Cancer Res Treat.  2013 Jun;45(2):134-144.

RASSF1A Suppresses Cell Migration through Inactivation of HDAC6 and Increase of Acetylated alpha-Tubulin

• Jung HY
• Jung JS
• Whang YM
• Kim YH

Abstract

PURPOSE
The RAS association domain family protein 1 (RASSF1) has been implicated in a tumor-suppressive function through the induction of acetylated alpha-tubulin and modulation of cell migration. However, the mechanisms of how RASSF1A is associated with acetylation of alpha-tubulin for controlling cell migration have not yet been elucidated. In this study, we found that RASSF1A regulated cell migration through the regulation of histon deacetylase 6 (HDAC6), which functions as a tubulin deacetylase.
MATERIALS AND METHODS
The cell migration was assessed using wound-healing and transwell assays. The role of RASSF1A on cell migration was examined by immunofluorescence staining, HDAC activity assay and western blot analysis.
RESULTS
Cell migration was inhibited and cell morphology was changed in RASSF1A-transfected H1299 cells, compared with controls, whereas HDAC6 protein expression was not changed by RASSF1A transfection in these cells. However, RASSF1A inhibited deacetylating activity of HDAC6 protein and induced acetylated alpha-tubulin expression. Furthermore, acetylated alpha-tubulin and HDAC6 protein were co-localized in the cytoplasm in RASSF1A-transfected H1299 cells. Conversely, when the endogenous RASSF1A expression in HeLa cells was blocked with RASSF1A siRNA treatment, acetylated alpha-tubulin was co-localized with HDAC6 protein throughout the whole cells, including the nucleus, compared with scramble siRNA-treated HeLa cells. The restoration of RASSF1A by 5-Aza-dC treatment also induced acetylated alpha-tubulin through inhibition of HDAC6 activity that finally resulted in suppressing cell migration in H1299 cells. To further confirm the role of HDAC6 in RASSF1A-mediated cell migration, the HDAC6 expression in H1299 cells was suppressed by using HDAC6 siRNA, and cell motility was found to be decreased through enhanced acetylated alpha-tubulin.
CONCLUSION
The results of this study suggest that the inactivation of HDAC6 by RASSF1A regulates cell migration through increased acetylated alpha-tubulin protein.

Keyword

Cell movement; HDAC6; Lung neoplasms; RASSF1A; Tumor suppressor genes

MeSH Terms

Acetylation
Blotting, Western
Cell Movement
Cytoplasm
Fluorescent Antibody Technique
Genes, Tumor Suppressor
HeLa Cells
Humans
Lung Neoplasms
RNA, Small Interfering
Transfection
Tubulin
RNA, Small Interfering
Tubulin

Figure

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  Fig. 1 The effects of RASSF1A on cell morphology and migration. (A) H1299 cells stably transfected with RASSF1A or pcDNA3. RASSF1A expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western bolt analysis. GAPDH and β-actin served as a loading control. (B) RASSF1A stable transfected H1299 cells and control cells were photographed before the use for the migration assay at 20× magnification. (C) The results of wound healing assay. Cells were grown to confluence, and the wound was made by scraping the cell monolayer with a pipette tip and left for 24 to 72 hours before photographing (D) the results of transwell assay. Cells were plated at 2×104 cells per upper chamber together, along with serum free medium and 10% fetal bovine serum placed in the low chamber. The cells were then allowed to migrate for 48 hours. The membrane inserts were stained with crystal violet stain and photographed at 10× magnification. Quantification of relative migration of RASSF1A stably transected H1299 cells in relation to vector control cells (p<0.05).

• 

  Fig. 2 The effects of RASSF1A on acetylated α-tubulin and histon deacetylase 6 (HDAC6) protein. (A) The expression of RASSF1A, HDAC6 and acetylated α-tubulin (Ac α-tubulin) protein, analyzed by western blot analysis. (B) The result of deacetylating activity of HDAC6 protein. Total cell lysates (200 µg) were immunoprecipitated with HDAC6 antibody, and it was then subjected to HDAC activity assay. Columns, mean absorbance value of three independent experiments; bars, standard error; p<0.05.

• 

  Fig. 3 The effects of RASSF1A on cellular localization of histon deacetylase 6 (HDAC6) and acetylated α-tubulin (Ac α-tubulin) protein. (A) The results of co-localization of acetylated α-tubulin and HDAC6 protein. Double-immunostaining with an anti-acetylated α-tubulin antibody (green) and anti-HDAC6 antibody (red) in RASSF1A-transfected H1299 cells and control H1299 cells. (B) Co-localization of RASSF1A with HDAC6 protein in cytoplasm. H1299 cells were transiently transfected with expression vectors carrying GFP-alone or GFP-tagged RASSF1A and immunostained with an anti-HDAC6 primary antibody followed by red fluorescence dye conjugated anti-rabbit secondary antibody. (C) The results on cytoplasmic and nuclear extract from RASSF1A-transfected H1299 cells and control cells. Cells were prepared and the proteins indicated were analyzed by western blot analysis.

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  Fig. 4 The effects of RASSF1A siRNA on cellular localization of histon deacetylase 6 (HDAC6) and acetylated α-tubulin (Ac α-tubulin). (A) HeLa cells, which display endogenous RASSF1A expression, were transiently transfected with RASSF1A siRNA. RASSF1A and Ac α-tubulin expressions were analyzed by western blot analysis. β-Actin served as a loading control. (B) The effects of RASSF1A knockdown on co-localization of acetylated α-tubulin and HDAC6 protein in HeLa cells.

• 

  Fig. 5 The effects of restoration of RASSF1A on deacetylating activity of histon deacetylase 6 (HDAC6) and cell migration in parent H1299 cells. (A) The restoration of RASSF1AmRNA expression in H1299 cells by 5-Aza-dC treatment was analyzed by reverse transcription polymerase chain reaction. GAPDH served as a loading control. (B) After H1299 cells were treated with 5-Aza-dC, the expressions of RASSF1A, HDAC6, and acetylated α-tubulin (Ac α-tubulin) protein were analyzed by western blot analysis. β-Actin served as a loading control. (C) The results of deacetylating activity of HDAC6 protein. Columns, mean absorbance value of three independent experiments; bars, standard error; p<0.05. (D) The results of wound-healing assay after the restoration of RASSF1A expression by 5-Aza-dC treatment. Cells were grown to confluence, and the wound was made by scraping the cell monolayer with a pipette tip and left for 48 to 72 hours before photographing.

• 

  Fig. 6 The effects of histon deacetylase 6 (HDAC6) siRNA on cell migration. (A) Control H1299 cells were treated with HDAC6 siRNA. The expressions of HDAC6, acetylated α-tubulin (Ac α-tubulin), and RASSF1A were analyzed by western blot analysis. β-Actin served as a loading control. (B) The results of deacetylating activity of HDAC6 protein. Columns, mean absorbance value of three independent experiments; bars, standard error; p<0.05. (C) The results of wound-healing assay. RASSF1A-transfected H1299 cells and control cells were transiently transfected with HDAC6 siRNA. Cells were grown to confluence, and the wound was made by scraping the cell monolayer with a pipette tip and left for 48 hours before photographing.

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