Elsevier

Human Pathology

Volume 42, Issue 12, December 2011, Pages 1888-1896
Human Pathology

Original contribution
Systematic analysis of proteins from different signaling pathways in the tumor center and the invasive front of colorectal cancer

https://doi.org/10.1016/j.humpath.2010.06.020Get rights and content

Summary

In colorectal cancer, the functional impact of proteins from different signaling pathways varies between tumor center and tumor front. Our objective was to identify differential protein expression profiles between the tumor center and the tumor front of colorectal cancer. Twenty proteins from different signaling pathways (epidermal growth factor receptor [EGFR], phosphorylated extracellular signal regulated kinase [pERK], receptor for hyalouronic acid mediated motility [RHAMM], Raf-1 kinase inhibitor protein [RKIP], β-catenin, E-cadherin, phosphorylated AK transforming [pAKT], p16, p21, Ki-67, B-cell Lymphoma-2 [BCL2], vascular endothelial growth factor, apoptosis protease activating factor 1 [APAF-1], mucin1 [MUC1], ephrin B2 receptor [EphB2], matrix metalloproteinase 7 [MMP7], phosphorylated mothers against decapentaplegic 2 [pSMAD2], caudal type homeobox transcription factor 2 [CDX2], Laminin5γ2, and mammalian sterile 20-like kinase 1 [MST1]) involved in colorectal cancer progression were studied immunohistochemically on 220 well-characterized patients using a multiple-punch tissue microarray including 437 and 430 samples from the tumor center and the invasive front, respectively. Mean expression between the tumor center and the tumor front varied statistically significantly for pSMAD2, pERK, Raf-1 kinase inhibitor protein, E-cadherin, pAKT, BCL2, vascular endothelial growth factor, EphB2, matrix metalloproteinase 7, CDX2, Laminin5γ2, MST1, and APAF-1. Overexpression of pAKT, BCL2, vascular endothelial growth factor, APAF-1, pERK, EphB2, Raf-1 kinase inhibitor protein, CDX2, E-cadherin, MST1 (P < .001 each), and pSMAD2 (P = .002) was more frequently observed in the tumor center, whereas matrix metalloproteinase 7 and Laminin5γ2 (P < .001 each) overexpression was associated with the invasive front. In multivariate analysis, vascular endothelial growth factor (P < .001), Raf-1 kinase inhibitor protein (P = .009), and Laminin5γ2 (P < .001) were the most relevant proteins with the multimarker phenotypes positive/positive/negative and negative/negative/positive being most discriminating between the tumor center and the tumor front. Moreover, the combination negative/negative/positive vascular endothelial growth factor/Raf-1 kinase inhibitor protein/Laminin5γ2 at the tumor front was associated with vascular/lymphatic invasion (P = .014), distant metastasis (P = .019), higher tumor grade (P < .001), and poorer survival (P = .05). Our findings show that, in colorectal cancer progression, vascular endothelial growth factor overexpression seems to play a role in the tumor center, whereas Laminin5γ2-positivity combined with Raf-1 kinase inhibitor protein loss is associated with tumor invasion at the front.

Introduction

The ability of invasion at the tumor front is due to both intrinsic features of the neoplastic cells, such as loss of epithelial differentiation [1], and favorable conditions of the tumor microenvironment [2]. Cells at the invasive front of colorectal carcinomas are considered to have a more aggressive behavior in comparison with more central regions [3]. It could thus be hypothesized that the neoplastic cells at the tumor front adapt their phenotype to satisfy the needs of their new role, which differs from the role of the neoplastic cells located in the main tumor body.

The expression and functional impact of proteins from various signaling pathways have been reported to be different between the tumor center and the tumor front in colorectal cancer (CRC) [3], [4], [5]. Indeed, the interaction between malignant epithelium and stromal elements, also called epithelial-mesenchymal transition, occurring at the invasive front is recognized as a crucial step in CRC carcinogenesis [6].

In CRC, TNM stage and other established morphological features provide the standard criterion for identifying patients with different risks of a specific outcome. However, the identification of reproducible and well-characterized predictive and prognostic biomarkers would help in defining subgroups in CRC.

The aim of the present study was to investigate potential differences in protein marker expression on 220 well-characterized patients with CRC with the ability to differentiate between the tumor center and the tumor front to identify prognostic factors for CRCs. Established and promising prognostic proteins selected to represent various signaling pathways including the wingless-INT signaling pathway (Wnt) (β-catenin, E-cadherin), extracellular signal regulated kinase/mitogen activated protein kinase (ERK/MAPK) (EGFR, pERK, Raf-1 kinase inhibitor protein [RKIP]), AKT (pAKT), transforming growth factor β (TGF-β) (pSMAD2), as well as proteins involved in cell cycle regulation, apoptosis, or proliferation (p21, p16, BCL2, Ki-67, apoptosis protease activating factor-1 [APAF-1], MST1) and in angiogenic or metastatic processes (RHAMM, vascular endothelial growth factor [VEGF], EphB2, matrix metalloproteinase [MMP7], Laminin5γ2, MUC1, CDX2) were evaluated using a multiple-punch tissue microarray.

Section snippets

Patients and clinicopathological features

Two hundred twenty nonconsecutive patients treated at the 4th Department of Surgery, University of Athens Medical School were entered into the study. Paraffin blocks were selected from the archives of the 2nd Department of Pathology, University of Athens Medical School (Attikon University Hospital), Greece. All selected cases had at least 1.2 cm tumor tissue thickness in at least 2 paraffin blocks to be included to the study.

Patients were treated between 2004 and 2006. All histomorphological

Immunohistochemistry

The number of evaluable tissue punches for each protein marker was as follows: EGFR, 791; RHAMM, 783; β-catenin, 799; p16, 788; p21, 800; Ki-67, 786; pAKT, 779; BCL2, 778; VEGF, 775; APAF-1, 766; MUC1, 792; pERK, 776; EphB2, 779; E-cadherin, 787; RKIP, 767; MMP-7, 781; pSMAD, 352; CDX2, 782; Laminin5γ2, 783; and MST1, 776. Immunohistochemical results are summarized in Table 2, Table 3, Table 4.

Differences in mean protein expression between the invasive tumor front and the tumor center

The mean protein expression between the tumor front and the tumor center varied significantly for

Discussion

Our results demonstrate that VEGF, RKIP, and Laminin5γ2 were the most relevant proteins, with the multimarker phenotypes positive/positive/negative and negative/negative/positive being most discriminating between the tumor center and the tumor front in CRC.

We found a differential VEGF expression between the tumor center and the tumor front in CRC, with loss of VEGF expression at the invasive tumor front. Vascular endothelial growth factor is a potent proangiogenic factor whose downstream

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