The PDZ protein TIP-1 facilitates cell migration and pulmonary metastasis of human invasive breast cancer cells in athymic mice

Abstract

Tax-interacting protein 1 (TIP-1, also known as Tax1bp3) inhibited proliferation of colon cancer cells through antagonizing the transcriptional activity of beta-catenin. However, in this study, elevated TIP-1 expression levels were detected in human invasive breast cancers. Studies with two human invasive breast cancer cell lines indicated that RNAi-mediated TIP-1 knockdown suppressed the cell adhesion, proliferation, migration and invasion in vitro, and inhibited tumor growth in mammary fat pads and pulmonary metastasis in athymic mice. Biochemical studies showed that TIP-1 knockdown had moderate and differential effects on the beta-catenin-regulated gene expression, but remarkably down regulated the genes for cell adhesion and motility in breast cancer cells. The decreased expression of integrins and paxillin was accompanied with reduced cell adhesion and focal adhesion formation on fibronectin-coated surface. In conclusion, this study revealed a novel oncogenic function of TIP-1 suggesting that TIP-1 holds potential as a prognostic biomarker and a therapeutic target in the treatment of human invasive breast cancers.

Fig. 1. TIP-1 expression levels are elevated in the invasive breast cancers

(A, B) Relative TIP-1 expression levels in pathologically validated clinical specimens of benign cystic lesions, invasive ductal/lobular breast carcinomas or adjacent normal mammary glands from two independent datasets. The data distribution was presented as box plot. N represents case number for each subgroup, * p<0.05, ANOVA. C) Kaplan-Meier survival curve to show survival time of breast cancer patient after diagnosis is stratified by the TIP-1 expression levels (log-rank analysis, p=0.0032).

Fig. 2. TIP-1 knockdown suppressed proliferation, migration and invasion of human breast cancer cells in vitro

(A) Western blot analyses of TIP-1 knockdown within BT549 (upper panel) and MDA-MB-231 (lower panel) cells with shRNAs. Actin was blotted as a loading control. (B) Cell proliferation assessed with Ki67 staining on monolayer cultures. The quantification was based on >500 cells for each group in three independent experiments. Cell migration (C) and invasion (D) were analyzed with the Boyden chamber-based system as described in the the Materials and Methods. Bar graph represents the number of cells that have migrated or invaded across the membrane. Results shown are mean ± SEM of three independent experiments. * p<0.05, the Student’s t-test.

Fig. 3. TIP-1 knockdown impaired the gene expression for focal adhesion formation and cell adhesion

(A) Western blot and semi-quantification of paxillin and integrin alpha-5 (ITGA5) protein levels in MDA-MB-231 cells with or without TIP-1 knockdown. Data were normalized to the levels of actin. (B) Flow cytometry analysis of integrin alpha-5 expression on the cell plasma surface of MDA-MB-231 cells. A control primary antibody was included. (C) Adhesion of MDA-MB-231 cells to different extracellular matrices including fibronectin, collagen IV or laminin. (D) Numbers of focal adhesion formed in cells were quantified based on phosphor-paxillin immunofluorescent staining of MDA-MB-231 cells on fibronectin-coated surface. All statistic analyses were based on quantitative measurements from three independent experiments. * p<0.05, the Student’s t-test.

Fig. 4. TIP-1 knockdown inhibited tumor growth in mammary fat pads and pulmonary metastasis of human breast cancer cells in athymic mice

(A) Tumor growth curve (left) of MDA-MB-231 xenografts in mammary fat pads, and statistics (right) of pulmonary metastases from the primary xenograft tumors in FoxN1-null mice. n=5. (B) Representative light images of pulmonary metastases by six weeks after the intravenous injection of MDA-MB-231 cells. (C) Quantitative measurements of lung weight, number and diameter of tumor nodules of the pulmonary metastases. * p<0.05, n=9, the Student’s t-test.