted only as strongly positive if reviewers independently defined them as such.

6 Statistical analysis. We used contingency tables to analyze the relationship of LY6K expression levels and clinicopathological variables of NSCLC patients. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for LY6K expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were performed with the Cox proportional-hazard regression model to determine associations between clinicopathological variables and cancer-related mortality. First, we analyzed associations between death and possible prognostic factors including age, gender, histological type, pt-classification, and pn-classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced LY6K expression into the model, along with any and all variables that satisfied an entry level of a P value smaller than As the model continued to add factors, independent factors did not exceed an exit level of P < ELISA. Serum levels of LY6K were measured by sandwich-type ELISA which had been originally constructed. In brief, for detection of soluble LY6K in serum, 96-well flexible microtiter plates (439454; NALGE NUNC International, Rochester, NY) were coated with 2 ng/ml of capturing polyclonal antibody to LY6K (TM38) overnight. Wells were blocked with 200 µl PBS (ph 7.4) containing 1% BSA, 5% sucrose, and 0.05% NaN 3 for 2 hours and then incubated for 2 hours with 3-fold diluted serum samples in PBS (ph 7.4) containing 1% BSA. After washing with

7 PBS (ph 7.4) containing 0.05% Tween 20, the wells were incubated for 2 hours with 200 ng/ml of biotin-conjugated polyclonal anti-ly6k antibody (MB44), followed by reaction with avidin-conjugated peroxidase (P347; Dako Cytomation, Glostrup, Denmark) for 30 minutes using a Substrate Reagent (R&D Systems). To prepare biotinylating rabbit polyclonal antibodies to LY6K (MB44), we used Biotin Labeling Kit-NH2 (LK03) according to the supplier s protocol (DOJINDO LABORATORIES, Kumamoto, Japan). The color reaction was stopped by adding 100 µl of 2N sulfuric acid. Color intensity was determined by a photometer at a wavelength of 450 nm, with a reference wave-length of 570 nm. A standard curve was drawn for each plate using recombinant LY6K proteins as a reference. Levels of CEA in serum were measured by ELISA with a commercially available enzyme test kit (HOPE Laboratories, Belmont, CA), according to the supplier s recommendations. Levels of CYFRA 21-1 in serum were measured by ELISA with a commercially available kit (DRG, Marburg, Germany). Differences in the levels of LY6K, CEA, and CYFRA 21-1 between tumor groups and a healthy control group were analyzed by Mann-Whitney U tests. The levels of LY6K, CEA, and CYFRA 21-1 were further evaluated by receiver-operating characteristic (ROC) curve analysis to determine cut-off levels with optimal diagnostic accuracy and likelihood ratios. The correlation coefficients between LY6K and CEA/CYFRA 21-1 as well as between CEA and CYFRA 21-1were calculated with Spearman rank correlation. Significance was defined as P < RNA interference assay. We had previously established a vector-based RNA

8 interference (RNAi) system, psih1bx3.0, to direct the synthesis of sirnas in mammalian cells (19, 21). We transfected 10 µg of sirna-expression vector, using 30 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA), into lung and esophageal cancer cell lines, which over-expressed LY6K. The transfected cells were cultured for five days in the presence of appropriate concentrations of geneticin (G418), and then cell numbers and viability were measured by Giemsa staining and triplicate MTT assays. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control 1 (EGFP: enhanced green fluorescent protein (GFP) gene, a mutant of Aequorea victoria GFP), 5 -GAAGCAGCACGACTTCTTC-3 ; control 2 (Scramble (SCR): chloroplast Euglena gracilis gene coding for 5S and 16S rrnas), 5 -GCGCGCTTTGTAGGATTCG-3 ; LY6K sirna-1 (si-ly6k-1), 5 -AACCTGACTGCGAGACAACGA-3 ; LY6K sirna-2 (si-ly6k-2), 5 -AAGGAGGTGCAAATGGACAGA-3. Down-regulation of LY6K protein expression by effective sirna (si-ly6k-2), but not by the two controls or si-ly6k-1, was confirmed with western-blotting in the cell lines used for this assay.