Correlation of Staining for LKB1 and COX-2 in Hamartomatous Polyps and Carcinomas from Patients with PeutzJeghers Syndrome
Departments of Epidemiology (CW,CIA,MS,LN,MLF) and Pathology (AR), University of Texas MD Anderson Cancer Center, Houston, Texas, and Department of Medicine (TJM), Milton S. Hershey Medical Center, Pennsylvania State University, Philadelphia, Pennsylvania
Correspondence to: Marsha L. Frazier, Dept. of Epidemiology, Unit 189, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail: mlfrazier{at}mail.mdanderson.org
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Summary |
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Key Words: PeutzJeghers syndrome LKB1 COX-2 germline mutation immunohistochemistry
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Introduction |
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Cyclo-oxygenase (COX) is the rate-limiting enzyme in the formation of prostaglandins from arachidonic acid. COX exists in two isoforms, COX-1 and COX-2. COX-2 is constitutively absent in normal tissues but is rapidly induced by certain inflammatory cytokines, tumor promoters, growth factors, and oncogenes (Eberhart and DuBois 1995; Smith et al. 1996
). Increased COX-2 levels have been identified in cancer of the colon, lung, breast, stomach, and prostate, and also in pancreatic adenocarcinomas (Prescott and Fitzpatrick 2000
). Overexpression of COX-2 has been shown to result in resistance to apoptosis, modulation of adhesion of epithelial cells to the extracellular matrix, and promotion of angiogenesis.
Because both LKB1 and COX-2 are involved in tumorigenesis and suppression of apoptosis, we wanted to determine if expression patterns of these two genes are related in PJPs and in carcinomas from PJS patients. Here we developed an antibody that specifically recognizes the LKB1 protein and examined the expression of LKB1 protein and COX-2 protein in PeutzJeghers polyps by immunostaining. We found that COX-2 was overexpressed in most of the PJPs, whereas LKB1 exhibited heterogeneity in its expression patterns. There is a statistically significant correlation between immunostaining of COX-2 and LKB1.
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Materials and Methods |
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DNA Extraction
Ten ml of blood was drawn in Vacutainer tubes containing EDTA (Becton Dickinson Vacutainer System; Rutherford, NJ) from each study subject. DNA was isolated with an Applied Biosystems 341 Nucleic Acid Purification System (Applied Biosystems; Foster City, CA) according to the manufacturer's instructions.
Mutation Analysis
Nine exons and the adjacent intronic regions of the LKB1 gene were amplified for analysis of germline mutation. The primers used in PCR were the same as those reported previously (Jenne et al. 1998), except for the primers used for exon 1. The primer pair of DJ698F (5'-GGTCCCCGAGGACGAAGTTGA-3') and STK11 exon 1R (5'-ATCAGGTACTTGCCGATGAG-3') was used for the first part of exon 1, and the primer pair of STK11 exon438 1F (5'-TCATCTACCAGCCGCGCCGCAA-3') and DJ673R (5'-ACCATCAGCACCGTGACTGG-3') was used for the second part of exon 1. The PCR products were treated using a PCR product Pre-Sequencing Kit (USB; Cleveland, OH) and were sequenced in both directions with the PCR primers. Positive results were confirmed in the CLIA-certified laboratory of GeneDx (GeneDx; Gaithersburg, MD; http://www.genedx.com/).
LKB1 Antibody Preparation
SigmaGenosys (The Woodlands, TX) generated an anti-peptide polyclonal antibody GN2733. The LKB1 peptide (KKKKLRRIPNGEAN) corresponding to the amino terminal region (AA81AA94) of human and murine LKB1 was synthesized and conjugated to keyhole limpet hemocyanin before injection into rabbits. The sequence-specific antibodies were then affinity-purified from these antisera using peptide columns. We also studied an additional polyclonal antibody in the carboxyl terminus region of the LKB1 but could not reliably detect expression in normal colon mucosa or in PJPs by immunobiochemistry.
Western Blotting
Total protein (20 µg) was analyzed by 10% SDS-PAGE and blotted according to standard protocols (Sambrook et al. 1989). GN2733 was used as primary antibody to detect the LKB1 protein in DiFi, a colon cancer cell line (Dolf et al. 1991
) and in mouse pancreas tissue lysates by an ECL Western blotting analysis system (Amersham Pharmacia Biotech; Fair Lawn, NJ)
Immunochemical Analysis
Slides were deparaffinized and endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 10 min at room temperature. Sections were then placed in PBS and microwaved for 10 min for antigen retrieval. The anti-LKB1 antibody was applied at 1:500 dilution and incubated overnight at 4C. Immunodetection was performed with an LSAB 2 system (DAKO; Carpinteria, CA). Hematoxylin was used as a counterstain. A rabbit anti-LKB1 polyclonal antibody GN2733 generated against the 14 amino acid sequences at the N-terminus of LKB1 described above was used for the anti-LKB1 immunostaining at a dilution of 1:500. A mouse monoclonal antibody against human COX-2 (Cayman Chemical Catalog no. 160112; Ann Arbor, MI) was used for anti-COX-2 immunostaining at a dilution of 1:1000. For every sample, immunostaining was repeated at least twice to confirm the results. Staining was graded for intensity as negative (-), low (+), intermediate (++), or high (+++) by two independent observers (C. Wei and A. Rashid) and there were no major discrepancies between the two observers.
Statistical Analysis
We used Spearman correlation to evaluate joint staining of LKB1 and COX-2 in polyps and cancers (analyzed both jointly and separately). We also used analysis of variance to compare the levels of staining, contrasting cancers with polyps and polyps in the small bowel to other sites. We used SAS (1999) version 8.1 for all analyses.
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Results |
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Statistical analysis of the expression of polyps and carcinomas listed in Table 2 showed a highly significant correlation in antibody expression. Over all the samples, the Spearman correlation between staining for cytoplasmic staining of LKB1 and COX-2 was 0.93. For nuclear staining of LKB1 vs COX-2 the correlation was 0.91, and for nuclear vs cytoplasmic staining of LKB1 the correlation was 0.92. All of these results are significant at p<0.0001. When the data were stratified by polyp vs carcinoma histology, the correlations remained over 90% for all comparisons and the correlation between cytoplasmic LKB1 staining and COX-2 staining in carcinomas was 100%. Analysis of variance showed no significant differences in antibody staining between carcinomas and polyps or between polyps of the small bowel and other locations.
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Discussion |
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We have identified seven types of germline mutations in eight patients with PJS from eight different families. According to Nezu et al. (1999), mutations that cause truncation of the LKB1 protein at amino acid 311 or before result in loss of kinase function. On this basis, four types of mutations found in families 1, 3, 4, 5, 6, and 7 resulted in disruption of the kinase domain and kinase deficiency. Tiainen et al. (2002)
demonstrated that kinase-deficient LKB1 mutants predominantly display nuclear immunostaining. This correlation between loss of kinase activity and nuclear accumulation of LKB1 suggests that the cytoplasmic localization is required for kinase activity. Here, in 10 (77%) of 13 PJPs and in carcinoma samples from these six families, the LKB1 protein displayed a localization pattern indistinguishable from that in the normal tissue samples. Our results indicate two possible explanations for these findings. One possibility is that the subcellular localization of LKB1 may not be solely related to kinase activity. If so, we would have detected more nuclear staining in those subjects with mutations before amino acid 311. The second possibility is that biallelic inactivation due to LOH is not a common event in PJPs, and so the other allele for these PJPs also contributes to the pattern of staining seen.
The nuclear localization pattern found for only specimen 9 from patient 7 is highly consistent with the inability of the mutant SL26 to retain cytoplasmic LKB1, as reported by Nezu et al. (1999). The SL26 mutation is a small in-frame deletion mutation that substitutes four amino acids (303IRQH), and was identified in a PJS patient reported by Hemminki et al. (1998)
. This small in-frame deletion disrupts the consensus sequence of the potential nuclear localization signal (NLS) (Robbins et al. 1991
), which consists of the pattern of two basic residues followed by 10-residue spacer, and another basic region in which at least three of five residues were basic residues. The splice error detected in patient 7 will cause exon 8 deletion (starting from codon 308), which disrupts the same potential NLS in the SL26 mutation, presumably altering the subcellular distribution of the LKB1 protein so that it may accumulate in the nucleus only, although we know the status of only one of the LKB1 alleles. The other allele needs to be further investigated.
We demonstrated that COX-2 was overexpressed in the epithelium of 85% (28/33) of the PJPs and carcinomas. This is a somewhat higher rate than the 70% (16/23) and 64% (7/11) recently reported in two smaller studies by Rossi et al. (2002) and McGarrity et al. (2003)
, respectively. By comparing the LKB1 and COX-2 immunostaining results, we found that the polyps that expressed LKB1 overexpressed COX-2, and the polyps that showed loss of LKB1 expression also showed lack of COX-2 expression. Our results indicate that COX-2 expression correlates strongly with LKB1 expression. An exception was the polyps from patient 7, where LKB1 protein was detected in the nucleus yet we failed to detect COX-2 expression. The regulatory mechanism of COX-2 overexpression in PJPs is unknown. A mouse model study (Rossi et al. 2002
) suggested that the Ras/Raf-1/MEK/ERK signal transduction pathway is most likely to mediate COX-2 induction in murine LKB1+/- polyposis. Further investigation to identify the physiological LKB1 substrates will facilitate a more comprehensive understanding of how LKB1 is involved in signaling pathways to mediate its role both in polyp formation and in COX-2 induction.
It has been reported that increased levels of COX-2 in intestinal epithelial cells increased their adhesion and simultaneously decreased their response to certain apoptotic stimuli (Tsujii and DuBois 1995). Another study showed that SC-58125, a selective COX-2 inhibitor, suppressed the growth of HCA-7 cells that had high levels of COX-2 expression in culture and in tumor implants in nude mice, but had no effect on HCT-116 cells that lack COX-2 expression in culture or when they were implanted in nude mice (Sheng et al. 1997
). Therefore, it is quite possible that the overexpression of COX-2 in PJPs and carcinoma samples will result in resistance to apoptosis, thus increasing the tumorigenic potential. Another report indicates that LKB1 is involved in p53-dependent apoptosis and that loss of LKB1 function in PJS leads to a deficiency in apoptosis, which results in the formation of multiple polyps and the subsequent development of malignant tumors (Karuman et al. 2001
). Our results of COX-2 overexpression in the majority of the PJ samples suggest that COX-2 may be involved in tumorigenesis in patients with PJS.
In summary, our results demonstrate that COX-2 is overexpressed in polyps and carcinomas from patients with PJS. Treatment with selective COX-2 inhibitors, such as celecoxib and NSAIDs, has been shown to induce apoptosis in a variety of cancer cells, including those of the colon, stomach, and prostate. Therefore, the finding of overexpression of COX-2 in PJPs and carcinomas provides insight into the development of chemopreventive strategies for this disease.
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Acknowledgments |
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We thank the patients and their families for their cooperation. We also thank Mariann Crapanzano for her editorial comments.
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Footnotes |
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