PIM3-mediated phosphorylation stabilizes myeloid leukemia factor 2 to promote metastasis in osteosarcoma
Cuiling Zeng, Xin Wang, Jinkun Zhong, Yu Zhang, Ju Deng, Wenqiang Liu, Weixuan Chen, Xinhao Yu, Dian Lin, Ruhua Zhang, Shang Wang, Jianpei Lao, Qi Zhao, Li Zhong, Tiebang Kang, Dan Liao
Cuiling Zeng, Xin Wang, Jinkun Zhong, Yu Zhang, Ju Deng, Wenqiang Liu, Weixuan Chen, Xinhao Yu, Dian Lin, Ruhua Zhang, Shang Wang, Jianpei Lao, Qi Zhao, Li Zhong, Tiebang Kang, Dan Liao
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Research Article Cell biology Oncology

PIM3-mediated phosphorylation stabilizes myeloid leukemia factor 2 to promote metastasis in osteosarcoma

Abstract

Osteosarcoma is the most common primary malignant bone cancer, characterized by a high incidence of lung metastasis and a lack of therapeutic targets. Here, by combining an in vivo CRISPR activation screen with the interactome of STUB1, a tumor suppressor in osteosarcoma, we identified that myeloid leukemia factor 2 (MLF2) promotes osteosarcoma metastasis. Mechanistically, MLF2 disrupted the interaction between BiP and IRE1α, thereby activating the IRE1α/XBP1-S-MMP9 axis. The E3 ligase STUB1 ubiquitinated MLF2 at Lys119 and targeted it for proteasomal degradation, whereas PIM3-mediated phosphorylation of MLF2 at Ser65 enhanced its stabilizing interaction with USP21. Our findings demonstrate that the PIM3/MLF2 axis is a critical regulator of osteosarcoma lung metastasis. We propose PIM3 as a potential therapeutic target for patients with osteosarcoma lung metastasis.

Authors

Cuiling Zeng, Xin Wang, Jinkun Zhong, Yu Zhang, Ju Deng, Wenqiang Liu, Weixuan Chen, Xinhao Yu, Dian Lin, Ruhua Zhang, Shang Wang, Jianpei Lao, Qi Zhao, Li Zhong, Tiebang Kang, Dan Liao

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Figure 6

The phosphorylation of MLF2 at S65 contributed to its binding with USP21.

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The phosphorylation of MLF2 at S65 contributed to its binding with USP21...
(A–C) HEK293T cells were cotransfected with the indicated plasmids for 48 hours and then subjected to IP using anti-FLAG antibody or anti-Myc antibody, followed by Western blotting. (D and E) Western blotting analysis and quantification of MLF2 and USP21 protein levels in the indicated U2OS or 143B cells stably expressing USP21-targeted shRNAs or overexpressing USP21. (F and G) The indicated U2OS stable cells with or without USP21 knockdown were incubated with 20 μg/mL cycloheximide (CHX) for the indicated periods and then analyzed by Western blotting (F). Quantitation of MLF2 protein levels was based on the Western blotting results (G). (H) U2OS cells stably expressing USP21-targeted shRNAs were cotransfected with MLF2-SFB and HA-ubiquitin (HA-Ub) for 48 hours and then subjected to IP using anti-FLAG antibody followed by Western blotting. (I and J) HEK293T cells cotransfected with MLF2-SFB and vector, USP21, or mutant USP21 (C221A) for 36 hours were incubated with 40 μg/mL CHX for the indicated periods and then analyzed by Western blotting (I). Quantitation of MLF2 protein levels was based on the Western blotting results (J). (K) HEK293T cells were cotransfected with the indicated plasmids for 48 hours and then subjected to IP using anti-FLAG antibody followed by Western blotting. Data in A–C, F, H, I, and K are representative of 3 independent experiments. Data in D, E, G, and J are presented as mean ± SD. n = 3 biologically independent experiments. P values were calculated using 1-way ANOVA with Dunnett’s test (D and J) and 2-tailed Student’s t test (E and G). NC, negative control; sh, short hairpin.