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[Cancer Research 59, 5488-5491, November 1, 1999]
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[Cancer Research 59, 5488-5491, November 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Tumorigenicity of Human Breast Cancer Is Associated with Loss of the Ca2+-activated Chloride Channel CLCA21

Achim D. Gruber and Bendicht U. Pauli2

Cancer Biology Laboratories, Department of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, New York 14853


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The human Ca2+-activated chloride channel-2 (CLCA2) is expressed in normal breast epithelium but not in breast tumors of different stages of progression. Northern analysis of nontransformed and transformed breast epithelial cell lines revealed CLCA2 expression in the nontransformed cell line MCF10A and the nontumorigenic cell line MDA-MB-453, whereas all tumorigenic cell lines were negative (MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7). When stably reintroduced into CLCA2-negative MDA-MB-231 and MDA-MB-435 cells, CLCA2 expression reduced Matrigel invasion in vitro and inducibility of s.c. and metastatic tumors of MDA-MB-231 cells in nude mice. Our results suggest that CLCA2 may act as a tumor suppressor in breast cancer.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The role of ion channels in carcinogenesis and tumor progression is poorly understood. In particular, little is known about involvement of channel proteins in processes such as growth regulation, apoptosis, invasion, or the complex path of metastasis. Recent studies have indicated that expression of certain channels may be linked to the biological behavior of specific tumor cell lines. For example, large voltage-dependent chloride currents are obligatory for migration and invasion of human malignant glioma cells (1) , whereas expression of the voltage-activated Na+ channel is associated with invasion and metastasis of human and rat prostate cancer cells (2, 3, 4) . In both of these malignancies, channel blockers inhibit migration, invasion, and metastasis in a dose-dependent manner (1 , 4) . Expression of other ion channels such as Ca2+-activated K+, voltage-activated K+, and volume-sensitive Cl- channels was associated with growth regulation of human breast cancer cells, proliferation of melanoma cells, and cervical carcinogenicity, respectively (5, 6, 7, 8) . Finally, transgenic initiation of morphologically distinct adenocarcinomas of the murine mammary gland with the activated oncogenes neu or ras was correlated with expression of an as yet uncharacterized cell surface-associated chloride channel (9) . Undoubtedly, additional studies are needed in establishing a clearer molecular concept of the contribution of ion channels to cancer than can presently be deduced from the few reports listed above.

Here, we report that human CLCA23 (10) , a member of a recently discovered, novel family of mammalian CLCA proteins (discussed in Refs. 10, 11, 12 ), is prominently expressed in normal human breast epithelium but is lost in breast cancer and in tumorigenic breast cancer cell lines. When stably reexpressed in CLCA2-negative human breast carcinoma cell lines, CLCA2 significantly reduced the tumorigenicity, invasiveness, and ability to colonize the lungs of nude mice of the transfected cancer cells. Reexpression of CLCA2 in these cells had no effect on cell growth and doubling in vitro.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Transfection.
The breast epithelial cell line MCF10A and the breast cancer cell lines MCF7, MDA-MB-435, MDA-MB-453, and MDA-MB-468, as well as NIH3T3 cells, were from American Type Culture Collection (Rockville, MD) and MDA-MB-231 from Dr. J. Price (M. D. Anderson Cancer Center, Houston, TX). MCF10A is a spontaneously immortalized, nontumorigenic, near-normal breast epithelial cell line (13) . All breast cancer cell lines used in this study are tumorigenic in nude mice (14) , except MDA-MB-453 (15) . All cells were grown in DMEM supplemented with 10% FBS. Wild-type or myc-tagged (construct m2) CLCA2 cDNA cloned into pcDNA3.1 (10) was transfected into MDA-MB-231 and MDA-MB-435 using LipofectAMINE Plus (Life Technologies), and individual clones were isolated by G418 selection. Two strong expressers of wild-type and myc-tagged CLCA2 were selected per cell line and used for further analyses. Clones of breast cancer cells stably transfected with vector alone were used as controls.

In Situ Hybridization.
Formalin-fixed, paraffin-embedded breast cancer biopsies from 10 patients with different stages of the disease (Cooperative Human Tissue Network) were hybridized with digoxigenin-labeled CLCA2 antisense RNA (2 ng/µl), complementary CLCA2 sense RNA (negative control), and EF-1{alpha} antisense RNA (positive control) as described in detail (12 , 16) .

Northern Blot Hybridization and RT-PCR.
Total RNA was isolated by the Trizol method (Life Technologies) from 70 to 80% confluent cell cultures and from tumors in nude mice, resolved on a formaldehyde/agarose gel (15 µg RNA/lane), and blotted onto nitrocellulose. Blots were hybridized with a [{gamma}32P]dCTP nick-labeled CLCA2 cDNA probe using ExpressHyb solution (Clontech) at 68°C. To exclude cross-hybridization of related family members, highly stringent washing conditions were used after the hybridization (two washes with 2x SSC, 0.1% SDS at 70°C for 30 min, followed by two washes with 0.1x SSC, 0.1% SDS at 70°C for 30 min). RT-PCR was performed to detect a 2838-bp fragment of CLCA2 mRNA using primers and PCR conditions as described (10) . All PCR products were gel-purified (QIAquick Gel Extraction kit; Qiagen), cloned into the pGem-T vector (Promega), and partially sequenced to verify amplification of CLCA2. In all RT-PCR assays, negative controls were included with water instead of RNA as template. For detection of the CLCA2 gene, a 270-bp DNA fragment of the CLCA2 promoter region (GenBank AF114429) was amplified from RNase A-treated genomic DNA extracted from all six cell lines (100 ng DNA/reaction; sense primer, 5'-CTGATTTCCAGCCCATATTTCC-3', and antisense primer, 5'-GGTTTCTTCCTGAACTTGCAAG-3').

Immunoblotting.
Extracts from MDA-MB-231 and MDA-MB-435 cells stably transfected with myc-tagged CLCA2 as well as from excised tumor samples from nude mice were resolved by SDS-PAGE (10% polyacrylamide) and electroblotted to nitrocellulose. Membranes were probed with mouse antihuman myc antibody 9E10 (Calbiochem) and developed by ECL (Amersham).

Cell Doubling Time.
Viable MDA-MB-231 and MDA-MB-435 cells transfected with vector alone, wild-type CLCA2, or construct m2 were seeded at 3 x 104 cells/100-mm dish. Cells were grown in DMEM supplemented with 10% FBS for 24, 48, 72, and 96 h (n = 5/time point) with changes to fresh medium every 24 h. The number of cells/dish was determined by counting cells with a hemocytometer (n = 8 data points/plate).

Anchorage-independent Growth.
Viable MDA-MB-231 and MDA-MB-435 cells transfected with vector alone, wild-type CLCA2, or construct m2 were suspended in soft (0.3%) or hard (0.9%) Bacto agar (Difco) and placed onto a bottom layer of 0.9% agar (3 x 104 cells/100-mm dish). Colonies were allowed to develop for 3 weeks. Average colony numbers were calculated after counting four random fields using an eyepiece with a grid at x200.

Migration and in Vitro Invasion Assay.
Migration was analyzed in a Neuro Probe 48-well chemotaxis chamber (Cabin John, MD), supplied with a polycarbonate membrane of 8-µm pore size (Poretics, Livermore, CA). The top chamber was seeded with 105 viable tumor cells in DMEM. The bottom chamber was filled with DMEM supplemented with 5% FBS as a chemoattractant. After 4 h (MDA-MB-231) or 16 h (MDA-MB-435) at 37°C, cells were removed from the upper side of the membrane with a cotton swab, and cells attached to the lower side were stained with Giemsa and counted in four different random fields using a light microscope at x200. In vitro invasion was assessed using the same chamber as above but with the polycarbonate membrane covered with a film of Matrigel (Collaborative Research). Cells (2 x 105 cells/ml) were seeded into the upper chamber in the presence of DMEM containing 5% FBS. The chambers were incubated at 37°C for 8 h (MDA-MB-231) or 24 h (MDA-MB-435), and the number of cells that had invaded through the Matrigel-coated membrane onto the lower membrane surface was counted as described above. Migration and invasion assays were performed in triplicate.

Tumorigenicity and Experimental Metastasis.
The tumorigenic and lung colonization potentials of MDA-MB-231 cells stably transfected with vector-alone, wild-type, or myc-tagged CLCA2 were assayed in female athymic nude mice (NCr-nu/nu), 6–8 weeks of age (Frederick Cancer Research Facility, National Cancer Institute). Cells were injected s.c. (lateral flank, 2 x 105 viable cells/0.1 ml DMEM) or i.v. (lateral tail vein, 4 x 105 viable cells/0.2 ml DMEM) in groups of eight mice. Growth rates of s.c. tumors were monitored by weekly caliper measurements of two tumor diameters. Lung colonies were counted grossly and histologically on formalin-fixed, H&E-stained serial lung sections (one sagittal section per mm) 8 weeks after i.v. injection of tumor cells (or when mice became moribund).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
CLCA2 Expression in Normal and Cancerous Breast Tissues and Cell Lines.
Using in situ hybridization, CLCA2 was detected in acini and small ducts of normal mammary gland but was absent in a series of 10 breast tumors, diagnosed by surgical pathologists as adenomas, invasive carcinomas, or metastases to lymph nodes or lungs (Fig. 1)Citation . Detection of EF-1{alpha} in consecutive tissue sections confirmed preservation of mRNA and appropriate experimental conditions. A similar pattern of CLCA2 expression was observed when cell lines derived from normal breast epithelium and various breast cancers were analyzed by Northern hybridization. CLCA2 was detected in the nontransformed human mammary epithelial cell line MCF10A as well as in the nontumorigenic cell line MDA-MB-453. However, CLCA2 was undetectable in all tumorigenic cell lines including MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7 (Fig. 2a)Citation . These results were confirmed by RT-PCR detection and sequencing of the PCR products from MCF10A and MDA-MB-453 cells, verifying identity with CLCA2 (data not shown). To test whether lack of CLCA2 expression in the tumorigenic cell lines was attributable to loss of the CLCA2 gene locus at 1p22–31 (17) , we amplified a DNA fragment within the CLCA2 gene promoter region from genomic DNA. Successful amplification indicated that the corresponding genomic CLCA2 locus was present in all cell lines (Fig. 2b)Citation .



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  		Fig. 1. Detection of CLCA2 mRNA in biopsies from normal human breast and breast cancer lesions by in situ hybridization using a digoxigenin-labeled antisense RNA probe (a and c–f). a, normal mammary gland tissue with CLCA2 expression in acini and small ducts (arrows). b, sense probe hybridization of a consecutive section confirmed specificity of the antisense probe hybridization. c–f, no CLCA2 hybridization signals were obtained from breast cancer samples (c, intraductal carcinoma; d, solid carcinoma with tubule formation; e, invasive tubulo-lobular carcinoma with fibrous stroma; f, lymph node metastasis of a medullary carcinoma with lymphocytes in the lower right corner). Bar, 100 µm.

 


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  		Fig. 2. Detection of CLCA2 in nonneoplastic and neoplastic human mammary epithelial cell lines. a, Northern blot hybridization of total RNA samples (15 µg/lane) revealed the presence of the 3.6-kb CLCA2 mRNA in cell lines MCA10A and MDA-MB-453 but not in cell lines MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7 (top panel). RNA loading amounts were compared by ethidium bromide-staining of 18S rRNA (bottom panel). b, PCR detection of a 270-bp DNA fragment of the CLCA2 gene promoter region in RNase A-treated genomic DNA samples indicated conservation of the CLCA2 gene locus in all cell lines. A negative control was included with sterile water as template (ethidium bromide-stained 1.3% agarose gel).

 
Effect of CLCA2 Expression on Breast Cancer Cells in Vitro.
To test the effect of CLCA2 expression on the biological behavior of breast cancer cells in vitro, we generated stable CLCA2 transfectants of cell lines lacking endogenous expression of CLCA2. As recipients, we chose the two cell lines MDA-MB-231 and MDA-MB-435 because of their high (MDA-MB-231) and low (MDA-MB-435) migratory, invasive, and metastatic capabilities (18) . In addition to transfection with wild-type CLCA2 cDNA, a human myc-tagged CLCA2 construct (construct m2; Ref. 10 ) was also stably transfected into a second set of the same cell lines to enable immunological detection of the CLCA2 protein in these cells. Stable vector alone-transfectants of the same cell lines were generated for control purposes. Expression of CLCA2 in the transfectants was assayed by Northern blot hybridization (wild-type and myc-tagged CLCA2) as well as by Western blotting (myc-tagged CLCA2; Fig. 3a and b).Citation The CLCA2 message detected by Northern blot hybridization was similar to those in MCF10A and MDA-MB-453 cells (Fig. 2a)Citation . Electrophysiological analyses confirmed that the constructs used for CLCA2 expression indeed generated a Ca2+-sensitive Cl- conductance in transfected HEK293 cells (10) . When wild-type and myc-tagged CLCA2 transfected cell lines were compared with vector-alone transfected cells in standard in vitro assays, no changes were seen with regard to growth rate (cell doubling time) and anchorage-independent growth in soft and hard agar (data not shown). However, when tested for their migratory phenotype, wild-type and myc-tagged CLCA2-transfected MDA-MB-435 expressed markedly decreased migration relative to cells transfected with vector alone (P < 0.01). CLCA2 expression in MDA-MB-231 cells, either untagged or myc-tagged, yielded only slightly reduced migratory activities of no statistical significance (Fig. 3c)Citation . However, both wild-type and myc-tagged CLCA2-transfected breast cancer cell lines exhibited significantly reduced invasion through Matrigel when compared with vector-alone transfected cells (P < 0.01; Fig. 3dCitation ).



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  		Fig. 3. Effect of stable transfection of breast cancer cell lines with wild-type and myc-tagged CLCA2. a, Northern blot hybridization with a [32P]dCTP-labeled CLCA2 probe (top panel) of total RNA (15 µg/lane) extracted from vector alone-transfected cell lines MDA-MB-231 and MDA-MB-435 (231 vector and 435 vector), wild-type CLCA2 transfectants (231 CLCA2 and 435 CLCA2), and myc-tagged CLCA2 transfectants (231 CLCA+ and 435 CLCA2+). RNA loading amounts were compared by ethidium bromide staining of 18S rRNA (bottom panel). b, immunodetection (anti-myc antibody 9E10) of the 34-kDa subunit of myc-tagged CLCA2 in stably transfected cell lines MDA-MB-231 and MDA-MB-453 (231 CLCA2+ and 435 CLCA2+). The weaker bands of different molecular weights are also visible in the vector alone-transfected cells, suggesting unspecific binding of the antibody. c, reduced migratory activities of wild-type and myc-tagged CLCA2-expressing MDA-MB-435 cells relative to vector alone-transfected cells (435 vector, 131 ± 11; 435 CLCA2, 41 ± 15; 435 CLCA2+, 58 ± 8; means; bars, SD, n = 6; *, P < 0.01, Student’s t test). Migration differences between MDA-MB-231 cells transfected with CLCA2 and vector alone were statistically not significant (231 vector, 165 ± 32; 231 CLCA2, 128 ± 12; 231 CLCA2+, 126 ± 21; n = 6). d, reduced invasion through Matrigel of MDA-MB-231 and MDA-MB-435 cells transfected with wild-type or myc-tagged CLCA2 relative to cells transfected with vector alone (231 vector, 232 ± 25; 231 CLCA2, 28 ± 7; 231 CLCA2+, 42 ± 13; 435 vector, 58 ± 14; 435 CLCA2, 23 ± 6; 435 CLCA2+, 15 ± 3; bars, SD; n = 6; *, P < 0.01).

 
Effect of CLCA2 Expression on Tumorigenicity and Lung Colonization in Nude Mice.
MDA-MB-231 cells, stably transfected with either wild-type or myc-tagged CLCA2 and used in the above experiments, were also tested for their tumorigenic and lung colonization potentials in nude mice. Cells were injected s.c., and tumor growth was monitored by weekly measurements of tumor sizes. A third set of animals injected with vector-alone transfected cells served as controls. All animals of the control group developed s.c. tumors, which were first visible 2–3 weeks after injection and increased in size up to 6 mm in diameter eight weeks after injection. However, both the number of animals with tumors and mean tumor sizes were significantly reduced in mice injected with wild-type or myc-tagged CLCA2-expressing cells (Table 1)Citation . When nude mice were i.v. injected with vector-alone transfected MDA-MB-231 cells, multiple tumor colonies were present in the lungs 8 weeks after injection or when moribund mice were euthanized. Animals injected with wild-type or myc-tagged CLCA2, however, had no or significantly fewer tumors in their lungs (Table 1)Citation . When s.c. or lung tumors were tested for expression of CLCA2 by Northern blot hybridization or immunoblotting, respectively, mRNA and protein expression levels were similar to those of the cell lines injected into the mice (data not shown). These results indicated that the mouse tumors did not originate from CLCA2-negative cells or from revertants that eliminated expression of the channel.


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  		Table 1 Tumorigenicity and lung colony formation of CLCA2-transfected MDA-MB-231 breast cancer cells in nude mice

 

   Discussion
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Loss of CLCA2 expression in human breast cancer appears to be closely associated with tumorigenicity, because neither in situ breast tumors nor tumorigenic breast cancer cell lines express CLCA2 and because normal mammary epithelial cells, spontaneously immortalized normal breast epithelial cells (MCF10A; Ref. 13 ), and nontumorigenic breast cancer cells (MDA-MB-453; Ref. 15 ) all express CLCA2. Mapping of the CLCA2 gene to a chromosomal region (1p22–31; Ref. 17 ), which is often deleted in the late stages of progression of human breast cancer (19) , suggested that loss of CLCA2 expression might be the result of this common chromosomal deletion. However, PCR amplification of a region within the CLCA2 promoter in all normal and cancerous breast tissues examined argues against this possibility. Thus, loss of CLCA2 expression seemed to happen at the transcriptional level and might be attributable to a transcriptional down-regulation of the gene, abnormal RNA processing, or both. Because loss of CLCA2 was most consistently associated with tumorigenicity, we tested whether reintroduction of CLCA2 into CLCA2-negative, tumorigenic breast cancer cells would abrogate their tumorigenic potential. Indeed, MDA-MB-231 tumor cell clones, which stably express CLCA2, produce s.c. tumors in nude mice with greatly reduced efficiency. The same is true when these cells are tested in a lung colony assay. These results were not caused by decreased cell doubling rates or an inability to form colonies in soft agar of the CLCA2-transfected breast cancer cells. The observed in vitro phenotypic changes of reduced invasiveness and migratory behavior also do not account for the loss of tumorigenicity, because neither of these capabilities is necessary for successful s.c. tumor growth. Contrasting the effects of other ion channels including chloride channels (1, 2, 3, 4) , decreased invasiveness in both of the CLCA2-transfected breast cancer cell lines, and the select down-regulation of migration in transfected MDA-MB-435 cells could be the result of a CLCA2-induced differentiation effect (20) and/or alteration in intracellular Ca2+ signaling (21) .

The role played by CLCA2 in the tumorigenicity of breast cancer cells is unknown. However, recent findings that stimulating the Fas pathway in lymphocytes induces an outwardly rectified chloride conductance prior to apoptosis and that channel blockers rescue Fas-stimulated lymphocytes from undergoing apoptosis (22) suggest that CLCA2 may play a similar role in breast cancer cells. This notion is especially pertinent when considering that an increase in intracellular Ca2+, which is responsible for CLCA gating, is known to occur early in the apoptotic cascade (23) . Reintroduction of the channel into tumorigenic breast cancer cells that had lost CLCA2 during carcinogenesis or early progression may make the transfectant tumor cells vulnerable to apoptotic stimuli upon injection into nude mice. Irrespective of the mechanisms that underlie the loss of CLCA2 in breast cancer disease, our data imply that CLCA2 is a tumor suppressor for breast cancer.


   FOOTNOTES
 
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.

1 This study was supported by USPHS Grants CA47668 and CA71626 from the National Cancer Institute, a grant from the American Boxer Club (to B. U. P.), and a fellowship from the German Research Council (to A. D. G.). Back

2 To whom requests for reprints should be addressed, at Cancer Biology Laboratories, Department of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, NY 14853-6401. Phone: (607) 253-3343; Fax: (607) 253-3708; E-mail: bup1{at}cornell.edu Back

3 The abbreviations used are: CLCA2, Ca2+-activated chloride channel-2; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR. Back

Received 8/10/99. Accepted 9/20/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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M. E. Loewen and G. W. Forsyth
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J. R. Beckley, B. U. Pauli, and R. C. Elble
Re-expression of Detachment-inducible Chloride Channel mCLCA5 Suppresses Growth of Metastatic Breast Cancer Cells
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Molecular and Functional Analyses of Two New Calcium-activated Chloride Channel Family Members from Mouse Eye and Intestine
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Calcium-activated Chloride Channel-2 in Human Epithelia
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M. Abdel-Ghany, H.-C. Cheng, R. C. Elble, H. Lin, J. DiBiasio, and B. U. Pauli
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Molecular and Functional Characterization of a Murine Calcium-activated Chloride Channel Expressed in Smooth Muscle
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T. J. Jentsch, V. Stein, F. Weinreich, and A. A. Zdebik
Molecular Structure and Physiological Function of Chloride Channels
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R. C. Elble and B. U. Pauli
Tumor Suppression by a Proapoptotic Calcium-activated Chloride Channel in Mammary Epithelium
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