Clinical & Experimental Metastasis

, Volume 22, Issue 8, pp 663–673

Enhanced cell surface CD44 variant (v6, v9) expression by osteopontin in breast cancer epithelial cells facilitates tumor cell migration: Novel post-transcriptional, post-translational regulation

  • Subarna A. Khan
  • Amy C. Cook
  • Maya Kappil
  • Ursula Günthert
  • Ann F. Chambers
  • Alan B. Tuck
  • David T. Denhardt
Original Research Paper

DOI: 10.1007/s10585-006-9007-0

Cite this article as:
Khan, S.A., Cook, A.C., Kappil, M. et al. Clin Exp Metastasis (2005) 22: 663. doi:10.1007/s10585-006-9007-0

Abstract

Osteopontin (OPN) is a glycosylated, secreted phosphoprotein that functions both as a cell attachment and chemotactic factor. Elevated expression of OPN confers enhanced metastatic ability on transformed cells, suggesting that OPN may contribute to the malignant progression of tumors. Migration of mammary carcinoma cells is stimulated by OPN via interactions with integrins and CD44 cell surface receptors. We hypothesized that OPN modulates specific CD44 isoform expression to facilitate breast cancer cell migration. The 21NT tumorigenic human breast cancer cell line was examined for regulation of CD44 expression at both the mRNA and protein levels in response to an engineered increase in OPN expression under CMV promoter control. Significant up-regulation of CD44s isoform mRNA expression was observed, but no change in CD44v6, v8, v9 or v10 mRNA levels. While there were elevated levels of CD44s, v6 and v9 protein at the cell surface, at the level of total cellular protein only CD44s and v6 were markedly increased. This suggests that OPN can regulate CD44 expression at both transcriptional and post-transcriptional (both amount and localization of protein) levels. To validate the functional consequence of OPN regulation of CD44 expression, we demonstrate that OPN-mediated cell migration was reduced by exposure to a anti-pan CD44 antibody, and to anti-CD44v6 and anti-CD44v9 function-blocking antibodies. Our data provide evidence that in 21NT cells OPN enhances CD44s mRNA expression, increases cell surface expression of CD44 variant forms without a change in mRNA levels, and stimulates cell migration.

Keywords

OsteopontinCD44 variantBreast cancerCell migration

Abbreviations

CD44

all forms of CD44

CD44s

the standard form of CD44 containing no variant exons

CD44vx

a variant of CD44 containing specific variant sequences indicated by the x

Ct

threshold cycle

FBS

fetal bovine serum

FC

flow cytometry

hrOPN

human recombinant osteopontin

HRP

horseradish peroxidase

MEM

minimal essential medium

OPN

osteopontin

PBS

phosphate-buffered saline

PE

phycoerythrin

RT-PCR

reverse transcriptase polymerase chain reaction

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Subarna A. Khan
    • 1
  • Amy C. Cook
    • 3
  • Maya Kappil
    • 1
  • Ursula Günthert
    • 4
  • Ann F. Chambers
    • 3
    • 5
  • Alan B. Tuck
    • 3
    • 5
    • 6
  • David T. Denhardt
    • 1
    • 2
  1. 1.Department of Cell Biology and Neuroscience, Nelson LaboratoriesRutgers UniversityPiscatawayUSA
  2. 2.Cancer Institute of New JerseyNew BrunswickUSA
  3. 3.Department of PathologyUniversity of Western OntarioLondonCanada
  4. 4.Institute for Medical MicrobiologyUniversity of BaselBaselSwitzerland
  5. 5.London Regional Cancer ProgramLondonCanada
  6. 6.London Health Sciences CentreLondonCanada