Cancer Letters

Cancer Letters

Volume 390, 1 April 2017, Pages 48-57
Cancer Letters

Original Article
The depletion of ATM inhibits colon cancer proliferation and migration via B56γ2-mediated Chk1/p53/CD44 cascades

https://doi.org/10.1016/j.canlet.2016.12.040Get rights and content

Highlights

  • Tumorigenic potential of ATM was studied in two ATM-depleted colon cancer cell lines.

  • ATM deficiency inhibits colon cancer cell proliferation, migration, and invasion.

  • Tumor-promoting function of ATM is implemented via B56γ2-mediated Chk1 signaling pathways.

  • CD44 was validated as a novel ATM target in tumor metastasis-promotion.

  • ATM act as an oncogenic factor in post-formed colon neoplasia.

Abstract

Ataxia-telangiectasia mutated (ATM) protein kinase is a major guardian of genomic stability, and its well-established function in cancer is tumor suppression. Here, we report an oncogenic role of ATM. Using two isogenic sets of human colon cancer cell lines that differed only in their ATM status, we demonstrated that ATM deficiency significantly inhibits cancer cell proliferation, migration, and invasion. The tumor-suppressive function of ATM depletion is not modulated by the compensatory activation of ATR, but it is associated with B56γ2-mediated Chk1/p53/CD44 signaling pathways. Under normal growth conditions, the depletion of ATM prevents B56γ2 ubiquitination and degradation, which activates PP2A-mediated Chk1/p53/p21 signaling pathways, leading to senescence and cell cycle arrest. CD44 was validated as a novel ATM target based on its ability to rescue cell migration and invasion defects in ATM-depleted cells. The activation of p53 induced by ATM depletion suppresses CD44 transcription, thus resulting in epithelial–mesenchymal transition (EMT) and cell migration suppression. Our study suggests that ATM has tumorigenic potential in post-formed colon neoplasia, and it supports ATM as an appealing target for improving cancer therapy.

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

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