Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis

Oncogene volume 31, pages 2237–2246 (2012)Cite this article

Abstract

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Agiostratidou G, Li M, Suyama K, Badano I, Keren R, Chung S et al. (2009). Loss of retinal cadherin facilitates mammary tumor progression and metastasis. Cancer Res 69: 5030–5038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I et al. (1997). Inhibition of Bax channel-forming activity by Bcl-2. Science 277: 370–372.

    Article  CAS  PubMed  Google Scholar 

  • Barriere H, Poujeol C, Tauc M, Blasi JM, Counillon L, Poujeol P . (2001). CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts. Am J Physiol Cell Physiol 281: C810–C824.

    Article  CAS  PubMed  Google Scholar 

  • Barry MA, Eastman A . (1992). Endonuclease activation during apoptosis: the role of cytosolic Ca2+ and pH. Biochem Biophys Res Commun 186: 782–789.

    Article  CAS  PubMed  Google Scholar 

  • Beckley JR, Pauli BU, Elble RC . (2004). Re-expression of detachment-inducible chloride channel mCLCA5 suppresses growth of metastatic breast cancer cells. J Biol Chem 279: 41634–41641.

    Article  CAS  PubMed  Google Scholar 

  • Cano A, Pérez-Moreno M, Rodrigo I, Locascio A, Blanco M, del Barrio M et al. (2000). The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76–83.

    Article  CAS  PubMed  Google Scholar 

  • Charafe-Jauffret E, Ginestier C, Monville F, Finetti P, Adélaïde J, Cervera N et al. (2006). Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene 25: 2273–2284.

    Article  CAS  PubMed  Google Scholar 

  • Cheng J, Ding M, Aribi A, Shah P, Rao K . (2006). Loss of RAB25 expression in breast cancer. Int J Cancer 118: 2957–2964.

    Article  CAS  PubMed  Google Scholar 

  • Creighton C, Chang J, Rosen J . (2010). Epithelial-mesenchymal transition (EMT) in tumor-initiating cells and its clinical implications in breast cancer. J Mammary Gland Biol Neoplasia 15: 253–260.

    Article  PubMed  Google Scholar 

  • D'Assoro AB, Leontovich A, Amato A, Ayers-Ringler JR, Quatraro C, Hafner K et al. (2010). Abrogation of p53 function leads to metastatic transcriptome networks that typify tumor progression in human breast cancer xenografts. Int J Oncol 37: 1167–1176.

    CAS  PubMed  Google Scholar 

  • Dhasarathy A, Kajita M, Wade P . (2007). The transcription factor snail mediates epithelial to mesenchymal transitions by repression of estrogen receptor-alpha. Mol Endocrinol 21: 2907–2918.

    Article  CAS  PubMed  Google Scholar 

  • Dontu G, Abdallah W, Foley J, Jackson K, Clarke M, Kawamura M et al. (2003). In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17: 1253–1270.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Elble RC, Walia V, Cheng HC, Connon CJ, Mundhenk L, Gruber AD et al. (2006). The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment. J Biol Chem 281: 29448–29454.

    Article  CAS  PubMed  Google Scholar 

  • Elenbaas B, Spirio L, Koerner F, Fleming M, Zimonjic D, Donaher J et al. (2001). Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev 15: 50–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Enerback C, Porter DA, Seth P, Sgroi D, Gaudet J, Weremowicz S et al. (2002). Psoriasin expression in mammary epithelial cells in vitro and in vivo. Cancer Res 62: 43–47.

    CAS  PubMed  Google Scholar 

  • Furlong I, Ascaso R, Lopez Rivas A, Collins M . (1997). Intracellular acidification induces apoptosis by stimulating ICE-like protease activity. J Cell Sci 110 (Pt 5): 653–661.

    CAS  PubMed  Google Scholar 

  • Gasco M, Shami S, Crook T . (2002). The p53 pathway in breast cancer. Breast Cancer Res 4: 70–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gautier L, Cope L, Bolstad B, Irizarry R . (2004). affy--analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20: 307–315.

    Article  CAS  PubMed  Google Scholar 

  • Grigoriadis A, Mackay A, Reis-Filho J, Steele D, Iseli C, Stevenson B et al. (2006). Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data. Breast Cancer Res 8: R56.

    Article  PubMed  PubMed Central  Google Scholar 

  • Gruber AD, Pauli BU . (1999). Tumorigenicity of human breast cancer is associated with loss of the Ca2+-activated chloride channel CLCA2. Cancer Res 59: 5488–5491.

    CAS  PubMed  Google Scholar 

  • Hajra KM, Chen DY, Fearon ER . (2002). The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res 62: 1613–1618.

    CAS  PubMed  Google Scholar 

  • Hamann M, Gibson A, Davies N, Jowett A, Walhin J, Partington L et al. (2009). Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents. J Physiol 587: 2255–2274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hennessy B, Gonzalez-Angulo A, Stemke-Hale K, Gilcrease M, Krishnamurthy S, Lee J et al. (2009). Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 69: 4116–4124.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hesketh T, Moore J, Morris J, Taylor M, Rogers J, Smith G et al. (1985). A common sequence of calcium and pH signals in the mitogenic stimulation of eukaryotic cells. Nature 313: 481–484.

    Article  CAS  PubMed  Google Scholar 

  • Hsu Y, Chen Y, Chou C, Tang M, Chen J, Wilkins R et al. (2007). KCl cotransporter-3 down-regulates E-cadherin/beta-catenin complex to promote epithelial-mesenchymal transition. Cancer Res 67: 11064–11073.

    Article  CAS  PubMed  Google Scholar 

  • Jiang Z, Deng T, Jones R, Li H, Herschkowitz JI, Liu JC et al. (2010). Rb deletion in mouse mammary progenitors induces luminal-B or basal-like/EMT tumor subtypes depending on p53 status. J Clin Invest 120: 3296–3309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kogan-Sakin I, Tabach Y, Buganim Y, Molchadsky A, Solomon H, Madar S et al. (2011). Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells. Cell Death Differ 18: 271–281.

    Article  CAS  PubMed  Google Scholar 

  • Kunzelmann K . (2005). Ion channels and cancer. J Membr Biol 205: 159–173.

    Article  CAS  PubMed  Google Scholar 

  • Li X, Cowell JK, Sossey-Alaoui K . (2004). CLCA2 tumour suppressor gene in 1p31 is epigenetically regulated in breast cancer. Oncogene 23: 1474–1480.

    Article  CAS  PubMed  Google Scholar 

  • Liao M, Zhang C, Zhou B, Zimonjic D, Mani S, Kaba M et al. (2007). Enrichment of a population of mammary gland cells that form mammospheres and have in vivo repopulating activity. Cancer Res 67: 8131–8138.

    Article  CAS  PubMed  Google Scholar 

  • Lin A, Lowe S . (2001). Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation. Proc Natl Acad Sci USA 98: 5025–5030.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Malo M, Li L, Fliegel L . (2007). Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771. J Biol Chem 282: 6292–6299.

    Article  CAS  PubMed  Google Scholar 

  • Mani S, Guo W, Liao M, Eaton E, Ayyanan A, Zhou A et al. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133: 704–715.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marshall A, Pai V, Sartor M, Horseman N . (2009). In vitro multipotent differentiation and barrier function of a human mammary epithelium. Cell Tissue Res 335: 383–395.

    Article  PubMed  Google Scholar 

  • Minn A, Gupta G, Padua D, Bos P, Nguyen D, Nuyten D et al. (2007). Lung metastasis genes couple breast tumor size and metastatic spread. Proc Natl Acad Sci USA 104: 6740–6745.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moolenaar W, Tsien R, van der Saag P, de Laat S . (1983). Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature 304: 645–648.

    Article  CAS  PubMed  Google Scholar 

  • Moustakas A, Heldin CH . (2007). Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci 98: 1512–1520.

    Article  CAS  PubMed  Google Scholar 

  • Onder T, Gupta P, Mani S, Yang J, Lander E, Weinberg R . (2008). Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 68: 3645–3654.

    Article  CAS  PubMed  Google Scholar 

  • Perona R, Serrano R . (1988). Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump. Nature 334: 438–440.

    Article  CAS  PubMed  Google Scholar 

  • Pharoah P, Day N, Caldas C . (1999). Somatic mutations in the p53 gene and prognosis in breast cancer: a meta-analysis. Br J Cancer 80: 1968–1973.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Polyak K, Weinberg R . (2009). Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 9: 265–273.

    Article  CAS  PubMed  Google Scholar 

  • Poulsen JH, Fischer H, Illek B, Machen TE . (1994). Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 91: 5340–5344.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pouyssegur J, Dayan F, Mazure NM . (2006). Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441: 437–443.

    Article  CAS  PubMed  Google Scholar 

  • Prat A, Parker J, Karginova O, Fan C, Livasy C, Herschkowitz J et al. (2010). Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12: R68.

    Article  PubMed  PubMed Central  Google Scholar 

  • Rao K, Bryant E, O′Hara Larivee S, McDougall J . (2003). Production of spindle cell carcinoma by transduction of H-Ras 61L into immortalized human mammary epithelial cells. Cancer Lett 201: 79–88.

    Article  CAS  PubMed  Google Scholar 

  • Rao K, Alper O, Opheim K, Bonnet G, Wolfe K, Bryant E et al. (2006). Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells. Cancer Cell Int 6: 15.

    Article  PubMed  PubMed Central  Google Scholar 

  • Riker A, Enkemann S, Fodstad O, Liu S, Ren S, Morris C et al. (2008). The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis. BMC Med Genomics 1: 13.

    Article  PubMed  PubMed Central  Google Scholar 

  • Sablina AA, Hector M, Colpaert N, Hahn WC . (2010). Identification of PP2A complexes and pathways involved in cell transformation. Cancer Res 70: 10474–10484.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sarrio D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J . (2008). Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 68: 989–997.

    Article  CAS  PubMed  Google Scholar 

  • Sontheimer H . (2008). An unexpected role for ion channels in brain tumor metastasis. Exp Biol Med (Maywood) 233: 779–791.

    Article  CAS  Google Scholar 

  • Tanaka H, Shirkoohi R, Nakagawa K, Qiao H, Fujita H, Okada F et al. (2006). siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells. Int J Cancer 118: 1680–1691.

    Article  CAS  PubMed  Google Scholar 

  • Taube J, Herschkowitz J, Komurov K, Zhou A, Gupta S, Yang J et al. (2010). Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci USA 107: 15449–15454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thiery J . (2002). Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2: 442–454.

    Article  CAS  PubMed  Google Scholar 

  • Thiery JP, Sleeman JP . (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7: 131–142.

    Article  CAS  PubMed  Google Scholar 

  • Walia V, Ding M, Kumar S, Nie D, Premkumar L, Elble R . (2009). hCLCA2 Is a p53-Inducible Inhibitor of Breast Cancer Cell Proliferation. Cancer Res 69: 6624–6632.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walia V, Elble R . (2010). Enrichment for breast cancer cells with stem/progenitor properties by differential adhesion. Stem Cells Dev 19: 1175–1182.

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Klijn J, Zhang Y, Sieuwerts A, Look M, Yang F et al. (2005). Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 365: 671–679.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the following grants: NIH grant 1R15CA151094-01 and Excellence in Academic Medicine grants 56252 and 56253 to RCE; and NIH grants DK065742 and DA028017 to LP.

Author information

Author notes

  1. V Walia

    Present address: 7Current address: Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health (NIH), Bethesda, MD, USA.,

Authors and Affiliations

  1. Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL, USA

    V Walia, D Cao, L Premkumar & R C Elble

  2. Simmons Cancer Institute, Southern Illinois University School of Medicine, Springfield, IL, USA

    V Walia, Y Yu, J Cheng, K Rao & R C Elble

  3. Committee on Genetics, Genomics and Systems Biology (GGSB), University of Chicago, Chicago, IL, USA

    M Sun

  4. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

    J R McLean

  5. Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia

    B G Hollier

  6. Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

    S A Mani

Authors
  1. V Walia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J R McLean
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B G Hollier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S A Mani
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. K Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. L Premkumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. R C Elble
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R C Elble.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Walia, V., Yu, Y., Cao, D. et al. Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis. Oncogene 31, 2237–2246 (2012). https://doi.org/10.1038/onc.2011.392

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2011.392

Keywords

This article is cited by

Search

Advanced search

Quick links