Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.     

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

Epithelial phenotypes in the developing human prostate.

An intermediate population has been identified among prostate glands called transiently amplifying (TA) cells, which are characterized by coexpression of basal and luminal cytokeratins (CKs), high proliferation, and lack of p27 expression. These cells are rare in the normal adult prostate and increase in pretumoral conditions, but their importance in the developing gland remains unknown. We analyzed fetal prostates for the expression of CKs (5/6, 18, 19) and factors involved in proliferation and apoptosis: p63, Ki67, p27, epidermal growth factor (EGFR), Bcl2, androgen receptor (AR). Immunostaining was performed on a tissue microarray, including 40 prostates from fetuses aged 13-42 weeks and normal prostate tissue from 10 adults. In both solid buds and the basal compartment of canalized glands, cells expressed p63, CK5/6, CK19, CK18, BCL2, EGFR and were p27 negative. Luminal cells of fetal canalized glands continue to express CK19, EGFR, and BCL2, without p27 expression. In contrast, adult epithelial luminal cells showed diffuse AR and p27 expression, without CK19, BCL2, and EGFR staining. Proliferation was high and diffuse in fetal glands and rare and restricted to basal cells in adult glands. These results indicate that most fetal epithelial prostatic cells exhibit the phenotype of TA cells, suggesting their regulatory function in prostate development.     

EPN: a novel epithelial cell line derived from human prostate tissue

This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo "Hayflike crisis" and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.     

Inhibition of CHOP accentuates the apoptotic effect of α-mangostin from the mangosteen fruit (Garcinia mangostana) in 22Rv1 prostate cancer cells

The mangosteen (Garcinia mangostana) fruit has been a popular food in Southeast Asia for centuries and is increasing in popularity in Western countries. We identified α-Mangostin as a primary phytochemical modulating ER stress proteins in prostate cancer cells and propose that α-Mangostin is responsible for exerting a biological effect in prostate cancer cells. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells procured from two patients undergoing radical prostatectomy were treated with α-Mangostin and evaluated by RT-PCR, Western blot, fluorescent microscopy and siRNA transfection to evaluate ER stress. Next, we evaluated α-Mangostin for microsomal stability, pharmacokinetic parameters, and anti-cancer activity in nude mice. α-Mangostin significantly upregulated ER stress markers in prostate cancer cells. Interestingly, α-Mangostin did not promote ER stress in prostate epithelial cells (PrECs) from prostate cancer patients. CHOP knockdown enhanced α-Mangostin-induced apoptosis in prostate cancer cells. α-Mangostin significantly suppressed tumor growth in a xenograft tumor model without obvious toxicity. Our study suggests that α-Mangostin is not the only active constituent from the mangosteen fruit requiring further work to understand the complex chemical composition of the mangosteen.     

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin alpha2beta1(hi) and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 microg/ml insulin (DMEM+10% FBS+Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.     

Ubiquitylation of ε-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the ε-subunit of coatmer complex, ε-COP. PIRH2 promotes the ubiquitylation of ε-COP in vitro and in vivo and consequently promotes the degradation of ε-COP. The interaction between PIRH2 and ε-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of ε-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.     

A mouse prostate cancer model induced by Hedgehog overexpression

Summary Hedgehog is a regulatory protein during embryonic development and its abnormal activation in adult tissues has been implicated in tumorigenesis within sites where epithelial–mesenchymal interactions take place. In the prostate, Hedgehog signaling activation was observed during advanced cancer progression and metastasis, but whether Hedgehog overexpression can initiate prostate tumorigenesis remains unknown. We introduced a Hedgehog-expressing vector by intra-prostate injection and electroporation to address the effects of Hedgehog overexpression. The manipulation caused lesions with characteristic prostatic intraepithelial neoplasia or even prostatic cancer (CaP) phenotypes within 30 days, with Hedgehog overexpression demonstrated by immunohistochemistry and Western blot detections. The tumorigenic phenotypes were confirmed by discontinuity of basal cell marker p63, mix-up of CK-8/CK-18 positive epithelial cells in the stoma as well as absence of α-SMA positive fibro-muscular sheath. Comparable Hedgehog overexpression was found in human CaP specimen. Thus, Hedgehog overexpression induced prostate tumorigenesis starting from the normal status. Furthermore, a mouse prostate cancer model induced by Hedgehog overexpression was established and may be used for testing novel therapeutical approaches targeting at Hedgehog signaling pathway.These authors have contributed equally to this work.     

PC-１基因表达增强C4-2B前列腺癌细胞生存

建立稳定表达外源PC-１基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC- １基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-１基因的真核表达载体pcDNA3.1PC-１稳定转染C4-2B细胞,Western 印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-１基因表达. MTT和软琼脂集落形成能力等一系列方法,研究PC-１基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化. 结果表明,PC-１基因的高表达能够诱导雄激素受体（AR）调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX31、Jagged1、EphA3、SGEF和 NOTCH3等表达发生变化. 实验结果初步证明,PC-１基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-１基因表达可调控一些重要信号通路.对PC-１基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.     

Molecular basis for the antiandrogen withdrawal syndrome

In patients with prostate cancer who manifest disease progression during combined androgen blockade therapy, discontinuation of antiandrogen treatment might result in prostate-specific antigen decline, often associated with clinical improvement. The response called antiandrogen withdrawal syndrome is thus acknowledged as a general phenomenon. However, molecular mechanisms responsible for this syndrome are not completely understood. This article outlines the proposed mechanisms, including alterations of androgen receptor gene and its coregulatory proteins and activation of the signal transduction pathway, and the potential therapeutic approaches based on the specific mechanisms.     

The application of the RT-PCR method for the staging of the prostate cancer progression

Molecular biology seems to bring more convincing markers for the detection of prostate cancer as well as the development of metastases than immunohistochemistry. The main goal of present work was to detect the expression of prostate specific antigen (PSA) and prostate-specific membrane antigen (PSM) genes in the micrometastases by the RT-PCR to assess the progression of prostate cancer. We analyzed 50 patients: 28 patients with clinically localized or locally advanced prostate cancer who underwent radical prostatectomy, 7 patients with clinically proven metastases, 8 patients with benign prostatic hyperplasia, and 7 healthy young men. The results of RT-PCR in the first group of 28 patients varied, however, they were in good correlation with the health status of the patients. Positive results of PSA and notably for PSM were good predictors of beginning metastasing process. Seven patients with metastatic disease had positive RT-PCR results both for PSA and PSM. All of the patients with benign prostatic hyperplasia and healthy young men had negative RT-PCR results for PSA and PSM. The study showed that positive RT-PCR results for PSA and especially for PSM correlated well with the progression of the disease and negative results reflected good health status of the patients.