Single-cell analysis reveals the Comma-1D cell line as a unique model for mammary gland development and breast cancer.

The mammary gland epithelial tree contains two distinct cell populations, luminal and basal. The investigation of how this heterogeneity is developed and how it influences tumorigenesis has been hampered by the need to perform studies on these populations using animal models. Comma-1D is an immortalized mouse mammary epithelial cell line that has unique morphogenetic properties. By performing single-cell RNA-seq studies, we found that Comma-1D cultures consist of two main populations with luminal and basal features, and a smaller population with mixed lineage and bipotent characteristics. We demonstrated that multiple transcription factors associated with the differentiation of the mammary epithelium in vivo also modulate this process in Comma-1D cultures. Additionally, we found that only cells with luminal features were able to acquire transformed characteristics after an oncogenic HER2 (also known as ERBB2) mutant was introduced in their genomes. Overall, our studies characterize, at a single-cell level, the heterogeneity of the Comma-1D cell line and illustrate how Comma-1D cells can be used as an experimental model to study both the differentiation and the transformation processes in vitro.

miR-424/503 modulates Wnt/ß-catenin signaling in the mammary epithelium by targeting LRP6.

During the female lifetime, the expansion of the epithelium dictated by the ovarian cycles is supported by a transient increase in the mammary epithelial stem cell population (MaSCs). Notably, activation of Wnt/ß-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a modulator of canonical Wnt signaling in the mammary epithelium. We show that mammary tumors of miR-424(322)/503-depleted mice exhibit activated Wnt/ß-catenin signaling. Importantly, we show a strong association between miR-424/503 deletion and breast cancers with high levels of Wnt/ß-catenin signaling. Moreover, miR-424/503 cluster is required for Wnt-mediated MaSC expansion induced by the ovarian cycles. Lastly, we show that miR-424/503 exerts its function by targeting two binding sites at the 3'UTR of the LRP6 co-receptor and reducing its expression. These results unveil an unknown link between the miR-424/503, regulation of Wnt signaling, MaSC fate, and tumorigenesis.

miR-424(322)/503 is a breast cancer tumor suppressor whose loss promotes resistance to chemotherapy.

The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of ∼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumorigenesis, and the phenomenon of chemoresistance.

Drugging MYCN through an allosteric transition in Aurora kinase A.

MYC proteins are major drivers of cancer yet are considered undruggable because their DNA binding domains are composed of two extended alpha helices with no apparent surfaces for small-molecule binding. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora A. We describe a class of inhibitors that disrupts the native conformation of Aurora A and drives the degradation of MYCN protein across MYCN-driven cancers. Comparison of cocrystal structures with structure-activity relationships across multiple inhibitors and chemotypes, coupled with mechanistic studies and biochemical assays, delineates an Aurora A conformation-specific effect on proteolytic degradation of MYCN, rather than simple nanomolar-level inhibition of Aurora A kinase activity.

Targeting MYCN in neuroblastoma by BET bromodomain inhibition.

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

Tumor dosimetry using [124I]m-iodobenzylguanidine microPET/CT for [131I]m-iodobenzylguanidine treatment of neuroblastoma in a murine xenograft model.

PURPOSE: [(124)I]m-iodobenzylguanidine ((124)I-mIBG) provides a quantitative tool for pretherapy tumor imaging and dosimetry when performed before [(131)I]m-iodobenzylguanidine ((131)I-mIBG) targeted radionuclide therapy of neuroblastoma. (124)I (T (1/2) = 4.2 days) has a comparable half-life to that of (131)I (T (1/2) = 8.02 days) and can be imaged by positron emission tomography (PET) for accurate quantification of the radiotracer distribution. We estimated expected radiation dose in tumors from (131)I-mIBG therapy using (124)I-mIBG microPET/CT imaging data in a murine xenograft model of neuroblastoma transduced to express high levels of the human norepinephrine transporter (hNET). PROCEDURES: In order to enhance mIBG uptake for in vivo imaging and therapy, NB 1691-luciferase (NB1691) human neuroblastoma cells were engineered to express high levels of hNET protein by lentiviral transduction (NB1691-hNET). Both NB1691 and NB1691-hNET cells were implanted subcutaneously and into renal capsules in athymic mice. (124)I-mIBG (4.2-6.5 MBq) was administered intravenously for microPET/CT imaging at 5 time points over 95 h (0.5, 3-5, 24, 48, and 93-95 h median time points). In vivo biodistribution data in normal organs, tumors, and whole-body were collected from reconstructed PET images corrected for photon attenuation using the CT-based attenuation map. Organ and tumor dosimetry were determined for (124)I-mIBG. Dose estimates for (131)I-mIBG were made, assuming the same in vivo biodistribution as (124)I-mIBG. RESULTS: All NB1691-hNET tumors had significant uptake and retention of (124)I-mIBG, whereas unmodified NB1691 tumors did not demonstrate quantifiable mIBG uptake in vivo, despite in vitro uptake. (124)I-mIBG with microPET/CT provided an accurate three-dimensional tool for estimating the radiation dose that would be delivered with (131)I-mIBG therapy. For example, in our model system, we estimated that the administration of (131)I-mIBG in the range of 52.8-206 MBq would deliver 20 Gy to tumors. CONCLUSIONS: The overexpression of hNET was found to be critical for (124)I-mIBG uptake and retention in vivo. The quantitative (124)I-mIBG PET/CT is a promising new tool to predict tumor radiation doses with (131)I-mIBG therapy of neuroblastoma. This methodology may be applied to tumor dosimetry of (131)I-mIBG therapy in human subjects using (124)I-mIBG pretherapy PET/CT data.