Engineered extrachromosomal oncogene amplifications promote tumorigenesis.

Focal gene amplifications are among the most common cancer-associated mutations but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs) in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures.

Immortalization and transformation of primary cells mediated by engineered ecDNAs.

Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general approach to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs, also known as "double minutes") in a spatiotemporally controlled manner in cancer cell lines and in primary cells derived from genetically engineered mice. With this strategy, ecDNA formation can be coupled with expression of fluorescent reporters or other selectable markers to enable the identification and tracking of ecDNA-containing cells.

ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus.

Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer.

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA.

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ER/Notch feed-forward loop responsible for the generation of CD133 CSCs.