Interplay between 15-lipoxygenase-1 and metastasis-associated antigen 1 in the metastatic potential of colorectal cancer

Abstract

Objectives: Metastasis-associated antigen 1 (MTA1) is implicated in metastasis while 15-lipoxygenase-1 (15-LOX-1) reduces cell motility, when re-expressed in colorectal cancer (CRC). We aimed to understand any potential interplay between MTA1 and 15-LOX-1 in CRC metastasis.

Materials and methods: ALOX15 and MTA1 expression in tumour and normal samples were analysed from TCGA RNA-seq data, microarray data sets and a human CRC cDNA array. Western blots, chromatin immunoprecipitation (ChIP), luciferase assays and electrophoretic mobility shift assays (EMSA) were carried out in HT-29 and LoVo cells re-expressing 15-LOX-1 to determine NF- κB activity at the MTA1 promoter. Functional assays in cells ectopically expressing either 15-LOX-1, MTA-1 or both, were carried out to determine adhesion and cell motility.

Results: Significantly higher expression of MTA1 was observed in tumours compared to normal tissues; MTA1 overexpression resulted in reduced adhesion in CRC cell lines. Re-expression of 15-LOX-1 in the CRC cell lines reduced expression of endogenous MTA1, corroborated by negative correlation between the two genes in two independent human CRC microarray data sets, with greater significance in specific subsets of patients. DNA binding and transcriptional activity of NF-κB at the MTA1 promoter was significantly lower in cells re-expressing 15-LOX-1. Functionally, the same cells had reduced motility, which was rescued when they overexpressed MTA1, and further corroborated by expressions of E-cadherin and vimentin.

Conclusions: Expression of MTA1 and 15-LOX-1 negatively correlated in specific subsets of CRC. Mechanistically, this is at least in part through reduced recruitment of NF-κB to the MTA1 promoter.

Figure 1

Expression analysis of MTA1 and ALOX15 in colon tumour data sets. (a) Analysis of MTA1 expression in tumour (n=416) and normal samples (n=50) in the TCGA colon adenocarcinoma and rectal adenocarcinoma data (left panel). MTA1 expression in a colon cancer cDNA RT‐qPCR array consisting of tumour (n=40) and normal samples (n=8) (right panel). Significantly higher expression was seen in the tumour compared to the normal samples. (b) Hanging drop assay showing significantly lower cell‐cell adhesion in HCT‐116 colon cancer cells transfected with 500 ng and 1 μg of an MTA1 vector when compared to the empty vector‐transfected cells. (****P<0.0001) (c) Comparative expressions of ALOX15 and MTA1 in the TCGA colon and rectal tumour data set. Lower expression with a significantly greater variability was seen in the expression of ALOX15 compared to MTA1 (n=408). (d) Correlation analysis showing a significant negative correlation in the expressions of ALOX15 and MTA1 in the GSE39582 data set showing patient stratification into subgroups C1‐C6 (left panel) (n=566 for tumour, n=19 for normal). Stronger negative correlation in the expression of MTA1 and ALOX15 in the patient subgroup C5 (n=152 right panel). (e) Correlation analysis showing a significant negative correlation in the expressions of ALOX15 and MTA1 in the GSE41258 microarray data set including 54 normal and 186 primary tumour samples. P value is based on Pearson's correlation coefficient

Figure 2

15‐LOX‐1 re‐expression in colon cancer cell lines results in a loss of MTA1 expression. Western blot analysis of 15‐LOX‐1 (72 kDa) and MTA1 (80 kDa) in 15‐LOX‐1‐ or pcDNA3.1 (empty vector)‐transfected HT‐29 and LoVo cells. Equal protein loading was shown by β‐actin (42 kDa) levels. The cells showed the loss in expression of MTA1 when 15‐LOX‐1 was ectopically expressed. Three independent experiments were performed and statistical comparisons were carried out using paired t‐test (*P < .05)

Figure 3

15‐LOX‐1 expression results in a loss of NF‐κB nuclear translocation in LoVo cells. Western blot analysis of nuclear and cytoplasmic extracts isolated from 15‐LOX‐1‐expressing LoVo cells. The lysates were probed with a p65 or an IκBα antibody. Control cells included the LoVo cells transfected with the empty pcDNA3.1 vector. Equal protein loading and lack of cross contamination between nuclear and cytoplasmic extracts was shown by TopoIIβ (180 kDa) and α‐Tubulin (55 kDa)

Figure 4

Analysis of NF‐κB binding to the promoter of MTA1. (a) Schematic diagram of NF‐κB‐binding sites in the region between chr14: 105884100‐105886185 on the MTA1 promoter. Two regions were identified: Region I with one and Region II with five NF‐κB‐binding consensus sequences. (b) ChIP assay showing decreased NF‐κB p65 recruitment on the MTA1 promoter at Region I and Region II in the presence of 15‐LOX‐1 expression in HT‐29, and LoVo cells. 15‐LOX‐1 re‐expression is shown by Western blot in the ChIP lysates from HT‐29 and LoVo cells. (c) Luciferase assays showing reduced MTA1 promoter activity in HT‐29 and LoVo cells, respectively after re‐expression of 15‐LOX‐1. The data are normalized to pLuc‐MCS‐EV (empty vector)/pHRLTK ratios. (d) EMSA showing the loss of p65 binding to the consensus sequences 1 and 6 on the MTA1 promoter in the presence of 15‐LOX‐1 expression. Nuclear lysates from pcDNA3.1 (empty vector)‐transfected HT‐29 and LoVo cells served as controls and showed binding to these regions. Incubation of the nuclear extracts from EV transfected cells with the mutated oligos resulted in a complete loss of binding, further confirming the specificity of the reaction. Three independent experiments were performed and statistical comparisons were carried out using paired t‐test (*P<.05, **P<0.01)

Figure 5

Lower motility of cells transfected with 15‐LOX‐1 that was partially rescued with MTA1 overexpression. (a) HCT‐116 cells stably expressing 15‐LOX‐1, the same cells transiently transfected with 500 ng of an MTA1 plasmid (15‐LOX‐1+MTA1), HCT‐116 cells transiently transfected with the MTA1 plasmid (MTA1) or the control (empty vector, EV transfected) cells were analysed for motility with a Transwell assay. (b) Proteins from the same cell models as in A were extracted and analysed for Western blots showed reduced expression of vimentin in cells expressing 15‐LOX‐1. This phenotype was completely rescued when the cells co‐expressed 15‐LOX‐1 and 1 μg of the MTA1 plasmid. (c) LoVo cells were transiently transfected individually with either the 15‐LOX‐1 plasmid, or the MTA1 plasmid, or a combination of the two plasmids for 24 hours. The cells were scratched and monitored every 24 hours for 48 hours for wound closure. (d) Western blots using LoVo protein lysates from cells used in the scratch assay showed higher E‐cadherin expression in cells expressing 15‐LOX‐1 alone compared to cells expressing MTA1 alone. The high expression of E‐cadherin was retained even when cells expressed both MTA1 and 15‐LOX‐1 (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05)