Osteopontin Induction of Hyaluronan Synthase 2 Expression Promotes Breast Cancer Malignancy

Amy C Cook,Ann F Chambers,Eva A Turley,Alan B Tuck

doi:10.1074/jbc.m602694200

Amy C Cook, Ann F Chambers + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m602694200

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Abstract

Osteopontin (OPN) is a tumor-associated, secreted phosphoprotein that has been implicated in breast cancer progression and metastasis. Research concerning how OPN functions in tumor progression has led to the identification of a limited number of genes that contribute functionally to OPN-induced cellular behaviors. Recent microarray analysis, comparing 21NT breast cancer cells transfected to constitutively overexpress OPN with control cells, revealed hyaluronan synthase 2 (HAS2) to be a gene highly up-regulated in OPN-overexpressing cells. In this study, we further examined the relationship between OPN and HAS2. We show that 21NT OPN-transfected cells express high levels of HAS2, which is associated with increased HA production and matrix retention and is necessary for tumor cell adhesion to bone marrow endothelial cells and anchorage-independent growth. Finally, stable transfection of antisense HAS2 into 21NT cells overexpressing OPN resulted in a reduction in HAS2 expression, HA production, and pericellular retention. Antisense-mediated down-regulation of HAS2 also resulted in a significant decrease in cellular proliferation and colony growth in soft agar. To our knowledge, this is the first report of the ability of OPN to regulate HAS2 expression and HA production in breast cancer cells and further illustrates a unique functional relationship by which enhanced HA production facilitates OPN-mediated cell behaviors.

Highlights

Osteopontin (OPN)3 is an extracellular phosphoglycoprotein that has been increasingly implicated in the progression and metastasis of various cancers, including breast cancer
We subsequently categorized those genes differentially regulated by OPN into the six “Hallmarks of Cancer” categories, as defined by Hanahan and Weinberg [11]. Of those genes grouped into the “Tissue Invasion and Metastasis” category, we identified hyaluronan synthase 2 (HAS2) as a gene highly upregulated by OPN
We show that overexpression of OPN in a tumorigenic human mammary epithelial cell line contributes to increased HAS2 expression and HA production to promote anchorage-independent growth and adhesion to bone marrow endothelial cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The NTOPbi (OPN-transfected) and NTCi (mock transfected) cell lines were cultured in standard growth medium (␣HE ϩ 10% fetal bovine serum ϩ 200 ␮g/ml G418) as described previously [10]. To determine the efficiency of the reaction and purity of the products, a melt curve analysis was performed at the end of each reaction by incubating the samples at 95 °C for 1 min and ramping down to 55 °C at a rate of 0.2 °C/s This was followed by a stepwise increase in temperature from 55 to 95 °C for 81 cycles, in which the temperature was increased by 0.5 °C/cycle (30 s/cycle), with fluorescent measurements taken at each temperature increment. Bone Marrow Endothelial Cell Adhesion Assay—21NT control (NTCi) and OPN-transfected cells (NTOPbi) were plated in 150-mm culture dishes in standard growth medium and grown to ϳ80% confluence. The cells were trypsinized, and cell number was determined using a hemacytometer on every second or third day of an 11-day growth period

RESULTS

Elevated Levels of HA Promote

DISCUSSION