Supplementary Material A novel EGFR isoform confers increased invasiveness to cancer cells Min Zhou, Hai Wang, Keke Zhou, Xiaoying Luo, Xiaorong Pan, Bizhi Shi, Hua Jiang, Jiqin Zhang, Kesang Li, Hua-Mao Wang, Huiping Gao, Shun Lu, Ming Yao, Ying Mao, Hong-Yang Wang, Shengli Yang, Jianren Gu, Chuanyuan Li, and Zonghai Li 1 List of contents Supplementary Figures .............................................................................................3 Figure S1. Related to Figure 1..................................................................................... 3 Figure S2. Related to Figure 2..................................................................................... 5 Figure S3. Related to Figure 3..................................................................................... 6 Figure S4. Related to Figure 4..................................................................................... 8 Figure S5. Related to Figure 5..................................................................................... 9 Figure S6. Related to Figure 6................................................................................... 11 Supplementary Tables ............................................................................................12 Table S1. Clinical characteristics of patients with low or high expression of EGFR or EGFRvA in 52 glioma patients....................................................................................12 Table S2. Higher expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells............................................................................................................................14 Table S3. Lower expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells ...........................................................................................................................18 Table S4. Primers used in this study .........................................................................22 Supplementary Materials and Methods ................................................................25 Supplementary References .................................................................................. 31 2 Figure S1. EGFRvA is widely expressed in various cancer cell lines and tissues. (A) Products of long distance reverse transcription PCR (RT-PCR) for EGFRvA. The complete open reading frame (ORF) was amplified and sequenced. (B) Detection of EGFR and EGFRvA mRNA in tumor cell lines by RT-PCR. β-actin was used as an internal control. (C) Quantitative analysis of the expression of EGFR and EGFRvA mRNA in normal tissues by qRT-PCR. Relative mean expression of EGFR and EGFRvA was normalized to their 3 expression level in brain. Data are means ± SEM. (D) Immunoblotting analysis of GFP, EGFR or EGFRvA-transfected NIH/3T3 cells. EGFRvA and EGFR were detected by 1F3-52 and 7F4, respectively. Polyclonal antibody SC-03 recognizing both EGFRvA and EGFR was used to detect total EGFR. (E) Immunocytochemical staining of EGFR and EGFRvA in A431 and MDA-MB-468 cells. NIH/3T3 EGFR and NIH/3T3 EGFRvA served as controls. Scale bar, 90 µm. 4 Figure S2. The upregulation of EGFRvA in glioma tissues compared with paired adjacent non-neoplastic brain tissues and a poor prognosis in patients with high-grade gliomas, (A) Western blot analysis of EGFR and EGFRvA in 6 pairs of glioma tissues (T) and corresponding adjacent noncancerous tissues (N). (B) A poor prognosis in patients with high-grade gliomas, 5 Figure S3. EGFRvA promotes cell migration and invasion in vitro and in vivo. (A-B) EGFRvA promotes cell proliferation in vitro. Growth curves of U87MG transfectants (A) and NIH/3T3 transfectants (B). * , P < 0.01 when compared with GFP control. Data are mean ± SEM. (C) 6 Representative images of transwell migration and invasion assays of U87MG transfectants. Scale bar, 100 µm. (D) Transwell migration and invasion assays of NIH/3T3 transfectants. Data are mean ± SEM. (E) Body weight change (Weight Day 18 - Weight Day 0) of the three group mice. Data are mean ± SEM. (F) Representative Micro-CT images of the thorax of mice at the mid-lung level. Scale bar, 5 mm. (G) Representative gross lung (upper panel) and H&E-stained lung sections (lower panel) from mice subcutaneously injected with indicated U87MG transfectants. Black arrowheads and dashed lines mark metastatic nodules. Scale bars, 5 mm. (H-M) The extrapulmonary metastatic foci induced by U87MG EGFRvA cells were observed adjacent to the spine (H), on the diaphragm (I), chest wall (J), or in the chest cavity (K), peritoneum lymph nodes (L) and mesenteric lymph nodes in the abdominal cavity (M). Scale bar, 5 mm. 7 Figure S4. EGFRvA caused the activation of STAT3 and increased expression of HB-EGF. (A) Key molecules of the EGFR signaling pathway were analyzed by Western blot using the indicated antibodies. (B) Immunofluorescence analysis of p-STAT3, HB-EGF, MMP-2 and MMP-9 in U87MG GFP, U87MG EGFR and U87MG EGFRvA cells. Scale bar, 40 µm. 8 Figure S5. The positive feedback regulation between HB-EGF and p-STAT3 in EGFRvA-expressing cells. (A) Western blot analysis of EGFR Y992 and Y1045 phosphorylation in HB-EGF treated U87MG EGFR and U87MG EGFRvA cells. Cells treated with EGF were used as positive controls. (B) Western blot analysis of p-STAT3 in the cell 9 lysates from U87MG cells treated with the culture supernatant of the indicated cells in the absence (upper and middle panel) or presence (lower panel) of 10 µg/ml HB-EGF neutralizing antibody for different times. IgG from mouse served as a negative control. (C) Human HB-EGF promoter. Human HB-EGF promoter sequences were obtained from NCBI MapViewer. Putative STAT3-binding sites are shown in red. The capital letters denote the 5' region of HB-EGF encoding the mRNA transcript. (D) ChIP assay showing STAT3 binding to the HB-EGF promoter. The chromatins were prepared from MD-MB-468 cells and the immunoprecipitated DNA fragments within HB-EGF promoter by STAT3 antibody were detected by PCR analysis. 10 Figure S6. Cell viability and cell adhesion of U87MG EGFRvA cells treated with STAT3 inhibitor AG490, and siRNA-mediated knockdown of STAT3 in U87MG transfectants. (A) Cell viability and cell adhesion assays of U87MG EGFRvA cells treated with increasing concentrations of AG490. Data are mean ± SEM. (B) siRNA-mediated knockdown of STAT3 was confirmed by qRT-PCR. Data are mean ± SEM. 11 Supplementary Tables Table S1. Clinical characteristics of patients with low or high expression of EGFR or EGFRvA in 52 glioma patients Low EGFR expression High EGFR expression Low EGFRvA expression High EGFRvA expression Characteristic (EGFR below median) (EGFR above median) P value (EGFRvA below median) (EGFRvA above median) P value n = 26 n = 26 n = 26 n = 26 Age (years，mean ± SD) 38.29 ± 17.46 47.31 ± 19.45 0.098a 43.52 ± 17.18 42.14 ± 20.59 0.803a Sex Male 16 14 0.575b 13 17 0.262b Female 10 12 13 9 Stage Ⅰ-Ⅱ 15 7 0.025c 17 5 0.001c Ⅲ-Ⅳ 11 19 9 21 Median survival 15.1 14.2 0.235c 17.4 12.5 0.005c (months after operation) 12 aP values were measured with student’s t test. bP values were derived from Pearson chi-square tests. cP values were derived from log rank test. 13 Table S2. Higher expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells No. Genes Ratio Description Calcium/calmodulin-dependent 3',5'-cyclic nucleotide 1 PDE1A 7.9042 phosphodiesterase 1A 2 HIST1H4I 7.2628 Histone H4 3 CBWD2 6.7816 COBW domain-containing protein 2 Shaw-related voltage-gated potassium channel protein 2 isoform 4 KCNC2 6.7431 KV3.2c 5 NTN4 5.9598 Netrin-4 precursor 6 CAND1 5.9092 Cullin-associated NEDD8-dissociated protein 1 7 ACTR3B 5.5942 actin-related protein 3-beta isoform 2 8 CACNB1 5.4914 Voltage-dependent L-type calcium channel subunit beta-1 9 ARL4C 5.2431 ADP-ribosylation factor-like protein 4C 10 PPARG 5.0289 Peroxisome proliferator-activated receptor gamma 11 WNK2 4.7088 Serine/threonine-protein kinase WNK2 12 SRPX2 4.4692 sushi-repeat-containing protein, X-linked 2 13 TCEAL1 4.2817 Transcription elongation factor A protein-like 1 14 CHP1 3.8001 Calcium-binding protein p22 15 PCSK2 3.6554 Neuroendocrine convertase 2 precursor 16 NP_689653.3 3.5664 CDNA FLJ32549 fis, clone SPLEN1000049 17 FGF1 3.5028 Acidic fibroblast growth factor (aFGF) 18 IGF1 3.4219 Insulin-like growth factor IA precursor 19 MYH1 3.3756 Myosin-1 14 20 KCNE4 3.3178 Potassium voltage-gated channel subfamily E member 4 21 SERPINB1 3.2563 Leukocyte elastase inhibitor 22 HB-EGF 3.0638 Heparin-binding EGF-like growth factor precursor 23 NP_001035160.1 3.0380 seven transmembrane helix receptor 24 OLFM1 3.0105 Noelin precursor 25 FNDC3A 2.9603 Fibronectin type-III domain-containing protein 3a 26 HSPB8 2.8863 Heat-shock protein beta-8 (HspB8) 27 OLFM3 2.8797 Noelin-3 precursor (Olfactomedin-3) 28 TIMP4 2.8509 Metalloproteinase inhibitor 4 precursor (TIMP-4) 29 C4orf32 2.8481 CDNA FLJ39370 fis, clone PEBLM2007437 30 HAS2 2.8358 Hyaluronan synthase 2 31 PLAUR 2.8165 Urokinase plasminogen activator surface receptor precursor (uPAR) 32 KLHL5 2.8134 Kelch-like protein 5 33 PDGFA 2.7252 Platelet-derived growth factor A chain precursor 34 Q8N8W3 2.7148 CDNA FLJ38763 fis, clone KIDNE2014119 35 RAB27B 2.7073 Ras-related protein Rab-27B (C25KG) 36 PENK 2.6660 Proenkephalin A precursor 37 RHPN1 2.6506 Rhophilin-1 (GTP-Rho-binding protein 1) 38 PBEF1 2.6335 Nicotinamide phosphoribosyltransferase 39 TADA2L 2.6295 Transcriptional adapter 2-like (ADA2-like protein) 40 FBLN2 2.6083 Fibulin-2 precursor 41 AMICA1 2.5796 Junctional adhesion molecule-like