Suppression of nonhomologous end joining repair by overexpression of HMGA2

Abstract

Understanding the molecular details associated with aberrant high mobility group A2 (HMGA2) gene expression is key to establishing the mechanism(s) underlying its oncogenic potential and effect on the development of therapeutic strategies. Here, we report the involvement of HMGA2 in impairing DNA-dependent protein kinase (DNA-PK) during the nonhomologous end joining (NHEJ) process. We showed that HMGA2-expressing cells displayed deficiency in overall and precise DNA end-joining repair and accumulated more endogenous DNA damage. Proper and timely activation of DNA-PK, consisting of Ku70, Ku80, and DNA-PKcs subunits, is essential for the repair of DNA double strand breaks (DSB) generated endogenously or by exposure to genotoxins. In cells overexpressing HMGA2, accumulation of histone 2A variant X phosphorylation at Ser-139 (gamma-H2AX) was associated with hyperphosphorylation of DNA-PKcs at Thr-2609 and Ser-2056 before and after the induction of DSBs. Also, the steady-state complex of Ku and DNA ends was altered by HMGA2. Microirradiation and real-time imaging in living cells revealed that HMGA2 delayed the release of DNA-PKcs from DSB sites, similar to observations found in DNA-PKcs mutants. Moreover, HMGA2 alone was sufficient to induce chromosomal aberrations, a hallmark of deficiency in NHEJ-mediated DNA repair. In summary, a novel role for HMGA2 to interfere with NHEJ processes was uncovered, implicating HMGA2 in the promotion of genome instability and tumorigenesis.

Figure 1. HMGA2 expression induces DNA-PKcs hyper-phosphorylation and reduces DNA end-binding of Ku80

(A) Relative levels (numbers in italic) of Dox-induced pDNA-PKcs-T2609 and pDNA-PKcs-S2056 in HeLa and HeLa/HMGA2 cells normalized against that of DNA-PKcs are shown. (B) Confocal images of pDNA-PKcs-T2609 foci (green) and Dox (red) distribution in HeLa and HeLa/HMGA2 cells 30 min post-Dox (5μM) treatment. Middle-panel is enlarged view of the selected pDNA-PKcs-T2609 foci (upper-panel) as indicated by the white boxes. The mean foci volume (lower-left-panel) and the average number of foci per cell in 75-100 cells (lower-right-panel) are shown. (C, top-panel) Relative levels of Dox-induced pDNA-PKcs-T2609 and pDNA-PKcs-S2056 in Hs578T and HCC1419 breast cancer cells. (bottom-panel) Hs578T cells were transduced with vector or shHMGA2 for 72 h prior to Dox treatment. Relative levels of pDNA-PKcs-T2609 and pDNA-PKcs-S2056 normalized against that of DNA-PKcs are italicized. (D) Relative DNA binding by Ku in HeLa, HeLa/HMGA2, and HeLa/HMGA2(4P/A) cells following Dox treatment. *, p<0.05; **, p<0.01, from three independent experiments.

Figure 2. HMGA2 induces a continued presence of DNA-PKcs at DSB sites in response to laser damage in living cells

(A) Short term (1 min, left-panel) and long term (2 h, right-panel) time-lapse imaging of YFP-DNA-PKcs-expressing V3/vector, V3/HMGA2, and V3/HMGA2(4P/A) cells before and after microirradiation. (B and C) Initial accumulation (B) and 2 h time-course accumulation (C) kinetics of YFP-DNA-PKcs of respective cells at DSB sites after microirradiation. Error bars, ± SD from 10 independent measurements.

Figure 3. HMGA2 delayed clearance of γ-H2AX post X-ray exposure

Western blot analysis of γ-H2AX elimination in (A) DNA-PK(+/+) M059K and DNA-PK(-/-) M059J cells; (B) HeLa, HeLa/vector, HeLa/HMGA2, and HeLa/HMGA2(4P/A) cells; (C) HMGA2-non-expressing CL48, HMGA2-expressing Hep3B and HepG2 hepatoma cells; and (D) HMGA2-non-expressing HCC1419 and HMGA2-expressing Hs578T breast cancer cells. Cells were either nonirradiated or irradiated with 3-Gy X-rays; protein lysates were prepared at different time points post X-ray exposure as indicated.

Figure 4. Ectopic expression of HMGA2 in WI-38 cells induces chromosomal aberrations

(A) A near 4n WI38/HMGA2 cell with part of chromosome 13 translocated to chromosome 10 (arrows), der(10)t(10;13)(q26;q12)×2. (B) Chromosomal breaks at chromosomes 15 and 16 (arrows), observed in WI-38/HMGA2 cells. (C) A dicentric chromosome with chromosomes 10 and X fused together (arrows). (D) Quantification of chromosome aberrations detected in ectopically expressed HMGA2 in WI-38 cells. Inset, expression of HMGA2.