Mass spectrometry-based expression profiling of clinical prostate cancer

The maturation of MS technologies has provided a rich opportunity to interrogate protein expression patterns in normal and disease states by applying expression protein profiling methods. Major goals of this research strategy include the identification of protein biomarkers that demarcate normal and disease populations, and the identification of therapeutic biomarkers for the treatment of diseases such as cancer (Celis, J. E., and Gromov, P. (2003) Proteomics in translational cancer research: Toward an integrated approach. Cancer Cell 3, 9-151). Prostate cancer is one disease that would greatly benefit from implementing MS-based expression profiling methods because of the need to stratify the disease based on molecular markers. In this review, we will summarize the current MS-based methods to identify and validate biomarkers in human prostate cancer. Lastly, we propose a reverse proteomic approach implementing a quantitative MS research strategy to identify and quantify biomarkers implicated in prostate cancer development. With this approach, the absolute levels of prostate cancer biomarkers will be identified and quantified in normal and diseased samples by measuring the levels of native peptide biomarkers in relation to a chemically identical but isotopically labeled reference peptide. Ultimately, a centralized prostate cancer peptide biomarker expression database could function as a repository for the identification, quantification, and validation of protein biomarker(s) during prostate cancer progression in men.     

Nuclear matrix localization of high mobility group protein I(Y) in a transgenic mouse model for prostate cancer

Nuclear shape and the underlying nuclear structure, the nuclear matrix in cancer cells. Since the NM composition is considered to maintain nuclear shape and architecture, nuclear matrix proteins (NMPs) may be involved in transformation. Our laboratory has recently characterized a subset of NMPs that are associated with prostate cancer development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. One of the identified NMPs, E3E, has a similar molecular weight (22 kDa) with a protein known as HMGI(Y). HMGI(Y) belongs to a group of non-histone and chromatin-associated proteins, high-mobility-group (HMG) proteins, and it has been shown to associate with the NM. HMGI(Y) has been reported to be elevated in different types of cancer including prostate cancer. In this study, we examined the expression of HMGI(Y) protein in the NMP composition of the TRAMP model during the progression from normal to neoplasia. The expression of HMGI(Y) in the NMP extracts of three prostatic epithelial cell lines derived from a 32-week TRAMP mouse: TRAMP-C1, TRAMP-C2, and TRAMP-C3 was also examined. Using both one-dimensional and high-resolution two-dimensional immunoblot analyses, we found that: (i) HMGI(Y) is a nuclear matrix protein expressed as two protein bands with MW of 22-24 kDa and (ii) HMGI(Y) expression is correlated with neoplastic and malignant properties in late stage TRAMP prostate tumors. Overall, these findings support the evidence that HMGI(Y) can be utilized as a marker and prognostic tool for prostate cancer.     

Essential Role for Activation of the Polycomb Group (PcG) Protein Chromatin Silencing Pathway in Metastatic Prostate Cancer

The Polycomb-group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of the BMI1 oncogene causes neoplastic transformation of lymphocytes and plays an essential role in the pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both the BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from the circulation which overexpress both the BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased the BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate important biological role of PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.     

False-positive TUNEL staining observed in SV40 based transgenic murine prostate cancer models

The TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) and LADY (Probasin-large T antigen transgenic mouse) mice are widely used autochthonous models of prostate cancer. Both models utilise probasin promoters to direct androgen-regulated expression of oncogenic SV40 specifically to epithelial cells of the mouse prostate. The oncogenic processes and phenotypes which result mimic many features of human prostate cancer, making these transgenic mouse models useful experimental systems. The terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP in situ nick end labelling (TUNEL) assay is a commonly used method for the detection of cells undergoing apoptosis. In this study, we demonstrate false-positive TUNEL staining in frozen prostate tissue from TRAMP and LADY mice, which was not observed in non-transgenic control animals and is not due to non-specific binding of labelled-dUTP substrate. The false-positive signal co-localised with large SV40 T-antigen expression. False-positive signal was apparent using multiple commercial TUNEL kits with different detection systems. These results caution against the use of the TUNEL assay for detection of apoptosis in frozen prostate tissue of large T-antigen based autochthonous transgenic models of prostate cancer.     

Accentuation of differentially expressed proteins using phage technology

Protein profiling is frequently used to elucidate disease-specific or differentially expressed proteins. While recent developments have resulted in improved differential profiling, alternative expression platforms that complement existing techniques are continually being explored. We developed a novel method utilizing the amplification and selection capabilities of random peptide-expressing M13 bacteriophage to accentuate differentially expressed proteins in biologic specimens. While the current study used this method to demonstrate differentially expressed proteins in lung cancer tissue in comparison to normal lung tissue, this approach is applicable to a wide range of sample types.     

Survey of genetically engineered mouse models for prostate cancer: analyzing the molecular basis of prostate cancer development, progression, and metastasis

Genetically engineered mouse models have been generated to study the molecular basis of prostate cancer (PCa) development, progression, and metastasis. Selection of a prostate-specific promoter, such as the probasin (PB) and prostate specific antigen (PSA) promoters, is critical for generating sufficient levels of transgene expression to elicit a phenotypic response. To date, target genes have included growth factors, cell cycle regulators, pro- and anti-apoptotic proteins, steroid hormone and growth factor receptors, oncogenes, tumor suppressors, and homeobox genes. The experimental approaches used to generate these mouse models include overexpression of the transgene, knock-out/knock-in of transgene expression and conditional regulation of expression using Cre/lox technology. This review summarizes the promoters, which have been utilized to create genetically engineered mouse models for PCa. Furthermore, the effects of gene disruption on promoting low- and high-grade intraepithelial neoplasia (LGPIN and HGPIN, respectively), locally invasive carcinoma and metastatic lesions will be discussed. To date, the PB-Cre4 x PTENloxp/loxp model appears to be the only model that represents the entire continuum of prostate adenocarcinoma development, tumor progression, and metastasis, although models that develop prostatic neuroendocrine (NE) cancer can be generated by disrupting one genetic event. Indeed, analysis of bigenic mouse models indicates that two genetic events are generally required for progression from HGPIN to locally invasive adenocarcinoma and that two to five genetic events can promote metastasis to distant sites. Studying the effects of genetic perturbation on PCa biology will increase our understanding of the disease process and potentially provide targets for developing novel therapeutic approaches.     

Protein expression profiling of breast cancer cells by dissociable antibody microarray (DAMA) staining

Dissociable antibody microarray (DAMA) staining is a technology that combines protein microarrays with traditional immunostaining techniques. It can simultaneously determine the expression and subcellular location of hundreds of proteins in cultured cells and tissue samples. We developed this technology and demonstrated its application in identifying potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among three normal breast cell lines and seven breast cancer cell lines and identified 10 differentially expressed proteins by the data analysis program DAMAPEP (DAMA protein expression profiling). Among those proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2 were confirmed by Western blot and statistical analysis to have higher expression levels in breast cancer cells than in normal breast cells. These proteins could be potential biomarkers for the diagnosis of breast cancer.     

Transgenic overexpression of PKCε in the mouse prostate induces preneoplastic lesions

It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCe, the product of the PRKCE gene, is up-regulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCa, PKCd, or PKCe in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCa and PB-PKCd mice did not show any evident phenotype, PB-PKCe mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6, and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCe overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCe by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCe overexpression and prostate cancer development.     

Expression of the ZNT (SLC30) family members in the epithelium of the mouse prostate during sexual maturation

A prostate contains ~10-fold higher zinc than other soft organs. The function of the prostate is to produce a zinc-enriched seminal fluid. To establish a protein expression profile for zinc transporters involved in zinc efflux and intracellular sequestration/storage in the mouse prostate during sexual maturation, ZNT expression were investigated by immunohistochemistry. Our study demonstrated that ZNT proteins were differentially expressed in the prostate during sexual maturation. ZNT1 was mainly detected on the lateral membrane of the epithelium. Other ZNTs examined resided intracellularly. Among differences were a staining of ZNT2/ZNT5 in the ER-rich area of the epithelium in the anterior lobe, a staining of ZNT2 along the lateral and apical membrane, a luminal border staining of ZNT4, a staining of ZNT5 in the Golgi area of the epithelium in the ventral lobe, a uniform expression of ZNT6 across the lobes and ages, and a staining of ZNT7 in all lobes across ages.     

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue‐specific transgene expression in the prostate epithelium has previously been achieved using short prostate‐specific promoters, rendering transgenic mouse lines susceptible to integration site‐dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre‐induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre‐ERT2 protein from small prostate‐specific promoters, these mouse lines will be useful in research focused on prostate‐specific disorders such as benign hyperplasia or cancer. genesis 47:757–764, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of pathways associated with invasive behavior by ovarian cancer cells using multidimensional protein identification technology (MudPIT)

Proteomic profiling has emerged as a useful tool for identifying tissue alterations in disease states including malignant transformation. The aim of this study was to reveal expression profiles associated with the highly motile/invasive ovarian cancer cell phenotype. Six ovarian cancer cell lines were subjected to proteomic characterization using multidimensional protein identification technology (MudPIT), and evaluated for their motile/invasive behavior, so that these parameters could be compared. Within whole cell extracts of the ovarian cancer cells, MudPIT identified proteins that mapped to 2245 unique genes. Western blot analysis for selected proteins confirmed the expression profiles revealed by MudPIT, demonstrating the fidelity of this high-throughput analysis. Unsupervised cluster analysis partitioned the cell lines in a manner that reflected their motile/invasive capacity. A comparison of protein expression profiles between cell lines of high (group 1) versus low (group 2) motile/invasive capacity revealed 300 proteins that were differentially expressed, of which 196 proteins were significantly upregulated in group 1. Protein network and KEGG pathway analysis indicated a functional interplay between proteins up-regulated in group 1 cells, with increased expression of several key members of the actin cytoskeleton, extracellular matrix (ECM) and focal adhesion pathways. These proteomic expression profiles can be utilized to distinguish highly motile, aggressive ovarian cancer cells from lesser invasive ones, and could prove to be essential in the development of more effective strategies that target pivotal cell signaling pathways used by cancer cells during local invasion and distant metastasis.     

Activated polyamine catabolism depletes acetyl-CoA pools and suppresses prostate tumor growth in TRAMP mice

The enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) regulates the catabolism and export of intracellular polyamines. We have previously shown that activation of polyamine catabolism by conditional overexpression of SSAT has antiproliferative consequences in LNCaP prostate carcinoma cells. Growth inhibition was causally linked to high metabolic flux arising from a compensatory increase in polyamine biosynthesis. Here we examined the in vivo consequences of SSAT overexpression in a mouse model genetically predisposed to develop prostate cancer. TRAMP (transgenic adenocarcinoma of mouse prostate) female C57BL/6 mice carrying the SV40 early genes (T/t antigens) under an androgen-driven probasin promoter were cross-bred with male C57BL/6 transgenic mice that systemically overexpress SSAT. At 30 weeks of age, the average genitourinary tract weights of TRAMP mice were approximately 4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were approximately 12 times greater indicating sustained suppression of tumor outgrowth. Tumor progression was also affected as indicated by a reduction in the prostate histopathological scores. By immunohistochemistry, SV40 large T antigen expression in the prostate epithelium was the same in TRAMP and TRAMP/SSAT mice. Consistent with the 18-fold increase in SSAT activity in the TRAMP/SSAT bigenic mice, prostatic N(1)-acetylspermidine and putrescine pools were remarkably increased relative to TRAMP mice, while spermidine and spermine pools were minimally decreased due to a compensatory 5-7-fold increase in biosynthetic enzymes activities. The latter led to heightened metabolic flux through the polyamine pathway and an associated approximately 70% reduction in the SSAT cofactor acetyl-CoA and a approximately 40% reduction in the polyamine aminopropyl donor S-adenosylmethionine in TRAMP/SSAT compared with TRAMP prostatic tissue. In addition to elucidating the antiproliferative and metabolic consequences of SSAT overexpression in a prostate cancer model, these findings provide genetic support for the discovery and development of specific small molecule inducers of SSAT as a novel therapeutic strategy targeting prostate cancer.     

PTOV1 is associated with UCH-L1 and in response to estrogen stimuli during the mouse oocyte development

To investigate the biological significance of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) involvement in oocyte maturation, we screened for proteins that bound to UCH-L1 in mouse ovaries, and we found that the prostate tumor overexpressed-1 (PTOV1) protein was able to bind to UCH-L1. PTOV1 is highly expressed in prostate cancers and considered as a potential marker for carcinogenesis and the progress of prostate cancer. It was reported that PTOV1 plays an important role in cell cycle regulation, but its role in mammalian oocyte development and meiosis is still unclear. In this paper, it was found that the expression levels of PTOV1 in mouse ovaries progressively increased from prepubescence to adulthood. And we found by immunohistochemistry that PTOV1 spreaded in both the cytoplasm and nuclei of oocytes during prepuberty, but in normal adult mouse oocytes, it concentrated not only in nuclei but also on the plasma membrane, though in some oocytes with abnormal shapes, PTOV1 did not display the typical distribution patterns. In granulosa cells, however, it was found to locate in the cytoplasm at all the selected ages. In postnatal mouse ovaries (28 days), estradiol treatment induced the adult-specific distribution pattern of PTOV1 in oocytes. In addition, UCH-L1 was shown to be associated with CDK1, which participated in the regulation of cell cycle and oocyte maturation. Therefore, we propose that the distribution changes of PTOV1 are age-dependent, and significant for mouse oocyte development and maturation. Moreover, the discovery that PTOV1 is associated with UCH-L1 in mouse oocytes supports the explanations for that UCH-L1 is involved in oocyte development and maturation, especially under the regulation of estrogen.     

Dynamics of expression of growth differentiation factor 15 in normal and PIN development in the mouse

Growth differentiation factor (GDF15) is a distant member of the transforming growth factor-beta superfamily, a diverse group of structurally related proteins that exert multiple effects on cell fate such as on cell growth and differentiation but little is known about GDF15 in these processes. Previously we observed the mature GDF15 to be associated with human prostate carcinogenesis hence prompting us to study GDF15 further. Here we report gdf15 expression both at the RNA and protein levels, in normal prostatic tissues of wild type (wt) and prostatic intraepithelial neoplasia (PIN) of transgenic (Tg) 12T-7s model mice during embryonic, postnatal, and adult prostate formation up to 15 weeks after birth. Dynamic changes in expression, at both the mRNA and protein level, correlated with cell proliferation and differentiation during distinct phases of normal mouse prostate development and alterations in the dynamics of gdf15 expression correlated with the changes in development resulting in PIN formation. Most notably mature gdf15 protein was significantly elevated during hyperplasia and PIN development. Changes in the protein levels did not always correlate well with the mRNA levels. This was more prominent during PIN than during normal prostate development suggesting that this may also be an indicator of disturbed regulation of gdf15 in PIN. We propose that gdf15 is a growth factor with dual function either promoting proliferation or growth arrest and differentiation due most likely to differences in cellular differentiation. Because of the differentiation defect in PIN its epithelium no longer responds to gdf15 by cellular growth arrest as does the normal epithelium and gdf may even stimulate proliferation. The data supports our hypothesis that GDF15 plays a role in the early stages of human prostate cancer.     

Tissue-specific microdissection coupled with ProteinChip array technologies: applications in cancer research

Analysis of whole genomes to monitor specific changes in gene activation or changes in gene copy number due to perturbation has recently become possible using DNA chip technologies. It is now becoming apparent, however, that knowing the genetic sequence encoding a protein is not sufficient to predict the size or biological nature of a protein. This can be particularly important in cancer research where posttranslational modifications of a protein can specifically lead to the disease. To address this area, several proteomic tools have been developed. Currently the most widely used proteomics tool is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can display protein expression patterns to a high degree of resolution. However, 2D-PAGE can be time consuming; the analysis is complicated and, compared with DNA techniques, is not very sensitive. Although some of these problems can be alleviated by using high-quality homogeneous samples, such as those generated using microdissection techniques, the quantity of sample is often limited and may take several days to generate sufficient material for a single 2D-PAGE analysis. As an alternative to 2D-PAGE, a preliminary study using a new technique was used to generate protein expression patterns from either whole tissue extracts or microdissected material. Surface-enhanced laser desorption and ionization allows the retention of proteins on a solid-phase chromatographic surface or ProteinChip Array with direct detection of retained proteins by time-of-flight mass spectrometry. Using this system, we analyzed tumor and normal tissue from head and neck cancer and microdissected melanoma to determine differentially expressed proteins. In particular, comparisons of the protein expression patterns from microdissected normal and tumor tissues indicated several differences, highlighting the importance of extremely defined tissue lysates for protein profiling.     

Androgen dependent regulation of protein kinase A subunits in prostate cancer cells

Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.