Nutrition and Health Sciences, Department of


Date of this Version

Winter 12-5-2013


A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of the Requirements For the Degree of Master of Science, Major: Nutrition, Under the Supervision of Professor Janos Zempleni. Lincoln, Nebraska: December, 2013

Copyright (c) 2013 Jie Zhou


Biotin feeding in a Balb/c mouse model of breast cancer suggest that the tumor load is ~100% greater in biotin-deficient mice compared with biotin-sufficient controls, and that the increase is associated with metastasis rather than new tumors. The oncogenes c-Myc and COX-2 are implicated in cancer metastasis, and their expressions are repressed by binding of MBP-1 to the promoter. Mass spectrometry studies, designed to discover novel biotin-containing proteins, suggest that lysine residues K10 and K12 in the c-Myc promoter binding protein (MBP-1) contains covalently bound biotin (MBP-1bio). We hypothesized that biotinylation of MBP-1 increases the binding of MBP-1 to the c-Myc and COX-2 promoter, thereby decreasing oncogene expression and metastasis. A novel antibody against MBP-1bio was used to confirm the existence of MBP-1bio in human mammary carcinoma MCF-7 cells; the abundance of MBP-1bio depended on the concentration of biotin in culture media: supplemented > sufficient > deficient. The activity of a c-Myc reporter gene was ~37% greater in biotin-deficient cells compared with sufficient and supplemented controls. Likewise, both c-Myc and COX-2 mRNA levels were significantly increased in deficient compared with sufficient cells. When K10 and K12 in MBP-1 were removed by site-directed mutagenesis, biotin supply no longer affected the expression of c-Myc and COX-2 in MCF-7 cells. Biotinylation of MBP-1 is an enzymatic process catalyzed by using recombinant holocarboxylase synthetase (HLCS), judged by using recombinant HLCS and recombinant MBP-1. Consistent with this observation, c-Myc and COX-2 are de-repressed in HLCS mutant WG2215 human fibroblasts compared with controls. We conclude that loss of MBP-1bio in biotin-depleted and HLCS-deficient organisms leads to an increased expression of oncogenes, thereby increasing cancer risk.

Advisor: Janos Zempleni