Functional analysis of the -2548G/A leptin gene polymorphism in breast cancer cells.
Leptin is overexpressed in human breast tumors and is produced by breast cancer cells in response to obesity-related stimuli. The leptin promoter polymorphism Lep-2548G/A can be associated with increased leptin secretion by adipocytes and elevated cancer risk. However, molecular mechanisms underlying the link between Lep-2548G/A and breast cancer have never been addressed. Lep-2548G/A is proximal to a binding site for the transcriptional factor Sp1. Furthermore nucleolin, a transcriptional repressor, can bind Sp1 or its consensus site. Consequently, we focused on the impact of Lep-2548G/A on Sp1- and nucleolin-dependent leptin transcription in breast cancer cells. The Lep-2548G/A was identified in a homozygous conformation in BT-474 and SK-BR-3 breast cancer cells, in a heterozygous conformation in MDA-MB-231 cells, and a wild-type Lep-2548G/G sequence was present in MCF-7 and ZR-75-1 cells. The occurrence of Lep-2548A/A and Lep-2548G/A coincided with high and intermediate leptin mRNA expression, respectively, while cells containing Lep-2548G/G expressed low leptin mRNA levels. We demonstrated that the existence of Lep-2548G/A improved efficient recruitment of Sp1 to DNA under insulin treatment, while Sp1 loading on DNA containing Lep-2548G/G was not insulin-dependent. In contrast, nucleolin binding to Lep-2548G/A was downregulated in response to insulin, while it was not regulated on Lep-2548G/G. The presence of Lep-2548G/A was studied in breast cancer epithelial cells by IHC and LCM. Interestingly, all 14 tumors expressing high leptin levels contained Lep-2548A/A. In conclusion, the occurrence of Lep-2548G/A can enhance leptin expression in breast cancer cells via Sp1- and nucleolin-dependent mechanisms and possibly contribute to intratumoral leptin overexpression.
Terrasi
Marianna
M
Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, USA.
Fiorio
Elena
E
Mercanti
Anna
A
Koda
Mariusz
M
Moncada
Camilo A
CA
Sulkowski
Stanislaw
S
Merali
Salim
S
Russo
Antonio
A
Surmacz
Eva
E
D015153:Blotting, Western; D015992:Body Mass Index; D001943:Breast Neoplasms / Q000150:complications / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D005838:Genotype; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D020738:Leptin / Q000235:genetics*; D009765:Obesity / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000097796:Nucleolin
D007004:Hypoglycemic Agents; D007328:Insulin; D020738:Leptin; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D016329:Sp1 Transcription Factor; D000097796:Nucleolin