Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.     

Prostate cancer vs hyperplasia: relationships with prostatic and adipose tissue fatty acid composition

The objective of the present study was to study whether adipose tissue and prostatic tissue fatty acid composition differentiates between prostate cancer and benign hyperplasia patients. In addition, the present investigation aimed at exploring the extent to which prostatic tissue fatty acid composition differentiates between prostate-confined cancer and extraprostatic disease including possible metastasis. The subjects were 71 male patients from the island of Crete. Half the patients (n=35) had been diagnosed with benign hyperplasia of the prostate, half with prostatic malignancy (n=36). Patients were examined at the outpatient clinic of the urology unit, University Hospital, Medical School, University of Crete. Relative to benign hyperplasia patients, cancer patients had elevated adipose tissue saturated and reduced monounsaturated fatty acid levels. Cancer patients had reduced prostate tissue stearic to oleic acid ratios and stearic acid levels as opposed to hyperplasia patients. The most pronounced difference between cancer patients and hyperplasia patients was a 3-fold elevated prostatic palmitoleic acid in the former group. Relative to benign hyperplasia patients, cancer patients had reduced prostate tissue arachidonic and docosahexaenoic acid levels. Finally, there was a significantly reduced omega-3/omega-6 polyunsaturated fatty acid ratio in the prostate cancer patient as opposed to the benign hyperplasia group. The pronounced elevations in prostatic tissue palmitoleic acid in cancer patients highlight a possible role of this fatty acid in neoplastic processes. The decreased arachidonic acid levels in cancer patients possibly stem from enhanced metabolism of arachidonic acid via lipoxygenase and cyclooxygenase pathways, and the formation of derivatives such as 5-HETE, 15-HETE, 12(S)-HETE and PGE(2).     

VEGF-C、FLt4和结核菌L型感染在前列腺癌中的表达及临床意义

目的探讨血管内皮细胞生长因子-C(VEGF-C)与血管内皮细胞生长因子受体VEGFR-3(Flt4),在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化和原位杂交和抗酸染色等方法检测65例前列腺癌(carcinoma of prostate,PCa)和30例良性前列腺增生(benign prostatic hyperplasia,BPH)中的VEGF-C、Fit4蛋白及mRNA的表达,以及结核菌L型的检出率。并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ2检验进行统计学处理。结果VEGF-C、Flt4蛋白及mRNA阳性表达率和结核菌L型检出率,前列腺癌明显高于前列腺增生(P0.001~0.05)。VEGF-C、Flt4蛋白及mRNA阳性表达率和结核菌L型检出率与前列腺癌的临床分期、病理分级有明显差异(P0.05)。淋巴结转移组中VEGF-C、Flt4蛋白及mRNA的阳性表达率明显高于非转移组(P0.01)。结核菌L型检出率淋巴结转移组明显高于非转移组(P0.05)。结论VEGF-C、Flt4的蛋白及mRNA在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移呈正相关,提示这2种基因均可作为判断前列腺癌生物学行为及患者预后参考指标;结核菌L型感染可能有协同致瘤作用,与前列腺癌的发生发展可能有一定关系。因此研究VEGF-C、Flt4的阳性表达和结核菌L型感染与前列腺癌的关系,具有重要的临床应用价值。     

Epithelial Na,K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

Background  

An important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na⁺ gradients it generates are utilized for the Na⁺ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression.     

Proteomic analysis of formalin-fixed prostate cancer tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.     

Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor β1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4(+) CD25(+(high)) CD127(-) FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4(+) CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV(+) PCa.     

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.     

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Background  

Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells.     

Adiponectin as a potential marker of prostate cancer progression: studies in organ-confined and locally advanced prostate cancer

Serum levels of adiponectin were measured in patients with benign prostatic hyperplasia and prostate cancer of pT2 and pT3 stage. Adiponectin ELISA assay, immunohistochemistry, and selected metabolic and biochemical parameters measurement was performed in 25 patients with benign prostatic hyperplasia and 43 with prostate cancer (17 patients with organ-confined and 26 patients with locally advanced disease). Serum adiponectin levels did not differ between prostate benign hyperplasia and cancer clinical stage T2, but was significantly higher in pT3 relative to pT2 group (14.51+/-4.92 vs. 21.41+/-8.12, P = 0.003). Tissue immunohistochemistry showed enhanced staining in neoplastic prostate glands and intraepithelial neoplasia relative to benign prostatic hyperplasia without distinction between disease grade and stage. Serum adiponectin levels are higher in locally advanced relative to organ-confined prostate cancer and may thus serve as an auxiliary marker providing further improvement for discrimination between pT2 and pT3 stages.     

The association of prostatic lipids with progression,racial disparity and discovery of biomarkers in prostate cancer

BackgroundIt remains under-investigated whether prostatic lipid profiles are associated with pathogenesis, progression, racial disparity, and discovery of biomarkers in prostate cancer (PCa).MethodsThe electrospray ionization-tandem mass spectrometry was applied to quantitate prostatic lipids in human and mouse PCa and non-cancer prostatic tissues. Biostatistics and bioinformatics were used to compare the concentrations of prostatic lipids at levels of total lipid, group, class and individual species between PCa and benign prostatic tissues, between races, and among pathological conditions of PCa.ResultsProstatic concentrations of total lipids as well as neutral lipids were significantly higher in PCa than in benign prostatic tissues in all population and Caucasian American population, but not in African American population. The prostatic phospholipid were not statistically different between PCa and benign prostatic tissues in all study populations. Cholesteryl ester is the only lipid class significantly higher in PCa than in benign prostatic tissues in all study populations. A panel of prostatic lipid parameters in each study population was identified as diagnostic and prognostic biomarkers with >60% of sensitivity, specificity and accuracy simultaneously. Lipid profiling on mouse prostatic tissues further confirmed correlation of prostatic lipid profiles to the pathogenesis and progression of PCa. In addition, a few prostatic lipids in mouse can serve as prognostic biomarkers in differentiation of indolent from aggressive PCa.ConclusionThe prostatic lipids are widely associated with the pathogenesis, progression and racial disparity of PCa. A panel of prostatic lipids can serve as diagnostic, prognostic and race-specific biomarkers for PCa.     

The association of prostatic lipids with progression,racial disparity and discovery of biomarkers in prostate cancer

BackgroundIt remains under-investigated whether prostatic lipid profiles are associated with pathogenesis, progression, racial disparity, and discovery of biomarkers in prostate cancer (PCa).MethodsThe electrospray ionization-tandem mass spectrometry was applied to quantitate prostatic lipids in human and mouse PCa and non-cancer prostatic tissues. Biostatistics and bioinformatics were used to compare the concentrations of prostatic lipids at levels of total lipid, group, class and individual species between PCa and benign prostatic tissues, between races, and among pathological conditions of PCa.ResultsProstatic concentrations of total lipids as well as neutral lipids were significantly higher in PCa than in benign prostatic tissues in all population and Caucasian American population, but not in African American population. The prostatic phospholipid were not statistically different between PCa and benign prostatic tissues in all study populations. Cholesteryl ester is the only lipid class significantly higher in PCa than in benign prostatic tissues in all study populations. A panel of prostatic lipid parameters in each study population was identified as diagnostic and prognostic biomarkers with >60% of sensitivity, specificity and accuracy simultaneously. Lipid profiling on mouse prostatic tissues further confirmed correlation of prostatic lipid profiles to the pathogenesis and progression of PCa. In addition, a few prostatic lipids in mouse can serve as prognostic biomarkers in differentiation of indolent from aggressive PCa.ConclusionThe prostatic lipids are widely associated with the pathogenesis, progression and racial disparity of PCa. A panel of prostatic lipids can serve as diagnostic, prognostic and race-specific biomarkers for PCa.     

Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients

Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.     

CyclinD1、CDK4、P16和结核菌L型感染在前列腺癌中的表达及临床意义

目的探讨CyclinD1、CDK4和P16在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化和抗酸染色等方法检测了65例前列腺癌（carcinoma of prostate,PCa）和30例良性前列腺增生（benignprostatic hyperplasia,BPH）中的CyclinD1、CDK4和P16的表达,以及结核菌L型的检出率。并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果 CyclinD1、CDK4阳性表达前列腺癌明显高于前列腺增生（P〈0.01）;并与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈正相关。P16阳性表达前列腺增生明显高于前列腺癌（P〈0.01）;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈负正相关。结核菌L型检出率前列腺癌明显高于前列腺增生;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.05）。结论 CyclinD1、CDK4和P16在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移相关,因此研究CyclinD1、CDK4和P16的阳性表达和结核菌L型感染与前列腺癌发生发展中可能有协同作用,具有重要的临床应用价值。     

骨形态发生蛋白7 在前列腺癌中的表达

目的:探讨骨形态发生蛋白( BMP-7)在前列腺癌组织中的表达及其与临床分期之间的关系.方法:应用免疫印迹法检测30例前列腺癌患者及30例前列腺良性增生患者前列腺组织中BMP-7的表达情况.结果:前列腺癌组织中BMP-7的表达显著高于前列腺良性增生组织,且BMP-7的表达随前列腺癌的临床分期、Gleason分级增高而增加.结论:BMP-7在前列腺癌中的表达明显增高,其表达量与临床分期相关,前列腺癌组织中BMP-7的表达增高提示预后不佳.     

Benign prostatic hyperplasia-associated prostate-specific antigen (BPSA) shows unique immunoreactivity with anti-PSA monoclonal antibodies.

We previously identified a modified molecular form of prostate-specific antigen that is significantly elevated in the nodular transition zone tissue of prostates with benign prostatic hyperplasia. This prostate-specific antigen form, designated BPSA, is inactive and contains clipped polypeptide bonds at amino-acid residues Lys145-146 and Lys182-183. BPSA is not elevated in prostate cancer tissues and may therefore be a prostate-specific antigen marker to better discriminate benign prostatic hyperplasia from early prostate cancer. In this work we characterize the immunoreactivity of BPSA in competition assays with prostate-specific antigen using anti-prostate-specific antigen mAb recognizing six different epitopes on the prostate-specific antigen molecule. One mAb showed > 50% loss of immunoreactivtiy with BPSA compared with prostate-specific antigen, while the binding of two mAbs was largely unaffected and three mAbs had intermediate reactivity. BPSA purified from prostate tissue and seminal plasma, as well as BPSA generated in vitro by mild trypsin-treatment were found to have a similar pattern of reactivity to the six mAbs. However, other forms of inactive seminal plasma prostate-specific antigen, either intact or clipped at Lys145 only, had immunoreactivity similar to total prostate-specific antigen. These results demonstrate that BPSA has unique immunological properties from other forms of prostate-specific antigen, which should allow the development of BPSA-specific mAbs for the study of benign prostatic hyperplasia. Measurement of BPSA levels in the serum may help discriminate benign prostatic hyperplasia from early prostate cancer.     

COX-2、Ki-67和结核菌L型感染在前列腺癌中的表达

目的探讨环氧合酶-2（COX-2）和Ki-67在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化、原位杂交和抗酸染色等方法检测了65例前列腺癌（carcinoma of prostate,PCa）和30例良性前列腺增生（benign prostatic hyperplasia,BPH）中的COX-2、Ki-67蛋白及mRNA的表达,以及结核菌L型的检出率;并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果COX-2、Ki-67蛋白及mRNA阳性表达和结核菌L型检出率,前列腺癌明显高于前列腺增生（P〈0.001～0.05）。COX-2、Ki-67蛋白及mRNA阳性表达和结核菌L型检出率与前列腺癌的临床分期、病理分级有明显差异（P〈0.01～0.05）。淋巴结转移组中COX-2、Ki-67蛋白及mRNA的阳性表达率明显高于非转移组（P〈0.01）。结核菌L型检出率淋巴结转移组明显高于非转移组（P〈0.05）。结论COX-2、Ki-67蛋白及mRNA在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移呈正相关,提示2种基因均可作为判断前列腺癌生物学行为及患者预后参考指标。结核菌L型感染极有可能导致基因的变异或过表达,成为诱发肿瘤因素之一,它们可能有协同致瘤作用。     

Evidence of Heavy Methylation in the Galectin 3 Promoter in Early Stages of Prostate Adenocarcinoma: Development and Validation of a Methylated Marker for Early Diagnosis of Prostate Cancer

Galectins, soluble intracellular and extracellular β-galactoside-binding proteins, are known to be involved in the progression and metastasis of various cancers, including prostate adenocarcinoma, but the detailed mechanism of their biological roles remains elusive. In the prostate cancer cell lines PC-3 and DU-145, galectin 3 (gal3) is present at normal levels, whereas in LNCaP, its expression is silenced. In LNCaP, the gal3 promoter was heavily methylated, whereas PC-3 or DU-145 cells showed negligible or no methylation in the gal3 promoter indicating a negative correlation between gal3 promoter methylation and its expression. On immunohistochemical analysis of normal and tumor prostate tissues, gal3 was found expressed both in nucleus and cytoplasm of benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, and stage I. The expression of the gal3 was found drastically downregulated in advanced stages and, interestingly, mostly in the cytoplasm. On methylation analysis, the gal3 promoter in stage II prostate adenocarcinoma (PCa) was found heavily methylated, whereas in stages III and IV, it was only lightly methylated. However, in stage I PCa, both heavy and light methylations were observed in the gal3 promoter. In normal and benign prostatic hyperplasia tissues, the gal3 promoter was almost unmethylated. The differential cytosine methylation in the gal3 promoter in stages I to IV PCa enabled us to develop and validate a methylation-specific polymerase chain reaction-based sensitive assay specific for stages I and II PCa. These stages are considered the critical stages for successful intervention, thus underscoring the significance of this diagnostic assay.     

Combination of Autoantibody Signature with PSA Level Enables a Highly Accurate Blood-Based Differentiation of Prostate Cancer Patients from Patients with Benign Prostatic Hyperplasia

Although an increased level of the prostate-specific antigen can be an indication for prostate cancer, other reasons often lead to a high rate of false positive results. Therefore, an additional serological screening of autoantibodies in patients’ sera could improve the detection of prostate cancer. We performed protein macroarray screening with sera from 49 prostate cancer patients, 70 patients with benign prostatic hyperplasia and 28 healthy controls and compared the autoimmune response in those groups. We were able to distinguish prostate cancer patients from normal controls with an accuracy of 83.2%, patients with benign prostatic hyperplasia from normal controls with an accuracy of 86.0% and prostate cancer patients from patients with benign prostatic hyperplasia with an accuracy of 70.3%. Combining seroreactivity pattern with a PSA level of higher than 4.0 ng/ml this classification could be improved to an accuracy of 84.1%. For selected proteins we were able to confirm the differential expression by using luminex on 84 samples. We provide a minimally invasive serological method to reduce false positive results in detection of prostate cancer and according to PSA screening to distinguish men with prostate cancer from men with benign prostatic hyperplasia.     

Detection of pre-neoplastic and neoplastic prostate disease by MALDI profiling of urine

The heterogeneous progression to the development of prostate cancer (PCa) has precluded effective early detection screens. Existing prostate cancer screening paradigms have relatively poor specificity for cancer relative to other prostate diseases, commonly benign prostatic hyperplasia (BPH). A method for discrimination of BPH, HGPIN, and PCa urine proteome was developed through testing 407 patient samples using matrix assisted laser desorption-mass spectrometry time of flight (MALDI-TOF). Urine samples were adsorbed to reverse phase resin, washed, and the eluant spotted directly for MALDI-TOF analysis of peptides. The processing resolved over 130 verifiable signals of a mass range of 1000-5000 m/z to suggest 71.2% specificity and 67.4% sensitivity in discriminating PCa vs. BPH. Comparing BPH and HGPIN resulted in 73.6% specificity and 69.2% sensitivity. Comparing PCa and HGPIN resulted in 80.8% specificity and 81.0% sensitivity. The high throughput, low-cost assay method developed is amenable for large patient numbers required for supporting biomarker identification.