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, 11 (6), R87

Human Mammary Cancer Progression Model Recapitulates Methylation Events Associated With Breast Premalignancy

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Human Mammary Cancer Progression Model Recapitulates Methylation Events Associated With Breast Premalignancy

Nancy Dumont et al. Breast Cancer Res.

Abstract

Introduction: We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.

Methods: HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).

Results: vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.

Conclusions: We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Figures

Figure 1
Figure 1
vHMEC expressing Ha-ras are resistant to proliferative arrest and display increased numbers of chromosomal abnormalities. (a) Immunoblot analysis demonstrating Ha-rasV12 expression in HMEC and vHMEC following retroviral transduction with pLXSP3-Ha-rasV12 (r) or the control pLXSP3 vector (v). Constructs were expressed in HMEC and vHMEC derived from five different individuals. A representative blot is shown along with actin as a loading control. (b) Growth curves demonstrating that HMEC underwent a proliferative arrest in response to oncogenic ras (orange line, left graph), while vHMEC continued to proliferate (orange line, right graph). (c) Cell cycle analysis of HMEC and vHMEC expressing Ha-rasV12 or control vector demonstrating that the number of cells in S-phase dropped from 33.8% to 8.8% following Ha-rasV12 expression in HMEC, but remained the same in vHMEC (37.9% and 37.6%, respectively). (d) Chromosomal analysis of vHMEC-vector and vHMEC-ras cells. Control vHMEC (vector) and vHMEC expressing oncogenic Ha-RasV12 (ras) were harvested at different passages (P+1, P+5, and P+8), as indicated, and processed for metaphase analysis. Standard G-banding karyotypic analysis was performed on at least fifty metaphase spreads for each cell population. Aneuploidy refers to additions or deletions of whole chromosomes. Structural abnormalities include all deletions, duplications, rings, marker chromosomes, chromatid exchanges and translocations. The total number of abnormalities includes all structural abnormalities and telomeric associations, not including numerical abnormalities.
Figure 2
Figure 2
Signals from the extracellular environment cooperate with ras to immortalize vHMEC and upregulate telomerase activity. (a) Growth curves of HMEC (red line), vHMEC expressing control vector (blue line), and vHMEC expressing Ha-rasV12 (orange line). Arrow indicates time at which vHMEC-ras cells were exposed to serum (day 220). These cells demonstrated increased population doublings within two passages (day 243) in serum-containing media, and are referred to as vHMEC-ras0.5 cells. Their growth curve is depicted in green. After 560 days, the vHMEC-ras0.5 cells were cultured in the absence of serum and continued to proliferate. These cells are referred to as vHMEC-ras0.5- >0 and their growth curve is depicted in black. (b) Telomerase activity assay depicting the amount of telomerase activity in lysates prepared from parental vHMEC (par), vHMEC-vector (vec), vHMEC-ras (ras), and vHMEC-ras0.5 (ras0.5) cells, as well as two clones isolated from the ras0.5 cell population (cl-1 and cl-2).
Figure 3
Figure 3
Immortalized variant human mammary epithelialcells expressing oncogenic ras are capable of anchorage-independent growth but are not tumorigenic in vivo. (a) Soft agar colony assay. Parental vHMEC (par), vHMEC-vector (vec), vHMEC-ras (ras), vHMEC-ras0.5 (ras0.5), clone 1 (cl-1), and clone 2 (cl-2), which were isolated from the vHMEC-ras0.5 cells, were plated in 35 mm dishes at a concentration of 50,000 cells per dish, in triplicate. After 14 days, colonies were counted manually in eight different fields. The data are presented as the average of the sum of eight different fields counted. (b) Bioluminescence imaging of SCID-Beige mice following orthotopic injection of 1 × 106 (top panel), 4 × 106 (middle panel), or 10 × 106 (bottom panel) vHMEC-ras clone 1 cells expressing GFP-luciferase into the left and right #4 mammary fat pad. Cell growth and survival was monitored weekly by bioluminescence imaging utilizing the Xenogen IVIS imaging system. Representative images of each experiment are shown.
Figure 4
Figure 4
Progression to malignancy is associated with DNA hypermethylation at several gene loci. MSP analysis of CCND2, RASSF1A, SFRP1, p57, MGMT, and THBS1 in the cells indicated using primer sets listed in Table 1 that specifically amplify either methylated (m) or unmethylated (u) DNA. Positive (+) and (-) controls for the methylated product, as well as a H2O negative control, were included in all experiments. All MSP experiments were performed on at least two independent cell populations. The data presented for the SFRP1-exon1 MSP are from two different experiments. Analysis of the primary cells (HMEC, vHMEC, vector, ras, and ras0.5) and the cell lines (184A1, MCF10A, MCF7, and MDA231) was done separately and merged. The data presented for all the other genes were obtained in the same experiment. Replicate experiments are available online in Additional file 2.
Figure 5
Figure 5
DNA methylation correlates with reduced gene expression. Quantitative RT-PCR for (a) RASSF1A; (b) SFRP1; and (c) MGMT. Methylation status as evaluated by MSP is indicated as follows: Unmethylated (U), partially methylated (U/M), or methylated (M). For SFRP1, the U/M labels refer to the methylation status of the CpG island that extends into exon 1.
Figure 6
Figure 6
Concomitant methylation of RASSF1A and SFRP1 in malignant and premalignant breast tissues. (a) MSP analysis of RASSF1A, SFRP1-exon1, p57, and MGMT in normal and malignant (invasive ductal carcinoma) breast tissues using primer sets that specifically amplify either methylated (m) or unmethylated (u) DNA, as in Figure 4 B and C. MSP analysis of RASSF1A and SFRP1 in normal, hyperplasia without atypia (H1 and H2), atypical ductal hyperplasia (ADH, H3), invasive ductal carcinoma (b), and ductal carcinomas in situ (c) tissues. Additional diagnostic information about these samples is available in Table 3.

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