LATS2 inhibits cell proliferation and metastasis through the Hippo signaling pathway in glioma

Abstract

As a core kinase in the Hippo pathway, large tumor suppressor kinase 2 (LATS2) regulates cell proliferation, migration and invasion through numerous signaling pathways. However, its functions on cell proliferation, migration and invasion in glioma have yet to be elucidated. The present study revealed that LATS2 was downregulated in glioma tissues and cells, as determined by reverse transcription‑quantitative polymerase chain reaction and immunohistochemistry. In addition, Cell Counting Kit‑8, scratch wound healing and Transwell assays revealed that overexpression of LATS2 in U‑372 MG cells inhibited cell proliferation, migration and invasion. Furthermore, western blot analysis indicated that the expression levels of phosphorylated (p)‑yes‑associated protein and p‑tafazzin were increased in cells with LATS2 overexpression. These results indicated that LATS2 is a potential tumor suppressor, and downregulation of LATS2 in glioma may contribute to cancer progression.

Figure 1.

LATS2 was downregulated in glioma. (A) Expression levels of LATS2 were significantly lower in astrocytomas, oligodendrogliomas and glioblastomas compared with in normal tissues, as detected by reverse transcription-quantitative polymerase chain reaction. (B) Representative immunohistochemistry images (magnification, ×200) of LATS2 in astrocytomas, oligodendrogliomas and primary glioblastomas. (C) Number of positive cells in immunohistochemistry was calculated. (D) mRNA expression levels of LATS2 in glioma cell lines (U-372 MG, LN-229, U-251 MG and A172) were lower than in the normal glial cell line (HEB); U-372 MG cells had the lowest expression of LATS2. Data are presented as the means ± standard deviation. **P<0.01; ***P<0.001 compared with the normal group or HEB cells. LATS2, large tumor suppressor kinase 2.

Figure 2.

Overexpression of LATS2 suppresses cell proliferation by delaying G₁/S transition in U-372 MG cells. (A) Efficiency of LATS2 plasmid and siLATS2 transfection was validated in U-372 MG cells. The mRNA and protein expression levels of LATS2 were significantly upregulated or downregulated in cells transfected with plasmids or siRNA, respectively. Empty vector or NC siRNA were used as controls. (B) Effects of LATS2 on cell growth were determined by Cell Counting Kit-8 assay. OD₄₅₀ was measured at 0, 24, 48 and 72 h post-transfection. Overexpression of LATS2 decreased cell growth (left), whereas LATS2 knockdown increased cell growth (right). (C) Effects of LATS2 on cell cycle progression were measured by flow cytometry using propidium iodide. Overexpression of LATS2 increased the percentage of cells in the G₁ phase and decreased the number of cells in the S phase compared with the control, whereas knockdown of LATS2 had the opposite effects; G₂/M phase was not affected. (D) Protein expression levels of cyclin D, CDK4 and CDK6 were determined by western blotting and β-actin was used as an internal reference. Expression levels of these proteins were decreased in LATS2-overexpressed cells and were increased in LATS2 knockdown cells. All data are presented as the means ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001 compared with control or siNC groups. CDK, cyclin-dependent kinase; LATS2, large tumor suppressor kinase 2; OD, optical density; NC, negative control; si/siRNA, small interfering RNA.

Figure 3.

LATS2 suppressed cell migration and invasion. (A) Scratch wound healing assay was performed in U-372 MG cells transfected with LATS2 (left) or siLATS2 (right) for 24 h. Wound repair rate was inversely related to the expression levels of LATS2. Representative images (magnification, ×200) of wound closure at 0 and 24 h are shown. Black dotted lines indicate initial injury at 0 h and injury closure at 24 h. (B) Cell migration was quantified as a percentage of the healed area. (C) Transwell invasion assay was performed in U-372 MG cells transfected with LATS2 (left) or siLATS2 (right). Representative images (magnification, ×400) of invasive cells are shown. The number of invasive cells was counted and was inversely related to the expression levels of LATS2. All data are presented as the means ± standard deviation (n=3). **P<0.01; ***P<0.001 compared with control or siNC groups. Each assay was performed in triplicate. LATS2, large tumor suppressor kinase 2; NC, negative control; si/siRNA, small interfering RNA.

Figure 4.

LATS2 regulates YAP and TAZ by phosphorylation and subcellular localization. (A) Levels of p- and total YAP and TAZ were determined by western blotting; β-actin was used as an internal reference. Overexpression (left) increased the phosphorylation of YAP and TAZ, whereas knockdown (right) of LATS2 decreased the phosphorylation of YAP and TAZ; no effects were detected on total protein levels. (B) Subcellular localization of YAP and TAZ was determined by immunofluorescence (magnification, ×800). YAP and TAZ [Alexa Fluor^® 488-conjugated secondary antibody (top)], nuclei [DAPI (middle)] and merged images (bottom) are presented. YAP and TAZ were translocated from the nucleus to the cytoplasm in cells with LATS2 overexpression. LATS2, large tumor suppressor kinase 2; NC, negative control; p, phosphorylated; si/siRNA, small interfering RNA; TAZ, tafazzin; YAP, yes-associated protein.

Figure 5.

YAP S127A mutant abolished the effects of LATS2 on cell proliferation, migration and invasion. (A) Expression levels of p- and total YAP were determined in wild-type YAP- and YAP S127A-transfected cells by western blotting. β-actin was used as an internal reference. Total levels of YAP were increased in both wild-type YAP- and YAP S127A-transfected cells compared with the control; however, p-YAP was only increased in wild-type YAP-transfected cells. (B) YAP S127A abolished LATS2-induced p-YAP upregulation. Expression levels of LATS2, total YAP and p-YAP were determined by western blotting. β-actin was used as an internal reference. (C) YAP S127A abolished LATS2-induced suppression of cell proliferation. Growth curves were plotted with the results of the Cell Counting Kit-8 assay, and OD₄₅₀ was measured at 24, 48 and 72 h. Cell growth was restored by cotransfection of cells with LATS2 and YAP S127A. (D) YAP S127A restored the migration of the LATS2-transfected cells. Cell migration in each group was determined using the scratch wound healing assay; representative images (magnification, ×200) are shown. Following cotransfection with YAP S127A and LATS2, cells had a similar wound repair rate to the control group, whereas transfection with LATS2 alone resulted in a significantly lower repair rate compared with cells cotransfected with LATS2 and YAP S127A. (E) Overexpression of YAP S127A restored the invasive ability of LATS2-transfected cells. Cell invasion in each group was determined by transwell invasion assay and the number of invasive cells was counted (magnification, ×800). Cells cotransfected with YAP S127A and LATS2 had a similar number of invasive cells to the control group, whereas transfection with LATS2 alone resulted in significantly fewer invasive cells compared with the control group. All data are presented as the means ± standard deviation (n=3). ***P<0.001 compared with cells cotransfected with LATS2 and YAP S127A. Each assay was performed in triplicate. LATS2, large tumor suppressor kinase 2; p, phosphorylated; si/siRNA, small interfering RNA; YAP, yes-associated protein.