Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 14, 2018; 24(34): 3884-3897
Published online Sep 14, 2018. doi: 10.3748/wjg.v24.i34.3884
Formin-like 3 regulates RhoC/FAK pathway and actin assembly to promote cell invasion in colorectal carcinoma
Yuan-Feng Zeng, Yi-Sheng Xiao, Yong Liu, Xiao-Jiang Luo, Li-Dan Wen, Qian Liu, Min Chen
Yuan-Feng Zeng, Yong Liu, Qian Liu, Min Chen, Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
Yi-Sheng Xiao, Teaching and Researching Section of Morphology, College of Basic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi Province, China
Xiao-Jiang Luo, Department of General Surgery, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
Li-Dan Wen, Clinical Medical Sciences Institute, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
Author contributions: Zeng YF, Xiao YS, Liu Y, Luo XJ, Wen LD, Liu Q and Chen M carried out the experiment; Zeng YF conceived experiments and analyzed data; all authors were involved in writing this paper and had final approval of the submitted and published versions.
Supported by the National Natural Science Foundation of China, No. 81201972; the China Postdoctoral Science Foundation, No. 2013M531555; and the Postdoctoral Science Foundation of Jiangxi province, No. 2013KY44.
Institutional review board statement: The study was reviewed and approved by Ethical Committee of Jiangxi Provincial People’s Hospital.
Conflict-of-interest statement: The authors declare no conflicts of interest in the present study.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Yuan-Feng Zeng, PhD, Chief Doctor, Department of Pathology, Jiangxi Provincial People’s Hospital, 152 Patriotic Road, Donghu District, Nanchang 330006, Jiangxi Province, China.
Telephone: +86-791-86895672 Fax: +86-791-86895672
Received: May 5, 2018
Peer-review started: May 7, 2018
First decision: May 23, 2018
Revised: June 16, 2018
Accepted: June 27, 2018
Article in press: June 27, 2018
Published online: September 14, 2018
Research background

Formin-like 3 (FMNL3) is a novel member of the diaphanous-related formins subfamily, which act as downstream effectors of Rho-GTPase signaling and regulate actin-dependent processes, such as cell motility and invasion. Increased expression of FMNL3 has been identified to contribute to metastasis and the poor prognosis of colorectal carcinoma (CRC). However, the exact molecular mechanism by which FMNL3 promotes CRC cell invasion and metastasis remains ambiguous. Therefore, elucidation of the underlying mechanism may help to block the metastatic progression and improve the survival rate of patients with CRC.

Research motivation

It is necessary to explore whether FMNL3 regulates Rho GTPase signaling to affect cytoskeletal organization and subsequent CRC cell invasion. Recent studies have demonstrated FMNL3 in reorganization of actin-dependent protrusions, such as filopodia and lamellipodia. The potential role of FMNL3 in tumor cell invasion and metastasis has also been reported in several tumor types. Moreover, FMNL3 acts as a downstream effector of RhoC to promote prostate cancer invasion by controlling lamellipodia. These findings give us a good lead for further study regarding the mechanism of FMNL3 regulation during CRC cell invasion and metastasis.

Research objectives

In this study, we investigate the effects of FMNL3 on CRC cell proliferation, invasion and migration in vitro by gain- and loss-of –function approaches. Moreover, we explore the role of FMNL3 in the RhoC-dependent signaling pathway and actin assembly dynamics, and the relation with CRC cell invasion. Our study provides significant insights into the signaling mechanism of FMNL3 during CRC invasion that may contribute to the future design of more effective metastasis-related therapies.

Research methods

Experiments using gene transfection or silencing were conducted to construct FMNL3 stably-expressed or –depleted cell lines to complete the following functional studies. A series of in vitro experiments, such as MTT, transwell chamber and scratch assays, were performed to explore the effects of FMNL3 on cell proliferation, invasion and migration. Rhodamine-conjugated phalloidin staining and confocal microscopy was used to display and observe F-actin dependent protrusions, such as filopodia and lamellipodia. Western blots and gelatin zymography assays were carried out to explore how the signaling pathway of FMNL3 is involved in CRC invasion. The key inhibitors of the RhoC/FAK pathway were used to treat CRC cells to verify the reliability of the signaling mechanism. In addition, the experiments involving immunofluorescence co-localization, co-immunoprecipitation and GST-pull downs were performed to unveil the partner of FMNL3 in the signaling pathway.

Research results

The results of in vitro experiments showed a positive role of FMNL3 in cell proliferation, invasion and migration of CRC. Rhodamine-conjugated phalloidin staining and confocal microscopy-based observations demonstrated that FMNL3 induced the elongation of filopodia, but inhibited the broadening of lamellipodia in a RhoC-dependent manner to enhance the invasive abilities of CRC cells. In addition, the results of western blots and gelatin zymography assays suggested that FMNL3 was involved in the RhoC/FAK signaling pathway and acted as an effector of RhoC to activate the downstream signaling of p-FAK as well as p-MAPK and p-AKT. This was followed by the increased expression of MMP-2, MMP-9 and VEGF, resulting in the promotion of CRC cell invasion. The results of inhibitor treatments confirmed the essential role of FMNL3 in the activation of the RhoC/FAK pathway and the subsequent promotion of CRC cell invasion. A direct interaction of FMNL3 with RhoC in vivo and in vitro was displayed by co-IP, co-localization and GST-pull down analyses.

Research conclusions

In conclusion, FMNL3 plays a positive role in CRC cell proliferation, invasion and migration. In addition, FMNL3 activates the RhoC/FAK signaling pathway via its interaction with RhoC. FMNL3 also regulates RhoC-dependent remodeling of actin-based protrusions, such as filopodia and lamellipodia, to promote CRC cell invasion. FMNL3 can be applied as a promising specific biomarker for CRC progression and metastasis.

Research perspectives

Our study illuminates the role and molecular mechanism of FMNL3 in the regulation of CRC invasion, and revealed RhoC’s involvement in the RhoC/FAK signaling pathway as the partner of FMNL3. Other groups reported the interaction of FMNL3 with Cdc42 or RhoJ, and proposed that FMNL3 acted as a downstream effector of Cdc42 or RhoJ to promote the filopodia outgrowth during endothelial lumen formation. There may be cross-talk between RhoC and other small GTPase proteins, such as Cdc42 and RhoJ, to coordinate the regulation of FMNL3-dependent CRC cell invasion. This, however, needs further investigation.