Figure 8. Blockade of UBE2O with ATO reduces tumorigenesis through AMPKα2 restoration.

(A) A diagram depicting the mechanism by which arsenite inhibits UBE2O (top). Recombinant UBE2O proteins were subjected to in vitro self-ubiquitination assay in the presence of ATO (10 μM) (bottom).

(B) Lysates from 293T cells expressing the indicated plasmids treated with ATO (1 μM) together with MG132 (10 μM) for 6 hr were subjected to metal-affinity purification for His-tagged ubiquitin then immunoblotting for ubiquitinated AMPKα2. Ni-NTA, Ni²⁺-nitrilotriacetic acid. # indicates nonspecific band.

(C) Lysates from HCT116 cells expressing UBE2O shRNA treated with ATO (0.5 μM) for the indicated times were subjected to immunoblotting (top). AMPKα2 or AMPKα1 protein levels were quantified by normalizing to the intensity of the Actin band (bottom).

(D) Tumor volumes of mouse allograft implanted with E1A+H-Ras^V12 MEFs overexpressing UBE2O treated with ATO (i.p. 2 mg/kg, daily) were measured at different day. n = 7, p value was determined by ANOVA (n.s., non-significant; ***p<0.001). Scale bars, 75 μm.

(E) Representative images, immunohistochemical analysis of Ki-67, and quantification of Ki-67 positive cells of mouse xenograft tumors from (D). Scale bars, 75 μm. n = 7, p value was determined by Student’s t test (n.s., non-significant; ***p<0.001).

(F) Lysates from (D) were subjected to immunoblotting.

(G) TFS analysis of PyVT;Ube2o^+/+ mice treated with vehicle (n = 15) or ATO (i.p. 2.5 mg/kg, every other day for 65 days) (n = 13). TFS curves of vehicle-treated PyVT;Ube2o^+/− mice (n = 15) are also shown. p value was determined by Log-rank (Mantel-Cox) test (n.s., non-significant; **p<0.01).

(H) The weight of mammary tumors isolated from 105 days old vehicle-treated PyVT;Ube2o^+/+ (n = 13), ATO-treated PyVT;Ube2o^+/+ (n = 13) and vehicle-treated PyVT;Ube2o^+/− (n = 7) mice were quantified. p value was determined by Student’s t test (*p<0.05; ***p<0.001).

(I) Sections of lungs isolated from 105 days old vehicle-treated PyVT;Ube2o^+/+, ATO-treated PyVT;Ube2o^+/+ and vehicle-treated PyVT;Ube2o^+/− mice were stained for H&E or PyV T Ag. Arrowheads indicate clusters of metastatic cells in the lung. Scale bars, 75 μm. The number of metastatic sites in lungs of mice was also quantified (right). n = 7, p value was determined by Student’s t test (n.s., non-significant; *p<0.05).

(J) H&E-stained sections of anterior prostate lobes isolated from 12 weeks old TRAMP;Ube2o^+/+ mice treated with ATO (i.p. 2.5 mg/kg, every other day for 4 weeks). Scale bars, 75 μm.

(K) Quantification of PIN in (J). Vehicle-treated TRAMP;Ube2o^+/+ mice cohort (n = 9); ATO-treated TRAMP;Ube2o^+/+ mice cohort (n = 6); vehicle-treated TRAMP;Ube2o^+/− mice cohort (n = 9). p value was determined by Student’s t test (n.s., non-significant; *p<0.05; **p<0.01).

(L) Prostate tissue weight of 12 weeks old vehicle-treated TRAMP;Ube2o^+/+ (n = 10), ATO-treated TRAMP;Ube2o^+/+ (n = 6) and vehicle-treated TRAMP;Ube2o^+/− (n = 9) mice. p value was determined by Student’s t test (n.s., non-significant; *p<0.05).

Error bars represent +/− SEM. See also Figure S8.