MLF2 Negatively Regulates P53 and Promotes Colorectal Carcinogenesis

Abstract Inactivation of the p53 pathway is linked to a variety of human cancers. As a critical component of the p53 pathway, ubiquitin‐specific protease 7 (USP7) acts as a deubiquitinase for both p53 and its ubiquitin E3 ligase mouse double minute 2 homolog. Here, myeloid leukemia factor 2 (MLF2) is reported as a new negative regulator of p53. MLF2 interacts with both p53 and USP7. Via these interactions, MLF2 inhibits the binding of USP7 to p53 and antagonizes USP7‐mediated deubiquitination of p53, thereby leading to p53 destabilization. Functionally, MLF2 plays an oncogenic role in colorectal cancer, at least partially, via the negative regulation of p53. Clinically, MLF2 is elevated in colorectal cancer and its high expression is associated with poor prognosis in patients with colorectal cancer. In wild‐type‐p53‐containing colorectal cancer, MLF2 and p53 expressions are inversely correlated. These findings establish MLF2 as an important suppressor of p53 function. The study also reveals a critical role for the MLF2–p53 axis in promoting colorectal carcinogenesis.


Supplementary
(A) HCT116 cells expressing Flag-p53 and HA-MLF2 were subjected to proximity ligation assay (PLA) using anti-Flag and anti-HA antibodies.The isotype-matched rabbit (R) and mouse (M) IgG were also used as negative control.The PLA signal was acquired using a Zeiss LSM 980 microscope (Top).Numbers of puncta were also counted (n = 100 cells) (Down).
(B) Schematic representation of wild-type MLF2 and its deletion mutants used for mapping experiments.
(C) HEK293T cells were transfected with Flag-p53 together with the indicated GFP-MLF2 constructs.Cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis.
(D) Schematic representation of wild-type p53 and its deletion mutants used for mapping experiments.
(E) HEK293T cells were transfected with HA-MLF2 alone or together with the indicated Flag-p53 constructs.Cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis.(A) HCT116 cells expressing Flag-USP7 and HA-MLF2 were subjected to proximity ligation assay (PLA) using anti-Flag and anti-HA antibodies.The isotype-matched rabbit (R) and mouse (M) IgG were also used as negative control.The PLA signal was acquired using a Zeiss LSM 980 microscope (Top).Numbers of puncta were also counted (n = 100 cells) (Down).
(C) Schematic representation of wild-type MLF2 and its deletion mutants.HEK293T cells were transfected with Flag-USP7 together with the indicated GFP-MLF2 constructs.Cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis.
(D and E) Schematic representation of wild-type USP7 and its deletion mutants.

Figure S2 .
Figure S2.Related to Figure 2. (A and B) SW480 cells were infected with lentiviruses expressing control shRNA, MLF2 shRNA#1, or MLF2 shRNA#2 (A).SW480 cells were infected with lentiviruses expressing control or Flag-MLF2 (B).Forty-eight hours later, cell lysates were analyzed by western blotting.(C and D) HCT116 cells were infected with lentiviruses expressing control shRNA or MLF2 shRNA (C).HCT116 cells were infected with lentiviruses expressing control or Flag-MLF2 (D).Twenty-four hours later, cells were transfected with the indicated luciferase reporter constructs.Reporter activity was then measured 24 h after transfection.***, p < 0.001; ns., no significance.(Eand F) HCT116 cells were infected with lentiviruses expressing control shRNA or

Figure S4 .
Figure S4.Related to Figure 5. (A and B) The growth curves of RKO cells expressing control or Flag-MLF2 (A).The expression of MLF2 was detected by western blot analysis (B).Data shown are mean ± SD (n=3).**, p < 0.01.(C and D) The growth curves of RKO cells expressing control shRNA, MLF2 shRNA, p53 shRNA, or MLF2 shRNA plus p53 shRNA (C).The knockdown efficiency of MLF2 and p53 was verified by western blot analysis (D).Data shown are mean ± SD (n=3).***, p < 0.001.(E) The representative images for Figure 5E.(F and G) (F) RKO cells expressing control or Flag-MLF2 were treated with doxorubicin (Dox; 0.5 μg/ml) for the indicated periods of time.Cells were co-stained with Annexin V-FITC and Hoechst 33342, and Annexin V-positive cells were counted as apoptotic cells.Data shown are mean ± SD (n=3).Cell lysates were analyzed by western blotting with the indicated antibodies.CL-PARP and CL-Cas-3 indicate cleaved PARP and cleaved caspase-3, respectively.***, p ˂ 0.001.(G) The representative images were also shown.(H) The representative images for Figure 5F.(I and J) (I) RKO cells expressing control shRNA, MLF2 shRNA, p53 shRNA, or MLF2 shRNA plus p53 shRNA were treated with doxorubicin (Dox; 0.5 μg/ml) for the indicated periods of time.Cells were co-stained with Annexin V-FITC and Hoechst 33342, and Annexin V-positive cells were counted as apoptotic cells.Cell lysates were analyzed by western blotting with the indicated antibodies.Data shown are mean ± SD (n=3).CL-PARP and CL-Cas-3 indicate cleaved PARP and cleaved caspase-3, respectively.***, p ˂ 0.001.(J) The representative images were also shown.(K-M) (K) The growth curves of SW480 cells expressing control or Flag-MLF2.(L)

Figure S5 .
Figure S5.Related to Figure 6.(A-D) A total of 2 × 10 6 HCT116 cells transduced with lentiviruses expressing control or Flag-MLF2 were individually injected into nude mice (n = 6 for each group).Xenograft tumors were taken four 24 days after injection (A).Excised tumors were weighed (B).Tumor sizes were measured at the indicated time points (C).Protein extracts from the excised xenografts were also analyzed by western blotting (D).**, p ˂ 0.01; ***, p < 0.001.