Epithelial cancers, such as breast cancer, are being more frequently identified at the early pre-invasive stage of tumor development [1
]. These pre-invasive mammary lesions originate from the luminal epithelial cells that line the ducts and lobules of the mammary glandular epithelium and have a disrupted epithelial architecture characterized by hyperproliferative cells occupying the normally hollow luminal spaces of the ducts and lobules [2
]. The amplification and overexpression of the receptor tyrosine kinase ErbB2 is observed in approximately 50% of pre-invasive lesions; however, in most cases, the genetic and epigenetic abnormalities that promote pre-invasive tumor growth are poorly understood [4
Since such a wide range of molecular perturbations can induce and enhance tumor growth, there are probably shared molecular signaling modules that integrate biochemical signals from the suite of genetic contexts found in epithelial tumors [5
]. To explain how normal cells become tumorigenic, a molecular framework that underpins the pre-invasive stage of tumor growth must be established. Such a molecular framework can assist in the identification of patients amenable to targeted therapeutics, in the development of novel therapeutics to treat pre-invasive cancer, and, in the future, in the introduction of preventative treatment [6
]. Attempts to identify the core signaling modules that promote these pre-invasive growth characteristics through the analysis of genetic abnormalities and gene expression patterns of pre-invasive tumor lesions have to date been unsuccessful [7
The Raf–MEK1/2–ERK1/2 mitogen-activated protein kinase signal transduction module transmits extracellular and oncogenic stimuli, resulting in cellular responses [10
]. In this module, Raf isoforms phosphorylate their primary substrates, the dual-specificity kinases MEK1/2. Once activated, MEK1/2 phosphorylate ERK1/2 on tyrosine and threonine residues, substantially increasing ERK1/2 catalytic activity [11
]. The Raf–MEK1/2–ERK1/2 module is activated by growth factors and proteins overexpressed in human breast cancer epithelium, by cytokines and hormones produced by fibroblasts and macrophages in the mammary stromal compartment, and by increased tissue stiffness observed during tumor progression [10
]. In addition, the sequencing of breast cancer patient genomes suggests that infrequent mutations may drive tumor progression through known signaling pathways, such as the Raf–MEK1/2–ERK1/2 cascade [5
]. Considering the array of stimuli known to activate the Raf–MEK1/2–ERK1/2 module, it may be complicit in tumorigenesis in a variety of contexts.
Consistent with a role for the Raf–MEK1/2–ERK1/2 module in mammary carcinogenesis, ERK1/2 are activated in primary breast cancer tissue and in associated lymph node metastases [13
]. The activation of ERK1/2 is not associated with a specific genetic signature, however, as ERK1/2 is active in ER-positive breast cancer, HER2-positive breast cancer and in triple-negative breast cancer [15
]. ERK1/2 phosphorylate transcription factors, kinases, proteases and non-enzymatic regulatory proteins, thus potentially integrating the Raf–MEK1/2–ERK1/2 module into a range of cellular activities associated with tumorigenesis [16
]. Accumulating evidence, however, has shown that results obtained in one cell type should not be generally applied across all classes of cancer without experimental validation [6
]. For example, the K-Ras2 oncogene has distinct effects on tumor progression depending on both the cell type of origin and the genetic context in which it is mutated [6
]. In addition, extrapolating the role of protein kinases in promoting breast cancer progression based on either their known substrate profile or biological behaviors induced in two-dimensional culture models has proven to be unreliable [17
]. For example, the chemically induced homodimerization of the epidermal growth factor receptor (EGFR) is sufficient to induce focus formation in Rat 1 cells and the proliferation of MCF-10A mammary epithelial cells in monolayer cultures [17
]. EGFR homodimerization of EGFR, however, is not sufficient to induce the proliferation of differentiated MCF-10A cells grown in organotypic culture [17
]. Considering the uncertainty in predicting the response of cells to the activation of a signaling pathway, determining the response of differentiated mammary epithelial cells to Raf–MEK–ERK activation can better define the early events of mammary tumorigenesis.
Three-dimensional organotypic culture models have been indispensable tools in deciphering the molecular and cell biological mechanisms underlying the disruption of differentiated epithelial architecture that is characteristic of pre-invasive mammary epithelial lesions. In organotypic culture models, individual mammary epithelial cells plated on reconstituted basement membrane proliferate to form a hollow sphere of polarized, growth-arrested cells (termed acini), thus recapitulating the salient features of the mammary gland [20
]. Since the mammary epithelial cells differentiate and form a hollow monolayer of cells, organotypic cultures provide a more accurate reconstitution of the biochemical and cell biological growth restraints found in mammary glandular epithelium than is achieved using traditional two-dimensional cell culture models [22
]. Once cells become proliferative, they are confronted with similar local environmental selection pressures to those found during tumorigenesis. Namely, cells are required to become resistant to cell death triggered by the induction of either apoptosis or autophagy when cells enter the luminal space [23
]. Organotypic culture models therefore provide both the biochemical signaling barriers that must be overcome for initial proliferation to occur, and the microenvironmental context in which pre-invasive tumor cells must survive and propagate.
We have previously developed a method for imaging cells in Raf:ER-induced acini at single-cell resolution through imaging a histone–green fluorescence protein (GFP) correct fusion protein, H2B-GFP [25
]. Using this unbiased discovery approach we have found that Raf:ER activation induces a disruption of epithelial architecture through promoting a non-invasive form of motility, cell proliferation and the survival of cells in the lumen. These findings suggest that ERK1/2 activation can promote the early events of tumorigenesis and that the induction of motility can, in principle, occur before tumor cell invasion. To determine how ERK1/2 signaling promotes the early events of tumorigenesis we have examined the intracellular signaling pathways that promote proliferation, cell survival and motility in response to ERK1/2 activation in mammary epithelial acini.