RRM2 induces NF-κB-dependent MMP-9 activation and enhances cellular invasiveness

doi:10.1016/j.bbrc.2006.12.177

Biochemical and Biophysical Research Communications

Volume 354, Issue 1, 2 March 2007, Pages 190-196

https://doi.org/10.1016/j.bbrc.2006.12.177 Get rights and content

Abstract

Ribonucleotide reductase is a dimeric enzyme that catalyzes conversion of ribonucleotide 5′-diphosphates to their 2′-deoxynucleotide forms, a rate-limiting step in the production of 2′-deoxyribonucleoside 5′-triphosphates required for DNA synthesis. The ribonucleotide reductase M2 subunit (RRM2) is a determinant of malignant cellular behavior in a range of human cancers. We examined the effect of RRM2 overexpression on pancreatic adenocarcinoma cellular invasiveness and nuclear factor-κB (NF-κB) transcription factor activity. RRM2 overexpression increases pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression in a NF-κB-dependent manner. RNA interference (RNAi)-mediated silencing of RRM2 expression attenuates cellular invasiveness and NF-κB activity. NF-κB is a key mediator of the invasive phenotypic changes induced by RRM2 overexpression.

Section snippets

Materials and methods

Cell lines and culture. Capan2 and MIAPaCa2 pancreatic ductal adenocarcinoma cells were obtained from ATCC (Rockville, MD). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco-BRL, Gaithersburg, MD). Cells were maintained in a humidified (37 °C, 5% CO₂) incubator and passaged upon reaching 80% confluence.

Expression constructs and transfection. RRM2 cDNA inserts were produced by PCR (primer 1: 5′-CACCATGCTCTCCCTCCGTGT-3′; primer 2:

RRM2 overexpression enhances cellular invasiveness and upregulates MMP-9 expression and activity

Pancreatic adenocarcinoma cells differentially express RRM2 [19]. Capan2 was selected for RRM2 overexpression studies, as this cell line inherently expresses a low level of RRM2. Two transfectant clones of Capan2 exhibiting stable RRM2 overexpression were established: Capan2-R2.1 and Capan2-R2.2. Overexpression of RRM2 protein was confirmed in each clone by Western blot analysis (Fig. 1a). Capan2-R2.1 and Capan2-R2.2 expressed levels of RRM2 7.2- and 8.2-fold higher than that of empty vector

Discussion

Although RR is recognized to have importance in maintaining DNA integrity, the results of this study support the hypothesis that RRM2 has additional functions, including participation in oncologically important signaling events that influence the invasive phenotype. High RR activity is associated with tumor progression and resistance to cellular stressors such as chemotherapeutic agents and ionizing radiation [12], [19], [27], [28], [29]. Increased RR activity is reported to be present in

Acknowledgments

The authors acknowledge the secretarial assistance of Jan Rounds. This study was supported by NIH Grant RO1 CA114103 and American Cancer Society Grant RSG-04421-01-CCE.

References (39)

O. Guittet et al.
Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells
J. Biol. Chem.
(2001)
S. Eriksson et al.
Ribonucleotide reductase in cultured mouse lymphoma cells. Cell cycle-dependent variation in the activity of subunit protein M2
J. Biol. Chem.
(1981)
M. Thelander et al.
Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells
J. Biol. Chem.
(1985)
M.S. Duxbury et al.
Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: a novel therapeutic strategy in pancreatic cancer
Surgery
(2004)
H. Ito et al.
Inhibition of tyrosine kinase Src suppresses pancreatic cancer invasiveness
Surgery
(2003)
C. Yan et al.
KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-kappa B binding to the promoter as a consequence of Ikappa Balpha -induced block of p65/p50 nuclear translocation
J. Biol. Chem.
(2001)
C. He
Molecular mechanism of transcriptional activation of human gelatinase B by proximal promoter
Cancer Lett.
(1996)
J.G. Cory et al.
Regulation of ribonucleotide reductase activity in mammalian cells
Mol. Cell. Biochem.
(1983)
J.G. Cory
Purine and pyrimidine nucleotide metabolism
Y.G. Goan et al.
Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line
Cancer Res.
(1999)

P. Reichard

From deoxynucleotides to DNA synthesis

Fed. Proc.

(1978)

P. Reichard

Ribonucleotide reductase and deoxyribonucleotide pools

Basic Life Sci.

(1985)

J. Shao et al.

In vitro characterization of enzymatic properties and inhibition of the p53R2 subunit of human ribonucleotide reductase

Cancer Res.

(2004)

H. Tanaka et al.

A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage

Nature

(2000)

S. Bjorklund et al.

S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs

Biochemistry

(1990)

B.S. Zhou et al.

Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential

Clin. Exp. Metastasis

(1998)

A.B. Raitano et al.

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells

Pancreas

(1990)

A.Y. Jong et al.

A simple and sensitive ribonucleotide reductase assay

J. Biomed. Sci.

(1998)

Z. Shen et al.

Multiple transcription factor profiling by enzyme-linked immunoassay

Biotechniques

(2002)

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RRM2 induces NF-κB-dependent MMP-9 activation and enhances cellular invasiveness

Abstract

Section snippets

Materials and methods

RRM2 overexpression enhances cellular invasiveness and upregulates MMP-9 expression and activity

Discussion

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Surgery

Surgery

J. Biol. Chem.

Cancer Lett.

Regulation of ribonucleotide reductase activity in mammalian cells

Mol. Cell. Biochem.

Purine and pyrimidine nucleotide metabolism

Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line

Cancer Res.

From deoxynucleotides to DNA synthesis

Fed. Proc.

Ribonucleotide reductase and deoxyribonucleotide pools

Basic Life Sci.

In vitro characterization of enzymatic properties and inhibition of the p53R2 subunit of human ribonucleotide reductase

Cancer Res.

A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage

Nature

S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs

Biochemistry

Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential

Clin. Exp. Metastasis

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells

Pancreas

A simple and sensitive ribonucleotide reductase assay

J. Biomed. Sci.

Multiple transcription factor profiling by enzyme-linked immunoassay

Biotechniques