Upregulation of MMP-2 by HMGA1 promotes transformation in undifferentiated, large-cell lung cancer

Abstract

Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we show that HMGA1 is an important oncogene that drives transformation in undifferentiated, large-cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large-cell human lung carcinoma cells. We also show that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target upregulated by HMGA1 in large-cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large-cell lung cancer cells, but not in squamous cell carcinoma cells. In large-cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large-cell carcinoma cells, indicating that the HMGA1-MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large-cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large-cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors.

FIGURE 1

HMGA1 is overexpressed in cultured human lung cancer cells and primary lung tumors. A. HMGA1 protein is increased in the cultured human lung cancer cell lines: H1299, SK-MES-1, H358, H125, and H82 compared to the control BEAS-2B cells derived from normal human bronchial lung. B. HMGA1a mRNA is increased in the cultured human human lung cancer cell lines: H1299, H460, H661, H125, U1752, SK-MES-1, and H82 compared to RNA from pooled, normal lung tissue from normal individuals. C. HMGA1a mRNA is increased in 71% of primary lung cancer tumors by qRT-PCR. C, Control RNA from normal lung derived from 3 normal individuals who died from causes other than lung cancer. Samples 1-15 are from primary lung tumors diagnosed histopathologically as adenocarcinoma and samples 16-24 are from primary lung tumors diagnosed as squamous cell carcinoma. The bottom panel shows tumor grade, stage, and lymph node metastases (+) if present. Additional clinical data is available in Supplementary Table S1. D. HMGA1 mRNA is elevated in this case of adenocarcinoma by in situ hybridization.

FIGURE 2

Inhibiting HMGA1 expression blocks transformation in cultured human lung cancer cells derived from metastatic disease. A. Western analysis shows decreased HMGA1 protein in the antisense cell lines. The lane marked HMGA1-AS-1 represents H1299 polyclonal cells transfected with the amino-terminal antisense HMGA1 construct, the lane marked HMGA1-AS-2 represents H1299 polyclonal cells transfected with the carboxyl-terminal antisense construct, and the lane marked vector control represents H1299 polyclonal cells transfected with control vector alone. The blot was probed with the HMGA1 antibody as well as an antibody to β-actin to control for protein loading. Western analysis of HMGA2 protein in the control and antisense cell lines shows no decrease in HMGA2 protein. This blot was also probed with a β-actin antibody as a loading control. B. Decreased anchorage-independent cell growth or foci formation in cells with decreased HMGA1 proteins (bar, 100 μm). C. Graphical representation of decreased foci formation in the cells with decreased HMGA1 proteins. (p = 0.0000995 for HMGA1-AS-1 and p = 0.0000531 for HMGA1-AS-2; student's t-test). This experiment was performed 3 times in duplicate and results are expressed as the average (bar) +/- the standard deviation. D. Cell growth rates in control cells and cells with decreased HMGA1 proteins were similar. This experiment was repeated 2 times and results are expressed as the average (bar) +/- the standard deviation.

FIGURE 3

HMGA1a induces a transformed phenotype with anchorage-independent cell growth in cultured cells derived from normal, human lung tissue. A. BEAS2-B cells transfected with the HMGA1a vector overexpress the HMGA1a protein compared to cells transfected with control, empty vector by Western analysis. Membranes were blotted with the HMGA1 antibody as well as a β-actin antibody to control for sample loading. B. Lung cells overexpressing HMGA1 exhibit anchorage-independent cell growth (foci formation) in soft agar. Results are expressed as the average (bar) +/- the standard deviations from 2 different experiments performed in duplicate. A representative plate from each condition is shown. C. Cell growth rates of the transfected BEAS-2B cell lines. This experiment was performed with duplicate plates and repeated twice. The error bars depict the standard deviations from a representative experiment.

FIGURE 4

The HMGA1-MMP-2 pathway contributes to anchorage-independent cell growth, migration, and invasion in metastatic lung cancer cells. A. Chromatin immunoprecipitation experiments with sheared chromatin from H1299 cells after cross-linking proteins bound to DNA with formaldehyde (15). The graph shows the quantity of immunoprecipitated DNA (bars) +/- the standard deviations with the following antibodies (all from Upstate, except for the HMGA1 antibody): HMGA1 (35), Polymerase II (Pol II or histone H3, both as positive controls), or rabbit IgG (as a negative control). Additional negative controls included no chromatin and no DNA. The HPRT promoter sequence was also used as a negative control because there are no HMGA1 DNA-binding sites in the region amplified and previous chromatin immunoprecipitation experiments showed no binding by HMGA1 to the amplified region (15, 35). The gel shows total input DNA compared with the DNA immunoprecipitated with the same antibodies. B. MMP-2 mRNA and protein are decreased in the H1299 cells transfected with siRNA to MMP-2 (20 nmol/L; Dharmacon) compared with cells transfected with the off-target control siRNA (siCONTROL nontargeting siRNA pool at 20 nmol/L; Dharmacon). The mRNA levels for MMP-2 and control β-actin (β-actin primer/probe set; Applied Biosystems) were assessed at 24 h, 48 h, 8 days, and 10 days after adding the siRNA. MMP-2 mRNA was normalized to β-actin as the loading control. MMP-2 mRNA was decreased at all time points; only the day 8 time point is shown here. (See Supplementary Figure 1 for the remaining time points.) All standard deviations were less than 5%. Protein was assessed for secreted MMP-2 by zymography at the same time points as the mRNA. The MMP-9 protein was also measured as a loading control. The zymogram shows decreased MMP-2 protein at 8 days; see Supplemental Figure 1 for the remaining time points. C. Anchorage-independent cell growth or foci formation are decreased in the H1299 cells with knock-down in MMP-2 expression. The bar graph shows the decreased number of foci in the cells incubated with siRNA to MMP-2 compared with the control siRNA. The bars represent the mean +/- the standard deviations from three experiments done in triplicate (100% +/- 41 % versus 49 % +/- 19 %; p =0.0019; student's t-test). The photograph shows actual foci from the experiment (bar, 100 μm). D. Migration and invasion were also assessed in the H1299 non-small cell lung carcinoma cells with and without MMP-2 knock-down. Twenty-four hours after transfection with the siRNA, 3 × 10⁴ cells/well were seeded onto the wells for migration/invasion insert. After 48 h, inserts were stained and counted for cells that migrated or invaded. There was a significant decrease in migration (p < 0.0001; student's t-test) and invasion (p = 0.0006; student's t-test). All experiments were performed in triplicate and repeated at least once. E. Growth curves showed that cells incubated with siRNA to MMP-2 or control siRNA proliferate at similar rates.