DNA polymerase iota (Pol ι ) promotes the migration and invasion of breast cancer cell via EGFR-ERK-mediated epithelial to mesenchymal transition

Abstract

BACKGROUND AND OBJECTIVE:

Dysregulation of DNA polymerase iota (Pol $\iota$ ) in breast cancer might contribute to the accumulation of genomic mutations and promotes breast cancer progression. In this study we explored the clinical relevance and biological function of Pol $\iota$ in breast cancer.

METHODS:

qRT-PCR was used to determine the expression levels of Pol $\iota$ in 31 breast cancer tissues. Then the stable overexpression of Pol $\iota$ and knockdown of Pol $\iota$ breast cancer cell lines were constructed. Wound-healing assay and transwell assay were performed to evaluate cell migratory and invasiveness, respectively. Signaling pathway was analyzed by western blot.

RESULTS:

The expression levels of Pol $\iota$ is overexpressed in breast cancer tissues and significantly higher in breast cancer tissues with lymph node metastasis compared to those without lymph node metastasis. Elevated Pol $\iota$ expression promoted migratory and invasiveness of breast cancer cells. Signaling pathway analysis indicated EGFR-ERK cascade works as a mediator of Pol $\iota$ -induced EMT of breast cancer cells.

CONCLUSIONS:

These data demonstrate the underlying mechanism by which Pol $\iota$ promotes breast cancer progression, suggesting that Pol $\iota$ may be a potential therapeutic target against breast cancer.

1. Introduction

Breast cancer is the most common malignancies and second leading cause of cancer death among women [1]. Poor prognosis in breast cancer patients is largely related to tumor metastasis, which is accounting for more than 90% of cancer-related mortality [2, 3]. Following years of research, many studies have focused on the mechanisms of metastasis in breast cancer, whereas the exact mechanism underlying breast cancer metastasis still remains unclear. Consequently, identifying novel biomarkers relating to breast cancer metastasis and illuminating the underlying mechanisms will greatly improve the treatment strategy for breast cancer patients.

The genome is continuously exposed to both endogenous and exogenous stimulus such as reactive oxygen species and UV radiation. As a result, DNA damage occurs at least 10,000 times/cell/day in human which has been proven to play a major role in tumorigenesis [4]. Consequently, living cells have evolved repair mechanisms to remove and repair lesion sites. Translesion DNA Synthesis (TLS) is a DNA damage tolerance (DDT) pathway which relies on specialized DNA polymerases that have active sites capable of accommodating damaged or distorted templates and directly bypass lesions [5, 6]. Since the error-prone character of the TLS polymerase, it can tolerate the errors in the replication process and cause the mutation of the genes, which increases the instability of the genome [6]. As one of the important TLS polymerases, DNA polymerase iota (Pol $\iota$ ) exhibits the lowest fidelity of any eukaryotic polymerase in vitro studies [7]. Previous studies revealed that Pol $\iota$ was overexpressed in several cancers cells [8, 9]. In our recent study, we demonstrated that the expression of Pol $\iota$ is upregulated in human esophageal squamous cell cancer tissues [10], and that elevated Pol $\iota$ was positively involved in the acquisition of aggressive phenotypes of esophageal squamous cell cancer by enhancing its invasiveness and metastasis [11]. Research from other group demonstrated that dysregulation of Pol $\iota$ in breast cancer might contribute to the accumulation of genomic mutations, subsequently increasing the frequency of a tumor acquiring phenotype [12]. However, the exact role of Pol $\iota$ in breast cancer remains unclear.

In this study, we showed that Pol $\iota$ is upregulated in breast cancer tissues. The expression levels of Pol $\iota$ in breast cancer tissues are positively associated with lymph node metastasis. Furthermore, we identified that Pol $\iota$ drives breast cancer cell migration and invasion in vitro via EGFR-ERK-mediated epithelial to mesenchymal transition (EMT).

2. Material and methods

2.1 Patient tissues and cell culture

Thirty-one human breast cancer tissues and paired normal tissues were obtained from The Nanjing Medical University Affiliated Suzhou Hospital (Jiangsu, China). Fresh tissues were immediately snap-frozen and stored at $-$ 80 ${}^{\circ }$ C for real-time PCR analysis. The characteristics of breast cancer patients included in this study were described in Table 1. This study was approved by the Institutional Ethics Committee of the Nanjing Medical University. Human breast cancer cell lines, including MCF-7 and MDA-MB-231 were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured in DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Clark Bioscience, Richmond, VA, USA). Cells were incubated in a humidified atmosphere with 5% CO ${}_2$ at 37 ${}^{\circ }$ C.

Table 1

Clinicopathological parameters of breast cancer patients

Clinicopathological parameters	Cases
Age
$<$ 60	12
$⩾$ 60	19
T stage
T1–2	15
T3–4	16
N stage
N0	21
N1–N3	10
Clinical stage
1	12
2–5	19

2.2 RNA extraction and quantitative RT-PCR

Total RNA was isolated using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (1 $\mu$ g) from each sample was subjected to first-strand cDNA synthesis using Superscript II reverse transcriptase (Invitrogen Life Technologies, USA). Real-time PCR (qRT-PCR) was conducted using SYBR Premix Ex Taq (TaKaRa, Kusatsu, Shiga, Japan) and a StepOne Plus instrument (Applied Biosystems, Rochester, NY, USA). The primers for human $\beta$ -actin, Pol $\iota$ and MMP-9 were as follows: $\beta$ -actin, Forward: 5’-AGCGAGCAT CCCCCAAAGTT-3’, Reverse: 5’-GGGCACGAAGG CTCATCATT-3’; Pol $\iota$ , Forward: 5’-ACAAACCGGG ATTTCCTACC-3’, Reverse: 5’-TCACACTTCCTTTC CCTTGAA-3’; MMP-9, Forward: 5’-TATGGTCCTC GCCCTGAACCT-3’, Reverse: 5’-GCACAGTAGTGG CCGTAGAAGG-3’. Relative Pol $\iota$ and MMP-9 mRNA expression levels were calculated using the 2 ${}^{-\mathrm{\Delta }\text{CT}}$ method and normalized to $\beta$ -actin expression levels.

Figure 1.

Pol $\iota$ expression is frequently elevated in breast cancer. (A and B) Pol $\iota$ expression in 31 breast cancer tissues and paired normal tissues. Pol $\iota$ expression was significantly elevated in breast cancer tissues compared to the paired normal tissues (* $P<$ 0.05). (C and D) Pol $\iota$ expression positively correlated with lymph node metastasis (C, * $P<$ 0.05), but not with T tumor stages (D, * $P>$ 0.05).

2.3 Generation of stable cell lines

The lentivirus expression vectors containing the human Pol $\iota$ coding region or Pol $\iota$ siRNA and control vector were obtained from GenePharm (Suzhou, China), and packaged into viral particles. The particles were used to infect MCF-7 cells and MDA-MB-231 cells. Then the cells were cultured in DMEM medium containing 1 $\mu$ g/ml Puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 7 days which could select stable cell lines.

2.4 Western blot analysis

Cells were harvested and lysed in RIPA buffer (Sigma, USA) containing protease inhibitors for 10 min at 4 ${}^{\circ }$ C. The proteins were separated by gradient gel 4–20% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies targeting Pol $\iota$ , E-cad, N-cad (Abcam, Cambridge, MA, USA), EGFR, p-EGFR, ERK, p-ERK (Cell Signaling Technology, Beverly, MA, USA) and $\beta$ -actin (Beyotime Biotechnology, Haimen, China). The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Biotechnology, China). The protein bands were visualized using chemiluminescence (ECL, Tanon, Shanghai, China).

Figure 2.

Generation of stable cell lines. Pol $\iota$ overexpression and knockdown breast cancer cell lines were constructed using lentivirus vectors. qRT-PCR (A) and western blot (B) were used to analyze Pol $\iota$ expression in these cell lines. $\beta$ -actin levels served as loading control.

Figure 3.

Pol $\iota$ promotes breast cancer cell migration in vitro. Cell migration was detected by wound-healing assay in vitro. Overexpression of Pol $\iota$ enhanced the cell migration of MCF-7 cells, whereas Pol $\iota$ knockdown repressed the cell migration of MDA-MB-231 cells (* $P<$ 0.05, ** $P<$ 0.01).

Wound-healing assay Cell migration was detected by wound-healing assay in vitro. Cells were seeded in 6-well plates at a density of 1 $\times$ 10 ${}^6$ cells per well. After 24 h incubation, a thin mark was drawn vertically with a 200 $\mu$ l pipette tip. Cells were then washed two times with PBS to remove the floating cells. Fresh serum-free medium was added, and photos were taken at 0, 24 and 48 h to assess cell migration using a light microscope (Leica Corporation, Wetzlar, Germany).

2.5 Transwell assay

Cell invasion was assessed using matrigel-coated transwell chambers (Corning, Corning, NY, USA). A total of 1 $\times$ 10 ${}^5$ cells were suspended in 200 $\mu$ l of serum-free media with 10 g/L BSA and seeded in the upper chamber, which was pre-coated with 60 $\mu$ l matrigel (1:6 dilution; BD Bioscience, Billerica, MA, USA). The lower chambers were filled with 600 $\mu$ l media containing 10% FBS. After 48 h incubation, the cells remaining in the upper chambers were scraped off. The cells invaded through the matrigel to the lower surface of the filter were fixed with 3.7% paraformaldehyde and stained with crystal violet. The penetration of cells through the membrane was photographed under a microscope.

2.6 Statistical analysis

All data were presented as mean values $\pm$ SEM. Statistical significance was considered to be a $P$ -value $<$ 0.05. Statistical analyses were performed using SPSS 19.0 software (IBM, USA). Differences between groups were estimated using the ${\chi }^2$ test or Student’s $t$ test. Pearson’s correlation was used to analysis the correlation between two genes expression.

Figure 4.

Pol $\iota$ promotes breast cancer cell invasion in vitro. Cell invasion was detected by matrigel transwell assay. Overexpression of Pol $\iota$ enhanced the cell invasion of MCF-7 cells, and Pol $\iota$ knockdown repressed the cell invasion of MDA-MB-231 cells (* $P<$ 0.05).

3. Results

3.1 Pol $\iota$ expression correlates with lymph node metastasis in human breast cancer

To compare expression differences between breast cancer tissues and normal tissues, qRT-PCR was used to evaluate the mRNA levels of Pol $\iota$ expression in 31 human breast cancer tissues and paired normal tissues. Pol $\iota$ expression was significantly upregulated in breast cancer tissues (58.1%) when compared with that in normal breast tissues ( $P<$ 0.05). Since lymph node metastasis is characterized as the single most important prognostic factor for breast cancer [12, 13]. We further evaluated the correlation between Pol $\iota$ expression and lymph node metastasis, and found that the expression of Pol $\iota$ is significantly higher in breast cancer with lymph node metastasis than those without lymph node metastasis ( $P<$ 0.05, Fig. 1C), while the expression level is not correlated with T stages ( $P>$ 0.05, Fig. 1D). These results suggest that Pol $\iota$ may promote tumor progression through enhancing breast cancer invasion and metastasis.

3.2 Pol $\iota$ expression is associated with breast cancer cell migration and motility in vitro

To investigate how Pol $\iota$ affects breast cancer metastasis, ectopic expression (MCF-7-LV5-Pol $\iota$ compared with MCF-7-LV5) and specific knockdown of Pol $\iota$ (MDA-MB-231-LV3-shPol $\iota$ compared with MDA-MB-231-LV3) in breast cancer cell lines were achieved. Quantitative real-time PCR and western blotting and confirmed the expression levels of Pol $\iota$ in these cell lines (Fig. 2A and B). As increased motility is essential for tumor metastasis, these cell models were subjected to in vitro wound-healing assays. Overexpression of Pol $\iota$ dramatically promoted the motility of MCF-7 cells. In contrast, Pol $\iota$ depletion slowed down the migration of MDA-MB-231 cells (Fig. 3). Since invasion is a crucial aspect contributing to cancer metastasis [14], we performed transwell assays to explore how altering Pol $\iota$ expression influences the invasiveness of breast cancer cells. The results revealed that cell invasion ability of breast cancer cells showed a loss of invasive ability with loss of Pol $\iota$ and increased invasive ability with Pol $\iota$ overexpression. In summary, these data indicated the competence of Pol $\iota$ to enhance migration and invasion of ESCC cells.

Figure 5.

Pol $\iota$ enhances EMT of breast cancer cell through activating the EGFR-ERK pathway. Western blot analysis of the expression levels of EMT markers, E-cad, N-cad, MMP-9 and EGFR-ERK signal pathway in Pol $\iota$ overexpression and knockdown cell lines. $\beta$ -actin levels served as loading control.

3.3 Pol $\iota$ induces EMT through activating the EGFR-ERK pathway in breast cancer

Epithelial to mesenchymal transition (EMT) contributes to cancer cell invasion and metastasis, and is marked by loss of E-cadherin and gain of N-cadherin [15]. To investigate the potential molecular mechanisms underlying the pro-invasive tendency of Pol $\iota$ in breast cancer. The specific alterations along the EMT markers, E-cad, N-cad and MMP-9 under the impact of Pol $\iota$ were examined. Overexpression of Pol $\iota$ increased N-cad, MMP-9 expression and decreased E-cad expression in MCF-7 cells as evidenced by western blot analysis. In contrast, knockdown of Pol $\iota$ markedly reduced N-cad, MMP-9 expression and enhanced E-cad expression in MDA-MB-231 cells. To further investigate the relationship between Pol $\iota$ and MMP-9 expression in breast cancer tissues, 31 breast cancer tissues was utilized. Notably, the expression of Pol $\iota$ and MMP-9 were positively associated with breast cancer ( $P<$ 0.05, Fig. 6).

Since EGFR-ERK signaling pathway has a critical role in EMT of various cancers [16]. We examined the effect of altered Pol $\iota$ levels on EGFR-ERK pathway in our model systems. Overexpression of Pol $\iota$ increased p-EGFR and p-ERK expression in MCF-7 cells. In contrast, knockdown of Pol $\iota$ significantly decreased p-EGFR and p-ERK expression in MDA-MB-231 cells. Taken together, these results revealed that Pol $\iota$ induces EMT via activating EGFR-ERK pathway, and then enhances the metastatic potential of breast cancer cells.

Figure 6.

The correlation between Pol $\iota$ expression and MMP-9 expression in breast cancer tissues. qPCR was used to detect MMP-9 expression in 31 breast cancer tissues. Pol $\iota$ expression positively correlated with MMP-9 expression ( $P<$ 0.05).

4. Discussion

As the most error-generating DNA polymerase, Pol $\iota$ could be commonly misincorporating G opposite a template T in an undamaged DNA strand [17]. Thus, ectopic expression of Pol $\iota$ in cancer cells may contribute to the accumulation of genomic mutation and tumor progression. In this study, we discovered a crucial role of Pol $\iota$ in breast cancer metastasis. By comparing the mRNA expression levels of Pol $\iota$ between breast cancer tissues with different $N$ stages (Fig. 1C), we found elevated Pol $\iota$ in tumors with lymph node metastasis, suggesting an important role of Pol $\iota$ in breast cancer metastasis. Using well established breast cancer cell model systems, we found that Pol $\iota$ could promote breast cancer cell migration and invasion in vitro (Figs 3 and 4). Moreover, our results revealed a potential molecular mechanism by which Pol $\iota$ promoted breast cancer migration and invasion through inducing EMT via activating EGFR-ERK pathway.

It is well established that EMT is correlated with tumor development and metastasis, during which tumor cells probably gain aggravative migration and invasion. EMT is a biological process that epithelial cells lose numerous epithelial characteristics and assume properties that are typical of mesenchymal cells [18, 19], which is marked by loss of E-cadherin and gain of N-cadherin [20]. In our study, we found that Pol $\iota$ increased N-cad and decreased E-cad expression in breast cancer cells (Fig. 5). This result provides a potential mechanism underlying Pol $\iota$ -induced breast cancer migration and invasion, which could be driven by EMT. Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that contributes to cell invasion and metastasis by enhancing the progression of ECM. Through EMT, cancer cells break down the collagen of basement membranes by MMPs, invading the lymph vessels or blood [21, 22]. We also found that Pol $\iota$ upregulated MMP-9 expression (Fig. 5), and that Pol $\iota$ levels positively correlated with that of MMP-9 in breast cancer tissues (Fig. 6). Token altogether, we are confident that overexpression of Pol $\iota$ promotes the migration and invasion of breast cancer cells through inducing EMT.

EGFR belongs to the ErbB family of tyrosine kinase receptors and exerts critical function in epithelial cell physiology [23]. It is frequently overexpressed and/or mutated in different types of human cancers and activates several signaling pathways, mainly the MAPK/ERK and PI3K/AKT pathways, promoting tumor cell migration and invasion [24]. Studies have revealed that EGFR has an important role in EMT [25] by downregulating of caveolin-1 which leads to loss of E-cadherin and enhancing transcriptional activation of $\beta$ -catenin [26]. Recently, more and more studies reveal a crucial role for EGFR downstream signaling, especially ERK signaling, in the regulation of EMT in various tumor types [27, 28, 29, 30]. In this study, we found that Pol $\iota$ promoted the phosphorylation of EGFR and ERK expression in breast cancer cells, indicating that Pol $\iota$ promotes EMT in breast cancer cells via EGFR-ERK signaling pathway. The classic EGFR signaling pathway includes ligand- and kinase-dependent activation. In fact, EGFR has also been shown to be activated by membrane depolarization, by various stress responses including hyperosmotic conditions, ultraviolet radiation and reactive oxygen species [31]. However, it remains unclear whether overexpression of Pol $\iota$ induces the genomic mutations, subsequently leading to the activation of EGFR-ERK signal pathway in breast cancer. The potential mechanism of Pol $\iota$ regulating EGFR-ERK signaling pathways merits further exploration.

Taken together, our results elucidate that elevated Pol $\iota$ expression enhances breast cancer migration and invasion through EGFR-ERK pathway-mediated EMT. These findings demonstrate an inevitable role for Pol $\iota$ in the progression of breast cancer, indicating that Pol $\iota$ may be a potential candidate for a therapeutic target for metastatic breast cancer.