A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1

Abstract

BackgroundThe molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation.MethodsMaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells.ResultsUtilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity.ConclusionsThis study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1187-z) contains supplementary material, which is available to authorized users.

Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging

“…Compositional differences between libraries were normalized using the trimmed mean of M-values (TMM) method (Robinson and Oshlack, 2010). The top 120 gene signatures for each of the adult mouse subsets (basal, luminal or stromal) derived from a previous RNA-seq study (Sheridan et al, 2015) were used for interrogating the expression profiles of the tumor clones. Log2-RPKM values displayed in heatmaps and line graphs were computed using the edgeR package with a prior.count of 2 to reduce variability at low counts.…”

“…To gain further insight into the cellular morphology of clones, we performed immunolabeling on confetti tumors for E-cadherin, a marker of normal luminal epithelial cells, and Zeb1, an EMT marker expressed by mesenchymal cells (Shibue and Weinberg, 2017;Thiery et al, 2009). Notably, Zeb1 mRNA is restricted to fibroblasts and myofibroblasts in the mouse mammary gland (Sheridan et al, 2015). Although E-cadherin is known to be variably expressed among breast tumor epithelial cells and is lost upon the EMT (Dongre and Weinberg, 2019;Thiery et al, 2009), this marker is useful for demarcating the perimeter of luminal epithelial cells and works effectively for 3D imaging.…”

Thymospheres Are Formed by Mesenchymal Cells with the Potential to Generate Adipocytes, but Not Epithelial Cells

“…The regulation of such a population might play a key role in the compositional changes associated with age-related involution (including increased fibroblast and adipocyte presence) and poor thymic function (Taub and Longo, 2005). That this population can be propagated as thymospheres in vitro opens up the possibility that they might be amenable to interrogation in a screening platform to better understand their requirements and role, as has been performed with other sphere-compatible systems (Saxe et al, 2007;Sheridan et al, 2015).…”

Covering all your bases: incorporating intron signal from RNA-seq data

“…134 libraries were examined for this dataset. Mouse mammary cells from female virgin mice with additional samples from mammosphere and the CommaD-βGeo (CommaD-bG) cell line were sequenced in a study by Sheridan et al (2015) (28) to obtain poly(A) RNA libraries; GEO accession number GSE63310. Mammary cell populations include mammary stem cell-enriched basal cells, luminal progenitor-enriched (LP) and mature luminal-enriched (ML) cell populations.…”