α3β1 integrin promotes radiation-induced migration of meningioma cells

Abstract

Cell motility is influenced by the microenvironment, signal transduction and cytoskeleton rearrangement. Cancer cells become resistant to these control mechanisms and gain the ability to move throughout the body and invade healthy tissues, which leads to metastatic disease. Integrins respond to context-dependent cues and promote cell migration and survival in cancer cells. In the present study, we analyzed the role of integrins in radiation-induced migration of meningioma cells. Migration and cell proliferation assays revealed that radiation treatment (7 Gy) significantly increased migration and decreased proliferation in two cell lines, IOMM-Lee and CH-157-MN. α3 and β1 integrins were overexpressed at both the protein and transcript levels after radiation treatment and a function-blocking α3β1 antibody inhibited the radiation-induced migration. Immunofluorescence studies illustrated the localization of α3 integrin and F-actin at the migration front of irradiated cells. Further, an increase in phosphorylation of FAK and ERK was observed, while both FAK phosphorylation inhibitor and FAK shRNA inhibited ERK phosphorylation and downregulated uPA and vinculin. In addition to the co-localization of FAK and ERK at the migration front, these FAK-inhibition results link the downstream effects of ERK to FAK. Correspondingly, U0126 quenched ERK phosphorylation and reduced the expression of molecules involved in migration. Furthermore, brain sections of the animals implanted with tumors followed by radiation treatment showed elevated levels of α3 integrin and active ERK. Taken together, our results show that radiation treatment enhances the migration of meningioma cells with the involvement of α3β1 integrin-mediated signaling via FAK and ERK.

Figure 1. Radiation induces migration and decreases proliferation of meningioma cells

A) IOMM-Lee and CH-157-MN cells (1×10⁵) were seeded in 6-well plates and irradiated at different doses of radiation as indicated. Then, cells were trypsinized and seeded at 1×10⁴ cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added and followed by another 4 hrs of incubation and addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. B) IOMM-Lee and CH-157-MN cells (1×10⁵) were seeded in 8-µm pore size transwell inserts, irradiated and allowed to migrate for 24 hrs. Later, cells that remained in the upper chamber and those that attached to the other side were fixed, stained and counted under a light microscope. The percentage of migrated cells was calculated. C) IOMM-Lee and CH-157-MN cells (2×10⁵) were seeded in 3-µm pore size transwell inserts, irradiated and allowed to migrate through the membrane for 24 hrs. Cells were then fixed and placed in crystal violet; cell bodies that remained on the top side of the membranes were scraped, the dye retained by the migration front on the lower side was extracted in 10% acetic acid, absorbance was measured at 590 nm, and the relative migration front was plotted. Values are mean ± S.D. from three independent experiments. * Statistically different compared to control and radiated groups (P<0.05)

Figure 2. α3 and β1 integrins are overexpressed and involved in radiation-induced migration

A) IOMM-Lee and CH-157-MN cells (1×10⁵) cells were seeded in 6-well plates, irradiated and incubated for 24 hrs. The cell lysates were subjected to western blot analysis using anti-α3, anti-β1 and GAPDH antibodies and quantified. Similarly, total RNA was extracted and subjected to RT-PCR for α3, β1, and GAPDH with specific primers. B). The expression levels of protein and RNA were quantified using image J software. C) IOMM-Lee and CH-157-MN cells (1×10⁵) were seeded in 8-µm pore size transwell inserts and treated with α3β1 antibody (10 µg/mL) one hour preceding the radiation treatment and allowed to migrate for 24 hrs. Cells that remained in the upper chamber and those that attached to the other side were fixed, stained and counted under a light microscope. The percent of migrated cells was calculated. D) IOMM-Lee and CH-157-MN cells (1×10⁵) cells were seeded in 6-well plates, irradiated and incubated for 24 hrs. The cell lysates were subjected to immunoprecipitation and followed by western blot analysis with the anti beta 1 and alpha 3 integrin antibodies. Values are mean ± S.D. from three independent experiments. * Statistically different (P<0.01)

Figure 3. F-actin and α3 integrin localized to the leading edge in migrating cells

Fluorescent micrograph of irradiated (7 Gy) IOMM-Lee and CH-157-MN cells that were grown for 24 hrs and stained with TRTc-labeled phalloidin and anti-α3 integrin antibody to mark localization. A) Pink fluorescence indicates where F-actin localized in the cell. B) Green fluorescence indicates the localization of α3 integrin, which is enriched at the leading front of migrating cells and in focal adhesions at the membrane edge of the extending lamellipodium. DAPI was used for nuclear staining. Images are representative pictures of several cells. Arrow indicates direction of cell movement.

Figure 4. FAK transduces signals in migrating cells

A) Immunoblot blot analysis of IOMM-Lee and CH-157-MN cell lysates treated with radiation (7 Gy). We checked for FAK and pFAK (Tyr 397), and GAPDH served as a loading control. B) The relative phosphorylation was quantified. C) IOMM-Lee and CH-157-MN cells (1×10⁵) cells were seeded in 8-µm pore size transwell inserts, irradiated, treated with FAK inhibitor as indicated, and allowed to migrate for 24 hrs. Cells that remained in the upper chamber and those that attached to the other side were fixed, stained and counted under a light microscope. The percent of migrated cells was calculated. Western blotting analysis of IOMM-Lee and CH-157-MN cell lysates treated with 10 µM FAK phosphorylation inhibitor followed by irradiation (7 Gy). GAPDH served as a loading control. C) Western blotting analysis of the IOMM-Lee and CH-157-MN cell lysates transfected with FAK shRNA for 24 hrs and treated with 7 Gy. GAPDH served as a loading control. All blots are representatives of three independent experiments. Values are mean ± S.D. from three independent experiments. * Statistically different (P<0.01)

Figure 5. ERK acts as a downstream effector of FAK

A) IOMM-Lee and CH-157-MN cells (1×10³) seeded in 2-well chamber slides were treated with FAK phosphorylation inhibitor (10 µM), subsequently irradiated (7 Gy) and allowed to grow for 24 hrs. Double immunostaining for co-localization was conducted with anti-FAK and anti-pERK antibodies followed by the secondary antibodies conjugated with fluorophores for green and red fluorescence respectively. Representative merged fluorescent microscopy (yellow) images of cells expressing FAK and pERK are shown. DAPI was used for the nuclear staining. Arrow indicates the direction of cell movement. B) IOMM-Lee and CH-157-MN cells (1×10⁵) were seeded in 6-well plates, irradiated and treated with U0126 inhibitor (10 µM). Cells were trypsinized and seeded at 1×10⁴ cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. C) Immunoblot analysis of the IOMM-Lee and CH-157-MN cell lysates treated with U0126 (10 µM) and radiation (7 Gy). GAPDH served as a loading control. All blots are representative of three independent experiments. D) Quantification of pERK levels in the inhibitor and radiation-treated cells. Values are mean ± S.D. from three independent experiments. * Statistically different (P<0.05)

Figure 6. Migration in vivo

IOMM-Lee and CH-157-MN cells were implanted intracranially in nude mice and treated with radiation as described in Materials and Methods. A total of 5 animals were studied in each group and 3 weeks after radiation treatment, animals were perfused and the brains were harvested and processed. A) Immunohistochemical analysis for α3 integrin and pERK in the paraffin-embedded sections of intracranial tumors implanted with IOMM-Lee and CH-157-MN cells. Slides were counterstained with hematoxylin for nuclear staining (40X). B) Tissue sections were stained with H&E as per standard protocol, and a representative tumor volume is shown (20X). C) IOMM-Lee and CH-157-MN luciferase-expressing stable cells (1×10⁵) were implanted into nude mice (4–6 weeks old). The first group was treated with cells irradiated at 7 Gy and the second group was infused with non-irradiated cells. Tumor progression was followed for one week with daily in vivo imaging. D) The luciferase activity from both groups of animals was quantified as photons per second. Values are mean ± S.D (n=5)