NUCKS1 promotes breast cancer cell proliferation and metastasis via PI3K/ AKT pathway

Purpose: To investigate the role of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in breast cancer. Methods: Breast cancer cells were maintained in RPMI-1640 medium containing 10 % fetal bovine serum in a incubator at 37 °C. Cell proliferation was determined by CCK8 and colony formation assays. Flow cytometry and Transwell assays were used to determine cell cycle and metastasis, respectively. Results: Expression of NUCKS1 was significantly elevated in breast cancer (p < 0.01). Overexpression of NUCKS1 significantly increased cell viability (p < 0.01), and promoted proliferation of breast cancer cells. Knockdown of NUCKS1 inhibited cell proliferation, and induced cell cycle arrest at G1 phase. However, overexpression of NUCKS1 promoted cell cycle progression via down-regulation of p21 and up-regulation of cyclin D1 and CDK1. Cell migration and invasion were induced by overexpression of NUCKS1, and suppressed by silencing of NUCKS1. Overexpression of NUCKS1 enhanced p-AKT and p-PI3K expression, while knockdown of NUCKS1 reduced the expression of p-AKT and p-PI3K in breast cancer cells. Conclusion: NUCKS1 promotes breast cancer cell proliferation and metastasis via activation of PI3K/AKT signaling. The silencing of NUCKS1 can be used as a strategy to develop therapies for the management of breast cancer.


INTRODUCTION
Breast cancer is the leading cause of death in women [1]. Although advanced strategies have improved the prognosis of breast cancer, the mortality of breast cancer is increasing and the overall survival is unsatisfactory [2]. Therefore, therapeutic targets are urgently needed for breast cancer treatment.
Nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) is a member of the high mobility group family and mediates gene transcription and chromatin remodeling [3]. It functions as a paralog of RAD51-associated protein 1 and is involved in genome stability and homologous recombination during DNA damage response and metabolism [4]. Moreover, NUCKS1 is also regarded as a transcriptional regulator for components of insulin signaling and has been implicated in the pathogenesis of metabolic and inflammatory diseases [3]. Increasing evidence has shown that NUCKS1 was related to poor prognoses of several cancers, and that it modulated tumor progression. For example, high levels of NUCKS1 in cervical squamous cell carcinoma have been associated with tumor recurrence [5] and predicted poor prognoses of colorectal cancer [6]. It binds to CDK1 and promotes nonsmall cell lung cancer cell proliferation and metastasis [7]. Knockdown of NUCKS1 reduces the viability of hepatocellular carcinoma cells, and inhibits tumor growth [8]. In breast cancer, the expressions of NUCKS1 in low-grade nonspecific invasive tumors were lower than those in high-grade invasive cancers [9]. Moreover, NUCKS1 is up-regulated in breast cancer tissues and might be involved in inflammation and cell cycling [10]. However, the specific role of NUCKS1 in breast cancer has not yet been clearly elucidated.
The PI3K signaling pathway is essential for cell proliferation and growth, and is involved in the progression of tumors [11]. The PI3K/AKT is abnormally activated in breast cancer, and inhibition of PI3K/AKT is currently in a clinical trial for breast cancer treatment [12]. Interestingly, NUCKS1 has been shown to be involved in miR-137-mediated inhibition of PI3K/AKT in lung cancer [13], and promoting the activation of PI3K/AKT signaling to induce cell aggressiveness of gastric cancer [14]. Therefore, NUCKS1/PI3K/AKT might be involved in breast cancer tumorigenesis.
The aim of this study was to investigate the effects of NUCKS1 on cell proliferation and cell metastasis of breast cancer, and the underlying mechanisms.

Cell culture
Breast cancer cells (MCF-7, MDA-MB-231, and MCF10A) were acquired from the Cell Center of the Institute of Basic Medical Sciences (Beijing, China). The cells were identified as mycoplasma-free by short tandem repeat profiling. The cells were maintained in RPMI-1640 medium containing 10 % fetal bovine serum (Gibco, Grand Island, NY, USA) in a 37 °C incubator.

Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
Cells were lysed in TRIzol (Invitrogen, Carlsbad, CA, USA) to isolate RNAs. The RNAs were then reverse transcribed into cDNAs, and a PreTaq II kit (Takara, Dalian, Liaoning, China) was used to determine the mRNA expression of NUCKS1. The relative expression of NUCKS1 was calculated using the 2 -∆∆Cq method through normalization to GAPDH. The primers used are shown in Table 1.

Cell proliferation and cell cycle assays
The MCF-7 and MDA-MB-231 cells were seeded in 6-well plates for 10 days and then fixed in methanol. The cells were stained with crystal violet and photographed using a light microscope (Olympus, Tokyo, Japan). For flow cytometry, MCF-7 and MDA-MB-231 cells were harvested 48 h post-transfections and then resuspended in the binding buffer from the BD Cycletest™ Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA). The cells were stained with propidium iodide (BD Biosciences) and analyzed in a FACS flow cytometer (Life Technologies, Darmstadt, Germany).

Transwell assay
The MCF-7 and MDA-MB-231 cells were harvested and suspended in serum-free RPMI-1640 medium. The cells were photographed using a light microscope (Olympus). For the cell migration assay, cells were also plated into a chamber without Matrigel coating, and the same above mentioned experiments were performed.

Statistical analysis
All data with at least triple replicates are expressed as the mean ± standard error of the mean (SEM), and analyzed by Student's t-test or one-way analysis of variance using SPSS statistical software for Windows (SPSS, Chicago, IL, USA). A value of p < 0.05 was considered statistically significant.

Elevation of NUCKS1 in breast cancer
The expression level of NUCKS1 was identified in breast cancers. Results based on The Cancer Genome Atlas database indicated up-regulation of NUCKS1 in breast cancer tissues (Figure 1 A  and B). Moreover, breast cancer cells (MCF-7 and MDA-MB-231) showed higher expression of NUCKS1 than MCF10A cells (Figure 1 C and D), indicating a possible correlation between NUCKS1 and breast cancer.

NUCKS1 promoted breast cancer cell cycle progression
The ratios of G1 phases in MCF-7 and MDA-MB-231 cells were decreased by overexpression of NUCKS1 (Figure 3 A and B). However, knockdown of NUCKS1 increased the ratio of cells in the G1 phase and promoted cell cycle arrest (Figure 3 A and B). Overexpression of NUCKS1 down-regulated protein expression of p21, while it up-regulated cyclin D1 and CDK1 expressions (Figure 3 C). Silencing of NUCKS1 enhanced p21 expression and reduced cyclin D1 and CDK1 expressions (Figure 3 C).

NUCKS1 contributed to activation of PI3K/AKT
Overexpression or knockdown of NUCKS1 did not affect protein expressions of PI3K and AKT ( Figure 5 A and B). However, NUCKS1 overexpression increased expressions of p-PI3K and p-AKT. The p-PI3K and p-AKT were reduced by knockdown of NUCKS1 ( Figure 5 A and B), indicating the suppressive effect of NUCKS1 silencing on PI3K/AKT signaling in breast cancer cells.

DISCUSSION
Immunohistochemical analysis showed that NUCKS was overexpressed in breast cancerous tissues when compared to non-cancerous tissues [15]. NUCKS functions as an oncogene to suppress autophagy and promote cell proliferation of gastric cancer [16]. This study, therefore, identified the oncogenic role of NUCKS1 in breast cancer.
This NUCKS1 was up-regulated in breast cancer tissues, and high expression of NUCKS1 was related to distant metastasis and lymph node involvement and predicted poor prognoses in patients with invasive breast carcinomas [9]. This study also showed increased expression of NUCKS1 in breast cancer. Overexpression of NUCKS1 promoted cell proliferation and metastasis of breast cancer; however, silencing of NUCKS1 suppressed the proliferation and metastasis.
NUCKS1 has been regarded as a transcription factor, and it up-regulates expression of S phase kinase-associated protein 2, resulting in degradation of p21 and p27 during cell cycle progression [17]. Moreover, knockdown of CDK1 attenuates NUCKS1 overexpression-induced non-small cell lung cancer cell proliferation and metastasis [7]. This study also found that NUCKS1 functioned as a cell cycle regulator, and shRNA-dependent NUCKS1 enhanced expression of p21, and reduced expression of cyclin D1 and CDK1 to induce cell cycle arrest in breast cancer. NUCKS1 is involved in homologous recombination during DNA damage response and metabolism [4], and DNA damage induces p53-dependent suppression of NUCKS1 and inhibits cell cycle progression [17]. Therefore, NUCKS1 might also regulate DNA damage repair during the progression of breast cancer.  NC or siNC PI3K/AKT signaling is essential for the metastasis, progression, and drug resistance of breast cancer [18]. Inhibitors of PI3K and AKT have been used in clinical trials to treat breast cancer [11]. NUCKS1 stimulated the upregulation of p-PI3K, p-AKT, and p-mTOR in gastric cancer, and promoted the aggressiveness of gastric cancer [14]. The results of the present study showed that knockdown of NUCKS1 reduced the expressions of p-PI3K and p-AKT in breast cancer

CONCLUSION
The NUCKS1 function as an oncogene in breast cancer. Silencing of NUCKS1 reduces cell proliferation and metastasis of breast cancer cells, while it induces cell cycle arrest via inactivation of PI3K/AKT signaling, suggesting that NUCKS1 silencing can be further investigated as a strategy to treat breast cancer.

Open Access
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