Myo9b is a key player in SLIT/ROBO-mediated lung tumor suppression
Ruirui Kong, … , Wei Feng, Jane Y. Wu
Ruirui Kong, … , Wei Feng, Jane Y. Wu
Published November 3, 2015
Citation Information: J Clin Invest. 2015;125(12):4407-4420. https://doi.org/10.1172/JCI81673.
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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 1

SLIT2 inhibits cell migration by regulating RhoA activity in lung cancer cells.

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SLIT2 inhibits cell migration by regulating RhoA activity in lung cancer...
(A) A wound-healing assay was performed using H1299 cells treated with control (Ctr), SLIT2 (SL), and ROBO1N (RN) media. At 0 and 13 hours after wound formation, phase-contrast images of migrating cells were obtained using an inverted microscope. Scale bar: 100 μm. Data represent 5 independent experiments. (B) Quantification of cell migration distance over a 13-hour period. Data represent the mean ± SEM of 3 independent experiments. ***P < 0.0001, Mann-Whitney U test. (C) H1299 cells were transfected with plasmids encoding either WT CDC42-Myc or Rac1-Myc and treated 48 hours after transfection with mock control or SLIT2 media for 5 or 15 minutes. Cell lysates were then prepared, a GST pull-down assay was performed using GST-PBD, and immunoblotting with anti-Myc Ab was done to measure active or total CDC42 or Rac1 levels in pull-down or total cell lysates, respectively. Active RhoA or total RhoA levels were measured by GST pull-down with GST-RBD and analyzed by immunoblotting with specific anti-RhoA Ab. SLIT protein levels in the culture media and actin (as an internal loading control) are also shown. (D and E) Cell migration in the H1299Ctr (Ctr) and H1299SLIT (SL) groups following transfection with a control vector or a Myc-tagged DN-RhoA plasmid as determined by wound-healing assay. Images were taken 0 and 12 hours after wound formation. Scale bar: 100 μm. n = 5. Data in E represent the mean ± SEM of 3 independent experiments. ***P < 0.0001, Mann-Whitney U test.