Email Alert | RSS

Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (6): 1113-1123. doi: 10.19723/j.issn.1671-167X.2025.06.015

Previous Articles     Next Articles

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

Yanhua LIU1, Min LU1, Xuyang ZHAO2, Kuan'gen ZHANG1, Rui WU1, Fang MEI1, Zhihao DAI1, Jiangfeng YOU1, Fei PEI1,*()   

  1. 1. Department of Pathology, School of Basic Medical Sciences Peking University / Peking University Third Hospital, Beijing 100191, China
    2. Peking University Institute of Systems Biomedicine, Beijing 100191, China
  • Received:2023-05-22 Online:2025-12-18 Published:2025-03-17
  • Contact: Fei PEI
  • Supported by:
    the National Natural Science Foundation of China(81572533); Beijing Natural Sciences Foundation(7182078)

RICH HTML

   

PDF (PC)

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

CLC Number: 

  • R737.25

Table 1

Nucleotide sequences of primers used in plasmid construct"

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′

Figure 1

Expression level of LASS2 in prostate cancer and adjacent tissue A, immunohistochemical staining of LASS2 in 90 prostate carcinoma samples with paired adjacent normal tissues. Representative sections from prostate cancer (Gleason score 6, 7, 8, 9) or adjacent normal tissue stained with HE and LASS2 antibody are presented (×200). B, LASS2 levels was significantly downregulated in prostate cancer tissues compared with paired adjacent normal tissues (P < 0.01); C, LASS2 levels decreased with increasing Gleason scores of prostate cancer tissues (P < 0.01). HE, hematoxylin-eosin; LASS2, homo sapiens longevity assurance homolog 2 of yeast LAG1."

Figure 2

Serine phosphorylation site at the C-terminus of LASS2 A, protein analysis using PROSITE (http://prosite.expasy.org/) predicted that LASS2 contained seven protein serine phosphorylation sites, and these phosphorylation sites are highly conserved among human, mouse and rat; B, FLAG-LASS2 was purified from transfected HEK 293T cells in the presence of phosphatase inhibitor and subjected to mass spectrometric analysis. Phosphoproteomic study has identified three serine phosphorylation sites in the C-terminal of LASS2. LASS2, homo sapiens longevity assurance homolog 2 of yeast LAG1."

Figure 3

Effects of LASS2-WT and its mutants on the growth, migration, and invasion ability of prostate cancer cell line PC-3M-1E8 A, growth curve assay and MTT test (n=3); B, cell cloning experiment (n=3); C, cell migration experiment (n=4); D, cell invasion experiment (n=3); E, cell cycle experiment; F, cell apoptosis experiment (n=3). These figures showed dephosphorylation of LASS2 at serine residue 348 promote growth, migration and invasion of PC-3M-1E8 cells. LASS2, homo sapiens longevity assurance homolog 2 of yeast LAG1; *P < 0.05, * *P < 0.01, * * *P < 0.001 vs. LASS2-WT."

Figure 4

Effects of LASS2-WT and its mutants on V-ATPase activity and extracellular H+ concentration in prostate cancer cell line PC-3M-1E8 A, Western blotting; B, co-immunoprecipitation; C, GST pull-down; D, V-ATPase activity detection experiment (n=3); E, extracellular H+ detection experiment (n=6); F, gelatin enzyme spectrum experiment. These figures showed that LASS2-S348A dephosphorylation mutant enhances V-ATPase activity and extracellular H+ concentration. LASS2, homo sapiens longevity assurance homolog 2 of yeast LAG1; WT, wild type; V-ATPase, vacuolar ATPase; ATP6V0C, C subunit of V0 domain of the V-ATPase; GST, glutathione-S-transferase; MMP, matrix metalloproteinase. *P < 0.05, * *P < 0.01, * * *P < 0.001."

Figure 5

Effects of calyculin A or taxol on proliferation, migration, and invasion of prostate cancer cell line PC-3M-1E8 A, growth curve assay; B, MTT test; C, cell migration experiment (n=8); D, cell invasion experiment (n=3). Dimethyl sulfoxide (DMSO) was the negative control. These figures showed that calyculin A significantly inhibits the proliferation, migration, and invasion of PC-3M-1E8 cells. *P < 0.05, * * *P < 0.001, vs. LASS2-WT."

1
Siegel RL , Miller KD , Fuchs HE , et al. Cancer statistics, 2022[J]. CA Cancer J Clin, 2022, 72 (1): 7- 33.
2
Feng RM , Zong YN , Cao SM , et al. Current cancer situation in China: Good or bad news from the 2018 Global Cancer Statistics?[J]. Cancer Commun (Lond), 2019, 39 (1): 22.
3
Ma C , Liu Y , Zheng J , et al. Identification of tumor metastasisrelated gene TMSG-1 by mRNA differential display[J]. Sci China C Life Sci, 2002, 45 (5): 553- 560.

doi: 10.1360/02yc9061
4
Pan H , Qin WX , Huo KK , et al. Cloning, mapping, and characterization of a human homologue of the yeast longevity assurance gene LAG1[J]. Genomics, 2001, 77 (1/2): 58- 64.
5
Laviad EL , Albee L , Pankova-Kholmyansky I , et al. Characte-rization of ceramide synthase 2: Tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate[J]. J Biol Chem, 2008, 283 (9): 5677- 5684.

doi: 10.1074/jbc.M707386200
6
Tidhar R , Ben-Dor S , Wang E , et al. Acyl chain specificity of ceramide synthases is determined within a region of 150 residues in the Tram-Lag-CLN8 (TLC) domain[J]. J Biol Chem, 2012, 287 (5): 3197- 3206.

doi: 10.1074/jbc.M111.280271
7
Zhang K , Wu R , Mei F , et al. Phosphorylated LASS2 inhibits prostate carcinogenesis via negative regulation of Wnt/β-catenin signaling[J]. J Cell Biochem, 2021, 122 (9): 1048- 1061.

doi: 10.1002/jcb.29926
8
Sassa T , Hirayama T , Kihara A . Enzyme activities of the ceramide synthases CERS2-6 are regulated by phosphorylation in the C-terminal region[J]. J Biol Chem, 2016, 291 (14): 7477- 7487.

doi: 10.1074/jbc.M115.695858
9
Winter E , Ponting CP . TRAM, LAG1 and CLN8: Members of a novel family of lipid-sensing domains?[J]. Trends Biochem Sci, 2002, 27 (8): 381- 383.

doi: 10.1016/S0968-0004(02)02154-0
10
Spassieva S , Seo JG , Jiang JC , et al. Necessary role for the Lag1p motif in (dihydro) ceramide synthase activity[J]. J Biol Chem, 2006, 281 (45): 33931- 33938.

doi: 10.1074/jbc.M608092200
11
Abbas YM , Wu D , Bueler SA , et al. Structure of V-ATPase from the mammalian brain[J]. Science, 2020, 367 (6483): 1240- 1246.

doi: 10.1126/science.aaz2924
12
Bott LC , Forouhan M , Lieto M , et al. Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy[J]. Brain Commun, 2021, 3 (4): fcab245.

doi: 10.1093/braincomms/fcab245
13
徐晓艳, 由江峰, 裴斐, 等. RNA沉默LASS2基因促进人前列腺癌细胞体外侵袭及其机制研究[J]. 北京大学学报(医学版), 2011, 43 (6): 814- 819.

doi: 10.3969/j.issn.1671-167X.2011.06.006
14
Xu X , Liu B , Zou P , et al. Silencing of LASS2/TMSG1 enhances invasion and metastasis capacity of prostate cancer cell[J]. J Cell Biochem, 2014, 115 (4): 731- 743.

doi: 10.1002/jcb.24716
15
Zou P , Yang Y , Xu X , et al. Silencing of vacuolar ATPase c subunit ATP6V0C inhibits the invasion of prostate cancer cells through a LASS2/TMSG1-independent manner[J]. Oncol Rep, 2018, 39 (1): 298- 306.
16
Cotter K , Capecci J , Sennoune S , et al. Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells[J]. J Biol Chem, 2015, 290 (6): 3680- 3692.

doi: 10.1074/jbc.M114.611210
17
Wang J , Chen D , Song W , et al. ATP6L promotes metastasis of colorectal cancer by inducing epithelial-mesenchymal transition[J]. Cancer Sci, 2020, 111 (2): 477- 488.

doi: 10.1111/cas.14283
18
Yu W , Wang L , Wang Y , et al. A novel tumor metastasis suppressor gene LASS2/TMSG1 interacts with vacuolar ATPase through its homeodomain[J]. J Cell Biochem, 2013, 114 (3): 570- 583.

doi: 10.1002/jcb.24400
19
张宽根, 周雨禾, 邵雅昆, 等. 肿瘤转移抑制基因LASS2/TMSG1 S248A突变体通过增加ATP6V0C表达促进前列腺癌的侵袭[J]. 北京大学学报(医学版), 2019, 51 (2): 210- 220.

doi: 10.19723/j.issn.1671-167X.2019.02.003
20
Cirak Y , Varol U , Atmaca H , et al. Zoledronic acid in combination with serine/threonine phosphatase inhibitors induces enhanced cytotoxicity and apoptosis in hormone-refractory prostate cancer cell lines by decreasing the activities of PP1 and PP2A[J]. BJU Int, 2012, 110 (11 Pt C): E1147- E1154.
[1] Xiaoyong YANG, Fan ZHANG, Lulin MA, Cheng LIU. Clinical characteristics and influencing factors of extraglandular invasion of prostatic ductal adenocarcinoma [J]. Journal of Peking University (Health Sciences), 2025, 57(5): 956-960.
[2] Jiaxin NING, Haoran WANG, Shuhang LUO, Jibo JING, Jianye WANG, Huimin HOU, Ming LIU. Multi-omics analysis of the relationship between oxidative stress-related gene and prostate cancer [J]. Journal of Peking University (Health Sciences), 2025, 57(4): 633-643.
[3] Zeyuan WANG, Shuanbao YU, Haoke ZHENG, Jin TAO, Yafeng FAN, Xuepei ZHANG. A preoperative prediction model for pelvic lymph node metastasis in prostate cancer: Integrating clinical characteristics and multiparametric MRI [J]. Journal of Peking University (Health Sciences), 2025, 57(4): 684-691.
[4] Zhicun LI, Tianyu WU, Lei LIANG, Yu FAN, Yisen MENG, Qian ZHANG. Risk factors analysis and nomogram model construction of postoperative pathological upgrade of prostate cancer patients with single core positive biopsy [J]. Journal of Peking University (Health Sciences), 2024, 56(5): 896-901.
[5] Yuxuan TIAN,Mingjian RUAN,Yi LIU,Derun LI,Jingyun WU,Qi SHEN,Yu FAN,Jie JIN. Predictive effect of the dual-parametric MRI modified maximum diameter of the lesions with PI-RADS 4 and 5 on the clinically significant prostate cancer [J]. Journal of Peking University (Health Sciences), 2024, 56(4): 567-574.
[6] Kaifeng YAO,Mingjian RUAN,Derun LI,Yuxuan TIAN,Yuke CHEN,Yu FAN,Yi LIU. Diagnostic efficacy of targeted biopsy combined with regional systematic biopsy in prostate cancer in patients with PI-RADS 4-5 [J]. Journal of Peking University (Health Sciences), 2024, 56(4): 575-581.
[7] Junyong OU,Kunming NI,Lulin MA,Guoliang WANG,Ye YAN,Bin YANG,Gengwu LI,Haodong SONG,Min LU,Jianfei YE,Shudong ZHANG. Prognostic factors of patients with muscle invasive bladder cancer with intermediate-to-high risk prostate cancer [J]. Journal of Peking University (Health Sciences), 2024, 56(4): 582-588.
[8] Yi LIU,Chang-wei YUAN,Jing-yun WU,Qi SHEN,Jiang-xi XIAO,Zheng ZHAO,Xiao-ying WANG,Xue-song LI,Zhi-song HE,Li-qun ZHOU. Diagnostic efficacy of prostate cancer using targeted biopsy with 6-core systematic biopsy for patients with PI-RADS 5 [J]. Journal of Peking University (Health Sciences), 2023, 55(5): 812-817.
[9] Chang-wei YUAN,De-run LI,Zhi-hua LI,Yi LIU,Gang-zhi SHAN,Xue-song LI,Li-qun ZHOU. Application of dynamic contrast enhanced status in multiparametric magnetic resonance imaging for prostatic cancer with PI-RADS 4 lesion [J]. Journal of Peking University (Health Sciences), 2023, 55(5): 838-842.
[10] Han LU,Jian-yun ZHANG,Rong YANG,Le XU,Qing-xiang LI,Yu-xing GUO,Chuan-bin GUO. Clinical factors affecting the prognosis of lower gingival squamous cell carcinoma [J]. Journal of Peking University (Health Sciences), 2023, 55(4): 702-707.
[11] Yu TIAN,Xiao-yue CHENG,Hui-ying HE,Guo-liang WANG,Lu-lin MA. Clinical and pathological features of renal cell carcinoma with urinary tract tumor thrombus: 6 cases report and literature review [J]. Journal of Peking University (Health Sciences), 2021, 53(5): 928-932.
[12] SUN Zheng-hui,HUANG Xiao-juan,DONG Jing-han,LIU Zhuo,YAN Ye,LIU Cheng,MA Lu-lin. Risk factors of renal sinus invasion in clinical T1 renal cell carcinoma patients undergoing nephrectomy [J]. Journal of Peking University (Health Sciences), 2021, 53(4): 659-664.
[13] Yi LIU,Zhi-jian LIU,Qi SHEN,Jing-yun WU,Yu FAN,De-run LI,Wei YU,Zhi-song HE. A clinical analysis of 14 cases of prostatic stromal tumor of uncertain malignant potential [J]. Journal of Peking University (Health Sciences), 2020, 52(4): 621-624.
[14] Yi-chang HAO,Ye YAN,Fan ZHANG,Min QIU,Lang ZHOU,Ke LIU,Jian LU,Chun-lei XIAO,Yi HUANG,Cheng LIU,Lu-lin MA. Surgical strategy selection and experience summary of prostate cancer with positive single needle biopsy [J]. Journal of Peking University (Health Sciences), 2020, 52(4): 625-631.
[15] Ye YAN,Hai-zhui XIA,Xu-sheng LI,Wei HE,Xue-hua ZHU,Zhi-ying ZHANG,Chun-lei XIAO,Yu-qing LIU,Hua HUANG,Liang-hua HE,Jian LU. Application of U-shaped convolutional neural network in auto segmentation and reconstruction of 3D prostate model in laparoscopic prostatectomy navigation [J]. Journal of Peking University(Health Sciences), 2019, 51(3): 596-601.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Copyright © Journal of Peking University (Health Sciences), All Rights Reserved.
Powered by Beijing Magtech Co. Ltd