Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs During Breast Cancer Progression

Research Article

Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs during Breast Cancer Progression

Lingbao Ai,1 Wan-Ju Kim,1 Tae-You Kim, 1 C. Robert Fields,1 Nicole A. Massoll,2 Keith D. Robertson,1 and Kevin D. Brown1

1Department of Biochemistry and Molecular Biology and University of Florida Shands Cancer Center Program in Cancer Genetics, Epigenetics, and Tumor Virology, and 2Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida

Abstract cellular phenotype result from dysregulation of gene expression Cystatin M is a secreted inhibitor of lysosomal cysteine and/or protein function attributable to genetic and/or epigenetic proteases. Several lines of evidence indicate that cystatin M is changes within the genome. a tumor suppressor important in breast malignancy; however, Numerous tumor suppressor or growth-regulatory genes under- the mechanism(s) that leads to inactivation of cystatin M go de novo methylation and transcriptional silencing in human during cancer progression is unknown. Inspection of the cancer cells. Aberrant cytosine methylation leading to gene ¶ ¶ human cystatin M locus uncovered a large and dense CpG silencing occurs at clustered 5 -CG-3 dinucleotides (called CpG island within the 5¶ region of this gene (termed CST6). islands) within the genome (2). In a broader sense, cancer- Analysis of cultured human breast tumor lines indicated that associated CpG methylation is part of a more complex set of cystatin M expression is either undetectable or in low epigenetic events that result in the transformation of chromatin abundance in several lines; however, enhanced gene expres- from a transcriptionally active to an inactive state (3). Epigenetic sion was measured in cells cultured on the DNA demethylat- gene silencing is now widely recognized as either a causative or a ing agent 5-aza-2¶-deoxycytidine (5-aza-dC). Increased correlative event in tumor development (4, 5). In breast cancer, a cystatin M expression does not correlate with a cytotoxic list of well-characterized tumor suppressors, including BRCA1, INK4a r response to 5-aza-dC; rather, various molecular approaches p16 , 14-3-3 , E-cadherin, and ATM, are targets for epigenetic indicated that the CST6 gene was aberrantly methylated in silencing (6–11). Site-specific DNA methylation is often an early these tumor lines as well as in primary breast tumors. event as shown in tumor types with a well-defined pattern of Moreover, 60% (12 of 20) of primary tumors analyzed progression, such as colon cancer, and is now widely regarded as displayed CST6 hypermethylation, indicating that this aber- one of the ‘‘hits’’ in the Knudsen hypothesis leading to tumor rant characteristic is common in breast malignancies. Finally, suppressor gene inactivation. preinvasive and invasive breast tumor cells were micro- Acquisition of an invasive cellular phenotype in breast cancer is dissected from nine archival breast cancer specimens. Of the a key event in progression and severity of this disease. The five tumors displaying CST6 gene methylation, four tumors majority of breast cancers are adenocarcinomas arising from the displayed methylation in both ductal carcinoma in situ and epithelial layers that line the mammary glands. These glands are invasive breast carcinoma lesions and reduced expression of composed of terminal mammary lobules and lactiferous ducts that cystatin M in these tumors was confirmed by immunohisto- connect the lobule units to the nipple. It is now widely recognized chemistry. In summary, this study establishes that the tumor that the preponderance of diagnosed cases of breast cancer arise suppressor cystatin M is a novel target for epigenetic silencing from ductal epithelium at the terminal lobular ductal unit (12). during mammary tumorigenesis and that this aberrant event The first recognizable premalignant change in ductal architecture can occur before development of invasive breast cancer. is epithelial hyperplasia (13). Usual ductal hyperplasia can occur (Cancer Res 2006; 66(16): 7899-909) normally, regresses spontaneously, and carries a modest risk (f2-fold) of developing into more severe disease (14). However, with continued cell proliferation and acquisition of characteristic Introduction morphologic hallmarks, a lesion termed ductal carcinoma in situ During the process of carcinogenesis, tumor cells arise from (DCIS) develops. Although considered a cancerous lesion and has a normal cells that have acquired abnormal proliferative capability higher risk (f10-fold) of developing into a more serous form of (1). This is a multistep process during which oncogene activity is the disease (14), the cell growth in DCIS is confined to the increased and tumor suppressor activity is constrained. Often mammary duct because the basement membrane and myoepithe- concomitant with uncontrolled cellular proliferation is acquisition lial cell layer that surrounds the duct remains intact. Further of enhanced migratory capability, evasion of immune surveillance, changes in cells comprising DCIS lesions may lead to the ability to and promotion of angiogenesis. Such disease-producing changes in escape the duct structure and invade the surrounding stroma. Such invasive breast carcinoma (IBC) lesions may continue to grow in size, destroying nearby normal breast architecture. Further Note: Supplementary data for this article are available at Cancer Research Online disease progression can result in far more serious metastatic (http://cancerres.aacrjournals.org/). disease, as malignant cells enter the lymphatics or vasculature and Requests for reprints: Kevin D. Brown, Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, spread to distal sites within the body. Gainesville, FL 32610. Phone: 352-273-5458; Fax: 352-392-1445; E-mail: kdbrown1@ Increased extracellular proteolytic activity can lead to destruc- ufl.edu. I2006 American Association for Cancer Research. tion of the extracellular glycoprotein scaffolds that maintain tissue doi:10.1158/0008-5472.CAN-06-0576 architecture, thus facilitating invasion of normal tissue and www.aacrjournals.org 7899 Cancer Res 2006; 66: (16). August 15, 2006

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2006 American Association for Cancer Research. Cancer Research promoting metastasis of cancer cells (15). In addition, extracellular Materials and Methods proteolytic activity can result in the activation of latent growth- Cell culture and drug treatment. The breast tumor cell lines BT-549, promoting molecules or inactivation of growth-inhibiting mole- MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3, and T-47D were obtained cules (16). For example, enhanced activity of several matrix from American Type Culture Collection (ATCC, Manassas, VA). The metalloproteinases (MMP) was observed in cultured breast tumor nontumor breast epithelium-derived lines MCF-10A and 184B5 were also cell lines (17), induced expression of MMP3 in mammary obtained from ATCC. Cell lines were maintained in medium supplemented epithelium promotes mammary tumors in mice (18), and MMP1 with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, has recently been identified as a possible early biomarker in breast CA), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37jCin tumors (19). In addition to MMPs, lysosomal cysteine proteases 5% CO2 humidified atmosphere. MCF-7, MDA-MB-231, MDA-MB-468, and are known to degrade extracellular matrix components in vitro SK-BR-3 were grown in Eagle’s MEM with 2 mmol/L L-glutamine, 1.5 g/L (20–22), and the cysteine proteinases cathepsins B and L have been sodium bicarbonate, 0.1 mmol/L nonessential amino acids, and 1.0 mmol/L sodium pyruvate. BT-549 and T-47D were cultured in RPMI 1640 with implicated in tumor cell invasion and metastasis (23–25). 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, As an alternative to increased expression or export of proteases, 10 mmol/L HEPES, and 1.0 mmol/L sodium pyruvate. For MCF-7 and decreased expression or activity of their normal endogenous T-47D cells, the medium was further supplemented with 0.01 mg/mL inhibitors will predictably result in a similar outcome. Indeed, an bovine insulin (Sigma, St. Louis, MO). Both MCF-10A and 184B5 cells were imbalance between proteases and their inhibitors is viewed as cultured on MEGM (Clonetics, San Diego, CA) supplemented with cholera important in driving tumorigenesis (26). Cystatins are a class of toxin (Biomol, Plymouth Meeting, PA). protease inhibitors originally shown to inhibit the activity of Unless otherwise indicated, 5-aza-dC treatment was conducted as cathepsins and other cysteine proteases (27). At present, cystatins outlined previously (35). Following a 24-hour incubation with 5 Amol/L are recognized as a superfamily of proteins and are classified into 5-aza-dC, the cells were rinsed with PBS, fresh culture medium was added, five families: stefins, cystatins, latexins, fetuins, and kininogens and the cells returned to the incubator for 24 hours. At the end of this A (28). Cystatin M (also known as cystatin E) was originally identified incubation, 5 mol/L 5-aza-dC was again added, and the cells were incubated with drug for another 24 hours until harvest. 5-Aza-dC (Sigma) by two groups. One group identified this protein by using was freshly prepared as 5 mmol/L stock solution in PBS before use. differential display to compare gene expression in primary and Reverse transcription-PCR. Total RNA was extracted from cultured metastatic breast tumor lines (29) and the other group used cells and tissues using TRIzol reagent (Invitrogen). This RNA was then used inspection of expressed sequence tag sequences obtained from in first-strand cDNA synthesis reactions using Stratascript first-strand cDNAs clones from a variety of tissue sources (30). Characteriza- synthesis system (Stratagene, La Jolla, CA) using supplied random hexamer tion of this protein determined that cystatin M is produced and primers. Cystatin M expression was subsequently analyzed by PCR. CST6- secreted in both an unglycosylated (14 kDa) form and a specific primers used are forward 5¶-GACTGCCGCAAGACC-3¶ and reverse glycosylated (17 kDa) form. Since the identification of cystatin M 5¶-GAAGTGCCCTCCACCA-3¶. Glyceraldehyde-3-phosphate dehydrogenase and the proposal that this gene could function as a tumor (GAPDH) served as a control for RNA integrity and was amplified using ¶ ¶ ¶ suppressor (29), several subsequent studies point toward a critical primers forward 5 -ACCACAGTCCATGCCATCAC-3 and reverse 5 -TCCAC- ¶ role for this protease inhibitor in cancer progression. For example, CACCCTGTTGCTGA-3 . PCR thermocycling conditions used are 1 cycle at 95jC for 5 minutes; 38 cycles at 94jC for 45 seconds, 56jC for 1 minute, the breast tumor line MDA-MB-435S is highly tumorigenic, and 72jC for 90 seconds; and final extension at 72jC for 10 minutes. invasive, and metastatic in nude mice (31). Shridhar et al. (32) Thermocycling conditions for GAPDH were the same as given for CST6, observed that this line expresses subdetectable levels of cystatin M except that an annealing temperature of 58jC was used and PCR was but that expression of recombinant cystatin M reduced cell conducted for 25 cycles. Following thermocycling, reactions were analyzed proliferation, migration, and Matrigel invasion. Further, although by electrophoresis on 10% polyacrylamide gels followed by staining with cystatin M expression was detectable in normal human mammary ethidium bromide. epithelium, reduced or subdetectable levels of cystatin M were Quantitative PCR. Total RNA was digested with RQ1 RNase-free DNase observed in IBC lesions (33). These workers also found that (Promega, Madison, WI) following the manufacturer’s protocol and DNA- expression of cystatin M in MDA-MB-435S cells delayed primary free total RNA (1.0 Ag) was used in a 20 AL cDNA synthesis. SYBR Green tumor growth and reduced the burden of lung metastases in scid PCR Master Mix (Applied Biosystems, Foster City, CA) was used for A A mice orthotopically implanted with this human breast cancer line. quantitative PCR (qPCR) and 2 L cDNA was added to final 20 LPCR solution using above-outlined reverse transcription-PCR (RT-PCR) primer Thus, ample evidence supports the view that cystatin M expression sets from cystatin M or GAPDH. Thermocycling conditions for qPCR are is down-regulated in breast cancer and that this event is likely to 1 cycle at 95jC for 10 minutes and total 40 cycles at 95jC for 15 seconds, contribute to development and/or progression of this tumor type; 56jC for 30 seconds, and 72jC for 30 seconds. Thermocycling was however, the mechanism underlying reduced cystatin M expression conducted using a Bio-Rad MiniOpticon System (Bio-Rad Laboratories, in breast malignancy remains unknown. Hercules, CA) and CT was calculated using supplied software. Cystatin M D Based on the gene structure, Keppler (28) first proposed that transcript abundance relative to GAPDH transcript abundance ( CT) was ÀDD cystatin M may be subject to epigenetic silencing through calculated and subsequently used to calculate changes (2 CT) in cystatin aberrant gene methylation. More recently, Rivenbark et al. (34) M expression. For analysis of gene expression following 5-aza-dC treatment, uncovered that cystatin M expression increased in cultured cystatin M transcript abundance in untreated cells was set at 1 and relative MCF-7 breast tumor cells on treatment with the global DNA transcript abundance in treated cells was subsequently calculated using demethylating agent 5-aza-2¶-deoxycytidine (5-aza-dC) and that this reference value. For analysis of matched breast tumor and normal samples, cystatin M transcript abundance in normal tissues was defined as this cell line displayed CpG methylation within a region directly 100% and relative transcript abundance in tumors was calculated upstream of the CST6 gene. Here, we show that the CST6 gene is accordingly. commonly subject to aberrant hypermethylation not only in SDS-PAGE and Immunoblotting. Culture medium from MDA-MB-231 cultured breast tumor cells but also in primary breast tumors. and MDA-MB-468 cells with or without 5-aza-dC treatment was collected. This study firmly establishes cystatin M as a target for epigenetic The medium was subsequently concentrated using iCON concentrators silencing in breast cancer. (Pierce, Rockford, IL) and protein concentration was assayed by BCA

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(Pierce). SDS-PAGE and immunoblotting was done using established 15 individual IBC or DCIS lesions (f10,000 cells) were microdissected from protocols (36). Biotinylated goat anti-human cystatin E/M antibody was the sections using a Leica AS LMD Laser Capture Microdissection (LCM) purchased from R&D Systems (Minneapolis, MN). Streptavidin-conjugated System (Leica, Wetzlar, Germany). Following microdissection, 45 AL lysis horseradish peroxidase (HRP; Kirkegaard and Perry Labs, Gaithersburg, buffer [10 mmol/L Tris-HCl (pH 8.0), 0.1% Tween 20] containing 5 AL MD) was used for antibody detection, and the blot was developed using proteinase K (100 mg/mL) was added and the cells were incubated at 55jC SuperSignal West Pico chemiluminescent substrate (Pierce) followed by for 12 hours. Subsequently, proteinase K was inactivated by incubation at exposure on X-ray film. Alternatively, SDS-PAGE gels were stained with 99jC for 10 minutes, and these samples were used without further Coomassie brilliant blue R250 (0.1%, w/v) dissolved in 10% methanol/10% processing in sodium bisulfite modification reactions. acetic acid. Immunohistochemistry. For cystatin M immunostaining, formalin- Human breast tumor and normal tissue genomic DNA extraction. fixed, paraffin-embedded human breast tumors were sectioned (5 Am thick) Twenty fresh-frozen archival breast tumors were obtained from the and mounted on glass microscope slides. Sections were subsequently University of Florida Shands Cancer Center Molecular Tissue Bank. All deparaffinized, rehydrated, and subsequently processed for antigen specimens and pertinent patient information were treated in accordance retrieval by steaming in citrate buffer [10 mmol/L sodium citrate, 0.05% with policies of the institutional review board of the University of Florida Tween 20 (pH 6.0)] for 20 minutes. After incubation in 0.3% H2O2 and Health Sciences Center. Tumors analyzed in this study were examined by a blocking with 5% bovine serum albumin in PBS, tissues were incubated in surgical pathologist and identified as invasive breast adenocarcinoma affinity-purified, biotinylated goat anti-human cystatin M antibody (R&D (stage II or III). Where indicated, matched normal breast tissue was Systems; working concentration, 10 Ag/mL) at room temperature for obtained from disease-free surgical margin. Genomic DNA (gDNA) was 2 hours. Following three washes with 0.2% Tween 20 in PBS, 1 to 2 drops of isolated from these samples, as well as cultured cell lines, using a Qiagen streptavidin-conjugated HRP solution were layered on the tissues for Blood and Cell Culture DNA kit (Qiagen, Inc., Valencia, VA) and stored at 30 minutes. Subsequently, the sections were washed again and 3,3¶- À20jC before use. diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA) Sodium bisulfite DNA modification. DNA was modified with EZ DNA was layered on the sections according to the manufacturer’s directions. Methylation kit (ZYMO Research Co., Orange, CA) according to the After this, sections were counterstained with hematoxylin. Control staining manufacturer’s instructions. Briefly, f1.5 Ag denatured gDNA was treated was carried out using the same procedure, except that cystatin M antibody with sodium bisulfite at 50jCforf16 hours in the dark. Following this, was replaced with biotinylated goat anti-rabbit IgG (KPL, Gaithersburg, samples were applied to supplied columns, columns were washed, MA) used at the same concentration. Stained sections were observed using desulfonation was conducted, samples were washed again, and DNA was a Zeiss Axioplan 2 microscope and photomicrographs were taken using a subsequently eluted with 20 AL elution buffer. Generally, 2 AL modified DNA CCD camera. was used in subsequent PCRs. Methylation-specific PCR. Qiagen HotStarTaq DNA polymerase was Results used for PCR. Reactions contained supplied 1Â PCR buffer supplemented CST6 with 0.1 mmol/L deoxynucleotide triphosphate, 2.5 mmol/L MgCl2, and Architecture of the human gene. Reduced expression of 0.5 Amol/L of each forward and reverse primers. The methylation-specific the cystatin M gene (CST6) is a commonly encountered event in PCR (MSP) primers specific for methylated CST6 gene is the forward both cultured human breast cancer lines and primary breast primer 5¶-TCGAGTTTCGTTTTAGTTTTAGGTC-3¶ and reverse primer malignancies (29, 33). As first proposed by Keppler (28) and 5¶-CATAACCGTCAATACCGTCG-3¶. The MSP primers specific for unmethy- consistent with a recent study using a genome-wide analysis ¶ lated CST6 gene are the forward primer 5 -TGAGTTTTGTTTTAGTTT- approach (34), we hypothesized that reduced expression of this ¶ ¶ ¶ TAGGTT-3 and reverse primer 5 -CCATAACCATCAATACCATCAA-3. gene may stem from aberrant methylation of CpG dinucleotides Thermocycling conditions used are 1 cycle at 95jC for 15 minutes; within the proximal gene region of the CST6 gene in breast cancer. 38 cycles at 94jC for 30 seconds, 60jC (for M primer set) or 55jC (for U primer set) for 1 minute, and 72jC for 1 minute; and final extension at To initially investigate this possibility, we examined the architec- 72jC for 10 minutes. PCR products were loaded 10% polyacrylamide gels ture of the human CST6 gene using public genome sequence and the gel was stained with ethidium bromide and directly visualized databases. Using the University of California at Santa Cruz (UCSC) under UV illumination. Human placental gDNA (Biochain Institute, genome browser (http://genome.ucsc.edu/) to search the May 2004 Hayward, CA) was used in primer validation experiments and methylated assembly (National Center for Biotechnology Information Build 35) in vitro with SssI methylase (NEB, Ipswich, MA) as outlined previously (11). of reposited human genome sequences, we observed that the CST6 Bisulfite genomic sequencing. Bisulfite genomic sequencing (BGS) gene maps to chromosome 11.q13.1 (Fig. 1A). This is consistent was conducted as outlined previously (11). A 362-bp segment (nucleotides with the location of this gene as determined previously by À 117 to +244; transcriptional start site, +1) was amplified from bisulfite- fluorescence in situ hybridization (FISH; ref. 37). Analysis of the modified gDNA. The primer sequences used for BGS are 5¶-GTTTTT- 35-kbp region of genome in which the CST6 gene resides shows TGGGTTTTTTGAATTT-3¶ and 5¶-TTACTACCCATATTATAACTAACCAC-3¶. The following thermocycling conditions were used: 1 cycle at 95jCfor this locus to contain a moderate number of genes (Fig. 1B). 15 minutes; 38 cycles at 94jC for 30 seconds, 55jC for 1 minute, and 72jC Telomerically flanking the CST6 gene is the CATSPER1 gene that for 1 minute; and final extension at 72jC for 10 minutes. Resultant encodes a voltage-gated calcium channel located in the plasma amplicons were subcloned into the plasmid pCR-TOPO (Invitrogen), and membrane of the sperm tail and is necessary for sperm motility recombinants were identified by EcoRI digestion and subsequently and fertility in mice (38). Centromerically flanking CST6 is the sequenced at the University of Florida ICBR DNA sequencing facility using barrier to autointegration factor-1 (BANF1)gene,knownto a vector-encoded primer (M13-Forw). function as a retroviral integration cofactor (39), and MGC11102, Laser capture microdissection and DNA extraction from paraffin- a theoretical protein with sequence homology to a putative embedded tumor samples. Archival blocks of formalin-fixed, paraffin- translation initiation factor in plants that shares limited homology embedded breast tumors (invasive ductal carcinomas) were obtained from with translation initiation factor 1A. the Department of Pathology of the University of Florida Shands Hospital in accordance with institutional review board policy. All tumors were Analysis of the CST6 gene shows it to be a rather compact gene confirmed by histologic examination to contain both IBC and DCIS lesions. contained within a 1,513-bp genomic interval (Fig. 1C). The gene is For each tumor, 10-Am-thick sections were mounted on PEN membrane composed of three exons: exon 1 (294 bp), exon 2 (126 bp), and slides (MicroDissect GmbH Co., Mittenaar, Germany). H&E staining of the exon 3 (188 bp). Exon 1 encodes the 5¶ untranslated region (UTR) sections was conducted according to a Leica protocol. After staining, 10 to of the cystatin M transcript as well as the translational initiation www.aacrjournals.org 7901 Cancer Res 2006; 66: (16). August 15, 2006

Figure 1. Genomic architecture of the human CST6 gene. The UCSC genome browser was used to search the May 2004 assembly of the human genome database to examine the CST6 locus. A, location of the CST6 gene within human chromosome 11. B, 35-kbp region flanking the human CST6 gene. Relative location of the MGC11102, BANF1, and CATSPER1 genes within this region of chromosome 11q.13.1. C, intron/exon structure of the human CST6 gene. Noted is the relative location of the three coding exons, the translational start (ATG) and stop (TGA) codons. D, structure of the 5¶ end of the CST6 gene. Graph of percent guanine (G) and cytosine (C) nucleotides across this region, location of the 70 CpG dinucleotides within this region, and boundaries of the CpG island (shaded region). Note also exon 1 and the transcriptional start site (TSS) of the CST6 gene.

codon (ATG) and exon 3 encodes the translational stop codon was either very low or subdetectable but was markedly increased (TGA) and the 3¶ UTR of the cystatin M transcript. Exons 1 and 2 following culture on 5-aza-dC. and exons 2 and 3 are separated by 541- and 365-bp introns, To independently confirm these results and quantify changes in respectively. Analysis of reposited transcript sequences revealed no gene expression, qPCR was done. We measured that culturing evidence for alternate splicing within the cystatin M transcript MCF-7 cells with 5-aza-dC resulted in a 13.1-fold (SD, 2.1; n =4) (data not shown). Closer inspection of the 5¶ end of the CST6 gene increase in cystatin M transcript abundance compared with showed this region of the genome to be an unusually CG rich untreated cultures of MCF-7 cells (Fig. 2B). Similarly, significant (Fig. 1D). As originally described by Gardiner-Garden and increases in cystatin M transcripts were measured in T-47D Frommer (40), a 507-bp segment of this region of the genome (27.5-fold; SD, 3.1; n = 4), BT-549 (57.3-fold; SD, 7.4; n = 4), SK-BR-3 (bp À186 to +320; transcriptional start site, +1) that contains 60 (35.3-fold; SD, 4.3; n = 4), and MDA-MB-468 (49.9-fold; SD, 6.8; CpG dinucleotides meets the criteria of a CpG island. Moreover, n = 4) cells. Consistent with our RT-PCR results, we measured an exon 1 of the CST6 gene is engulfed by this CpG island. Because insignificant (1.1-fold; SD, 0.2; n = 4) increase in cystatin M mRNA aberrant cytosine methylation within CpG islands that encompass in 5-aza-dC treated MDA-MB-231 cells by qPCR. both the 5¶ flanking region and the transcriptional start site is To examine cystatin M protein expression, the MDA-MB-468 cell commonly observed in cancer cell genomes (41, 42), the line was cultured on 5-aza-dC, and because the cystatin M protein architecture of the CST6 gene makes this gene a likely candidate is a secreted protein, tissue culture medium was collected and for epigenetic silencing. concentrated. Subsequently, concentrated medium from untreated Decreased cystatin M expression in cultured breast cancer MDA-MB-468 cells as well as cells treated with 5-aza-dC for 1 or 2 cells correlates with CST6 gene methylation. To further explore days was immunoblotted with anti-cystatin M. For these experi- the possibility that the CST6 gene is a target for epigenetic ments, an affinity-purified rabbit anti-cystatin M antiserum found silencing in breast cancer, we analyzed a panel of cultured human to be specific for the unglycosylated (14 kDa) and glycosylated breast cancer cell lines for cystatin M expression. Cells were (17 kDa) forms of cystatin M by immunoblot analysis of tissue cultured in the presence or absence of the DNA demethylating culture medium and whole-cell extracts (see Supplementary drug 5-aza-dC and total RNA was harvested. Following this, cDNA Fig. S1A) was used. The results (Fig. 2C) clearly show that no was synthesized from total RNA and used in RT-PCR assays to cystatin M was detected in the medium of untreated cells or cells examine expression of the cystatin M transcript. Our results cultured on 5-aza-dC for 1 day. However, MDA-MB-468 cells indicated that the line MDA-MB-231 displayed expression of the cultured on 5-aza-dC for 2 days showed strong expression of both cystatin M transcript and that the relative abundance of this unglycosylated and glycosylated forms of cystatin M. Analysis of transcript was unaltered by culture on 5-aza-dC (Fig. 2A). In medium from untreated MDA-MB-231 cells displayed detectable contrast, cystatin M transcript abundance in five other breast levels of cystatin M expression (Fig. 2C) and expression of this tumor lines (MCF-7, T-47D, SK-BR-3, MDA-MB-468, and BT-549) protein in this line did not change on treatment with 5-aza-dC

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(data not shown). Additional studies indicate that increased We next sought to directly assess CpG methylation in human cystatin M transcript abundance in response to 5-aza-dC does not breast cancer cell lines. To accomplish this, we developed a set of correlate with a cytotoxic response to this drug in MCF-7 cells and oligonucleotide primers useful in analysis of the methylation status that staurosporine, a potent inducer of apoptosis (43), does not of the CST6 gene by MSP (44) and BGS (ref. 45; Fig. 3A). For MSP, promote increased cystatin M expression (Supplementary Fig. S2). oligonucleotide primers were designed to amplify a segment of the Taken together, these experiments indicate that the majority of CST6 gene existing in either a methylated or an unmethylated state breast cancer cell lines analyzed in this study display reduced based on the modification status of CpG dinucleotides following cystatin M expression and that this defect can be complemented sodium bisulfite modification. The primers designed to amplify by culture on the global DNA demethylating drug 5-aza-dC. unmethylated CST6 gene sequence yield a 137-bp amplicon Moreover, these findings clearly suggest that cystatin M expression spanning from nucleotides À78 to +58, and the methylated primer is controlled, either directly or indirectly, by DNA methylation. set yield a 134-bp amplicon spanning from nucleotides À76 to +57 (numbering system based on location of the cystatin M transcriptional start site). Amplification of the CST6 gene from bisulfite-modified human placental gDNA was observed using the unmethylated specific primer set but not with the methylated specific primer set and this amplification was restricted to bisulfite-modified DNA (Fig. 3B). Conversely, when this gDNA was methylated in vitro using SssI methylase and subsequently subjected to bisulfite modification before PCR, we observed amplification of the methylated specific amplicon but not the unmethylated specific product. Sequence analysis of both unmethylated and methylated amplicons indicated that these primer sets specifically amplify the targeted portion of the CST6 gene (data not shown). These validation experiments indicate that our MSP primers amplify gDNA in a manner that clearly ascertains the methylation status of the CST6 gene. MSP analysis was subsequently conducted on two nontumor, mammary epithelium-derived cell lines. MSP analysis of 184B5 and MCF-10A cells determined that the CST6 gene is unmethylated in both of these cell lines (Fig. 3C). RT-PCR analysis of 184B5 and MCF-10A cells indicated that each of these lines express detectable levels of cystatin M transcript (Fig. 3C). gDNA was harvested from our panel of breast cancer cell lines either cultured in the absence or presence of 5-aza-dC and was subsequently bisulfite modified and used in MSP assays. We observed amplification of the unmethylated specific amplicon in modified DNA from MDA-MB-231, indicating that the CST6 gene is unmethylated in this cell line (Fig. 3D). Analysis of DNA from T-47D and SK-BR-3 cells not cultured on 5-aza-dC showed amplification with both unmethylated and methylated specific amplicons. Although amplification of both unmethylated and methylated specific MSP products is a common finding in MSP analysis (44) potentially attributable to heterogeneous gene methylation within the tumor cell population, this result indicates the presence of methylated CpG within the CST6 gene in at least a subset of cells of each line. In nontreated cultures of MCF-7, MDA-MB-468, and BT-549, we observed exclusive amplification Figure 2. Culturing human breast tumor cells with the DNA demethylating with the methylated specific primer set, indicating CST6 gene agent 5-aza-dC increases cystatin M expression. A, six human breast cancer cell lines (T-47D, MDA-MB-231, MDA-MB-468, MCF-7, BT-549, and SK-BR-3) methylation in the lines. Further, MSP analysis conducted on these were either cultured in the presence (+) or absence (-) of the DNA demethylating lines after treatment with 5-aza-dC indicated a clear increase in drug 5-aza-dC for 2 days. Subsequently, total RNA was harvested and used the abundance of the unmethylated specific amplicon, consistent in RT-PCRs with cystatin M–specific (top) or GAPDH-specific (bottom) primers. with demethylation of the CST6 gene and increased expression of B, thermocycle threshold (C T) values for cystatin M and GAPDH transcripts were measured by qPCR in each indicated cell line and relative cystatin M the cystatin M transcript. expression as a function of GAPDH transcript abundance was calculated. Fold To independently assess CST6 gene methylation, bisulfite- increase in relative cystatin M transcript abundance in 5-aza-dC-treated cells compared with untreated cells (defined as 1.0). Bars, 1 SD. **, P < 0.01. modified gDNA from several breast cancer cell lines was analyzed C, MDA-MB-468 cells were cultured with 5-aza-dC for the indicated length of by BGS. This entailed sequencing an amplicon obtained using time (lanes 1-3). At each time point, medium from cells was removed, concentrated, and analyzed by immunoblot analysis with anti-cystatin M (top). primers designed to amplify a segment of the CST6 gene in a Medium used to culture MDA-MB-231 cells (lane 4) was included in the analysis methylation-independent manner (see Fig. 3A). Specifically, PCR as a positive control. Alternatively, concentrated medium was subjected to primers were designed to regions of the CST6 gene devoid of CpG SDS-PAGE and staining of the gel with Coomassie brilliant blue R250 (bottom) to confirm equal protein loading. Note the 14-kDa unglycosylated and 17-kDa dinucleotides; thus, amplification proceeds in a manner unbiased glycosylated forms of human cystatin M. by gene methylation status. The 362-bp BGS amplicon, which www.aacrjournals.org 7903 Cancer Res 2006; 66: (16). August 15, 2006

Figure 3. Analysis of CST6 gene methylation in cultured human breast tumor cell lines. A, location of methylated specific (M-F and M-R) and unmethylated specific (U-F and U-R) oligonucleotide primers used for MSP analysis of the CST6 gene. Note also the primers used in BGS analysis (UB-F and UB-R). Location of these primers relative to the transcriptional start, the translational start, and the CpG island present within the human CST6 gene. B, MSP primers were verified by amplifying human placental DNA that was either methylated in vitro with SssI methylase or not methylated before bisulfite modification and PCR analysis. Also tested was methylated and unmethylated DNA before bisulfite modification. Presence of the 134-bp methylated specific and 137-bp unmethylated specific amplicon as well as reaction specificity is evident. C, gDNA from the nontumor, breast epithelium- derived cell lines MCF-10A and 184B5 was bisulfite modified and subjected to MSP (left). Alternatively, total RNA was harvested from these lines and used in a multiplexed RT-PCR containing both cystatin M–specific and GAPDH-specific primers (right). D, gDNA was harvested from breast cancer cell lines cultured in the presence (+) or absence (-) of 5-aza-dC for 2 days. This material was subsequently bisulfite modified and used in MSP with methylated specific (M) or unmethylated specific (U) primers. E, BGS analysis was conducted on bisulfite-modified gDNA from MDA-MB-231, BT-549, MDA-MB-468, and MDA-MB-468 cultured in the presence of 5-aza-dC. Result of sequence analysis of five independent clones for each cell line; percent methylation across all 46 CpG dinucleotides within the 362-bp amplicon. ., methylated CpG; o, unmethylated CpG.

contains 46 CpG dinucleotides, was subsequently subcloned into a Aberrant methylation of the CST6 gene is common in vector, recombinants were identified, and multiple clones were human breast tumors. We next analyzed CST6 methylation in a subjected to automated DNA sequencing. These sequences were panel of four patient-matched breast tumor/normal mammary compared with nonmodified CST6 gene sequence and the tissue samples. Fresh-frozen IBC tumors (stage II or III) and normal methylation status of CpG dinucleotides was determined by breast tissue from the same patient were obtained from the characteristic chemical changes associated with cytosines existing University of Florida Shands Cancer Center Molecular Tissue Bank. in either a methylated or an unmethylated state before bisulfite gDNA was extracted from these samples (designated BrCA- treatment (45). Results from BGS analysis of modified gDNA from 11-BrCA-14), bisulfite modified, and used in MSP (Fig. 4A). In all the cell lines MDA-MB-231, BT-549, and MDA-MB-468 (with and normal breast tissue samples, we observed exclusive amplification without culture on 5-aza-dC) are shown (Fig. 3E). MDA-MB-231 of the unmethylated specific amplicon, indicating that the CST6 cells, which contain an unmethylated CST6 gene as judged by MSP, gene is not methylated in normal mammary tissue. In the tumor showed very limited CpG methylation by BGS. Alternatively, the sample of BrCA-13, we also observed only amplification of the majority of clones from analysis of cell lines BT-549 and MDA-MB- unmethylated specific amplicon, indicating that the CST6 gene is 468 displayed dense methylation of the portion of the CST6 gene not methylated in this tumor sample. In tumors BrCA-11 and BrCA- analyzed. This finding is consistent with the observation that both 12, amplification of the methylated specific amplicon was clearly of these lines are positive for CpG methylation as judged by MSP. detectable, whereas BrCA-14 displayed low levels of the methylated Further, only sparse methylation of the CST6 gene was observed specific amplicon. This analysis indicates that, like cultured breast when gDNA was harvested and analyzed from MDA-MB-468 cells cancer cell lines, CpG methylation of the CST6 gene occurs in cultured in the presence of 5-aza-dC. Thus, based on both MSP and primary breast tumors. Moreover, because CST6 gene methylation BGS analyses, we conclude that the CST6 gene is a target for is not observed in normal breast tissue, CST6 gene methylation in aberrant methylation in numerous breast cancer cell lines and that tumor tissues is an aberrant, disease-associated event. reduced expression of cystatin M correlates with this epigenetic To examine cystatin M gene expression, total RNA was event. harvested from these frozen tumor/normal tissue samples.

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Subsequently, cDNA was synthesized and first-strand reactions BrCA-13 tumor, the results obtained from this qPCR analysis used in PCRs with cystatin M–specific or GAPDH-specific primers. indicate that reduced cystatin M expression correlates with This analysis indicated that, in tumors BrCA-11 and BrCA-12, aberrant methylation of the CST6 gene in breast tumors. cystatin M transcript was either undetectable or at very low levels Figure 4D shows representative MSP data obtained from seven when compared with matched normal tissues (Fig. 4B). In tumors additional IBC tumor samples for which no matched normal BrCA-14 and BrCA-13, cystatin M transcript abundance seemed to samples were available. In tumor samples BrCA-2, BrCA-6, and be roughly equivalent in tumor and matched normal tissues. To BrCA-18, we observed amplification with only the unmethylated quantify differences in cystatin M gene expression in these specific primer sets, indicating a lack of CST6 gene hyper- matched normal and tumor samples, qPCR analysis was done. methylation in these tumor samples. Tumor samples BrCA-0, Analysis of this data set was conducted by comparing relative BrCA-4, BrCA-16, and BrCA-17 show amplification with both cystatin M transcript abundance in tumor samples with transcript unmethylated and methylated specific primer sets, indicating the abundance in its matched normal tissue sample. The results of this presence of methylated CST6 gene in these tumors. Of the 20 IBC analysis indicated that the BrCA-11 tumor displayed extremely low tumors analyzed during this study, we observed that 12 (60%) levels of cystatin M expression. Specifically, we measured that this displayed aberrant methylation of the CST6 gene as judged by MSP, tumor contains 0.09% (SD, 0.01%; n = 3) of the level of cystatin M leading us to conclude that methylation of the CST6 gene is a transcript measured in the matched normal tissue (Fig. 4C). common event in invasive breast tumors. Similarly, the BrCA-12 tumor sample showed 6.7% (SD, 1.2%; n =3) BGS was again used to independently confirm our MSP results of the level of cystatin M measured in its matched normal tissue and examine CST6 gene methylation in a more detailed manner sample. Analysis of tumor BrCA-13 showed that this tumor than that afforded by MSP. In normal breast tissue, we observed no expressed slightly higher (109.4%; SD, 12.1%; n = 3), whereas tumor methylation of any of the 46 CpG dinucleotides within the portion BrCA-14 expressed slightly lower (95.9%; SD, 7.5%; n = 3) levels of of the CST6 gene examined (Fig. 4E). Similarly, we observed only cystatin M compared with their respective matched normal tissues. sparse methylation of the CST6 gene in tumor samples BrCA-13 Because MSP analysis clearly detected methylation in the BrCA-11 and BrCA-2. This result is in accordance with MSP results and BrCA-12 tumor samples, weak amplification of the methyla- observed on these tumors. In tumor BrCA-4, two of five clones tion-specific amplicon in BrCA-14 tumor and no methylation in sequenced display extensive CpG methylation. This finding

Figure 4. Analysis of CST6 gene methylation and expression in human primary breast tumors. A, four frozen human breast tumors (BrCA-11-BrCA-14) and patient-matched normal mammary tissue were obtained from the University of Florida Shands Cancer Center Molecular Tissue Bank. gDNA was extracted from these tissues, bisulfite modified, and subjected to MSP analysis. B, total RNA was also extracted from these matched tumor/normal breast tissue samples and RT-PCR assays were used for assessment of cystatin M (top) and GAPDH (bottom) expression. C, qPCR analysis of relative cystatin M transcript abundance in tumor tissues was calculated relative to cystatin M transcript abundance in matched normal tissue (defined as 100%). Columns, mean of three independent measurements; bars, 1 SD. **, P < 0.01. D, MSP analysis of a panel of seven other human primary breast tumors. E, BGS analysis of normal human breast tissue and five primary breast tumors. Result of sequence analysis of five independent clones for each tissue sample. ., methylated CpG; o, unmethylated CpG.

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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2006 American Association for Cancer Research. Cancer Research suggests that either a subset of cells within this tumor display Finally, four tumors displayed CST6 gene methylation in both CST6 gene methylation or there is partial allelic methylation within IBC and DCIS lesions (Fig. 5E; Supplementary Fig. S3). The results individual cells of this tumor. In tumors BrCA-12 and BrCA-0, we of this experiment indicate that CST6 methylation may precede observed dense, nearly saturating methylation of the CST6 gene in the escape of tumor cells from the ducts of the mammary gland three to four of the five clones sequenced. The results of this and invasion of surrounding normal stroma. In tumor BrCA-109, experiment clearly indicate that the CpG island present at the which displayed CST6 methylation in both DCIS and IBC lesions, 5¶ end of the human CST6 gene is subject to dense methylation in we also observed prominent staining of normal ductal epithelium breast tumors. but did not observe strong staining in either DCIS or IBC lesions Aberrant hypermethylation of the CST6 gene can occur within this tumor (Fig. 5F). In general, CST6 methylation-positive before tumor cell invasion. During breast cancer progression, tumors, such as BrCA-109, display low-level immunohistochemical tumor cells may acquire an invasive phenotype allowing progres- staining, indicating a sharp reduction in cystatin M expression in sion from DCIS to the more serious IBC tumor type. To examine neoplasms of this type. Of note, the cystatin M–positive DCIS epigenetic silencing of cystatin M at these phases of breast tumor lesion shown in BrCA-101 (Fig. 5B) is a low-grade lesion, whereas development, we microdissected DCIS and IBC lesions from the cystatin M–negative DCIS lesion in BrCA-109 (Fig. 5F)isa archival tumor samples where histologic examination revealed high-grade lesion. Although our study was not designed to contain both lesion types. Nine tumor samples were selected for specifically examine cystatin M expression in various classifica- study based on these criteria, and 10 to 15 individual DCIS and IBC tions and grades of DCIS lesions, this result may suggest that lesions (f10,000 cells per lesion type) were microdissected from more aggressive types of cancer lesions have a higher likelihood of each tumor using LCM. Following LCM, DNA was extracted from silencing cystatin M. Clearly, a broader study is required to the dissected cells, bisulfite modified, and used in MSP assays. In adequately address this issue. Nevertheless, these results support parallel, anti–cystatin M antiserum was used for immunohisto- the conclusions we have drawn using other approaches to chemical analysis in each of the tumors studied. Sections were investigate cystatin M expression in cultured breast cancer cell sequentially incubated with biotinylated goat anti–cystatin M lines and surgically obtained breast tumors; specifically, aberrant antibody, strepavidin-conjugated HRP, developed with a buffered methylation of the CST6 gene correlates with reduced cystatin M solution containing DAB, and stained with hematoxylin. Charac- expression. terization of this antibody indicates specific detection of cystatin M in immunohistochemical analysis of human breast tumors (see Supplementary Figs. S1B and S1C). Discussion Of the nine tumors studied, four tumors were found to be The central focus of this study was to examine the CST6 gene, negative for CST6 gene methylation in both IBC and DCIS lesions which encodes the cysteine protease inhibitor cystatin M, as a (Fig. 5A; Supplementary Fig. S3). As shown for tumor BrCa-101 potential target for epigenetic silencing in breast cancer. The (Fig. 5B) and other methylation-negative tumors (Supplementary rationale behind this hypothesis is that expression of cystatin M is Fig. S3), we observed intense staining of normal ductal epithelium decreased in breast cancer cells and that available evidence within the surgical margin of this tumor, in clear agreement with implicates this molecule as a tumor suppressor in breast cancer. the epithelial expression pattern observed previously for cystatin The first evidence in support of our hypothesis came from M (46, 47). We also observed strong staining of both DCIS and IBC inspection of CST6 genomic architecture. This analysis showed a lesions within the tumor mass, consistent with the lack of gene large (f500 bp) and dense CpG island superimposed on the 5¶ end methylation observed in this tumor sample. A general lack of of this gene. This CpG island engulfs the entirety of the first exon chromagen in tissue sections stained with nonspecific biotinylated of the CST6 gene, which encodes both transcriptional and goat IgG confirmed the specificity of DAB staining. Of note, no or translational start sites as well as a significant portion of region limited staining was observed within the fibrous stroma of this upstream of the CST6 coding region. Such molecular architecture tumor. We have conducted MSP analysis on cultured normal is entirely consistent with other genes known to be targets for human fibroblasts and found them to be negative for CST6 epigenetic silencing during tumorigenesis (41). We subsequently methylation (data not shown). This suggests that mechanisms determined that cystatin M expression was low in many cultured other than gene methylation may control cystatin M expression in breast cancer lines and that this expression could be markedly this cell type. increased by culturing the cells on the global DNA demethylating One tumor, BrCA-107, presented weak methylation in DCIS agent 5-aza-dC. Further, analysis of the methylation status of the lesions, but a strong methylated specific amplicon was detected in CpG island in the CST6 gene showed that this region of the IBC lesions from the same tumor (Fig. 5C), suggesting that CST6 genome contains multiple methylated CpG in many tumor cell methylation occurred during or following stromal invasion. By lines, that often this pattern of methylation is quite dense, and that immunohistochemistry, we observed strong staining of normal methylation of this CpG island corresponds to reduced cystatin M ductal epithelium and DCIS lesions in BrCA-107 (Fig. 5D), expression. In primary breast tumors, we also observed methyla- consistent with the general lack of CST6 gene methylation in tion of the CST6-associated CpG island and observed that normal mammary tissue in general and DCIS lesions in this tumor expression correlates with methylation and that methylation of in particular. In this sample, we observed areas of the tumor this gene is encountered quite frequently in this disease. Moreover, containing IBC lesions that stain strongly and areas that show because dense methylation was not observed in normal breast weak staining. This finding is in agreement with our MSP analysis, tissue, we conclude that this epigenetic event is aberrant and is which indicates the presence of both unmethylated and methyl- disease associated. Finally, we used LCM to dissect noninvasive ated CST6 gene within microdissected IBC lesions taken from DCIS and IBC cells from the same tumor and subjected DNA from BrCA-107 and moreover indicates that cystatin M silencing can be these lesions to methylation analysis. We observed in many of the heterogeneous within a single breast tumor. tumors that methylation of the CST6-associated CpG island occurs

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Figure 5. MSP and immunohistochemical analysis of cystatin M expression in DCIS and IBC lesions. Paraffin-embedded breast adenocarcinoma tumors were sectioned and stained, and DCIS and IBC lesions were dissected by LCM. Subsequently, DNA was extracted from the pooled DCIS and IBC lesions, bisulfite modified, and used in MSP. In addition, tumor sections were incubated with anti–cystatin M or nonspecific goat IgG, processed for immunohistochemistry using HRP- conjugated secondary antibody and diaminobenzidine, and counterstained with hematoxylin. A, MSP analysis of tumor BrCA-101. B, micrographs of normal ductal epithelium and DCIS and IBC lesions within the BrCA-101 tumor incubated with either anti–cystatin M (top row)or control IgG (bottomrow ). C, MSP analysis of tumor BrCA-107. D, micrographs of normal ductal epithelium and DCIS and IBC lesions within the BrCA-107 tumor. Two regions of IBC lesion within this tumor: one shows clear anti–cystatin M staining (left) and one shows limited staining for this protein (right). E, MSP analysis of tumor 109. F, micrographs of normal ductal epithelium and DCIS and IBC lesions within the BrCA-109 tumor. Weak staining with anti–cystatin M is observable in DCIS and IBC lesions in this tumor.

in noninvasive DCIS cells, suggesting that epigenetic silencing of silencing during the process of tumorigenesis remain unclear, it this gene may contribute to acquisition of an invasive cellular phe- is well recognized that epigenetic gene silencing is an often notype during breast cancer progression. When taken together, the encountered event in breast and other tumor types (4, 5). For conclusion drawn from these studies is that the CST6 gene is a novel example, our group recently showed that the ATM gene, which and frequent target for epigenetic silencing in breast cancer and that encodes a DNA damage-activated protein kinase critical in the this event has a high likelihood of affecting disease progression. activation of cellular response to genotoxic insult and maintenance Analysis of the CST6 locus determined that this gene possesses of genomic integrity (49, 50), is epigenetically silenced in breast compact genomic architecture. The coding region of the gene is tumors (11). Indeed, several other genes are subject to epigenetic contained within a f1.5-kb genomic interval on chromosome silencing in breast cancer, such as BRCA1, p16INK4a, 14-3-3r, 11.q13.1, in agreement with previous mapping studies conducted E-cadherin, WTH3, estrogen receptor-a,TIMP-3,anddeath- using a hybridization approach (37). Our analysis indicated that associated protein kinase (6–10, 51–54). Moreover, silencing of the CST6 gene is composed of three coding exons of modest size genes, such as these, will, for example, result in the relaxation (294, 12, and 188 bp) separated by two moderately sized introns of cellular controls that serve to restrain acquisition and fixation of (541 and 365 bp). Further, the genomic region flanking the CST6 mutations, control cellular growth, restrict invasion of normal gene contains several other functionally unrelated genes. Using tissue, and maintain drug sensitivity. Because alterations, such as gene-specific FISH probes, Dickinson et al. (48) mapped a 905-kb these, can provide a powerful stimulus to cancer development region that contains the genes encoding most members of the and/or progression, it is abundantly clear that epigenetic gene family 2 cystatins (i.e., C, D, S, SN, and SA) and two pseudogenes silencing is a critical aberrant event during breast tumorigenesis. within 20p11.2. Thus, the cystatin M gene is decidedly distinct Cystatin M is a characterized inhibitor of lysosomal cysteine from the majority of family 2 cystatin genes that exist within a proteases (27, 29). This class of proteases is capable of degrading tight gene cluster on human chromosome 20. extracellular matrix components (20–22); thus, cysteine proteases We observed that the CpG island present within the 5¶ end of the have long been considered of potential importance during tumor human CST6 gene often undergoes methylation in breast cancer cell invasion and metastasis (55, 56). Cystatin M is a secreted cells. Further, this aberrant molecular event correlates with protein and, when expressed at appropriate levels, functions to reduced cystatin M expression, leading us to the straightforward constrain extracellular cysteine protease activity (27). Consistent conclusion that cystatin M is targeted for epigenetic silencing in with this notion, restoration of cystatin M expression in breast breast tumors. Although the mechanisms that lead to gene cancer cells resulted in reduced cell proliferation and cellular www.aacrjournals.org 7907 Cancer Res 2006; 66: (16). August 15, 2006

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2006 American Association for Cancer Research. Cancer Research invasion in vitro (32). Thus, silencing of cystatin M expression has cystatin M protein, this result suggests that silencing of cystatin clear implication in the acquisition of an invasive cellular M could facilitate escape of tumors from the duct structure. phenotype. Similarly, the TIMP-3 gene, which encodes another Similarly, epigenetic silencing of E-cadherin, a molecule that inhibitor of extracellular proteolytic activity, also undergoes promotes homotypic cell-cell adhesion (57), is often observed in epigenetic silencing in breast cancer (54), indicating that genes microdissected DCIS lesions (58), leading these authors to similarly with functions similar to cystatin M are silenced by this propose that E-cadherin silencing allows tumor cells to dissociate mechanism. Beyond a theorized role in tumor suppression, from the primary tumor and invade surrounding tissue. extracellular proteolytic activity can result in inactivation of In summary, we document that the CST6 gene is targeted for growth-inhibiting molecules or the production of molecular epigenetic silencing in human breast cancer. We observed that fragments with growth-promoting properties (16), suggesting that hypermethylation of the CST6 gene occurs in 60% of the primary silencing of cystatin M and other secreted protease inhibitors breast tumors investigated and can occur in noninvasive DCIS could potentially affect diverse aspects of the tumorigenic process. lesions as well as IBC lesions. In light of the function of the Dysregulation of mechanisms that constrain cellular invasive- encoded cystatin M protein in inhibiting extracellular protease ness have a distinctly important role in breast cancer. The majority activity, this often-encountered aberrant epigenetic event is likely of breast cancers are adenocarcinomas that develop from the to be of importance in the progression of breast malignancies. ductal epithelium of the mammary gland. Although the cells that comprise a DCIS lesion are frank tumor cells free to infiltrate within the ductal structures, this type of lesion is physically Acknowledgments constrained by the myoepithelium and basement membrane that Received 2/14/2006; revised 5/16/2006; accepted 6/20/2006. comprises the outer layers of the duct. However, should cells of a Grant support: NIH grants R21CA102220 (K.D. Brown) and R01CA114229 (K.D. DCIS lesion acquire an invasive capability, they can break through Robertson). The costs of publication of this article were defrayed in part by the payment of page these barriers and invade surrounding stroma resulting in an IBC. charges. This article must therefore be hereby marked advertisement in accordance We have documented that methylation of the CST6-associated with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Drs. William Cance and Martha Campbell-Thompson (University of CpG island and diminished expression of cystatin M can occur in Florida) for providing tumor samples and Ms. Marda Scott-Jorgensen for expert DCIS lesions. Considering the biochemical properties of the technical assistance.

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