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

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 ∘ 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 ∘ C.

Total RNA was isolated using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (1 μ 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 β -actin, Pol ι and MMP-9 were as follows: β -actin, Forward: 5’-AGCGAGCAT CCCCCAAAGTT-3’, Reverse: 5’-GGGCACGAAGG CTCATCATT-3’; Pol ι , Forward: 5’-ACAAACCGGG ATTTCCTACC-3’, Reverse: 5’-TCACACTTCCTTTC CCTTGAA-3’; MMP-9, Forward: 5’-TATGGTCCTC GCCCTGAACCT-3’, Reverse: 5’-GCACAGTAGTGG CCGTAGAAGG-3’. Relative Pol ι and MMP-9 mRNA expression levels were calculated using the 2 - Δ ⁢ CT method and normalized to β -actin expression levels.

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The lentivirus expression vectors containing the human Pol ι coding region or Pol ι 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 μ 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 ∘ 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 ι , E-cad, N-cad (Abcam, Cambridge, MA, USA), EGFR, p-EGFR, ERK, p-ERK (Cell Signaling Technology, Beverly, MA, USA) and β -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).

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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 × 10 6 cells per well. After 24 h incubation, a thin mark was drawn vertically with a 200 μ 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).

Cell invasion was assessed using matrigel-coated transwell chambers (Corning, Corning, NY, USA). A total of 1 × 10 5 cells were suspended in 200 μ l of serum-free media with 10 g/L BSA and seeded in the upper chamber, which was pre-coated with 60 μ l matrigel (1:6 dilution; BD Bioscience, Billerica, MA, USA). The lower chambers were filled with 600 μ 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 ± 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 χ 2 test or Student’s t test. Pearson’s correlation was used to analysis the correlation between two genes expression.

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3.1 Pol ι 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 ι expression in 31 human breast cancer tissues and paired normal tissues. Pol ι expression was significantly upregulated in breast cancer tissues (58.1%) when compared with that in normal breast tissues ( 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 ι expression and lymph node metastasis, and found that the expression of Pol ι is significantly higher in breast cancer with lymph node metastasis than those without lymph node metastasis ( 0.05, Fig. 1C), while the expression level is not correlated with T stages ( $P>$ 0.05, Fig. 1D). These results suggest that Pol ι may promote tumor progression through enhancing breast cancer invasion and metastasis.

3.2 Pol ι expression is associated with breast cancer cell migration and motility in vitro

To investigate how Pol ι affects breast cancer metastasis, ectopic expression (MCF-7-LV5-Pol ι compared with MCF-7-LV5) and specific knockdown of Pol ι (MDA-MB-231-LV3-shPol ι 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 ι 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 ι dramatically promoted the motility of MCF-7 cells. In contrast, Pol ι 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 ι 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 ι and increased invasive ability with Pol ι overexpression. In summary, these data indicated the competence of Pol ι to enhance migration and invasion of ESCC cells.

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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 ι in breast cancer. The specific alterations along the EMT markers, E-cad, N-cad and MMP-9 under the impact of Pol ι were examined. Overexpression of Pol ι 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 ι markedly reduced N-cad, MMP-9 expression and enhanced E-cad expression in MDA-MB-231 cells. To further investigate the relationship between Pol ι and MMP-9 expression in breast cancer tissues, 31 breast cancer tissues was utilized. Notably, the expression of Pol ι and MMP-9 were positively associated with breast cancer ( 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 ι levels on EGFR-ERK pathway in our model systems. Overexpression of Pol ι increased p-EGFR and p-ERK expression in MCF-7 cells. In contrast, knockdown of Pol ι significantly decreased p-EGFR and p-ERK expression in MDA-MB-231 cells. Taken together, these results revealed that Pol ι induces EMT via activating EGFR-ERK pathway, and then enhances the metastatic potential of breast cancer cells.

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