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Cleavage of ß-Catenin by Calpain in Prostate and Mammary Tumor Cells

¹ Cellular and Molecular Biology Graduate Program, ² Department of Urology, ³ Michigan Urology Center, ⁴ Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan; and the ⁵ Department of Urology, University of Ulm, Ulm, Germany

	ABSTRACT

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Mutations in the NH₂-terminal regulatory domain of the ß-catenin gene lead to aberrant stabilization and accumulation of the protein and increased TCF/LEF-dependent transcription. Although these mutations are common in some cancers, they are infrequent in prostate and breast cancer. We have found that metastatic prostate cancer specimens, obtained through a rapid autopsy tissue procurement program, expressed a novel M_r 75,000 proteolytic fragment of ß-catenin (ß-cat⁷⁵). ß-Cat⁷⁵ was also expressed in multiple prostate and breast cancer cell lines and was closely associated with the activity of the calcium-dependent protease, calpain. In a prostate cancer cDNA microarray, m-calpain RNA levels were found to be significantly increased in metastatic disease compared with normal prostate. We showed calpain-dependent generation of ß-cat⁷⁵ in cell culture and in vitro. Molecular mapping revealed that calpain cleavage removed the NH₂-terminal regulatory domain of the ß-catenin protein. Treatment of MCF-7 cells with ionomycin led to increased accumulation of ß-cat⁷⁵ in the nucleus and TCF-dependent transcriptional activity. Overexpression of a similar ß-catenin fragment that lacks the NH₂-terminal 132 amino acids and has transforming potential activated TCF-dependent transcription. Given the low frequency of mutation-induced activation of ß-catenin in prostate and breast cancers, proteolytic cleavage of ß-catenin by calpain may represent a novel mechanism by which the protein is activated during tumorigenesis.

	Introduction

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In addition to participating in the adherens junction with E-cadherin, ß-catenin is known to mediate intra-cellular signaling through Wnt pathway activation and glycogen synthase kinase-3ß inhibition (1) . Wnt signaling results in the stabilization of ß-catenin, nuclear accumulation, and association with TCF/LEF factors whereby ß-catenin acts as a transcriptional coactivator (2) . In the absence of Wnt signaling, glycogen synthase kinase-3ß, when in complex with APC (adenomatous polyposis coli) and axin, phosphorylates ß-catenin at critical residues at its NH₂ terminus, resulting in its ubiquitination and degradation by the proteasome (2) . Mutations in the NH₂-terminal region result in increased stabilization of ß-catenin and association with TCF/LEFs, leading to the constitutive transcriptional coactivation of tumor-promoting genes (1 , 3 , 4) .

Mutations in the APC/ß-catenin pathway have been found in numerous cancers; however, in some cancers, such as breast and prostate cancer, where mutation of ß-catenin is infrequent, overexpression of ß-catenin target genes has been observed (5, 6, 7, 8, 9, 10, 11) . In these tissues in which mutation of the ß-catenin gene is uncommon, other mechanisms may exist by which ß-catenin is abnormally activated and promotes tumorigenesis. Our laboratory has been investigating proteolytic mechanisms that target the lateral adhesion complex in prostate epithelium and has recently identified calpain as a mediator of proteolytic cleavage of E-cadherin in prostate cancer (12) . On the basis of these previous findings, we hypothesized that calpain may target other components, such as ß-catenin, in the lateral adhesion complex. In this study, we investigated the role of calpain in the proteolytic cleavage of ß-catenin and its implications in prostate and mammary tumorigenesis.

	Materials and Methods

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Tissue Culture Cell Lines, Chemicals, and Transfections.
MCF-7, LNCaP, DU145, PC-3, 293T, SKBR3, and PrEC LgT cells were maintained in 7.5% fetal bovine serum supplemented with 2 mmol/L L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml Fungizone. The SUM 52, 102, 149, 185, 225, 229, and 1315 cell lines were maintained as described previously (13) . Cells were incubated at 37°C and subcultured weekly. Calpain inhibitor III and calpeptin were used at a final concentration of 20 µmol/L (Calbiochem, La Jolla, CA). Ionomycin was used at a final concentration of 10 µmol/L (Calbiochem). Transfection of MCF-7 and SKBR3 cells was done with the FuGene 6 reagent according to the manufacturer’s protocol (Roche, Indianapolis, IN).

Plasmids.
The pTOPFLASH and pFOPFLASH reporter constructs were kindly provided by Dr. Bert Vogelstein. The ß-catenin construct, which contains an in-frame deletion of amino acids 29–48, was kindly provided by Dr. Frank McCormick. The ß-catenin construct with a deletion of 132 amino acids at the NH₂ terminus was kindly provided by Dr. Eric Fearon.

Western Analyses and Antibodies.
Western analyses of tissue and cultured cells were done as described previously (12) . Subcellular fractionation was done with the ProteoExtract kit (Calbiochem). An antibody mapped to amino acids 768–781 at the COOH-terminus of ß-catenin was used for Western blotting at 1:4,000 dilution (C2206, Sigma, St. Louis, MO). An NH₂-terminal antibody to ß-catenin (1:100) was also used (Calbiochem). The -catenin antibody was used at 1:500 dilution (Zymed Laboratories, South San Francisco, CA). The c-myc antibody (9E10) was used at a 1:200 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). The -spectrin antibody was used at 1:1,000 dilution, and the hsp-70 antibody was used at 1:200 (Chemicon International, Temecula, CA). The actin antibody was used at 1:1,000 dilution (Sigma). The anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were used at 1:5,000 and 1:3,000 dilutions, respectively (The Jackson Laboratory, West Grove, PA).

In vitro Calpain Assay.
The assay was done as described previously (12) . A COOH-terminal specific antibody (Zymed Laboratories) was used to immunoprecipitate ß-catenin from LNCaP whole cell lysates. The reactions were boiled in reducing sample buffer for 5 minutes, and the supernatants were loaded onto 6% NOVEX gels for Western analysis.

Ionomycin and Calpain Inhibitor Experiments.
MCF-7 cells (1.5 x 10⁵) were plated in 6-well plates. Four days after plating, cells were pretreated with 20 µmol/L calpain inhibitor III or calpeptin in fresh serum-containing media for 15 minutes. Cells were then treated with 10 µmol/L ionomycin for 90 minutes. Attached and detached cells were harvested, lysed, quantitated, and 35 µg of protein per sample were loaded onto 6% NOVEX gels for Western Analysis.

Reporter Gene Assays.
MCF-7 cells (1.5 x 10⁵) were plated in 6-well plates. Forty-eight hours later, cells were transfected with 1 µg of the pTOPFLASH reporter construct, which contains three copies of optimal TCF-4–binding sites upstream of a luciferase reporter gene and 100 ng of the pSV-ß-gal control vector per well (Promega, Madison, WI). Forty-eight hours after transfection, cells were pretreated with 20 µmol/L of the calpain inhibitors in fresh serum-containing media for 15 minutes. Cells were then treated with 10 µmol/L ionomycin for 90 minutes. Attached and detached cells were harvested, and luciferase levels were measured according to the manufacturers protocol (Promega). For reporter gene assays using 293T cells, 1.5 x 10⁵ cells were plated in 6-well plates. Forty-eight hours later they were transfected with 0.5 µg of expression plasmid, 0.5 µg of pTOPFLASH or pFOPFLASH, and 100 ng of pSV-ß-gal. Forty-eight hours after transfection, cells were harvested and luciferase levels were measured. Luciferase values were normalized to ß-galactosidase levels to control for transfection efficiency (Applied Biosystems, Foster City, CA). Statistical analysis was done using the paired Student’s t test with two-tailed distribution.

Tissue Procurement.
Metastatic prostate cancer samples were obtained with a rapid autopsy protocol that has been described previously (12 , 14) .

cDNA Microarray Analysis.
The analysis was done as described previously (12) .

	Results

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Detection of a M_r 75,000 a ß-Catenin Fragment in Epithelial Tumor Cells.
Multiple metastatic prostate cancer samples obtained through the University of Michigan rapid autopsy program were analyzed for ß-catenin expression by Western blot. All metastatic samples expressed the M_r 95,000, full-length ß-catenin, as well as a smaller M_r 75,000 fragment (ß-cat⁷⁵; Fig. 1A ). We also detected a M_r 90,000 ß-catenin band in some of the samples (see Discussion). Analysis of five normal liver samples did not express ß-cat⁷⁵; two representative samples are shown (Fig. 1A , Lanes 1and 2). The ß-cat⁷⁵ fragment was also detectable in various prostate and breast cancer cell lines (Fig. 1B) . These observations showed that prostate cancer tissue as well as multiple prostate and breast cancer cell lines expressed a M_r 75,000 ß-catenin fragment.

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A Mr 75,000 ß-catenin fragment in prostate and mammary tumor cells is associated with increased calpain activity. A, protein extracts from metastatic prostate cancer tissue specimens from different patients were analyzed for ß-catenin expression. Four prostate cancer metastases from the liver (Lanes 3–6), two from the lymph node (Lanes 7–8), two from the stomach (Lanes 9–10), one from the adrenal gland (Lane 11), and two normal liver samples (Lanes 1–2) were resolved by 6% SDS-PAGE and immunoblotted with a ß-catenin antibody (top). Identical quantities of cell lysate were resolved by 10% SDS-PAGE and immunoblotted with an actin antibody (bottom). B, protein extracts from various breast and prostate epithelial cell lines were resolved by 8% SDS-PAGE and immunoblotted with a ß-catenin antibody. C, protein extracts from immortalized and cancer cell lines were resolved by 6% SDS-PAGE and immunoblotted with an -spectrin antibody (top) and a ß-catenin antibody (bottom). D, a cDNA microarray was done with 21 benign and 19 metastatic prostate samples. Benign and metastatic tissue samples had a median cDNA expression of m-calpain of 0.98 (95% confidence interval, 0.9–1.1) and 1.4 (confidence interval, 1.3–1.4) respectively. Pairwise comparison with the Mann-Whitney test revealed a P value of <0.001. The horizontal line in each box denotes the value of the median, and the upper and lower borders of the box denote the 75th and 25th percentile, respectively. The extended bars represent the maximum and minimum expression values.

Calpain Generates ß-Cat⁷⁵ in Cell Culture and In vitro.
Because calpain is a calcium-dependent protease, we investigated whether the generation of ß-cat⁷⁵ could be induced by calcium influx. Treatment of MCF-7 cells with the calcium ionophore, ionomycin, resulted in a dose-dependent accumulation of ß-cat⁷⁵ (Fig. 2A , top). The generation of ß-cat⁷⁵ was also extremely rapid, being clearly detectable in <3 minutes after ionomycin treatment (data not shown). An -spectrin immunoblot revealed the parallel dose-dependent accumulation of the M_r 145,000/150,000 -spectrin cleavage fragments, which implicated calpain as a mediator of ß-catenin cleavage (Fig. 2A , middle). Western blot analysis using a COOH-terminal antibody to -catenin showed that it was not cleaved in response to ionomycin (Fig. 2A , bottom). We also failed to detect poly(ADP-ribose) polymerase cleavage, which argues against the involvement of caspases and suggests MCF-7 cells are viable after 90 minutes of ionomycin treatment (data not shown). Pretreatment of MCF-7 cells with calpain inhibitors before treatment with ionomycin were effective in blocking the generation of ß-cat⁷⁵, as well as the calpain-specific M_r 145,000/150,000 -spectrin doublet (Fig. 2B) . A caspase-3 inhibitor failed to block ß-catenin cleavage after ionomycin treatment in LNCaP cells (data not shown). These results strongly implicate calpain as a mediator of ß-catenin cleavage in MCF-7 cells.

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. ß-Cat75 is generated by calpain in cultured cells and in vitro. A, MCF-7 cells were treated with increasing doses of ionomycin, then harvested after 90 minutes. Protein extracts were resolved by 6% SDS-PAGE and immunoblotted with a ß-catenin antibody (top), an -spectrin antibody (middle) and an -catenin antibody (bottom). B, MCF-7 cells were pretreated with 20 µmol/L calpain inhibitor III or calpeptin for 15 minutes, then treated with 10 µmol/L ionomycin for 90 minutes. Thirty-five micrograms of protein extracts were resolved on a 6% SDS-PAGE gel. The membrane was immunoblotted with a ß-catenin antibody (top) then stripped and reprobed with an -spectrin antibody (middle). Thirty-five micrograms of protein extracts from the same experiment were resolved on a separate 8% SDS-PAGE gel and immunoblotted with an actin antibody (bottom). C, ß-catenin, immunoprecipitated from LNCaP cells was pretreated with EGTA before the addition of µ-calpain for the indicated times. The cleavage reactions were resolved by 6% SDS-PAGE and immunoblotted with a ß-catenin antibody.

Calcium Influx Induces ß-Cat⁷⁵ Nuclear Accumulation and Increased TCF-Dependent Transcription.
Protein mapping using NH₂- and COOH-terminal specific antibodies to ß-catenin revealed that the NH₂ terminus of ß-catenin is removed by calpain proteolysis, and based on molecular weight calculations, we predicted that ß-cat⁷⁵ must consist of at least residues 100–781 (Fig. 3) . Previous studies have shown that NH₂-terminal deletion mutants of ß-catenin, which lack the regulatory domain, are more stable than wild-type, leading to accumulation of the protein (16, 17, 18) . Analysis of nuclear and cytosolic fractions of ionomycin-treated MCF-7 cells showed increased accumulation of ß-cat⁷⁵ in both compartments (Fig. 4A) . Reprobing for the nuclear protein, c-myc, and the cytosolic protein, hsp70, demonstrated that there was efficient separation of the two fractions. These results showed that ß-cat⁷⁵ can accumulate in the nucleus in ionomycin-treated cells. Ionomycin treatment of MCF-7 cells also exhibited an increase in TCF-dependent transcriptional activity, and this increase was blocked by pretreatment of cells with calpain inhibitor III or calpeptin, which paralleled their ability to block the generation of ß-cat⁷⁵ (Fig. 4B) . These results show that the increase in TCF-dependent transcriptional activity correlated with the generation of ß-cat⁷⁵ by calpain and suggested that ß-cat⁷⁵ is a stable and transcriptionally active fragment.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Functional domains of ß-catenin and antibody mapping of ß-cat75. A schematic of the functional domains of ß-catenin is shown. E-cadherin, APC, and TCF-4 bind to the core region of ß-catenin whereas the two transactivation domains are at the NH2 and COOH terminus of the molecule. The regulatory domain is located at the NH2 terminus and consists of amino acids 29–49. An asterisk indicates the amino acids that are most frequently mutated in human cancer. An NH2-terminal antibody (antibody A) failed to detect ß-cat75, in contrast with a COOH-terminal antibody (antibody B).

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Accumulation of ß-cat75 in the nucleus and association with increased TCF/LEF- dependent transcription. A, MCF-7 cells were treated with 10 µmol/L ionomycin for 90 minutes, harvested, and cytosolic and nuclear fractions were prepared. Protein extracts from untreated and ionomycin-treated cells were resolved by 6% SDS-PAGE and immunoblotted with a ß-catenin antibody (top). The membrane was stripped and reprobed with a c-myc antibody (middle), and an hsp-70 antibody (bottom). B, MCF-7 cells were transfected with the pTOPFLASH construct and a control ß-gal plasmid. Cells were pretreated with the calpain inhibitors and then treated with 10 µmol/L ionomycin for 90 minutes. Cells were harvested, and luciferase levels were normalized to ß-galactosidase levels. P < 0.05 was considered statistically significant. C, protein extracts from SKBR3 cells transiently transfected with ß-catenin constructs lacking either amino acids 29–48, or the NH2-terminal 132 amino acids were resolved by SDS-PAGE and immunoblotted with a ß-catenin antibody. The SUM 225 cell line is shown as a control. D, the indicated plasmids were transiently transfected into 293T cells for 48 hours. The ratio of the luciferase activities of the pTOPFLASH reporter construct compared with the pFOPFLASH construct is shown.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The APC/ß-catenin pathway has been shown to play a key role in cancer (1) . Mutations in the NH₂ terminus of ß-catenin pathway result in the stabilization of the ß-catenin protein and aberrant transcriptional activity that is thought to contribute to tumorigenesis (3 , 4 , 19) . Apart from mutations of proteins in the APC/ß-catenin pathway, additional mechanisms that may contribute to the deregulation of ß-catenin have not been fully explored. We have identified a novel mechanism by which the NH₂ terminus of ß-catenin is removed through proteolytic processing by calpain. Treatment of MCF-7 cells with the calcium ionophore, ionomycin, induced the rapid, calpain-dependent generation of ß-cat⁷⁵. ß-Cat⁷⁵ accumulated in the nucleus and cytosol that coincided with increased TCF-dependent transcriptional activity of endogenous ß-catenin. Calpain inhibitors blocked the increase in TCF-dependent transcription induced by ionomycin, which strongly correlated with the ability of these inhibitors to block ß-catenin cleavage.

Overexpression of a ß-catenin fragment that closely resembled ß-cat⁷⁵ activated TCF-dependent transcription in 293T cells; however, similar experiments done in prostate and breast cancer cell lines did not show significant activation of TCF-dependent transcription by N132 ß-catenin (data not shown). We hypothesize that other mechanisms of ß-catenin regulation exist in these cell lines, compared with 293T cells, which are highly sensitive to the ß-catenin-TCF reporter assay. Although studies have shown that ß-catenin fragments that lack the NH₂-terminal 132 amino acids can transform cells (18 , 20) , the signaling activity of this construct does not necessarily correlate with transforming potential (20) . In addition, ß-catenin fragments that lack the NH₂-terminal 89 or 90 amino acids have been shown to induce mammary tumors in mice (21 , 22) . These studies show that ß-catenin NH₂-terminal deletion mutants, which are similar to calpain-generated ß-cat⁷⁵, have transforming potential. The common characteristic that the engineered NH₂-terminal deletion mutants share is that they are more stable than wild-type ß-catenin (16, 17, 18) . Thus, we conclude that the role of ß-cat⁷⁵ in breast and prostate tumorigenesis may be attributable more to its enhanced stability and not necessarily to enhanced TCF-dependent transcription.

We and others have detected a M_r 90,000 ß-catenin fragment by Western blot analysis (Fig. 2, A and C ; ref. 6 , 23 , 24 ). We believe that the M_r 90,000 fragment is calcium-induced; however, generation of this fragment was not inhibited by calpain inhibitors after ionomycin treatment (Fig. 2B) . Thus, we believe that this fragment may be generated by another calcium-sensitive protease. Our finding that ß-cat⁷⁵ accumulated in the nucleus after calpain-mediated cleavage of ß-catenin contrasts with that in a prior study in which it was reported that activation of the Gq signaling pathway induces ß-catenin nuclear export and subsequent cytoplasmic degradation by calpain in cells that contain APC mutation (25) . We speculate that this difference may be attributable to the presence of wild-type APC in MCF-7 cells used in our study (6) .

In prostate and breast cancer, mutations in ß-catenin or APC are infrequent (6 , 7 , 26) . Despite this, ß-catenin/TCF-regulated target genes have been shown to be overexpressed in prostate and breast cancer cells (8, 9, 10, 11) . In addition, nuclear ß-catenin has been observed in metastatic prostate cancer lesions that contain wild-type ß-catenin (27) . It is possible that calpain-mediated cleavage of ß-catenin in tumor cells that lack APC or ß-catenin mutations may contribute to the higher expression of TCF/LEF target genes or may exert other oncogenic activities because of its enhanced stability. The significant increase of m-calpain expression in metastatic prostate cancer by cDNA microarray correlated very closely with the cleavage of ß-catenin in metastatic prostate samples of patients who had died of metastatic disease in our study (Fig. 1) . These findings implicate calpain as a potential target in the development of anticancer therapies. The clinical relevance of this mechanism should be a subject of additional study.

Calpain-mediated cleavage of proteins might be prevalent in cancers where the expression of the gene is not altered by transcriptional mechanisms, gene amplification, allelic loss, or the function of the protein altered by point mutation. We propose that calpain-mediated cleavage of ß-catenin may lead to aberrant stabilization of the protein and increased tumorigenic potential in prostate and breast cancer cells.

	ACKNOWLEDGMENTS

 
We thank Kathleen Day and Erin Sargent for helpful technical assistance.

	FOOTNOTES

 
Grant support: RO1 DK56137 from the NIH.

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.

Requests for reprints: Mark L. Day, Box 0944, Room 6219 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0944. Phone: 734-763-9968; Fax: 734-647-9271; E-mail: mday{at}umich.edu

Received 3/24/04. Revised 8/23/04. Accepted 8/25/04.

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