We recently reported that a high level of hepatoma-derived growth factor (HDGF) expression in tumors correlates with a high incidence of tumor relapse or distant metastasis and shortened survival time in patients with non–small cell lung cancer (NSCLC). However, the mechanisms of the HDGF-associated aggressive biological behavior are unknown. In this study, we knocked down HDGF expression in NSCLC cells to determine the biological consequences. Transfection with HDGF-specific small interfering RNA (siRNA) resulted in down-regulation of HDGF expression in four NSCLC cell lines. Down-regulation of HDGF resulted in no detectable effect on anchorage-dependent cell growth as determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a microelectronic cell sensor system, and flow cytometry. In contrast, cells transfected with HDGF-siRNA grew more slowly and formed significantly fewer colonies in soft agar than did cells treated with LipofectAMINE alone or transfected with negative control siRNA. In an in vitro invasion assay, significantly fewer cells transfected with HDGF-siRNA than cells treated with LipofectAMINE alone were able to invade across a Matrigel membrane barrier. In an in vivo mouse model, A549 cells treated with HDGF-siRNA grown significantly slower than the cells treated with LipofectAMINE alone or negative control siRNA. Morphologically, HDGF-siRNA–treated tumors exhibited markedly reduced blood vessel formation and increased necrosis, whereas the Ki67 labeling indices were similar in tumors treated with controls. Our results suggest that HDGF is involved in anchorage-independent growth, cell invasion, and formation of neovasculature of NSCLC. These qualities may contribute to the HDGF-associated aggressive biological behavior of NSCLC. (Cancer Res 2006; 66(1): 18–23)
Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor originally purified from media conditioned with the human hepatoma cell line HuH-7 and can stimulate proliferation of Swiss 3T3 cells (1). Its precise function is unclear, but HDGF is known to be highly expressed during the early development of many tissues, including cardiovascular (2), kidney (3), and liver (4). Although lacking the secretory sequence present in most secretory proteins (5), HDGF has been shown to act as a potent exogenous mitogen for HuH-7 hepatoma cells (6), COS-7 kidney cells (6), aortic vascular smooth muscle cells (7), and endothelial cells (3). As deduced from the cDNA clone of HDGF, the amino acid sequence contains 240 residues with a motif homologous to the consensus sequences of a bipartite nuclear localization sequence and a DNA-binding PWWP motif, suggesting that the protein translocates to the nucleus and binds to DNA. In fact, HDGF is found mainly in nucleus, and its role as a transcription factor has been postulated (8, 9).
In a recent study, we investigated the role of HDGF in non–small cell lung cancer (NSCLC) and found that the protein is frequently overexpressed in these tumors (10). In patients with early-stage NSCLC, poorer clinical outcome was significantly correlated with higher HDGF expression, suggesting that HDGF is involved in the determination of aggressive biological behavior of NSCLC cells. To elucidate the mechanism of HDGF-mediated aggressiveness in NSCLC cells, we down-regulated HDGF expression in these cells using small interfering RNA (siRNA) technology and studied effects of the down-regulation in cell proliferation and invasion. Our results suggest that HDGF is involved in anchorage-independent growth and cell invasion of NSCLC. These qualities may contribute to the HDGF-associated aggressive biological behavior of NSCLC.
Materials and Methods
Cell culture. NSCLC cell lines H226, H1944, H292, H157, A549, H596, H460, and H358 were obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS). The normal human bronchial epithelial cell lines HBE1 and HBE3 (kindly provided by Dr. John Minna of The University of Texas Southwestern Medical Center, Dallas, TX) were cultured in keratinocyte serum-free medium with 25 μg/mL bovine pituitary extract and 0.2 ng/mL recombinant epidermal growth factor (Invitrogen, Carlsbad, CA).
siRNA and knockout of HDGF expression. We selected two sites in the HDGF mRNA sequence as siRNA targets based on principles described previously (11). The targeted HDGF sequences, based on which the siRNAs were chemically synthesized by Ambion (Austin, TX), were 5′-AACCGGCAGAAGGAGUACAAA-3′ (siRNA-1) and 5′-AAAUCAACAGCCAACAAAUAC-3′ (siRNA-2). The negative control siRNAs were purchased from Ambion. In vitro transfections were done using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) following manufacturer's protocols.
Cell proliferation analysis. Cells were plated onto 96-well plates at a density of 1 × 104 per well with medium containing 10% FBS and incubated for 15 hours. Cell numbers were determined at 0, 24, 48, and 72 hours after transfection using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)–based CellTiter96 cell proliferation assay (Promega, Madison, WI). ACEA RT-CES (ACEA Biosciences, San Diego, CA), a microelectronic cell sensor system, was used to confirm the number of living cells. NSCLC cells (1 × 104) were seeded into each sensor-containing well (19.6-mm2 surface with 150 μL of medium) of the microtiter plates. The electronic sensors provided a continuous (every 6 hours), quantitative measurement of the cell index (reflect to the surface area covered by the cells) in each well. After 15 hours of culture, the cells were transfected as described above. Cell growth was measured every 6 hours for 72 hours, and cell indexes were recorded for each well at all time points.
Cell cycle analysis. A549 cells were harvested by trypsinization 72 hours after transfection and fixed with 70% ethanol. After RNase treatment, the cell cycle distribution was determined using a BD FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson, San Jose, CA).
Anchorage-independent growth assay. Twenty-four hours after transfection with siRNA, ∼2,000 cells in 1 mL of 0.3% agarose with DMEM were plated in each well on the top of existing 0.6% bottom agarose in six-well tissue culture plates in triplicate for each treatment condition. The plates were covered with 1 mL of medium with 10% FBS and incubated at 37°C in a 5% CO2 incubator for 3 weeks. The covering medium was replaced every week. At the end of 3 weeks, cell colonies >0.1 mm in diameter were counted under a microscopic field at ×40 magnifications. Means were based on numbers from triplicate wells for each treatment condition and were analyzed using two-sided Student's t test.
In vitro cell invasion assay. The in vitro invasion assay was carried out in BD BioCoat Matrigel invasion chambers (Becton Dickinson). After rehydration of the chambers, 1.1 × 104 cells in 100 μL of the growth medium with 10% FBS were added into each of the upper chambers. Cells in the chambers were transfected with LipofectAMINE alone or 100 nmol/L HDGF-siRNA-1 in serum-free condition. Four hours later, the medium was replaced with fresh growth medium containing 10% FBS in the upper chambers, whereas the lower wells contained serum-free medium. After 20 hours, the medium in each of the lower wells was replaced with 750 μL of serum-free medium containing 30 μg/mL laminin (Sigma-Aldrich, St. Louis, MO). After an additional 24 hours of incubation, the noninvading cells on the upper side of the chamber membranes were removed. The invading cells to the opposite side of the chamber membranes were examined. The invading cells on each of triplicate membranes were counted. Means were based on the numbers from the triplicate wells for each treatment condition and were analyzed using two-sided Student's t test.
Western blot analysis. Total proteins were loaded into each well on 10% SDS-polyacrylamide gels, separated by electrophoresis, and transferred to Hybond-P polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, United Kingdom). The membranes were probed with a rabbit polyclonal anti-HDGF antibody (gift from Dr. Allen Everett of Johns Hopkins Hospital, Baltimore, MD) followed by incubating with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (Amersham Biosciences). Immunodetection was done using the enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences). β-Actin was used as protein loading control monoclonal anti-β-actin antibody (Sigma-Aldrich).
Global gene expression analysis. Total RNA was isolated from cells using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA). Ten micrograms of total RNA were reverse-transcribed into double-stranded cDNA and then transcribed in the presence of biotin-labeled ribonucleotides, using the BioArray High-Yield RNA transcript labeling kit (Enzo Laboratories, Farmingdale, NY) as described by the manufacturer. The biotin-labeled cRNA was purified using RNeasy mini-column (RNeasy kit, Qiagen) and fragmented at 94°C for 35 minutes in 1× fragmentation buffer [40 mmol/L Tris-acetate (pH 8.0), 100 mmol/L KOAc, 30 mmol/L MgOAc]. Affymetrix U133A chips were used for gene expression analysis using the Affymetrix GeneChip system (Affymetrix, Santa Clara, CA).
The expression levels were extracted from positional-dependent nearest-neighbor model developed by Zhang et al. (12). Genes that are absent or always expressed at low levels were excluded from further analysis because variation in gene expression at low levels are usually not reproducible. The removed genes have mean log expression level of <7.1. Genes with SD <0.06 were also removed. Comparisons were then done between cells treated with LipofectAMINE alone and cells treated with 100 nmol/L HDGF-siRNA-1.
Northern blot analysis. Total RNA (10 μg) was loaded in each lane. cDNA probes corresponding to HDGF, GLO1, SERPINE2, AXL, and actin were prepared using reverse transcription-PCR followed by cDNA purification and labeling.
In vivo tumor model. Athymic Swiss nu/nu/Ncr nude (nu/nu) mice, bred and maintained in our institutional specific pathogen-free mouse colony, were used. Briefly, 4-week-old male nude mice were injected s.c. with 106 A549 cells in 100 μL of PBS at a single dorsal site. Three groups (five each) of mice were tested. Group 1 were injected with A549 cells treated with LipofectAMINE alone; group 2 were injected with A549 cells treated with LipofectAMINE plus 100 nmol/L HDGF-siRNA-1; group 3 was injected with A549 cells treated with LipofectAMINE plus 100 nmol/L negative control siRNA. Tumor size was measured every 2 days for 20 days. Tumor growth was quantified by measuring the tumors in three dimensions with calipers. The results were expressed as the mean tumor volume (n = 5) with 95% confidence intervals. The statistical significance of differences in tumor growth was analyzed using Wilcoxon rank sum test.
Tumor morphology and Ki67 immunohistochemistry. Formalin-fixed and paraffin-embedded tissue sections were stained with H&E for morphologic examination. For Ki67 immunohistochemistry, an anti-Ki67 antibody (Lab Vision, Fremont, CA) was used. The expression signal was detected using standard avidin-biotin immunohistochemical techniques according to the manufacturer's recommendations (Vector Laboratories, Burlingame, CA).
Results and Discussion
HDGF is highly expressed in NSCLC. Western blot analysis using a polyclonal anti-HDGF antibody revealed that most of the NSCLC cell lines expressed high levels of HDGF, whereas the immortalized normal bronchial epithelial cell lines (HBE1 and HBE3) expressed low levels of HDGF (Fig. 1A). We selected four cell lines (A549, H358, H226, and H1944) for further investigation.
HDGF-siRNA-1 knocks out HDGF in NSCLC cells. To determine the role of HDGF in NSCLC, we used RNA interference (RNAi) strategy to down-regulate the molecule. In A549 cells, the HDGF protein level was substantially reduced 48 hours after transfection with 100 nmol/L HDGF-siRNA-1, whereas 100 nmol/L HDGF-siRNA-2 induced only a slight reduction of the protein; these effects lasted up to at least 72 hours after transfection (Fig. 1B). Using 100 nmol/L concentration of HDGF-siRNA-1, the protein level was similarly down-regulated in H1944, H358, and H226 cells. These results indicate that HDGF-siRNA-1 effectively and specifically down-regulated HDGF protein expression in a panel of NSCLC cells.
Down-regulation of HDGF has minimal effect on anchorage-dependent growth of NSCLC cells. We next examined the growth curves of A549 cells transfected with 2 or 100 nmol/L HDGF-siRNA-1 in the presence of 5% bovine serum. Results of the MTT assay showed that the growth curves of these cells were comparable to those of cells treated with LipofectAMINE alone or transfected with negative control siRNA (Fig. 2A). These observations were confirmed by using a microelectronic cell sensor system (Fig. 2B). Similar results were obtained in H1944, H358, and H226 cells (data not shown). These data suggest that HDGF plays a minimal role in controlling anchorage-dependent growth in NSCLC cells in our culture condition.
To determine a role of HDGF in cell cycle regulation, we did flow cytometry analysis in A549 cells 72 hours after transfection with 2 or 100 nmol/L HDGF-siRNA-1. The cell cycle distributions of these cells were similar to those of cells treated with LipofectAMINE alone or transfected with negative control siRNA (Fig. 2C).
Although HDGF can stimulate DNA synthesis and cell proliferation in vascular or bronchial epithelial cells has been previously reported (8, 9, 13), our results are consistent with our clinical observation that the expression levels of HDGF was not associated with Ki67 labeling indices in primary NSCLC (10). In fact, HDGF-mediated cell growth was observed only when the cells were cultured in serum-free condition (13, 14); the presence of serum would have masked HDGF stimulation because of the effect of other growth stimulators in serum.
Down-regulation of HDGF reduces anchorage-independent growth of NSCLC cells. The effect of HDGF on anchorage-independent growth of the four cell lines was analyzed using the soft agar growth assay. Three weeks after seeding, cells transfected with 100 nmol/L HDGF-siRNA-1 produced significantly fewer and smaller colonies than did cells treated with LipofectAMINE alone or transfected with negative control siRNA (Fig. 3A). The numbers (average of triplicate wells with three randomly selected fields per well) of colonies visible in a microscopic field at ×40 magnifications for the four cell lines are presented in an attached table (Fig. 3A). These results suggest that HDGF is involved in anchorage-independent cell growth, a feature of malignant transformation, of NSCLC cells.
Down-regulation of HDGF reduces NSCLC cells' capability to invade. We then used Matrigel invasion chambers to determine the effect of HDGF on the invasion potential of the four cell lines. Transfection with 100 nmol/L HDGF-siRNA-1 resulted in significantly fewer invasive cells in A549 and H226 cell lines (Fig. 3B). The number of cells transfected with HDGF-siRNA-1 that were invasive averaged 140 (140 ± 73.65), whereas the number of cells treated with LipofectAMINE alone that were invasive averaged 759 (759 ± 156.79; P = 0.004) for A549; 126 (126 ± 28) versus 516 (516 ± 19; P = 0.0001) for H226. Because H1944 and H358 did not invade in both controls and treated cells in these chambers, we were unable to determine the effect of HDGF-siRNA-1 in these cell lines.
Together with the soft agar experiments, these results may explain the increased rates of tumor relapse and distant metastasis in patients whose primary NSCLC tumors had a high level of HDGF after surgical removal of the tumors (10). An association between higher HDGF and poor clinical outcome has also been observed in patients with NSCLC by another group (15) and in patients with primary hepatocellular carcinoma (16) and melanoma (17).
Genes down-regulated by HDGF-siRNA-1 treatment. We did global gene expression analysis using Affymetrix U133A gene chip, which can measure expression of >16,000 unique genes, to elucidate potential mechanism of HDGF-siRNA-1–induced growth inhibition in soft agar and inhibition of invasion in the invasion chambers. We compared gene expression profiles between A549 cells treated with LipofectAMINE alone and A549 cells treated with 100 nmol/L HDGF-siRNA-1 at 48-hour time point. Among 10,938 unique genes with expression level qualifying our analysis as specified in Materials and Method, 15 genes were down-regulated ≥2-fold in HDGF-siRNA-1–treated cells compared with cells treated with LipofectAMINE alone (attached table in Fig. 4), whereas none of the genes were up-regulated ≥2-fold. Among the 15 genes, the expression of HDGF was down-regulated most dramatically (>4-fold) as expected. The next two genes are GLO1 and SERPINE2. GLO1 has been shown elevated in lung cancers (18), whereas SERPINE2 has been suggested to play a role in invasion of pancreatic cancer cells (19). AXL, a receptor tyrosine kinase also in the list, has been reported overexpressed in multiple types of cancers (20–22) and linked to adverse clinical outcome in patients with cancer (23). To confirm the down-regulation of GLO1, SERPINE2, and AXL in HDGF-siRNA-1 treated cells, we did Northern blot analysis to compare the gene expression levels in A549 and H226 cells treated with HDGF-siRNA-1, siRNA control, and LipofectAMINE alone. The results are consistent with the microarray experiment (Fig. 4) and agree with the notion that HDGF is involved in regulation of expression of these genes.
Down-regulation of HDGF inhibits tumorigenicity of NSCLC cells in vivo. To further determine a role of HDGF in progression of NSCLC, we did an in vivo animal experiment. We found that A549 cells transfected with 100 nmol/L HDGF-siRNA-1 formed substantially smaller tumors in nude mice compared with those transfected with LipofectAMINE alone or negative control siRNA (Fig. 5, left). The tumor volume for mice with cells transfected with the HDGF-siRNA was 76.27 ± 39.06 mm3 compared with 345.64 ± 135.67 or 295.33 ± 80.53 mm3 for mice with cells treated with LipofectAMINE alone or negative control siRNA, respectively (P = 0.037 or P = 0.018, respectively). Under light microscopy, we observed a substantially reduced blood vessels in the HDGF-siRNA–transfected tumors compared with the tumors derived from cells treated with LipofectAMINE or negative control siRNA (Fig. 5A,-D, right). Substantial tumor necrosis was observed only in tumors derived from cells treated with the HDGF-siRNA (Fig. 5C, and D, right). Interestingly, Ki67 expression index, an indicator of cell proliferation, was similar in tumors of the three animal groups (Fig. 5E and F, right).
The in vivo animal experiment provides a strong support for the importance of HDGF in NSCLC and suggests that HDGF may be a target for treating NSCLC or preventing the development of lung cancer. The finding of reduced blood vessel formation in the HDGF-siRNA–treated tumors suggests that HDGF plays a role in the neovasculature formation in vivo, which may be an important mechanism of HDGF in tumor development and progression of NSCLC. This is consistent with previous reports supporting the role of HDGF in angiogenesis as a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from those used by vascular endothelial growth factor (14, 24). The observed tumor necrosis is likely a consequence of the poor blood supply in these tumors. Consistent with our in intro and clinical observations, the lack of change in cell proliferation in the tumors indicates that HDGF plays a minimal role in the tumor cell proliferation for patients with NSCLC. Future studies will focus on the molecular mechanisms of HDGF-induced tumor development and progression as well as on strategies to down-regulate the protein or inhibit its function for potential therapeutic applications.
Grant support: Department of Defense grant DAMD17-01-1-01689-1 and National Cancer Institute grants PO1 CA106451, PO1 CA91844, and U01 CA86390.
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