Table 1.
Association between CHD1L expression and clinical features of invasive ductal carcinoma patients.
Fig 1.
CHD1L is specifically upregulated in human breast cancer cell lines and tissues.
(A) Left, Expression of CHD1L protein in cultured human normal mammary epithelial cell line (MCF10A) and various breast cancer cell lines (MDA-MB-231, T-47D, SK-BR-3, and MCF-7). Right, Expression of CHD1L protein in paired breast cancer tissues (T) and adjacent non-tumor tissues (ANT), with each pair obtained from the same patient. β-Actin was used as a loading control. Quantification of relative protein levels is shown below the blots. Results are representative of at least three repeated experiments. (B) Positive expression of CHD1L protein in normal mammary glands (a), negative expression of CHD1L protein in normal mammary glands (b), positive expression of CHD1L protein in carcinoma with lymph node metastasis (c), positive expression of MMP-2 protein in carcinoma with positive expression of CHD1L (d), positive expression of MMP-9 protein in carcinoma with positive expression of CHD1L (e), negative expression of CHD1L protein in carcinoma without lymph node metastasis (f), negative expression of MMP-2 protein in carcinoma with negative expression of CHD1L (g), and negative expression of MMP-9 protein in carcinoma with negative expression of CHD1L (h) were examined using immunohistochemical staining. Scale bar: 20 μm. (C) Kaplan–Meier analysis was performed between the CHD1L level and overall survival of breast cancer patients with positive (n = 112) and low (n = 156) CHD1L expression.
Fig 2.
CHD1L promotes chemotaxis of breast cancer cells.
(A) Expression of CHD1L protein in MDA-MB-231 cells (MDA231), MDA-MB-231 cells transfected with control scrambled siRNA (Scr/MDA231), and MDA-MB-231 cells transfected with two sets of stable siRNA-targeting CHD1L (SiCHD1L#1/MDA231 and SiCHD1L#2/MDA231) was detected using Western blot. β-Actin was used as a loading control. Quantification of relative protein levels on three different Western blot analyses is shown below the blots. (B) Comparison of cell proliferation in Scr/MDA231 and SiCHD1L/MDA231 (SiCHD1L#1/MDA231). Each data point was an average of triplicate assays. (C) Cell chemotaxis ability was assessed using chemotaxia assay in MDA-MB-231, Scr/MDA231, and SiCHD1L/MDA231 (SiCHD1L#1/MDA231) with EGF stimulation. Columns, mean of triplicate measurements. Bars, standard deviation. *, P < 0.05 (Student’s t test, versus MDA-MB-231 or Scr/MDA231). (D) Expression of CHD1L protein in MCF-7 cells, MCF-7/Con cells (transfected with GV230 vector), and MCF-7/CHD1L cells (stably transfected with GV230-CHD1L, stable clone 4) was detected using Western blot. β-Actin was used as a loading control. Quantification of relative protein levels on three different Western blot analyses is shown below the blots. (E). Comparison of cell proliferation in MCF-7/Con and MCF-7/CHD1L. Each data point was an average of triplicate assays. (F) Comparison of chemotactic abilities with EGF stimulation in MCF-7, MCF-7/Con, and MCF-7/CHD1L. The data collected in this set of figures are representative of at least three independent experiments. Columns, mean of triplicate measurements. Bars, standard deviation. *, P < 0.05 (Student’s t test, versus MCF-7 or MCF-7/Con).
Fig 3.
CHD1L promotes invasion of breast cancer cells.
(A) Invasion of breast cancer cell line MDA-MB-231 with EGF stimulation was analyzed. Left, Quantification of penetrated cells was analyzed. Bars, standard deviation. *, P < 0.05 (Student’s t test, versus Scr/MDA231 with EGF stimulation). Right, Images were taken at a magnification. Scale bar: 20 μm. (B) Invasion of breast cancer cell line MCF-7 with EGF stimulation was analyzed. Left, Quantification of penetrated cells was analyzed. Bars, standard deviation, *, P < 0.05 (Student’s t test, versus MCF-7/Con with EGF stimulation). Right, Images were taken at a magnification. Scale bar: 20 μm. (C) Cell migration was assessed using the wound healing assay. Left, Cell motility rates of SiCHD1L/MDA231 and Scr/MDA231. Bars, standard deviation. *, P < 0.05 (Student’s t test, versus Scr/MDA231 with EGF stimulation). EGF, 10 ng/mL. Right, Images were taken at 0 and 24 h. (D) MMP-2 level in culture media of SiCHD1L/MDA231 and Scr/MDA231 cells was detected using ELISA. EGF, 10 ng/mL. Bar, standard deviation. *, P < 0.05 (Student’s t test, versus Scr/MDA231 with EGF stimulation). (E) MMP-9 level in culture media of SiCHD1L/MDA231 and Scr/MDA231 cells was assessed using ELISA assay, EGF, 10 ng/mL. Bars, standard deviation. *, P < 0.05 (Student’s t test, versus Scr/MDA231 with EGF stimulation). (F) MMP-2 and MMP-9 proteins were extracted in Scr/MDA231 and SiCHD1L/MDA231 cells with or without EGF stimulation, and Western blot was performed using an antibody to MMP-2 and MMP-9. β-Actin was used as a loading control. (G) Protein levels of pAkt, Akt, pARK5, ARK5, pmTOR, and mTOR were detected using Western blot. Each result is representative of at least three independent experiments. Akt was used as a loading control. Quantification of relative protein levels on three different Western blot analyses is shown below the blots.
Fig 4.
CHD1L promotes lung colonization of human breast cancer in vivo.
(A) Comparison of tumor cell colonies in mouse lungs between groups. (B) Tumor foci in mouse lungs were visualized using H&E staining. Scale bar: 50 μm. (C) Lung metastatic nodules were counted (n = 10). (D) Expression of CHD1L, MMP-2, and MMP-9 was detected in mice xenograft tumors by Western blot analysis. β-Actin was used as a loading control. Quantification of relative protein levels on three different Western blot analyses is shown below the blots.