Affiliations of authors: Divisions of Diagnostic Molecular Oncology (AO, BZ, TM) and Surgical Oncology (KY, MM, TM), Cancer Research Institute, Kanazawa University, Kanazawa, Japan; Molecular Laboratory, Division of Urology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan (VB); Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia (SYF)
Correspondence to: Toshinari Minamoto, MD, PhD, Division of Diagnostic Molecular Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan (e-mail: minamot{at}kenroku.kanazawa-u.ac.jp)
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ABSTRACT |
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INTRODUCTION |
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-catenin is a multifunctional protein involved in cellcell adhesion, normal embryonic development, cell differentiation, and malignant transformation (6,7). The oncogenic properties of
-catenin are associated with its function as a transcription factor in the Wnt/
-catenin/T-cell factor (Tcf) signaling pathway. In normal cells,
-catenin is associated with glycogen synthase kinase 3
(GSK3
) in a complex that includes the APC protein. GSK3
phosphorylates
-catenin, which results in APC-mediated
-catenin degradation via the ubiquitinproteasome pathway. The binding of Wnt proteins to their receptor inhibits GSK3
phosphorylation activity and subsequently stabilizes
-catenin. GSK3
phosphorylation activity can also be inhibited by phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) signaling. In tumor cells, mechanisms that inhibit GSK3
-induced phosphorylation of
-catenin block its interaction with the E3 ubiquitin ligase receptor,
-transducin repeatcontaining protein (
-TrCP), which prevents
-catenin ubiquitination and degradation, and ultimately leads to
-catenin activation. These mechanisms include mutations in the phosphorylation recognition motif of
-catenin, mutations in the APC gene that result in failure to recruit GSK3
to the complex, and unregulated PI3K/Akt signaling. In both normal and transformed cells, stabilized
-catenin is translocated to the nucleus, where it interacts with Tcf/lymphoid enhancer factor (Lef) proteins and activates the transcription of a number of target genes including c-myc, cyclin D1, and matrix metalloproteinase 7 (810). Therefore,
-catenin translocated into the nucleus is predominantly in an active and oncogenic form (6,810).
Recently, several studies have reported that the nuclear accumulation of -catenin (i.e.,
-catenin activation) is an early event in colorectal cancers (11,12). However, little was known regarding the clinical relevance of oncogenic
-catenin activation until we determined two distinct types of
-catenin activation (13,14). We found that
-catenin activation was detectable in 50%65% of colorectal cancers and that, in 40%50% of colorectal cancers,
-catenin accumulated in nuclei throughout the tumor (referred to as the diffuse type), but in 10%15% of colorectal cancers,
-catenin accumulated in nuclei only in tumor cells that formed the invasion edge (referred to as the invasion edge type). The latter type of
-catenin activation was strongly associated with advanced tumor stage and recurrence. Consequently, this type of oncogenic
-catenin activation is an independent and reliable factor that can identify a subset of patients with colorectal cancer who are highly susceptible to tumor recurrence and thus have a less favorable survival rate (13,14).
The observation of different types of -catenin activation is supported by evidence that
-catenin is activated in most colorectal cancers as a result of mutational inactivation of APC and that mutations of its gene were detected in a minority of colorectal cancers with wild-type APC (15,16). Recently, we found that the diffuse type of
-catenin activation was closely associated with loss of heterozygosity in the APC loci, whereas the invasion edge type occurred independently of APC inactivation (Ougolkov A: unpublished data). Thus, our findings indicate that the diffuse type and the invasion edge type of
-catenin activation are early (i.e., APC inactivationdependent) and presumably late (i.e., APC inactivationindependent) events, respectively, in colorectal carcinogenesis. It is therefore important to clarify mechanisms underlying differences in
-catenin activation types to aid in the identification and development of new diagnostic tools and molecular therapeutic targets.
Oncogenic activation of -catenin occurs primarily as a consequence of its stabilization by escaping ubiquitin-mediated proteasomal degradation, and the effects of activated
-catenin depend on a growing number of effectors and regulators (810). A major regulator of
-catenin stability and activity is the
-TrCP subfamily of F-box proteins, including
-TrCP1 and
-TrCP2 (1721). Human
-TrCPs are components of the Skp1Cul1F-box protein complex that regulates cellular levels of various proteins by functioning as the E3 ubiquitinprotein ligase that targets such substrates as
-catenin, I
B
(the inhibitor of nuclear factor kappaB [NF-
B]), and Emi1 cell-cycle regulator protein for degradation via the ubiquitinproteasome pathway (22,23). The gene encoding
-TrCP has been mapped to chromosome 10q24 (24). We demonstrated that, in 293T cells harboring wild-type CTNNB1 and APC genes,
-catenin/Tcf signaling increased levels of
-TrCP1 mRNA and protein in a Tcf-dependent manner but without increased
-TrCP1 gene transcription (25). We also showed that increased induction of
-TrCP1 expression by the
-catenin/Tcf pathway resulted in accelerated degradation of wild-type
-catenin protein, suggesting that a negative feedback loop may control the
-catenin/Tcf pathway under physiologic conditions (25). In addition, induction of
-TrCP was associated with the activation of the NF-
B pathway, which is known to promote cell survival (25). Whether
-catenin-dependent increases in
-TrCP levels, activation of NF-
B, and inhibition of cell death are linked in colorectal cancers is unknown.
We hypothesized that -TrCP participates in tumorigenesis by modifying not only the activation of the
-catenin signaling pathway but also the activation of other oncogenic signaling pathways. However, because little is known about
-TrCP in cancer, we examined whether
-TrCP alters the distinct type of oncogenic
-catenin activation in colorectal cancer and how these distinct types of
-catenin activation affect the malignant potential of the tumors and patient outcome. We also investigated the relationship between
-TrCP expression, NF-
B, and the incidence of apoptosis.
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PATIENTS AND METHODS |
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From all consecutive patients who underwent surgery for the removal of colorectal cancer in our institute between 1998 and 2002, 45 patients, for whom a complete set of samples were available (described below), agreed to be enrolled in the present study. All patients signed written informed consent forms. The Institutional Review Board of Kanazawa University Graduate School of Medical Science approved all study protocols. Of the 45 patients, 23 were men and 22 were women. The mean patient age was 68 years (range = 4488 years). Seventeen patients had cancer of the cecum and ascending colon, three patients had cancer of the transverse colon, two patients had cancer of the descending colon, nine patients had cancer of the sigmoid colon, and 14 patients had cancer of the rectum. The Japanese Classification of Colorectal Carcinoma criteria (26) were used to describe the gross appearance and histologic characteristics of the primary tumors. Regarding gross appearance, five tumors had a protruding appearance (type 1), 39 tumors had localized and expansive growth with ulceration (type 2), and one tumor had infiltrative proliferation (type 3). On histologic examination, all tumors were classified as adenocarcinomas, of which 19 were well differentiated, 23 were moderately differentiated, and three were poorly differentiated (including mucinous adenocarcinoma). Carcinoma cells had invaded lymph vessels in 19 (42%) patients and invaded blood vessels in 24 (53%) patients. According to the TumorNodeMetastasis (TNM) classification system (27), routine clinical examination and pathology diagnosis indicated the following: one patient had stage I disease, 26 had stage II disease, and 18 had stage III disease. Follow-up examination after surgery disclosed metastases to distant organs (i.e., liver, lung) in eight patients.
Tissue Samples and Cell Lines
Samples from normal mucosa and tumor tissues were taken from each patient's fresh surgical specimen, immediately snap-frozen, and stored at 80 °C. After sampling, the surgical specimens were then fixed with neutral buffered 10% formalin, embedded in paraffin, and processed for routine histopathology. We used serial sections of paraffin-embedded tumors to ensure that all studies used samples that contained the same histopathologic characteristics of the respective tumor. Tissue sections were prepared on silica-coated glass slides (DAKO Cytomation, Kyoto, Japan) for immunohistochemical analysis of -TrCP and NF-
B expression and
-catenin activation (described below). Patients with esophageal cancer or rheumatoid arthritis provided written informed consent to allow use of normal esophageal mucosa and synovitis tissues, respectively, which were subjected to routine pathology examination.
Colorectal cancer cell lines (SW480, SW620, HCT116, HT29, and LoVo) and the 293T line, a human renal epithelial cell line that expresses simian virus 40 large T antigen, were obtained from American Type Culture Collection (Manassas, VA) and grown in the specified media. Cells were harvested during the exponential growth phase, subjected to centrifugation to pellet the cells, and stored as cell pellets at 80 °C until use. These colorectal cancer cell lines were chosen because they contain genetic alterations that have resulted in -catenin accumulation and activation, including mutational inactivation of the APC tumor suppressor gene in the SW480 and SW620 (codon 1338: CAGGln to TAGstop codon), HT29 (codon 853: CAGGln to TAGstop codon; and codon 1555: GAAAAAACT to GAAAAAAACT), and LoVo (codon 1114: CGAArg to TAGstop codon; and codon 1430: AAACCAT to AAACAT) cell lines, and mutation in the phosphoacceptor site of CTNNB1 (
-catenin gene) in the HCT116 cell line (codon 45: deletion of three bases and loss of the serine residue) (28).
Total RNA was isolated from frozen tissue samples and cell pellets by using acid guanidinium thiocyanate (RNA-Bee; Tel-Test, Friendswood, TX) and phenolchloroform (29,30). Cellular proteins were extracted from fresh surgical specimens and pellets of cell lines using a lysis buffer (CelLytic-MT, Sigma-Aldrich, St. Louis, MO). After the protein concentration in each sample was measured by using the Bradford method with the Coomassie Protein Assay Reagent (Pierce, Rockford, IL), the total protein extracts were mixed with a cocktail of protease inhibitors (Sigma-Aldrich) according to the manufacturer's recommendation, divided into aliquots, and stored at 80 °C.
Reverse TranscriptionPolymerase Chain Reaction
Expression of -TrCP1 mRNA was quantitatively determined by reverse transcriptionpolymerase chain reaction (RT-PCR). Complementary DNA (cDNA) was generated from 1 µg of total RNA by reverse transcription using a Reverse Transcription System Kit (Promega, Madison, WI). To quantify
-TrCP1 expression in each cDNA sample, the target (i.e.,
-TrCP1) was amplified by PCR in parallel with an internal control (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). To amplify a 211-base-pair (bp) fragment of
-TrCP1 that contains the downstream portion of exon 9 and the upstream portion of exon 10, the following set of primers was designed: upstream primer 5'-ATGCAAGCGAATTCTCACAGG-3' and downstream primer 5'-GGAACGATCTTTGGAGCAGGT-3'. Amplification for
-TrCP1 was performed by using a thermal cycling program with varying numbers of cycles, in which each cycle consisted of 94 °C for 1 minute, 59 °C for 1 minute, and 72 °C for 1 minute. An expression vector containing the
-TrCP1 gene (31) was used as a positive control for the PCR. A 598-bp GAPDH fragment was amplified following a program with varying numbers of cycles, in which each cycle consisted of 94 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 1 minute, and using the forward primer 5'-CCACCCATGGCAAATTCCATGGCA-3' and the reverse primer 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. All PCR products were subjected to electrophoresis through a native 10% polyacrylamide gel and were visualized by staining with SYBRGreen 1 (Nippon Gene, Tokyo, Japan).
Before we amplified and quantified -TrCP1 expression, we performed densitometric analysis using National Institutes of Health (NIH) Image Program software, version 1.62 (NIH, Bethesda, MD) to measure and compare the levels of the GAPDH PCR products amplified by varying numbers of cycles to determine the number of PCR cycles that would permit us to quantify the target product within a linear range.
-TrCP1 was then amplified using the quantitative parameters indicated by the comparison (Fig. 1). Finally, we adjusted the densitometric value of
-TrCP1 mRNA in each sample to that of GAPDH amplified during the same number of cycles to determine the level of
-TrCP1 expression. For each sample, we repeated all steps (from generating cDNA to measuring the PCR product by densitometry) to confirm reproducibility. Increased expression of
-TrCP1 was defined as the level of expression in the primary tumor that exceeded the level of constitutive expression in the matched normal tissue by more than 50%. Because no tumor had lower levels of expression than those in the matched normal tissue, all tumors were found to have either constitutive or increased levels of expression.
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Protein extracts (100 µg) from tissue samples or cell pellets were thawed and separated by sodium dodecyl sulfate8% polyacrylamide gel electrophoresis, and electro-transferred to a nitrocellulose membrane (Amersham, Buckingham, U.K.). Transferred proteins were analyzed by immunoblotting with a rabbit polyclonal antibody against -TrCP proteins [described in (32); diluted 1 : 1000] or a mouse monoclonal antibody to
-catenin [epitope described in (13); diluted 1 : 500] (Transduction Laboratories, Lexington, KY). Primary antibodies were diluted in a buffer containing 0.1% Tween-20 and 1% nonfat milk in Tris-buffered saline. Blotted membranes were incubated with primary antibodies overnight at 4 °C with gentle shaking. Signals were developed using enhanced chemiluminescence (ECL; Amersham, Little Chalfont, Buckinghamshire, U.K.). Protein extract (100 µg) from 293T cells was used as positive control for detecting
-TrCP and
-catenin proteins.
Expression of the NF-B active form in the tumor was confirmed by immunoblotting with the same antibody used for immunohistochemistry (described below). The antibody detects two bands: the p65 subunit of NF-
B (65 kd) and the splicing variant of p65, delta2 (63 kd). The epidermoid carcinoma cell line A431, which has constitutively active NF-
B, was used as a positive control for the detection of the p65 subunit of NF-
B by immunoblotting.
To examine the association between -TrCP and
-catenin in colorectal cancer cell lines, a 250-µg aliquot of protein extract from each cell line was immunoprecipitated with the antibody to
-catenin, according to the method reported previously (33). Immunoprecipitated materials were probed serially with antibodies to
-TrCP and
-catenin by the immunoblotting procedure. Protein extract (250 µg) from 293T cells was used as a positive control.
Immunohistochemistry
The distinct patterns of subcellular localization of -catenin and localization of
-TrCP and the p65 subunit of NF-
B were determined immunohistochemically using the avidinbiotinperoxidase complex method in microwaved tissue sections, as described (13,14). Sections were incubated with antibodies to
-catenin (diluted to 1 : 100 in phosphate-buffered saline [PBS] containing 5% normal horse serum),
-TrCP (diluted 1 : 200 in PBS containing 5% normal goat serum), or the p65 subunit of NF-
B (diluted 1 : 100 in PBS containing 5% normal horse serum) overnight at 4 °C in a moist chamber. We immunostained normal esophageal mucosa from a specimen used in our previous study (13,14) as a positive control for membranous expression of
-catenin, and we immunostained normal colorectal mucosa adjacent to the tumor as an internal positive control. The monoclonal antibody to the p65 subunit of NF-
B (Chemicon, Temecula, CA) recognizes an epitope (CDTDDRHRIEEKRKRKT) within the nuclear localization signal for p65, the DNA binding subunit mainly responsible for the strong gene transactivating potential. Synovitis tissues removed from patients with rheumatoid arthritis were fixed in 10% buffered formalin, processed for immunohistochemistry, and stained as a positive control for the NF-
B p65 subunit. Creating negative controls for the immunohistochemistry involved replacing the primary antibodies with nonimmune mouse and rabbit immunoglobulin G1 (IgG1) (DAKO, Glostrup, Denmark).
-catenin subcellular expression in the primary tumor was classified into one of three patterns: membranous expression, similar to that found in normal crypts; diffuse nuclear accumulation, defined as
-catenin-positive nuclei in cells distributed throughout the tumor; and nuclear accumulation in invasion edge, defined as
-catenin-positive nuclei only in tumor cells at the invasion edge adjacent to the stromal tissue with membranous
-catenin expression in the remaining tumor cells (Fig. 2) (13,14). Because
-catenin translocated into the nucleus is predominantly in an active and oncogenic form (6,810), we refer to the nuclear accumulation of
-catenin as
-catenin activation. Two well-trained investigators (B. Zhang and K. Yamashita) who were blinded to the histopathologic characteristics of the primary tumors or the patients clinical outcomes independently reviewed the types of
-catenin activation and the expression of
-TrCP and the NF-
B p65 subunit in each tumor specimen. The investigators agreed on the expression patterns for all samples.
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Carcinoma cells undergoing apoptosis were detected in tissue sections by the terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatebiotin nick-end labeling (TUNEL) method (34), using the in situ apoptosis detection TUNEL kit (Takara, Shiga, Japan) according to the manufacturer's recommended protocol. The frequency of apoptosis was calculated as an apoptotic index, in which the proportion of cells undergoing apoptosis was expressed as a percentage of all carcinoma cells observed. The apoptotic index of each primary tumor was calculated as the number of TUNEL-positive cells and bodies (35) per 2500 carcinoma cells counted in five randomly selected fields in each tumor; each field was subjected to two independent counts. The in situ apoptosis detection TUNEL kit contained tissue sections that served as a positive control.
Statistical Analysis
Differences in expression levels, as determined by densitometry between paired normal and tumor tissues, were analyzed by the Wilcoxon signed rank test. Expression levels (i.e., densitometric values) of -TrCP1 were compared between groups of patients by using the MannWhitney U test (for comparison between two unpaired groups) and the KruskalWallis test with a posttest (for comparisons among three unpaired groups). Associations between
-TrCP1 expression and clinical and histopathologic variables were determined using Fisher's exact test for 2 x 2 tables and the exact test for large tables for 2 x 3 tables. Student's t test was used to determine whether differences in the apoptotic index (mean value and 95% confidence intervals [CIs]) between tumors with different levels of
-TrCP expression and between those with different types of
-catenin activation were statistically significant. In all statistical analyses, a P value of less than .05 was considered statistically significant. All statistical analyses were two-sided and were computed using StatView (version 5.0) software for Macintosh (SAS Institute, Cary, NC).
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RESULTS |
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Constitutive expression (i.e., similar expression levels between the tumor and respective normal tissue) of -TrCP1 was observed in 20 (44%) of 45 samples (Fig. 1, A). Increased expression of
-TrCP1 was observed in the other 25 (56%) samples (representative sample shown in Fig. 1, B). The mean intensity of
-TrCP1 expression level in all tumors combined (0.759 arbitrary units, 95% CI = 0.683 to 0.835 arbitrary units) was statistically significantly higher than that in the respective normal tissues (0.484 arbitrary units, 95% CI = 0.408 to 0.560 arbitrary units; P<.001 by the Wilcoxon signed rank test). Moreover, the mean intensity level of
-TrCP1 expression in tumors with increased expression (0.835 arbitrary units, 95% CI = 0.735 to 0.935 arbitrary units) was statistically significantly higher than that in tumors with constitutive expression (0.663 arbitrary units, 95% CI = 0.559 to 0.767 arbitrary units; P = .015 by the MannWhitney U test). All tumors that had increased
-TrCP1 expression had increased or overexpressed
-TrCP protein (Fig. 2, A). No tumor lost expression of
-TrCP1 mRNA or
-TrCP protein relative to the expression in its normal counterpart.
Next, we examined the types of -catenin activation in the 45 colorectal tumor samples. Nuclear accumulation of
-catenin protein, indicative of oncogenic activation, was detected in 22 (49%) of the 45 samples. Of these 22 tumors, 12 showed the diffuse type of
-catenin activation [i.e., nuclear accumulation in cells throughout the tumor (13)] (Fig. 2, B), and 10 showed the invasion edge type of
-catenin activation [i.e., nuclear accumulation in cells only along the invasion edge (13)] (Fig. 2, C). Increased
-TrCP1 mRNA or
-TrCP protein expression in the tumor was associated with
-catenin activation (P = .023) (Table 1). The association was stronger for tumors with the invasion edge type of
-catenin activation (P = .026) than for tumors with the diffuse type of
-catenin activation (P = .314). The expression level of
-TrCP1 was higher in tumors with
-catenin activation than in tumors without
-catenin activation, although the difference between the groups was not statistically significant (P = .6).
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DISCUSSION |
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Phosphorylation of -catenin protein within the Asp-Ser-Gly-Iso-His-Ser sequence by GSK3
, which is tethered to the complex with
-catenin by APC and axin, is essential for
-TrCP-mediated ubiquitination and degradation of
-catenin (1719). Genetic alterations in axin or CTNNB1 are rarely detected in colorectal cancers, whereas genetic alterationsgene deletion or mutationthat inactivate APC are frequently detected (2,3). Inactivation of APC abrogates phosphorylation of
-catenin and thereby prevents its interaction with
-TrCP, rendering
-catenin resistant to ubiquitination. Therefore, the strong association between increased
-TrCP expression and oncogenic
-catenin activation in the same tumors suggests that, unlike 293T cells (25), the negative feedback loop between this E3-ubiquitin ligase receptor (
-TrCP) and its target oncoprotein (
-catenin) is defective in colorectal cancer.
The selective and intentional degradation of proteins in the ubiquitinproteasome system is important in the turnover of regulatory proteins (4042). The E3 ubiquitin ligase receptors are responsible for substrate specificity (4042). Loss of expression or function of certain types of ubiquitin ligase has been observed in several human diseases, both inherited and acquired, including neurologic and neoplastic diseases (4346). Various aberrations in the ubiquitinproteasome systemmediated control of signaling by oncogenes products (or oncoprotein) and tumor suppressors have been implicated in development of human cancers (43,44). One such mechanism involves uncontrolled and accelerated removal of a substrate (i.e., loss-of-function), whereas another involves the stabilization of a substrate resulting from an inactivation of an enzyme in the system or from a mutation in a targeting motif in the substrate (i.e., gain-of-function) (43). Mdm2/Hdm2 functions as an E3 ubiquitin ligase, and inactivation of p53 resulting from Mdm2/Hdm2-mediated degradation is an example of a loss-of-functiontype aberration (47,48). Similarly, low expression levels of the cyclin-dependent kinase inhibitor p27kip1, a tumor suppressor protein (49), have been associated with progression of a variety of human cancers (5055). In particular, a decrease or loss of p27kip1 expression in colorectal cancer is associated with aggressive tumor behavior and unfavorable patient outcome (56,57). This decrease is attributed to ubiquitination of p27kip1 mediated by its cognate E3 ubiquitin ligase, Skp2 (5861), which has recently been reported to have oncogenic activity (62,63). By contrast, somatic mutations either in the APC gene or in the -catenin phosphoacceptor sites that result in the stabilization and subsequent oncogenic activation of
-catenin by escaping the ubiquitinproteasome system represent one of the best documented gain-of-functiontype aberrations (43).
Several proteins that are regulated via the ubiquitin-proteasome system and are involved in colorectal carcinogenesis are affected by changes in -TrCP expression. Although
-TrCP is not a direct participant in the gain-of-functiontype aberration of the ubiquitin system associated with
-catenin activation,
-TrCP is implicated in colorectal carcinogenesis because the negative feedback loop regulating
-catenin oncoprotein expression is defective. In addition,
-TrCP targets I
B
(31,64,65), the inhibitor of NF-
B (66), for ubiquitination. Increased
-TrCP expression is associated with increased I
B
ubiquitination, which in turn results in the translocation of NF-
B to the nucleus and the transactivation of antiapoptosis genes important for cell survival (38,6769). Consistent with this scenario, we found an association between increased
-TrCP expression and decreased apoptosis in the colorectal cancers. Furthermore,
-TrCP also plays a critical role in the inducible processing that converts NF-
B into an active form (70,71). By immunohistochemistry, we detected expression of the p65 subunit of NF-
B and found that its nuclear localization was closely associated with the presence of
-TrCP and a low incidence of apoptosis in tumor cells. Thus, our results suggest that stabilizing NF-
B and processing it for activation is one of the important molecular mechanisms that may explain how increased
-TrCP functions against apoptosis of tumor cells in colorectal cancer.
Inhibition of apoptosis in tumors expressing high levels of -TrCP may contribute to their propensity to metastasize. Indeed, we noted that patients with metastases expressed higher levels of
-TrCP than patients without metastases (Table 3). This observation is important because it demonstrates that one of the key molecules in the ubiquitin system that regulates the oncogenic activity of
-catenin is associated with biologic behavior or malignant behavior and/or the potential of colorectal cancer. Such relevance to human cancer has not been previously demonstrated, in part because
-TrCP1 gene mutations or alterations in protein expression were rarely found in stomach cancer (72), prostate cancer (73), or melanoma (74). Investigating and understanding the clinical implications of the pathologic alterations in the ubiquitinproteasome system may lead to the development of molecular cancer therapies that target
-catenin- and/or NF-
B-mediated oncogenic signaling pathways by modulating the E3 family of ubiquitin ligases (75,76).
In summary, increased expression of -TrCP participates in colorectal cancer development and progression by coupling and integrating oncogenic
-catenin- and NF-
B-dependent cell survival signaling. To strengthen the clinical associations we found, multivariable analyses from larger prospective studies will be required. Greater understanding of the involvement of
-catenin in human carcinogenesis may lead to the development of molecular therapies targeting this oncogene (and its product oncoprotein), its regulators, and/or its effectors.
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NOTES |
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We thank Dr. Naofumi Mukaida (Division of Molecular Bioregulation, Kanazawa University Cancer Research Institute) for assistance in preparation of the -TrCP1 plasmid, Atsuko Kaneda-Shimizu for technical assistance, and Michael Meyer for editorial assistance.
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Manuscript received October 29, 2003; revised June 3, 2004; accepted June 15, 2004.
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