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 2

Myo9b is a key player in SLIT/ROBO signaling in lung cancer cells.

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Myo9b is a key player in SLIT/ROBO signaling in lung cancer cells. (A) I...
(A) Interaction of endogenous ROBO1 and Myo9b proteins in H1299 lung cancer cells as demonstrated by co-IP experiments. IP was performed using the control IgG (Ctr) or anti-Myo9b Ab, followed by Western blotting. ROBO1 protein was detected in proteins immunoprecipitated by the specific anti-Myo9b, but not by the control Ab. (B) Interaction of the purified Myo9b RhoGAP domain (GST-GAP) with ROBO-ICD proteins as shown by GST pull-down experiments. (C) H1299Ctr and H1299SLIT (stably expressing SLIT2) cells transfected with siRNA against Myo9b (siMyo9b-1) or control siRNA (siCtr) were examined for cell migration using a wound-healing assay. Representative images at 0 and 12 hours after wound formation. Scale bar: 100 μm. (D) Cell migration distance (μm) for the different treatment groups shown in C was quantified and is presented as the mean ± SEM. ***P < 0.0001, Mann-Whitney U test. Data are representative of 5 independent experiments. (E) H1299 cells stably expressing SLIT2 were transfected with siMyo9b-1 or siCtr. GST pull-down experiments and Western blotting were performed to measure active and total RhoA levels.