Somatic mutation and functional polymorphism of a novel regulatory element in the HGF gene promoter causes its aberrant expression in human breast cancer
Jihong Ma, … , Robert Ferrell, Reza Zarnegar
Jihong Ma, … , Robert Ferrell, Reza Zarnegar
Published February 2, 2009
Citation Information: J Clin Invest. 2009;119(3):478-491. https://doi.org/10.1172/JCI36640.
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Research Article Oncology

Somatic mutation and functional polymorphism of a novel regulatory element in the HGF gene promoter causes its aberrant expression in human breast cancer

Abstract

The HGF gene is transcriptionally silenced in normal differentiated breast epithelial cells, but its repression fails to occur in mammary carcinoma tissues and cell lines. The molecular mechanisms underpinning aberrant HGF expression in breast cancer cells are unknown. Here we report the discovery of a DNA element located 750 bp upstream from the transcription start site in the human HGF promoter that acts as a transcriptional repressor and is a target of deletion mutagenesis in human breast cancer cells and tissues. This HGF promoter element consists of a mononucleotide repeat of 30 deoxyadenosines (30As), which we have termed “deoxyadenosine tract element” (DATE). Functional studies revealed that truncation mutations within DATE have profound local and global effects on the HGF promoter region by modulating chromatin structure and DNA-protein interactions, leading to constitutive activation of the HGF promoter in human breast carcinoma cell lines. We found that 51% of African Americans and 15% of individuals of mixed European descent with breast cancer harbor a truncated DATE variant (25As or fewer) in their breast tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression. Notably, breast cancer patients with the truncated DATE variant are substantially younger than those with a wild-type genotype. We also suggest that DATE may be used as a potential genetic marker to identify individuals with a higher risk of developing breast cancer.

Authors

Jihong Ma, Marie C. DeFrances, Chunbin Zou, Carla Johnson, Robert Ferrell, Reza Zarnegar

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Figure 1

Truncation of DATE in the HGF gene promoter region correlates with aberrant HGF expression in human carcinoma cell lines.

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Truncation of DATE in the HGF gene promoter region correlates with aberr...
(A) Detection of HGF gene expression by RT-PCR. HGF gene expression was noted in HTB25, HTB128, and C33A cells but not in other cells. (B) Detection of HGF, Met, and phosphorylated Met protein expression by western blot assay. HGF and phospho-Met protein expression were observed in HTB25, HTB128, and C33A cells. Met expression was observed in all cells. (C) Detection of HGF and Met protein expression by immunofluorescence microscopy in C33A and HeLa cells. Scale bar: 40 μm. HGF protein expression was notable only in C33A cells but not HeLa cells, whereas Met protein expression occurred in both cells. Nuclei were stained blue with VECTASHIELD Mounting medium with DAPI. (D) Schematic representation of the human HGF promoter and the location and DNA sequence of wild-type DATE (30As). (E) Representative PCR genotype analysis of DATE from 11 human breast cancer cell lines and 12 human cervical carcinoma cell lines. The 70-bp PCR product was fractionated on 15% denaturing polyacrylamide gel containing 8 M urea. Markers used were cloned and sequenced cell-derived 70-bp DNA fragments containing 28As, 26As, 21As, 17As, or 14As in DATE. Arrows indicate truncated DATE band. (F) Representative DNA sequencing of the major band corresponding to the truncated DATE (arrows in E) confirmed that the shorter alleles of truncated DATE in HTB25, HTB128, and C33A cell lines were 22As, 25As, and 14As, respectively, while HeLa cells showed wild-type DATE with 30As.