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The Osteoclast-Associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1

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The Osteoclast-Associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1 CANCER RESEARCH57. 5386-5390. December I, 19971 The Osteoclast-associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1 Amanda J. Littlewood-Evans,' Graeme Bilbe, Wayne B. Bowler, David Farley, Brenda Wlodarski, Toshio Kokubo, Tetsuya Inaoka, John Sloane, Dean B. Evans, and James A. Gallagher Novartis Pharina AG, CH-4002 Base!. Switzerland (A. I. L-E., G. B., D. F., D. B. El; Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology 1W. B. B., B. W., J. A. G.J, and Department of Pathology If. S.), University of Liverpool, Liverpool 5.69 3BX, United Kingdom [W. B. B., B. W., J. S., J. A. G.]; and Takarazuka Research Institute, Novartis Pharma lid., Takarazuka 665, Japan IT. K., T. LI ABSTRACT (ZR75—l,Hs578Tduct, BT474, ZR75—30,BT549, and T-47D), adenocarci noma (MDA-MB-231, SK-BR3, MDA-MB-468, and BT2O), and breast car Human cathepsin K is a novel cysteine protease previously reported cinoma[MVLNandMTLN,transfectedMCF-7-derivedclonesobtainedfrom to be restricted in its expression to osteoclasts. Immunolocalization of Dr. J. C. Nicolas (Unit de Recherches sur Ia Biochemie des Steroides Insern cathepsin K in breast tumor bone metastases revealed that the invading U58, Montpelier, France), and MCF-7]. The MCF-7 cells were obtained from breast cancer cells expressed this protease, albeit at a lower intensity two different sources, ATCC and Dr. Roland Schuele (Tumor Klinik, Freiburg, than in osteoclasts. In situ hybridization and immunolocalization stud Germany). These are indicated in the figure legend as clone 1 (ATCC) and ies were subsequently conducted to demonstrate cathepsin K mRNA clone 2 (Dr. Schuele). Other breast lines consisted of HBL-lOObreast epithe and protein expression in samples of primary breast carcinoma. Ex hal line isolated from the milk of a nursing mother, two different cultures of pression of cathepsin K mRNA was confirmed by reverse transcription MCF1OA(from two laboratories in Novartis Pharma AG) derived from mam PCR and Southern analysis in a number of human breast cancer cell mary tissue of a patient with fibrocystic breast disease, the human mammary lines and in primary human breast tumors and their metastases. As epithelial line HMEC4144 (Clonetics, San Diego, CA), and MTSVI.7neo, a this protease is known to degrade extracellular matrix, including bone nonmalignant human breast-derived cell line (6). RNA samples from various matrix proteins, it is possible that cathepsin K may contribute to the primary breast tumors and their metastases were obtained from the Cancer Invasive potential of breast cancer cells, Including those that metasta Tissue Bank Research Center (University of Liverpool). All primary carci size to bone. Thus, cathepsin K may be a potential target leading to the noma tissues were graded by the modified Bloom and Richardson system (7). design of novel drugs for cancer therapy. Immunohistochemistry. Blocks of tissue were fixed with formaldehyde for 24 h prior to paraffin embedding and were further subjected to fixation in INTRODUCTION methacarn. Paraffin sections (5 @.tm)weremounted onto Vectabond (Vector Laboratories, Petersborough, United Kingdom)-treated slides and dried over Cathepsins are a family of proteases that have been proposed to night in a 50°Covenbefore dewaxing with xylene. The immunohistochemistry be involved in the progression of a number of disease processes was performed as described previously (5). A specific chicken anticathepsin K including rheumatoid arthritis (1) and tumor invasion (2). Re antibody was used for the immunostaining. This antibody has been character cently, a novel cysteine protease, cathepsin K, was cloned from ized and validated for selectivity by Western blot analysis, which revealed that rabbit and human osteoclast-derived libraries (3, 4) and was found the antibody reacted only with cathepsin K and not with any other cathepsins to exhibit a variable amino acid homology to other members of the (5, 8). cathepsin family, ranging from 17% with cathepsin D to 48% in Situ Hybridization. An EcoRlJSall cDNA fragment corresponding to amino acid homology with cathepsin S. the first 455 bp from the ATGof the humancathepsin K gene was cloned We have previously demonstrated the osteoclast-specific expres into Bluescript KS+ and checked in GenBank to ensure that there was no sion of cathepsin K using immunolocalization and in situ hybridiza homology to other sequences that would result in cross-hybridization of the tion techniques in various normal and bone disease conditions and probes. Sense and antisense RNA probes for in situ hybridization were labeled with the SP6, T7, T3 in vitro transcription kit with DIG-labeled also in multinucleated cells in giant cell tumor of bone (5). However, UTP (Boehringer Mannheim) according to the manufacturer's instructions. in sections of human bone in which skeletal breast cancer metastases The RNA probes were stored at —20°C. were present, we have also observed a positive immunoreactivity, Freshly excised tissue from surgical procedures was submerged in pre albeit oflower intensity, in the breast cancer cells that had invaded the chilled n-hexane, transferred into sterile 50-mi falcon tubes, and stored at bone tissue. This unexpected observation has lead us to investigate —70°Cuntil use. A cryostat was cooled to —35°Cand 10 micron sections were further the possible association of the expression of cathepsin K in cut and mounted onto 3-aminopropyltriethoxysilane-coated slides. The in situ other human breast cancer tissues and cells. In the current study, we hybridization procedure was performed as described by Komminoth (9). For therefore analyzed the expression of cathepsin K using immunocyto the detection of the DIG-labeled probes, the protocol of the manufacturer's kit chemistry, in situ hybridization, and RT-PCR2 in surgically removed was used (Boehringer Mannheim). The reaction was stopped in 10 mMTris, 1 primary breast tumors, bone metastases, and human breast cancer cell mM EDTA, pH 8.1. Slides were dipped briefly in water, counterstained with lines cultured in vitro. 1% (w/v) light green in 1% acetic acid, mounted, and photographed. RT-PCR Analysis. Total RNA was reverse transcribedaccordingto the manufacturer's instructions (Promega). For the primary breast tumor samples MATERIALSAND METHODS and their metastasis, 2.5 .tg of total RNA were used for cDNA synthesis in a 50-pi reaction volume. Two primers for cathepsin K were designed and used Materials. Breastcancer cell lines (purchasedfrom ATCC unless other to amplify an expected product of 617 bp (sense, 5'-TFC CCG CAG TAA wise stated) were classified into subtypes: infiltrating ductal carcinoma TGA CAC C; antisense, 5'-lTF CCC CAG iTT TCT CCC C). In a [email protected] reaction volume, the following components were added: 10 @.dofPCR buffer Received 7/10/97; accepted 10/1/97. (lOX stock, Boehringer Mannheim), S @tlofDMSO, 2 pi ofdNTP mmxture (10 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 mM stock), 0.5 i@gof each primer, 0.5 @tlof Taq polymerase (Boehringer 18 U.S.C. Section 1734 solely to indicate this fact. Mannheim), water to 97 pJ, and 3 @dofreverse-transcribed RNA. The PCR 1 To whom requests for reprints should be addressed. at Friedrich Miescher-Institute was performed so that the amplification was in the log phase. The conditions R1066.4. 16, Maulbeer Strasse, Basel. CH4002, Switzerland. Phone: 061-6977876; Fax: of denaturation, annealing, and extension used were 94°Cfor 3 mm followed 061-6973976; E-mail: liulaml @fmi.ch. 2 The abbreviations used are: RI-PCR, reverse transcription PCR: ATCC, American by 35 cycles of 94°CforI mm, 48°Cfor1.5 mm, and 72°Cfor2.5 mm. At Type Culture Collection; DIG, digoxygenin. the end of the cycling phase, a further extension period of 10 mm at 72°Cwas 5386 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. @@@@@@@@@ C @1@t'@:@-@@: ‘1 CAThEPSIN K IN BREAST CARCINOMA @ A@' %:@ B @@@ “*s.• @‘ -a, @. I “• %4@@@,;;lT;s@1 @. ;‘.-‘‘s@tai11:ss(.,...@ J.;@ P@ @@@ @@‘,‘:• V ‘•@t'@rAl@@@:? a 1:5. @ 5• • ‘ •%@ 40 @ A ., tt. ‘.. @.@: # , (5*1@. @a D .S a A@. E L@' F S 4' I ‘a ‘A (@‘ @ ., , @ A .‘a5'@' ‘S @ . @ G :@ ;. : .. H @ , .. •,@@:‘;@. @ : ‘@ @% @ .•;. -a@t#4 5' ; —. I . • _ @@@@@ . : -. :@ :,@ Fig. 1. Immunoreactivity of cathepsin K in breast cancer cells. A, immunolocalization of cathepsin K in a skeletal metastasis of breast tumor. Positive staining can be seen in osteoclasts (medium arrows), adjacent to bone matrix (small arrow), and in breast cancer cells (large arrow), present in the marrow space. B, invasive carcinoma of no special type (grade 3) showing strong cytoplasmic positivity. The stromal cells are negative. C, intravascular, probably intralymphatic, tumor showing strong cytoplasmic positivity (arrow). The adjacent arteriole shows positivity of the smooth muscle coat. D, nonneoplastic terminal duct lobular units showing microcystic change. There is cytoplasmic positivity that is restricted to the myoepithelial cells. E, a normal terminal duct lobular unit showing no positivity. F, ductal carcinoma in situ of high nuclear grade and comedo growth pattern showing strong cytoplasmicpositivity.Thereisweakstainingofthestromalmyofibroblasts.G,sametumorasshowninB,exhibitingnegativityforcathepsinKinthisparticulararea.Thereis vascular invasion in this field, and the intravascular tumor is also negative. H. invasive carcinoma of no special type (grade 2) showing strong cytoplasmic positivity. The stroma is negative except for the pericytes of small blood vessels. 5387 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. @@@ II 0 @0 . t@ CAThEPSIN K IN BREAST CARCINOMA A B A @ . S @ , @: @A @ @I. I-@; . .,.@ @ A . @_.,%, @ @‘I,. “a .* @ ‘ @1 ‘ Fig.
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