Donghui Cheng

Isolation and functional characterization of murine prostate stem cells.

The ability to isolate prostate stem cells is essential to explore their role in prostate development and disease. In vitro prostate colony- and sphere-forming assays were used to quantitatively measure murine prostate stem/progenitor cell enrichment and self-renewal. Cell surface markers were screened for their ability to positively or negatively enrich for cells with enhanced growth potential in these assays. Immunohistochemical and FACS analyses demonstrate that specific cell surface markers can be used to discriminate prostate stromal (CD34+), luminal epithelial (CD24+CD49f−), basal epithelial (CD24+CD49f+), hematopoietic (CD45+, Ter119+), and endothelial (CD31+) lineages. Sorting for cells with a CD45−CD31−Ter119−Sca-1+CD49f+ antigenic profile results in a 60-fold enrichment for colony- and sphere-forming cells. These cells can self-renew and expand to form spheres for many generations and can differentiate to produce prostatic tubule structures containing both basal and luminal cells in vivo. These cells also localize to the basal cell layer within the region of the gland that is proximal to the urethra, which has been identified as the prostate stem cell niche. Prostate stem cells can be isolated to a purity of up to 1 in 35 by using this antigenic profile. The remarkable similarity in cell surface profile between prostate and mammary gland stem cells suggests these markers may be conserved among epithelial stem cell populations.

Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics

The epithelium of the adult prostate contains 3 distinct cell types: basal, luminal, and neuroendocrine. Tissue-regenerative activity has been identified predominantly from the basal cells, isolated by expression of CD49f and stem cell antigen-1 (Sca-1). An important question for the field is whether all basal cells have stem cell characteristics. Prostate-specific microarray databases were interrogated to find candidate surface antigens that could subfractionate the basal cell population. Tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1/GA733-1) was identified because it was enriched after castration, in prostate sphere cells and in the basal fraction. In the murine prostate, Trop2 shows progenitor characteristics such as localization to the region of the gland proximal to the urethra and enrichment for sphere-forming and colony-forming cells. Trop2 subfractionates the basal cells into 2 populations, both of which express characteristic basal cell markers by quantitative PCR. However, only the basal cells expressing high levels of Trop2 were able to efficiently form spheres in vitro. In the human prostate, where Sca-1 is not expressed, sphere-forming progenitor cells were also isolated based on high expression of Trop2 and CD49f. Trop2-expressing murine basal cells could regenerate prostatic tubules in vivo, whereas the remaining basal cells had minimal activity. Evidence was found for basal, luminal, and neuroendocrine cells in prostatic tubules regenerated from Trop2hi basal cells. In summary, functionally distinct populations of cells exist within the prostate basal compartment and an epithelial progenitor can give rise to neuroendocrine cells in vivo.

Self‐Renewal and Multilineage Differentiation In Vitro from Murine Prostate Stem Cells

Murine prostate stem cells express integrin α6, which modulates survival, proliferation, and differentiation signaling through its interaction with the extracellular protein laminin. When plated in vitro in laminin containing Matrigel medium, 1 of 500–1,000 murine prostate cells can grow and form clonogenic spheroid structures that we term prostate spheres. Prostate spheres can be serially passaged individually or in bulk to generate daughter spheres with similar composition, demonstrating that sphere‐forming cells are capable of self‐renewal. Spheres spontaneously undergo lineage specification for basal and transit‐amplifying cell types. P63‐expressing cells localized to the outer layers of prostate spheres possess higher self‐renewal capacity, whereas cells toward the center display a more differentiated transit‐amplifying phenotype, as demonstrated by the expression of the prostate stem cell antigen. When dihydrotestosterone is added to the medium, the androgen receptor is stabilized, is imported to the nucleus, and drives differentiation to a luminal cell‐like phenotype. A fraction of sphere cells returned to an in vivo environment can undergo differentiation and morphogenesis to form prostate tubular structures with defined basal and luminal layers accompanied by prostatic secretions. This study demonstrates self‐renewal and multilineage differentiation from single adult prostate stem/progenitor cells in a specific in vitro microenvironment.

Isolation, cultivation and characterization of adult murine prostate stem cells

The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we describe step-by-step procedures on the basis of previous work in our laboratory for the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; and the cultivation of prostate stem cells in vitro and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally, it will take approximately 8 h to harvest prostate cells, isolate the stem cell–enriched population and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo.

N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells

MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.

Stem‐Like Epithelial Cells Are Concentrated in the Distal End of the Fallopian Tube: A Site for Injury and Serous Cancer Initiation

The reproductive role of the fallopian tube is to transport the sperm and egg. The tube is positioned to act as a bridge between the ovary where the egg is released and the uterus where implantation occurs. Throughout reproductive years, the fallopian tube epithelium undergoes repetitive damage and regeneration. Although a reservoir of adult epithelial stem cells must exist to replenish damaged cells, they remain unidentified. Here, we report isolation of a subset of basally located human fallopian tube epithelia (FTE) that lack markers of ciliated (β‐tubulin; TUBB4) or secretory (PAX8) differentiated cells. These undifferentiated cells expressed cell surface antigens: epithelial cell adhesion molecule, CD44, and integrin α 6. This FTE subpopulation was fivefold enriched for cells capable of clonal growth and self‐renewal suggesting that they contain the FTE stem‐like cells (FTESCs). A twofold enrichment of the FTESC was found in the distal compared to the proximal end of the tube. The distal fimbriated end of the fallopian tube is a well‐characterized locus for initiation of serous carcinomas. An expansion of the cells expressing markers of FTESC was detected in tubal intraepithelial carcinomas and in fallopian tubes from patients with invasive serous cancer. These findings suggest that FTESC may play a role in the initiation of serous tumors. Characterization of these stem‐like cells will provide new insight into how the FTE regenerate, respond to injury, and may initiate cancer. STEM CELLS2012;30:2487–2497

Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

A basal stem cell signature identifies aggressive prostate cancer phenotypes

Significance Aggressive cancers often possess functional and molecular traits characteristic of normal stem cells. It is unclear if aggressive phenotypes of prostate cancer molecularly resemble normal stem cells residing within the human prostate. Here, we transcriptionally profiled epithelial populations from the human prostate and show that aggressive prostate cancer is enriched for a prostate basal stem cell signature. Within prostate cancer metastases, histological subtypes had varying enrichment of the stem cell signature, with small cell neuroendocrine carcinoma being the most stem cell-like. We further found that small cell neuroendocrine carcinoma and the prostate basal stem cell share a common transcriptional program. Targeting normal stem cell transcriptional programs may provide a new strategy for treating advanced prostate cancer. Evidence from numerous cancers suggests that increased aggressiveness is accompanied by up-regulation of signaling pathways and acquisition of properties common to stem cells. It is unclear if different subtypes of late-stage cancer vary in stemness properties and whether or not these subtypes are transcriptionally similar to normal tissue stem cells. We report a gene signature specific for human prostate basal cells that is differentially enriched in various phenotypes of late-stage metastatic prostate cancer. We FACS-purified and transcriptionally profiled basal and luminal epithelial populations from the benign and cancerous regions of primary human prostates. High-throughput RNA sequencing showed the basal population to be defined by genes associated with stem cell signaling programs and invasiveness. Application of a 91-gene basal signature to gene expression datasets from patients with organ-confined or hormone-refractory metastatic prostate cancer revealed that metastatic small cell neuroendocrine carcinoma was molecularly more stem-like than either metastatic adenocarcinoma or organ-confined adenocarcinoma. Bioinformatic analysis of the basal cell and two human small cell gene signatures identified a set of E2F target genes common between prostate small cell neuroendocrine carcinoma and primary prostate basal cells. Taken together, our data suggest that aggressive prostate cancer shares a conserved transcriptional program with normal adult prostate basal stem cells.

Normal immune development and glucocorticoid-induced thymocyte apoptosis in mice deficient for the T-cell death-associated gene 8 receptor.

ABSTRACT T-cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor transcriptionally upregulated by glucocorticoids (GCs) and implicated by overexpression studies in psychosine-mediated inhibition of cytokinesis and in GC-induced apoptosis. To examine the physiological function of TDAG8, we generated knockout (KO) mice by homologous recombination. An enhanced green fluorescent protein reporter was knocked into the disrupted tdag8 locus to allow the analysis of TDAG8 expression in living cells. Interestingly, we found that during thymocyte development, TDAG8 expression resembled the dynamic regulation described for known modulators of GC-induced apoptosis, including Bcl-2, Notch1, and GC receptor. TDAG8 was expressed in double-negative cells, was downregulated at the double-positive transition, and was upregulated in single-positive thymocytes. However, despite this striking expression pattern, maturation and selection of thymocytes, as well as major immune functions, were not affected in TDAG8 KO mice. In contrast to previous overexpression results, TDAG8 was dispensable for psychosine-induced formation of multinucleated cells. Furthermore, TDAG8 KO thymocytes showed normal apoptosis following in vivo and in vitro GC treatment. These results, while establishing a useful reporter strain to study T-lymphocyte maturation, argue against a critical role for TDAG8 in immune development, psychosine-mediated inhibition of cytokinesis, and GC-induced cell death.

Sustained suppression of Bcr-Abl-driven lymphoid leukemia by microRNA mimics

Many cancers and leukemias are associated with strong dominant oncogenic mutations that activate tyrosine kinases and other classes of molecules, including transcription factors and antiapoptotic mechanisms. Some of these events can be targeted with small molecules or antibody-based therapeutics, but many remain intractable. In addition, cancer-related enzyme targets can often mutate, and drug-resistant variants are selected. Therapies directed at the mRNA encoding dominant oncogenes could provide a more global set of technologies for cancer treatment. To test this concept, we have used the model of transformation of hematopoietic cells by the chimeric Bcr-Abl oncogene, a highly activated tyrosine kinase. Our results show that tandem arrays of miRNA mimics, but not single miRNA mimics, directed against the Abl portion of the mRNA and introduced by lentiviral vectors can effectively alter the leukemogenic potency when the degree of suppression of expression of Bcr-Abl is reduced >200-fold from control levels. Only methods capable of such dramatic sustained reduction in the level of expression of highly activated kinase oncogenes are likely to be effective in controlling malignant cell populations.