Silencing of KIF3B Suppresses Breast Cancer Progression by Regulating EMT and Wnt/β-Catenin Signaling

Breast cancer is the most common malignant tumors in women. Kinesin family member 3B (KIF3B) is a critical regulator in mitotic progression. The objective of this study was to explore the expression, regulation, and mechanism of KIF3B in 103 cases of breast cancer tissues, 35 metastatic lymph nodes and breast cancer cell lines, including MDA-MB-231, MDA-MB-453, T47D, and MCF-7. The results showed that KIF3B expression was up-regulated in breast cancer tissues and cell lines, and the expression level was correlated with tumor recurrence and lymph node metastasis, while knockdown of KIF3B suppressed cell proliferation, migration, and invasion both in vivo and in vitro. In addition, UALCAN analysis showed that KIF3B expression in breast cancer is increased, and the high expression of KIF3B in breast cancer is associated with poor prognosis. Furthermore, we found that silencing of KIF3B decreased the expression of Dvl2, phospho-GSK-3β, total and nucleus β-catenin, then subsequent down-regulation of Wnt/β-catenin signaling target genes such as CyclinD1, C-myc, MMP-2, MMP-7 and MMP-9 in breast cancer cells. In addition, KIF3B depletion inhibited epithelial mesenchymal transition (EMT) in breast cancer cells. Taken together, our results revealed that KIF3B is up-regulated in breast cancer which is potentially involved in breast cancer progression and metastasis. Silencing KIF3B might suppress the Wnt/β-catenin signaling pathway and EMT in breast cancer cells.


INTRODUCTION
Breast cancer is the most common cancer and the leading cause of death in women around the world (1). Over the past decades, therapeutic management, including surgical resection in combination with hormonal therapy, chemo-radiotherapy and biological therapy, has achieved great progress for this disease. However, the survival of advanced-stage breast cancer is still very poor (2). Moreover, the efficiency of the currently available chemotherapy is limited and the development of drug resistance among breast cancer cells further complicates the problem (3). Therefore, the detection of novel therapeutic targets may provide better treatment to such breast cancer patients.
It has been known that Wnt signaling pathway plays a critical role in cell proliferation, differentiation, and migration (4). Dvl2 (Disheveled 2), GSK-3b and b-catenin are the main regulators in Wnt signaling (5,6). During Wnt/b-catenin pathway activation, Dvl2 protein is up-regulated and GSK-3b is inhibited by phosphorylation at the Ser9 site, which decreased b-catenin degradation and led to b-catenin accumulating in the cytosol. b-catenin then travels to the nucleus and interacts with the T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate specific Wnt target genes, including C-myc, CyclinD1, MMP-2, MMP-7 and MMP-9, resulting in tumorigenesis and metastasis (7)(8)(9)(10). Moreover, Wnt/b-catenin signaling is also associated with the regulation of epithelial cell phenotype and the maintenance of tissue homeostasis. Impaired Wnt signaling pathway could contribute to epithelial mesenchymal transition (EMT) (11). It has also been shown that EMT plays a vital role in tumor progression and metastasis (12)(13)(14). EMT is a crucial event whereby epithelial cells lose their epithelial characteristics and acquire mesenchymal phenotype (15). At the molecular level, it's manifested with reduced E-cadherin expression but increased mesenchymal marker (Vimentin) and transcription factors' (Slug and Snail) expression (16)(17)(18). During EMT, epithelial cells lose cell junction and cell polarity, becoming isolated and motile (19,20). The significance of EMT and Wnt/b-catenin signaling in breast cancer proliferation and metastasis has been reported in previous studies (21)(22)(23).
Kinesin super family proteins (KIFs) were first discovered in the 1980s. As a class of conserved microtubule-dependent molecular motor proteins, KIFs transport intracellular cargo along microtubules (24,25). KIFs are also involved in multiple cellular processes, such as mitosis, meiosis, and vesicle transport. Mitosis is a complex and highly controlled process of cell division. Any abnormality during mitosis can lead to cell death, gene deletion, chromosome translocation, duplication, and even carcinogenesis (26,27). KIF3 is a subfamily of KIFs, including KIF3A/3B heterodimer and kinesin-associated protein KAP3 (28,29). This complex is involved in the intracellular transport of membrane-bound complexes and organelles in epithelial cells, neurons and other cells (30)(31)(32). KIF3B consists of a group of molecular motors and function in vesicle transport, spermatogenesis, mitotic progression, and intravasation of cancer cells for metastasis (33). Recently, the role of KIF3B in cancer progression has been widely studied. KIF3B has been shown to be over-expressed in multiple human cancers, such as gastric cancer, oral squamous cell carcinoma, pancreatic cancer, prostate cancer, seminoma, hepatocellular carcinoma, and acute lymphoblastic leukemia (34)(35)(36)(37)(38)(39)(40). Silencing of KIF3B in an avian embryo model significantly inhibited vasculotropism and metastasis in prostate cancer cell PC3 and other cancer cells (33). Increased expression of KIF3B was correlated with poor survival in patients with hepatocellular carcinoma while its inhibition decreased cancer growth and induced tumor apoptosis (39). Therefore, KIF3B is a novel therapeutic target to block cancer metastasis and inhibit cancer development. However, the role of KIF3B in breast cancer with Wnt signaling and the EMT process in breast cancer remains subtle.
In this study, we studied the expression level of KIF3B in breast cancer tissues and metastatic lymph nodes and its association with cancer progression. We also explored the role of KIF3B in EMT and in regulation of Wnt/b-catenin signaling pathway and identified the downstream targets CyclinD1, Cmyc, MMP-2, MMP-7 and MMP-9 in vitro and in vivo. Our results indicate that silencing of KIF3B could suppress breast cancer progression by regulating Wnt/b-catenin signaling and EMT, providing support that KIF3B could serve as a potential therapeutic target for the treatment of breast cancer.

METHODS
Database Searching for Gene Expression and Survival Analysis UALCAN (http://ualcan.path.uab.edu/) is an interactive web resource for analyzing transcriptome data of cancers from TCGA database. Using UALCAN, we evaluated the mRNA expression level of KIF3B in breast cancer from various angles

Cell Proliferation Assays
The procedure of MTT assay has been described previously (42). For colony formation assays, 1 × 10 4 cells were added to 1 ml of the growth medium with 0.3% agar after transfection with KIF3B shRNA lentivirus then layered onto 2 ml of 0.6% agar beds in 6well culture plate. After three weeks of culture, cells were then stained with crystal violet (0.05%) for 30 min and photographed. Colonies were counted using Image-ProPlus 6.0 software (Media Cybernatics, Inc.,Bethesda, USA).

Cell Cycle Analysis
Cells were fixed in 70% ethanol for 1 h at 4°C overnight and then incubated with 1 mg/ml RNaseA for 30 min at 37°C. Cells were stained with propidium iodide (50 mg/ml) in PBS containing 0.5% Tween-20 and analyzed using flow cytometry (BD Accuri C6, USA).

Transwell Migration and Invasion Assays
Transwell chambers (Corning, 8 um poly carbonate membrane) were used according to the manufacturer's protocol. Briefly, 600 ul of medium containing 15% FBS was added into the lower chamber. 4 × 10 4 cells (for migration) were plated in the upper chamber containing 200 ul of serum-free medium. After 24 h, the cells in the upper chamber were removed with a cotton swab. 8 × 10 5 cells (for invasion) were added to the upper chamber with Matrigel (Corning, 1:8). After 48 h, the cells in the upper chamber were removed with a cotton swab. The migratory and invasive cells on the lower filters were fixed with methanol and then stained with Giemsa. Cells in five random view fields were counted under the microscope.

Tumor Growth and Metastasis Assays in Nude Mice
All studies using animal were approved by the Animal Ethics Committee of Qingdao University, China. In short, 4 × 10 6 Scr-shRNA and KIF3B-shRNA MDA-MB-231 cells were subcutaneously implanted into BABL/c nude mice with five mice in each group. Size and weight of tumors were recorded every 7 days, and the tumor volume was measured using the following formula: volume (mm 3 ) = (width 2 × length)/2. Twenty BABL/c mice were randomly divided into two groups (10 in each group) and injected with Scr-shRNA and KIF3B-shRNA MDA-MB-231 cells (2 × 10 6 ) via tail vein. Six weeks later, mice were euthanized. The lungs were stained with HE. The whole lung tissue of each mouse was sectioned, and metastatic nodules were counted in high-power fields under a microscope.

Statistical Analysis
All statistical analyses were performed using SPSS 23.0 software. All values were presented as mean ± SD. Wilcoxon's test was used for non-normal distributed data. The Student's t test was used for data that were normally distributed. Differences were considered statistically significant at P < 0.05 and P < 0.01.

Bioinformatic Analysis of KIF3B Expression and Prognostic Value in Breast Cancer Patients
We evaluated KIF3B expression and prognostic value in breast cancer patients by using the UALCAN database. The results showed that KIF3B is over-expressed in primary breast tumors (n = 1097) compared to normal tissues (n = 114) ( Figure 1A, P < 0.01). We observed that KIF3B expression increased in breast cancer stages 1-3 compared to normal samples ( Figure 1B, P < 0.01). The expression of KIF3B is increased in breast cancer N0-N2 status, but decreased in N3 status ( Figure 1C, P < 0.01). Furthermore, breast cancer patients with high-expression of KIF3B (n = 271) displayed worse survival compared to the patients with low/medium-expression of KIF3B (n = 810) ( Figure 1D, P < 0.01).

Over-Expression of KIF3B in Breast Cancer
Real time RT-PCR and western blot assays were performed to examine the expression level of KIF3B in breast cancer tissues and nine pairs of fresh tissues. The results showed that both the mRNA (Figures 2A, B, P < 0.01) and protein levels ( Figures 2C, D, P < 0.01) of KIF3B in the tumor tissues were markedly higher than that of the corresponding adjacent tissues, demonstrating that KIF3B was up-regulated in breast cancer. We further detected KIF3B expression by immunohistochemistry and confirmed that KIF3B expression was higher in breast cancer than in corresponding adjacent tissues (P < 0.01) (Figures 2E, F; Table 1).

Relationship Between KIF3B Expression and Clinicopathological Factors of Breast Cancer Patients
By analyzing the relationship between the expression of KIF3B and the clinicopathological features of the patients, we found that KIF3B expression in lymph node metastasis was significantly higher than in primary focus (P < 0.01) ( Figures 2G, H, Table 1). Also, we found that the expression of KIF3B in the primary tumors with lymph node metastasis was higher than those without lymph node metastasis ( Table 1, P < 0.01). Moreover, patients with recurrent carcinoma showed higher expression of KIF3B than those without recurrence ( Table 1, P < 0.01). No significant differences in age, tumor size, grade or vascular invasion were observed ( Table 1).

Silencing of KIF3B Suppresses Cell Proliferation and Induces G2/M Arrest
MTT assay and colony-formation assay were performed to explore the effect of KIF3B depletion on cell proliferation. The results showed that the growth rate of the KIF3B-shRNA group was markedly slower than the Scr-shRNA group in MDA-MB-231 and MCF-7 cells. And the growth rate of KIF3B overexpression group was significantly faster than that of the NC group in MDA-MB-453 cells ( Figure 4A Figure 4B). These findings indicate that KIF3B depletion could inhibit cell growth.

Depletion of KIF3B Suppresses Cell Migration and Invasion Through Inhibiting EMT
We also discovered that silencing of KIF3B may cause morphological changes in the breast cancer cells. MDA-MB-231and MCF-7 became shortened and more adherent to each other due to KIF3B knockdown. However, over-expression of KIF3B in MDA-MB-453 cells led to elongated morphological appearances and mesenchymal-like properties ( Figure 5E), suggesting that these cells were undergoing EMT.
To further explore the effect of KIFB in EMT, KIF3B, Ecadherin, Vimentin, MMP-2, MMP-9, Slug and Snail were  (Figures 5F-H). These findings suggest that silencing of KIF3B might inhibit cell migration and invasion through suppressing EMT in breast cancer cells.

Silencing of KIF3B Suppresses Wnt/b-Catenin Signaling Pathway in Breast Cancer Cells
To investigate the role of KIF3B on Wnt/b-catenin signaling in breast cancer cells, we examined the expression levels of  Figures 6A, B), which were consistent with the results in xenografts. On the contrary, over-expression of KIF3B in MDA-MB-453 cells increased the expression level of bcatenin, b-catenin (Nucleus), Dvl2, p-GSK-3b ser9 , CyclinD1, Cmyc and MMP-7 ( Figure 6C). We also examined the effect of KIF3B on the expression of another two target genes of Wnt/bcatenin signaling, MMP-2 and MMP-9 ( Figures 5F-H). All these data indicated that deletion of KIF3B inhibited proliferation and invasion of breast cancer cells probably by regulating the Wnt/b-catenin signaling pathway.

Depletion of KIF3B Suppresses Tumor Growth in Nude Mice
We next explored the tumor-forming capacity of KIF3Bsilencing MDA-MB-231 cells in nude mice. The results showed that the tumor size (812.67 ± 74.78 mm 3 vs. 1370.67 ± 66.01 mm 3 , P < 0.05, Figures 7A, B) and weight (196.10 ± 46.78 mg vs. 431.70 ± 57.78 mg, P < 0.01, Figure 7C) in KIF3B-shRNA cell xenografts were significantly reduced compared with the Scr-shRNA group; however, hematoxylin and eosin (HE) staining of the xenografts showed no significant difference ( Figure 7D, left: ×100 Scr-shRNA group ×400; right: KIF3B-shRNA group ×400). Subsequently, the expressions of KIF3B, b-catenin, Cyclin peaked at 48h after KIF3B transfection (*P < 0.05; **P < 0.01). b-actin was used as the control. Scr-shRNA or NC was used as a negative control. Data were expressed as the gray-scale ratio of KIF3B protein relative to that of b-actin. All data were presented as mean ± SD. All the experiments were repeated three times.

Silencing of KIF3B Inhibits Metastasis In Vivo
To explore the effect of KIF3B on tumor migration and invasion in vivo, KIF3B-shRNA MDA-MB-231 cells were intravenously injected into nude mice. There was no significant difference in body weight of the mice after six weeks ( Figure 8A). However, the weight of the lungs in the KIF3B-shRNA group was  directed motor and is an important regulator in multiple cellular processes, such as mitosis, intracellular transport and cilium assembly (46,47). In the past few years, a large body of data has shown the association of KIF3B abnormality with tumor proliferation or invasion in several human cancers (34)(35)(36)(37)(38)(39)(40). Recently, Li et al. found that KIF3B is highly expressed in breast cancer, and over-expression of tumor-related KIFs correlates with worse outcome of breast cancer patients by bioinformatic analysis (48). However, the expression and related mechanisms of KIF3B in breast cancer have not been experimentally verified.
Our data firstly provided evidence that KIF3B expression was significantly increased at both the mRNA and protein levels in breast cancer tissues compared with the corresponding adjacent tissues. IHC analysis also showed that KIF3B was highly expressed in breast cancer tissues than the adjacent tissues, which confirmed our UALCAN analysis and the result published by Li et al. (48). Furthermore, we found that KIF3B expression in lymph node metastasis was significantly higher than in the primary focus. And the increased expression of KIF3B was correlated with lymph node metastasis and tumor recurrence, suggesting a significant association of high KIF3B expression with tumor growth and metastasis of breast cancer.
Previous study has shown that Wnt/b-catenin signaling pathway activation becomes dominant in basal-like breast cancers and predicts poor prognosis, and inhibiting Wnt/bcatenin signaling can effectively suppress breast cancer development (22,23,49), but the underlying mechanisms are poorly understood. KIF3A,another subunit of the KIF3 subfamily proteins, has been shown to promote proliferation and invasion via Wnt signaling in advanced prostate cancer (50). Since KIF3B and KIF3A are similar in sequence and function (28), we investigated whether KIF3B plays a role in regulating Wnt signaling. In this study, we firstly revealed that KIF3B is a potent activator of Wnt signaling in human breast cancer. We found that up-regulation of KIF3B activates the Wnt signaling via increasing the expression of Dvl2, p-GSK-3b ser9 , total and nucleus b-catenin, then up-regulation of Wnt signaling target genes such as CyclinD1, Cmyc and MMP-7, which are highly expressed in breast cancer (51)(52)(53)(54). Meanwhile, silencing of KIF3B could down-regulate CyclinD1 and C-myc and induce cell cycle arrest at G2/M phase. These results indicate the presence of KIF3B/GSK-3b/b-catenin axis and depletion of KIF3B might be one of the molecular mechanisms inhibiting the Wnt signaling pathway in breast cancer.
EMT could promote breast cancer cell stemness, invasion, and metastasis (49). Wnt/b-catenin signaling pathway has been shown to regulate EMT in several types of cancers, including breast cancer (55). In addition, during tumor invasion and metastasis, MMP-2, MMP-7, and MMP-9, the target genes of Wnt/b-catenin signaling, function to degrade the ECM and basement membrane so that tumor cells can detach, invade, and metastasize (7,56). E-cadherin, Vimentin, Slug and Snail were the main factors of EMT (57). In our study, high expression of KIF3B was shown to be associated with lymph node metastasis. Depletion of KIF3B resulted in both morphological changes and suppressed migration and invasion via inhibiting EMT in MDA-MB-231 and MCF-7 cells. Furthermore, the upregulated expression of E-cadherin was accompanied by the down-regulation of Vimentin, MMP-2, MMP-9, MMP-7, Slug and Snail in the KIF3B-shRNA group. These results were further confirmed in the xenografts. From these results, we conclude that depletion of KIF3B might repress migration and invasion of MDA-MB-231 and MCF-7 cells through inhibiting EMT as a result of suppressed Wnt/b-catenin signaling.
Collectively, our results provided support for the first time that KIF3B was highly expressed in breast cancer, and the high level expression was closely associated with lymph node metastasis and tumor recurrence. In addition, KIF3B knockdown might repress proliferation, migration, and invasion through regulating Wnt/b-catenin signaling and EMT in breast cancer cells (Figure 9), suggesting that KIF3B plays a key regulatory role in cell proliferation and metastasis in breast cancer.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethics committee of the Affiliated Hospital of Qingdao University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Ethics Committee of Qingdao University, China.