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FMNL1 promotes growth and metastasis of breast cancer by inhibiting BRCA1 via upregulation of HMGA1

Purpose: To investigate the role and mechanism of formin-like protein 1 (FMNL1) in breast cancer progression. Methods: Expression of FMNL1 in breast cancer cells was evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. Colony formation and CCK8 assays were performed to assess cell proliferation. Cell migration and invasion were determined using wound-healing and Transwell assays, respectively. Results: Data from UALCAN prediction (http://ualcan.path.uab.edu/analysis.html) showed that FMNL1 was significantly upregulated in primary breast cancer tissue compared to normal tissue (p < 0.01). Enhanced FMNL1 mRNA and protein expression was also identified in breast cancer cells. shRNAmediated FMNL1 knockdown decreased viability of breast cancer cells and reduced cell proliferation, migration, and invasion. Expression of protein high mobility group AT-hook 1 (HMGA1) was reduced, whereas breast cancer gene 1 (BRCA1) expression was enhanced, in breast cancer cells transfected with shRNA-FMNL1. Overexpression of HMGA1 attenuated FMNL1-knockdown–induced decreased HMGA1 expression and increased BRCA1 expression in breast cancer cells. BRCA1 knockdown counteracted the suppressive effects of FMNL1 silencing on breast cancer cell proliferation, migration, and invasion. Conclusion: FMNL1 promotes breast cancer cell growth and metastasis by inhibiting BRCA1 via upregulation of HMGA1, providing a potential therapeutic target for breast cancer.


INTRODUCTION
Breast cancer is one of the most common malignancies among women worldwide [1]. Early diagnosis and improvement of therapeutic strategies has reduced the death rate among breast cancer patients [1]. However, metastasis is still the major cause of death in breast cancer patients, accounting for more than 90 % of cancer-related mortality [1]. Therefore, strategies to suppress breast cancer metastasis are important for the treatment of breast cancer.
Formin-like protein 1 (FMNL1) is expressed in hematopoietic and lymphoid tissues, binds to actin filaments, and participates in cellular processes, such as phagocytosis, cell adhesion, and cell migration [2]. FMNL1 has been reported to be overexpressed in human hematological malignancies, including leukemic and Non-Hodgkin's lymphoma cell lines [3], and contributes to leukemic cell proliferation and migration [4]. Downregulation of FMNL1 suppressed bone metastasis in non-small cell lung cancer through reduction of transforming growth factor beta 1 (TGFβ1) [5]. FMNL1 was overexpressed in basal type breast cancer [6], and the invasiveness of breast carcinoma cells was suppressed through downregulation of FMNL1 [7]. However, the mechanism involved in FMNL1-mediated breast cancer progression has not yet been reported.
High mobility group AT-hook 1 (HMGA1) interacts with the transcription machinery to regulate transcription of target genes through alteration of chromatin structure and participates in benign and malignant neoplasias [8].
Overexpression of HMGA1 promoted breast cancer invasion and metastasis [9]. Silencing of HMGA1 slowed breast cancer growth and metastasis [10]. FMNL1 was hypothesized to mediate HMGA1 during the progression of breast cancer.
The involvement of FMNL1 in breast cancer cell growth and metastasis was investigated in this study, and targeting the downstream pathway involved in the metastatic pattern of FMNL1mediated breast cancer might provide a novel strategy for treatment.

Cell proliferation
Two days post-transfection, MCF-7 cells were plated in a 6-well plate. The DMEM was replaced every 3 days, and 14 days later formaldehydefixed cells were stained with 0.4 % crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The cells were counted under a light microscope (Olympus, Tokyo, Japan).

Wound-healing assay
Two days post-transfection, MCF-7 cells were plated in a 6-well plate, and a scratch wound was made in the monolayer with a pipette. Twentyfour hours later, cellular debris was removed, and wound width was photographed under a microscope (Olympus).

Transwell assay
The upper chamber of a transwell chamber (BD Biosciences, San Jose, CA, USA) was coated with Matrigel (Clontech, Mountain View, CA, USA), and 100 μL MCF-7 cell suspension in serum-free DMEM was added to the chamber. The lower chamber was filled with DMEM with 15 % FBS (400 μL). Forty-eight hours later, formaldehyde-fixed cells in the lower chamber were stained with 0.1 % crystal violet (Sigma-Aldrich) and counted under a microscope (Olympus).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
RNA was isolated from the breast cancer cells using Trizol (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized using the Reverse Transcription System (Thermo Fisher Scientific), and expression of FMNL1 mRNA was determined using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with β-actin as the endogenous control. The primer sequences used are shown in Table 1.

Western blotting
Cells were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific), and the protein concentration was determined using the bicinchoninic acid protein kit (Thermo Fisher Scientific). SDS-PAGE was used to separate the samples, and the samples were then electrotransferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5 % bovine serum albumin and probed overnight with primary antibodies: anti-FMNL1 (1:2000, Cell Signaling Technology, Danvers, MA, USA), anti-HMGA1 and anti-BRCA1 (1:2500, Cell Signaling Technology), and anti-β-actin (1:4000, Cell Signaling Technology). Following incubation with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000; Cell Signaling Technology), the density of immunoreactive bands on the membranes was detected using enhanced chemiluminescence (KeyGen, Nanjin, China).

Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM), and comparison between groups was determined by one-way analysis of variance (ANOVA) or Student's t-test. The p < 0.05 was considered statistically significant.

FMNL1 contributed to breast cancer cell metastasis
In addition to the anti-proliferative effect on breast cancer cells, silencing of FMNL1 inhibited MCF-7 cell migration (Figure 3 A) and invasion (Figure 3 B), indicating that knockdown of FMNL1 played an anti-invasive role in breast cancer cells.

FMNL1 inhibited BRCA1 through downregulation of HMGA1
Western blotting was used to assess the underlying mechanism involved in FMNL1mediated breast cancer progression. Expression of HMGA1 protein was reduced, whereas BRCA1 expression was enhanced, in MCF-7 cells transfected with shFMNL1 ( Figure 4). However, overexpression of HMGA1 downregulated expression of BRCA1 protein and upregulated HMGA1 protein in MCF-7 cells transfected with shFMNL1 (Figure 4), revealing that silencing of FMNL1 increased BRCA1 expression through downregulation of HMGA1 in breast cancer cells.

DISCUSSION
Formins regulate cytoskeletal remodeling through binding to actin filaments, thus modulating the migration and invasion of adherent cancer cells [11]. Inhibition of formin homology 2 domain containing 1 interfered with breast cancer cell proliferation and invasion [12]. Because a previous study showed that FMNL1 was overexpressed in basal type breast cancer [6], the role of FMNL1 in breast cancer progression was investigated in this study.
Firstly, FMNL1 was overexpressed in breast cancer cells. Arjonen et al showed that FMNL1 was upregulated in breast cancer with poor prognosis [13]. The relation between FMNL1 expression and clinicopathological parameters of patients with breast cancer should be investigated to explore the diagnostic and prognostic roles in breast cancer. Consistent with the findings of a previous study that downregulation of FMNL1 suppressed the invasiveness of breast carcinoma cells [7], knockdown of FMNL1 in this study reduced viability of breast cancer cells and repressed cell proliferation, migration, and invasion.
Results of this study demonstrated that expression of HMGA1 protein was reduced by knockdown of FMNL1. Ectopic expression of HMGA1 attenuated the FMNL1 silencing-induced decrease of HMGA1. Suppression of FMNL1 blocked the TGFβ1 pathway to reduce bone metastasis in non-small cell lung cancer [5]. TGFβ1 has been shown to induce specificity protein 1 and enhance the promoter activity of HMGA1 to contribute to breast cancer cell growth and metastasis [14]. Hence, silencing of FMNL1 in this study might suppress the promoter activity of HMGA1 through reduction of TGFβ1 to decrease HMGA1 expression and suppress breast cancer progression. A previous study has shown that HMGA1 binds to the promoter of BRCA1, and negatively regulates BRCA1 expression in sporadic breast carcinoma [15]. Expression of BRCA1 protein was enhanced by knockdown of FMNL1 in breast cancer cells, and ectopic expression of HMGA1 attenuated the FMNL1 silencing-induced increase in BRCA1. BRCA1 has a pleiotropic biological function and may play a role in transcriptional regulation, chromatin remodeling, DNA damage repair, cell cycle regulation, and checkpoint control [16]. Reduced BRCA1 expression is often observed in sporadic breast cancer, and decreased BRCA1 expression is positively correlated with the invasion of human breast cancer [17]. High expression levels of BRCA1 inhibited the proliferation of breast cancer cells [18].
Knockdown of BRCA1 in this study attenuated FMNL1-induced inhibition of breast cancer progression. Therefore, silencing of FMNL1 might suppress breast cancer cell growth and metastasis through downregulation of HMGA1 and upregulation of BRCA1.

CONCLUSION
This study provides evidence that FMNL1 contributes to breast cancer cell growth and metastasis. Furthermore, knockdown of FMNL1 in vitro decreases HMGA1 and increases BRCA1 to suppress breast cancer cell growth and metastasis. However, a clinically relevant animal model of breast cancer should be used to confirm the suppressive role of FMNL1 silencing on breast cancer growth.

Conflict of interest
No conflict of interest is associated with this work.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Qian Zhang and Hua Yang designed the study and supervised the data collection. Caiyi Tang and Qian Wang analyzed and interpreted the data. Lili Ren, Chao Jia, and Hui Zou prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Open Access
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.