The Prostate Basal Cell (BC) Heterogeneity and the p63-Positive BC Differentiation Spectrum in Mice

The prostate epithelium is composed of basal (BC), luminal (LEC), and neuroendocrine (NEC) cells. It is unclear how many subtypes of BCs in the prostate and which subtype of BCs contains the main stem cell niche in the adult prostate. Here we report seven BC subpopulations according to their p63, cytokeratin 14 (K14) and K5 expression patterns, including p63-positive/K14-negative/K5-negative (p63+/K14-/K5-), p63-/K14+/K5-, p63-/K14-/K5+, p63+/K14+/K5-, p63+/K14-/K5+, p63-/K14+/K5+, and p63+/K14+/K5+ BCs. We generated a p63-CreERT2 knock-in mouse line that expresses tamoxifen-inducible Cre activity in the p63-expressing cells, including the prostate BCs. We then crossbred this line with ROSA26R mice, and generated p63-CreERT2×ROSA26R bi-genic mice harboring the Cre-activated β-galactosidase reporter gene. We treated these bi-genic mice with tamoxifen to mark the p63+ BCs at different ages or under different hormonal conditions, and then traced the lineage differentiation of these genetically labeled BCs. We discovered that these p63+ BCs contain self-renewable stem cells in culture and efficiently differentiated into LECs, NECs and BCs in the postnatal, adult and re-generating mouse prostates. Therefore, BC population contains heterogeneous BCs that express different combinations of the p63, K14 and K5 differentiation markers. Because K14+ and K5+ BCs were previously shown to be extremely inefficient to produce LECs in adulthood, we propose that the p63+/K5-/K14- subpopulation of BCs contains most stem-like cells, especially in adult animals.     

Spatial-Temporal Lineage Restrictions of Embryonic p63+ Progenitors Establish Distinct Stem Cell Pools in Adult Airways

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Aberrant p63 and WT-1 expression in myoepithelial cells of pregnancy-associated breast cancer: implications for tumor aggressiveness and invasiveness

Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly associated with tumor aggressiveness and invasiveness.     

Stem cells (p63(+)) in keratinocyte cultures from human adult skin

Epidermal stem cells (ESC) are responsible for maintaining skin cellular homeostasis, as they give rise to fast-dividing transit amplifying cells committed to terminal differentiation, while retaining their self-renewal capacity. However, no pure ESC cultures are available and no highly specific cytochemical marker was identified. We report here the experimental conditions allowing the selective enrichment in ESC, using cultured adult human keratinocytes. The main step was the selection of cells able to rapidly adhere to human collagen type IV in vitro . Thus, an increased proportion of putative ESC of about 65% was obtained, as demonstrated by p63 expression.     

p63基因的结构与功能

p63基因是肿瘤抑制基因p53家族成员之一,与p53基因表现出高度同源性.较之p53基因,p63基因更为复杂.p63的两个不同启动子和多种内含子剪接方式,导致p63基因编码产生多种亚型P63蛋白.这些P63亚型蛋白,在不同的组织不同的发育阶段发挥不同的生物学功能.本文就p63基因结构、p63在细胞周期和凋亡中的作用,以及在表皮发育中的功能等方面的研究进展作一概述.     

p63 is a prosurvival factor in the adult mammary gland during post-lactational involution,affecting PI-MECs and ErbB2 tumorigenesis

In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/− glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/− glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/− mice again exhibit normal epithelial architecture by conventional histology. However, using Rosa^LSL-LacZ;WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/− glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/− ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.     

The Notch pathway in prostate development and cancer

Abstract The Notch family of transmembrane receptors are important mediators of cell fate determination. Accordingly, Notch signaling is intimately involved in the development of numerous tissues. Recent findings have highlighted a critical role for Notch signaling in normal prostate development. Notch signaling is required for embryonic and postnatal prostatic growth and development, for proper cell lineage specification within the prostate, as well as for adult prostate maintenance and regeneration following castration and hormone replacement. Evidence for Notch as a regulator of prostate cancer development, progression, and metastasis has also emerged. This review summarizes our current understanding of the role of Notch pathway elements, including members of the Jagged, Delta-like, hairy/enhancer-of-split, and hairy/enhancer-of-split related with YRPW motif families, in prostate development and tumorigenesis. Data supporting Notch pathway elements as oncogenes and tumor suppressors in prostate tumors, as well as data implicating Notch receptors and ligands as potential markers of normal prostate stem/progenitor cells and prostate cancer stem/initiating cells, are also presented.     

p63与p21在鼻咽癌细胞中的表达及相互作用的初步研究

鼻咽癌是中国南方地区常见的恶性肿瘤,p63是新发现的p53家族成员.本研究利用免疫组化方法对p63、p53及p21基因在鼻咽癌中的表达进行了检测,发现三者在鼻咽癌中均有表达;并利用染色质免疫共沉淀(ChIP)的方法对p63及p21的相互作用进行了初步研究,发现P63蛋白能与p21基因启动子区域的一个位点结合.     

p63亚型及其与疾病的相关性

p63足p53家族成员的核转录因子,根据N端及C端的不同,已经发现TAp630α、TAp63β、rap63y、ANp630α、△Np63β、△Np63β、△Np63δ、△Np63δ种亚型。p63的表达受到多种转录因子的调控,其mRNA的稳定性由RNPCI调节,蛋白的稳定性主要由HECT家族成员Itch／AIP4、WWPI调节。p63在上皮细胞分化、组织发育过程中起着关键性作用,因此,p63基因突变可以导致外胚层发育不良的相关疾病,同时,p63在肿瘤的形成和转移的过程中具有重要的调控作用。     

p63 is a suppressor of tumorigenesis and metastasis interacting with mutant p53

p53 mutations, occurring in two-thirds of all human cancers, confer a gain of function phenotype, including the ability to form metastasis, the determining feature in the prognosis of most human cancer. This effect seems mediated at least partially by its ability to physically interact with p63, thus affecting a cell invasion pathway, and accordingly, p63 is deregulated in human cancers. In addition, p63, as an 'epithelial organizer', directly impinges on epidermal mesenchimal transition, stemness, senescence, cell death and cell cycle arrest, all determinant in cancer, and thus p63 affects chemosensitivity and chemoresistance. This demonstrates an important role for p63 in cancer development and its progression, and the aim of this review is to set this new evidence that links p63 to metastasis within the context of the long conserved other functions of p63.     

Differential effects of p63 mutants on transactivation of p53 and/or p63 responsive genes

p63, known to play a role in development, has more recently also been implicated in cancer progression. Mutations in p63 have been shown to be responsible for several human developmental diseases. Differential splicing of the p63 gene gives rise to p63 isoforms, which can act either as tumor suppressors or as oncogene. In this report, we studied the effects of naturally occurring TAp637 mutants on the regulation of p53/p63 and p63 specific target genes. We observed significant differences among p63 mutants to regulate the p53/p63 and p63 specific target genes. Additionally, we observed a differential effect of p63 mutants on wildtype-p63-mediated induction ofp53/p63 and p63 specific target genes. We also demonstrated that these mutants differentially regulate the binding of wildtype p63 to the promoter of target genes. Furthermore, the effects of these mutants on cell death and survival were consistent with their ability to regulate the downstream targets when compared to wildtype TAp63T. In summary, our data demonstrate that p63 mutants exhibit differential effects on p63 and p53/p63 specific target genes and on the induction of apoptosis, and provide further insight into the function of p63.     

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.     

Deletion of p21/Cdkn1a confers protective effect against prostate tumorigenesis in transgenic adenocarcinoma of the mouse prostate model

Cyclin-dependent kinase inhibitors (CDKIs) p21^Cip1/Waf1 (p21) and p27^Kip1 (p27) play a determining role in cell cycle progression by regulating CDK activity; however, p21 role in prostate cancer (PCa) is controversial. Whereas p21 upregulation by anticancer agents causes cell cycle arrest in various PCa cell lines, elevated p21 levels have been associated with higher Gleason score, poor survival and increased PCa recurrence. These conflicting findings suggest that more studies are needed to examine p21 role in PCa. Herein, employing genetic approach, transgenic mice harboring p21/Cdkn1a homozygous deletion (p21^−/−) were crossed with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice to characterize in vivo consequences of p21 deletion on prostate tumorigenesis. Lower urogenital tract weight of p21^−/−/TRAMP mice was significantly lower than those of p21^+/−/TRAMP and TRAMP mice. Histopathology further supported these observations, showing less aggressiveness in prostates of p21^−/−/TRAMP. Furthermore, a significantly higher incidence of low-grade prostatic intraepithelial lesions (PIN) with a concomitant reduction in adenocarcinoma incidence was observed in p21^−/−/TRAMP mice compared with TRAMP mice. In addition, whereas TRAMP mice showed the presence of poorly differentiated adenocarcinoma lesions, no such lesions were observed in p21/TRAMP transgenic mice. Specifically, there was a significant reduction in the severity of lesions in both p21^−/−/TRAMP and p21^+/−/TRAMP mice compared with TRAMP mice. Together, our data showed that p21 deletion reduces prostate tumorigenesis by slowing-down progression of PIN (pre-malignant) to adenocarcinoma (malignant), suggesting that intact p21 expression is associated with PCa aggressiveness, while its decreased levels may in fact confer protection against prostate tumorigenesis.     

p63 and p73 expression in extrahepatic bile duct carcinoma and their clinical significance

p53 plays a pivotal role in the prevention of human tumor formation. p73 and p63 are new members of the p53 tumor suppressor family, which are becoming increasingly recognized as important players in human tumorigenesis. However, the roles of these proteins are not well elucidated in extrahepatic bile duct (EBD) carcinoma. We examined expressions of the p63 and p73 genes and proteins in normal biliary epithelia, biliary dysplasias, and EBD carcinomas using immunohistochemistry and RT-PCR analysis. p63 and p73 proteins were overexpressed in 26.3 and 41.0% of EBD carcinomas, respectively. p63 protein expression was more frequent in tumors with vascular invasion (P = 0.002) and distal location (P = 0.04), while p73 expression was more common in cancers with deeper tumor invasion (P = 0.04). Patients with tumors co-expressing both p63 and p73 were found to have a significantly worse overall survival rate compared to those with either p63 or p73 expression (P < 0.05) as determined in univariate and multivariate analyses. Our results strongly imply that the p53 family members have different functions in EBD carcinomas. Our data also indicate that interactions between p63 and p73 play an important role in tumorigenesis of EBD carcinoma.