Embigin,a developmentally expressed member of the immunoglobulin super family,is also expressed during regression of prostate and mammary gland

Cell adhesion molecules (CAMs) are intimately involved in a variety of cellular processes, including development, cell growth, apoptosis, and differentiation. Interaction of CAMs with components of the extracellular matrix (ECM), growth factors, and other CAMs provides an intricate regulatory mechanism for a diverse range of cellular responses. Embigin is a developmentally expressed protein that is a member of the immunoglobulin superfamily (IgSF) class of CAMs. We have identified embigin as a gene expressed during tissue regression in rat prostate and lactating mammary gland following hormonal ablation. In the absence of the appropriate hormone, the secretory epithelial cells of these two tissues undergo successive waves of apoptotic cell death co-incident with extensive reorganization of the surviving tissue. Using Northern analysis, in situ hybridization analysis, RT-PCR, and Western analysis, we have characterized the expression of embigin mRNA and protein in both regressing prostate and mammary gland. During development of the prostate gland, increased expression of embigin is correlated with the appearance of highly organized lumenal and ductal structures. Embigin is also expressed in a variety of adult tissues including heart, liver, lung, and brain. Zoo-blot analysis with the rat embigin cDNA indicates that embigin homologs exist in species as diverse as Homo sapiens and Drosophila melanogaster, suggesting that it has been highly conserved during evolution. Embigin protein is expressed at readily detectable levels in a variety of prostate and mammary cancer cell lines, and in some cell lines the expression of embigin appears to be down-regulated in the presence of ECM. Our data have led us to propose a model in which embigin functions as a regulator of cell/ECM interactions during development and in the homeostasis of normal adult tissues. Dev. Genet. 21:268–278, 1997. © 1997 Wiley-Liss, Inc.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Cell fate regulation by reticulon-4 in human prostate cancers

Reticulon-4 (RTN4), a reticulon family protein localized in the endoplasmic reticulum, is reported to be involved in multiple physiological processes like neuroendocrine secretion and membrane trafficking in neuroendocrine cells. Previous studies have presented a great potential of RTN4 for the treatment of autoimmune-mediated demyelinating diseases and spinal cord injury regeneration. While interaction with Bcl-2 and Bcl-2-like family in apoptosis modulation implicated its possible role in various human cancers. However, the investigation of this gene in prostate cancer is mainly ignored. Here in our current study, we focused on its role in prostate cancer and found that RTN4 DNA copy numbers were higher in prostate cancer than normal prostate gland while its RNA and protein expressions were relatively lower. Chromosomal neighbor gene EML6 had similar expression patterns with RTN4 in prostate cancer tissues and cell lines, and further research found that they could be both targeted by miR-148a-3p. Lentivirus-mediated RTN4 overexpression potently inhibited DU145 and LNCaP cells proliferation. Cell cycle was blocked in G2/M phase and significant cell senescence was observed in RTN4 overexpressed prostate cancer cells. Finally, interaction networks in the normal prostate gland and cancer tissues further revealed that RTN4 maybe phosphorylated by MAPKAPK2 and FYN at tyrosine 591 and serine 107, respectively. All these results implied that RTN4 might somehow participate in prostate tumor progression, and this elicits possibility to develop or identify selective agents targeting RTN4 for prostate cancer therapy.     

Prostate epithelial stem cell culture

The prostate gland is the site of the second most common cancer in men in the UK, with 9,280 deaths recorded in 2000. Another common disease of the prostate is benign prostatic hyperplasia and both conditions are believed to arise as a result of changes in the balance between cell proliferation and differentiation. There are three types of prostatic epithelial cell, proliferative basal, secretory luminal, and neuroendocrine. All three are believed to be derived from a common stem cell through differentiation along different pathways but the mechanisms behind these processes is poorly understood. In particular, there has until recently been very little information about prostate stem cell growth and differentiation. This review will discuss ways of distinguishing these prostate cell types using markers, such as keratins. Methods available for the culture of prostate epithelial cells and for the characterisation of stem cells both in monolayer and three-dimensional models are examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Frequent HIN-1 promoter methylation and lack of expression in multiple human tumor types

HIN-1 (high in normal-1) is a candidate tumor suppressor identified as a gene silenced by methylation in the majority of breast carcinomas. HIN-1 is highly expressed in the mammary gland, trachea, lung, prostate, pancreas, and salivary gland, and in the lung, its expression is primarily restricted to bronchial epithelial cells. In this report, we show that, correlating with the secretory nature of HIN-1, high levels of HIN-1 protein are detected in bronchial lavage, saliva, plasma, and serum. To determine if, similar to breast carcinomas, HIN-1 is also silenced in tumors originating from other organs with high HIN-1 expression, we analyzed its expression and promoter methylation status in lung, prostate, and pancreatic carcinomas. Nearly all prostate and a significant fraction of lung and pancreatic carcinomas showed HIN-1 hypermethylation, and the majority of lung and prostate tumors lacked HIN-1 expression. In lung carcinomas, the degree of HIN-1 methylation differed among tumor subtypes (P = 0.02), with the highest level of HIN-1 methylation observed in squamous cell carcinomas and the lowest in small cell lung cancer. In lung adenocarcinomas, the expression of HIN-1 correlated with cellular differentiation status. Hypermethylation of the HIN-1 promoter was also frequently observed in normal tissue adjacent to tumors but not in normal tissue from noncancer patients, implying that HIN-1 promoter methylation may be a marker of premalignant changes. Thus, silencing of HIN-1 expression and methylation of its promoter occurs in multiple human cancer types, suggesting that elimination of HIN-1 function may contribute to several forms of epithelial tumorigenesis.     

New gene expressed in prostate: a potential target for T cell-mediated prostate cancer immunotherapy

New gene expressed in prostate (NGEP) is a prostate-specific gene encoding either a small cytoplasmic protein (NGEP-S) or a larger polytopic membrane protein (NGEP-L). NGEP-L expression is detectable only in prostate cancer, benign prostatic hyperplasia and normal prostate. We have identified an HLA-A2 binding NGEP epitope (designated P703) which was used to generate T cell lines from several patients with localized and metastatic prostate cancer. These T cell lines were able to specifically lyse HLA-A2 and NGEP-expressing human tumor cells. NGEP-P703 tetramer binding assays demonstrated that metastatic prostate cancer patients had a higher frequency of NGEP-specific T cells when compared with healthy donors. Moreover, an increased frequency of NGEP-specific T cells was detected in the peripheral blood mononuclear cells of prostate cancer patients post-vaccination with a PSA-based vaccine, further indicating the immunogenicity of NGEP. These studies thus identify NGEP as a potential target for T cell-mediated immunotherapy of prostate cancer.     

Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.

A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.     

Three mouse monoclonal antibodies to human differentiation antigens: reactivity with two mucin-like antigens and with connective tissue fibers

Three human differentiation antigens (MU78, MT334, and MQ49) have been defined by mouse monoclonal antibodies developed from mice immunized with ovarian carcinoma cell lines. Their distribution was determined on 148 cultured cell lines of various histologic types and on frozen sections of 16 normal tissues. MU78 was found in fibrillar structures in soft connective tissue with a distribution resembling that of elastin fibers; however, elastin fibers in elastic cartilage and in the aorta were nonreactive. MU78 was detected in cultured carcinoma cells of various histologic types, where it had a nonfibrillar, cytoplasmic distribution, but was not detected in normal epithelial cells in frozen sections. Cultured fibroblasts, astrocytomas, melanomas, and lymphomas did not contain MU78. In cell lines, MU78 appears to be a protein of 2000-5000 daltons. The other two antigens, MT334 and MQ49, are both mucin-like molecules, and the determinants are probably carbohydrate in nature. Of the normal tissues examined, MT334 was detected only in goblet cells of the colon, though it was present in a variety of carcinomas in culture. It was detected as both a cytoplasmic and secreted component. MQ49 was detected in various secretory epithelial cells, in Hassall's corpuscles in the thymus, and in cultured carcinomas of various histologic types. It was found on the cell surface as well as in the cytoplasm and is present on a glycolipid as well as on a sulfated mucin. These results, and results of other recent studies, demonstrate the importance of mucin-like molecules as antigens in epithelial cells and secretions.     

Molecular cloning and characterization of STAMP1, a highly prostate-specific six transmembrane protein that is overexpressed in prostate cancer

We have identified a novel gene, six transmembrane protein of prostate 1 (STAMP1), which is largely specific to prostate for expression and is predicted to code for a 490-amino acid six transmembrane protein. Using a form of STAMP1 labeled with green fluorescent protein in quantitative time-lapse and immunofluorescence confocal microscopy, we show that STAMP1 is localized to the Golgi complex, predominantly to the trans-Golgi network, and to the plasma membrane. STAMP1 also localizes to vesicular tubular structures in the cytosol and colocalizes with the early endosome antigen 1 (EEA1), suggesting that it may be involved in the secretory/endocytic pathways. STAMP1 is highly expressed in the androgen-sensitive, androgen receptor-positive prostate cancer cell line LNCaP, but not in androgen receptor-negative prostate cancer cell lines PC-3 and DU145. Furthermore, STAMP1 expression is significantly lower in the androgen-dependent human prostate xenograft CWR22 compared with the relapsed derivative CWR22R, suggesting that its expression may be deregulated during prostate cancer progression. Consistent with this notion, in situ analysis of human prostate cancer specimens indicated that STAMP1 is expressed exclusively in the epithelial cells of the prostate and its expression is significantly increased in prostate tumors compared with normal glands. Taken together, these data suggest that STAMP1 may have an important role in the normal prostate cell as well as in prostate cancer progression.     

Testosterone stimulates growth and secretory activity of the female prostate in the adult gerbil (Meriones unguiculatus)

The prostate of the female gerbil (Meriones unguiculatus) is similar to the human female prostate (Skene gland) and, despite its reduced size, it is functional and shows secretory activity. However, virtually nothing is known about its physiological regulation. This study was thus undertaken to evaluate the behavior of the gerbil female prostate in a hyperandrogenic condition. Adult females received subcutaneous injections of testosterone cypionate (1 mg/kg body weight every 48 h) up to 21 days. Circulating levels of testosterone and estradiol were monitored, and the prostate and ovaries subjected to structural and immunocytochemical analyses. The treatment resulted in sustained high levels of circulating testosterone, and caused a transient increase in estradiol. There was an increase in epithelial cell proliferation accompanied by significant reorganization of the epithelium and an apparent reduction in secretory activity, followed by a progressive increase in luminal volume density and accumulation of secretory products. Immunocytochemistry identified the expression of androgen receptor and a prostate-specific antigen (PSA)-related antigen in prostatic epithelial cells. A circulating PSA-related antigen was also found, and its concentration showed strong negative correlation with circulating estrogen. Epithelial dysplasia was detected in the prostate of treated females. Analysis of the ovaries showed the occurrence of a polycystic condition and stromal cell hyperplasia. The results indicate that testosterone has a stimulatory effect on the female prostate, inducing epithelial cell proliferation, differentiation, secretory activity, and dysplasia. The results also suggest that prostatic growth and activity, polycystic ovaries, and ovarian stromal cell hyperplasia are related to a hyperandrogenic condition in females.     

Selective Expression of Myosin IC Isoform A in Mouse and Human Cell Lines and Mouse Prostate Cancer Tissues

Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.     

Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

Prostate cancer expression profiling by cDNA sequencing analysis.

Prostate cancer is a frequently diagnosed solid tumor that is originated mostly from prostate epithelium. One of the key issues in prostate cancer research is to develop molecular markers that can effectively detect and distinguish the progression and malignancy of prostate tumors. Automated, single-pass cDNA sequencing was utilized to rapidly identify expressed genes in a number of cDNA libraries constructed from various normal and tumor prostatic tissues. These included cell lines as well as short-term epithelial culture. A total of 6604 expressed sequence tags (ESTs) were generated and searched against on-line nucleotide and protein databases. A relational database centric software system was constructed to process, store, and analyze EST data rapidly. cDNA contigs were also obtained by assembly of multiple EST sequences. Protein structural signatures were annotated using motif analysis tools including BLOCKS and an in-house-designed neural network. Cross-library comparisons revealed their unique gene expression profiles. Several differentially expressed cDNA clones were identified, and their expression patterns were confirmed by RNA dot blot and RT-PCR analyses.     

MicroRNA-4723 Inhibits Prostate Cancer Growth through Inactivation of the Abelson Family of Nonreceptor Protein Tyrosine Kinases

The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer.