Original ArticleThe depletion of ATM inhibits colon cancer proliferation and migration via B56γ2-mediated Chk1/p53/CD44 cascades
Introduction
Ataxia-telangiectasia mutated (ATM) is a homonymous Ser/Thr protein kinase encoded by the gene mutated in Ataxia Telangiectasia (A-T). A-T is a rare autosomal recessive disorder characterized by cerebellar neurodegeneration, immunodeficiency, hypersensitivity to ionizing radiation, and a strong predisposition for cancer [1]. ATM belongs to the phosphatidylinositol-3 kinase-related kinase (PIKK) family, which has six members. The family members share four conserved domains, including the FRAP-ATM-TRRAP (FAT) domain, kinase domain, PIKK regulatory domain, and FAT-C-terminal (FATC) domain. ATM is a key modulator of the DNA damage response (DDR) elicited by double strand breaks (DSBs). Under non-stressed conditions, ATM is inactive as a dimer or multimer. In response to DSBs induced by ionizing radiation (IR) and/or other agents, ATM undergoes autophosphorylation at S1981, resulting in dimer dissociation that is followed by its recruitment to the DNA damage lesion and its subsequent activation [2]. Activated ATM then sends signals to downstream targets to initiate signaling for the cell cycle checkpoint and DNA repair. Therefore, ATM participates in a variety of cellular processes related to DNA damage, such as the cell cycle, apoptosis, senescence, and autophagy [3], [4], [5].
Given the central role of ATM in the maintenance of genome stability, ATM mutations or deletions are associated with increased genetic alterations that may drive malignant transformation. One example of this notion is that A-T patients display an increased susceptibility to the development of malignancies, particularly leukemia and lymphoma [1]. In addition, ATM deficiency in mice also results in increased predisposition to various tumors, including lymphoid tumors, intestinal tumors, and epithelial tumors [6], [7], [8]. Moreover, ATM heterozygosity has been reported to increase the incidence of breast cancer in TP53 heterozygous mice [9]. Similarly, the conditional deletion of ATM in a mouse model of pancreatic ductal adenocarcinoma (PDAC) accelerates cancer formation [10], and the knockdown of ATM is linked to an enhancement of neuroblastoma [11]. Together, these studies provide evidence that ATM acts as a tumor suppressor in tumor initiation and tumorigenicity. However, recent studies have suggested that in post-formed tumors, ATM can have a very different function. Chen et al. reported a new cancer-promoting role for ATM. Their experiments revealed that reducing the amount of ATM in breast cancer cells made them less migratory and invasive. They identified interleukin (IL)-8 as a target of ATM, which ATM uses to promote the migration of mutant p53-containing cancer cells [12]. Similarly, Stagni et al. reported that ATM expression and activity are essential for HER2-dependent breast tumorigenicity [13]. In addition, ATM has been reported to activate several signaling pathways involved in cell migration and proliferation, such as the AKT, ERK, and Wnt signaling pathways [14], [15]. Yin et al. showed that Wip1 suppresses ovarian cancer metastasis by negatively regulating the ATM-mediated AKT/Snail cascade [14]. Taken together, these findings suggest that ATM may exert a more complex role than a role as a pure tumor suppressor during cancer progression.
Current studies on the function of ATM mainly utilize siRNA-mediated knockdown techniques and are performed under stress conditions, such as ionizing radiation, treatment with DSB-inducing drugs, or oxidative stress. In contrast, the functional role of ATM in cancer cells under normal physiological conditions has not been characterized. Therefore, we used an ATM knockout cancer cell model, which was established via targeted homologous recombination. With two isogenic sets of human colon cancer cell lines that differed only in their ATM status, we demonstrated that ATM deficiency inhibits cancer cell proliferation, migration, and invasion under normal growth conditions. Moreover, we revealed that the inhibitory effect of ATM depletion on tumorigenicity is associated with B56γ2/PP2A-mediated Chk1/p53/CD44 signaling pathways. Our study provides reliable evidence that ATM plays a tumorigenic role in post-formed colon neoplasia.
Section snippets
Cell culture
The human colon cancer cell lines DLD1, HCT116, and HEK293T were obtained from the American Type Culture Collection (ATCC, USA). HCT116 p53−/− cells were kindly provided by Dr. Bert Vogelstein of Johns Hopkins University, Baltimore, USA. DLD1 cells were cultured in RPMI-1640 medium (HyClone) supplemented with 10% FBS (HyClone). HCT116 cells were cultured in McCoy's 5A growth medium (Gibco) supplemented with 10% FBS. During selection for genetic targeting events, the cells were cultured in
Targeted deletion of ATM in colon cancer cells
Recombinant adeno-associated virus (rAAV) vectors are an efficient means of permanently inactivating genes in human cancer cells [19], [20]. Using rAAV vectors, we disrupted the endogenous ATM gene in two near-diploid colon cancer cell lines, DLD1 and HCT116 (Fig. 1A). Targeting was initially evaluated via PCR of the genomic DNA. Following the disruption of the first allele, Cre recombinase was used to remove the drug-resistance cassette integrated at the targeted locus. This process of
Discussion
Despite knowledge of the well-established function of ATM in DSB signaling and DNA repair, the exact role of ATM in cancer is controversial [33]. To more comprehensively understand the involvement of ATM in cancer progression, we developed two isogenic sets of human colon cell lines that differed only in their ATM gene status. Utilizing these cell lines, we demonstrated that ATM has cancer-promoting activity in the neoplastic state. We showed that the deletion of ATM significantly inhibits
Acknowledgments
We thank Véronique Orian-Rousseau of the Karlsruhe Institute of Technology for the CD44s expression plasmid. We thank Michael B. Kastan of Saint Jude Children's Research Hospital for the ATM expression plasmid. We thank Dr. Yuezhen Deng of Chinese Academy of Sciences for technical assistance. This work was supported by the National Natural Science Foundation of China (No. 81572826 and 81372490), Zhejiang Provincial Natural Science Foundation (No. LZ14H160003) and Zhejiang Provincial Program for
References (39)
- et al.
Ataxia-telangiectasia
Handb. Clin. Neurol.
(2012) - et al.
The ATM protein kinase and cellular redox signaling: beyond the DNA damage response
Trends Biochem. Sci.
(2012) - et al.
ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans
Blood
(2015) PP2A: unveiling a reluctant tumor suppressor
Cell
(2007)- et al.
MacroH2A1 and ATM play opposing roles in paracrine senescence and the senescence-associated secretory phenotype
Mol. Cell
(2015) - et al.
Tumor suppressor p53 and its mutants in cancer metabolism
Cancer Lett.
(2015) - et al.
DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation
Nature
(2003) - et al.
The ATM protein kinase: regulating the cellular response to genotoxic stress, and more
Nat. Rev. Mol. Cell Biol.
(2013) - et al.
ATM functions at the peroxisome to induce pexophagy in response to ROS
Nat. Cell Biol.
(2015) - et al.
ATM promotes apoptosis and suppresses tumorigenesis in response to Myc
Proc. Natl. Acad. Sci. U. S. A.
(2006)
Atm is a negative regulator of intestinal neoplasia
Oncogene
Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice
Breast Cancer Res.
Loss of ATM accelerates pancreatic cancer formation and epithelial-mesenchymal transition
Nat. Commun.
Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism
Oncotarget
ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion
Elife
ATM kinase sustains HER2 tumorigenicity in breast cancer
Nat. Commun.
Wip1 suppresses ovarian cancer metastasis through the ATM/AKT/Snail mediated signaling
Oncotarget
Oxidized ATM promotes abnormal proliferation of breast CAFs through maintaining intracellular redox homeostasis and activating the PI3K-AKT, MEK-ERK, and Wnt-beta-catenin signaling pathways
Cell Cycle
RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer
Nat. Commun.
Cited by (14)
Discovery of novel ataxia telangiectasia mutated (ATM) kinase modulators: Computational simulation, biological evaluation and cancer combinational chemotherapy study
2022, European Journal of Medicinal ChemistryCitation Excerpt :Besides, A41 exhibited comparable antiproliferation abilities with KU55933 and KU60019 in MCF-7, SW480, and NCI–H1299 cells, which might serve as a good starting point for the further optimization of ATM inhibitors. Meanwhile, there are few studies on the chemotherapy sensitization of ATM inhibitors compared with that of radiotherapy sensitization [21,34,52,53], and some findings concentrating on chemotherapy sensitization had finally come to different conclusions [19,54]. Facing this dilemma, we combined ATM modulators, including inhibitors A41, KU55933, KU60019, and AZD0156, and agonist A22, with chemotherapeutic agents, Etoposide and Irinotecan, in MCF-7 and SW480 cells, and studied the mechanism of action involved in these combination effects.
ATM in DNA repair in cancer
2019, Pharmacology and TherapeuticsCitation Excerpt :In response to oxidative stress, ATM facilitates interleukin-8 release to enhance breast and lung cancer cell migration (Chen et al., 2015). In colon cancer cells, ATM deficiency leads to inhibition of B56γ2 ubiquitination and degradation, which further downregulates Chk1-p53-p21 signals to reduce cell migration (Liu et al., 2017). Chromatin remodeling is highly associated with the DDR process because the chromatin relax can increase sensitivity to DDR-targeting agents (Sulli, Di Micco, & d'Adda di Fagagna, 2012).
Inhibitory effects of piperine on proliferation, migration, and invasion of human colon cancer SW480 cells
2021, World Chinese Journal of Digestology
- 1
These authors contributed equally to this work.