35 research outputs found
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Evidence for a multipotent mammary progenitor with pregnancy-specific activity
Introduction: The mouse mammary gland provides a powerful model system for studying processes involved in epithelial tissue development. Although markers that enrich for mammary stem cells and progenitors have been identified, our understanding of the mammary developmental hierarchy remains incomplete. Methods: We used the MMTV promoter linked to the reverse tetracycline transactivator to induce H2BGFP expression in the mouse mammary gland. Mammary epithelial cells (MECs) from virgin mice were sorted by flow cytometry for expression of the mammary stem cell/progenitor markers CD24 and CD29, and H2BGFP. Sorted populations were analyzed for in vivo repopulation ability, expression of mammary lineage markers, and differential gene expression. Results: The reconstituting activity of CD24+/CD29+ cells in cleared fat pad transplantation assays was not distinguished in GFP+ compared to GFP- subpopulations. However, within the CD24+/CD29lo luminal progenitor-enriched population, H2BGFP+, but not H2BGFP-, MECs formed mammary structures in transplantation assays; moreover, this activity was dramatically enhanced in pregnant recipients. These outgrowths contained luminal and myoepithelial mammary lineages and produced milk, but lacked the capacity for serial transplantation. Transcriptional microarray analysis revealed that H2BGFP+/CD24+/CD29lo MECs are distinct from H2BGFP-/CD24+/CD29lo MECs and enriched for gene expression signatures with both the stem cell (CD24+/CD29+) and luminal progenitor (CD24+/CD29lo/CD61+) compartments. Conclusions: We have identified a population of MECs containing pregnancy-activated multipotent progenitors that are present in the virgin mammary gland and contribute to the expansion of the mammary gland during pregnancy
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Identification of cancer genes that are independent of dominant proliferation and lineage programs
Large, multidimensional cancer datasets provide a resource that can be mined to identify candidate therapeutic targets for specific subgroups of tumors. Here, we analyzed human breast cancer data to identify transcriptional programs associated with tumors bearing specific genetic driver alterations. Using an unbiased approach, we identified thousands of genes whose expression was enriched in tumors with specific genetic alterations. However, expression of the vast majority of these genes was not enriched if associations were analyzed within individual breast tumor molecular subtypes, across multiple tumor types, or after gene expression was normalized to account for differences in proliferation or tumor lineage. Together with linear modeling results, these findings suggest that most transcriptional programs associated with specific genetic alterations in oncogenes and tumor suppressors are highly context-dependent and are predominantly linked to differences in proliferation programs between distinct breast cancer subtypes. We demonstrate that such proliferation-dependent gene expression dominates tumor transcriptional programs relative to matched normal tissues. However, we also identified a relatively small group of cancer-associated genes that are both proliferation- and lineage-independent. A subset of these genes are attractive candidate targets for combination therapy because they are essential in breast cancer cell lines, druggable, enriched in stem-like breast cancer cells, and resistant to chemotherapy-induced down-regulation
Profiling Y561-Dependent and -Independent Substrates of CSF-1R in Epithelial Cells
Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK
ERK and p38 MAPK Activities Determine Sensitivity to PI3K/mTOR Inhibition via Regulation of MYC and YAP
Aberrant activation of the PI3K/mTOR pathway is a common feature of many cancers and an attractive target for therapy, but resistance inevitably evolves as is the case for any cancer cell-targeted therapy. In animal tumor models, chronic inhibition of PI3K/mTOR initially inhibits tumor growth, but over time, tumor cells escape inhibition. In this study, we identified a context-dependent mechanism of escape whereby tumor cells upregulated the proto-oncogene transcriptional regulators c-MYC and YAP1. This mechanism was dependent on both constitutive ERK activity as well as inhibition of the stress kinase p38. Inhibition of p38 relieved proliferation arrest and allowed upregulation of MYC and YAP through stabilization of CREB. These data provide new insights into cellular signaling mechanisms that influence resistance to PI3K/mTOR inhibitors. Furthermore, they suggest that therapies that inactivate YAP or MYC or augment p38 activity could enhance the efficacy of PI3K/mTOR inhibitors.National Institutes of Health (U.S.) (Grant R01CA103866)National Institutes of Health (U.S.) (Grant AI47389
PTK6 Regulates IGF-1-Induced Anchorage-Independent Survival
Background: Proteins that are required for anchorage-independent survival of tumor cells represent attractive targets for therapeutic intervention since this property is believed to be critical for survival of tumor cells displaced from their natural niches. Anchorage-independent survival is induced by growth factor receptor hyperactivation in many cell types. We aimed to identify molecules that critically regulate IGF-1-induced anchorage-independent survival. Methods and Results: We conducted a high-throughput siRNA screen and identified PTK6 as a critical component of IGF-1 receptor (IGF-1R)-induced anchorage-independent survival of mammary epithelial cells. PTK6 downregulation induces apoptosis of breast and ovarian cancer cells deprived of matrix attachment, whereas its overexpression enhances survival. Reverse-phase protein arrays and subsequent analyses revealed that PTK6 forms a complex with IGF-1R and the adaptor protein IRS-1, and modulates anchorage-independent survival by regulating IGF-1R expression and phosphorylation. PTK6 is highly expressed not only in the previously reported Her2 breast cancer subtype, but also in high grade ER, Luminal B tumors and high expression is associated with adverse outcomes. Conclusions: These findings highlight PTK6 as a critical regulator of anchorage-independent survival of breast and ovarian tumor cells via modulation of IGF-1 receptor signaling, thus supporting PTK6 as a potential therapeutic target for multiple tumor types. The combined genomic and proteomic approaches in this report provide an effective strategy for identifying oncogenes and their mechanism of action
Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities
<p>Abstract</p> <p>Background</p> <p>Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups.</p> <p>Results</p> <p>We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects.</p> <p>Conclusions</p> <p>The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities.</p
Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages
Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk
Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours
Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy
Statistical methods for analysis of high-throughput RNA interference screens
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery, and in both of these areas large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however, small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow
CRB3 and the FERM protein EPB41L4B regulate proliferation of mammary epithelial cells through the release of amphiregulin.
Numerous observations have suggested a connection between the maintenance of cell polarity and control of cell proliferation; however, the mechanisms underlying these connections remain poorly understood. Here we found that ectopic expression of CRB3, which was previously shown to restore tight junctions and membrane polarity in MCF-10A cells, induced a hyperproliferative phenotype, with significantly enlarged acini in basement membrane culture, similar to structures induced by expression of proliferative oncogenes such as cyclinD1. We found that CRB3-induced proliferation is epidermal growth factor (EGF)-independent and occurs through a mechanism that involves secretion of the EGF-family ligand, amphiregulin (AREG). The increase in AREG secretion is associated with an increase in the number and size of both early and late endosomes. Both the proliferative and endocytic phenotypes associated with CRB3 expression require the FERM-binding domain (FBD) but not the PDZ-binding domain of CRB3, arguing that this proliferative phenotype is independent of the PDZ-dependent polarity signaling by CRB3. We identified the FBD-containing protein, EPB41L4B, as an essential mediator of CRB3-driven proliferation and observed that the CRB3-dependent changes in endocytic trafficking were also dependent on EPB41L4B. Taken together, these data reveal a previously uncharacterized role for CRB3 in regulating proliferation in mammalian cells that is associated with changes in the endocytic trafficking machinery