Microtubule-associated protein Mdp3 promotes breast cancer growth and metastasis

Abstract

Breast cancer is the most prevalent cancer in women worldwide with a high mortality rate, and the identification of new biomarkers and targets for this disease is greatly needed. Here we present evidence that microtubule-associated protein (MAP) 7 domain-containing protein 3 (Mdp3) is highly expressed in clinical samples and cell lines of breast cancer. The expression of Mdp3 correlates with clinicopathological parameters indicating breast cancer malignancy. In addition, Mdp3 promotes breast cancer cell proliferation and motility in vitro and stimulates breast cancer growth and metastasis in mice. Mechanistic studies reveal that γ-tubulin interacts with and recruits Mdp3 to the centrosome and that the centrosomal localization of Mdp3 is required for its activity to promote breast cancer cell proliferation and motility. These findings suggest a critical role for Mdp3 in the growth and metastasis of breast cancer and may have important implications for the management of this disease.

Keywords: breast cancer; cell motility; cell proliferation; growth; metastasis..

Figure 1

Mdp3 is highly expressed in human breast cancer tissues and cell lines. (A and B) Immunohistochemical staining of Mdp3 in breast cancer tissues (A) and adjacent tissues (B), with negative (-), low (+), medium (++), and high (+++) expression. (C) Quantitative analysis of Mdp3 expression in 85 breast cancer tissue samples and 49 adjacent tissue samples. (D-H) Analysis of the correlations between Mdp3 expression and clinicopathological parameters indicating the malignancy of breast cancer, including the pathological tumor node metastasis (pTNM) stage (D), histological grade (E), lymph node metastasis (F), estrogen receptor (G), and progesterone receptor (H). (I) Immunoblot analysis of Mdp3 and β-actin expression in MCF-10A normal breast epithelial cells and breast cancer cell lines.

Figure 2

Mdp3 promotes the proliferation of breast cancer cells. (A) Immunoblot analysis of Mdp3 and β-actin expression in MCF7 and BT549 cells transfected with control or Mdp3 siRNAs for 48 hours. (B and C) Determination of cell proliferation by SRB (B) and MTT (C) assays in cells transfected with control or Mdp3 siRNAs. (D) Quantification of colonies formed from control or Mdp3 siRNA-transfected MCF7 cells. (E) Immunoblot analysis of Mdp3 and β-actin expression in MCF-10A and BT549 cells transfected with control or Mdp3 for 48 hours. (F) Quantification of colonies formed from control or Mdp3-transfected cells. (G) MCF7 cells were transfected with control or Mdp3 siRNAs, and the DNA content was analyzed by flow cytometry. The M1, M2, and M3 gates indicate cells in G₁, S, and G2/M phases, respectively. (H) Quantification of the percentages of cells in G₁, S, and G2/M phases.

Figure 3

Mdp3 stimulates the motility of breast cancer cells. (A) Immunoblot analysis of Mdp3 and β-actin expression in MDA-MB-231 and Hs578T cells transfected with control or Mdp3 siRNAs for 48 hours. (B) Control or Mdp3 siRNA-transfected cells were scratched, and wound margins were imaged 0 or 24 hours later. (C) Experiments were performed as in B, and the extent of wound closure was analyzed. (D) Control or Mdp3 siRNA-transfected cells were plated into inserts precoated with matrigel, and cells underside the porous membrane were stained with crystal violet 24 hours later. (E) Experiments were performed as in D, and the extent of cell invasion was quantified. (F) Experiments were performed as in D, except that the inserts were precoated with fibronectin (FN), poly-D-lysine (PDL), or gelatin (GEL). (G) Movement tracks of MDA-MB-231 cells transfected with control or Mdp3 siRNAs. (H) Experiments were performed as in G, and the Euclidean distance and the accumulated distance were determined. (I) Experiments were performed as in panel G, and the velocity of cell movement was calculated. (J) Immunoblot analysis of Mdp3 and β-actin expression in MCF-10A and MDA-MB-231 cells transfected with control or Mdp3 for 48 hours. (K) Control or Mdp3-transfected cells were scratched, and the extent of wound closure was analyzed 24 hours later. (L) Control or Mdp3-transfected cells were plated into inserts precoated with matrigel, and the extent of cell invasion was quantified by crystal violet staining 24 hours later.

Figure 4

Mdp3 increases breast cancer growth and metastasis in mice. (A) MDA-MB-231 cells were transfected with control or Mdp3 siRNAs, and the transiently-silenced cells were injected subcutaneously into the right flanks of female athymic nude mice (7 mice per group). Tumor volume was measured each week. (B) Experiments were performed as in A, and mice were sacrificed 5 weeks post-injection. Tumors were then isolated and weighed. (C) MDA-MB-231 cells were transfected with control or Mdp3, and the transiently-overexpressed cells were injected subcutaneously into the right flanks of female athymic nude mice (6 mice per group). Tumor volume was measured every 4 days. (D) Experiments were performed as in C, and mice were sacrificed 16 days post-injection. Tumors were then isolated and weighed. (E) Immunohistochemical staining of Ki67 in tumors. (F) Experiments were performed as in E, and the intensity of Ki67 was measured. (G) Immunoblot analysis of Mdp3 and β-actin expression in 4T1-luc cells transfected with control or mouse Mdp3 siRNAs. (H) 4T1-luc cells transiently silenced with control or mouse Mdp3 siRNAs were injected into BALB/c mice (4 mice per group), and the mice were examined by a bioluminescence imaging instrument 8 days later. (I) Experiments were performed as in H, and mice were sacrificed at the endpoint of day 8. Tumors were isolated and analyzed with bioluminescence imaging. (J) Experiments were performed as in I, and photon flux was determined. (K) Hematoxylin and eosin staining of lung sections in mice injected with control or mouse Mdp3 siRNA-transfected 4T1-luc cells. (L) Experiments were performed as in K, and the percentage of metastasis area was quantified. (M) 4T1-luc cells transiently transfected with control or Mdp3 were injected into BALB/c mice (6 mice per group). Lung sections were examined by hematoxylin and eosin staining as in K, and the percentage of metastasis area was quantified.

Figure 5

Interaction of Mdp3 with γ-tubulin is required for its ability to promote breast cancer cell proliferation and motility. (A) Immunofluorescence microscopy of microtubules (red), Mdp3 (green), and nuclei (blue) in MDA-MB-231 and MCF7 cells. Scale bar, 10 μm. (B) Immunofluorescence microscopy of γ-tubulin (red), Mdp3 (green), and nuclei (blue) in MDA-MB-231 and MCF7 cells. Scale bar, 10 μm. (C) Cells were transfected with GST, GST-Mdp3 full-length (FL), or GST-Mdp3 C-terminus (CT). GST pull-down and immunoblotting were then performed with anti-γ-tubulin and anti-GST antibodies. (D) Examination of the interaction between endogenous Mdp3 and γ-tubulin by immunoprecipitation and immunoblotting. (E) Immunofluorescence staining of γ-tubulin (red) and nuclei (blue) in cells transfected with GFP-Mdp3-FL or GFP-Mdp3-CT (green). Scale bar, 10 μm. (F) Immunofluorescence staining of γ-tubulin (red), Mdp3 (green), and nuclei (blue) in BT549 cells transfected with control, γ-tubulin, or Mdp3 siRNAs. Scale bar, 10 μm. (G and H) BT549 cells were transfected with Mdp3 siRNA and GFP, GFP-Mdp3-FL, or GFP-Mdp3-CT. Cell proliferation was then determined with MTT assays (G), and cell migration was determined with wound healing assays (H). (I and J) BT549 cells were transfected with GFP, GFP-Mdp3-FL, or GFP-Mdp3-ΔCC1 (lacking the first coiled coil domain). Cell proliferation was then determined with MTT assays (I), and cell migration was determined with wound healing assays (J).