Mammary epithelial cell transformation: insights from cell culture and mouse models

Abstract

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely -- a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16INK4a, p21, p27 and p57), p14ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.

Figure 1

Structure of the mammary gland. Terminal ductal–lobular unit (TDLU), composed of ductal cells, is the unit thought to be the origin of most breast cancer. The stroma is composed of fatty tissue (adipocytes) and fibroblasts. Also shown are the two primary types of cells in normal ducts: outer contractile myoepithelial and inner columnar luminal cells. A putative progenitor/stem cell is also indicated.

Figure 2

Establishment of mammary epithelial cells from reduction mammoplasty/mastectomy specimens. The tissue is chopped, digested with collagenase and hyaluronidase, and plated in medium as organoids. Over a week or so, multiple types of epithelial cells and fibroblasts emerge; fibroblasts are removed by differential trypsinization (fibroblasts are loosely attached), remaining epithelial cells grow for 10–15 population doublings (PDs) followed by senescence of the majority of cells. Occasionally, an homogenous population of cells emerges that continue to proliferate for an additional 30–60 PDs, and eventually these cells also senesce (this step is referred to as agonescence).

Figure 3

Morphological heterogeneity of cells before and after selection. (a–d) Two views of mammary epithelial preselection cells (original magnifications: panels a and c, 40×; panels b and d, 100×). Cells shown in panel a grow as compact clusters and are relatively uniform, whereas cells in panel b grow more dispersed and exhibit different types of cells (small and large). (e,f) Views of postselection human mammary epithelial cells with relatively uniform morphology (original magnifications: panel e, 40×; panel f, 100×).

Figure 4

Current consensus: normal HMECs can be fully transformed in definable serial steps. The first step, bypass of senescence, is achieved by inactivation of p53 and pRb by SV40 large T, human papillomavirus (HPV) E6 and E7, or by inhibition of p53 and pRb expression by the RNAi approach (or expression of dominant-negative mutants in the case of p53). The second step, immortalization, is achieved through the expression of hTERT. Alternatively, expression of HPV E6 or overexpression of Bmi-1, mutant p53, or ZNF217 can be used to induce immortalization of HMECs. The third step, anchorage-independent growth, can be achieved by SV40 small t mediated modulation of PI3K and/or other signaling pathways or by overexpression of activated Rac1 and AKT. The fourth step, full transformation, requires the introduction of activated H-ras, which can be substituted by Raf and Ral-GEFs. Although the current model systems have utilized the serial schemes depicted, other combinations and/or schemes of oncogene introduction are likely also to be effective. Adapted from Elenbaas [9], Zhao [10], and Rangarajan [11] and coworkers. HMEC, human mammary epithelial cell; HPV, human papillomavirus; hTERT, catalytic subunit of human telomerase; PI3K, phosphatidylinositol 3-kinase; Ral-GEF, Ral guanine nucleotide exchange factor; RNAi, RNA interference.