Upregulation of ATG4D promotes anoikis resistance of tumor cells by autophagy enhancement

MA Xiaozhuang; ZHANG Yueting; SUN Liangbo; YAN Xiaojing; YANG Mingzhen; LIAN Jiqin

doi:10.16016/j.2097-0927.202602096

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Upregulation of ATG4D promotes anoikis resistance of tumor cells by autophagy enhancement

Basic Medicine | 更新时间：2026-07-15

- Upregulation of ATG4D promotes anoikis resistance of tumor cells by autophagy enhancement
- Journal of Army Medical University Vol. 48, Issue 13, Pages: 1854-1862(2026)
- 作者机构：
  
  陆军军医大学（第三军医大学）药学与检验医学系临床生物化学教研室，重庆
- 作者简介：
  
  LIAN Jiqin， E-mail：lianjiqin@sina.com
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(82273114);the Key Project of Innovation and Development Joint Fund of Natural Science Foundation of Chongqing(CSTB2022NSCQ-LZX0018)
- DOI：10.16016/j.2097-0927.202602096
  CLC： R394.2;R73-37;R730.23
- Received：27 February 2026，
  
  Revised：2026-05-12，
  
  Accepted：17 May 2026，
  
  Published：15 July 2026
- 稿件说明：
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马晓壮，张越婷，孙梁博，等.肿瘤细胞通过上调ATG4D表达增强自噬而抵抗失巢凋亡［J］.陆军军医大学学报，2026，48（13）：1854-1862.DOI：10.16016/j.2097-0927.202602096.

MA X Z，ZHANG Y T，SUN L B，et al.Upregulation of ATG4D promotes anoikis resistance of tumor cells by autophagy enhancement［J］.J Army Med Univ，2026，48（13）：1854-1862.DOI：10.16016/j.2097-0927.202602096.
马晓壮，张越婷，孙梁博，等.肿瘤细胞通过上调ATG4D表达增强自噬而抵抗失巢凋亡［J］.陆军军医大学学报，2026，48（13）：1854-1862.DOI：10.16016/j.2097-0927.202602096. DOI：

MA X Z，ZHANG Y T，SUN L B，et al.Upregulation of ATG4D promotes anoikis resistance of tumor cells by autophagy enhancement［J］.J Army Med Univ，2026，48（13）：1854-1862.DOI：10.16016/j.2097-0927.202602096. DOI：

摘要

目的

失巢凋亡抵抗（anoikis resistance，AR）是转移性肿瘤细胞所具备的关键特征，而自噬水平增强则是引起AR的重要原因，自噬相关蛋白4D（autophagy related 4D，ATG4D）是调控细胞自噬的关键酶，本研究着重探讨肿瘤细胞是否通过上调ATG4D表达增强自噬而促进AR的作用及分子机制。

方法

将肝癌HepG2细胞和胃癌AGS细胞分为AR组和对照组。AR组用超低黏附细胞培养板悬浮培养48 h，对照组用常规细胞培养板贴壁培养。Western blotting检测自噬标志蛋白Ⅱ型微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3 Ⅱ， LC3-Ⅱ）与p62表达变化并进行定量分析。使用30 μg/mL氯喹（chloroquine，CQ）处理对照组或AR组肿瘤细胞抑制细胞自噬，然后使用凋亡检测试剂盒结合流式细胞术分析细胞凋亡水平变化。qRT-PCR结合Western blotting检测ATG4D的mRNA与蛋白水平变化。在AR肿瘤细胞中通过脂质体法转染对照siRNA或

ATG4D

siRNA，Western blotting检测ATG4D蛋白含量及自噬标志物LC3-Ⅱ与p62表达变化，使用凋亡检测试剂盒结合流式细胞术分析细胞凋亡水平变化。构建包含

ATG4D

基因启动子的报告质粒，将报告质粒分别转入对照组及AR组肿瘤细胞，24 h后使用荧光素酶报告基因实验检测

ATG4D

基因启动子活性变化。使用放线菌素D（5 μg/mL）处理对照组或AR组肿瘤细胞，分别于处理后0、2、4、6、8 h收取细胞，通过qRT-PCR检测

ATG4D

的mRNA在不同时间点水平变化。使用环乙酰亚胺（50 μg/mL）处理对照组或AR组肿瘤细胞，分别于处理后0、2、4、6、8 h收取细胞，通过Western blotting检测ATG4D蛋白在不同时间点水平变化。

结果

与对照组肿瘤细胞相比，AR组肿瘤细胞自噬水平显著增强，LC3-Ⅱ水平显著

升高（

0.01），p62水平明显降低（

0.01）。使用CQ抑制自噬后，AR+CQ处理组肿瘤细胞的凋亡水平显著增加（

0.01）。qRT-PCR及Western blotting检测发现，AR组肿瘤细胞中

ATG4D

mRNA与ATG4D蛋白水平较对照组均显著增强（

0.01）。使用

ATG4D

siRNA能够明显抑制

ATG4D

的表达，并下调LC3-Ⅱ表达水平、增强p62表达水平、促进AR肿瘤细胞凋亡（

0.01）。与对照组肿瘤细胞相比，AR组肿瘤细胞中

ATG4D

基因启动子活性并无明显改变。经放线菌素D处理后，AR组肿瘤细胞在各处理时间点

ATG4D

的mRNA表达量均显著升高（

0.001），表明

ATG4D

mRNA的稳定性在AR肿瘤细胞中显著增强。经环乙酰亚胺处理后，AR组肿瘤细胞在各处理时间点ATG4D 的蛋白表达量无显著变化，表明ATG4D蛋白的稳定性在AR肿瘤细胞中无明显变化。

结论

ATG4D表达上调引起的自噬水平增强促进肿瘤细胞的AR。

Abstract

Objective

Anoikis resistance (AR) is a critical characteristic of metastatic tumor cells

and enhanced autophagy is an important contributor to AR. Autophagy-related 4D (ATG4D) is a key enzyme regulating autophagy. This study aims to investigate whether tumor cells promote AR by upregulating ATG4D expression to enhance autophagy and elucidate the underlying molecular mechanisms.

Methods

Hepatocellular carcinoma HepG2 cells and gastric cancer AGS cells were divided into an AR group and a control group. The AR group was cultured in ultra-low adhesion plates for 48 h

while the control group was cultured in conventional plates. Changes in the expression of autophagy markers LC3-Ⅱ and p62 were determined by Western blotting. Chloroquine (CQ

30 μg/mL) was used to inhibit autophagy in control or AR tumor cells

followed by flow cytometry for apoptosis levels with an apoptosis detection kit. The expression of ATG4D at mRNA and protein levels were assessed by qRT-PCR and Western blotting

respectively. AR tumor cells were transfected with control siRNA or

ATG4D

siRNA using Lipofectamine. Subsequently

the protein expression levels of ATG4D

LC3-Ⅱ and p62 were detected by Western blotting

and apoptotic rates were analyzed w

ith flow cytometry. A reporter plasmid containing the ATG4D promoter was constructed and transfected into control and AR tumor cells

respectively; after 24 h

luciferase reporter gene assay was employed to detect the changes in the activity of ATG4D promoter. To assess cell stability

the control or AR tumor cells were treated with actinomycin D (5 μg/mL) or cycloheximide (50 μg/mL)

and then harvested at 0

and 8 h after treatment. The mRNA and protein levels of

ATG4D

were determined by qRT-PCR and Western blotting

respectively.

Results

Compared with the control tumor cells

the AR tumor cells showed significantly enhanced autophagy

with significantly elevated LC3-Ⅱ levels (

0.01) and markedly reduced p62 levels (

0.01). Inhibition of autophagy using CQ significantly increased apoptosis in AR tumor cells (

0.01). qRT-PCR and Western blotting revealed significantly elevated

ATG4D

mRNA and protein levels in AR tumor cells compared with the control cells (

0.01).

ATG4D

siRNA transfection significantly inhibited

ATG4D

expression

downregulated LC3-Ⅱ

upregulated p62

and promoted apoptosis in AR tumor cells (

0.01).

ATG4D

promoter activity did not differ significantly between AR and control tumor cells. However

after actinomycin D treatment

ATG4D

mRNA expression was significantly higher in AR tumor cells at all time points (

0.001)

indicating enhanced mRNA stability. In contrast

cycloheximide treatment showed no significant change in ATG4D protein levels over time in AR tumor cells

suggesting unaltered protein stability.

Conclusion

Enhanced autophagy caused by

ATG4D

upregulation promotes AR in tumor cells.

关键词

Keywords

references

D’AMORE T ， BRAVOCO D ， DI PAOLA G ， et al . Anoikis resistance in gastric cancer： a comprehensive review ［J］. Cell Death Dis ， 2025 ， 16 （ 1 ）： 528 . DOI： 10.1038/s41419-025-07860-1 http://dx.doi.org/10.1038/s41419-025-07860-1 .

MEI J ， JIANG X Y ， TIAN H X ， et al . Anoikis in cell fate， physiopathology， and therapeutic interventions ［J］. MedComm ， 2024 ， 5 （ 10 ）： e718 . DOI： 10.1002/mco2.718 http://dx.doi.org/10.1002/mco2.718 .

FRISCH S M ， HU G Q . Anoikis resistance and cancer ［J］. BMC Cancer ， 2025 ， 25 ： 1764 . DOI： 10.1186/s12885-025-15178-6 http://dx.doi.org/10.1186/s12885-025-15178-6 .

WANG H Y ， FENG X R ， HE H L ， et al . Crosstalk between autophagy and other forms of programmed cell death ［J］. Eur J Pharmacol ， 2025 ， 995 ： 177414 . DOI： 10.1016/j.ejphar. 2025.177414 http://dx.doi.org/10.1016/j.ejphar.2025.177414 .

WANG H ， SUN P ， YUAN X J ， et al . Autophagy in tumor immune escape and immunotherapy ［J］. Mol Cancer ， 2025 ， 24 （ 1 ）： 85 . DOI： 10.1186/s12943-025-02277-y http://dx.doi.org/10.1186/s12943-025-02277-y .

FULLER A D ， BORDNER T H ， STAUB A J ， et al . Autophagy reprogramming in cancer ［J］. J Cell Physiol ， 2025 ， 240 （ 11 ）： e70107 . DOI： 10.1002/jcp.70107 http://dx.doi.org/10.1002/jcp.70107 .

WANG S Y ， LV Y Y ， ZHOU Y Y ， et al . Acidic extracellular pH induces autophagy to promote anoikis resistance of hepatocellular carcinoma cells via downregulation of miR-3663-3p ［J］. J Cancer ， 2021 ， 12 （ 12 ）： 3418 - 3426 . DOI： 10.7150/jca.51849 http://dx.doi.org/10.7150/jca.51849 .

MCMANN E ， GORSKI S M . Last but not least： emerging roles of the autophagy-related protein ATG4D ［J］. Autophagy ， 2024 ， 20 （ 9 ）： 1916 - 1927 . DOI： 10.1080/15548627.2024. 2369436 http://dx.doi.org/10.1080/15548627.2024.2369436 .

TAMARGO-GÓMEZ I ， MARTÍNEZ-GARCÍA G G ， SUÁREZ M F ， et al . ATG4D role in mAtg8s delipidation and neuroprotection ［J］. Autophagy ， 2021 ， 17 （ 6 ）： 1558 - 1560 . DOI： 10.1080/15548627.2021.1922979 http://dx.doi.org/10.1080/15548627.2021.1922979 .

LIU P F ， YANG S W ， YANG W H ， et al . Autophagy protease ATG4D facilitates proliferation and malignancy of osteosarcoma cells ［J］. FASEB J ， 2025 ， 39 （ 17 ）： e70990 . DOI： 10.1096/fj.202501321RR http://dx.doi.org/10.1096/fj.202501321RR .

ZHAO J Y ， LI X Y ， LIU T D ， et al . Silencing of ATG4D suppressed proliferation and enhanced cisplatin-induced apoptosis in hepatocellular carcinoma through Akt/Caspase-3 pathway ［J］. Mol Cell Biochem ， 2021 ， 476 （ 11 ）： 4153 - 4159 . DOI： 10.1007/s11010-021-04224-z http://dx.doi.org/10.1007/s11010-021-04224-z .

WANG M Y ， JING J J ， LI H ， et al . The expression characteristics and prognostic roles of autophagy-related genes in gastric cancer ［J］. PeerJ ， 2021 ， 9 ： e10814 . DOI： 10.7717/peerj.10814 http://dx.doi.org/10.7717/peerj.10814 .

NIE S ， SHI Z ， SHI M Y ， et al . PPARγ/SOD2 protects against mitochondrial ROS-dependent apoptosis via inhibiting ATG4D-mediated mitophagy to promote pancreatic cancer proliferation ［J］. Front Cell Dev Biol ， 2021 ， 9 ： 745554 . DOI： 10.3389/fcell.2021.745554 http://dx.doi.org/10.3389/fcell.2021.745554 .

KHAN S U ， FATIMA K ， MALIK F . Understanding the cell survival mechanism of anoikis-resistant cancer cells during different steps of metastasis ［J］. Clin Exp Metastasis ， 2022 ， 39 （ 5 ）： 715 - 726 . DOI： 10.1007/s10585-022-10172-9 http://dx.doi.org/10.1007/s10585-022-10172-9 .

WU Y R ， CHEN Y ， YAN X J ， et al . Lopinavir enhances anoikis by remodeling autophagy in a circRNA-dependent manner ［J］. Autophagy ， 2024 ， 20 （ 7 ）： 1651 - 1672 . DOI： 10.1080/15548627.2024.2325304 http://dx.doi.org/10.1080/15548627.2024.2325304 .

GAO X Y ， ZHANG Z W ， LI Q Y ， et al . GDF15 enhances anoikis resistance and metastasis of gastric cancer through protective autophagy ［J］. Cell Signal ， 2024 ， 124 ： 111457 . DOI： 10.1016/j.cellsig.2024.111457 http://dx.doi.org/10.1016/j.cellsig.2024.111457 .

YU Y ， SONG Y R ， CHENG L L ， et al . CircCEMIP promotes anoikis-resistance by enhancing protective autophagy in prostate cancer cells ［J］. J Exp Clin Cancer Res ， 2022 ， 41 （ 1 ）： 188 . DOI： 10.1186/s13046-022-02381-7 http://dx.doi.org/10.1186/s13046-022-02381-7 .

YU Y ， LIU B ， LI X X ， et al . ATF4/CEMIP/PKCα promotes anoikis resistance by enhancing protective autophagy in prostate cancer cells ［J］. Cell Death Dis ， 2022 ， 13 （ 1 ）： 46 . DOI： 10.1038/s41419-021-04494-x http://dx.doi.org/10.1038/s41419-021-04494-x .

URADE R ， CHANG W T ， KO C C ， et al . A fluorene derivative inhibits human hepatocellular carcinoma cells by ROS-mediated apoptosis， anoikis and autophagy ［J］. Life Sci ， 2023 ， 329 ： 121835 . DOI： 10.1016/j.lfs.2023.121835 http://dx.doi.org/10.1016/j.lfs.2023.121835 .

ZHANG C L ， WANG Y ， ZHEN Z F ， et al . mTORC1 mediates biphasic mechano-response to orchestrate adhesion-dependent cell growth and anoikis resistance ［J］. Adv Sci （Weinh）， 2024 ， 11 （ 6 ）： e2307206 . DOI： 10.1002/advs.202307206 http://dx.doi.org/10.1002/advs.202307206 .

FU X T ， SHI Y H ， ZHOU J ， et al . MicroRNA-30a suppresses autophagy-mediated anoikis resistance and metastasis in hepatocellular carcinoma ［J］. Cancer Lett ， 2018 ， 412 ： 108 - 117 . DOI： 10.1016/j.canlet.2017.10.012 http://dx.doi.org/10.1016/j.canlet.2017.10.012 .

NUCERA F ， DI STEFANO A ， RICCIARDOLO F L M ， et al . Role of ATG4 autophagy-related protein family in the lower airways of patients with stable COPD ［J］. Int J Mol Sci ， 2024 ， 25 （ 15 ）： 8182 . DOI： 10.3390/ijms25158182 http://dx.doi.org/10.3390/ijms25158182 .

VASUKUTTY A ， BHATTARAI P Y ， CHOI H S . Enhancer regulation in cancer： from epigenetics to m6A RNA modification ［J］. Arch Pharmacal Res ， 2025 ， 48 （ 7 ）： 706 - 735 . DOI： 10.1007/s12272-025-01561-1 http://dx.doi.org/10.1007/s12272-025-01561-1 .

JUNGFLEISCH J ， GEBAUER F . RNA-binding proteins as therapeutic targets in cancer ［J］. RNA Biol ， 2025 ， 22 （ 1 ）： 1 - 8 . DOI： 10.1080/15476286.2025.2470511 http://dx.doi.org/10.1080/15476286.2025.2470511 .

CUI D ， FENG Y ， QIAN R L . Up-regulation of microRNA miR-101-3p enhances sensitivity to cisplatin via regulation of small interfering RNA （siRNA） anti-human AGT4D and autophagy in non-small-cell lung carcinoma （NSCLC）［J］. Bioengineered ， 2021 ， 12 （ 1 ）： 8435 - 8446 . DOI： 10.1080/21655979.2021.1982274 http://dx.doi.org/10.1080/21655979.2021.1982274 .

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