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Overexpression of Focal Adhesion Kinase in Primary Colorectal Carcinomas and Colorectal Liver Metastases

Immunohistochemistry and Real-Time PCR Analyses¹

Departments of Pathology [A. L. L., C. A. L.], Surgery [B. C., L. C., X. Y.], Biostatistics [D. T. M.], and Lineberger Comprehensive Cancer Center [B. C., L. C., X. Y., D. T. M.], University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7210; Department of Surgery [W. G. C.], University of Florida, Gainesville, Florida 32610

	ABSTRACT

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Purpose: Focal adhesion kinase (FAK), a protein tyrosine kinase that functions in signaling events between cells and their extracellular matrix, is overexpressed in a variety of human solid tumors. To determine whether FAK expression is up-regulated in colorectal cancer, we analyzed FAK mRNA and protein levels in primary colorectal tumors and colorectal liver metastases.

Experimental Design: p125^FAK expression in formalin-fixed paraffin-embedded (FFPE) tissue was studied using immunohistochemical assays on 24 matched primary colorectal carcinomas and colorectal liver metastases as well as 18 different colorectal liver metastases using monoclonal anti-FAK 4.47. FAK mRNA expression was quantitated by real-time PCR on 39 matched normal colorectal mucosa and primary colorectal carcinomas as well as on 17 separate liver metastases.

Results: Elevated levels of p125^FAK expression were demonstrated in both primary colorectal tumors and colorectal liver metastases compared with normal colorectal mucosa. Immunohistochemistry experiments demonstrated equivalent FAK expression in matched samples of colorectal primary tumors and liver metastases. Using real-time PCR in 39 matched samples, FAK mRNA copy number was significantly higher in primary colorectal tumors compared with normal colorectal mucosa. FAK expression was analyzed by both real-time PCR and immunohistochemistry in a separate set of colorectal liver metastases. Immunohistochemistry demonstrated high levels of FAK expression in 89% of samples. Furthermore, FAK mRNA copies in these unmatched liver metastases were significantly higher than the primary tumor FAK mRNA copies.

Conclusion: These experiments have shown that both primary colorectal cancers and colorectal liver metastases express high levels of FAK mRNA and p125^FAK protein. Furthermore, the majority of colorectal liver metastases demonstrated robust FAK expression equivalent to or greater than that in the primary colorectal tumor.

	INTRODUCTION

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Originally isolated from v-src-transformed chicken embryo fibroblasts (1) , FAK³ is a protein tyrosine kinase involved in signaling events between cells and their extracellular matrix. The M_r 125,000 FAK protein functions in cellular motility (2) , integrin-signaling pathways (3, 4, 5, 6) , and apoptosis (7, 8, 9) . In human tumors, FAK has been shown to function as a survival signal for tumor cells to resist the apoptotic stimuli of invasion and metastasis. Previously, our group has examined a variety of tumor cell lines and shown that loss of adhesion and subsequent apoptotic cell death resulted when FAK expression was attenuated by antisense oligonucleotides (7) or dominant-negative gene constructs that disrupt FAK signaling (10) .

The human FAK gene was first isolated from a primary human sarcoma (11) . Initial studies of FAK expression in human tumors linked elevated levels of FAK mRNA to the progression of epithelial and mesenchymal tumors to invasive and metastatic phenotypes (12) . Studies at the protein level consistently demonstrated that FAK overexpression as a neoplastic tissue developed the capacity for invasion and metastasis (13 , 14) . In addition, FAK has been shown to be overexpressed in a variety of tumors, including breast (13 , 15) , thyroid (13) , ovarian (16) , head and neck (17) , and colon (13 , 15 , 18) .

Although several studies have demonstrated that primary and metastatic colorectal tumors overexpress FAK (12 , 13 , 18) , a recent report by Ayaki and colleagues reported reduced levels of FAK expression in colorectal liver metastases (19) . To clarify these disparate findings, we examined FAK expression at both the mRNA and protein level in matched samples of colorectal primary tumors with corresponding liver metastases in addition to a separate group of liver metastases from known colorectal carcinoma. Using real-time PCR as well as immunohistochemical analyses, we have demonstrated elevated levels of FAK expression in colorectal liver metastases at both mRNA and protein levels.

	MATERIALS AND METHODS

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Tissue Specimens
FFPE as well as snap-frozen colon and liver tissue were obtained from the University of North Carolina Department of Pathology and University of North Carolina-Lineberger Comprehensive Cancer Center Tissue Procurement and Analysis Facility. FFPE-matched tissue samples of primary colorectal carcinoma and liver metastasis from the same patient were obtained from 24 patients. FFPE and snap-frozen tissue samples of liver metastasis from a known primary colorectal carcinoma were obtained from 18 and 17 patients, respectively. Snap-frozen matched tissue samples of normal colorectal mucosa and primary colorectal carcinoma were obtained from 39 patients. Serial 5-µm sections were cut from each paraffin block, placed on positive coated slides, and stored at 4°C.

Immunohistochemistry
Immunohistochemical analyses were performed as described previously (20 , 21) with the following modifications: slides were heated at 60°C for 1 h before deparafinization, rehydration, and quenching of endogenous peroxidase activity (3% hydrogen peroxide in methanol). The hydration process was completed by rinsing for 5 min in DAKO Tris buffered saline buffer (DAKO Corporation, Carpinteria, CA) containing 0.05% Tween 20 (DAKO Corporation). During heat-induced epitope recovery (22 , 23) , sections were steam-heated in a standard steamer (Black and Decker) while submerged in antigen-retrieval Citra buffer (BioGenex, San Ramon, CA) for 30 min. Sections were blocked in normal horse serum (Vectastatin Elite kit; Vector Laboratories, Burlingame, CA) for 15 min and then incubated with anti-FAK 4.47 monoclonal antibody (1:250; Upstate Biotechnology, Lake Placid, NY) overnight at 4°C in a humidity chamber. Sections were washed in DAKO Tris buffered saline buffer containing 0.05% Tween 20 and then loaded into the DAKO Autostainer (DAKO Corporation) for application and incubation of the biotinylated horse antimouse IgG (Vectastatin Elite kit, Vector Laboratories) for 30 min at room temperature followed by avidin-biotin complex (Vectastatin Elite kit) for 30 min at room temperature. The chromogenic reaction was performed with SG (Vector Laboratories) for 10 min at room temperature. Slides were removed from the autostainer and counterstained with Nuclear Fast Red (Vector Laboratories) for 10 min and coverslipped with Permount (Richard-Allan Scientific, Kalamazoo, MI). Positive and negative controls were included with each run.

To illustrate anti-FAK 4.47 specificity and block immunoreactivity in paraffin-embedded samples, we used a GST-FAK fusion protein (GST-FAK-NT), containing the NH₂-terminal portion of FAK that generated the monoclonal antibody 4.47 (15) . GST-FAK-NT (1 µg/µl) and GST (1 µg/µl) control were incubated with the primary antibody, anti-FAK 4.47, for 1 h. To remove antibody reactivity, the GST antibody mixture was centrifuged three times at 6000 rpm with glutathione Sepharose 4B (Amersham Pharmacia Biotech, Buckinghamshire, England, United Kingdom) before application of the primary antibody to the tissue sections.

Immunohistochemistry Scoring
A single board-certified pathologist (C.A.L.) scored each tissue section for FAK expression based on a scoring system that measured intensity (0, none; 1, borderline; 2, weak; 3, moderate; 4, strong), percentage positive cells (0–100), cellular localization (cytoplasm, nucleus, membrane, or combination thereof), and overall distribution (homogeneous or heterogeneous).

Western Blot Analysis
Lysates of colorectal liver metastases were prepared as described previously (13) , and 50 µg of cell lysate were analyzed for FAK and ß-actin expression by Western blotting using anti-FAK 4.47 monoclonal antibody (1:1000 dilution) and anti-ß-actin AC-15 monoclonal antibody (1:2000 dilution; Sigma, St. Louis, MO). To demonstrate specificity of anti-FAK 4.47, we used recombinant FAK protein GST-FAK-NT to block immunoreactivity. Anti-FAK 4.47 monoclonal antibody (1:1000 dilution) was incubated with GST (1 µg/µl) and GST-FAK-NT (1 µg/µl) for 30 min on ice. The membrane was then probed with the antibody/fusion protein mixtures for 1 h at room temperature with shaking. After washing the membrane with washing buffer, the membrane was probed with goat antimouse IgG-horseradish peroxidase (1:5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Protein was visualized by enhanced Chemiluminescence detection system (Amersham, Arlington Heights, IL).

Measurement of FAK mRNA Using Real-Time Quantitative PCR
Synthesis of a FAK-specific Probe.
A FAK-specific and nonextendable oligonucleotide probe was designed from a cDNA sequence obtained from GenBank using the GCG analysis programs (University of Wisconsin). FAK-specific 5' and 3' oligonucleotides and an intervening fluorescent dye-labeled probe were fashioned using Primer Express (ABI/Perkin-Elmer). The probe was synthesized, labeled with 3' TAMRA (6-carboxy-tetramethylrhodamine; Ref. 24 ) and 5' FAM 6carboxyfluorescein, and purified by high-performance liquid chromatography (Integrated DNA Technologies, Coralville, IA).

Real-Time PCR Conditions.
The probe was hybridized to the target cDNA between the 5' and 3' oligonucleotides and, as amplification proceeded, the 5'-fluorophor was cleaved off the probe by 5' nuclease activity of the polymerase. Free in solution, the 5'-fluorophor was no longer quenched by TAMRA, resulting in increased fluorescence at 518 nm. Ribo-green (Molecular Probes, Eugene, OR) fluorescence was used to accurately quantitate starting levels of 10 ng/µl total-RNA extracted from the tumor samples. Fluorescence intensity produced during the PCR amplifications in each of 96 wells was monitored every 8.5 s using the 96-well thermal cycler ABI PRISM 7700 (Perkin-Elmer-ABI, Foster City, CA). A real-time amplification plot was generated for each well, for which the number of amplification cycles was plotted on the X axis and the log of change in fluorescence over baseline (Rn = fluorescence-baseline fluorescence) on the Y axis. The instrument’s software calculated a threshold cycle number (Ct) at which each PCR amplification reached a significant threshold level. This threshold cycle was directly proportional to the number of specific FAK template copies present in the sample.

FAK sRNA was used as positive control and absolute standard in all assays. In brief, full-length FAK cDNA was subcloned into the pcDNA3 vector bearing the T7 promoter. This linearized construct was used in vitro to transcribe FAK sRNA using the MEGAscript kit (Ambion, Austin, TX). Amplification of 2-fold serial dilutions of the FAK sRNA was used to construct standard curves and determine the dynamic range of the assay. Using this absolute standard, FAK message levels were measured accurately over the five-log range from 200 copies to 90 million template copies (0.1 femtograms to 2 x 10⁵ femtograms).

RNA Isolation and Quantitation of FAK mRNA Expression.
Total RNA was isolated using the guanidinium isothiocyanate-based system (RNeasy; Qiagen, Valencia, CA). Snap-frozen tumor specimens were power homogenized (Power Gen 125; Fisher Scientific, Pittsburgh, PA) before chloroform extraction. Any DNA contamination was resolved by treating all total-RNA isolates with DNase (RNase-free; Ambion). Each mRNA unknown was tested for DNA contamination by including reactions lacking the reverse transcriptase as a control.

All of the colorectal carcinomas and liver metastases were analyzed for FAK expression levels in individual experiments using the same master reaction mix to minimize experimental variability. Each tissue sample was tested in triplicate and the mean femtogram expression level calculated and converted to copy number using the formula (6.02 x 10²³ copies/mol) x (measured FAK grams)/(molecular weight of FAK message). The molecular weight of the linearized FAK sRNA calculated from its nucleotide sequence was M_r 1266501.

Statistical Methods
We used the nonparametric Wilcoxon signed-rank test on calculated difference scores (colorectal primary tumor FAK mRNA copies minus normal colorectal mucosa FAK mRNA copies, and log10 colorectal primary tumor FAK mRNA copies minus log10 normal colorectal mucosa FAK mRNA copies). We used the Wilcoxon two-group test (using normal scores) for group comparisons between unmatched primary colorectal tumors and liver metastases. P-values adjusting for multiple comparisons were calculated using the Bonferroni method. We used Fisher’s exact test for data categorized into two-by-two frequency count tables. All of the analyses were performed with SAS statistical software (version 8.2; SAS Institute, Cary, NC).

	RESULTS

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Immunohistochemical Analysis of p125^FAK Expression in Colorectal Carcinoma
Validation of p125^FAK Immunohistochemistry.
Initially, we performed experiments to validate specific immunostaining for the M_r 125,000 FAK protein. Using the FAK-specific monoclonal antibody 4.47 that has been shown to recognize FAK on both Western blots as well as FFPE sections (15) , we performed immunohistochemistry and Western blot experiments. Normal colorectal mucosa expressed minimal levels of p125^FAK (Fig. 1A) , whereas FAK was up-regulated in both the matched primary invasive colorectal carcinoma (Fig. 1B) and the liver metastasis (Fig. 1C) . The specificity of the FAK staining was demonstrated by a complete abrogation of immunostaining in the adjacent section of liver metastasis, when the antibody was preincubated with GST-FAK-NT fusion protein (Fig. 1D) . The specificity of the antibody reactions was further demonstrated biochemically in Western blot experiments using this liver metastasis sample. The M_r 125,000 FAK band was detected in the liver metastasis (Fig. 2 , Lane 1) but was completely blocked by incubation of the monoclonal antibody 4.47 with GST-FAK-NT (Fig. 2 , Lane 2). These results confirm, at both the immunohistochemical and the Western blot levels, that the staining pattern on immunohistochemistry was specific for p125^FAK.

View larger version (119K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Immunohistochemical analysis of FAK expression in a matched sample of primary colorectal carcinoma and colorectal liver metastasis. A and B, in this paired sample of normal colorectal mucosa and primary colorectal cancer from the same patient, little detectable p125FAK expression is evident in the normal colorectal mucosa. However, intense FAK staining is demonstrated in the primary colorectal cancer. C, this colorectal liver metastasis from the same patient as in A and B illustrates strong FAK immunoreactivity that is blocked (D) in the presence of GST-FAK-NT fusion protein. Blocking FAK expression confirms anti-FAK 4.47 specificity for p125FAK.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Western blot of p125FAK expression in a colorectal liver metastasis. Lane 1, Mr 125,000 band detected in this colorectal liver metastasis lysate that was probed with anti-FAK 4.47 (1:1000 dilution; Upstate Biotechnology) and GST (1 µg/µl) control. Lane 2, the same colorectal liver metastasis sample as in Lane 1 was probed with anti-FAK 4.47 (1:1000 dilution) and GST-FAK-NT (1 µg/µl) fusion protein. The presence of GST-FAK-NT with anti-FAK 4.47 blocks FAK protein expression and illustrates that monoclonal antibody 4.47 is specific to p125FAK. Anti-ß-actin AC-15 monoclonal antibody (1:2000 dilution; Sigma) loading controls were included with each blot.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Real-time PCR analysis of FAK expression in 39 matched samples of normal colorectal mucosa and colorectal primary tumor as well as a different set of colorectal liver metastases (n = 17). In the matched samples, FAK mRNA copies are significantly higher in the primary colorectal tumors compared with the normal colorectal mucosa (adjusted P = 0.001, paired Wilcoxon signed-rank test). Although the liver metastases are not matched to the primary colorectal tumors, FAK mRNA copies in these liver metastases is significantly higher compared with the primary tumor (adjusted P = 0.002, Wilcoxon two-group test).

View larger version (102K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Immunohistochemical and real-time PCR analysis of FAK expression in colorectal liver metastasis. A, representative examples of immunohistochemical staining that demonstrated different levels of FAK protein expression in four colorectal liver metastases samples. B, real-time PCR analysis for FAK mRNA in the same four colorectal liver metastases as in A. A1 and B, patient 31 illustrates the least amount of FAK protein expression in this set (n = 18) of colorectal liver metastases with FAK mRNA copy number slightly below the mean, 21,454. A2 and B, patient 39 represents the lowest mRNA FAK expression, 6,491, with high p125FAK expression (>50% of cells positive). A3 and B, patient 38 has high FAK protein expression with the highest FAK mRNA copy number, 85,584. A4 and B, patient 36 illustrates FAK up-regulation at both the message and protein with the majority of colorectal liver metastasis cells staining strongly and 29,967 copies of FAK mRNA.

	DISCUSSION

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In contrast to these results, Ayaki et al. (19) recently reported lower levels of FAK expression in colorectal liver metastases than in the primary colorectal tumors. These investigators used a different monoclonal anti-FAK antibody for Western blot experiments and a polyclonal anti-FAK antibody for immunohistochemical studies and found that both FAK and paxillin expression were generally lower in liver metastases than in primary colorectal specimens. Nonetheless, FAK immunohistochemical expression was positive in 8 (88%) of 9 liver metastases, compared with 2 (20%) of 10 normal mucosal specimens. It is not clear whether these investigators used antigen-retrieval techniques in their immunohistochemical experiments (22 , 23 , 25) . We have found that the FAK epitope is masked by the formalin fixation and paraffin embedding processes, and epitope retrieval was necessary for optimum immunohistochemical analyses (15) . Although this technique has the possibility of introducing false-positive staining (26) , we demonstrated the specificity of our analyses by abrogating immunoreactivity in the tissue specimens by incubating the monoclonal antibody with recombinant FAK protein.

Within our series of immunohistochemical experiments, we observed variability between FAK expression in the matched sample of liver metastases as compared with the unmatched liver metastases. The biological nature of genetic variability may explain this observation; however, we have also observed that the FAK antigen deteriorates as the duration of formalin fixation and paraffin embedding increases (data not shown). In fact, the unmatched liver metastases were older samples (at least 4 years of storage), and we hypothesize that the variability that we observed is secondary to some deterioration of the FAK antigen. Similarly, the variability that we observed between the unmatched liver metastases p125^FAK and their corresponding FAK mRNA expression was likely secondary to this FAK deterioration in the immunohistochemistry samples.

The up-regulation of FAK in primary and metastatic colorectal tumors is consistent with the role of FAK in suppressing apoptosis in tumor cells (10 , 27) . Evidence for reduced apoptosis in metastatic colorectal tumors has been presented by Hashimoto et al., (28) who examined intranuclear DNA strand breaks localized with in situ nick translation to measure the frequency of apoptosis in human colorectal cancer specimens. They found that the labeling indices of carcinomas that did metastasize to lymph nodes or the liver were lower than that of carcinomas that did not metastasize. Thus, liver metastases were more likely to have less apoptotic cells (28) . These findings support the conceptual framework whereby tumor cells that become invasive and metastatic are subjected to intense apoptotic pressure. This would select for cells that are resistant to apoptosis so that they could successfully grow and become sites of distant metastasis. Thus, our finding that FAK expression is up-regulated in colorectal liver metastases fits into this framework of enhanced survival signaling through FAK to promote continued growth of the metastatic colorectal tumor cells (7) .

	ACKNOWLEDGMENTS

 
We thank the Immunohistochemistry Laboratory and Tissue Procurement and Analysis Core Facility of the University of North Carolina Lineberger Comprehensive Cancer Center for tissue specimens, the use of laboratory equipment, and technical support; Jinli Chang for producing fusion proteins; and Bob Bagnell for assistance with photomicroscopy.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Cancer Institute Grant CA65910.

² To whom requests for reprints should be addressed, at University of Florida, Department of Surgery, PO Box 100286, 1600 SW Archer Road, Room 6172, Gainesville, FL 32610. Phone: (352) 265-0622; Fax: (352) 338-9809; E-mail: cance{at}surgery.ufl.edu

³ The abbreviations used are: FAK, focal adhesion kinase; FFPE, formalin-fixed paraffin-embedded; GST, glutathione S-transferase; sRNA, synthetic RNA.

Received 4/26/02; revised 7/31/02; accepted 8/ 6/02.

	REFERENCES

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