Role of Insulin-Like Growth Factor Binding Protein 2 in Lung Adenocarcinoma
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090500
ISSN1525-2191
AutoresToshiro Migita, Tadahito Narita, Reimi Asaka, Erika Miyagi, Hiroko Nagano, Kimie Nomura, Masaaki Matsuura, Yukitoshi Satoh, Sakae Okumura, Ken Nakagawa, Hiroyuki Seimiya, Yuichi Ishikawa,
Tópico(s)Ubiquitin and proteasome pathways
ResumoInsulin-like growth factor (IGF) signaling plays a pivotal role in cell proliferation and mitogenesis. Secreted IGF-binding proteins (IGFBPs) are important modulators of IGF bioavailability; however, their intracellular functions remain elusive. We sought to assess the antiapoptotic properties of intracellular IGFBP-2 in lung adenocarcinomas. IGFBP-2 overexpression resulted in a decrease in procaspase-3 expression; however, it did not influence the phosphorylation status of either IGF receptor or its downstream targets, including Akt and extracellular signal-regulated kinase. Apoptosis induced by camptothecin was significantly inhibited by IGFBP-2 overexpression in NCI-H522 cells. Conversely, selective knockdown of IGFBP-2 using small-interfering RNA resulted in an increase in procaspase-3 expression and sensitization to camptothecin-induced apoptosis in NCI-H522 cells. LY294002, an inhibitor of phosphatidyl-inositol 3-kinase, caused a decrease in IGFBP-2 levels and enhanced apoptosis in combination with camptothecin. Immunohistochemistry demonstrated that intracellular IGFBP-2 was highly expressed in lung adenocarcinomas compared with normal epithelium. Intracellular IGFBP-2 and procaspase-3 were expressed in a mutually exclusive manner. These findings suggest that intracellular IGFBP-2 regulates caspase-3 expression and contributes to the inhibitory effect on apoptosis independent of IGF. IGFBP-2, therefore, may offer a novel therapeutic target and serve as an antiapoptotic biomarker for lung adenocarcinoma. Insulin-like growth factor (IGF) signaling plays a pivotal role in cell proliferation and mitogenesis. Secreted IGF-binding proteins (IGFBPs) are important modulators of IGF bioavailability; however, their intracellular functions remain elusive. We sought to assess the antiapoptotic properties of intracellular IGFBP-2 in lung adenocarcinomas. IGFBP-2 overexpression resulted in a decrease in procaspase-3 expression; however, it did not influence the phosphorylation status of either IGF receptor or its downstream targets, including Akt and extracellular signal-regulated kinase. Apoptosis induced by camptothecin was significantly inhibited by IGFBP-2 overexpression in NCI-H522 cells. Conversely, selective knockdown of IGFBP-2 using small-interfering RNA resulted in an increase in procaspase-3 expression and sensitization to camptothecin-induced apoptosis in NCI-H522 cells. LY294002, an inhibitor of phosphatidyl-inositol 3-kinase, caused a decrease in IGFBP-2 levels and enhanced apoptosis in combination with camptothecin. Immunohistochemistry demonstrated that intracellular IGFBP-2 was highly expressed in lung adenocarcinomas compared with normal epithelium. Intracellular IGFBP-2 and procaspase-3 were expressed in a mutually exclusive manner. These findings suggest that intracellular IGFBP-2 regulates caspase-3 expression and contributes to the inhibitory effect on apoptosis independent of IGF. IGFBP-2, therefore, may offer a novel therapeutic target and serve as an antiapoptotic biomarker for lung adenocarcinoma. Insulin-like growth factor-I and -II (IGF-I and -II) are important regulators of cellular metabolism, growth, and survival. When IGFs bind to their receptors, the type I and type II IGF receptors (IGF-IR or IGF-IIR), they activate the downstream signaling cascades via the phosphorylation of tyrosine kinase. Activated IGF-1R transmits signals to the major distinct pathways mitogen-activated protein kinase and phosphatidyl inositol 3-kinase (PI3K), signaling pathways that are highly implicated in the development and progression of neoplasia. IGF's bioavailability is regulated by six high affinity IGF binding proteins (IGFBPs). Secreted IGFBPs by cancer cells interfere primarily with IGF-I or -II through the formation of IGF-IGFBPs complex, which in turn exert an inhibitory effect on IGF-mediated biological functions. IGF-independent functions of extracellular IGFBPs have long been discussed. 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Our aim for this study is to examine the effect of intracellular IGFBP-2 on apoptosis in lung cancer cells and elucidate its molecular mechanism. We also examine the significance of intracellular IGFBP-2 and procaspase-3 in clinical samples and explore the therapeutic implications. The human lung adenocarcinoma cell lines A549, NCI-H460, NCI-H23, NCI-H522, HOP62, COR-L105, and PC14 were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 media supplemented with 10% fetal bovine serum (both medium and serum were from Gibco-BRL, Tokyo, Japan) and 1% penicillin/streptomycin in an atmosphere of 5% CO2 at 37°C, as previously described.26Migita T Narita T Nomura K Miyagi E Inazuka F Matsuura M Ushijima M Mashima T Seimiya H Satoh Y Okumura S Nakagawa K Ishikawa Y ATP citrate lyase: activation and therapeutic implications in non-small cell lung cancer.Cancer Res. 2008; 68: 8547-8554Crossref PubMed Scopus (289) Google Scholar We also analyzed the mRNA and protein expression in 24 pairs of primary lung adenocarcinomas and corresponding normal lung tissues. All experiments were performed by using a protocol approved by the Institutional Review Board of the Japanese Foundation for Cancer Research (number 2007-1058). IGFBP-2 cDNA expression construct in pcDNA3.1/Neo (Invitrogen, Carlsbad, CA) was a generous gift from Dr. Hiroaki Kataoka (Section of Oncopathology and Regenerative Biology, Department of Pathology, University of Miyazaki, Japan).27Fukushima T Tezuka T Shimomura T Nakano S Kataoka H Silencing of insulin-like growth factor-binding protein-2 in human glioblastoma cells reduces both invasiveness and expression of progression-associated gene CD24.J Biol Chem. 2007; 282: 18634-18644Crossref PubMed Scopus (75) Google Scholar Cells were plated at 7 × 105 per well in 60-mm dishes and transfected in triplicate by using the FuGENE 6 Transfection Reagent according to the manufacturer's protocol (Roche Diagnostics, Inc., Indianapolis, IN). We established stable cell lines COR-L105, NCI-H522, and HOP62 overexpressing IGFBP-2 after 4 weeks of selection in 400 μg/ml of neomycin. The cells and frozen tissue were collected for RNA extraction by using an RNeasy Kit (Qiagen, Valencia, CA), and total RNA was applied for first-strand cDNA synthesis with a high capacity cDNA Reverse Transcriptase kit (Applied Biosystems, Foster City, CA). Gene-specific probes and primer were obtained from Universal ProbeLibrary (number 25, Roche Applied Science, Tokyo, Japan), and primer sequences were as follows: 5′-TTGCAGACAATGGCGATGACC-3′ (IGFBP-2 forward); 5′-GGGATGTGCAGGGAGTAGAGG-3′ (IGFBP-2 reverse). PCR was performed in 96-well plates by using the LightCycler 480 System (Roche Applied Science). All reactions were performed at least in triplicate. The relative amounts of all mRNAs were calculated by using the comparative threshold cycle (CT) method after normalization to human β2 microglobulin. To obtain total protein lysates, frozen tissue and cells were homogenized and dissolved in radioimmunoprecipitation assay buffer (150 mmol/L of NaCl, 1.0% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L of Tris, pH 7.6) containing proteinase inhibitors and phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan). The protein concentration of each lysate was determined by using a protein assay reagent kit (BioRad, Hercules, CA). The total cell lysate was applied on 4% to 12% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred electrophoretically from the gel to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were then blocked for 1 hour in blocking buffer (5% low-fat dried milk in Tris-buffered saline) and probed with the primary antibodies overnight. After being washed, the protein content was made visible with horseradish-peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence (Amersham, Piscataway, NJ). Signal densities were quantitatively determined by ImageJ 1.36 b software (NIH, Bethesda, MD). The primary antibodies used were raised against IGFBP-2 (C-18, Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3, phosphorylated (Tyr1135/1136) and total IGF-1R β, phosphorylated (Ser 473) and total Akt, phosphorylated (Thr 202/Tyr 204) and total Erk1/2, cleaved poly ADP-ribose polymerase (PARP; all obtained from Cell Signaling Technology, Danvers, MA), and β-actin (Sigma, St. Louis, MO). LY294002 was purchased from Sigma. Caspase activities were measured by using the Caspase-Glo 3/7 assay kit according to the manufacturer's instruction (Promega, Madison, WI). Cells (5 × 103 cells/well) were placed in a 96-well culture plate, followed by treatment with dimethyl sulfoxide (DMSO) vehicle or 200 nmol/L of camptothecin for 24 hours. One hundred microliters of Caspase-Glo 3/7 reagent was added to each well and incubated for 1 hour at room temperature. The culture media with the reagent served as blank, and blank control value was subtracted from each sample value. Luminescence of all samples was measured by using a Tecan Spectrafluor Plus (Wako, Osaka, Japan). IGFBP-2 concentrations in media of cell culture were determined with IGFBP-2 Duoset enzyme-linked immunosorbent assay (ELISA) Development system (R and D Systems, Minneapolis, MN) according to the manufacturer's protocol. Briefly, capture antibody was plated in a 96-well microplate and incubated overnight at room temperature. One hundred microliters of supernatant of culture media or IGFBP-2 standard were added into plate and incubate for 2 hours at room temperature, followed by the immunoreaction with IGFBP-2 detection antibody. IGFBP-2 concentration was calculated from the standard curve. All experiments were performed in duplicate or triplicate. Small-interfering RNA (siRNA) oligonucleotides for IGFBP-2 (Santa Cruz Biotechnology) and a negative control (Invitrogen) were transfected into the cells. Transfection was performed by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. Briefly, 60 pmol of siRNA and 10 μl of Lipofectamine RNAiMAX were mixed in 1 ml of Opti-MEM medium (10 nmol/L of final siRNA concentration). After 20 minutes of incubation, the mixture was added to the suspended cells and these were plated on dishes. Cells were harvested at 24-hour intervals until 72 hours after transfection. Cell proliferation was measured as the number of viable cells, as evaluated at 450 nm optical density by using Cell Count reagent SF (Nacalai Tesque). Apoptotic cells were determined by Hoechst 33342 staining, and the apoptosis rate (percent of total population) was evaluated by counting apoptotic and nonapoptotic cells in at least three randomly selected fields. Tissue microarrays were constructed from 169 paraffin-embedded lung adenocarcinomas. Briefly, H&E-stained sections containing representative tumor regions were selected. Tissues were punched from cancer areas of each donor block by using tissue cylinders with a diameter of 2 mm and then brought into a recipient paraffin block. Three tumor cores were taken per patient. Immunohistochemistry was performed on 5-μm thick, formalin-fixed, paraffin-embedded sections by using primary antibodies for IGFBP-2 (C-18, Santa Cruz Biotechnology) and procaspase-3 (Cell Signaling Technology). Antigen retrieval was performed for 30 minutes in citrate buffer for each antibody. The slides were developed by using the labeled streptavidin biotinylated peroxidase method (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. 3,3′-Diaminobenzidine tetrahydro-chloride was used as the chromogen, and hematoxylin was used as the counterstain. A549 xenografts in nude mice were previously established26Migita T Narita T Nomura K Miyagi E Inazuka F Matsuura M Ushijima M Mashima T Seimiya H Satoh Y Okumura S Nakagawa K Ishikawa Y ATP citrate lyase: activation and therapeutic implications in non-small cell lung cancer.Cancer Res. 2008; 68: 8547-8554Crossref PubMed Scopus (289) Google Scholar and were used as a positive control. The primary antibody was omitted for negative controls. All immunohistochemical staining was accomplished with a Dako Autostainer (DakoCytomation, Carpenteria, CA) under the same conditions. The staining intensity of IGFBP-2 and procaspase-3 was scored semiquantitatively: positive in less than 25% of cancer cells (weak), positive in 25% to 50% of cancer cells (moderate), and positive in more than 50% of cancer cells (strong). Representative score of each patient was defined as the highest score across three cores. For in vitro experiments, statistical analysis was performed by using Welch's t-tests. Comparisons of IGFBP-2 mRNA levels in clinical samples were made by using paired t-test analysis. Dose/time dependency of drugs was determined by the confidence interval (CI) based test of slope of the linear regression. Concentrations of drugs that suppressed cell proliferation to 50% of levels exhibited by control cells (IC50) were derived from the dose-response curve. Correlation between IGFBP-2 and caspase-3 expression in immunohistochemistry was evaluated by performing the Fisher's exact test. For all analyses, P ≤ 0.05 was considered statistically significant. Statistical analyses were performed by using the statistical programming language of R (http://www.R-project.org; accessed February 1, 2010) and Statistika (Statsoft, Inc., Tulsa, OK). At first, intracellular IGFBP-2 expression levels were examined in various lung cancer cell lines by the use of Western blot. IGFBP-2 was highly expressed in A549, NCI-H460 cells, but expressed at very low levels in HOP62 and COR-L105 cells (Figure 1A). The levels of secreted IGFBP-2 in media were measured by ELISA. Secreted IGFBP-2 levels correlated with intracellular protein levels obtained by Western blot (Figure 1B). IGFBP-2 expression is physiologically up-regulated by the energy restriction or insulin-dependent diabetes mellitus.28Rajaram S Baylink DJ Mohan S Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions.Endocr Rev. 1997; 18: 801-831Crossref PubMed Scopus (966) Google Scholar, 29Kaaks R Lukanova A Energy balance and cancer: the role of insulin and insulin-like growth factor-I.Proc Nutr Soc. 2001; 60: 91-106Crossref PubMed Scopus (497) Google Scholar To determine whether the supplement of nutrients can alter IGFBP-2 expression in lung cancer cells, we examined the effects of glucose or serum depletion on IGFBP-2 expression in A549 cells. Glucose depletion significantly reduced IGFBP-2 levels at both protein and mRNA levels (P = 0.0017), whereas serum depletion did not (P = 0.311; Figure 2A). IGFBP-2 protein and mRNA levels were dependent on glucose concentration (Figure 2B). These findings suggest that IGFBP-2 expression in cancer cells is glucose-dependent and is regulated by a mechanism that is distinct from normal cells. It has been reported that IGFBP-2 expression is regulated by the PI3K-PTEN (phosphatase and tensin homolog deleted on chromosome 10) pathway in prostate and glioblastoma cells.30Mehrian-Shai R Chen CD Shi T Horvath S Nelson SF Reichardt JK Sawyers CL Insulin growth factor-binding protein 2 is a candidate biomarker for PTEN status and PI3K/Akt pathway activation in glioblastoma and prostate cancer.Proc Natl Acad Sci USA. 2007; 104: 5563-5568Crossref PubMed Scopus (158) Google Scholar Thus, extracellular and intracellular IGFBP-2 levels were evaluated in lung cancer cells treated with LY294002, a PI3K inhibitor. PTEN protein was detected in all cell lines, except PC14, as described previously.26Migita T Narita T Nomura K Miyagi E Inazuka F Matsuura M Ushijima M Mashima T Seimiya H Satoh Y Okumura S Nakagawa K Ishikawa Y ATP citrate lyase: activation and therapeutic implications in non-small cell lung cancer.Cancer Res. 2008; 68: 8547-8554Crossref PubMed Scopus (289) Google Scholar Secretion of IGFBP-2 protein was suppressed in all cell lines by the treatment of LY294002 to varying degrees (Figure 2C). The effect of LY294002 on IGFBP-2 expression showed a significant dose dependence (P = 0.0048) and time course dependence (95% CI: 0.134 to 0.18, control; 0.029 to 0.043, LY294002) in A549 cells (Figure 2, D and E). Intracellular IGFBP-2 levels were also decreased with LY294002 (Figure 2F). Interestingly, a fraction of IGFBP-2 protein was degraded into approximately 20 kDa after treatment with LY294002 (Figure 2F). Conversely, IGFBP-2 mRNA was significantly increased with LY294002 (P < 0.005; Figure 2G), suggesting the existence of a compensatory feedback mechanism. To address whether IGFBP-2 is involved in apoptotic event, IGFBP-2 was enforced in cells with low endogenous IGFBP-2 levels, and then caspase expression was examined. IGFBP-2 overexpression resulted in a remarkable increase in intracellular IGFBP-2 levels in COR-L105, NCI-H522, and HOP62 cells compared with vector control (Figure 3A). Secreted IGFBP-2 levels of these cells were also increased corresponding to the levels of intracellular IGFBP-2 (Figure 3B). Intriguingly, IGFBP-2 overexpression resulted in a substantial decrease in procaspase-3 expression (Figure 3A). However, caspase-9 was not decreased (Figure 3A), suggesting IGFBP-2 specifically inhibits caspase-3 expression. Despite a higher amount of IGFBP-2 secretion into media, no significant changes were found in the IGF signaling pathway including phosphorylation statuses of IGF-1R, Akt, or Erk1/2 (Figure 3A). These findings suggest that IGFBP-2-mediated caspase-3 inhibition occurs in an IGF-independent manner. Next, to examine whether IGFBP-2 involves in apoptotic event, we compared the sensitivity of IGFBP-2 overexpressing cells and vector control cells to an apoptosis inducer, camptothecin. IGFBP-2 overexpressing and vector control H522 cells were exposed to 20 to 1000 nmol/L of camptothecin for 24 hours, and the cell proliferation and caspase-3 activity were analyzed. The results indicated that IGFBP-2 overexpressing H522 cells were significantly resistant to camptothecin (EV, IC50 = 686 nmol/L; BP-2, IC50 > 1000 nmol/L; Figure 3C). As expected, caspase-3 activity was significantly decreased in IGFBP-2 overexpressing cells compared with vector control cells on treatment with camptothecin (P < 0.02; Figure 3D). Apoptosis was evaluated by Hoechst 33342 staining and PARP cleavage. Enforced IGFBP-2 significantly inhibited PARP cleavage, as determined by Western blot (Figure 3E), and reduced camptothecin-induced apoptotic cells in H522 cells (P = 0.003; Figure 3F). Similar results were obtained with the treatment of cis-platin or etoposide (data not shown). To further elucidate the effects of IGFBP-2 on caspase-3, gene silencing for IGFBP-2 was performed in A549 and H522 cells. IGFBP-2 knockdown induced an increase in procaspase-3 expression until 72 hours after siRNA treatment in both cell lines (Figure 4A). No significant active form of cleaved caspase-3 was identified (data not shown). As is the results with IGFBP-2 overexpression, no substantial change was found in caspase-9. In addition, IGFBP-2 siRNA also decreased the phosphorylation status of IGF-1R. This effect might be because of a rapid decrease in both intracellular and extracellular IGFBP-2. Although IGFBP-2 knockdown resulted in morphological changes such as shrinkage in A549 cells, no substantial increase in apoptosis was identified by Hoechst 33342 staining or PARP cleavage (data not shown). We now asked whether IGFBP-2 inhibition sensitizes cells for drug-induced apoptosis. Figure 4B shows the cell proliferation of IGFBP-2 knockdown and negative control cells with a treatment of camptothecin. IGFBP-2 knockdown cells were more sensitive to campthothecin rather than vector control cells (95% CI: −2.7 × 10−4 to −1.6 × 10−4 vs. −4.5 × 10−4 to −3.0 × 10−4, in negative control and IGFBP-2 siRNA, respectively; Figure 4B). In caspase-3 activity assay (Figure 4C), there were no significant changes in caspase-3 activity between negative control and IGFBP-2 siRNA with DMSO treatment (white bars). When cells were treated with camptothecin, IGFBP-2 siRNA significantly increased caspase-3 activity than negative control siRNA (black bars). The sensitivity to camptothecin was significantly potentiated by IGFBP-2 inhibition (P < 0.0001). Apoptosis was significantly increased in cells with IGFBP-2 siRNA compared with negative control siRNA (P = 0.0009; Figure 4D). Cleaved PARP was more substantial in IGFBP-2 siRNA treated cells compared with vector control H522 cells (Figure 4E). As a PI3K inhibitor induced IGFBP-2 degradation (Figure 2F), we examined whether a PI3K inhibitor has an additive effect on apoptosis with camptothecin. As expected, combination therapy of LY294002 and campthothecin enhanced PARP cleavage in H522 cells when compared with campthothecin or LY294002 alone. IGFBP-2 levels were inversely correlated with the increase in the levels of cleaved PARP (Figure 4F, left panels). In contrast, there were no substantial effects of LY294002 on PARP cleavage in COR-L105 cells, which have low IGFBP-2 levels (Figure 4F, right panels). These data strongly suggest that IGFBP-2 regulates apoptosis via caspase-3. Moreover, IGFBP-2 becomes a the
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