[Effect of dephosphorylation of tumor metastasis suppressor gene LASS2 on vacuolar ATPase activity and invasiveness of prostate cancer].

OBJECTIVE

To explore the effects and the molecular mechanisms of homo sapiens longevity assurance homolog 2 of yeast LAG1(LASS2) dephosphorylation on the biological functions of prostate cancer cells.

METHODS

Firstly, we examined the expression profiles of LASS2 by immunohistochemical staining using microarray sections from 90 human patients with prostate cancer; then FLAG-tagged LASS2 plasmid was transferred into HEK 293T cells and phosphorylation sites was detected by mass spectrometry. Furthermore, we constructed five phosphorylation-deficient mutants of LASS2 and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. The cell biology functions of LASS2 and its five mutants were studied using growth curve, MTT assay, plate colony formation assay, wound migration assay, matrigel invasion study and flow cytometry; and the effect of LASS2 and its phosphorylation-deficient mutants on the physical interaction between LASS2 and ATP6V0C (C subunit of V0 domain of the vacuolar ATPase), ATP6V0C expression, vacuolar ATPase (V-ATPase) activity, extracellular hydrogen ion concentration and secretion of active matrix metalloproteinase 2(MMP-2) was detected. Finally, we examined the effect of protein phosphatase inhibitor calyculin A on growth, migration and invasion of aggressive prostate cancer cells.

RESULTS

LASS2 levels decreased with increasing Gleason scores of prostate cancer tissues by immunohistochemical staining; moreover, proteome analysis by mass spectrometry had identified that three residues in the C-terminal region of LASS2(Ser-341, Ser-348, and Ser-349) were phosphorylated. Dephosphorylation of LASS2 at serine residue 348 significantly enhanced growth, migration (from 49.11%±5.62% to 74.28%±8.77%, P < 0.001) and invasion (from 129.67±13.65 to 206.67±13.50, P < 0.001) of prostate cancer cells, decreased S phase arrest (from 44.17% to 37.90%, P < 0.05) and inhibited cell apoptosis (from 48.540%±0.269% to 29.700%±0.778%, P < 0.05) in vivo through increasing V-ATPase activity, extracellular hydrogen ion concentration and secretion of active MMP-2. Calyculin A significantly reduced growth and invasion of metastatic human prostate cancer cells.

CONCLUSION

Phosphorylation of LASS2 is essential for regulation of V-ATPase activity, and serine residue 348 of LASS2 is illustrated to be a key phosphorylation site. Phosphorylated LASS2 inhibits prostate cancer cell invasion via negative regulation of V-ATPase activity and protein phosphatase inhibitors are potential therapeutic strategy in aggressive prostate cancer.