NCK-associated protein 1 regulates metastasis and is a novel prognostic marker for colorectal cancer

Abstract

Metastatic colorectal cancer (CRC) remains a substantial problem for mortality and requires screening and early detection efforts to increase survival. Epithelial-mesenchymal transition (EMT) and circulation of tumor cells in the blood play important roles in metastasis. To identify a novel target for metastasis of CRC, we conducted a gene microarray analysis using extracted RNA from the blood of preclinical models. We found that NCK-associated protein 1 (NCKAP1) was significantly increased in the blood RNA of patient-derived xenograft (PDX) models of colon cancer. In the NCKAP1 gene knockdown-induced human colon cancer cell lines HCT116 and HT29, there was a reduced wound healing area and significant inhibition of migration and invasion. As the result of marker screening for cytoskeleton and cellular interactions, CRC treated with siRNA of NCKAP1 exhibited significant induction of CDH1 and phalloidin expression, which indicates enhanced adherent cell junctions and cytoskeleton. In HCT116 cells with a mesenchymal state induced by TGFβ1, metastasis was inhibited by NCKAP1 gene knockdown through the inhibition of migration, and there was increased CTNNB1 expression and decreased FN expression. We established metastasis models for colon cancer to liver transition by intrasplenic injection shRNA of NCKAP1-transfected HCT116 cells or by implanting tumor tissue generated with the cells on cecal pouch. In metastasis xenograft models, tumor growth and liver metastasis were markedly reduced. Taken together, these data demonstrate that NCKAP1 is a novel gene regulating EMT that can contribute to developing a diagnostic marker for the progression of metastasis and new therapeutics for metastatic CRC treatment.

Fig. 1. Correlation of NCKAP1 expression level and CRC.

A The mRNA levels of NCKAP1 were confirmed by qRT-PCR analysis in blood cells derived from four colon cancer PDX models. Data are reported as the mean ± SEM. Student’s t-test *p < 0.05, **p < 0.01 vs. Intact. (Intact: n = 4, PDX1: n = 5, PDX2: n = 4, PDX3: n = 5, PDX4: n = 2). B IHC staining was performed with anti-NCKAP1. Bar, 40 μm. C Linear regression analysis was subsequently performed between relative mRNA levels of NCKAP1 and tumor weights. Data are reported as the mean ± SEM. Student’s t-test. p = 0.0005. (n = 9).

Fig. 2. Inhibition of metastasis characteristics by reduction of NCKAP1.

A HCT116-Luc and HT29-Luc cells were transfected with 20 nM siControl or 20 nM siNCKAP1 for 72 h. Transfected cells were observed under a phase-contrast microscope. Bar, 50 μm. B A wound healing assay was performed. HCT116-Luc cells were transfected with 20 nM siControl or 20 nM siNCKAP1 for 24 h and further seeded on Culture-Insert 2 Well for 24 h. After scratching, cell migration was monitored with a phase-contrast microscope for 24 h. Bar, 100 μm. C, D A migration and invasion assay of HCT116-Luc cells transfected with 20 nM siControl or 20 nM siNCKAP1 for 72 h was performed. C The migrated cells were stained with crystal violet and observed using a fluorescence microscope (10 X). Quantitative results on the right: Data are reported as the mean ± SEM. Student’s t-test. *p < 0.05 vs. siControl. (n = 3). D The invaded cells were stained with crystal violet and counted. Quantitative results on the right: Data are reported as the mean ± SEM. Student’s t-test. *p < 0.05 vs. siControl. (n = 3). E Transfected HT-29-Luc cells were subjected to immunocytochemistry of CDH1 and DAPI. Representative pictures of cells are shown by confocal microscope. Bar, 20 μm and 50 μm. Quantitative results on the right: Data are reported as the mean ± SEM. Student’s t-test. ***p < 0.001 vs. siControl. (n = 3).

Fig. 3. Inhibition of TGFβ1-induced EMT by reduction of NCKAP1.

A HCT116-Luc cells were transfected with 20 nM siControl or 20 nM siNCKAP1 for two days and further treated with 5 ng/ml TGFβ1 for two days. Phase-contrast microscopy was performed. Bar, 50 μm. B Transfected HCT116-Luc cells were further treated with 5 ng/ml TGFβ1 for 24 h. After scratching, cell migration was monitored at the indicated time points and observed under a phase-contrast microscope. Bar, 100 μm. C The graphs quantitatively show the area of wound recovery in B. Data are reported as the mean ± SEM. One-way ANOVA and Tukey’s post hoc test. ***p < 0.0001 vs. TGFβ1 untreated siControl or TGFβ1 treated. (n = 3). D HT-29-Luc Cells were transfected with 20 nM siControl or 20 nM siNCKAP1 and further induced EMT with 5 ng/ml TGFβ1. Immunocytochemistry of CTNNB1 and DAPI was performed. Cells were observed under a confocal microscope. Bar, 40 μm. E The expression levels of mesenchymal related mRNA on HCT116-Luc cells were measured by qRT-PCR. Data are reported as the mean ± SEM. Two-way ANOVA and Bonferroni’s post hoc test. ***p < 0.0001 vs. siControl, *p < 0.05 vs. untreated TGFβ1 or siControl. (n = 3).

Fig. 4. Blockade of metastasis by NCKAP1 knockdown in animal models.

A A liver metastasis animal model was developed via intrasplenic injection of shNCKAP1-transfected HCT116-Luc cells (5 × 10⁶ cells/50 μl), and splenectomy was performed after 5 min of circulation in BALB/c nude mice. Images of metastatic tumor growth were monitored by the IVIS® Spectrum at 0, 7, and 19 days. Mice with metastatic livers were sacrificed at the endpoint of 22 days. Quantitative results on the right: the graph shows the fold change of liver weight/body weight ratio, and data are reported as the mean ± SEM. One-way ANOVA and Bonferroni’s post hoc test. *p < 0.05 vs. Control. (Control n = 4, shNCKAP1 (1) n = 3, shNCKAP1 (2) n = 2). B Tumor tissues for orthotopic transplantation were prepared from shScramble or shNCKAP1-transfected HCT116-Luc cells (5 × 10⁶ cells/50 μl) tumor tissues grown subcutaneously in the BALB/c nude mice. The tumor growth was monitored by for 38 days. Livers were isolated on day 38, and images of liver metastasis were measured by the IVIS® Spectrum.

Fig. 5. Validation of the possibility of NCKAP1 as a cancer diagnostic marker.

A IHC was performed with anti-NCKAP1 from TMA. Bar, 100 μm. Quantitative results on the right: the graph represents the score of NCKAP1. Data are reported as the mean ± SEM. One-way ANOVA and Bonferroni’s post hoc test. ns p > 0.05 vs. Stage I. (Stage I: n = 5, II: n = 16, III: n = 6, IV:3, Liver: n = 4, LN: n = 22). B The mRNA expression of NCKAP1 was confirmed by qRT-PCR analysis in blood buffy coats from patients with colon cancer. The bar graph below the dot graph represents the mean. Data are reported as the mean ± SEM. Student’s t-test. ns: non-significant (n = 10).