Suppression of breast cancer metastasis through the inactivation of ADP-ribosylation factor 1

Abstract

Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.

Keywords: ARF1; LM11; breast cancer; metastasis; zebrafish.

Figure 1. High-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer

(A) Summary graph of genetic alterations of the ARF genes in individual studies deposited in the cBioPortal. The amplification frequency of ARFs in breast cancer is shown in the inset. (B) A plot of the correlation between copy number alterations and mRNA expression of the ARF1 gene. (C) Analysis of ARF1 expression in breast normal and cancer tissues using Oncomine database. (D) Kaplan-Meier plot of RFS shown for breast cancer patients with high (red) and low (black) expression levels of the ARF1 gene.

Figure 2. ARF1 is upregulated in human breast cancer tissues

(A) Representative IHC results for ARF1 expression in breast cancer tissue arrays. (B) Quantitative data of staining intensity presented as integrated optical density (IDO). **p < 0.01; ***p < 0.001.

Figure 3. Knockdown of ARF1 leads to reduced cell invasion and metastasis in breast cancer

(A) The effect of shRNA-mediated ARF1 knockdown on MDA-MB-231 cells. (B) The effect of ARF1 depletion on cell invasion. (C, D, E, F) The effect of ARF1 depletion by shRNA on pulmonary metastases in the orthotopic mice model of breast cancer. (C, D) Quantitative data of lung weigh and gross surface pulmonary metastases. (E) Representative H&E stained lung sections from tumor-bearing mice. Six weeks after injection with MDA-MB-231 cells, the lungs from the NSG mice sacrificed were excised for pathological and histological analysis. Black arrows indicate representative metastatic foci. (F) The effect of ARF1 depletion by shRNA on metastatic dissemination in zebrafish. White arrows indicate disseminated MDA-MB-231 cells in the fish body. Quantitative data are shown in right panel. **p < 0.01.

Figure 4. LM11 inhibits ARF1 activation in breast cancer cells

(A) The relative expression levels of ARF1 in breast normal and cancer cells. (B) The activation of ARF1 in breast normal and cancer cells. (C) The effect of LM11 on the activation of ARF1 in breast cancer. Quantitative data of relative ARF1 activation (LM11 vs DMSO) are shown in right panel.

Figure 5. LM11 exhibits potent in vitro cytotoxicity and inhibits migration and invasion of breast cancer cells

(A, B) The effect of LM11 on cell viability in breast cancer cells. MCF7, MDA-MB-231 and Hs578T breast cancer cell lines were treated with different doses of LM11 (10–100 μM) for 24 hours and cell viability was determined by CellTiter-Glo^® Luminescent cell viability kit (A). LM11-treated MDA-MB-231 and Hs578T cells were stained with Zombie Aqua^TM dye and cell viability was determined by flow cytometry (B). (C, D, E) The effect of LM11 on cell migration and invasion in breast cancer cells. Representative images of these assays shown in (C) and quantitative data shown in (D) and (E). MDA-MB-231 and Hs578T cells were treated with LM11 for 24 hours and cell migration and invasion were determined by Gap closure and Boyden chamber, respectively. *p < 0.05; **p < 0.01.

Figure 6. LM11 effectively suppresses breast cancer metastasis in the zebrafish model

Tumor-bearing zebrafish were treated with 1 μM of LM11 for 4 days, and LM11 efficacy in metastatic dissemination was determined by confocal microscopy. White arrows indicate disseminated MDA-MB-231 cells in the fish body. Quantitative data are shown in right panel. **p < 0.01.