AF1q inhibited T cell attachment to breast cancer cell by attenuating Intracellular Adhesion Molecule-1 expression

Abstract

Aim: To investigate whether AF1q, overexpressed in metastatic cells compared with the primary tumor cells, plays a pivotal role in breast cancer metastasis.

Methods: To investigate whether AF1q has a responsibility in the acquisition of a metastatic phenotype, we performed RNA-sequencing (RNA-Seq) to identify the gene signature and applied the Metacore direct interactions network building algorithm with the top 40 amplicons of RNA-Seq.

Results: Most genes were directly linked with intercellular adhesion molecule-1 (ICAM-1). Likewise, we identified that ICAM-1 expression is attenuated in metastatic cells compared to primary tumor cells. Moreover, overexpression of AF1q attenuated ICAM-1 expression, whereas suppression of AF1q elicited the opposite effect. AF1q had an effect on ICAM-1 promoter region and regulated its transcription. Decreased ICAM-1 expression affected the attachment of T cells to a breast cancer cell monolayer. We confirmed the finding by performing the analysis on Burkitt's lymphoma.

Conclusion: Attenuation of ICAM-1 by AF1q on tumor cells disadvantages host anti-tumor defenses through the trafficking of lymphocytes, which affects tumor progression and metastasis.

Keywords: AF1q; MLLT11; RNA-sequencing; breast cancer; intercellular adhesion molecule-1; metastasis.

Figure 1.

A: AF1q expression status in breast cancer cell lines (1: MDA-MB-231, 2: MDA-MB-231-luc2-LN); B: validation of relative expression of gene obtained from RNA-seq by qPCR. qPCR analyses were performed as described in the method section using randomly selected 20 genes (upregulated genes 10 and downregulated genes 10). Relative expression values are presented as an average ± SD of three biological replicates; C: significantly expressed genes. 1,485 genes are significantly expressed in both cell lines with three biological replicates, respectively. Among those, transcripts of 762 genes increased in MDA-MB-231LN cell line and those of 723 genes decreased

Figure 2.

Functional interaction network analysis. A: 1,485 significantly selected genes were further analyzed for pathway process using Metacore. Top 40 network objects were, then, used for building direct interactions. ICAM-1 is directly linked with most genes and positioned at the end of the pathways. Red line represents suppression, blue line represents activation; B: RNA-seq shows that expression level of ICAM-1 is drastically decreased in MDA-MB-231LN cell line compared to MDA-MB-231

Figure 3.

Transcriptional regulation of ICAM-1 expression by AF1q in breast cancer. A: ICAM-1 mRNA expression was quantified using qPCR in MDA-MB-231LN cells engineered to overexpress or suppress AF1q; B: the blot shows ICAM-1 protein expression; C: MDA-MB-231LN cells were stained with ICAM-1 mAb and flow cytometry analysis were performed using FACS Calibur. On left panel, Red is profile of the MDA-MB-231LN/Ctrl and Blue is MDA-MB-231LN/AF1q. On the right panel, Red is MDA-MB-231LN/shCtrl and Blue is shAF1q cells. Representative data of three similar experiments are shown; D: Luciferase activity in HEK293T cells transfected with reporter constructs of the proximal promoter of ICAM-1. Renillar Luciferase activity was used to normalize the promoter activity

Figure 4.

ICAM-1 is important for attachment and cytotoxicity of T cells to breast cancer. A: Labelled T cells attached to MDA-MB-231LN cells monolayer. AF1q significantly reduced the number of attached T cells to the monolayer. When AF1q expression was suppressed, the number of attached T cells was increased. The Bar graph represents the % adhesion cells of total cells; B: ICAM-1 blocking antibody significantly reduced the number of attached T cells to the monolayer; C: a 72 h cytotoxicity assay was performed in 24 well plates where co-cultured at 10:1 or 20:1 E:T ratio with MDA-MB-231LN cells

Figure 5.

AF1q reciprocally regulates the expression of ICAM-1 in Burkitt’s lymphoma. A: ICAM-1 mRNA expression was quantified using qPCR in MDA-MB-231LN cells engineered to overexpress or suppress AF1q; B: Western blot analysis of ICAM-1 and AF1q in Burkitt’s lymphoma cell lines; C: representative images of AF1q and ICAM-1 staining in human Burkitt’s lymphoma tumor metastasis tissues. High IHC staining of AF1q in tissue samples show low ICAM-1 expression