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 1

In vivo genome-wide CRISPR activation screen identifies genes promoting lung metastasis of osteosarcoma.

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In vivo genome-wide CRISPR activation screen identifies genes promoting ...
(A) Diagram of in vivo CRISPR activation screening in U2OS/MTX300 cells. (B) Venn diagram indicates the overlaps among in vivo CRISPR activation screening, STUB1 interactome, and R2 database analysis. (C and D) Western blot analysis of MLF2 protein levels in U2OS/MTX300 and 143B cells stably expressing MLF2-targeted shRNAs or overexpression of MLF2. Data are representative of 3 independent experiments. (E–G) Quantification analyses of migration and invasion assays using the indicated U2OS/MTX300 or 143B cells stably expressing MLF2-targeted shRNAs or overexpression of MLF2. The data are presented as mean ± SD. n = 3 biologically independent experiments. P values were calculated using 1-way ANOVA with Dunnett’s test (E and F) and 2-tailed Student’s t test (G). (H–K) The procedure for the in vivo orthotopic model of osteosarcoma metastasis. Representative bioluminescence images of mice orthotopically transplanted with the indicated luciferase-transduced U2OS/MTX300 or 143B cells stably expressing MLF2-targeted shRNAs or overexpression of MLF2 (left) and quantification analyses of the metastasis of cancer cells in the lung based on the left (right). n = 6 mice per group. Data are presented as mean ± SD. P values were calculated using 2-tailed Student’s t test (J and K) and 2-tailed Student’s t test followed by Benjamini-Hochberg correction (H and I). NC, negative control; p, photon; sh, short hairpin; sr, steradian.