Opposing Roles of IGFBP-3 and Heparanase in Regulating A549 Lung Cancer Cell Survival

Abstract

In this study, we examined the roles of heparanase and IGFBP-3 in regulating A549 and H1299 non-small-cell lung cancer (NSCLC) survival. We found that H1299 cells, known to be p53-null with no expression of IGFBP-3, had higher heparanase levels and activity and higher levels of heparan sulfate (HS) in the media compared to the media of A549 cells. Inhibiting heparanase activity or its expression using siRNA had no effect on the levels of IGFBP-3 in the media of A549 cells, reduced the levels of soluble HS fragments, and led to decreased interactions between IGFBP-3 and HS in the media. HS competed with HA for binding to IGFBP-3 or IGFBP-3 peptide (²¹⁵-KKGFYKKKQCRPSKGRKR-²³²) but not the mutant peptide (K228AR230A). HS abolished the cytotoxic effects of IGFBP-3 but not upon blocking HA-CD44 signaling with the anti-CD44 antibody (5F12). Blocking HA-CD44 signaling decreased the levels of heparanase in the media of both A549 and H1299 cell lines and increased p53 activity and the levels of IGFBP-3 in A549 cell media. Knockdown of p53 led to increased heparanase levels and reduced IGFBP-3 levels in A549 cell media while knockdown of IGFBP-3 in A549 cells blocked p53 activity and increased heparanase levels in the media.

Keywords: CD44; IGFBP-3; extracellular; heparan sulfate; heparanase; hyaluronan; lung cancer; p53; signaling.

Figure 1

Higher heparanase levels and activity and higher levels of HS are found in H1299 cell media than in the media of A549 cells. Cells (0.2 × 10⁵) were grown in 10% FBS-supplemented media for 24 h then serum-starved overnight. The cell monolayers were then incubated in serum-free media for 72 h then the levels of heparanase (A) and activity (B) along with the levels of HS (C) were measured as described in the Materials and Methods section. Data were processed using the GraphPad Prism 9.4.1 software and presented as the mean ± S.D. of three independent assays, each performed in triplicate. Asterisks indicate a statistically significant difference from the control using media not incubated with cells. Mann–Whitney test, ** p < 0.01.

Figure 2

Blocking heparanase activity or expression using siRNA decreased HS level in the media and the amount of IGFBP-3 bound to HS. Cells (0.2 × 10⁵) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h, then treated as indicated for 72 h with OGT 2115 (100 µM) or with siRNA as described in the Methods section. The same concentration of total protein (15 µL of 600 µg/mL) of the cell lysates (A) was used for Western blotting using the indicated antibodies. As a loading control, anti α-tubulin antibodies were used. The levels of IGFBP-3 (B,E) and HS (C,D) were measured on the same amount of protein (3 µL of 600 µg/mL total protein) in the media as described in the Materials and Methods section. The graphs summarize the results expressed as means ± SD (n = 5) using the GraphPad 9.4.1 software. Asterisks indicate a statistically significant difference from the corresponding samples without inhibitor treatment or those treated with control siRNA of each cell line as indicated, Mann–Whitney test. The absence of asterisks indicates no significance, ** p < 0.0l.

Figure 3

Increasing HS concentrations blocked binding of HA to IGFBP-3 and WT- but not the mutant-peptide. IGFBP-3 protein, WT-, or mutant-peptide (50 nM) were bound to the plate wells. A single concentration of biotinylated-HA (35 µg/mL) was incubated for 1h without or with increasing concentrations of HS and then loaded into the coated wells. The signal was then processed, and the bound biotinylated-HA was then detected as described in the Materials and Methods section. Prior to data analysis, the OD were corrected for non-specific binding by subtracting the mean background absorbance for the negative controls prepared with all components except biotinylated-HA. Optical density measurements (450 nm) were normalized by expressing each point in relation to the best-fitted Emax value for IGFBP-3 (set to 100%). The data were then plotted as a function of increasing HS concentrations. Using the GraphPad Prism 9.4.1 software, the data were analyzed with a nonlinear regression curve fitting approach then expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.

Figure 4

HS blocked the cytotoxic effects of IGFBP-3 but not upon inhibition of HA–CD44 signaling with the 5F12 antibody. Cells were seeded in 96-well plates at 0.2 × 10⁵ cells per well in 10% FBS-supplemented media. The following day, the cell monolayers were incubated in serum-free medium for 24 h. Fresh serum-free media was then added and the cells were treated for 72 h with the IGFBP-3 protein (50 nM), the CD44 antibody (5F12, 5 μg/mL) added either separately or 2 h prior to addition of IGFBP-3, or in combination, without or with increasing HS concentrations. Cell viability of A549 (A) and H1299 (B) was then assessed by the MTT assay as described in the Materials and Methods section. Optical density measurements (570 nm) were normalized by expressing each point in relation to the best-fitted Emax value of cells without added IGFBP-3 or 5F12 (set to 100%). The data were then plotted as a function of increasing HS concentrations. Using the GraphPad Prism 9.4.1 software, the data were analyzed with a nonlinear regression curve fitting approach then expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.

Figure 5

Blocking HA–CD44 signaling decreases the levels of heparanase in the media of both cell lines and increases p53 activity and the levels of IGFBP-3 in A549 cell media. Cells (0.2 × 10⁵) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h, then treated as indicated for 72 h with 50 nM of IGFBP-3 protein/peptide/mutant, the CD44 antibody (5F12, 5 μg/mL) added either separately or 2 h prior to addition of IGFBP-3/peptide/mutant, or in combination. The levels of heparanase (A) and IGFBP-3 (B) in the media, using the same amount of protein (3 µL of 600 µg/mL total protein), and the p53 activity (C) in cell lysates were measured as described in the Materials and Methods section. The graphs summarize the results expressed as means ± SD (n = 5) using the GraphPad 9.4.1 software. Fold change was calculated relative to the control of each cell line (A) or to the A549 control (B,C). Asterisks indicate a statistically significant difference from the corresponding negative control of each cell line, Mann–Whitney test. Statistical differences between different groups were analyzed by a non-parametric Kruskal–Wallis test. Absence of asterisks indicates no significance, ** p < 0.01.

Figure 6

Knockdown of p53 led to increased heparanase levels and decreased IGFBP-3 levels in the media of A549 cells. Cells (0.2 × 10⁵) were grown in 10% FBS-supplemented media for 24 h then serum-starved overnight. The cells were then treated as indicated for 72h with control siRNA or p53 siRNA. The same concentration of total protein (15 µL of 600 µg/mL) of the cell lysates (A) was used for Western blotting using the indicated antibodies. As a loading control, anti α-tubulin antibodies were used. Transfected cells were treated with 50 nM of IGFBP-3 protein/peptide/mutant, the CD44 antibody (5F12, 5 μg/mL) added either separately or 2h prior to addition of IGFBP-3/peptide/mutant, or in combination (B–D). The levels of heparanase (B,C) and IGFBP-3 (D) were measured in the media using the same amount of protein (3 µL of 600 µg/mL total protein) as described in the Materials and Methods section. The graphs summarize the results expressed as means ± SD (n = 5) using the GraphPad 9.4.1 software. Fold change was calculated relative to the A549 control siRNA (B,D) or H1299 control siRNA (C). Asterisks indicate a statistically significant difference from the corresponding negative control of each cell line, Mann–Whitney test. Statistical differences between different groups were analyzed by a non-parametric Kruskal–Wallis test. Absence of asterisks indicates no significance, ** p < 0.0l.

Figure 7

Knockdown of IGFBP-3 led to increased heparanase levels in the media of A549 cells and decreased p53 activity. Cells (0.2 × 10⁵) were grown in 10% FBS-supplemented media for 24 h then serum starved overnight. The cells were then treated as indicated for 72 h with control siRNA or IGFBP-3 siRNA. The same concentration of total protein (15 µL of 600 µg/mL) of the cell lysates (A) was used for Western blotting using the indicated antibodies. As a loading control, anti α-tubulin antibodies were used. Cell transfectants were treated without or with 50 nM IGFBP-3 protein/peptide/mutant, the CD44 antibody (5F12, 5 μg/mL) added either separately or 2 h prior to addition of IGFBP-3 protein/peptide/mutant, or in combination (B–D). The media levels of IGFBP-3 (B) and heparanase (C) using the same amount of protein (3 µL of 600 µg/mL total protein), and the p53 activity in cell lysates (D) were measured as described in the Materials and Methods section. The graphs summarize the results expressed as means ± SD (n = 5) using the GraphPad 9.4.1 software. Fold change was calculated relative to the A549 control siRNA transfectants (Control). Asterisks indicate a statistically significant difference from the corresponding A549 negative control, Mann–Whitney test. Statistical differences between different groups were analyzed by a non-parametric Kruskal–Wallis test. Absence of asterisks indicates no significance, ** p < 0.0l.

Figure 8

Representation of the main hypothesis and findings from this study. Binding of IGFBP-3 to HA blocks HA–CD44 signaling leading to p53 activation which in turn results in increased IGFBP-3 levels. IGFBP-3 is now able to continue disrupting HA–CD44 signaling further increasing its own levels. Increased p53 activation resulting from disruption of HA–CD44 signaling by IGFBP-3 also leads to decreased heparanase levels and activity, blocking the enzyme’s ability to cleave cell-surface HS chains, decreasing soluble HS fragments, and formation of the IGFBP-3-HS complex. IGFBP-3 not bound to HS is now able to bind HA disrupting HA–CD44 signaling decreasing cell survival. Green lines/arrows indicate activation while red lines indicate inhibition.