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PAD1 Promotes Epithelial-Mesenchymal Transition and Metastasis in Triple-Negative Breast Cancer Cells by Regulating MEK1-ERK1/2-MMP2 Signaling

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PAD1 Promotes Epithelial-Mesenchymal Transition and Metastasis in Triple-Negative Breast Cancer Cells by Regulating MEK1-ERK1/2-MMP2 Signaling

Hao Qin et al. Cancer Lett.

Abstract

Peptidylargininedeiminase 1 (PAD1) catalyzes protein for citrullination, and this activity has been linked to the epidermal cornification. However, a role for PAD1 in tumorigenesis, including breast cancers has not been previously explored. Here we first showed that PAD1 is overexpressed in human triple negative breast cancer (TNBC). In cultured cells and xenograft mouse models, PAD1 depletion or inhibition reduced cell proliferation, suppressed epithelial-mesenchymal transition, and prevented metastasis of MDA-MB-231 cells. These changes were correlated with a dramatic decrease in MMP2/9 expression. Furthermore, ERK1/2 and P38 MAPK signaling pathways are activated upon PAD1 silencing. Treatment with MEK1/2 inhibitor in PAD1 knockdown cells significantly recovered MMP2 expression, while inhibiting P38 activation only slightly elevated MMP9 levels. We then showed that PAD1 interacts with and citrullinates MEK1 thereby disrupting MEK1-catalyzed ERK1/2 phosphorylation, thus leading to the MMP2 overexpression. Collectively, our data indicate that PAD1 appears to promote tumorigenesis by regulating MEK1-ERK1/2-MMP2 signaling in TNBC. These results also raise the possibility that PAD1 may function as an important new biomarker for TNBC tumors and suggest that PAD1-specific inhibitors could potentially be utilized to treat metastatic breast cancer.

Keywords: MEK/ERK; MMP2; Metastasis; PAD1; Triple-negative breast cancer.

Conflict of interest statement

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1. PAD1 mRNA expression in breast cancer microarray database
(A) Analysis of PAD1 mRNA levels in human breast carcinoma tissues compared with normal breast tissues in the Oncomine cancer microarray database and in the TCGA datasbase. Images were displayed as a boxplot (log2 median-centered). The sample numbers are shown. P values were calculated using the Student t test. NB: normal breast tissue; BC: breast carcinoma. (B) PAD1 mRNA expression in triple negative breast cancer (TNBC) and non-TNBC tissues was analyzed in Gluck Oncomine database. (C) Expression levels of PAD1 were examined by immunohistochemistry staining. Representative sections from patients (TNBC, #1 and #2) who stained positive for PAD1 and negative for ER, PR, and HER2, and patients (non-TNBC, #3and #4) who stained negative for PAD1 and positive for ER, PR, and HER2. Black arrow head indicating the positive signals. Original magnification, ×100
Figure 2
Figure 2. PAD1 enhances TNBC cell proliferation, migration and invasion
(A) Endogenous PAD1 mRNA levels in indicated breast cancer cell lines were determined by qRT-PCR. The mRNA levels of PAD1 in other cell lines were normalized to that in MCF-10A cells. GAPDH served as loading control. (B) Endogenous PAD1–4 mRNA levels in MDA-MB-231 cells were determined by qRT-PCR. The mRNA levels of PAD2–4 were normalized to that in MDA-MB-231 cells. (C) The representative immunoblot shows efficient PAD1 knockdown in MDA-MB-231 cells and the decreased PCNA expression upon depletion of PAD1. β-Actin served as loading control. (D) PAD1 knockdown or shRNA control MDA-MB-231 cells were cultured in regular medium, at indicated times, cell numbers were counted under light microscope. **P < 0.01. (E) PAD1 knockdown or shRNA control MDA-MB-231 cells were plated in soft agar and assayed for colony number after 3 weeks. The representative image was shown in the upper panel. Scale bar: 100 µm. The data were presented as the mean ± SD from three independent experiments (lower panel). *P < 0.05. (F) Wound healing assay was performed to detect the migratory capacity of MDA-MB-231 cells after PAD1 depletion. Representative images of wound closure assays at indicated hours after scratching were presented (upper panel). The data were presented as the mean ± SD from three independent experiments (lower panel). *P < 0.05. (G) Transwell assay was performed to evaluate the invasive potential of PAD1 KD cells or the shRNA control MDA-MB-231 cells. The representative image shows invasive cells that were fixed and stained with crystal violet (upper panel). The optical density of the invaded cells from shRNA control MDA-MB-231 cells was used as the normal 100%. The data were presented as the mean ± SD from three independent experiments (lower panel). *P < 0.05.
Figure 3
Figure 3. Silencing PAD1 reverses the epithelial-mesenchymal transition in TNBC cells, and correlates with a decrease in MMP2/9 expression
(A) qRT-PCR was used to detect changes in the mRNA expression of EMT-associated genes in PAD1 KD MDA-MB-231 and the control cells. Values are means ± SD. The mRNA for each gene in PAD1 KD cells was normalized to that in shRNA control MDA-MB-231 cells. (B) Western blot was performed to detect changes in the protein expression of EMT-associated genes in PAD1 knockdown or shRNA control MDA-MB-231 cells. (C) Immunofluorescence staining was performed to detect changes in the protein expression of EMT-associated genes in PAD1 knockdown or shRNA control MDA-MB-231 cells. Nuclei were counterstained with DAPI (×40). (D) Immunoblotting was used to detect changes in the protein expression of E-Cadherin (E-CAD) in MDA-MB-468 cells transiently silencing PAD1 (PAD1-siRNA) or control cells. (E) Immunoblotting was used to detect changes in the protein expression of E-CAD in MDA-MB-468 cells after the de novo expression of PAD1. (F and G) PAD1 knockdown or shRNA control MDA-MB-231 cells were analyzed for MMP2/9 mRNA by real-time RT-PCR (F) and for protein expression by immunoblot (G). (H and I) MDA-MB-231 cells were treated with 200 µM Cl-amidine. At indicated time, cells were collected and analyzed for MMP2/9 mRNA expression by real-time RT-PCR (H) and for protein expression by immunoblot (I). *P < 0.05. **P < 0.01.
Figure 4
Figure 4. PAD1 represses MMP2 and MMP9 expression through inhibition of MAPK signaling
(A) Western blot analysis of p-ERK1/2, ERK1/2, p-P38, P38, p-JNK, JNK in MDA-MB-231 cells depletion of PAD1 or the shRNA control cells. (B) Western blot analysis of MDA-MB-231 cells treated with D-Cl-amidine using anti-p-ERK1/2, anti-ERK1/2, anti-p-P38 and anti-P38 antibodies. β-Actin served as loading control. (C) Western blot analysis of PAD1-depleted or the control MDA-MB-231 cells treated for 4 hours with 10 µM U0126. Immunoblotting of cell lysates were then performed using anti-p-ERK1/2 and anti-ERK1/2 antibodies. β-Actin served as loading control. (D) Western blot analysis of PAD1-depleted or the control MDA-MB-231 cells treated for 4 hours with 10 µM SB203580. Immunoblotting of cell lysates were then performed using anti-p-P38 and anti-P38 antibodies. β-Actin served as loading control. (E and F) PAD1 knockdown or shRNA control MDAMB231 cells were treated for 4 hours with (E) 10 µM U0126, (F) 10 µM SB203580 and then analyzed for MMP2 and MMP9 mRNA expression by real-time RT-PCR. All values shown are mean ± SD. *P < 0.05.
Figure 5
Figure 5. Citullination of MEK1 by PAD1 inhibits MEK-ERK1/2 signaling activation
(A) Western blot showing that the shRNA-mediated depletion of PAD1 in MDA-MB-231 cells did not affect MEK activation versus shRNA control cells. (B and C) Reciprocal co-immunoprecipitation analysis in HEK293 cells revealed that PAD1 interacts with MEK1. (D) Immunofluorescence analysis showing that PAD1 and MEK1 colocalized in the cells. Nuclei were stained with DAPI (scale bar, 20µm). (E) Citrullination of MEK1 by enzymatically active PAD1 in vitro. His-tagged MEK1 proteins were treated with either WT or C645S mutant PAD1 (Flag-tagged). The reactions were assessed by western blot using anti-Pan-Cit antibody. His-MEK1 protein without PAD1 treatment was used as a negative control. Anti-His and anti-Flag western blots confirmed equal protein loading. (F) Co-immunoprecipitation analysis showed decreased levels of MEK1 binding to C645S PAD1. Cells transfected with Flag-PAD1 only was used as a negative control. Anti-Flag and anti-MEK1 western blots confirmed equal protein loading. (G) Immunofluorescence analysis showed that C645S PAD1 led to activation of ERK1/2. MDA-MB-231 cells transfected with Flag-PAD1 (WT) or Flag-PAD1 mutant (CS) were stained against the Flag-tag (red) and p-ERK1/2 (green). Nuclei were stained with DAPI. Signals were quantified (**P < 0.01 PAD1 CS vs. PAD1 WT). Scale bar, 20µm.
Figure 6
Figure 6. PAD1 depletion or inhibition reduces tumor growth and metastasis of MDA-MB-231 cells in nude mice
(A) The represented tumors formed by PAD1 KD MDA-MB231 cell injection (n=6) or the shRNA control cell injection (n=5) at harvest time. The lower panel shows the volume of tumors at indicated time after cell implantation. The volume of each tumor was quantified, and the average volume was plotted. **P < 0.01. (B) The represented tumors after treatment with D-Cl-amidine or PBS. The lower panel shows growth curves of tumors in nude mice. Data are shown as the mean ± SD. *P < 0.05. (C) Western blotting was performed to confirm the knockdown efficiency of PAD1 in established tumors. β-Actin was used as loading control. (D) Immunohistochemistry staining for Ki67 was performed in PAD1 KD MDA-MB-231 or control cells using anti-Ki67 antibody (Santa Cruz Biotechnology). Representative micrographs were presented from three independent experiments (40X). (E) Images of metastatic liver nodules spreading throughout the live tissues in mice injected with shRNA control MDA-MB-231cells. PAD1 knockdown clearly abolished the metastasis. Arrows indicate tumor foci. (F) Hematoxylin and eosin (H&E) staining was used to visualize the metastatic nodules in tumors from mice injected with shRNA control MDA-MB-231cells. Arrows indicate tumor foci. Scale bar = 200 µm.

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