肿瘤转移抑制基因LASS2去磷酸化对液泡型ATP酶活性及前列腺癌侵袭性的影响

doi:10.19723/j.issn.1671-167X.2025.06.015

摘要/Abstract

摘要：

目的: 探讨人源酵母LAG1长寿同源物2(homo sapiens longevity assurance homolog 2 of yeast LAG1, LASS2)去磷酸化对前列腺癌细胞生物学功能的影响及其分子机制。方法: 对90例前列腺癌患者的石蜡组织芯片标本进行LASS2免疫组织化学染色, 并构建FLAG标签的LASS2全长pcDNA3真核表达载体, 转染至HEK 293T细胞后行免疫共沉淀和蛋白质谱检测, 分析LASS2的磷酸化位点。根据LASS2磷酸化位点结果构建LASS2 C末端5个去磷酸化突变体的pcDNA3真核表达载体, 并稳定转染到高转移潜能前列腺癌PC-3M-1E8细胞系中, 通过生长曲线、MTT掺入实验、平板克隆形成实验、细胞划痕实验、Transwell侵袭实验和流式细胞术检测LASS2及其去磷酸化突变体对前列腺癌细胞生物学功能的影响。检测LASS2及其突变体对液泡型ATP酶V0复合体中c亚基(ATP6V0C)表达量及两者相互结合的影响, 以及其对液泡型ATP酶活性、细胞外H⁺浓度和基质金属蛋白酶2(matrix metalloproteinase 2, MMP-2)活性的影响, 并探索蛋白磷酸酶抑制剂calyculin A对前列腺癌细胞增殖、迁移和侵袭能力的影响。结果: 免疫组织化学染色显示前列腺癌组织中LASS2的表达与Gleason分级呈负相关; 质谱分析发现LASS2的C末端存在3个磷酸化位点(Ser-341、Ser-348和Ser-349);与野生型LASS2相比, LASS2 S348位点的去磷酸化突变体(LASS2-S348A)能显著增强前列腺癌细胞的增殖能力、锚着不依赖生长能力、细胞迁移能力(细胞迁移率从49.11%±5.62%增加到74.28%±8.77%, P < 0.001)和侵袭能力(穿膜细胞数从129.67±13.65增加到206.67±13.50, P < 0.001), 并显著降低S期比例(从44.17%降低到37.90%, P < 0.05)和细胞凋亡率(从48.540%±0.269%降低到29.700%±0.778%, P < 0.05)。LASS2-S348A去磷酸化突变体虽然不影响ATP6V0C的蛋白表达量, 但会明显抑制LASS2与ATP6V0C的结合能力, 并进而显著增强液泡型ATP酶活性和细胞外H⁺浓度, 最终促进MMP-2的活性; 而蛋白磷酸酶抑制剂calyculin A能够显著抑制前列腺癌细胞的增殖、迁移和侵袭。结论: LASS2蛋白C末端S348位点的磷酸化对于LASS2的肿瘤抑制功能至关重要, 其分子机制可能是LASS2 S348位点的磷酸化通过增强其与ATP6V0C的结合而抑制液泡型ATP酶活性, 进而降低细胞外H⁺浓度和抑制MMP-2的活性, 并最终抑制前列腺癌细胞的侵袭; 蛋白磷酸酶抑制剂calyculin A有望成为侵袭性前列腺癌潜在的治疗药物。

关键词: 前列腺肿瘤, 肿瘤浸润, 基因表达调控, 肿瘤, 空泡质子转运ATP酶, LASS2基因

Abstract:

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.

Key words: Prostatic neoplasms, Neoplasm invasiveness, Gene expression regulation, neoplastic, Vacuolar proton-translocating ATPases, LASS2 gene

中图分类号:

R737.25

刘艳华, 陆敏, 赵旭阳, 张宽根, 武睿, 梅放, 戴志豪, 由江峰, 裴斐. 肿瘤转移抑制基因LASS2去磷酸化对液泡型ATP酶活性及前列腺癌侵袭性的影响[J]. 北京大学学报（医学版）, 2025, 57(6): 1113-1123.

Yanhua LIU, Min LU, Xuyang ZHAO, Kuan'gen ZHANG, Rui WU, Fang MEI, Zhihao DAI, Jiangfeng YOU, Fei PEI. Effect of dephosphorylation of tumor metastasis suppressor gene LASS2 on vacuolar ATPase activity and invasiveness of prostate cancer[J]. Journal of Peking University (Health Sciences), 2025, 57(6): 1113-1123.

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Gene	Primer
LASS2	Forward: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTCCAGACCTTGTATGATTA-3′
	Reverse: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTAGTCATTCTTACGATGGTTGT-3′
LASS2-T332A	Forward: 5′-TACCAGCTTTCCAGCTATGAACTTGTGGGCCATG-3′
	Reverse: 5′-CATGGCCCACAAGTTCATAGCTGGAAAGCTGGTA-3′
LASS2-S341A	Forward: 5′-TCTGTTTCTTCCCGGTCAGCGCGTTCATCTTCTACCAG-3′
	Reverse: 5′-CTGGTAGAAGATGAACGCGCTGACCGGGAAGAAACAGA-3′
LASS2-T346A	Forward: 5′-CTCTGAGCTCTCTGCTTCTTCCCGGTCACTGC-3′
	Reverse: 5′-GCAGTGACCGGGAAGAAGCAGAGAGCTCAGAG-3′
LASS2-S348A	Forward: 5′-TCCTCCCCCTCTGAGGCCTCTGTTTCTTCCCG-3′
	Reverse: 5′-CGGGAAGAAACAGAGGCCTCAGAGGGGGAGGA-3′
LASS2-S349A	Forward: 5′-CCTCCTCCCCCTCTGCGCTCTCTGTTTCTTC-3′
	Reverse: 5′-GAAGAAACAGAGAGCGCAGAGGGGGAGGAGG-3′