Only a subset of breast cancer patients will require chemotherapy for long-term management of the disease. Unfortunately, the parameters that determine the malignancy of tumor cells are not well understood. Overexpression of the epidermal growth factor receptor (EGFR) occurs in 20-30% of breast tumors and is an indicator of poor prognosis, but the mechanism of overexpression is unclear, as the EGFR gene is rarely amplified in breast cancer. The acquisition of growth factor independence of human mammary epithelial (HME) cells in vitro correlates with increasing tumor aggressiveness in vivo.
To explore the relationship between EGF independence and overexpression of the EGFR, the Ethier lab at the University of Michigan developed an expression cloning strategy aimed at the functional identification of genes that mediate growth factor autonomy in human breast cancer cells (Figure 1). Using this approach, we have demonstrated that the EGFR ligand amphiregulin acts in an autocrine fashion through the EGFR in the donor breast cancer cell line SUM-149PT and confers EGF independence to HME cells. We isolated a second cDNA candidate in a similar screening experiment, which corresponded to NSEP1/YB-1 (Nuclease-sensitive element-binding protein 1/Y-box binding protein 1), a multifunctional regulator of gene transcription and translation. We found that NSEP1 expression in human mammary epithelial cells is sufficient to confer EGF autonomy in vitro, in an EGFR-dependent fashion.
Interestingly, NSEP1 was recently shown to be elevated in breast cancer and to have prognostic value. The current focus of my laboratory is to delineate the functional relationship between NSEP1 and EGFR in HME and breast cancer cells, and to determine if NSEP1 contributes to tumor development. In addition, the prognostic significance of NSEP1 co-expression with EGFR and other markers is being investigated.
Figure Legend: Expression cloning strategy. A cDNA expression library is constructed with mRNA from breast cancer cells that overexpress EGFR and are EGF-independent for growth in serum-free medium. As a vector, we use the pBMN plasmid, which yields high retroviral titers when transfected into FNX-Ampho packaging cells. The resulting retroviral library is harvested from the culture medium and used to infect EGF-dependent HME cells. Infected cells are selected for several weeks in serum-free, EGF-free medium. Under these conditions, control HME cells fail to proliferate. EGF-independent colonies are isolated, from which library inserts are rescued either by PCR or by biological rescue with MMLV. The role of the rescued cDNAs in EGF independence is verified by transducing them into HME cells and determining if they form EGF independent clones at increased frequency.
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