ICANCERRESEARCH56, 4841-4845, November 1, 1996] Advances in Brief Widespread Loss of Gelsolin in Breast Cancers of Humans, Mice, and Rats1 Harold L. Asch,2 Karen Head, Yan Dong, Farah Natoli, Janet S. Winston, James L. Connolly, and Bonnie B. Asch Departments of Experimental Pathology [H. L A., K. L H., Y. D., F. N., B. B. A.] and Pathology [J. S. WI, Roswell Park Cancer Institute, Buffalo, New York 14263, and Department of Pathology, Beth Israel Hospital, Boston, Massachusetts 02215 Ii. L C.) Abstract Materialsand Methods Down-regulation of gelsolin, an actin-binding protein, is frequently found Mammary Tissues and Cell Lines. Human breast tissues included 2 in several types of transformedcells and tumors.The presentstudy demon reduction mammoplasties, 1 of which had proliferative changes without atypia, strates that gelsolin protein and RNA were absent or markedly reduced in 3 nonmalignant tissues immediately adjacent to cancers, 29 sporadic, primary human breast cancer celllines relative to “normal―mortalhuman mammary invasive breast cancers (1 tubular and 28 poorly differentiated ductal carcino epithelial cells and benign, immortalized cell lines. Moreover, actin filaments mas), and 1 metastasis to chest wall. Normal mouse mammary tissues were were usually attenuated coincident with the reduction in gelsolin. GeISOIIn from pregnant BALB/c mice, and mammary tumors were from BALB/cfC3H was also missingor greatlydecreasedin 70% of3Ohumansporadic,invasive animals infected with mouse mammary tumor virus. Normal rat mammary breast carcinomas examined by immunocytochemistry and in 100% of virally tissues were provided by Dr. M. Ip from virgin Sprague-Dawley females, and induced mouse and chemically induced rat mammary carcinomas evaluated rat mammary carcinomas induced by DMBA treatment were a gift from Dr. C. by Northernanalysis.Southernanalysisrevealedno nutjormutationsin the Ip. HMECs from a reduction mammoplasty (Clonetics, Inc., San Diego, CA) gelsolin gene of human breast cancer cells. Our results show that partial or were grown in short-term culture (9—12passages) and represent human nor total lossofgelsolin expression is common to the majority ofbreast cancers of mal, mortal mammary cells. Benign immortal breast epithelial cell lines were diverse etiologiesin three animal species and point to gelsolin as a candidate HBL100, MCF1OA,and l84AlN4. Tumor cell lines included 184A1N4TH, suppress@w of breast cancer. SKBR3, MCF7, MDA-MB-231, and T47D. Immunocytochemistry and Analysis by Western, Northern, and South Introduction em Blots. Acetone-fixed,frozenbreasttissue sections (4—6p@m)wereincu bated in anti-gelsolin MAb (Sigma Chemical Co., St. Louis, MO), washed, and The AF3 cytoskeleton is directly or indirectly involved in many vital cell incubated with a fluorescein-tagged secondary antibody for IF or with a functions such as cell shape, motility, cytokinesis, endocytosis, mRNA local biotin-tagged secondary antibody for the ABC IP technique (Vector Labora ization, and growth regulation (1—3).The AF network has been implicated as tories, Burlingame, CA) using the diaminobenzidine color reagent. Additional both a target and mediator of signal transduction initiated through receptor breast tissues were fixed in formalin and embedded in paraffin; 4-,im-thick tyrosine kinases and extracellular matrix-integrin systems (4—8).In response sections were microwaved for 10 mm, followed by the ABC IP protocol using to many extracellular signals, cells change their shape, their adhesion to matrix a Ventana 320 Automated Slide staining system (Ventana Medical Systems, components, and their interactions with adjacent cells (5—9).Cells possess a Tucson, AZ). Cells grown on glass coverslips were fixed in 2% paraformal complex army of proteins that bind to actin in its filamentous or monomeric dehyde, stained by IF with the anti-gelsolin MAb, and subsequently stained globular forms to regulate the assembly/disassembly, architecture, and distri with rhodamine phalloidin (30 units/ml) to detect filamentous actin in the same bution ofAFs (1, 2, 9). A key determinant of AF length is gelsolin, a Mr90,000 cells. Negative primary antibody controls for tissue and cultured cell staining actin-binding protein that severs AFs, caps the fast-growing (barbed) ends, and promotes nucleation of polymerization (10). included use of an anti-ras MAb (Chemicon International, Inc., Temecula, CA), which was unreactivewith humanbreastcells (datanot shown). Evidence in support of gelsolin as a tumor suppressor has been steadily accruing.Gelsolin was markedlydiminishedin H-ms-transformedmouse fibro Immunohistochemistry staining was evaluated independently by two or three investigators (H. L. A. and B. B. A., and in some cases, J. S. W.), and blasts (1 1), human fibroblasts, and epitheial cells transformed by SV4O virus (12), gastric caitinoma cell lines (13), bladder cancer cell lines, and the majority of cancers were categorized as having reduced gelsolin if two-thirds or more of bladder cancers (14). Transfection and mutation studies have confirmed the tumor tumor cells in the sample were either negative or weakly stained. The percent suppressor function ofgelsolin for both epitheial and fibroblastic tumor cells (11, age of reactive and unreactive cells was estimated from examination of the 14). Two reports on mammary cancers indicated gelsolin was greatly reduced in entire tissue section. The results for the great majority of cancers were all tumorsexamined.The proteinwas undetectableby immunocytochemistryin12 clear-cut. The rare disagreements that arose about a staining pattern were of 12 human breast cancers (15), and gelsolin RNA was reduced 4—5-foldin resolved by simultaneous examination and discussion of the sample by all mouse mammary tumors (16). The key findings of the present study are: (a) three investigators. gelsolin expression was drastically reduced in the majority of human, mouse, and Cultured mammary cells were harvested in an antiprotease cocktail and rat mammarytumorsexaminedas well as in most humanbreastcancercell lines; solubilized in Laemmli buffer for Western blotting (17). Equal protein loads of (b) down-regulation of gelsolin protein in the human tumor cell lines was due to cell extracts (Dot/Metric protein assay; Geno Technology, St. Louis, MO) were decreased gelsolin RNA and may not involve major mutations of the gene; (c) in run on 7.5% SDS-PAGE minigels, and Western blots probed with anti-gelsolin most breast cancer cells, reduced gelsolin protein expression was associated with MA], were visualized by chemiluminescence (Pierce, Rockford, IL). Immu decreasedAFs. noreactive bands were quantitated by volume densitometry. Equal protein transfer for each lane was monitored by India ink staining of blots after immunostaining. The anti-gelsolin MAb was specific for a Mr 90'000 band. Received 8/8/96; accepted 9/18/96. The costs of publication of this article were defrayed in part by the payment of page Parallel Western blots were probed with an anti-actin MAb (Sigma), which charges. This article must therefore be hereby marked advertisement in accordance with reacts with the carboxy terminus of all known mammalian actins. For Northern 18 U.S.C. Section 1734 solely to indicate this fact. analysis, total RNA was extracted from cultured cells or tissues by ThI reagent I Supported by the Roswell Park Alliance Foundation and Grants CA56609 and CA62014 from the National Cancer Institute, NIH. (Molecular Research Center, Cincinnati, OH). Genomic DNA was isolated 2 To whom requests for reprints should be addressed. from cultured cells and digested with an endonuclease for Southern analysis. 3The abbreviations used are: ÀY,actin filament; DMBA, 7,l2-dimethylbenzanthra Northern and Southern blots were probed with a full-length cDNA clone of cene; HMEC, human mammary epitheial cell; MAb, monoclonal antibody; IF, immuno fluorescence; IP, immunoperoxidase; PIP2, phosphatidylinositol 4,5-trisphosphate; PLD, human cytoplasmic gelsolin (a gift of Dr. D. Kwiatkowski) following standard phospholipase D. procedures. 4841 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. @@@@@@@ / ‘@ @J@:-.S •@ “ @, @‘ LOSS OF GELSOLININ BREASTCANCER Results (Fig. 1, b and d), positive (Fig. le) or weakly stained (Fig. lg), whereas some tumors were heterogeneous, with tumor cell reactions Gelsolin Expression in Normal and Malignant Human Breast ranging from negative to positive (Fig. 1, f and h). In a few tumors, Tissues. Immunocytochemistry on frozen sections of human reduc distinct regions of positive tumor cells were adjacent to regions of tion mammoplasty epithelium or of normal-appearing epithelium ad negative tumor cells (Fig. lfj; in these cases, the stroma stained jacent to or within breast cancers produced moderate-to-strong stain equally in both regions, thereby demonstrating the evenness of the ing in luminal cells and an intense reaction in myoepithelial cells specific immunoreaction. Gelsolin reactivity was primarily cytoplas (Figs. 1, a, c, and h). The moderate-to-strong reactivity of stromal cells provided a positive control for the staining procedure (Fig. 1). mic, but occasionally, nuclei appeared to stain (Fig. 1). Expression of gelsolin in the epithelium of benign breast tissue with Gelsolinand Actin Expression in Malignant and Nonmalignant proliferative disease was also strong (data not shown). In contrast, Human Breast Cell Cultures. Gelsolin expression in cultured