Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant

Abstract

The Myc transcription factor is commonly dysregulated in many human cancers, including breast carcinomas. However, the precise role of Myc in the initiation and maintenance of malignancy is unclear. In this study we compared the ability of wild-type Myc (wt Myc) or Myc phosphorylation deficient mutants (T58A, S62A or T58A/S62A) to immortalize and transform human mammary epithelial cells (HMECs). All Myc constructs promoted cellular immortalization. As previously reported in other cells, the Myc T58A mutant tempered apoptotic responses and increased Myc protein stability in HMEC cells. More importantly, we now show that HMECs overexpressing the Myc T58A mutant acquire a unique cellular phenotype characterized by cell aggregation, detachment from the substrate and growth in liquid suspension. Coincident with these changes, the cells become anchorage-independent for growth in agarose. Previous studies have shown that wt Myc can collaborate with hTERT in inducing HMEC anchorage-independent growth. We have verified this observation and further shown that Myc T58A was a stronger facilitator of such co-transformation. Thus, our findings indicate that differences in Myc protein phosphorylation modulate its biological activity in human breast epithelial cells and specifically that the T58A mutation can facilitate both cellular immortalization and transformation. Finally, we used the isogenic cell lines generated in this study to identify a subset of genes whose expression is greatly altered during the transition from the immortal to the anchorage-independent states.

Fig. 1. Wt Myc and mutant Myc overexpression leads to immortalization of HMECs

Growth curves illustrate the immortalization of HMECs from two individual donors (a and b) and in two separate experiments with each donor (top and bottom panels). The cumulative number of days was plotted against the number of population doublings to obtain the growth curves. Two population doublings represent one tissue culture passage.

Fig. 2. Myc T58A affects apoptosis in HMECs

HMECs overexpressing either wt Myc or Myc T58A were subjected to 0 Gy or 24 Gy of ionizing radiation. After 72 hours, cells were fixed in ethanol, stained with propidium iodide and analyzed via flow cytometry for DNA content. a: Data shown represent the average percent +/− SD of cells in sub-G1 from three experiments. b: Representative DNA histograms showing the sub-G1 peaks for both cell lines.

Fig. 3. Myc T58A affects protein stability but not protein level in HMECs

a: HMECs overexpressing either wt Myc or Myc T58A were labeled with ³⁵S for one hour and then chased with ³⁵S-free media. Lysates were made at the indicated timepoints, immunoprecipitated with Myc antibody and subjected to electrophoresis. b: Densitometry was performed on the films from (a) and the data was used in linear regression analyses. Following linear regression, the Myc half-life for each experiment was calculated. Data in the graph represent the average half-life +/− SD for each Myc construct from three experiments. c: Western blot analysis was performed to detect Myc and phospho-Myc protein from whole cells lysates in immortalized HMECs, with β-actin as a loading control. LXSN is the empty vector control and HeLa is the positive control. hT = hTERT.

Fig. 4. Myc T58 overexpression results in a unique morphological change in HMEC lines

Representative phase-contrast micrographs at 32x magnification of HMEC lines were taken at timepoints corresponding to immortalization (top panel) and to the soft agar assays seen in Figure 5 (bottom panel). The inset in the Myc T58A bottom panel illustrates cells growing in suspension in the liquid medium. White size bars at the bottom of the micrographs represents 20 μm.

Fig. 5. HMECs overexpressing wt Myc and Myc T58A form colonies in soft agar

a: Immortalized HMECs and HeLa cells were grown in soft agar in 6-well plates for four weeks. Experiments were done in triplicate. Representative 1x photographs (left column) and 10x phase-contrast micrographs (right column) are shown. White size bars at bottom of micrographs for HeLa, wt Myc and Myc T58A colonies represent 0.2 mm. b: Soft agar assays were repeated in 60 mm tissue culture dishes with wt Myc, Myc T58A and hTERT HMECs at a later passage number than in (a). Colonies were stained with crystal violet after 15 days and counted.

Fig. 6. Wt Myc and Myc T58A overexpression facilitate anchorage-independent growth in HMECs immortalized with hTERT

a: Immortalized HMECs were grown in soft agar in 60 mm tissue culture dishes. Experiments were done in triplicate. Colonies were stained with crystal violet after 23 days and counted. b: Stained colonies were counted, and the average number of colonies was calculated and graphed. Error bars represent +/− SD of the average.