Immortalization and transformation of human mammary epithelial cells by c-Myc and c-Myc phosphorylation deficient mutants.

摘要

The c-Myc transcription factor is commonly dysregulated in breast cancer. c-Myc has been shown to regulate a number of genes, including human telomerase reverse transcriptase (hTERT), the catalytic subunit of the enzyme telomerase. Telomerase activity is characteristic of most immortalized and transformed cells, and immortalization is considered an early event in cancer development. The c-Myc N-terminal domain contains several phosphorylation sites, including threonine-58 (T58) and serine-62 (S62). Previous studies have utilized rodent fibroblast systems to show an impact of altering phosphorylation at these two sites on protein stability, apoptosis and transformation. In this study we compared the ability of wild-type c-Myc or c-Myc phosphorylation deficient mutants---c-Myc T58A, S62A and T58A/S62A---to immortalize and transform human mammary epithelial cells (HMECs). The c-Myc phosphorylation deficient mutants appear to be more efficient at promoting cellular immortalization than wild-type c-Myc. All c-Myc constructs were functional in HMECs, as evidenced by their ability to induce telomerase activity. The immortalized HMEC lines exhibited near-diploid karyotypes with specific chromosomal abnormalities, suggesting clonality. Most importantly, the immortalized lines overexpressing c-Myc T58A (and to lesser extent wild-type c-Myc) acquired anchorage-independent growth after further cell passaging. The ability of c-Myc T58A HMECs to form colonies in soft agar was accompanied by a unique clustering morphology that included detachment from the tissue culture substrate and free-floating growth in the media. When HMECs previously immortalized with hTERT were transduced with c-Myc T58A, limited growth in soft agar was seen, indicating that c-Myc T58A expression played a partial role in anchorage-independent growth. Furthermore, the c-Myc T58A protein displayed an increased half-life in HMECs compared to the wild-type protein, and cells overexpressing c-Myc T58A exhibited reduced apoptosis in response to ionizing radiation. Finally, we identified 149 genes that were differentially expressed between c-Myc T58A HMECs before and after acquiring the ability for anchorage-independent growth. Our findings suggest that differences in c-Myc protein phosphorylation impact c-Myc biological activity in human breast epithelial cells. In addition, these cell lines provide a unique tool for identifying the molecular and genetic changes during transition from the immortal to anchorage-independent states.