TM4SF4 overexpression in radiation-resistant lung carcinoma cells activates IGF1R via elevation of IGF1

Abstract

Transmembrane 4 L six family member 4 (TM4SF4) is a member of the tetraspanin L6 domain family. Other members of this family, TM4SF1 (also known as L6-Ag) and TM4SF5, have been shown to be upregulated in multiple tumors and involved in epithelial-to-mesenchymal transition and cell migration. However, unlike its homologs, little is known about TM4SF4. Here, we show that TM4SF4 was highly expressed in radiation-resistant lung adenocarcinoma cells, such as A549 and Calu-3 cells, and its expression activated cell growth, migration, and invasion. Overexpression of TM4SF4 in A549 cells increased the activation of PI3K, AKT, and NF-kappaB and the expression of PTEN. IGF1R was clearly activated by overexpression of TM4SF4, although EGFR was also slightly activated. TM4SF4 expression was correlated with the increased expression of IGF1, consequently resulting in IGF1R activation. Tumorigenic activity of TM4SF4 in lung adenocarcinoma cells was also demonstrated by xenograft assay; however, this activity was almost completely suppressed by treatment with anti-TM4SF4 antibody. Our results suggest that TM4SF4 overexpression in lung carcinoma cells results in resistance to radiotherapy via IGF1-induced IGF1R activation and blocking the activity of TM4SF4 using specific antibody can be a promising therapeutics against TM4SF4-overexpressing lung adenocarcinoma.

Figure 1. TM4SF4 expression in lung cancer cell lines is regulated by methylation

(A) RT-PCR and Western blot analysis of TM4SF4 expression in the indicated lung cancer cell types. Band intensities were measured using Image J software (National Institute of Health, Bethesda, MD, USA), normalized to β-actin and fold increase were indicated. (B) Bisulfite-converted sequence of TM4SF4. Each Y of underlined sequences indicates a methylated position. (C) Pyrosequencing diagram of A549 and H460 cells and comparison of methylation percentages at each CpG position. Each gray box in the diagram indicates the position of the two Y’s shown in panel B. (D) Methylation percentage of TM4SF4 gene in indicated lung cancer cells.

Figure 2. Suppression of TM4SF4 expression in A549 cells retarded cell growth, migration, and invasiveness

(A) RT-PCR and Western blot analysis of TM4SF4 expression in A549 cells transfected with siRNA targeting TM4SF4 (siTM4SF4): RNA and proteins were extracted from cells 72 h post-transfection. (B) Colony-forming assay of siTM4SF4-treated A549 cells: 24 h after siRNA transfection, cells were irradiated with a single dose of 2 Gy and, 24 h later, plated for colony-forming assay. Cells were incubated for 10 days and colonies stained with crystal violet were counted, and relative colony forming percentage (RCF) was plotted. (C) Wound-healing assay of siTM4SF4-transfected A549 cells: A549 cells transfected with siTM4SF4 or control siRNA (siCtl) 72 h after transfection were used in assay. At each period of assay, cells were photographed, wound size was determined, and size relative to initial size was represented as plotted. (D) Assay of migration (upper panel) and invasion (lower panel) of siTM4SF4-transfected A549 cells: A549 cells transfected with siTM4SF4 or siCtl were used for assay 48 h after transfection. Twenty-four hours later, cells were stained, photographed under a phase contrast microscope. Number of stained cells were counted and relative number of cells per field was plotted.

Figure 3. Overexpression of TM4SF4 in A549 cells enhanced cell growth, migration, and invasiveness

(A) RT-PCR and Western blot analysis of TM4SF4 expression in pcDNA-TM4SF4-transfected A549 cells: RNA and proteins were extracted from cells 72 h post-transfection. (B) Colony-forming assay of TM4SF4-overexpressing A549 cells: 24 h after transfection, cells were irradiated with a single dose of 2 Gy and, 24 h later, plated for colony-forming assay. (C) Wound-healing assay of TM4SF4-overexpressing A549 cells: A549 cells transfected with pcDNA-TM4SF4 or control vector were used for assay 72 h after transfection. At each period of assay, cells were photographed and wound size was determined. (D) Assay of migration (upper panel) and invasion (lower panel) of TM4SF4-overexpressing A549 cells: A549 cells transfected with pcDNA-TM4SF4 or control vector were used for assay 48 h after transfection.

Figure 4. PI3K, AKT, NF-kappaB, PTEN, and MMPs were regulated by TM4SF4 expression in A549 cells

(A) Western blot analysis of levels of phosphorylated or total PI3K, AKT, NF-kappaB, and ERK in siTM4SF4- or pcDNA-TM4SF4–transfected A549 cells. PTEN expression was also analyzed. Beta-actin served as internal control. (B) Western blot analysis of metalloproteinases 2, 7, and 9, N-cadherin, and C-cadherin in siTM4SF4- or pcDNA-TM4SF4–transfected A549 cells. (C) Phosphorylation of EGFR and IGF1R in siTM4SF4- and pcDNA-TM4SF4–transfected A549 cells.

Figure 5. IGF1R activation of TM4SF4-overexpressing A549 cells is induced by enhanced expression of IGF1 via NF-kappaB activation

(A) RT-PCR analysis of components of IGF1R signaling pathway and IL1 beta in TM4SF4- knockdown or overexpressing A549 cells. HGF served as negative control and beta-actin as internal control. Band intensities were measured using Image J software, normalized to β-actin and fold increase were indicated. (B) Colony formation assay and RT-PCR analysis of effect of blocking of TM4SF4 by anti-TM4SF4 antibody in TM4SF4-overexpressing A549 cells. (C) Colony formation assay of IGF1-knockdown A549 cells. (D) Effects of IGF1R, PI3K, or NF-kappaB inhibition on the expression of IGF1 and IL1 beta. TM4SF4-overexpressing A549 cells were treated with AG1024 (10 μM for 24 h), LY294002 (50 μM for 48 h) and SN50 (20 μM for 24 h) and analyzed by RT-PCR.

Figure 6. TM4SF4 expression in lung adenocarcinoma but not in large cell carcinoma

Non-neoplastic tissue (n=3, N1~N3), large cell carcinoma (n=4, L1~L3), and lung adenocarcinoma (n=6, A1~A6) tissue microarrays were stained with anti-TM4SF4 antibody and secondary reagent (brown). Nuclei were counterstained with hematoxylin (blue). Scale bar 50μm.

Figure 7. Anti-TM4SF4 antibody treatment inhibited the growth of xenograft tumor established by injection of TM4SF4-overexpressing A549 cells

(A) Anti-TM4SF4 antibody was injected into the implantation site of TM4SF4-overexpressing A549 cells at six time points (19, 21, 23, 26 and 28 d) after tumor cell implantation in mice and tumor volume was measured. (B) Colony-forming assay of TM4SF4-overexpressing A549 cells treated with anti-TM4SF4 antibody. Gamma-irradiated cells were also treated with anti-TM4SF4 antibody. Ten days after plating, colonies were counted and relative cell viability was plotted. (C) TM4SF4 expression in gamma-irradiated A549 cells. A549 cells gamma irradiated with a single dose of 2 or 4Gy, or three dose of 2Gy weekly and radiation-selected cells (RSC) after three dose of irradiation were analyzed by Western blot analysis.