Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells

Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
Genes & Development (Impact Factor: 10.8). 02/2001; 15(1):50-65.
Source: PubMed


A number of genetic mutations have been identified in human breast cancers, yet the specific combinations of mutations required in concert to form breast carcinoma cells remain unknown. One approach to identifying the genetic and biochemical alterations required for this process involves the transformation of primary human mammary epithelial cells (HMECs) to carcinoma cells through the introduction of specific genes. Here we show that introduction of three genes encoding the SV40 large-T antigen, the telomerase catalytic subunit, and an H-Ras oncoprotein into primary HMECs results in cells that form tumors when transplanted subcutaneously or into the mammary glands of immunocompromised mice. The tumorigenicity of these transformed cells was dependent on the level of ras oncogene expression. Interestingly, transformation of HMECs but not two other human cell types was associated with amplifications of the c-myc oncogene, which occurred during the in vitro growth of the cells. Tumors derived from the transformed HMECs were poorly differentiated carcinomas that infiltrated through adjacent tissue. When these cells were injected subcutaneously, tumors formed in only half of the injections and with an average latency of 7.5 weeks. Mixing the epithelial tumor cells with Matrigel or primary human mammary fibroblasts substantially increased the efficiency of tumor formation and decreased the latency of tumor formation, demonstrating a significant influence of the stromal microenvironment on tumorigenicity. Thus, these observations establish an experimental system for elucidating both the genetic and cell biological requirements for the development of breast cancer.

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Article: Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells

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    • "RESULTS EMT and Stem-Cell-like Traits Induced by Transient Twist1 Activation We utilized immortalized HMLEs (Elenbaas et al., 2001) retrovirally transduced with Twist1 fused to a mutated estrogen receptor (ER) ligand binding domain (Casas et al., 2011). To prevent selection of pre-existing mesenchymal cells (Scheel et al., 2011), we sorted bulk HMLE cells into purified epithelial subpopulations with high or low levels of the surface marker CD24 (24 hi and 24 lo ; Figures 1A–1C). "
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    ABSTRACT: Master regulators of the epithelial-mesenchymal transition such as Twist1 and Snail1 have been implicated in invasiveness and the generation of cancer stem cells, but their persistent activity inhibits stem-cell-like properties and the outgrowth of disseminated cancer cells into macroscopic metastases. Here, we show that Twist1 activation primes a subset of mammary epithelial cells for stem-cell-like properties, which only emerge and stably persist following Twist1 deactivation. Consequently, when cells undergo a mesenchymal-epithelial transition (MET), they do not return to their original epithelial cell state, evidenced by acquisition of invasive growth behavior and a distinct gene expression profile. These data provide an explanation for how transient Twist1 activation may promote all steps of the metastatic cascade; i.e., invasion, dissemination, and metastatic outgrowth at distant sites.
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    • "journal homepage: oncogenic allele of the HRAS gene (expressing HRAS V12G ) [2] were cultured in HuMEC ready medium (12752010, life technologies). Dharmafect 2 reagent (Dharmacon) was used to transfect NIH3T3 cells with siRNA at 40 nM according to the manufacturer's protocol. "
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    ABSTRACT: The upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. We recently reported in Sanij et al.(2014) [1] that the two isoforms of UBF (UBF1/2) are enriched at Pol II-transcribed genes throughout the mouse and human genomes. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) of UBF1/2, Pol I, Pol II, H3K9me3, H3K4me4, H3K9ac and H4 hyperacetylation, we reported a correlation of UBF1/2 binding with enrichments in Pol II and markers of active chromatin. In addition, we examined a functional role for UBF1/2 in mediating Pol II transcription by performing expression array analysis in control and UBF1/2 depleted NIH3T3 cells. Our data demonstrate that UBF1/2 bind highly active Pol II-transcribed genes and mediate their expression without recruiting Pol I. Furthermore, we reported ChIP-sequencing analysis of UBF1/2 in immortalized human epithelial cells and their isogenically matched transformed counterparts. Here we report the experimental design and the description of the ChIP-sequencing and microarray expression datasets uploaded to NCBI Sequence Research Archive (SRA) and Gene Expression Omnibus (GEO).
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    • "Previous studies involving in vitro transformation of HMECs have led to the establishment of model systems to study different types of breast cancers. The HMLERs derived from adherent HMECs generated squamous cell carcinomas (Elenbaas et al., 2001), which represent o5% of naturally occurring breast cancers. The BPLERs derived from HMECs cultivated in specialized media called WIT generated malignant adenocarcinomas (Ince et al., 2007); however, both these models showed an absolute requirement for oncogenic Ras for transformation , while Ras mutations are found in o5% of naturally arising breast cancers. "

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