Published online Sep 14, 2018. doi: 10.3748/wjg.v24.i34.3884
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
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.
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.
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.
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.
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.
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.
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.