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Zhao B, Ye DM, Li S, Zhang Y, Zheng Y, Kang J, Wang L, Zhao N, Ahmad B, Sun J, Yu T, Wu H. FMNL3 Promotes Migration and Invasion of Breast Cancer Cells via Inhibiting Rad23B-Induced Ubiquitination of Twist1. J Cell Physiol 2025; 240:e31481. [PMID: 39582466 DOI: 10.1002/jcp.31481] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2024] [Revised: 10/18/2024] [Accepted: 10/24/2024] [Indexed: 11/26/2024]
Abstract
Breast cancer is a heterogeneous malignant tumor, and its high metastasis rate depends on the abnormal activation of cell dynamics. Formin-like protein 3 (FMNL3) plays an important role in the formation of various cytoskeletons that participate in cell movement. The objective of this study was to explore the function of FMNL3 in breast cancer progression and endeavor to reveal the molecular mechanism of this phenomenon. We found that FMNL3 was abnormally highly expressed in aggressive breast cancer cells and tissues, and it significantly inhibited E-cadherin expression. FMNL3 could specifically interact with Twist1 rather than other epithelial-mesenchymal transition transcription factors (EMT-TFs). We also found that FMNL3 enhanced the repressive effect of Twist1 on CDH1 transcription in breast cancer cells. Further mechanism studies showed that FMNL3 suppressed the ubiquitin degradation of Twist1 by inhibiting the interaction between Twist1 and Rad23B, the ubiquitin transfer protein of Twist1. In vitro functional experiments, it was confirmed that FMNL3 promoted the migration and invasion of breast cancer cells by regulating Twist1. Furthermore, Twist1 could directly bind to the fmnl3 promoter to facilitate FMNL3 transcription. To conclude, this study indicated that FMNL3 acted as a pro-metastasis factor in breast cancer by promoting Twist1 stability to suppress CDH1 transcription.
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Affiliation(s)
- Binggong Zhao
- School of Bioengineering and Key Laboratory of Protein Modification and Disease, Dalian University of Technology, Dalian, Liaoning, China
| | - Dong-Man Ye
- Department of Medical Imaging, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning, China
| | - Shujing Li
- School of Bioengineering and Key Laboratory of Protein Modification and Disease, Dalian University of Technology, Dalian, Liaoning, China
| | - Yong Zhang
- Department of Medical Imaging, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning, China
| | - Yang Zheng
- Department of Medical Imaging, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning, China
| | - Jie Kang
- School of Bioengineering and Key Laboratory of Protein Modification and Disease, Dalian University of Technology, Dalian, Liaoning, China
| | - Luhong Wang
- School of Bioengineering and Key Laboratory of Protein Modification and Disease, Dalian University of Technology, Dalian, Liaoning, China
| | - Nannan Zhao
- Department of Medical Imaging, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning, China
| | - Bashir Ahmad
- School of Bioengineering and Key Laboratory of Protein Modification and Disease, Dalian University of Technology, Dalian, Liaoning, China
| | - Jing Sun
- Department of Medical Imaging, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning, China
| | - Tao Yu
- Department of Medical Imaging, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning, China
| | - Huijian Wu
- School of Bioengineering and Key Laboratory of Protein Modification and Disease, Dalian University of Technology, Dalian, Liaoning, China
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Zeng YF, Xiao YS, Liu Y, Luo XJ, Wen LD, Liu Q, Chen M. Formin-like 3 regulates RhoC/FAK pathway and actin assembly to promote cell invasion in colorectal carcinoma. World J Gastroenterol 2018; 24:3884-3897. [PMID: 30228782 PMCID: PMC6141330 DOI: 10.3748/wjg.v24.i34.3884] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2018] [Revised: 06/16/2018] [Accepted: 06/27/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To clarify the underlying mechanism of formin-like 3 (FMNL3) in the promotion of colorectal carcinoma (CRC) cell invasion.
METHODS The in vitro biological function analyses of FMNL3 were performed by gain- and loss-of function approaches. Changes in the F-actin cytoskeleton were detected by the technologies of phalloidin-TRITC labeling and confocal microscopy. The signaling pathway mediated by FMNL3 was explored by western blot, gelatin zymograph assay, co-immunoprecipitation (co-IP), immunofluorescence co-localization, and glutathione S-transferase (GST) pull-down assay.
RESULTS The in vitro experimental results showed that FMNL3 significantly promoted the proliferation, invasion, and migration of CRC cells (P < 0.05 and P < 0.01). Moreover, FMNL3 regulated the remodeling of actin-based protrusions such as filopodia and lamellipodia in a RhoC-dependent manner. The western blot and gelatin zymograph assay results indicated that FMNL3 was involved in the RhoC/ focal adhesion kinase (FAK) 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 resulted in the increased expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF), and the subsequent promotion of CRC cell invasion. The results of TAE226, U0126 or Ly294002 treatment confirmed an essential role of FMNL3 in activation of the RhoC/FAK pathway and the subsequent promotion of CRC invasion. Co-IP, co-localization and GST pull-down assays showed the direct interaction of FMNL3 with RhoC in vivo and in vitro.
CONCLUSION FMNL3 regulates the RhoC/FAK signaling pathway and RhoC-dependent remodeling of actin-based protrusions to promote CRC invasion.
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Affiliation(s)
- Yuan-Feng Zeng
- 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
| | - Yong Liu
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, 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
| | - Qian Liu
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
| | - Min Chen
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
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Péladeau C, Heibein A, Maltez MT, Copeland SJ, Copeland JW. A specific FMNL2 isoform is up-regulated in invasive cells. BMC Cell Biol 2016; 17:32. [PMID: 27578625 PMCID: PMC5006604 DOI: 10.1186/s12860-016-0110-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2016] [Accepted: 08/23/2016] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Formins are a highly conserved family of cytoskeletal remodeling proteins. A growing body of evidence suggests that formins play key roles in the progression and spread of a variety of cancers. There are 15 human formin proteins and of these the Diaphanous-Related Formins (DRFs) are the best characterized. Included in the DRFs are the Formin-Like proteins, FMNL1, 2 & 3, each of which have been strongly implicated in driving tumorigenesis and metastasis of specific tumors. In particular, increased FMNL2 expression correlates with increased invasiveness of colorectal cancer (CRC) in vivo and for a variety of CRC cell-lines in vitro. FMNL2 expression is also required for invasive cell motility in other cancer cell-lines. There are multiple alternatively spliced isoforms of FMNL2 and it is predicted that the encoded proteins will differ in their regulation, subcellular localization and in their ability to regulate cytoskeletal dynamics. RESULTS Using RT-PCR we identified four FMNL2 isoforms expressed in CRC and melanoma cell-lines. We find that a previously uncharacterized FMNL2 isoform is predominantly expressed in a variety of melanoma and CRC cell lines; this isoform is also more effective in driving 3D motility. Building on previous reports, we also show that FMNL2 is required for invasion in A375 and WM266.4 melanoma cells. CONCLUSIONS Taken together, these results suggest that FMNL2 is likely to be generally required in melanoma cells for invasion, that a specific isoform of FMNL2 is up-regulated in invasive CRC and melanoma cells and this isoform is the most effective at facilitating invasion.
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Affiliation(s)
- Christine Péladeau
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada
| | - Allan Heibein
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada
| | - Melissa T Maltez
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada
| | - Sarah J Copeland
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada
| | - John W Copeland
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada.
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Zeng YF, Xiao YS, Lu MZ, Luo XJ, Hu GZ, Deng KY, Wu XM, Xin HB. Increased expression of formin-like 3 contributes to metastasis and poor prognosis in colorectal carcinoma. Exp Mol Pathol 2015; 98:260-7. [PMID: 25758200 DOI: 10.1016/j.yexmp.2015.03.008] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2015] [Accepted: 03/06/2015] [Indexed: 02/06/2023]
Abstract
Formin-like 3 (FMNL3), a member of diaphanous-related formins subfamily, plays an important role in cytoskeleton reorganization, cell adhesion and cancer cell invasion in vitro. This study aimed to explore the expression of FMNL3 in colorectal carcinoma (CRC) cell-lines and tissues, and further evaluate its prognostic value and correlation with the clinicopathological parameters, and also investigate the effects of FMNL3 gene silencing on the growth and metastasis of CRC in vivo. Immunohistochemical analysis showed that FMNL3 protein was distributed in a punctuate aggregation pattern and located mainly in the cytoplasm of glandular cavity side, close to the nucleus of CRC cells. The positive rate of FMNL3 expression was 87.5% (84/96) in CRC, which was significantly higher than that in adjacent normal mucosa (30%, 9/30). Moreover, FMNL3 protein expressed far more in primary CRC with metastasis and corresponding lymph nodes metastatic CRC than in primary CRC without metastasis. Increased expression of FMNL3 was closely correlated with tumor size, differentiation, serosal invasion, and both lymph node metastasis and distant metastasis. However, it was not correlated with patients' age and gender. According to Kaplan-Meier survival analyses, patients with FMNL3 high expression level had lower overall survival rate than that with FMNL3 low expression level. Univariate and multivariate analyses revealed that high FMNL3 expression was a significant and independent prognostic predictor of patients with CRC. In addition, FMNL3 mRNA and protein levels were substantially up-regulated in CRC-metastasis-derived cell lines, as compared to those in primary-CRC-derived ones. FMNL3 gene silencing suppressed the growth and metastasis of CRC in vivo. In conclusion, FMNL3 plays an important role in the progression and metastasis of CRC and may be a novel potential prognostic predictor and therapeutic target for patients with CRC.
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Affiliation(s)
- Yuan-Feng Zeng
- Department of Pathology, Jiangxi Provincial People's Hospital, Nanchang, China; Institute of Translational Medicine, Nanchang University, Nanchang, China
| | - Yi-Sheng Xiao
- Teaching and Researching Section of Morphology, College of Basic Medicine, Jiangxi University of Chinese Traditional Medicine, Nanchang, China
| | - Ming-Zhi Lu
- Department of Pathology, Jiangxi Provincial People's Hospital, Nanchang, China
| | - Xiao-Jiang Luo
- Department of General Surgery, Jiangxi Provincial People's Hospital, Nanchang, China
| | - Guo-Zhu Hu
- Institute of Clinical Medical Sciences, Jiangxi Provincial People's Hospital, Nanchang, China
| | - Ke-Yu Deng
- Institute of Translational Medicine, Nanchang University, Nanchang, China
| | - Xiao-Mu Wu
- Jiangxi Institute of Neurology, Jiangxi Provincial People's Hospital, Nanchang, China
| | - Hong-Bo Xin
- Institute of Translational Medicine, Nanchang University, Nanchang, China.
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Abstract
Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells.
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Affiliation(s)
- Sonja Kühn
- Center of Advanced European Studies and Research (caesar); Group Physical Biochemistry; Bonn, Germany
| | - Matthias Geyer
- Center of Advanced European Studies and Research (caesar); Group Physical Biochemistry; Bonn, Germany
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Protein N-myristoylation is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3. Biosci Biotechnol Biochem 2012; 76:1201-9. [PMID: 22790947 DOI: 10.1271/bbb.120069] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-myristoylation by replacement of Gly2 with Ala or by the use of N-myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.
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Vega FM, Fruhwirth G, Ng T, Ridley AJ. RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. ACTA ACUST UNITED AC 2011; 193:655-65. [PMID: 21576392 PMCID: PMC3166870 DOI: 10.1083/jcb.201011038] [Citation(s) in RCA: 208] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Although closely related, RhoA and RhoC have distinct molecular targets and functional roles in cell migration and invasion. Several studies suggest that RhoA and RhoC, despite their sequence similarity, have different roles in cell migration and invasion, but the molecular basis for this is not known. Using RNAi, we show that RhoA-depleted cells became elongated and extended multiple Rac1-driven narrow protrusions in 2D and 3D environments, leading to increased invasion. These phenotypes were caused by combined but distinct effects of the Rho-regulated kinases ROCK1 and ROCK2. Depletion of ROCK2 induced multiple delocalized protrusions and reduced migratory polarity, whereas ROCK1 depletion selectively led to cell elongation and defective tail retraction. In contrast, RhoC depletion increased cell spreading and induced Rac1 activation around the periphery in broad lamellipodia, thereby inhibiting directed migration and invasion. These effects of RhoC depletion are mediated by the formin FMNL3, which we identify as a new target of RhoC but not RhoA. We propose that RhoA contributes to migratory cell polarity through ROCK2-mediated suppression of Rac1 activity in lamellipodia, whereas RhoC promotes polarized migration through FMNL3 by restricting lamellipodial broadening.
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Affiliation(s)
- Francisco M Vega
- Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, England, UK
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Marc Rhoads J, Wu G. Glutamine, arginine, and leucine signaling in the intestine. Amino Acids 2009; 37:111-122. [PMID: 19130170 DOI: 10.1007/s00726-008-0225-4] [Citation(s) in RCA: 234] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2008] [Accepted: 12/09/2008] [Indexed: 12/14/2022]
Abstract
Glutamine and leucine are abundant constituents of plant and animal proteins, whereas the content of arginine in foods and physiological fluids varies greatly. Besides their role in protein synthesis, these three amino acids individually activate signaling pathway to promote protein synthesis and possibly inhibit autophagy-mediated protein degradation in intestinal epithelial cells. In addition, glutamine and arginine stimulate the mitogen-activated protein kinase and mammalian target of rapamycin (mTOR)/p70 (s6) kinase pathways, respectively, to enhance mucosal cell migration and restitution. Moreover, through the nitric oxide-dependent cGMP signaling cascade, arginine regulates multiple physiological events in the intestine that are beneficial for cell homeostasis and survival. Available evidence from both in vitro and in vivo animal studies shows that glutamine and arginine promote cell proliferation and exert differential cytoprotective effects in response to nutrient deprivation, oxidative injury, stress, and immunological challenge. Additionally, when nitric oxide is available, leucine increases the migration of intestinal cells. Therefore, through cellular signaling mechanisms, arginine, glutamine, and leucine play crucial roles in intestinal growth, integrity, and function.
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Affiliation(s)
- J Marc Rhoads
- Department of Pediatrics, University of Texas Medical School at Houston, Houston, TX 77030, USA.
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Glutamine in neoplastic cells: focus on the expression and roles of glutaminases. Neurochem Int 2009; 55:71-5. [PMID: 19428809 DOI: 10.1016/j.neuint.2009.01.008] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2008] [Revised: 01/09/2009] [Accepted: 01/14/2009] [Indexed: 01/21/2023]
Abstract
Glutamine is an important source of energy for neoplastic tissues, and products of its metabolism include, among others, glutamate (Glu) and glutathione (GSH), the two molecules that play a key role in tumor proliferation, invasiveness and resistance to therapy. Glutamine hydrolysis in normal and transforming mammalian tissues alike, is carried out by different isoforms of glutaminases, of which the two major are liver-type glutaminase (LGA) and kidney-type glutaminase (KGA). This brief review summarizes available data on the expression profiles and activities of these isoenzymes in different neoplastic tissues as compared to the tissues of origin, and dwells on recent work demonstrating effects of manipulation of glutaminase expression on tumor growth. A comment is devoted to the emerging evidence that LGA, apart from degrading Gln for metabolic purposes, is involved in gene transcription; its enforced overexpression in glioma cells was found to reduce their proliferation and migration.
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