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.
View: Text | PDF
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

×

Figure 5

SLIT/ROBO signaling suppresses Myo9b RhoGAP activity.

Options: View larger image (or click on image) Download as PowerPoint
SLIT/ROBO signaling suppresses Myo9b RhoGAP activity. (A) Addition of pu...
(A) Addition of purified ROBO-ICD suppressed Myo9b RhoGAP activity in a dose-dependent manner. Extracts from HEK293 cells transfected with a Myc-RhoA plasmid were incubated with different combinations of purified Myo9b-GAP or control (MBP only) or ROBO-ICD at a molar ratio of 1:1, 1:2, or 1:3. GTP-RhoA levels were measured by GST pull-down, coupled with Western blotting. (B) Expression of ROBO1 suppressed Myo9b RhoGAP activity. H1299 cells were cotransfected with Flag-tagged Myo9b GAP and HA-tagged ROBO1 at different doses. GTP-RhoA levels were measured as described in A. (C) Purified ROBO-ICD blocked Myo9b RhoGAP-RhoA interaction in a concentration-dependent manner. Lysates from HEK293 cells transfected with a Myc-RhoA plasmid were incubated with GST-GAP in the presence of different concentrations of ROBO-ICD protein. RhoGAP and RhoA interaction was examined using GST pull-down. (D) SLIT overexpression led to increased GTP-RhoA levels by suppressing Myo9b RhoGAP activity. The parental H1299 cell line or H1299 cells that stably expressed SLIT2 (shown as SL– or SL+, respectively, in the corresponding lanes) were transfected with the control vector or full-length Myo9b, or different Myo9b deletion mutants (Myo9bGAP or Myo9bΔGAP). Extracts were subjected to GST pull-down experiments, and immunoblot analyses of the pull-down products are shown. (E) GTP-RhoA levels were quantified and are shown as the mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001, Mann-Whitney U test. (F) Expression of a dominant-negative mutant form of ROBO1 lacking its ICD (DN-ROBO) eliminated SLIT-induced activation of RhoA in H1299 cells. GST pull-down experiments, coupled with Western blotting, were performed to detect GTP-RhoA levels in H1299 cells following cotransfection with GFP-tagged DN-ROBO and Flag-tagged Myo9b-RhoGAP plasmids and treatment with the control or SLIT media.