HER-2/neu Represses the Metastasis Suppressor RECK via ERK and Sp Transcription Factors to Promote Cell Invasion

Abstract

Matrix metalloproteinase (MMP) inhibitory proteins may negatively regulate MMP activity to suppress tumor metastasis. In this study, we demonstrate that the HER-2/neu oncogene inhibits the expression of the MMP inhibitor RECK to promote cell invasion. RECK was inhibited via transcriptional repression in B104-1-1 cells, which express constitutively active HER-2/neu. Overexpression of HER-2/neu in NIH/3T3 or HaCaT cells also suppressed RECK expression. Deletion and mutation assays showed that HER-2/neu repressed RECK via the Sp1-binding site localized in the ؊82/؊71 region from the translational start site. DNA affinity precipitation and chromatin immunoprecipitation assays indicated that binding of Sp1 and Sp3 to this consensus site was increased in B104-1-1 cells. We also found that HER-2/neu inhibited RECK via the ERK signaling pathway. Sp1 proteins phosphorylated at Thr 453 and Thr 739 by ERK bound preferentially to the RECK promoter, and this binding was reversed by HER-2/neu and ERK inhibitors. Furthermore, our data indicate that HER-2/neu obviously increased HDAC1 binding to the Sp1-binding site localized in the ؊82/؊71 region of the RECK promoter. The histone deacetylase inhibitor trichostatin A reversed HER-2/neu-induced inhibition of RECK. HER-2/neu activation was associated with increased MMP-9 secretion and activation. Re-expression of RECK in HER-2/neu-overexpressing cells inhibited MMP-9 secretion and cell invasion. Taken together, our results suggest that HER-2/neu induces the binding of Sp proteins and HDAC1 to the RECK promoter to inhibit RECK expression and to promote cell invasion. Restoration of RECK provides a novel strategy for the inhibition of HER-2/neu-induced metastasis.The HER-2/neu oncogene (also known as erbB2) encodes a transmembrane glycoprotein that belongs to the human epidermal growth factor receptor family (1, 2). Structural analysis of HER-2/neu and the epidermal growth factor receptor revealed significant sequence homology and identical gross structural organization between these two proteins (3-5). Amplification and overexpression of HER-2/neu have been found in breast, ovarian, lung, gastric, and cervical carcinomas (6 -10). Up-regulation of this oncogene is frequently linked with increased metastasis and poor prognosis. The mechanism underlying HER-2/neuinduced metastasis is a field of intensive study, and several candidates involved in this process have been identified recently. The first candidate is the vascular endothelial growth factor. HER-2/neu may stimulate vascular endothelial growth factor expression, and neutralizing antibody against HER-2/neu suppresses vascular endothelial growth factor production and metastasis of cancer cells (11,12). The second is the urokinase-type plasminogen activator. Induction of the urokinase-type plasminogen activator by HER-2/neu has been shown to be correlated with increased metastasis (13). The third is cyclooxygenase-2. Cyclooxygenase-2 plays a critical role in tumor metastasis and may be a potential target for chemoprevention (14)...

The tumor suppressor RECK interferes with HER-2/Neu dimerization and attenuates its oncogenic signaling

“…Among the 27 cases with high HER-2/Neu, RECK expression is reduced in 59.3% (16/27). This finding is in consistent with our previous results that HER-2/Neu over-expression inhibits RECK gene transcription [5]. Although our cell-based study demonstrates the growth-inhibitory function of RECK, no correlation between RECK and tumor size was found.…”

“…We have addressed the transcriptional regulation of RECK gene in various types of human cancer and find that RECK is a molecular target for HER-2/Neu oncogene and Epstein-Barr virus oncoprotein latent membrane protein 1 [5,6]. Our results suggest the involvement of epigenetic mechanism in the control of RECK expression by oncogenes [7,8].…”

“…Cellular proteins were prepared, separated by 10% SDS-PAGE or native PAGE and subjected to Western blotting as described previously [5].…”

“…Total RNA was isolated by Total RNA Mini Kit (Geneaid Biotech Ltd, Taipei, Taiwan) and 1 lg RNA was subjected RT-PCR as described previously [5]. The primers used for analysis are RECK-forward: 5 0 -TCTGCAGGGGAAGTTGGTTG-3 0 ; RECK-reverse: 5 0 -CAGTTACAGGGCAGACCTGT-3 0 , HER-2/Neu-forward: 5 0 -CCATAA CACCCACCTCTGCT-3 0 ; HER-2/Neu-reverse: 5 0 -ACTGGCTGCAGTTTT GACACAC-3 0 ; VEGF-forward: 5 0 -AAGGAGGAGGGCAGAATCAT-3 0 ; VEGF-reverse: 5 0 -ATCTGCATGGTGATGTTGGA-3 0 ; cyclin D1-forward: 5 0 -TCTAAGATGAAGGAGACCATC-3 0 ; cyclin D1-reverse 5 0 -GCGGTAGTAGGACAGGAAGTTGTT-3 0 ; GAPDH-forward: 5 0 -AAG-GCTGGGGCTCATTTGC-3 0 and GAPDH-reverse: 5 0 -GCTGATGATCTT-GAGGCTGTTG-3 0 .…”

Jab1 is overexpressed in human breast cancer and is a downstream target for HER-2/neu

“…24 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. cDNA synthesis was performed at 501C for 30 min and the condition for PCR was 25 cycles of denaturation (941C/1 min), annealing (601C/1 min), extension (721C/1 min) and 1 cycle of final extension (721C/10 min).…”