Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy.

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.

Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers.

The active acquisition of epigenetic changes is a poorly understood but important process in development, differentiation, and disease. Our work has shown that repression of the p16/pRb pathway in human epithelial cells, a condition common to stem cells and many tumor cells, induces dynamic epigenetic remodeling resulting in the targeted methylation of a selected group of CpG islands. We hypothesized that cells in this epigenetically plastic state could be programmed by the microenvironment to acquire epigenetic changes associated with tumorigenesis. Here, we describe an in vitro model system where epigenetically plastic cells were placed in an environment that induced epithelial to mesenchymal transition (EMT) and led to a program of acquired de novo DNA methylation at targeted sites. In this model, we found that repression of E-cadherin transcription preceded the subsequent acquisition of methylated CpG sites. Furthermore, the induction of EMT was accompanied by de novo methylation of several other gene promoters, including those of the estrogen receptor and Twist. These data demonstrate that signals from the microenvironment can induce phenotypic and gene expression changes associated with targeted de novo epigenetic alterations important in tumor progression, and that these alterations occur through a deterministic, rather than stochastic, mechanism. Given the dynamic epigenetic reprogramming that occurs in these cells, DNA methylation profiles observed in human tumors may reflect the history of environmental exposures during the genesis of a tumor.

Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity.

Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.

Tumor suppressor p16INK4A regulates polycomb-mediated DNA hypermethylation in human mammary epithelial cells.

Alterations in DNA methylation are important in cancer, but the acquisition of these alterations is poorly understood. Using an unbiased global screen for CpG island methylation events, we have identified a non-random pattern of DNA hypermethylation acquired in p16-repressed cells. Interestingly, this pattern included loci located upstream of a number of homeobox genes. Upon removal of p16(INK4A) activity in primary human mammary epithelial cells, polycomb repressors, EZH2 and SUZ12, are up-regulated and recruited to HOXA9, a locus expressed during normal breast development and epigenetically silenced in breast cancer. We demonstrate that at this targeted locus, the up-regulation of polycomb repressors is accompanied by the recruitment of DNA methyltransferases and the hypermethylation of DNA, an endpoint, which we show to be dependent on SUZ12 expression. These results demonstrate a causal role of p16(INK4A) disruption in modulating DNA hypermethylation, and identify a dynamic and active process whereby epigenetic modulation of gene expression is activated as an early event in breast tumor progression.

Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis.

Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Recently, we found that kinase-defective Hck blocks Bcr-Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by Bcr-Abl. To investigate the contribution of Src kinases to Bcr-Abl signaling in vivo, we used the pyrrolo-pyrimidine Src kinase inhibitors PP2 and A-419259. Treatment of the Ph+ CML cell lines K-562 and Meg-01 with either compound resulted in growth arrest and induction of apoptosis, while the Ph- leukemia cell lines TF-1 and HEL were unaffected over the same concentration ranges. Suppression of Ph+ cell growth by PP2 and A-419259 correlated with a decrease in Src kinase autophosphorylation. Both inhibitors blocked Stat5 and Erk activation, consistent with the suppressive effects of the compounds on survival and proliferation. In contrast, the phosphotyrosine content of Bcr-Abl and its endogenous substrate CrkL was unchanged at inhibitor concentrations that induced apoptosis, blocked oncogenic signaling and inhibited Src kinases. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr-Abl, and identify myeloid-specific Src kinases as potential drug targets in CML.