Overexpression of NBS1 Contributes to Transformation through the Activation of Phosphatidylinositol 3-Kinase/Akt

Abstract

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40 -52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3␤ and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt. Nijmegen breakage syndrome (NBS)3 is an autosomal recessive hereditary disorder characterized by microcephaly, a "bird-like" facial appearance, growth retardation, immunodeficiency, radiosensitivity, chromosomal instability, and predisposition to tumor formation (1-3). The gene defective in NBS has been cloned, and the gene product, NBS1 (p95, nibrin), is a member of the DNA double strand break repair complex (hMre11 complex) including hMre11, hRad50, and NBS1 (1, 3). Increased radiation sensitivity and radioresistant DNA synthesis of NBS fibroblasts are similar to the cellular features of AT (ataxia-telangiectasis) cells (2, 4), demonstrated by the recent results that ATM (ataxiatelangiectasis-mutated) protein phosphorylates NBS1 (5-7), linking these two proteins in the same pathway. NBS1 is a putative tumor suppressor gene as shown by the existence of NBS patients and some mutations discovered in different tumors (1, 2). However, NBS1 is expressed in highly proliferating tissues developmentally (8) and is located at sites of DNA synthesis through interaction with E2F (9). In addition, Mre11 complex is able to prevent double strand break accumulation during chromosomal DNA synthesis to ensure cell cycle progression (10). Nbs1 knock out in mouse embryonic stem cells shows the phenotype of diminished expansion of the inner cell mass of mutant blastocysts (Nbs1 null) (11, 12). Cellular proliferation defects are shown in Nbs1 m/m mouse embryonic fibroblasts (13). Obviously, the roles of NBS1 are multiple, and some of them are still subject to intensive investigation.Phosphatidylinositol (PI) 3-kinase is a major signaling component downs...

Increased NBS1 Expression Is a Marker of Aggressive Head and Neck Cancer and Overexpression of NBS1 Contributes to Transformation

“…In addition, we showed previously that c-MYC, a dominant oncoprotein, directly activates NBS1 expression and that overexpression of NBS1 increases cell proliferation (31). Our recent results show that overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase (PI3K)/Akt (32). All these results implicated a role of NBS1 in the process of cell proliferation and tumorigenesis.…”

“…Real-time PCR analysis was done using 1 Ag total RNA isolated from tumor and neighboring normal tissues. Quantitative real-time PCR was done in a PRISM ABI7700 Sequence Detection System (Applied Biosystems, Foster City, CA) with the preset PCR program, and TBP (TATA box binding protein) was selected as an internal control (32). The primer sequences used in real-time PCR were NBS1 5V -ATGGAGGCCATATTTCCATGAC-3V and 5V -CAAGCAGCCA-GAACTTGGAAG-3V and TBP 5V -TGGTGGTGTTGTGAGAAGATGG-3V and 5V -CGGTGGGCACTTACAGAAGG-3V .…”

Combined single nucleotide polymorphism-based genomic mapping and global gene expression profiling identifies novel chromosomal imbalances, mechanisms and candidate genes important in the pathogenesis of T-cell prolymphocytic leukemia with inv(14)(q11q32)

“…Increased NBS1 expression has been observed in non-small cell lung carcinoma, hepatoma, melanoma and esophageal cancer samples. [36][37][38] Furthermore, Chen et al 36 have recently demonstrated that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation through stimulation of phosphatidylinositol (PI)3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as mammalian target of rapamycin. It is thus tempting to speculate that increased NBS1 expression contributes to TCL1-induced enhanced Akt signaling in T-PLL cells, which has been suggested to be causal for disease progression.…”

“…Potentially important, this set of genes included NBS1 or nibrin, the overexpression of which has T-cell prolymphocytic leukemia J Dürig et al recently been shown to contribute to the oncogenic transformation of head and neck cancer and non-small cell lung cancer cells through the activation of the PI3/Akt pathway. [36][37][38] Furthermore, chromosome 8 aberrations were associated with aberrant expression of the erythrocytic structural membrane protein ankyrin-1, which was absent in the eight normal control samples and the T-PLL case lacking iso(8q) (see T-PLL 3 in Figure 4). Aberrant expression of ankyrin-1 was confirmed at the protein level using western blot ( Figure 5).…”

Genetic Variants of NBS1 Predict Clinical Outcome of Platinum-based Chemotherapy in Advanced Non-small Cell Lung Cancer in Chinese

“…Studies in mice have indicated that heterozygous for NBS1-null mutations develop tumors in lung, liver, mammary gland and prostate (Dumon-Jones et al, 2003;Zhang et al, 2006). NBS1 overexpression has been observed in several smoking-related cancer sites including NSCLC, hepatoma, esophageal cancer and head and neck squamous cell carcinoma (HNSCC) (Chen, 2005;Yang, 2006). Furthermore, it was also correlated with recurrence/ metastasis of oral squamous cell carcinoma and the poor prognosis of HNSCC (Yang, 2006;Hsu et al, 2010).…”