ARTICLE |
Correspondence to: Richard V. Benya, Dept. of Medicine, U. of Illinois at Chicago, 840 South Wood Street (M/C 716), Chicago, IL 60612. E-mail: rvbenya@uic.edu
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Summary |
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Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.
(J Histochem Cytochem 51:10411048, 2003)
Key Words: bombesin, colorectal cancer, gastrin-releasing peptide, receptor
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Introduction |
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FOCAL ADHESION KINASE (FAK) is a 125-kD cytoplasmic protein localized to focal adhesions, structures primarily responsible for tethering cells to the extracellular matrix. FAK serves both as a scaffolding protein that recruits signaling molecules containing Src homology (SH2 and SH3) domains and mediates a variety of cellular functions including enhancing motility and inhibiting apoptosis (reviewed in
A number of studies have shown increased expression of FAK in colon cancers compared to that observed in adjacent non-malignant mucosal epithelial cells (
Tumor cell differentiation is another important parameter that has yet to be considered in studies of FAK expression in colon cancer. In the context of solid tumors, differentiation describes the degree to which the tumor cell(s) resemble the non-malignant tissues whence they originated. Clinically, the most important implication of tumor differentiation relates to the fact that it correlates with the development of metastases for all tumors studied (
We have recently proposed that gastrin-releasing peptide (GRP) and its receptor (GRP-R) are morphogens, critically involved in regulating colon cancer differentiation. Although mature epithelial cells lining the colon do not normally express GRP/GRP-R, they are upregulated in post-neoplastic transformation where they act to retain tumor cells in a better-differentiated state (reviewed in
We therefore undertook this study to evaluate FAK expression, as well as FAK activity as measured by the presence of various phosphorylated forms of this enzyme, in human colon cancers as a function of the differentiation of individual tumor cells. Using our novel algorithm for quantitative immunohistochemistry (
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Materials and Methods |
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Materials
Anhydrous ethanol (100%, 95%) and xylene were purchased from Pharmco Products (Brookfield, CT). Wash Buffer, Target Retrieval Solution, Protein Block Serum, Antibody Diluent, LSAB2 System-HRP, Liquid DAB Substrate-Chromogen System, EnVision+ HRP (DAB)Rabbit System, and Automated Hematoxylin were all from DAKO (Carpinteria, CA). Auto/Iodine, Redusol, and all other reagents were obtained from Fisher Scientific (Pittsburgh, PA).
As previously described (
Tumor Specimens and Histological Grading
The GI Cancer Tumor Bank contains surgical pathology specimens from all patients undergoing surgical resection for colon cancer between 1980 and 1999. Ten tumors were randomly selected equally divided across all four Dukes' stages. The University of Illinois Institutional Review Board approved this study. Differentiation was assessed non-subjectively across a 5-stage scale as previously defined (
Image Capture
All photomicrographs were obtained using a SPOT RT Digital Scanning Camera from Diagnostic Instruments (Sterling Heights, MI) attached to a Nikon E600 microscope system. Images were acquired at x1000 magnification and were saved in TIFF format for further evaluation.
Quantitative Immunohistochemistry
IHC was performed on 10 tumor specimens randomly selected from the UIC GI Cancer Tumor Bank and which had been originally resected between 1985 and 1997. FAK antibody conditions were established using sections of human prostate cancer, with tumors randomly selected from the UIC Cancer Center Tumor Bank. This study was approved by the UIC Institutional Review Board (IRB). In all instances, IHC was performed using antibodies resuspended in DAKO Antibody Diluent.
A modified indirect immunoperoxidase technique was performed on 4-µm-thick paraffin-embedded sections that were hydrated in graded alcohols and rinsed in a running water bath. Slides were incubated for 5 min in a 3% hydrogen peroxide solution to quench endogenous peroxidase activity. Mercuric pigments were removed by incubating in Auto/Iodine for 1 min, followed by incubation in Redusol two times for 2 min each. Hydration was completed by rinsing in DAKO 1 x Wash Buffer three times for 3 min. Antigen retrieval was used for tissue sections submerged in 1 x DAKO Target Retrieval Solution for 30 min at 100C in a steamer. Slides were allowed to cool to RT and rinsed in 1 x DAKO Wash Buffer. Sections were blocked for 15 min with Protein Block Serum and then incubated with antibody overnight at 4C. After rinsing with Wash Buffer, slides treated with GRP (1:125), GRP-R (1:750), and total FAK antibodies were incubated with biotinylated IgG for 10 min, rinsed, and then incubated with streptavidin conjugated to horseradish peroxidase (LSAB2 kit from DAKO) for 10 min. These same sections were then rinsed and incubated with Liquid DAB Substrate-Chromogen System for 5 min to identify bound antibody. Tissue sections treated with phosphospecific FAK antibodies were incubated with labeled polymer for 30 min, rinsed, and incubated with Liquid DAB+ for 10 min to identify bound antibody (EnVision+ HRPDAB kit from DAKO). After a final rinse in 1 x Wash Buffer, all slides were counterstained with DAKO Automated Hematoxylin for 2 min, dehydrated in alcohol, and mounted with a coverslip using Permount. Control tissues were processed simultaneously as the treated slides, with the exception that primary antibody was not applied.
Chromogen abundance was quantified by determining the cumulative signal strength of the digital image file of a histologically relevant region of interest (
Statistical Analysis
All data reported herein are valueless and are reported as energy units per pixel (eu/pix). Statistical evaluations were performed using StatView (Abacus Concepts; Berkeley, CA). In all instances, data are expressed as the mean ± SEM.
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Results |
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As we (
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We next established the conditions necessary for performing IHC using our antibodies to total and the various phosphorylated forms of FAK. To do this, we used sections of human prostate cancer because this tumor type ubiquitously expresses FAK (
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Using these concentrations, we evaluated human colon cancer sections that had been sequentially sectioned adjacent to those used to quantify GRP and GRP-R expression. In this way, FAK expression could be determined as a function both of tumor cell differentiation and as a function of the amount of GRP/GRP-R expressed in each region of defined histology. Similar to what we previously observed in murine colon cancer (
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The amount of total FAK and of pY397 and pY407 was not only associated with tumor cell differentiation but was also tightly correlated with the amount of GRP/GRP-R co-expression (Fig 3 and Fig 5). To determine this, we plotted the amount of total GRP and GRP-R chromogen present in each region of defined histological differentiation with the amount of total FAK (Fig 5, left) or the amount of pY397 and pY407 (Fig 5, middle). The tightness of this correlation was striking, with correlation co-efficient's possessing r 2 values in excess of 0.9. In contradistinction, there was no correlation between GRP/GRP-R co-expression and the amount of Y576, Y577, Y861, and Y925 present (Fig 5).
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The amount of chromogen generated by antibodies to the phosphorylated forms of FAK other than Y397 and Y407 varied considerably and showed no correlations with the degree of tumor cell differentiation (Table 1; Fig 5, right). Specifically, the amount of pY576-specific chromogen was less for moderately poorly differentiated cells (41.8 ± 17.0 eu/pix) than for those that were more or less well differentiated (82.6 ± 6.6 eu/pix and 80.1 ± 15.8 eu/pix for tumor cells that were moderate and poorly differentiated, respectively). A similar roller-coaster pattern was observed for pY577, with lower chromogen quantities for moderately well-differentiated tumor cells (35.6 ± 2.8 eu/pix) than for those that were well (73.2 ± 31.9 eu/pix) or moderately (87.6 ± 35.4 eu/pix) differentiated. In contrast, the least amount of chromogen for pY861 was for well (27.7 ± 7.9 eu/pix) and poorly differentiated (44.5 ± 5.0 eu/pix) tumor cells, whereas intermediate stages contained up to threefold more chromogen. But for Y925, similar low amounts of chromogen (range 3450 eu/pix) were observed for all tumor cells, irrespective of their stage of differentiation. Finally, the very large standard errors generally observed for chromogen generated using antibodies to Y576, Y577, Y861, and Y925 (Table 1) further reflects their lack of association with differentiation.
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Discussion |
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The role of FAK in colon cancer is not clear. Previous IHC studies performed on resected human colon cancers indicated that total FAK is present in greater amounts in invasive compared to non-invasive cancers (
Although the assessment of differentiation of entire colon cancers is not good for predicting patient outcome, this reflects the fact that this type of malignancy is unusual insofar as they are heterogeneously differentiated. However, when the differentiation of specific cell populations within a colon cancer is considered, this parameter does become an important prognostic parameter (
We previously showed that GRP/GRP-R upregulation in colon cancer recapitulates, albeit dysfunctionally, their normal role in regulating intestinal development during organogenesis (
But FAK has traditionally been viewed, perhaps because of its ability to promote the motility of cancer cell lines studied in vitro (
Studies of murine fibroblast cell lines have shown that GRP binding to its receptor causes FAK to be phosphorylated (
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Acknowledgments |
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Supported by NIH grant DK-07788 (to S. Glover) and CA-094346 and a VA Merit Review award (to R.V. Benya).
Received for publication October 16, 2002; accepted March 20, 2003.
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