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 3

MLF2 activates IRE1α/XBP1-S signaling via interfering in the binding between BiP and IRE1α.

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MLF2 activates IRE1α/XBP1-S signaling via interfering in the binding bet...
(A and B) HEK293T cells were cotransfected with MLF2-HA and BiP-Flag for 48 hours and then subjected to IP using anti-FLAG antibody (A) or anti-HA antibody (B) followed by Western blotting analysis. (C) The endogenous interaction between MLF2 and BiP in 143B cells was measured by co-IP with anti-MLF2 antibody, and isotype-matched IgG was used as a control. (D) The nuclear and cytoplasmic locations of MLF2 in 143B cells were analyzed by the NE-PER Nuclear and Cytoplasmic Extraction Kit. (E) The ER location of MLF2 in 143B cells was analyzed with the ER enrichment kit. (F) Schematic illustration of BiP structure. (G and H) The domain structure of BiP, which interacts with MLF2, was measured by co-IP. HEK293T cells were cotransfected with the indicated plasmids for 48 hours and then subjected to IP using anti-FLAG antibody (G) or anti-V5 antibody (H), followed by Western blotting analysis. (I and J) Lysates from U2OS cells stably overexpression of MLF2 (I) and 143B cells stably expressing MLF2-targeted shRNAs (J) were subjected to IP using anti-IRE1α antibody or anti-IgG antibody followed by Western blotting, as indicated. Data in A–E and G–J are representative of 3 independent experiments. NC, negative control; sh, short hairpin; NBD, nucleotide-binding domain; SBD, substrate-binding domain; WCL, whole cell lysate.