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Aging is a significant risk factor for disease in several tissues, including the prostate. Defining the kinetics of age-related changes in these tissues is critical for identifying regulators of aging and evaluating interventions to slow the aging process and reduce disease risk. An altered immune microenvironment is characteristic of prostatic aging in mice, but whether features of aging in the prostate emerge predominantly in old age or earlier in adulthood has not previously been established. Using highly multiplexed immune profiling and time-course analysis, we tracked the abundance of 29 immune cell clusters in the aging mouse prostate. Early in adulthood, myeloid cells comprise the vast majority of immune cells in the 3-month-old mouse prostate. Between 6 and 12 months of age, there is a profound shift towards a T and B lymphocyte-dominant mouse prostate immune microenvironment. Comparing the prostate to other urogenital tissues, we found similar features of age-related inflammation in the mouse bladder but not the kidney. In summary, our study offers new insight into the kinetics of prostatic inflammaging and the window when interventions to slow down age-related changes may be most effective.

Prostate cancer cells that survive clinical androgen receptor (AR) blockade mediate disease progression and lethality. Reprogrammed metabolic signaling is one mechanism by which tumor cells can survive treatment. However, how AR inhibition reprograms metabolism, and whether altered metabolism can be exploited to eradicate cells that survive AR blockade, remains unclear. Here, we comprehensively characterized the effect of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. AR inhibition maintains oxidative mitochondrial metabolism and reduces glycolytic signaling, through hexokinase II downregulation and decreased MYC activity. Robust elongation of mitochondria via reduced DRP1 activity supports cell fitness after AR blockade. In addition, AR inhibition enhances sensitivity to complex I inhibitors in several models, suggesting that AR blockade increases reliance on oxidative mitochondrial metabolism. Our study provides an enhanced understanding of how AR inhibition alters metabolic signaling and highlights the potential of therapies that target metabolic vulnerabilities in AR-inhibited cells.

The corneal endothelium plays a critical role in maintaining corneal clarity. There is an expected decline in cell density with age and disease, and maintaining the health of this cell layer is important as corneal endothelial cells generally are amitotic in vivo. Diabetes mellitus is a highly prevalent disease that damages the corneal endothelium. Diabetes causes structural and functional impairments in the corneal endothelium that decrease cellular reserve in response to stress. These effects have implications to consider for diabetic patients undergoing anterior segment surgery, and for corneal surgeons who use diabetic donor tissue and treat diabetic patients. In this review, we discuss the specifics of how diabetes mellitus impacts the corneal endothelium including alterations in cell morphology, cell density, ultrastructure, pump and barrier function, cataract surgery outcomes, and corneal transplant outcomes with attention to the use of diabetic donor tissue and diabetic transplant recipients.

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and down-regulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions.

Many organs experience a loss of tissue mass and a decline in regenerative capacity during aging. In contrast, the prostate continues to grow in volume. In fact, age is the most important risk factor for prostate cancer. However, the age-related factors that influence the composition, morphology and molecular features of prostate epithelial progenitor cells, the cells-of-origin for prostate cancer, are poorly understood. Here, we review the evidence that prostate luminal progenitor cells are expanded with age. We explore the age-related changes to the microenvironment that may influence prostate epithelial cells and risk of transformation. Finally, we raise a series of questions about models of aging and regulators of prostate aging which need to be addressed. A fundamental understanding of aging in the prostate will yield critical insights into mechanisms that promote the development of age-related prostatic disease.

Age is a significant risk factor for disease of the prostate. However, the mechanisms by which age increases disease risk have not been well described. We previously reported age-related changes within the inflammatory and luminal compartments of the mouse prostate. Old mouse prostates exhibit an expansion of the population of Trop2+ luminal progenitor cells and a reduction in the frequency and functional capacity of Trop2- luminal cells, indicating that different cell-types have distinct responses to aging. Whether distinct cell-types in the prostate share a common signature of aging has not been established. We transcriptionally profiled four distinct cell-types in young adult and old mouse prostates: stromal, basal, Trop2+ luminal progenitor and Trop2- luminal cells. Motif analysis of genes upregulated in old prostate cell-types pointed to transcriptional regulators of inflammatory and hypoxia-related signaling. Glutathione metabolism and the antioxidant response emerged as a common signature of aging across prostatic lineages. Expression of genes implicated in mouse prostate aging, including the antioxidant response gene Hmox1, correlates with age of diagnosis in primary prostate tumors from the TCGA cohort. These findings reveal a common signature shared by distinct cell-types in the old prostate reflective of age-associated metabolic reprogramming.

To determine how the Rho kinase inhibitor, ripasudil, affects metabolic function and cell viability in donor human corneal endothelial cells (HCECs).Endothelial cell-Descemet membrane (EDM) tissues were treated with 10 μM ripasudil and assayed for mitochondrial and glycolytic activity using extracellular flux analysis and then compared to untreated controls. In addition, EDM tissues with a 24-h ripasudil treatment and control tissues were exposed to 1 μM staurosporine to induce apoptosis and then analyzed for cell viability using apoptosis and necrosis assays.Mitochondrial respiration metrics, specifically maximal respiration (P = 0.758) and spare respiratory capacity (P = 0.777), did not differ among the 1-h ripasudil treatment, 24-h treatment, and untreated tissues. Glycolytic activity assays showed an increase in glycolytic capacity at 1 h compared to the 24-h exposure group (P = 0.049) and controls (P = 0.009). Following exposure to staurosporine, the percentage of apoptotic HCECs was lower (P = 0.009) in ripasudil-treated tissues (2.473%, standard error of the mean [SEM] 0.477%) compared to untreated controls (3.349%, SEM 0.566%). In contrast, the percentage of necrotic HCECs decreased but did not differ statistically (P = 0.158) between ripasudil-treated (3.789%, SEM 0.487%) and untreated (4.567%, SEM 0.571%) tissues.Exposures to ripasudil did not result in any detectable reduction in metabolic function for HCECs in an ex vivo donor tissue model, and an increase in glycolytic activity at the 1-h time point was detected. In addition, HCECs treated with ripasudil gained a protective effect against induced apoptosis, suggesting that ripasudil may help improve the integrity of the corneal endothelium.

Hormonal therapy targeting androgen receptor (AR) is initially effective to treat prostate cancer (PCa), but it eventually fails. It has been hypothesized that cellular heterogeneity of PCa, consisting of AR(+) luminal tumor cells and AR(−) neuroendocrine (NE) tumor cells, may contribute to therapy failure. Here, we describe the successful purification of NE cells from primary fresh human prostate adenocarcinoma based on the cell surface receptor C-X-C motif chemokine receptor 2 (CXCR2). Functional studies revealed CXCR2 to be a driver of the NE phenotype, including loss of AR expression, lineage plasticity, and resistance to hormonal therapy. CXCR2-driven NE cells were critical for the tumor microenvironment by providing a survival niche for the AR(+) luminal cells. We demonstrate that the combination of CXCR2 inhibition and AR targeting is an effective treatment strategy in mouse xenograft models. Such a strategy has the potential to overcome therapy resistance caused by tumor cell heterogeneity.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

SummaryDifferentiation involves bifurcations between discrete cell states, each defined by a distinct gene expression profile. Single-cell RNA profiling allows the detection of bifurcations. However, while current methods capture these events, they do not identify characteristic gene signals. Here we show that BioTIP – a tipping-point theory-based analysis – can accurately, robustly, and reliably identify critical transition signals (CTSs). A CTS is a small group of genes with high covariance in expression that mark the cells approaching a bifurcation. We validated its accuracy in the cardiogenesis with known a tipping point and demonstrated the identified CTSs contain verified differentiation-driving transcription factors. We then demonstrated the application on a published mouse gastrulation dataset, validated the predicted CTSs using independent in-vivo samples, and inferred the key developing mesoderm regulator Etv2. Taken together, BioTIP is broadly applicable for the characterization of the plasticity, heterogeneity, and rapid switches in developmental processes, particularly in single-cell data analysis.HighlightsIdentifying significant critical transition signals (CTSs) from expression noiseA significant CTS contains or is targeted by key transcription factorsBioTIP identifies CTSs accurately and independent of trajectory topologiesSignificant CTSs reproducibly indicate bifurcations across datasets

Background Cancer cell metabolism requires sustained pools of intracellular nicotinamide adenine dinucleotide (NAD+) which is maintained by a balance of NAD+ hydrolase activity and NAD+ salvage activity. We recently reported that human prostate cancer can be initiated following oncogene expression in progenitor-like luminal cells marked by low expression of the NAD+-consuming enzyme CD38. CD38 expression is reduced in prostate cancer compared to benign prostate, suggesting that tumor cells may reduce CD38 expression in order to enhance pools of NAD+. However, little is known about how CD38 expression is repressed in advanced prostate cancer and whether CD38 plays a role in regulating NAD+ levels in prostate epithelial cells. Methods CD38 expression, its association with recurrence after prostatectomy for clinically localized prostate cancer, and DNA methylation of the CD38 promoter were evaluated in human prostate tissues representing various stages of disease progression. CD38 was inducibly over-expressed in benign and malignant human prostate cell lines in order to determine the effects on cell proliferation and levels of NAD+ and NADH. NAD+ and NADH were also measured in urogenital tissues from wild-type and CD38 knockout mice. Results CD38 mRNA expression was reduced in metastatic castration-resistant prostate cancer compared to localized prostate cancer. In a large cohort of men undergoing radical prostatectomy, CD38 protein expression was inversely correlated with recurrence. We identified methylation of the CD38 promoter in primary and metastatic prostate cancer. Over-expression of wild-type CD38, but not an NAD+ hydrolase-deficient mutant, depleted extracellular NAD+ levels in benign and malignant prostate cell lines. However, expression of CD38 did not significantly alter intracellular NAD+ levels in human prostate cell lines grown in vitro and in urogenital tissues isolated from wild-type and CD38 knockout mice. Conclusions CD38 protein expression in prostate cancer is associated with risk of recurrence. Methylation results suggest that CD38 is epigenetically regulated in localized and metastatic prostate cancer tissues. Our study provides support for CD38 as a regulator of extracellular, but not intracellular, NAD+ in epithelial cells. These findings suggest that repression of CD38 by methylation may serve to increase the availability of extracellular NAD+ in prostate cancer tissues.

Research in the area of stem/progenitor cells has led to the identification of multiple stem-like cell populations implicated in prostate homeostasis and cancer initiation. Given that there are multiple cells that can regenerate prostatic tissue and give rise to prostate cancer, our focus should shift to defining the signaling mechanisms that drive differentiation and progenitor self-renewal. In this article, we will review the literature, present the evidence and raise important unanswered questions that will help guide the field forward in dissecting critical mechanisms regulating stem-cell differentiation and tumor initiation.

Prostate cancer remains a leading cause of cancer death in the United States (1). In 2017, it was estimated that over 160,000 prostate cancer cases were diagnosed in the United States, and ∼26,000 men died of prostate cancer (2). Because tumor growth in prostate cancer is predominantly dependent on the androgen receptor (AR), a homodimer-forming transcription factor, the disease is commonly treated via therapies targeting the AR-axis. While this approach has proven to be initially effective, a majority of prostate cancer patients eventually develop resistance to AR-targeting therapies, leading to castration-resistant prostate cancer (CRPC), the lethal stage of prostate cancer. Measuring prostate-specific antigen levels in patient serum is regularly used for evaluating the efficacy of prostate cancer treatment. The serum prostate-specific antigen value, however, does not always correlate with the malignant state of CRPC (3). Therefore, it is of high priority to discover therapeutic targets and biomarkers that can accurately assess the efficacy of treatment in patients who have become resistant to therapies targeting the AR-axis. Various mechanisms of resistance to AR targeting have been identified, such as AR bypass signaling, AR alternative splicing, and AR gene amplification (4). Notably, aberrant AR splicing seems to be heavily associated with resistance to AR-targeting … [↵][1]1To whom correspondence should be addressed. Email: agoldstein{at}mednet.ucla.edu. [1]: #xref-corresp-1-1

SUMMARY Aging is associated with loss of tissue mass and a decline in adult stem cell function in many tissues. In contrast, aging in the prostate is associated with growth-related diseases including benign prostatic hyperplasia (BPH). Surprisingly, the effects of aging on prostate epithelial cells have not been established. Here we find that organoid-forming progenitor activity of mouse prostate basal and luminal cells is maintained with age. This is caused by an age-related expansion of progenitor-like luminal cells that share features with human prostate luminal progenitor cells. The increase in luminal progenitor cells may contribute to greater risk for growth-related disease in the aging prostate. Importantly, we demonstrate expansion of human luminal progenitor cells in BPH. In summary, we define a Trop2+ luminal progenitor subset and identify an age-related shift in the luminal compartment of the mouse and human prostate epithelium., In Brief Aging is a significant risk factor for BPH and prostate cancer, but how aging increases disease risk in the prostate remains poorly defined. Crowell et al. show that progenitor-enriched luminal cells are expanded with aging in the mouse and human prostate, and may contribute to BPH., Graphical Abstract

Despite several recent studies addressing the cells of origin for prostate cancer, there is still considerable discussion in the field regarding the most relevant target populations for transformation. Tissue regeneration studies have pointed to a basal cell origin for mouse and human prostate cancer. In contrast, genetically engineered mouse models demonstrate that cells within both the basal and luminal layers can initiate murine prostate cancer. Based on differences between these two approaches, we propose that further work should address the requirement for microenvironmental components such as immune or mesenchymal cells on epithelial cell types of origin for prostate cancer.

Prostate cancer is a common malignancy among men in Western countries. Recently the morbidity and mortality of prostate cancer increase dramatically in several oriental countries including China. Rapidly evolving technology in molecular biology such as high-throughput sequencing and integrative analysis of genomic and transcriptomic landscapes have enabled the identification of key oncogenic events for prostate cancer initiation, progression and resistance to hormonal therapy. These surging data of prostate cancer genome also provide insights on ethnic variation and the differences in histological subtype of this disease. In this review, differences in the incidence of prostate cancer and the prevalence of main genetic alterations between Asian and Western populations are discussed. We also review the recent findings on the mechanisms underlying neuroendocrine differentiation of prostate cancer and the development of small cell neuroendocrine carcinoma after androgen deprivation therapy.

Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here, we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multilineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Coadministration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore, their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium.

While chronic inflammation has been causally associated with several epithelial malignancies, whether it causally contributes to the development of prostate cancer has remained unclear. We recently reported that progenitor-like inflammation-associated luminal cells marked by low expression of Cluster of Differentiation 38 (CD38) can initiate human prostate cancer and predict poor outcome.

SUMMARY Inflammation is a risk factor for prostate cancer, but the mechanisms by which inflammation increases that risk are poorly understood. Here, we demonstrate that low expression of CD38 identifies a progenitor-like subset of luminal cells in the human prostate. CD38lo luminal cells are enriched in glands adjacent to inflammatory cells and exhibit epithelial nuclear factor κB (NF-κB) signaling. In response to oncogenic transformation, CD38lo luminal cells can initiate human prostate cancer in an in vivo tissue-regeneration assay. Finally, the CD38lo luminal phenotype and gene signature are associated with disease progression and poor outcome in prostate cancer. Our results suggest that prostate inflammation expands the pool of progenitor-like target cells susceptible to tumorigenesis., In Brief Chronic inflammation of the prostate is a risk factor for cancer, but how inflammation increases disease risk remains poorly defined. Liu et al. show that luminal progenitor cells expressing low levels of CD38 are expanded around inflammation, and these progenitors are target cells that can initiate human prostate cancer.

We consider here multivariate data which we understand as the problem where each data point i is measured for two or more distinct variables. In a typical situation there are many data points i while the range of the different variables is more limited. If there is only one variable then the data can be arranged as a rectangular matrix where i is the index of the rows while the values of the variable label the columns. We begin here with this case, but then proceed to the more general case with special emphasis on two variables when the data can be organized as a tensor. An analysis of such multivariate data by a maximal entropy approach is discussed and illustrated for gene expressions in four different cell types of six different patients. The different genes are indexed by i, and there are 24 (4 by 6) entries for each i. We used an unbiased thermodynamic maximal-entropy based approach (surprisal analysis) to analyze the multivariate transcriptional profiles. The measured microarray experimental data is organized as a tensor array where the two minor orthogonal directions are the different patients and the different cell types. The entries are the transcription levels on a logarithmic scale. We identify a disease signature of prostate cancer and determine the degree of variability between individual patients. Surprisal analysis determined a baseline expression level common for all cells and patients. We identify the transcripts in the baseline as the “housekeeping” genes that insure the cell stability. The baseline and two surprisal patterns satisfactorily recover (99.8%) the multivariate data. The two patterns characterize the individuality of the patients and, to a lesser extent, the commonality of the disease. The immune response was identified as the most significant pathway contributing to the cancer disease pattern. Delineating patient variability is a central issue in personalized diagnostics and it remains to be seen if additional data will confirm the power of multivariate analysis to address this key point. The collapsed limits where the data is compacted into two dimensional arrays are contained within the proposed formalism.

Men with advanced prostate cancer are typically treated with hormonal therapy, which leads to tumour shrinkage. However, tumours relapse and develop into the lethal form of the disease, termed castration-resistant prostate cancer (CRPC). Two distinct, but not mutually exclusive, models have been proposed in the literature to describe the onset of CRPC: adaptation and selection. Although some studies indicate that tumour cells acquire new alterations that enable them to survive in the castrated state (adaptation), other research points to the outgrowth of rare, pre-existing cells capable of surviving hormonal therapy (selection). Targeting the cells that survive hormonal therapy--by either adaptation or selection--is necessary to prevent the development of CRPC. Current research is focused on not only understanding the cellular mechanisms of CRPC, but also defining critical pathways that can be targeted with combinatorial therapies in castration-resistant cancer cells.

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.

Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without previous immortalization via genetic influences such as SV40 T-antigen, thus limiting our knowledge of the events that can transform naive human epithelium. Here we describe a system developed in our laboratory to directly transform freshly isolated primary human prostate epithelial cells without previous culture or immortalization. Prostate tissue is obtained from patients and benign tissue is separated from malignant tissue. Benign and malignant tissues are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated on the basis of differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. At 8-16 weeks after transplantation into immune-deficient mice, the development of lesions, histologically classified as benign prostate, prostatic intraepithelial neoplasia and adenocarcinoma, can be evaluated.

Epithelial-specific activation of the PI3-kinase pathway is the most common genetic alteration in type I endometrial cancer. In the majority of these tumors, PTEN expression is lost in the epithelium but maintained in tumor stroma. Currently reported PTEN knockout mouse models initiate type I endometrial cancer concomitant with loss of PTEN in both uterine epithelium and stroma. Consequently, the biologic outcome of selectively activating the PI3-kinase pathway in the endometrial epithelium remains unknown. To address this question, we established a malleable in vivo endometrial regeneration system from dissociated murine uterine epithelium and stroma. Regenerated endometrial glands responded to pharmacologic variations in hormonal milieu similar to the native endometrium. Cell-autonomous activation of the PI3-kinase pathway via biallelic loss of PTEN or activation of AKT in adult uterine epithelia in this model was sufficient to initiate endometrial carcinoma. AKT-initiated tumors were serially transplantable, demonstrating permanent genetic changes in uterine epithelia. Immunohistochemistry confirmed loss of PTEN or activation of AKT in regenerated hyperplastic glands that were surrounded by wild-type stroma. We demonstrate that cell-autonomous activation of the PI3-kinase pathway is sufficient for the initiation of endometrial carcinoma in naive adult uterine epithelia. This in vivo model provides an ideal platform for testing the response of endometrial carcinoma to targeted therapy against this common genetic alteration.

Another Cell Culprit in Prostate Cancer A recent controversial hypothesis about the cellular origins of human cancer, the so-called “cancer stem cell hypothesis,” has fueled interest in identifying the specific cell types that give rise to common epithelial cancers. A single, well-defined cell of origin could, in principle, lead to more effective targeted therapies. Based on histological evidence and/or studies of mouse tumors, luminal cells are believed to be the cell of origin in prostate cancer. Now, using functional assays of cells derived from benign human prostate tissue, Goldstein et al. (p. 568 ) find that a different cell type, basal cells, can give rise in mice to prostate tumors that closely resemble human prostate tumors. Thus, the cellular origin of prostate cancer may be more complex than anticipated.

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.

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 provide 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; 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 hours 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.

BACKGROUND Prostate stem/progenitor cells function in glandular development and maintenance. They may be targets for tumor initiation, so characterization of these cells may have therapeutic implications. Cells from dissociated tissues that form spheres in vitro often represent stem/progenitor cells. A subset of human prostate cells that form prostaspheres were evaluated for self-renewal and tissue regeneration capability in the present study.

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 Trop2 hi 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.

© 2015 Cold Spring Harbor Laboratory Press. Cleared mammary fat pad (MFP) transplantation has been a standard technique for studies of mammary development and cancer for several decades. The mammary gland is comprised of several fundamental components: The epithelial compartment contains basal/myoepithelial cells and luminal cells, and the stromal compartment (called the MFP) contains adipocytes, smooth muscle cells, fibroblasts, and immune cells. In 3- to 4-wk-old female mice, the mammary epithelium is concentrated very close to the nipple and has not yet grown beyond the mammary lymph node to penetrate the bulk of the MFP. This developmental feature provides an anatomical fixed point, and enables one to cut away the portion of the MFP from the nipple to the lymph node, leaving behind the majority of the MFP free of epithelium. The “cleared” MFP can serve as a supportive native microenvironment fully sufficient for the organogenesis of injected donor epithelium. Normal mammary epithelial donor cells will produce histologically and functionally normal mammary ductal epithelium several weeks posttransplant, with the exception that the ducts will not be connected to the nipple. The assay described here provides a powerful platform for assessing the developmental and tumorigenic potential of engineered cells of interest.

© 2015 Cold Spring Harbor Laboratory Press. Animal models of cancer provide fundamental insight into the cellular and molecular mechanisms of human cancer development. As an alternative to genetically engineered mouse models, increasing evidence shows that tissue recombination and transplantation models represent an efficient approach to faithfully recapitulate solid epithelial cancer in mice. Cancer can be rapidly initiated through lentiviral delivery of defined genetic alterations into target cells that are grown in a physiological milieu with an appropriate epithelial–stromal interaction. Through genetic manipulation of distinct subpopulations of epithelial cells and mesenchymal cells, this powerful system can readily test both cell-autonomous roles of genetic events in the epithelial compartment and the paracrine effects of the microenvironment. Here we review the recent advances in mouse models of several epithelial cancers achieved using orthotopic transplantation and tissue recombination strategies, with an emphasis on the dissociated cell in vivo prostate regeneration model to investigate prostate cancer.

© 2015 Cold Spring Harbor Laboratory Press. An appropriate microenvironment provided by the mesenchyme is important for establishing tissue recombination models for epithelial cancer. Urogenital sinus mesenchymal (UGSM) cells derived from embryonic rodent show potent inductive effects for prostate regeneration. Genetic manipulation of these mesenchymal cells allows us to define the contribution of the tumor microenvironment to prostate cancer development. This protocol describes preparation and propagation of murine UGSM cells in culture.

© 2015 Cold Spring Harbor Laboratory Press. Tissue recombination models are useful for studying cancer initiation, progression, and metastasis. They also provide an in vivo environment in which to investigate the functional role of stem cells in tissue repair. In this protocol, we describe in detail the dissociated prostate regeneration assay. Dissociated adult murine prostate cells are combined with embryonic urogenital sinus mesenchymal cells and implanted under the renal capsule. Morphological tissue structures with appropriate epithelial–stroma interactions are reconstituted in the grafts.

Inflammation promotes the initiation of various malignancies by inducing genetic and epigenetic changes. Here we show that bacterial infection-induced prostatitis results in microenvironmental changes that enhance the differentiation of prostate basal cells into luminal cells, a cellular process that rarely occurs under normal physiological conditions. Previously, we showed in a mouse model that disease initiation for prostate cancer with a basal cell origin requires and is limited by basal-to-luminal differentiation and that prostatic inflammation induced by bacterial infection accelerates disease initiation by enhancing basal-to-luminal differentiation. Collectively, our results show that inflammation-induced microenvironmental changes alter the prostate epithelial lineage differentiation program, and we propose this alteration as a distinct and complementary process through which inflammation promotes tumor initiation.

The cells that initiate and propagate cancer are important therapeutic targets. However, the progression from cells of origin to tumor-propagating cells is poorly defined for most human cancers. Mouse models indicate that both basal and luminal cells can initiate prostate cancer, while studies with human prostate tissue have demonstrated a role for basal cells in transformation. Our recent study provides evidence that a common cell of origin can produce alternative variants of human epithelial cancer. Our findings also reveal that the cell of origin that initiates cancer is not continuously required to maintain and propagate the disease. Importantly, the cells responsible for initiating human prostate cancer can have a distinct cellular phenotype from the cells needed to maintain it.

Objective To determine accuracy of a human immunodeficiency virus type 1 (HIV-1) antibody testing system using a device to collect and stabilize oral mucosal transudate (OMT), a fluid with increased levels of IgG; an enzyme immunoassay (EIA) screening test optimized for OMT; and a Western blot confirmatory test designed for use with OMT. Design The OMT specimens were tested by EIA and, if indicated, confirmatory Western blot according to a standard testing algorithm. The OMT results were compared with true HIV status as determined by serum testing and/or clinical diagnosis. Patients Specimens from 3570 subjects (2382 at low risk, 698 at high risk, 242 with acquired immunodeficiency syndrome [AIDS], and 248 "nonspecificity" [persons with diseases associated with an increased frequency of false-positive results in HIV testing]) were collected at 11 geographically diverse sites (including blood banks, public health clinics, general medical clinics, HIV clinics, sexually transmitted disease clinics, and a hemophilia center) in the United States. Main outcome measures Overall accuracy of testing OMT for HIV-1 antibodies compared with true HIV-1 antibody status; sensitivity and specificity of OMT EIA and Western blot. Results Sensitivity of OMT EIA testing in 673 true-positive subjects was 99.9% (672/673). The OMT Western blot results in the 673 true-positive subjects were positive in 665 and indeterminate in 8. The EIA followed by Western blot (if EIA was repeatedly reactive) yielded a negative result in 99.9% (2893/2897) of OMT samples from true negatives and an indeterminate result in 4. The OMT testing system provided the correct result or would trigger appropriate follow-up testing in 3569 (>99.9%) of 3570 cases. Conclusion HIV-1 antibody testing of OMT samples is a highly accurate alternative to serum testing.

The adult mouse prostate has a seemingly endless capacity for regeneration, and sonic hedgehog (SHH) signaling has been implicated in this stem cell-driven process. However, it is not clear whether SHH acts on the epithelium or stromal cells that secrete factors required for epithelial expansion. Because little is known about stromal stem cells compared with their epithelial counterparts, we used in vivo mouse genetics tools to characterize four prostate stromal subtypes and their stem cells. Using knockin reporter alleles, we uncovered that SHH signals from prostate basal epithelial cells to adjacent stromal cells. Furthermore, the SHH target gene Gli1 is preferentially expressed in subepithelial fibroblast-like cells, one of four prostate stromal subtypes and the subtype closest to the epithelial source of SHH. Using Genetic Inducible Fate Mapping to mark adult Gli1- or Smooth muscle actin-expressing cells and follow their fate during regeneration, we uncovered that Gli1-expressing cells exhibit long-term self-renewal capacity during multiple rounds of androgen-mediated regeneration after castration-induced involution, and depleted smooth muscle cells are mainly replenished by preexisting smooth muscle cells. Based on our Genetic Inducible Fate Mapping studies, we propose a model where SHH signals to multiple stromal stem cells, which are largely unipotent in vivo.

The relationship between the cells that initiate cancer and the cancer stem-like cells that propagate tumors has been poorly defined. In a human prostate tissue transformation model, basal cells expressing the oncogenes Myc and myristoylated AKT can initiate heterogeneous tumors. Tumors contain features of acinar-type adenocarcinoma with elevated eIF4E-driven protein translation and squamous cell carcinoma marked by activated beta-catenin. Lentiviral integration site analysis revealed that alternative histological phenotypes can be clonally derived from a common cell of origin. In advanced disease, adenocarcinoma can be propagated by self-renewing tumor cells with an androgen receptor-low immature luminal phenotype in the absence of basal-like cells. These data indicate that advanced prostate adenocarcinoma initiated in basal cells can be maintained by luminal-like tumor-propagating cells. Determining the cells that maintain human prostate adenocarcinoma and the signaling pathways characterizing these tumor-propagating cells is critical for developing effective therapeutic strategies against this population.

Based on the unique capacity of the rodent prostate to undergo seemingly endless rounds of androgen cycling in response to castration and androgen add-back, the prostate has been proposed to contain long-term self-renewing stem cells. However the prospective isolation and characterization of stem-like cells from rodent and human prostate tissue has only been described over the last 2 decades. Several models of epithelial homeostasis in the adult prostate have been proposed based on either the presence of a multipotent tissue stem cell that differentiates through a series of intermediate developmental stages or the coexistence of multiple unipotent lineage-restricted stem cells. The isolation of cells with stem and progenitor activity is an important first step to delineate the epithelial hierarchy of the prostate. In addition, isolation of stem cells allows characterization of their functional capacities and the molecular programs regulating their activity. These studies will enable detection or targeting of stem and progenitor cells during various stages of neoplastic transformation and tumor progression, including the lethal phase of the disease, castration-resistant prostate cancer.

New generation of anti-androgen treatments such as enzalutamide and abiraterone are improving survival of men with advanced and metastatic prostate cancers, but resistance to these inevitably arises. The molecular mechanisms governing the emergence of treatment resistance in castration resistant prostate cancer (CRPC) patients are not well understood. Recent experience suggests that tumor regeneration from castration-resistant stem-like cells induce resistance to hormonal therapy. Therefore, elucidating novel targets essential for driving stem-like activity is critical to prevent and defeat CRPC. It has been shown that normal and cancer stem cells exploit normal development process of epithelial-mesenchymal transition (EMT) to survive and metastasize, and that EMT confer stem cell properties to more differentiated cancer cell progeny in breast and other cancers. However, it is unclear if EMT is linked with stem cells in normal and malignant prostate. We previously showed that N-cadherin, a marker of EMT, promotes CRPC progression and that there was a correlation between N-cadherin expression and the expression of stem cell associated markers. This leads to hypothesis that N-cadherin may promote CRPC by enhancing tumorige esis/tumor initiating properties of cancer cells in a castrate environment. N-cadherin may confer tumor initiating or stem-like properties to prostate cancer cells perhaps uniquely or specific to in a castrate environment. Here, we verified that EMT is linked to stem cells in both normal prostate and CRPC. We further demonstrate that N-cadherin is involved in increased stem cell properties in normal mouse and human prostates from clinical samples in an androgen-deprived environment. Castration elevates the expression of N-cadherin and stem cell marker Trop2, accompanied with enhanced stem cell properties of luminal population in normal mouse prostates. N-cadherin upregulates stem cell properties in prostate cancer cells in the absence of androgen and CRPC tumors. N-cadherin-positive cells from CRPC tumors behave as stem cells. Overexpression of N-cadherin enhances ALDH activity in a panel of prostate cancer cell lines. Our findings reveal an important role of N-cadherin in prostate cancer and stem cells, and provide useful information of EMT-related biomarkers for preventing and developing efficient therapeutics to combat the treatment resistance to next-generation AR antagonists. Citation Format: Shu Lin, Tanya Stonyanova, Evelyn Kono, Jaclyn Matsuura, Naoko Kobayashi, Joyce Yamashiro, Dai Hong, Owen N. Witte, Andrew S. Goldstein, Robert E. Reiter. N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-279.

Computers are organized into hardware and software. Using a theoretical approach to identify patterns in gene expression in a variety of species, organs, and cell types, we found that biological systems similarly are comprised of a relatively unchanging hardware-like gene pattern. Orthogonal patterns of software-like transcripts vary greatly, even among tumors of the same type from different individuals. Two distinguishable classes could be identified within the hardware-like component: those transcripts that are highly expressed and stable and an adaptable subset with lower expression that respond to external stimuli. Importantly, we demonstrate that this structure is conserved across organisms. Deletions of transcripts from the highly stable core are predicted to result in cell mortality. The approach provides a conceptual thermodynamic-like framework for the analysis of gene-expression levels and networks and their variations in diseased cells.

Dominant mutations or DNA amplification of tyrosine kinases are rare among the oncogenic alterations implicated in prostate cancer. We demonstrate that castration-resistant prostate cancer (CRPC) in men exhibits increased tyrosine phosphorylation, raising the question of whether enhanced tyrosine kinase activity is observed in prostate cancer in the absence of specific tyrosine kinase mutation or DNA amplification. We generated a mouse model of prostate cancer progression using commonly perturbed non-tyrosine kinase oncogenes and pathways and detected a significant up-regulation of tyrosine phosphorylation at the carcinoma stage. Phosphotyrosine peptide enrichment and quantitative mass spectrometry identified oncogene-specific tyrosine kinase signatures, including activation of EGFR, ephrin type-A receptor 2 (EPHA2), and JAK2. Kinase:substrate relationship analysis of the phosphopeptides also revealed ABL1 and SRC tyrosine kinase activation. The observation of elevated tyrosine kinase signaling in advanced prostate cancer and identification of specific tyrosine kinase pathways from genetically defined tumor models point to unique therapeutic approaches using tyrosine kinase inhibitors for advanced prostate cancer.