Basal Prostate Epithelial Cells Research Articles

Phospholipase D2 in prostate cancer: protein expression changes with Gleason score

BackgroundPhospholipases D1 and D2 (PLD1/2) are implicated in tumorigenesis through their generation of the signalling lipid phosphatidic acid and its downstream effects. Inhibition of PLD1 blocks prostate cell growth and colony formation. Here a role for PLD2 in prostate cancer (PCa), the major cancer of men in the western world, is examined.MethodsPLD2 expression was analysed by immunohistochemistry and western blotting. The effects of PLD2 inhibition on PCa cell viability and cell motility were measured using MTS, colony forming and wound-healing assays.ResultsPLD2 protein is expressed about equally in luminal and basal prostate epithelial cells. In cells from different Gleason-scored PCa tissue PLD2 protein expression is generally higher than in non-tumorigenic cells and increases in PCa tissue scored Gleason 6–8. PLD2 protein is detected in the cytosol and nucleus and had a punctate appearance. In BPH tissue stromal cells as well as basal and luminal cells express PLD2. PLD2 protein co-expresses with chromogranin A in castrate-resistant PCa tissue. PLD2 inhibition reduces PCa cell viability, colony forming ability and directional cell movement.ConclusionsPLD2 expression correlates with increasing Gleason score to GS8. PLD2 inhibition has the potential to reduce PCa progression.

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.

Abstract B20: Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin

Abstract Overexpression of Myc and loss of Pten are very common oncogenic events in prostate cancer. However, why these specific oncogenic events, as opposed to others, are involved in prostate cancer develop remains unclear. While the impact of Myc and Pten on cell proliferation is well-characterized, their role in differentiation and the resulting impact on tumorigenesis is poorly understood. We recently developed an in vitro differentiation model in which basal prostate epithelial cells (PrECs) can be differentiated into secretory luminal cells. Our model offers several advantages, in that it is human-specific and readily manipulated genetically and biochemically; providing a rare opportunity to dissect the specific genetic and biochemical events associated with human PrEC differentiation and determining how it is impacted by specific oncogenic events. Using this model, we tested the hypothesis that overexpression of Myc and loss of Pten induce prostate cancer because their dysregulation impairs differentiation of an intermediate progenitor cell population that marks the tumor cell of origin. Transient induction of both Pten and Myc are required for normal PrEC differentiation. Constitutive Myc overexpression initially accelerates differentiation, but the differentiated cells ultimately die via a p53-independent mechanism. Overexpression of Myc in combination with loss of Pten (Myc+shPten), is required for tumorigenesis because it rescues cell survival and blocks terminal differentiation. We identified the chromatin binding protein, ING4, as a Myc target required for PrEC terminal differentiation. ING4 is lost in over 60% of human prostate tumors and in the Myc+shPten cells, ING4 expression is blocked. Loss of ING4 in Myc overexpressing cells is sufficient to replace loss of Pten; ING4 loss, like Pten loss, is required for tumorigenesis to block terminal differentiation and keep cells alive. We further identified Miz1, a component of the Myc repressor complex, as a direct target of ING4 required for terminal PrEC differentiation. Myc/Miz1 is required to suppress integrin α6 and β1 expression during terminal PrEC differentiation; in Myc+shPten cells, Miz1 fails to be induced and integrin α6β1 is aberrantly co-expressed with AR in an intermediate progenitor and tumorigenic cell population. Conclusions: Prostate cancer oncogenesis requires dysregulation of Myc and Pten, because they are required for the normal PrEC terminal differentiation pathway. Pten loss is required to remove the ING4/Miz1-mediated terminal differentiation program initiated by Myc overexpression, to maintain the intermediate progenitor cells in a partially differentiated, yet proliferative state, and to evade cell death. Citation Format: Penny Berger, McLane Watson, Mary Winn, Cindy K. Miranti. Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B20.

Abstract B18: Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development

Abstract Myc is overexpressed in the majority of human prostate cancers and is a known determinant of cell fate, yet the cell of origin from which prostate cancers arise is controversial. Furthermore, the mechanisms by which oncogenes such as Myc disrupt prostate epithelial cell fate are poorly understood. Using a novel human in vitro differentiation model in which prostate basal epithelial cells are induced to differentiate into lumenal cells, we previously demonstrated that Myc-driven prostate cancer develops in an intermediate progenitor cell population whose full differentiation is derailed upon oncogenic transformation (Berger et al, Cancer Res 74:3357-68, 2014). In basal prostate epithelial cells, Myc is required for transient expression of the chromatin-binding protein ING4, which is required for lumenal cell differentiation. In human tissues, ING4 expression is lost in >60% of primary prostate tumors. Loss of ING4 prevented differentiation and was necessary for Myc-dependent tumorigenesis in vivo. ING4 loss generated Myc-dependent tumor cells co-expressing basal and lumenal markers, indicating Myc-dependent oncogenesis disrupted an intermediate step in the prostate epithelial differentiation program. Our objective for this study was to further elucidate the mechanisms by which Myc controls prostate epithelial cell fate. Myc is a known downstream target of Notch1, and several studies suggest Notch signaling is aberrant in prostate cancer; although the mechanistic details are vague. We found that Notch3 is required for lumenal cell differentiation and hypothesized that Notch3 expression is directly controlled by Myc. Inhibition of total Notch signaling with a γ-secretase inhibitor (RO4929097) prevented differentiation. Total Notch1 mRNA and protein levels change very little during differentiation; whereas both Notch3 mRNA and protein increase dramatically. Knock-down of Notch3 by shRNA blocked differentiation, while over expression of active Notch3 (NCID3) induced spontaneous differentiation. Less than 15% of the increase in Notch3 mRNA was attributable to increased mRNA stability, and required new protein synthesis. Temporally, Myc mRNA and protein levels increase prior to Notch3. Blocking Myc expression prevented Notch3 induction. The 2kb proximal promoter region of Notch3 lacked the elements that promote Notch3 induction. We identified a Notch3 enhancer element with Myc binding motifs that support differentiation-induced luciferase reporter activity. We further determined that p38α-MAPK is required for Myc and Notch3 induction. We are currently determining how Myc-dependent regulation of Notch3 influences Myc-dependent regulation of ING4. Thus, our studies demonstrate that at least 2 targets of Myc, ING4 and Notch3, control prostate epithelial cell fate, and that disruption of at least one of them is required for Myc-driven human prostate cancer development. In depth understanding of Myc-driven differentiation pathways will provide new insights into how oncogenic transformation by Myc in intermediate progenitor prostate epithelial cells gives rise to prostate cancer. Funding was provided by the Association for International Cancer Research, NIH/NCI CA154835, Department of Defense W81XWH-14-10479, and the Van Andel Research Institute. Citation Format: Sander B. Frank, Penny L. Berger, Cindy K. Miranti. Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development. [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 B18.

Induced Pluripotency of Human Prostatic Epithelial Cells

Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract C63: Understanding prostate cancer initiation and development through differentiation: Role of ING4 and Myc in prostate differentiation and cancer development

Abstract Introduction: Genes involved in differentiation and cell fate determination are frequently altered in cancers. Understanding how normal differentiation is controlled, and how its deregulation leads to cancer development is paramount to developing better diagnostic and therapeutic strategies for prostate cancer. Our laboratory recently developed an in vitro differentiation model in which human AR-negative basal prostate epithelial cells can be differentiated in to AR-positive secretory cells. Loss of integrin expression and cell-matrix adhesion is crucial to generating stable AR-expressing cells. This is consistent with the observation that integrins are only expressed in basal cells and AR is only expressed in secretory cells. However, in prostate cancer basal cells are lost and AR and integrin α6β1 are co-expressed in the tumor cells. The mechanism by which this occurs is unknown. Our hypothesis is that oncogenic conversion disrupts an intermediate step in normal prostate epithelial differentiation, causing retention of integrin α6β1 and AR expression in the same cell. The earliest alterations in cell adhesion during differentiation are the loss of the extracellular matrix laminin 5 and its respective ligand-specific integrin subunits, α3 and β4. This results in the pairing of the α6 and β1 subunits to generate α6β1, which is not lost until several days later. Several lines of evidence suggest Myc is required to suppress α6β1 expression. Misregulated expression of Myc may contribute to prostate cancer development through up-regulation of integrin α6β1. ING4, a PHD finger containing nuclear protein, is a tumor suppressor that blocks Myc-induced hyperplasia. We hypothesize that ING4 controls Myc, is vital for the normal prostate epithelial differentiation, and loss of ING4 is required for tumorigenesis. Methods: We manipulated Myc and ING4 expression in differentiating human prostate epithelial cells and measured the effects on differentiation. We generated tumorigenic variants by over expressing Myc and Erg, and suppressing Pten. We measured ING4 expression in prostate cancer tissues. Results: Myc over expression resulted in faster differentiation, i.e. loss of integrin expression and induction of AR-responsive genes. However, the differentiated Myc over expressing cells eventually died while control cells did not. ING4 expression increased transiently during differentiation. Loss of ING4 expression did not affect early differentiation; however, cells could not complete differentiation. Myc over expression resulted in an increase in ING4 expression, and ING4 over expression, like Myc, initially augmented differentiation, but induced death of the differentiated cells. Knock-down of ING4 reversed the effect of Myc over expression. Epithelial cells over expressing Myc, Erg, and shPten were tumorigenic in mice, but failed to properly differentiate in vitro. They initiated the AR program, but failed to switch off integrin expression. Over-expression of ING4 in the tumorigenic cells restored normal differentiation. In a cohort of 50 patients, ING4 expression was lost in 67% of primary tumors. Conclusions: These data indicate Myc stimulates ING4 expression in mid differentiation and ING4 is required transiently for proper differentiation and loss of integrin expression. Sustained over expression of ING4, directly or indirectly by Myc, results in death of the differentiated cells. In tumorigenic cells, the differentiation program is altered such that Myc over expression no longer promotes full differentiation, ING4 is not turned on, and cells survive. We hypothesize that ING4 loss is required for prostate cancer development to overcome the death-inducing effects of Myc over expression. Funding: Association for International Cancer Research and VARI. Citation Format: Penny Berger, Suwon Kim, Cindy K. Miranti. Understanding prostate cancer initiation and development through differentiation: Role of ING4 and Myc in prostate differentiation and cancer development [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C63.

Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells

Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.

KGF suppresses α2β1 integrin function and promotes differentiation of the transient amplifying population in human prostatic epithelium

Prostate epithelial stem cells are self-renewing cells capable of differentiation into prostate epithelium, and are thought to contribute towards both benign and malignant conditions in the human prostate. We have previously demonstrated that prostate epithelial basal cells express high levels of integrin alpha2beta1 and this population can be subdivided into stem (alpha2beta1(hi) CD133+) and transient-amplifying population (TAP) cells (alpha2beta1(hi) CD133-). However, the molecular mechanism(s) controlling the commitment and regulation of these cells towards differentiated epithelium remains unclear. Here, we demonstrate that beta1 integrin function is required for the maintenance of basal prostatic epithelial cells and suppression of its function by either methylcellulose or, more specifically, beta1-blocking antibody (80 microg/ml) induces differentiation, with associated expression of the differentiation-specific markers prostate acid phosphatase (PAP) and cytokeratin 18 (CK18). Keratinocyte growth factor (KGF), a stromal-derived growth factor, has previously been implicated in prostate organogenesis using in vitro tissue recombination experiments. We show that treatment with KGF (10 ng/ml) potently induces epithelial differentiation with concomitant suppression of alpha2beta1 integrin expression as well as the induction of androgen receptor expression. Specifically, p38-MAPK appears to be involved and the presence of SB202190, a p38 inhibitor, significantly blocks KGF-induced differentiation. Furthermore, the expression of the high-affinity receptor tyrosine kinase to KGF (FGFR2) is predominantly detectable in alpha2beta1(hi) CD133- TAP cells when compared with stem cells (alpha2beta1(hi) CD133+), which would therefore be relatively unresponsive to the differentiating effect of KGF. Taken together, using a human primary culture model, we have demonstrated key roles for interactions between KGF and integrin-mediated function in the regulation of prostate epithelial differentiation.

Basal prostate epithelial cells stimulate the migration of prostate cancer cells.

Carcinoma cells in PIN are situated above a layer of basal epithelial cells, which shield the tumor cells from stimulation by factors from the prostate stroma. During progression to invasive carcinoma, the basal cell layer becomes disrupted and tumor cells adhere to the basement membrane. The close proximity of basal epithelial cells to tumor cells in the early stages of prostate oncogenesis raises the possibility that basal epithelial cells participate in tumor cell invasion. Here, we investigated the migration-promoting activity of secreted factors from basal epithelial cells on BPH-1 cells, which we used as an in vitro model of preinvasive prostate cancer cells. We showed that the conditioned medium of basal epithelial cells (PEC-CM) contains adhesion proteins and chemotactic factors that stimulate adhesion, planar polarization, migration, and phosphorylation of Akt and that LY294002 and Wortmannin partially inhibit PEC-CM-triggered migration. We identified laminin-5 as a major migration-stimulating protein for BPH-1 cells in PEC-CM. Laminin-5 induced migration is completely inhibited by LY294002 or Wortmannin. In addition, antibody-depletion of laminin-5 from PEC-CM significantly diminishes the migration of BPH-1 cells. These results demonstrated, that laminin-5 is secreted by basal prostate epithelial cells in vivo and in vitro and stimulates migration of BPH-1 cells through a PI3-kinase dependent mechanism. Altogether, the possibility that basal epithelial cells assist in the invasion of in situ carcinoma cells is supported by the results from our in vitro system.

Regulation of trespin expression by modulators of cell growth, differentiation, and apoptosis in prostatic epithelial cells

We recently identified a novel rat ov-serpin, Trespin, which inhibits the trypsin-like serine proteinase plasmin and is expressed in several tissues, including prostate. In this report Trespin expression was studied in prostatic cell lines, NRP-152, NRP-154, and DP-153, derived from the Lobund-Wistar rat. Northern blots revealed Trespin mRNA is expressed in NRP-152 and DP-153 basal epithelial cell lines but not in the luminal line, NRP-154. Similarly, Trespin levels drop >30-fold following transdifferentiation of NRP-152 cells toward a luminal variant, further suggesting Trespin expression is specific for basal prostatic epithelial cells. Trespin expression in NRP-152 cells is up-regulated by dexamethasone (Dex) and insulin-like growth factor-I (IGF-I), each of which stimulate growth and prevent differentiation and apoptosis. However, Dex (alone) facilitates loss of Trespin by TGF-β, yet enhances the ability of LR 3-IGF-I to reverse such loss, similar to the pattern of apoptosis induced by TGF-β. Likewise, several apoptosis inducers markedly decrease Trespin mRNA levels. HEK293 cells stably overexpressing Trespin display increased cell proliferation and partial resistance to growth inhibition and phosphorylation of c-Jun induced by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA). Together these data strongly suggest that Trespin has critical functions tied to the regulation of growth, differentiation, and apoptosis of prostatic epithelial cells.

Transdifferentiation of NRP-152 rat prostatic basal epithelial cells toward a luminal phenotype: regulation by glucocorticoid, insulin-like growth factor-I and transforming growth factor-beta.

The role of basal epithelial cells in prostatic function, development and carcinogenesis is unknown. The ability of basal prostatic epithelial cells to acquire a luminal phenotype was explored in vitro using the NRP-152 rat dorsal-lateral prostate epithelial cell line as a model system. NRP-152, which was spontaneously immortalized and clonally derived, is an androgen-responsive and nontumorigenic cell line that has a basal cell phenotype under normal growth conditions. However, when placed in mitogen-deficient media, these cells undergo a dramatic morphological change to a luminal phenotype. Under these growth-restrictive conditions, immunocytochemical analysis shows that NRP-152 cells acquire the luminal markers Z0-1 (a tight-junction associated protein), occludin (integral tight-junction protein), and cytokeratin 18, and lose the basal markers cytokeratins 5 and 14. Total protein and mRNA levels of cytokeratins 8, 18, c-CAM 105 (the calcium-independent cell adhesion molecule) and Z0-1, as detected by western and/or northern blot analyses, respectively, are induced, while cytokeratin 5 and 15 are lost, and occludin is unchanged. Concomitant with this differentiation, expression of transforming growth factor-beta2 (TGF-beta2), TGF-beta3, and TGF-beta receptor type II (TbetaRII) is induced, while those of TGF-beta1 and TbetaRI remain essentially unchanged. Mitogens, such as insulin-like growth factor-I and dexamethasone inhibit luminal differentiation, while exogenous TGF-beta induces such differentiation. These data together with TGF-beta neutralization experiments using pan-specific antibody implicate an important role for autocrine TGF-beta in the induction of the luminal differentiation.

Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens

We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA. Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.