New specific molecular targets for radio-chemotherapy of rectal cancer

Abstract

Patients with locally advanced rectal cancer often receive preoperative radio-chemotherapy (RCT). The mechanisms of tumour response to radiotherapy are not understood. The aim of this study was to identify the effects of RCT on gene expression in rectal tumour and normal rectal tissue. For that purpose tissue samples from 21 patients with resectable adenocarcinomas were collected for use in whole genome-microarray based gene expression analysis. A factorial experimental design allowed us to determine the effect of RCT on tumour tissue alone by removing the effect of radiation on normal tissue. This resulted in 1327 differentially expressed genes in tumour tissue with p<0.05. In addition to known markers for radio-chemotherapy, a Gene Set Enrichment Analysis (GSEA) showed a significant enrichment in gene sets associated with cell adhesion and leukocyte transendothelial migration. The profound change of cell adhesion molecule expression in rectal tumour tissue could either increase the risk of metastasis, or decrease the tumour's invasive potential.

Figure 1

Comparison of treatment groups of rectal cancer patients. The Venn diagram shows the number of all differentially expressed genes across different comparisons: NR‐NN, radiated normal rectal tissue vs. non‐radiated normal rectal tissue; NR‐NN, radiated rectal tumour tissue vs. non‐radiated rectal tumour tissue; and comparison of NR‐NN with TR‐TN. The number of differential genes with p<0.05 of each comparison is indicated, respectively.

Figure 2

Principal component analysis (PCA) and bridge partial least square analysis (PLS) on different rectal cancer patient samples. (A) Two‐dimensional PCA of differentially expressed genes (p<0.05), derived from 20 patients with rectal cancer, before and after radio‐chemotherapy (RCT), showing separation of the different sample groups. (B) The plot depicts the scores of the PLS model. Components 1 and 2 (left) look similar to PCA analysis shown in (A), but component 3 (right) clearly separates the sample groups for normal rectal tissue. All samples are colour coded according to group. Black: NN (normal rectal tissue); green: NR (irradiated normal rectal tissue samples); blue: TN (rectal tumour tissue samples); red: TR (irradiated rectal tumour samples). *Potential non‐responders.

Figure 3

Genes involved in cell adhesion are specifically affected by RCT in rectal cancer tissue samples. The difference in the cell adhesion gene expression between radiated rectal tumour (TR, red) and rectal tumour (TN, green), normal rectal tissue (NN, black) and radiated normal rectal tissue (NR, blue) is shown. Gene expression values are shown in a spectrum where down‐regulated is green, no change is black, and up‐regulated is red. Only genes (47) within the PANTHER BP00124 cell adhesion gene set (618 genes) and with two‐sided t‐test p<0.001 (TR vs. TN, NN, NR) are shown in the figure. Top panel of the heat map depicts patient sample groups. Black, non‐radiated normal rectal tissue; blue, radiated normal rectal tissue; green, non‐radiated rectal tumour tissue; red, radiated tumour rectal tissue.

Figure 4

Cell adhesion pathway and ECM–receptor interaction pathways are affected by RCT. The signalling pathways for cell adhesion and ECM–receptor interaction, generated with KEGG comparing non‐radiated rectal tumour samples with radiated rectal tumour samples, is shown. Each bar (blue or red) represents a gene. Red bars indicate an increase in irradiated rectal tumour gene expression as compared to non‐irradiated rectal tumour samples. Blue bars indicate a decrease. Bar height is inversely related to p‐value; a larger bar represents a smaller p‐value up to a maximum height at p‐value less then 10−8.

Figure 5

Leukocyte transendothelial migration pathway is affected by RCT. The pathway for leukocyte transendothelial migration generated with KEGG is shown. Each bar (blue or red) represents a gene. Red bars indicate an increase in irradiated rectal tumour gene expression as compared to non‐irradiated rectal tumour samples. Blue bars indicate a decrease. Bar height is inversely related to p‐value; a larger bar represents a smaller p‐value up to a maximum height at p‐value less then 10−8.

Figure 6

Immunohistological staining of different rectal tissue samples. The immunoreactivity of phospho‐Histone H2A.X (C, D) and CD31 (G, H) was significantly increased in cancer tissue after receiving RCT compared to untreated samples. The immunoreactivity of COL6A1 (K, L) and LAMA4 (O, P) was slightly increased in cancer patients after RCT. There were no changes observed for the expression pattern of these proteins in tumour‐free adjacent mucosa (E, F: CD31; I, J: COL6A1; M, N: LAMA4) except for phospho‐Histone H2A.X, which was increased (A, B). The slides were counterstained with haematoxylin, and the original magnification for images was 400×.