Human Prostate Basal Cells Research Articles

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

Single-cell RNA sequencing of human prostate basal epithelial cells reveals zone-specific cellular populations and gene expression signatures.

Despite evidence of genetic signatures in normal tissue correlating with disease risk, prospectively identifying genetic drivers and cell types that underlie subsequent pathologies has historically been challenging. The human prostate is an ideal model to investigate this phenomenon because it is anatomically segregated into three glandular zones (central, peripheral, and transition) that develop differential pathologies: prostate cancer in the peripheral zone (PZ) and benign prostatic hyperplasia (BPH) in the transition zone (TZ), with the central zone (CZ) rarely developing disease. More specifically, prostatic basal cells have been implicated in differentiation and proliferation during prostate development and regeneration; however, the contribution of zonal variation and the critical role of basal cells in prostatic disease etiology are not well understood. Using single-cell RNA sequencing of primary prostate epithelial cultures, we elucidated organ-specific, zone-specific, and cluster-specific gene expression differences in basal cells isolated from human prostate and seminal vesicle (SV). Aggregated analysis identified ten distinct basal clusters by Uniform Manifold Approximation and Projection. Organ specificity compared gene expression in SV with the prostate. As expected, SV cells were distinct from prostate cells by clustering, gene expression, and pathway analysis. For prostate zone specificity, we identified two CZ-specific clusters, while the TZ and PZ populations clustered together. Despite these similarities, differential gene expression was identified between PZ and TZ samples that correlated with gene expression profiles in prostate cancer and BPH, respectively. Zone-specific profiles and cell type-specific markers were validated using immunostaining and bioinformatic analyses of publicly available RNA-seq datasets. Understanding the baseline differences at the organ, zonal, and cellular level provides important insight into the potential drivers of prostatic disease and guides the investigation of novel preventive or curative treatments. Importantly, this study identifies multiple prostate basal cell populations and cell type-specific gene signatures within prostate basal epithelial cells that have potential critical roles in driving prostatic diseases. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract A073: Human prostate-on-chip models to define stromal and epithelial interactions in normal and cancerous prostate

Abstract Background: The secretion of morphogenic factors from the stroma, whose production is dependent on stromal androgen receptor (AR) action, is crucial for maintaining glandular structure and homeostatic function of the prostate gland. However, the precise nature of the factors involved, their regulation, and how they impact homeostasis in the human prostate is not clear. We hypothesize that loss of stromal AR function as seen in prostate cancer progression and during androgen-deprivation therapy likely contribute to tumor progression through disruption of this homeostasis. It is important to understand these interactions in the human context to better aid in improving patient outcomes. Approach: Microfluidic-based technology was used to develop the first human Prostate-on-Chip (PoC) where human prostate fibroblasts are co-cultured with human prostate basal epithelial cells under flow. Addition of androgen to the stromal cells is sufficient to induce luminal cell differentiation on top of the neighboring basal cells – recapitulating the cellular architecture of the human gland. To study prostate cancer in this context, we replaced the normal epithelial cells with tumor cells to interrogate the tumor-stromal interactions; thus, generating a human Prostate Cancer-on-Chip model (PCoC). Cytokine arrays and RT-qPCR was used to identify androgen-induced stromal morphogens and tumor cytokines involved in tumor/stromal interactions. Molecular biology approaches were used to identify the mechanisms involved. Results: Within the PCoC model, tumor cells invaded into the stroma and conversely stromal cells invaded into the tumors. This was accompanied by localized CAF conversion of the stroma. Invasion of tumor cells into the stroma could be blocked with an integrin-α6 binding peptide. Strikingly, the level of AR expression in the stroma was also reduced, recapitulating the loss of stromal AR expression seen in patients. A cytokine array identified TNFα and TGFβ as tumor secreted factors responsible for suppressing stromal AR protein and mRNA expression. TNFα suppressed stromal AR expression in the absence of CAF conversion via NF-κB binding to the AR promoter. On the other hand, p38-MAPK signaling suppressed AR expression independent of TNFα. FGF10 and Wnt16, but not KGF, were identified as androgen-induced stromal secreted morphogens. Loss of stromal AR expression, led to suppression of FGF10 and Wnt16, but not KGF expression. Thus, stromal AR inhibition by tumor secreted TNFα or p38-MAPK signaling suppresses the stromal morphogens required to maintain normal prostate homeostasis. Conclusions: We have developed the first human Prostate-on-Chip and Prostate Cancer-on-Chip models which recapitulate the biology of both the normal human prostate as well as prostate cancer stromal interactions. These models can be used to address a range of clinical problems in cancer development, drug response, and biomarker identification in the context of the tumor microenvironment. This model can easily be adapted for personalized medicine approaches. Citation Format: Shekha K. Tahsin, Linan Jiang, Neha Sane, Kailie Szewczyk, Hunain Khawaja, Yitshak Zohar, Cindy K. Miranti. Human prostate-on-chip models to define stromal and epithelial interactions in normal and cancerous prostate [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A073.

LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation

Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).

Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.

Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC.

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc.

Abstract 1824: Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes

Abstract Background: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells. Androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Cancer cell escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. Methods: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen and AR dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial EP156T-AR cells, but did not exert a positive feedback on endogenous AR expression. Mesenchymal type prostate EPT3-AR cells with exogenous AR expression, in contrast to epithelial type EP156T-AR cells, were androgen non-responsive and were unable to produce detectable PSA in the culture supernatants even with higher levels of exogenous AR protein than in EP156T-AR and LNCaP cells for up to 2 weeks in androgen containing growth medium. The restricted PSA expression in the mesenchymal context suggests that if ADT increases the pool of mesenchymal type prostate cancer cells, then this might go undetected during PSA monitoring of disease progression. Citation Format: Jan Roger Olsen, Waqas Azeem, Margrete R. Hellem, Kristo Marvyin, Yaping Hua, Yi Qu, Lisha Li, Biaoyang Lin, XiSong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes. [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 1824.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes.

BackgroundExpression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.MethodsImmortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.ResultsA variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).ConclusionsRegulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users.

Transcriptome profiling links the intrinsic properties of human prostate basal cells to prostate cancer aggressiveness

ABSTRACTGenome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations reveals the unexpected findings that basal cells preferentially express gene categories associated with stem cells, neurogenesis, and rRNA biogenesis. Importantly, the basal cell gene expression profile is enriched in clinically advanced, anaplastic, castration-resistant, and metastatic prostate cancers.

Prostate epithelial cell of origin determines cancer differentiation state in an organoid transformation assay

The cell of origin for prostate cancer remains a subject of debate. Genetically engineered mouse models have demonstrated that both basal and luminal cells can serve as cells of origin for prostate cancer. Using a human prostate regeneration and transformation assay, our group previously demonstrated that basal cells can serve as efficient targets for transformation. Recently, a subpopulation of multipotent human luminal cells defined by CD26 expression that retains progenitor activity in a defined organoid culture was identified. We transduced primary human prostate basal and luminal cells with lentiviruses expressing c-Myc and activated AKT1 (myristoylated AKT1 or myrAKT1) to mimic theMYCamplification andPTENloss commonly detected in human prostate cancer. These cells were propagated in organoid culture before being transplanted into immunodeficient mice. We found that c-Myc/myrAKT1-transduced luminal xenografts exhibited histological features of well-differentiated acinar adenocarcinoma, with strong androgen receptor (AR) and prostate-specific antigen (PSA) expression. In contrast, c-Myc/myrAKT1-transduced basal xenografts were histologically more aggressive, with a loss of acinar structures and low/absent AR and PSA expression. Our findings imply that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types subjected to the same oncogenic insults. This study provides a platform for the functional evaluation of oncogenes in basal and luminal epithelial populations of the human prostate. Tumors derived in this fashion with defined genetics can be used in the preclinical development of targeted therapeutics.

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

The long‐term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin−Sca‐1+ CD49f+ Trop2high‐phenotype) and human (Lin− CD49f+ TROP2high) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti‐human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single‐cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f+/TROP2high phenotype of basal prostate progenitor cells and characterized by in vivo sandwich‐transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9+/CD24+/CD29+/CD44+/CD47+/CD49f+/CD104+/CD147+/CD326+/Trop2high of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan‐1 and stage‐specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f+ TROP2+ basal prostate progenitor cells. Transplantation experiments suggest that CD49f+ TROP2high SSEA‐4high human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f+ TROP2high or CD49f+ TROP2high SSEA‐4low cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA‐4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage.

A basal stem cell signature identifies aggressive prostate cancer phenotypes

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.

Abstract A28: N-Myc drives small cell neuroendocrine cancer initiated from human prostate basal cells

Abstract Human small cell prostate carcinoma (SCPC) represents up to 25% of lethal castration-resistant prostate cancer. Molecular characterization of a panel of SCPCs recently identified frequent genomic amplification of both MYCN and AURKA. The oncogenic function of N-Myc has been characterized in many cancer types but its role in the initiation of human SCPC has not been defined. To investigate the function of N-Myc in human prostate transformation, we utilized a tissue regeneration model of human prostate cancer. Human prostate basal epithelial cells were isolated from benign primary prostate tissue from four independent human donors, infected with lentiviruses expressing the oncogenes N-Myc and activated (myristoylated) AKT1, mixed with mouse urogenital sinus mesenchyme, and grafted subcutaneously in immunodeficient NOD-SCID-IL2Rγnull mice. Palpable tumors were appreciated within 2-3 months and histological evaluation revealed regions of mixed high-grade adenocarcinoma and small cell carcinoma. The small cell carcinoma histology showed typical findings of high nuclear-to-cytoplasmic ratio, nuclear molding, stippled chromatin, and frequent mitotic and apoptotic features. Immunohistochemical analysis demonstrated low to absent androgen receptor (AR) staining in both the adenocarcinoma and small cell carcinoma. Further, we found heterogeneous staining for neuroendocrine markers including neural cell adhesion molecule 1, chromogranin A, synaptophysin, thyroid transcription factor 1, and forkhead box A2 in the regions of small cell carcinoma in support of the histological diagnosis. To assess the androgen dependence of tumor growth, regenerated N-Myc and activated AKT1 tumors derived from the lentiviral infection of primary human prostate basal cells were passaged in either intact or castrate immunodeficient mice. Tumors were propagated both in the presence and absence of androgens and showed a small but progressive enrichment of the small cell carcinoma over the adenocarcinoma with successive passage in castrate hosts. Molecular profiling by RNA-Seq of three regenerated N-Myc and activated AKT1 tumors showed a significant resemblance to a subset of human SCPC clinical specimens. Further, the adenocarcinoma and small cell carcinoma components of each tumor were remarkably similar at the transcriptional level despite their differences in histological appearance. In conclusion, we have established a novel model of human small cell prostate cancer arising from benign human prostate basal cells. Overexpression of N-Myc and activated AKT1 in basal cells produces tumors that recapitulate the histology, immunophenotype, and transcriptional profile of human SCPC. These findings support a direct role for N-Myc in the neuroendocrine transformation of basal cells in the human prostate. Further studies are ongoing to elucidate the mechanisms by which N-Myc induces a neuroendocrine differentiation program in prostate cancer. Citation Format: John K. Lee, Bryan A. Smith, John W. Phillips, Jungwook Park, Tanya I. Stoyanova, Robert Baertsch, Artem Sokolov, Colleen Mathis, Donghui Cheng, Joshua M. Stuart, Jiaoti Huang, Owen N. Witte. N-Myc drives small cell neuroendocrine cancer initiated from human prostate basal cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A28.

Deep RNA-Seq analysis reveals unexpected features of human prostate basal epithelial cells

Abstract Prostate cancer is the second leading cause of cancer-related deaths among American men [ 1 ]. The prostate gland mainly contains basal and luminal cells, which are constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here, for the first time, we describe a whole-genome transcriptome analysis of human benign prostatic basal and luminal populations by using deep RNA sequencing (GSE67070) [2]. Combined with comprehensive molecular and biological characterizations, we show that the differential gene expression profiles account for their distinct functional phenotypes. Strikingly, in contrast to luminal cells, basal cells preferentially express gene categories associated with stem cells, neural and neuronal development, and RNA processing. Of clinical relevance, the treatment failed castration-resistant and anaplastic prostate cancers molecularly resemble a basal-like phenotype. We also identified genes associated with patient clinical outcome. Therefore, we provide a gene expression resource for understanding human prostate epithelial lineages, and link the cell-type specific gene signatures to subtypes of prostate cancer development.

A developmental stage-dependent switch of the mechanisms for prostate epithelial maintenance

A developmental stage-dependent switch of the mechanisms for prostate epithelial maintenance

Epcam, CD44, and CD49f distinguish sphere-forming human prostate basal cells from a subpopulation with predominant tubule initiation capability.

BackgroundHuman prostate basal cells expressing alpha-6 integrin (CD49fHi) and/or CD44 form prostaspheres in vitro. This functional trait is often correlated with stem/progenitor (S/P) activity, including the ability to self-renew and induce differentiated tubules in vivo. Antigenic profiles that distinguish tubule-initiating prostate stem cells (SCs) from progenitor cells (PCs) and mature luminal cells (LCs) with less regenerative potential are unknown.Methodology/Principle FindingsProstasphere assays and RT-PCR analysis was performed following FACS separation of total benign prostate cells based upon combinations of Epcam, CD44, and/or CD49f expression. Epithelial cell fractions were isolated, including Epcam+CD44+ and Epcam+CD44+CD49fHi basal cells that formed abundant spheres. When non-sphere-forming Epcam+CD44− cells were fractionated based upon CD49f expression, a distinct subpopulation (Epcam+CD44−CD49fHi) was identified that possessed a basal profile similar to Epcam+CD44+CD49fHi sphere-forming cells (p63+ARLoPSA−). Evaluation of tubule induction capability of fractionated cells was performed, in vivo, via a fully humanized prostate tissue regeneration assay. Non-sphere-forming Epcam+CD44− cells induced significantly more prostate tubular structures than Epcam+CD44+ sphere-forming cells. Further fractionation based upon CD49f co-expression identified Epcam+CD44−CD49fHi (non-sphere-forming) basal cells with significantly increased tubule induction activity compared to Epcam+CD44−CD49fLo (true) luminal cells.Conclusions/SignificanceOur data delineates antigenic profiles that functionally distinguish human prostate epithelial subpopulations, including putative SCs that display superior tubule initiation capability and induce differentiated ductal/acini structures, sphere-forming PCs with relatively decreased tubule initiation activity, and terminally differentiated LCs that lack both sphere–forming and tubule-initiation activity. The results clearly demonstrate that sphere-forming ability is not predictive of tubule-initiation activity. The subpopulations identified are of interest because they may play distinct roles as cells of origin in the development of prostatic diseases, including cancer.

A Tale of Two Cells: Discovering the Origin of Prostate Cancer

I n July, researchers in the lab of Owen Witte, M.D., at the University of California, Los Angeles, reported in Science that human prostate basal cells, transformed and then transplanted into mice, could form tumors. But in September 2009, a different group reported in Nature that prostate cancer in mice originated from a rare population of luminal cells. Basal versus luminal: irreconcilable? Apparently not. “Neither of them contradicts each other at all,” said Lynette Wilson, Ph.D., a professor in the departments of cell biology and urology at New York University. It’s possible that there are different prostate cancer cells of origin, she said. And the two papers are together a big step forward for a fi eld badly lacking such basic knowledge. That’s because the cellular origins of prostate cancer are still largely unknown. “The prostate cancer fi eld is struggling because, unlike [for] the breast, there is not a clear consensus about prostatic cell lineage,” said Gail Risbridger, Ph.D., a professor in the department of anatomy and developmental biology at Monash University in Melbourne, Australia. In breast cancer, six different subtypes have been mapped out, each arising from a distinct step in the differentiation of breast tissue from a common precursor cell. But we do not yet have such a map for prostate cancer. “This whole fi eld is still in its infancy,” Wilson said. Closing this knowledge gap could be the key to distinguishing between aggressive and indolent cancers and in developing individualized treatments — research that’s much further along in some other cancers. “It is conceivable that by identifying cell types of origin, you could distinguish different types of prostate cancer that differ in terms of their prognosis and/or treatment response,” said Michael Shen, Ph.D., a professor of medicine and genetics and development at Columbia University in New York. Shen led the group that reported in Nature last year that a rare population of luminal cells in mice gave rise to prostate cancer. That study and the recent report in Science open the door to new experiments that should pin down the exact cell types in which human prostate cancer originates.

Global Reactivation of Epigenetically Silenced Genes in Prostate Cancer

Transcriptional silencing associated with aberrant promoter hypermethylation is a common mechanism of inactivation of tumor suppressor genes in cancer cells. To globally profile the genes silenced by hypermethylation in prostate cancer, we screened a whole genome expression microarray for genes reactivated in the LNCaP, DU-145, PC-3, and MDA2b prostate tumor cell lines after treatment with the demethylating drug 5-aza-2-deoxycytidine and the histone deacetylation-inhibiting drug trichostatin A. A total of 2,997 genes showed at least 2-fold upregulation of expression after drug treatment in at least one prostate tumor cell line. For validation, we examined the first 45 genes, ranked by upregulation of expression, which had a typical CpG island and were known to be expressed in the normal cell counterpart. Two important findings were, first, that several genes known to be frequently hypermethylated in prostate cancer were apparent, and, second, that validation studies revealed eight novel genes hypermethylated in the prostate tumor cell lines, four of which were unmethylated in normal prostate cells and hypermethylated in primary prostate tumors (SLC15A3, 66%; KRT7, 54%; TACSTD2, 17%; GADD45b, 3%). Thus, we established the utility of our screen for genes hypermethylated in prostate cancer cells. One of the novel genes was TACSTD2/TROP2, a marker of human prostate basal cells with stem cell characteristics. TACSTD2 was unmethylated in prostatic intraepithelial neoplasia and may have utility in emerging methylation-based prostate cancer tests. Further study of the hypermethylome will provide insight into the biology of the disease and facilitate translational studies in prostate cancer.

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 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.