Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress

Abstract

Loss of function of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene is associated with many human cancers. In the cytoplasm, PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K) signaling pathway. PTEN also accumulates in the nucleus, where its function remains poorly understood. We demonstrate that SUMOylation (SUMO, small ubiquitin-like modifier) of PTEN controls its nuclear localization. In cells exposed to genotoxic stress, SUMO-PTEN was rapidly excluded from the nucleus dependent on the protein kinase ataxia telangiectasia mutated (ATM). Cells lacking nuclear PTEN were hypersensitive to DNA damage, whereas PTEN-deficient cells were susceptible to killing by a combination of genotoxic stress and a small-molecule PI3K inhibitor both in vitro and in vivo. Our findings may have implications for individualized therapy for patients with PTEN-deficient tumors.

Fig. 1. SUMOylation of PTEN at lysine 254 in vivo

(A) Multiple cell lines express a 75 kDa form of PTEN. Whole cell lysates were immunoblotted with 3 PTEN antibodies: 6H2.1 mouse mAb (top), rabbit mAb CST #9559 (middle), or a rabbit pAb (bottom) CST #9552. PTEN-H is indicated (arrow). (B) Transfection of minimal PTEN cDNA leads to PTEN-H formation. pcDNA3.1-HA-PTEN (WT) or pcDNA3.1-HA (EV) were transfected into HEK293 cells and HA-immunoprecipitates immunoblotted for PTEN. (C) SUMOylation of endogenous PTEN. PTEN was immunoprecipitated from HEK293 lysates with protein A beads alone or PTEN antibody (CST #9559) and immunoblotted with the PTEN 6H2.1 antibody (left) then stripped and re-probed with antibodies to Sumo2/3 (right). (D) Formation of SUMO-PTEN requires Ubc9. HEK293 cells were transfected with scrambled or Ubc9 siRNAs, and harvested at 48 (middle lane) or 72 (right lane) hrs post-transfection. Lysates were immunoblotted for PTEN, Ubc9 and GAPDH, as indicated. Arrow indicates SUMO-PTEN. (E) Sensitivity of PTEN-H to deSUMOylases. Immunoprecipitated Flag-PTEN was incubated with GST, GST-SENP1 or GST-SENP2, as indicated, in the presence or absence of 20 mM NEM. Reactions were immunoblotted with anti-PTEN 6H2.1 (top panel) or anti-Sumo2 (lower panel) antibodies. (F) PTEN is SUMOylated on lysine 254 in vivo. HEK293 cells were transfected with FLAG-PTEN-wt or FLAG-PTEN-K254R, together with His-SUMO2 and myc-His-Ubc9, as indicated. Flag immunoprecipitates were immunoblotted for PTEN.

Fig. 2. PTEN SUMOylation regulates nuclear retention, which is sensitive to genotoxic stress

(A) Exclusion of the SUMO-deficient mutant PTEN K254R from the nucleus. Flag-FITC immunofluorescence images of HEK293 cells transfected as indicated. Insets show DAPI staining. (B) Nuclear retention of SUMOylated PTEN. Immunofluorescence as in (A). Cells were treated with 10 ng/ml of leptomycin B for 4 hours. (C) Decreased nuclear PTEN localization following genotoxic stress. Immunofluorescence as in (A) of PTEN-FITC U87MG cells transfected as indicated, 4 hours post-IR. Bar graph represents percentage of cells with nuclear PTEN in control or IR-treated cells (p<0.001, t test, n=5, bars represent SEM). (D) Decreased nuclear PTEN following genotoxic stress. HeLa cells were treated as indicated, harvested after 4 hours and separated into cytoplasmic and nuclear fractions followed by immunoblotting for PTEN. Fractionation was monitored by immunoblotting for PARP (nuclear protein) and GAPDH (cytoplasmic protein). Arrow indicates SUMO-PTEN.

Fig. 3. Requirement of nuclear SUMO-PTEN for homologous recombination repair of DNA double-strand breaks

U87MG cells (panels A to F) were reconstituted the indicated proteins. Cells treated with IR (5 Gy) were immunostained at the indicated times with antibodies to γH2AX, 53BP1, BRCA1 or RAD51. Cells containing >5 foci were scored as positive. Bars represent SEM. PTEN SUMOylation is required for the resolution of 53BP1 foci (p=0.0014, t test, n=3) (A), γH2AX foci (p=0.007, t test, n=3) (B) and BRCA1 foci (p=0.0136, t test, n=3) (C). (D) PTEN SUMOylation is required for RAD51 focus formation. (p=0.0016, t test, n=3). (E) Impaired homologous recombination-based DNA repair in PTEN- and SUMO-PTEN-deficient cells. U87MG cells stably expressing a DR-GFP reporter were reconstituted with the indicated PTEN proteins and their HR-mediated repair assessed as previously described (16). HR efficiency is expressed relative to that of wt. (p=0.012, t test, n=2). (F) Lack of SUMO-PTEN increases radiosensitivity. Surviving fraction was determined by Sulforhodamine B staining 6 days following exposure to 3 Gy of IR (p=0.02, t test, n=3).

Fig. 4. PTEN is part of a ATM-regulated signaling cascade governing sensitivity to genotoxic agents

(A) Decreased SUMO-PTEN after DNA damage. Lysates from HeLa cells treated as indicated were immunoblotted for PTEN. A ratio of SUMO-PTEN to PTEN immunoreactivity relative to the non-irradiated control is indicated. (B) ATM Regulation of SUMO-PTEN abundance by ATM. Lysates from HeLa cells treated as indicated were immunoblotted for PTEN and quantified as in (A). (C) ATM phosphorylates serine/threonine 398 of PTEN in vitro. The indicated GST-fusion proteins were incubated with active ATM in the presence of ³²P-ATP. 32P incorporation was quantified by PhosphorImager and normalized to protein loading. (D) Phosphorylation of PTEN S398 in vivo by ATM. HA immunoprecipitates from HEK293 cells transfected and treated as indicated, were immunoblotted with antibody against phospho-ATM substrates (CST #9607). (E) SUMO-PTEN-T398A is not sensitive to IR. Flag immunoprecipitates from HEK293 cells transfected and treated as indicated were immunoblotted for PTEN. (F) PTEN-T398A remains in the nucleus after IR. Cells were treated and imaged as in Fig. 2C. (G-H) PTEN loss sensitizes cells to combination treatment with IR and a pan-PI3K inhibitor. Bars represent SEM. (G) U87MG cells reconstituted with empty vector or PTEN-wt were treated with either 2 Gy of IR, 500 nM BKM120 or both, and apoptosis was measured by annexin V⁺/7-AAD⁺ staining (p=0.0019, t test, n=3). (H) HCT116 PTEN-null and parental PTEN-WT cells were treated with either IR (2 Gy) or 1 mM cisplatin alone or in combination with 500 nM BKM120, as indicated. Apoptosis was measured as in (G) (IR+BKM120 p=0.0025, t test, n=3) (Cis+BKM120 p=0.0475, t test, n=3). (I) PTEN-deficient cells are sensitive to the Cisplatin/PI3K inhibitor combination in vivo. HCT116 parental or HCT116 PTEN^−/− cells were injected subcutaneously into NOD/SCID mice (n=10). Mice were treated with cisplatin, BKM120, or both. Data points represent mean tumor volume ± SEM. Tumor growth difference was measured between day 1 and 16 and one way ANOVA was performed (p=0.0321 for HCT116 parental and p<0.0001 for HCT116 PTEN^−/−) followed by Dunnet’s multiple comparison test (HCT116 PTEN^−/− BKM120 + Cisplatin vs Ctr, p<0.0001).