Myo9b is a key player in SLIT/ROBO-mediated lung tumor suppression
Ruirui Kong, Fengshuang Yi, Pushuai Wen, Jianghong Liu, Xiaoping Chen, Jinqi Ren, Xiaofei Li, Yulong Shang, Yongzhan Nie, Kaichun Wu, Daiming Fan, Li Zhu, Wei Feng, Jane Y. Wu
Ruirui Kong, Fengshuang Yi, Pushuai Wen, Jianghong Liu, Xiaoping Chen, Jinqi Ren, Xiaofei Li, Yulong Shang, Yongzhan Nie, Kaichun Wu, Daiming Fan, Li Zhu, Wei Feng, Jane Y. Wu
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Research Article Oncology

Myo9b is a key player in SLIT/ROBO-mediated lung tumor suppression

Abstract

Emerging evidence indicates that the neuronal guidance molecule SLIT plays a role in tumor suppression, as SLIT-encoding genes are inactivated in several types of cancer, including lung cancer; however, it is not clear how SLIT functions in lung cancer. Here, our data show that SLIT inhibits cancer cell migration by activating RhoA and that myosin 9b (Myo9b) is a ROBO-interacting protein that suppresses RhoA activity in lung cancer cells. Structural analyses revealed that the RhoGAP domain of Myo9b contains a unique patch that specifically recognizes RhoA. We also determined that the ROBO intracellular domain interacts with the Myo9b RhoGAP domain and inhibits its activity; therefore, SLIT-dependent activation of RhoA is mediated by ROBO inhibition of Myo9b. In a murine model, compared with control lung cancer cells, SLIT-expressing cells had a decreased capacity for tumor formation and lung metastasis. Evaluation of human lung cancer and adjacent nontumor tissues revealed that Myo9b is upregulated in the cancer tissue. Moreover, elevated Myo9b expression was associated with lung cancer progression and poor prognosis. Together, our data identify Myo9b as a key player in lung cancer and as a ROBO-interacting protein in what is, to the best of our knowledge, a newly defined SLIT/ROBO/Myo9b/RhoA signaling pathway that restricts lung cancer progression and metastasis. Additionally, our work suggests that targeting the SLIT/ROBO/Myo9b/RhoA pathway has potential as a diagnostic and therapeutic strategy for lung cancer.

Authors

Ruirui Kong, Fengshuang Yi, Pushuai Wen, Jianghong Liu, Xiaoping Chen, Jinqi Ren, Xiaofei Li, Yulong Shang, Yongzhan Nie, Kaichun Wu, Daiming Fan, Li Zhu, Wei Feng, Jane Y. Wu

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Figure 4

The Myo9b RhoGAP domain contains a unique region that specifically recognizes RhoA.

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The Myo9b RhoGAP domain contains a unique region that specifically recog...
(A) Ribbon diagram for the structural model of the Myo9b RhoGAP/RhoA complex. The Myo9b RhoGAP domain and RhoA are shown in green and red, respectively. The Myo9b RhoGAP domain interacts with RhoA through the 3 patches to form a stable complex. The switch I, switch II, and A3 helix of RhoA are responsible for the binding to the RhoGAP domain and are labeled. (B) “Open-book” view of the interaction interfaces between the Myo9b RhoGAP domain and RhoA by a surface representation. Here, the residues are colored as in Figure 3E. (C) Combined ribbon-stick model to illustrate in detail the interaction interface between patch II and the A3 helix. The side chains of the residues involved in the interface packing between patch II and the A3 helix are represented as sticks and are shown in magenta and orange, respectively. GDP and MgF3 are shown as sticks and spheres, respectively. (D) Mutations inside patch II impaired Myo9b RhoGAP activity in the inactivation of RhoA. H1299 cells were transfected with the control vector (Ctr) or with plasmids encoding either the WT Myo9b RhoGAP domain (WT) or the indicated mutants. Cell extracts were subjected to GST pull-down assays to measure RhoA activity. (E) Mutations inside patch II disrupted binding between the Myo9b RhoGAP domain and RhoA. GST pull-down experiments were performed using recombinant WT or mutant forms of the GST-Myo9b RhoGAP domain and the cell lysates from HEK293 cells transfected with a Myc-RhoA plasmid.