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1.  Immunohistochemical staining of radixin and moesin in prostatic adenocarcinoma 
Some members of the Protein 4.1 superfamily are believed to be involved in cell proliferation and growth, or in the regulation of these processes. While the expression levels of two members of this family, radixin and moesin, have been studied in many tumor types, to our knowledge they have not been investigated in prostate cancer.
Tissue microarrays were immunohistochemically stained for either radixin or moesin, with the staining intensities subsequently quantified and statistically analyzed using One-Way ANOVA or nonparametric equivalent with subsequent Student-Newman-Keuls tests for multiple comparisons. There were 11 cases of normal donor prostates (NDP), 14 cases of benign prostatic hyperplasia (BPH), 23 cases of high-grade prostatic intraepithelial neoplasia (HGPIN), 88 cases of prostatic adenocarcinoma (PCa), and 25 cases of normal tissue adjacent to adenocarcinoma (NAC) analyzed in the microarrays.
NDP, BPH, and HGPIN had higher absolute staining scores for radixin than PCa and NAC, but with a significant difference observed between only HGPIN and PCa (p = < 0.001) and HGPIN and NAC (p = 0.001). In the moesin-stained specimens, PCa, NAC, HGPIN, and BPH all received absolute higher staining scores than NDP, but the differences were not significant. Stage 4 moesin-stained PCa had a significantly reduced staining intensity compared to Stage 2 (p = 0.003).
To our knowledge, these studies represent the first reports on the expression profiles of radixin and moesin in prostatic adenocarcinoma. The current study has shown that there were statistically significant differences observed between HGPIN and PCa and HGPIN and NAC in terms of radixin expression. The differences in the moesin profiles by tissue type were not statistically significant. Additional larger studies with these markers may further elucidate their potential roles in prostatic neoplasia progression.
PMCID: PMC3029218  PMID: 21235778
2.  Immunohistochemical Staining of Slit2 in Primary and Metastatic Prostatic Adenocarcinoma1 
Translational Oncology  2011;4(5):314-320.
BACKGROUND: Conflicting roles for Slit2, a protein involved in mediating the processes of cell migration and chemotactic response, have been previously described in prostate cancer. Here we use immunohistochemistry to evaluate the expression of Slit2 in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets). METHODS: Tissue microarrays were immunostained for Slit2. The staining intensities were quantified using automated image analysis software. The data was statistically analyzed using one-way analysis of variance with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Eleven cases of NDP, 35 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 106 cases of PCa, and 37 cases of Mets were analyzed. RESULTS: Specimens of PCa and HGPIN had the highest absolute staining for Slit2. Significant differences were seen between PCa and NDP (P < .05), PCa and NAC (P < .05), HGPIN and NDP (P < .05), and HGPIN and NAC (P < .05). Whereas the average Mets staining was not significantly different from NDP or NAC, several individual Mets cases featured intense staining. CONCLUSIONS: To our knowledge, this represents the first study comparing the immunohistochemical profiles of Slit2 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC. These findings suggest that Slit2 expression can be increased in HGPIN, PCa, and Mets, making it a potentially important biomarker for prostate cancer.
PMCID: PMC3162306  PMID: 21966548
3.  Immunohistochemical profiles of claudin-3 in primary and metastatic prostatic adenocarcinoma 
Diagnostic Pathology  2011;6:12.
Claudins are integral membrane proteins that are involved in forming cellular tight junctions. One member of the claudin family, claudin-3, has been shown to be overexpressed in breast, ovarian, and pancreatic cancer. Here we use immunohistochemistry to evaluate its expression in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets).
Tissue microarrays were immunohistochemically stained for claudin-3, with the staining intensities subsequently quantified and statistically analyzed using a one-way ANOVA with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Fifty-three cases of NAC, 17 cases of BPH, 35 cases of PIN, 107 cases of PCa, and 55 cases of Mets were analyzed in the microarrays.
PCa and Mets had the highest absolute staining for claudin-3. Both had significantly higher staining than BPH (p < 0.05 in both cases) and NAC (p < 0.05 in both cases). PIN had a lower, but non-significant, staining score than PCa and Mets, but a statistically higher score than both BPH and NAC (p < 0.05 for both cases). No significant differences were observed between PCa, Mets, and PIN.
To our knowledge, this represents one of the first studies comparing the immunohistochemical profiles of claudin-3 in PCa and NAC to specimens of PIN, BPH, and Mets. These findings provide further evidence that claudin-3 may serve as an important biomarker for prostate cancer, both primary and metastatic, but does not provide evidence that claudin-3 can be used to predict risk of metastasis.
PMCID: PMC3033791  PMID: 21255442
4.  Nicotinamide N-methyltransferase is overexpressed in prostate cancer and correlates with prolonged progression-free and overall survival times 
Oncology Letters  2014;8(3):1175-1180.
Nicotinamide N-methyltransferase (NNMT) has been identified to be associated with tumorigenesis and the malignant transformation of numerous types of cancer. The aim of the present study was to explore the expression and prognostic significance of NNMT in prostate cancer (PCa). Immunohistochemical NNMT expression was examined in 26 cases of benign prostate hyperplasia (BPH), 18 cases of high-grade prostatic intraepithelial neoplasia (HGPIN) and 120 cases of PCa. While rarely expressed in BPH (8/26 cases; 30.8%), NNMT was found to be significantly upregulated in HGPIN (15/18 cases; 83.3%) and PCa (77/120 cases; 64.2%). Clinicopathological analysis revealed that NNMT expression was negatively correlated with Gleason score (P<0.001). Furthermore, Kaplan-Meier survival curves revealed that high NNMT expression was associated with prolonged progression-free survival (PFS) and overall survival (OS) times in patients with advanced PCa. Multivariate analysis showed that NNMT was an independent prognostic marker of PFS and OS in patients with advanced PCa. The results of the present study suggested that NNMT may contribute to the development of PCa and may potentially be a favorable prognostic marker for the survival of patients with advanced PCa.
PMCID: PMC4114624  PMID: 25120681
nicotinamide N-methyltransferase; prostate cancer; immunohistochemistry; prognosis
5.  Oxidative Stress Measured by Urine F2-Isoprostane Level Is Associated With Prostate Cancer 
The Journal of urology  2011;185(6):2102-2107.
Oxidative stress is implicated in prostate cancer (PCa) by several lines of evidence. We studied the relationship between the level of F2-Isoprostanes (F2IP), a validated biomarker of oxidative stress, and PCa and high-grade prostatic intraepithelial neoplasia (HGPIN).
This case-control analysis within the Nashville Men’s Health Study (NMHS) included men recruited at prostate biopsy. Body morphometrics, health history and urine were collected on over 2000 men prior to biopsy. F2-isoprostanes were measured by gas chromatography/mass spectrometry within an age-matched sample of NMHS participants that included 140 patients with HGPIN, 160 biopsy-negative controls, and 200 PCa cases. Multivariable linear and logistic regression were used to determine the associations between F2IP level and HGPIN and PCa.
Mean age was 66.9 (SD 7.2) and 10.1% were non-white. Adjusted geometric mean F2IP levels were higher in patients with PCa (1.82, 95% CI[1.66-2.00]) or HGPIN (1.82, 95% CI[1.68-1.96]) than in controls (1.63, 95% CI[1.49-1.78]), p<0.001, but were similar across Gleason scores (p=0.511). The adjusted odds of HGPIN and PCa increased with increasing F2IP quartile (p-trend = 0.015 and 0.047, respectively) and the highest F2IP quartile was associated with a significantly increased odds of PCa (OR 2.44, 95% CI [1.17-5.09], p=0.017).
Pre-diagnosis urine F2IP level is elevated in men with HGPIN or PCa, suggesting urinary F2IP provides a biomarker for the role for oxidative stress in prostate carcinogenesis. F2IP may also serve to estimate the efficacy of interventions targeting oxidative stress mechanisms in prostate cancer prevention or treatment.
PMCID: PMC3093434  PMID: 21496850
prostate cancer; oxidative stress; isoprostanes; high-grade prostatic intraepithelial neoplasia; biopsy
6.  Prostate stem cell antigen (PSCA) expression in human prostate cancer tissues and its potential role in prostate carcinogenesis and progression of prostate cancer 
Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. The purpose of the present study was to examine the expression status of PSCA protein and mRNA in clinical specimens of human prostate cancer (Pca) and to validate it as a potential molecular target for diagnosis and treatment of Pca.
Materials and Methods
Immunohistochemical (IHC) and in situ hybridization (ISH) analyses of PSCA expression were simultaneously performed on paraffin-embedded sections from 20 benign prostatic hyperplasia (BPH), 20 prostatic intraepithelial neoplasm (PIN) and 48 prostate cancer (Pca) tissues, including 9 androgen-independent prostate cancers. The level of PSCA expression was semiquantitatively scored by assessing both the percentage and intensity of PSCA-positive staining cells in the specimens. Then compared PSCA expression between BPH, PIN and Pca tissues and analysed the correlations of PSCA expression level with pathological grade, clinical stage and progression to androgen-independence in Pca.
In BPH and low grade PIN, PSCA protein and mRNA staining were weak or negative and less intense and uniform than that seen in HGPIN and Pca. There were moderate to strong PSCA protein and mRNA expression in 8 of 11 (72.7%) HGPIN and in 40 of 48 (83.4%) Pca specimens examined by IHC and ISH analyses, with statistical significance compared with BPH (20%) and low grade PIN (22.2%) samples (p < 0.05, respectively). The expression level of PSCA increased with high Gleason grade, advanced stage and progression to androgen-independence (p < 0.05, respectively). In addition, IHC and ISH staining showed a high degree of correlation between PSCA protein and mRNA overexpression.
Our data demonstrate that PSCA as a new cell surface marker is overexpressed by a majority of human Pca. PSCA expression correlates positively with adverse tumor characteristics, such as increasing pathological grade (poor cell differentiation), worsening clinical stage and androgen-independence, and speculatively with prostate carcinogenesis. PSCA protein overexpression results from upregulated transcription of PSCA mRNA. PSCA may have prognostic utility and may be a promising molecular target for diagnosis and treatment of Pca.
PMCID: PMC420493  PMID: 15132743
Prostate; Neoplasm; Prostate stem cell antigen (PSCA)
7.  Morphoproteomic Confirmation of a Constitutively Activated mTOR Pathway in High Grade Prostatic Intraepithelial Neoplasia and Prostate Cancer 
Preclinical studies have implicated the mammalian target of rapamycin (mTOR) pathway in the cell cycle progression and growth of prostate cancer cells. Downstream signaling from PI3′-K/Akt leads to phosphorylation (p) of mTOR at serine 2448 and to activation of its substrate, p70S6Kinase (p70S6K), phosphorylated on threonine 389. This promotes translation and cell cycle progression. Morphoproteomic analysis, that combines both the application of phosphospecific probes directed against putative sites of activation on protein analytes and cellular compartmentalization [1] was carried out on tissue microarray (TMA) slides from 64 cases of primary, previously untreated adenocarcinomas of the prostate. Gleason scores ranged from 6 to 10. High grade prostatic intraepithelial neoplasia (HGPIN), which accompanied the invasive cancer in 20 cases, and 15 non-neoplastic controls from benign prostatic hypertrophy specimens in a separate TMA were also included. Ninety-three percent (93%) of tumors exhibited moderate to strong cytoplasmic/plasmalemmal expression of p-mTOR and eighty-five percent (85%) showed similar staining intensity for p-p70S6K. HGPIN demonstrated comparable and occasionally, stronger expression levels for these protein analytes. Quantitative digital imaging revealed an overall increase in the mean expression levels in HGPIN, reaching statistical significance for p-mTOR (Ser 2448) at p<0.05. Morphoproteomic analysis confirms the constitutive activation of the mTOR pathway in prostate cancer and HGPIN, with relative overexpression of p-mTOR in HGPIN. These findings coincide with preclinical studies in supporting a role for the mTOR pathway in the biology and development of prostate cancer through its putative precursor lesion, HGPIN and in suggesting a potential therapeutic target.
PMCID: PMC2480543  PMID: 18787612
mTOR pathway; prostate cancer; high grade PIN; morphoproteomics; tissue microarray
8.  Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer 
Journal of Clinical Pathology  2004;57(8):872-876.
Aims: Retinoids are involved in cell growth, differentiation, and carcinogenesis. Their effects depend on cytosolic transport and binding to nuclear receptors. CRBP1 encodes a protein involved in this process. Because altered CRBP1 expression and promoter hypermethylation occur in several tumours, these changes were investigated in prostate tumorigenesis.
Methods: The CRBP1 promoter was assessed by methylation specific polymerase chain reaction on tissue samples from 36 radical prostatectomy specimens (paired normal tissue, adenocarcinoma, and high grade prostatic intraepithelial neoplasia (HGPIN)), 32 benign prostatic hyperplasias (BPHs), and 13 normal prostate tissue samples from cystoprostatectomies. Methylation of DNA extracted from microdissected tissue was examined blindly. CRBP1 expression was assessed by immunohistochemistry on formalin fixed, paraffin wax embedded tissue.
Results: Loss of CRBP1 expression was seen in 15 of 36 adenocarcinomas and 18 of 36 HGPINs. Fifteen adenocarcinomas and nine HGPINs showed overexpression, whereas the remainder showed normal expression. BPH displayed normal expression. No significant associations were found between CRBP1 expression and Gleason score or stage. CRBP1 promoter hypermethylation was found in 17 of 36 adenocarcinomas, three of 35 HGPINs, one of 36 normal prostate tissues from the same patients, none of 32 BPHs, and none of 13 normal prostate tissues from cystoprostatectomies. Loss of expression and hypermethylation of CRBP1 were not significantly associated.
Conclusions: Altered CRBP1 expression and hypermethylation are common in prostate carcinoma, although CRBP1 hypermethylation is not an early event in tumorigenesis. Moreover, both adenocarcinoma and HGPIN show frequent CRBP1 overexpression. The molecular mechanisms underlying altered CRBP1 expression in prostate cancer deserve further study.
PMCID: PMC1770387  PMID: 15280411
CRBP1; prostate cancer; PIN; methylation; immunoexpression
9.  Caveolin-1 Upregulation Contributes to c-Myc–Induced High-Grade Prostatic Intraepithelial Neoplasia and Prostate Cancer 
Molecular cancer research : MCR  2011;10(2):218-229.
Previously we reported caveolin-1 (Cav-1) overexpression in prostate cancer (PCa) cells and demonstrated that it promotes PCa progression. Here, we report that Cav-1 was overexpressed in 41.7% (15 of 36) of high-grade prostatic intraepithelial neoplasia (HGPIN) specimens obtained during radical prostatectomies. Positive correlations exist between Cav-1–positive (Cav-1+) HGPIN and Cav-1+ primary PCa (rho = 0.655, P< 0.0001) and between Cav-1 and c-Myc expression in HGPIN (rho = 0.41, P = 0.032). To determine whether Cav-1 cooperates with c-Myc in development of premalignant lesions and PCa in vivo, we generated transgenic mice with c-Myc overexpression driven by the ARR2PB promoter. In this ARR2PB–c-myc model, Cav-1 overexpression was found in mouse PIN (mPIN) lesions and PCa cells and was associated with a significantly higher ratio of proliferative to apoptotic labeling in mPIN lesions than in the Cav-1–negative epithelia adjacent to those lesions (10.02 vs 4.34; P = 0.007). Cav-1 overexpression was also associated with increased levels of P-Akt and VEGF-A, which were previously associated with Cav-1–induced PCa cell survival and positive-feedback regulation of cellular Cav-1 levels, respectively. In multiple PCa cell lines, Cav-1 protein (but not mRNA) was induced by c-Myc transfection, whereas VEGF siRNA transfection abrogated c-Myc–induced Cav-1 overexpression, suggesting a c-Myc–VEGF–Cav-1 signaling axis. Overall, our results suggest that Cav-1 is associated with c-Myc in the development of HGPIN and PCa. Further, Cav-1 overexpression in HGPIN is potentially a biomarker for early identification of patients who tend to develop Cav-1+ primary PCa.
PMCID: PMC3908884  PMID: 22144662
Caveolin-1; c-Myc; prostatic intraepithelial neoplasia; prostate cancer; metaplasia
10.  Periostin identified as a potential biomarker of prostate cancer by iTRAQ-proteomics analysis of prostate biopsy 
Proteome Science  2011;9:22.
Proteomics may help us better understand the changes of multiple proteins involved in oncogenesis and progression of prostate cancer(PCa) and identify more diagnostic and prognostic biomarkers. The aim of this study was to screen biomarkers of PCa by the proteomics analysis using isobaric tags for relative and absolute quantification(iTRAQ).
The patients undergoing prostate biopsies were classified into 3 groups according to pathological results: benign prostate hyperplasia (BPH, n = 20), PCa(n = 20) and BPH with local prostatic intraepithelial neoplasm(PIN, n = 10). Then, all the specimens from these patients were analyzed by iTRAQ and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS). The Gene Ontology(GO) function and the transcription regulation networks of the differentially expressed were analyzed by MetaCore software. Western blotting and Immunohistochemical staining were used to analyze the interesting proteins.
A total of 760 proteins were identified from 13787 distinct peptides, including two common proteins that enjoy clinical application: prostate specific antigen (PSA) and prostatic acid phosphatase(PAP). Proteins that expressed differentially between PCa and BPH group were further analyzed. Compared with BPH, 20 proteins were significantly differentially up-regulated (>1.5-fold) while 26 were significantly down-regulated in PCa(<0.66-fold). In term of GO database, the differentially expressed proteins were divided into 3 categories: cellular component(CC), molecular function (MF) and biological process(BP). The top 5 transcription regulation networks of the differentially expressed proteins were initiated through activation of SP1, p53, YY1, androgen receptor(AR) and c-Myc The overexpression of periostin in PCa was verified by western blotting and immunohistochemical staining.
Our study indicates that the iTRAQ technology is a new strategy for global proteomics analysis of the tissues of PCa. A significant up-regulation of periostin in PCa compared to BPH may provide clues for not only a promising biomarker for the prognosis of PCa but also a potential target for therapeutical intervention.
PMCID: PMC3100237  PMID: 21504578
iTRAQ; Periostin; Proteomics; Prostate cancer
11.  Comparison of annexin II, p63 and α‐methylacyl‐CoA racemase immunoreactivity in prostatic tissue: a tissue microarray study 
Journal of Clinical Pathology  2006;60(7):773-780.
Current ancillary markers for diagnosis in prostate biopsies include p63 and α‐methylacyl‐CoA racemase (AMACR). Annexin II (ANXII), a calcium and phospholipid binding protein, is lost in prostate cancer.
To investigate ANXII expression in order to assess its utility as a novel diagnostic marker in comparison to p63 and AMACR.
Using immunohistochemistry on six tissue microarrays, ANXII, p63, and AMACR expression was analysed from 210 radical prostatectomy cases. Staining was evaluated in benign and atrophic glands, high‐grade prostatic intraepithelial neoplasia (HGPIN), and prostatic adenocarcinoma. Separate scores were given for ANXII, AMACR and p63 expression.
Diffuse cytoplasmic expression of ANXII correlated with p63 reactivity in basal cells. Benign glands were positive for ANXII in 286/292 cores (98%) and negative for AMACR in all 292 cores. HGPIN showed heterogeneous expression of AMACR and ANXII. A significantly larger proportion of HGPIN glands were correctly identified as ANXII negative than as positive for AMACR. ANXII loss in prostate cancer was found in 282/320 cores (88%) and correlated with positive AMACR expression (272/320 cores, 85%), which was not statistically significant. There was no statistically significant correlation between ANXII scores and the clinical parameters examined.
Immunohistochemical staining for ANXII is a consistent and reliable marker of prostatic neoplasia. The findings of this study suggest the potential utility of ANXII as a diagnostic aid in prostate cancer histopathology.
PMCID: PMC1995785  PMID: 16916997
annexin A2; α‐methylacyl‐CoA racemase; diagnostic markers, prostatic intraepithelial neoplasia; prostatic neoplasms
12.  Diagnostic utility of p501s (prostein) in comparison to prostate specific antigen (PSA) for the detection of metastatic prostatic adenocarcinoma 
Diagnostic Pathology  2007;2:41.
Immunohistochemical detection of prostate specific antigen (PSA) is widely used to identify metastatic prostatic adenocarcinoma. However, PSA may not be expressed in some poorly differentiated prostatic carcinomas and its immunoreactivity has been found in some non-prostatic tissues. P501s (prostein) is a prostate-specific marker that is expressed in the cytoplasm of benign and malignant prostatic glandular cells. It has not been detected in any other normal or malignant tissues. The purpose of this study was to evaluate the expression of P501s in metastatic prostatic adenocarcinoma and compare its expression with PSA.
Immunohistochemical stains with anti-P501s antibodies were performed on 5-micron sections of tissue microarray (TMA) specimens. The TMA is constructed with normal donor prostates (NDP), prostatic adenocarcinoma (PRCA), non-neoplastic prostatic tissues adjacent to malignant glands (NAT), benign prostatic hyperplasia (BPH), high-grade prostatic neoplasia (PIN), metastatic adenocarcinoma to lymph nodes (MLN), metastatic adenocarcinoma to other sites (MC), and samples of benign testis, colon, adrenal and kidney. The two groups of metastatic lesions were also subjected to stains with antibodies to PSA. A composite score (ranging from 0 to 3) was assigned to score intensity of staining.
Granular staining pattern of p501s was seen in all benign glands (score = 1.77 – 2.1) and malignant acini (score = 1.52) at the apical aspect of cytoplasm, predominantly adjacent to the nuclei. No staining was observed in controls including testis, colon, adrenal and kidney. The MLN group received a score of 1.0, with 10% of cases negative for p501s. The MC cases had a score of 0.64, with 16.7% of case showing loss of p501s expression. Although the metastatic lesions demonstrated similar rate of negative expression with PSA antibody, only 2 MC cases (3.3%) showed simultaneous negative stains for both P501S and PSA.
P501s is an organ specific marker for benign and malignant prostatic epithelial cells. Its characteristic cytoplasmic stain pattern provides an additional valuable immunomarker for detection of metastatic prostatic malignancy, even though the intensity of its expression is reduced, as in the case with PSA. Simultaneous stains with P501S and PSA will greatly improve the detection rate and identify a significant majority of the metastases.
PMCID: PMC2174437  PMID: 17963516
13.  Initial High-grade Prostatic Intraepithelial Neoplasia With Carcinoma on Subsequent Prostate Needle Biopsy: Findings at Radical Prostatectomy 
There are only a few small studies on men with an initial biopsy showing high-grade prostatic intraepithelial neoplasia (HGPIN) who later have cancer on repeat biopsy and then undergo radical prostatectomy. It is unknown whether this scenario impacts the prognosis of subsequent radical prostatectomy. We compared radical prostatectomy findings in 45 men with an initial diagnosis of HGPIN who subsequently were diagnosed with cancer with 18,494 men diagnosed with cancer who lacked an earlier diagnosis of HGPIN. All cases were retrieved from our institution between 1993 and 2008. The mean patient age was 60.2 years, and the mean serum prostate-specific antigen value was 9.0 ng/mL. For the 45 men with an initial HGPIN diagnosis, 21 of 45 (46.7%) men were found to have cancer within 6 months and 29 of 45 (64.4%) within 1 year after the diagnosis of HGPIN. Cancer involved a single core in 32 of 45 (71.1%) cases, and the maximum tumor volume was ≤5% in 57.8% of the 45 cases. Men with initial HGPIN had 84.4% organ-confined cancer, whereas cases without HGPIN had 65.4% organ-confined cancer (P=0.007) at radical prostatectomy. For the RPs performed in men with an earlier diagnosis of HGPIN followed by cancer on biopsy, the mean and median tumor volumes were 0.3 cm3 and 0.12 cm3 (0.003 cm3 to 1.46 cm3). Favorable pathologic stage was maintained even when we restricted the analysis to men with only Gleason score 6 cancer on biopsy. In men with Gleason score 6 cancer on biopsy, men with an initial diagnosis of HGPIN had 88.9% organ confined versus 73.2% for men with no earlier biopsy diagnosis of HGPIN, (P=0.03). At radical prostatectomy, although men with an earlier HGPIN diagnosis had less adverse findings in terms of Gleason score, surgical margin involvement, seminal vesicle involvement, and lymph node metastasis, the differences did not reach statistical significance. This was possibly due to the relatively small number of positive events in the men with no earlier HGPIN and due to the relatively small number of cases with earlier HGPIN. Prostatic adenocarcinomas discovered after an initial HGPIN diagnosis on biopsy are more likely to be organ confined, yet of similar grade, compared with cases diagnosed as cancer on the first biopsy. These findings likely reflect cancers associated with HGPIN, in which the cancers were missed on the initial biopsy as a result of smaller size.
PMCID: PMC4417348  PMID: 21716083
prostatic intraepithelial neoplasia; prostate cancer; needle biopsy
14.  Elevated Jagged-1 and Notch-1 expression in high grade and metastatic prostate cancers 
Background: Emerging evidence has suggested that Notch signaling pathway may be involved in the development, progression and metastasis of prostate cancer (PCa). In the present study, we investigated the expression levels of Jagged-1 and Notch-1 in human prostate tumors and their associations with PCa progression and metastasis. Methods: Immunohistochemistry (IHC) for Jagged-1 and Notch-1 was performed on tissue microarray (TMA) slides containing 286 formalin-fixed and paraffin-embedded (FFPE) tissue specimens with various prostatic pathologies, including benign changes, high grade prostatic intraepithelial neoplasia (HGPIN), low- and high-grade PCas as well as metastatic PCa. Results: Cytoplasmic and membranous IHC scores for Jagged-1 in both metastatic PCa and high grade PCa were significantly higher than those in low grade PCa and in benign prostatic tissues. Similarly, cytoplasmic IHC scores of Notch-1 in both metastatic PCa and high grade PCa were significantly elevated compared with those observed in low grade PCa and in benign prostatic tissues. A statistically significant correlation was identified between the expression of Jagged-1 and Notch-1 in human prostatic tissues. Furthermore, significantly more highly expressed Jagged-1 in membrane was observed in Caucasian patients with high-grade or metastatic PCa (vs. African Americans) and in PCa patients with positive surgical margins (vs. negative surgical margins). Conclusion: Our results provide strong evidence that up-regulation of Jagged1-Notch1 signaling plays a role in PCa progression and metastasis and suggest that Jagged-1 and Notch-1 may be useful markers in distinguishing indolent and aggressive PCas.
PMCID: PMC3633979  PMID: 23634247
Prostate cancer (PCa); cancer metastasis; Jagged-1; Notch-1; tissue microarray (TMA); immunohistochemistry (IHC)
15.  Prognostic significance of peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 in cholangiocarcinoma 
Human pathology  2012;43(10):1719-1730.
We performed a comparative proteomic analysis of protein expression profiles in four cholangiocarcinoma (CCA) cell lines: K100, M156, M213, and M139. The H69 biliary cell line was used as a control. Peroxiredoxin 1 (PRX1) and ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) were selected for further validation by immunohistochemistry (IHC) using a CCA tissue microarray (n=301) to assess their prognostic value in this cancer. Both PRX1 and EBP50 were overexpressed in CCA tissues compared with normal liver tissues. Of the 301 CCA cases, overexpression of PRX1 in 103 (34.3%) was associated with an age-related effect in young patients (P = 0.011) and the absence of cholangiocarcinoma in lymphatic vessels and perineural tissues (P = 0.004 and P = 0.037, respectively). Expression of EBP50 correlated with histopathologic type, being higher in 180 (59.8%) of moderately or poorly differentiated tumors (P = 0.039) and was associated with the presence of cholangiocarcinoma in lymphatic and vascular vessels (P< 0.001 and P< 0.001, respectively). The high expression of EBP50 and the low expression of PRX1 correlated with reduced survival by univariate analysis (P = 0.017 and P = 0.048, respectively). Moreover, the impact of PRX1 and EBP50 expression on patient survival was an independent predictor in multivariate analyses (P = 0.004 and P = 0.025, respectively). Therefore, altered expression of PRX1 and EBP50 may be used as prognostic markers incholangiocarcinoma.
PMCID: PMC3386378  PMID: 22446018
Cholangiocarcinoma; EBP50; Proteomics; PRX1; Prognostic marker; Tumor marker
16.  Epigenetic regulation of MDR1 gene through post-translational histone modifications in prostate cancer 
BMC Genomics  2013;14:898.
Multidrug resistance 1 (MDR1) gene encodes for an ATP binding cassette transporter - P-glycoprotein (P-gp) - involved in chemoresistance to taxanes. MDR1 promoter methylation is frequent in prostate carcinoma (PCa), suggesting an epigenetic regulation but no functional correlation has been established. We aimed to elucidate the epigenetic mechanisms involved in MDR1 deregulation in PCa.
MDR1 promoter methylation and P-gp expression were assessed in 121 PCa, 39 high-grade prostatic intraepithelial neoplasia (HGPIN), 28 benign prostatic hyperplasia (BPH) and 10 morphologically normal prostate tissue (NPT) samples, using quantitative methylation specific PCR and immunohistochemistry, respectively. PCa cell lines were exposed to a DNA methyltransferases inhibitor 5-aza-2′deoxycytidine (DAC) and histone deacetylases inhibitor trichostatin A (TSA). Methylation and histone posttranscriptional modifications status were characterized and correlated with mRNA and protein expression. MDR1 promoter methylation levels and frequency significantly increased from NPTs, to HGPIN and to PCa. Conversely, decreased or absent P-gp immunoexpression was observed in HGPIN and PCa, inversely correlating with methylation levels. Exposure to DAC alone did not alter significantly methylation levels, although increased expression was apparent. However, P-gp mRNA and protein re-expression were higher in cell lines exposed to TSA alone or combined with DAC. Accordingly, histone active marks H3Ac, H3K4me2, H3K4me3, H3K9Ac, and H4Ac were increased at the MDR1 promoter after exposure to TSA alone or combined with DAC.
Our data suggests that, in prostate carcinogenesis, MDR1 downregulation is mainly due to histone post-translational modifications. This occurs concomitantly with aberrant promoter methylation, substantiating the association with P-gp decreased expression.
PMCID: PMC3878566  PMID: 24344919
CpG island hypermethylation; Epigenetic regulation; Histone post-translational activation/repression marks; MDR1; P-gp; Prostate
17.  Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion 
Cancer letters  2009;283(1):29-35.
Chemokines and corresponding receptor interactions have been shown to be involved in prostate cancer (PCa) progression and organ-specific metastasis. We have recently shown that PCa cell lines and primary prostate tumors express CXCR5, which correlates with PCa grade. In this study, we present the first evidence that CXCL13, the only ligand for CXCR5, and IL-6 were significantly elevated in PCa patient serum compared to serum from subjects with benign prostatic hyperplasia (BPH), or high-grade prostatic intraepithelial neoplasia (HGPIN) as well as normal healthy donors (NHD). Serum CXCL13 levels significantly (p < 0.0001) correlated with serum prostate-specific antigen (PSA), whereas serum IL-6 levels significantly (p < 0.0003) correlated with CXCL13 serum levels. CXCL13 was found to be a better predictor of PCa than PSA. In addition, CXCL13 was highly expressed by human bone marrow endothelial (HBME) cells and osteoblasts (OBs), but not osteoclasts (OCs), following treatment with physiologically relevant levels of interleukin-6 (IL-6). We further demonstrate that CXCL13, produced by IL-6-treated HBME cells, was able to induce PCa cell invasion in a CXCR5-dependent manner. CXCL13-mediated PCa cell αvβ3-integrin clustering and adhesion to HBME cells was abrogated by CXCR5 blockade. These results demonstrate that the CXCL13-CXCR5 axis is significantly associated with PCa progression.
PMCID: PMC3600557  PMID: 19375853
chemokine; prostate; integrin; adhesion; invasion
18.  Chinese and Western prostate cancers show alternate pathogenetic pathways in association with ERG status 
We have previously reported genetic differences between Western and Chinese prostate cancers, including different frequencies of ERG rearrangements. We investigated further ERG expression and rearrangements in prostate cancers and high-grade prostatic intraepithelial neoplasia (HGPIN) from the UK and China to determine differences between these two populations by tissue microarray based immunohistochemistry and fluorescence in situ hybridization. In keeping with our previous observation, that ERG was rearranged at a higher frequency in UK prostate cancer samples (38%, 58/155) than Chinese ones (8%, 7/93), ERG rearrangements were also found in 21% (4/19) and 0% (0/19) foci of HGPIN in UK and Chinese samples respectively. ERG nuclear expression in UK cancers (34%, 54/160) was significantly higher than that in Chinese ones (10%, 9/88) (p<0.001). ERG nuclear expression in UK HGPIN (28%, 11/39) was higher than that in Chinese HGPIN (0%, 0/9), but without statistical significance (p=0.193). ERG nuclear expression was correlated to ERG rearrangements in both UK (Kappa=0.686) and Chinese (Kappa=0.565) cancers. These data demonstrate that ERG rearrangement and expression frequencies are different in prostate cancers from UK and China as early as the precursor lesion, HGPIN. The nuclear expression is associated with ERG rearrangements which mainly occur in the Western samples. UK and Chinese prostate cancers may be the result of different genetic mechanisms.
PMCID: PMC3512186  PMID: 23226619
ERG; prostate cancer; high-grade prostatic intraepithelial neoplasia; genomic rearrangement; protein expression
19.  Tumor suppressor function of ezrin-radixin-moesin-binding phosphoprotein-50 through β-catenin/E-cadherin pathway in human hepatocellular cancer 
AIM: To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) in hepatocellular carcinoma (HCC).
METHODS: Three human HCC cell lines, i.e., SM-MC7721, HepG2 and Hep3B, were used. We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50. Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells. In vitro cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution was assessed with flow cytometry. Invasion and migration ability of before and after the transfection were determined with a transwell assay. Cell apoptosis was demonstrated with Annexin V-FITC. The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.
RESULTS: The transfection efficiency was confirmed with Western blotting (1.36 ± 0.07 vs 0.81 ± 0.09, P < 0.01). The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells (P < 0.01). Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50 (61.3% ± 3.1% vs 54.0% ± 2.4%, P < 0.05). The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells (5.8 ± 0.8 vs 21.6 ± 1.3, P < 0.01). Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells (14.8% ± 2.7% vs 3.4% ± 1.3%, P < 0.05). The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells (0.28 ± 0.07 vs 0.56 ± 0.12, P < 0.05; 0.55 ± 0.08 vs 0.39 ± 0.07, P < 0.05). In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells (28.9 ± 7.2 vs 70.1 ± 7.2, P < 0.01).
CONCLUSION: The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin, E-cadherin. EBP50 may serve asa potential therapeutic target in HCC.
PMCID: PMC3587489  PMID: 23483729
Hepatocellular carcinoma; Ezrin-radixin-moesin-binding phosphoprotein-50; Growth; Migration; Invasion
20.  High-grade prostatic intraepithelial neoplasia and atypical small acinar proliferation on initial 21-core extended biopsy scheme: incidence and implications for patient care and surveillance 
World Journal of Urology  2009;27(5):587-592.
To evaluate the incidence of high-grade prostatic intraepithelial neoplasia (HGPIN) and atypical small acinar proliferation (ASAP) in initial 21-core extended biopsy scheme and to determine the prostate cancer detection rate in the repeated biopsy.
Between 2002 and 2008, 2,006 patients underwent a first 21-core extended biopsy scheme. Incidences of cancer, ASAP and HGPIN were studied. Cancer detection rate in the repeated 21-core extended biopsy for ASAP and HGPIN was reported and compared with those obtained on repeated biopsy for clinico-biological indications.
Incidences of HGPIN and ASAP were 1.7% and 1.1%, respectively. The 6-core and 12-core biopsy schemes detecting HGPIN would have missed the diagnosis of cancer in 10% and 3.6% of cases, compared to a 21-core biopsy protocol, respectively. The cancer detection rate on repeated biopsy for HGPIN was 19% and not significantly different compared with the detection rate on repeated biopsy for clinico-biological indications (16.8%, p=0.77). Seven prostate cancers were found among the 17 re-biopsies for ASAP revealing a detection rate of 41.2% (p=0.01). All detected cancers were organ-confined. No clinico-pathological data was independent predictor of cancer on repeated biopsy.
Our report demonstrates the different risk profiles for HGPIN and ASAP in a 21-core extended biopsy scheme. Presence of HGPIN does not imply a higher risk for cancer detection at immediate re-biopsy compared to other patients for who repeated biopsies were indicated for increasing or persistently increased PSA levels. Repea biopsy is warranted when ASAP is diagnosed because of a high risk of prostate cancer.
PMCID: PMC2825569  PMID: 19373471
Aged; Biopsy; Humans; Incidence; Male; Middle Aged; Population Surveillance; Prostatic Intraepithelial Neoplasia; epidemiology; pathology; Prostatic Neoplasms; epidemiology; pathology; atypical small acinar proliferation; high grade intraepithelial neoplasia; prostate cancer; saturation biopsy
21.  Periostin: a promising target of therapeutical intervention for prostate cancer 
In our recent study, Periostin was up-regulated in prostate cancer(PCa) compared with benign prostate hyperplasia (BPH) by proteomics analysis of prostate biopsies. We investigated the effect of sliencing Periostin by RNA interference (RNAi) on the proliferation and migration of PCa LNCap cell line.
All the prostate biopsies from PCa, BPH and BPH with local prostatic intraepithelial neoplasm(PIN) were analyzed by iTRAQ(Isobaric tags for relative and absolute quantification) technology. Western blotting and immunohistochemical staining were used to verify Periostin expression in the tissues of PCa. Periostin expression in different PCa cell lines was determined by immunofluorescence staining, western blotting and reverse transcription PCR(RT-PCR). The LNCap cells with Periostin expression were used for transfecting shRNA-Periostin lentiviral particles. The efficancy of transfecting shRNA lentiviral particles was evaluated by immunofluorescence, western blotting and Real-time PCR. The effect of silencing Periostin expression by RNAi on proliferation of LNCap cells was determined by MTT assay and tumor xenografts. The tissue slices from theses xenografts were analyzed by hematoxylin and eosin(HE) staining. The expression of Periostin in the xenografts was deteminned by Immunohistochemical staining and western blotting. The migration of LNCap cells after silencing Periostin gene expression were analyzed in vitro.
Periostin as the protein of interest was shown 9.12 fold up-regulation in PCa compared with BPH. The overexpression of Periostin in the stroma of PCa was confirmed by western blotting and immunohistochemical staining. Periostin was only expressed in PCa LNCap cell line. Our results indicated that the transfection ratio was more than 90%. As was expected, both the protein level and mRNA level of Periostin in the stably expressing shRNA-Periostin LNCap cells were significantly reduced. The stably expressing shRNA-Periostin LNCap cells growed slowly in vitro and in vivo. The tissues of xenografts as PCa were verificated by HE staining. Additionally, the weak positive Periostin expressed tumor cells could be seen in the tissues of 6 xenografts from the group of down-regulated Periostin LNCap cells which had a significant decrease of the amount of Periostin compared to the other two group. Furthermore, our results demonstrated that sliencing Periostin could inhibit migration of LNCap cells in vitro.
Our data indicates that Periostin as an up-regulated protein in PCa may be a promising target of therapeutical intervention for PCa in future.
PMCID: PMC3146429  PMID: 21714934
Periostin; Prostate cancer; RNAi; Proliferation; Migration
22.  Characterization of TMPRSS2-ERG fusion high-grade prostatic intraepithelial neoplasia and potential clinical implications 
More than 1,300,000 prostate needle biopsies are performed annually in the U.S. with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer (PCA) on subsequent needle biopsies in PSA screened populations. In contemporary series, PCA is detected in about 20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histological subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in PCA has also been demonstrated to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion PCA is associated with a more aggressive clinical course.
Experimental Design
In this study we assessed a series of HGPIN lesions and paired PCA for the presence of TMPRSS2-ERG gene fusion.
Fusion positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances the matching cancer shared the same fusion pattern. 60% of TMPRSS2-ERG fusion PCA had fusion negative HGPIN.
Given the more aggressive nature of TMPRSS2-ERG PCA, the findings of this study raise the possibility that gene fusion positive HGPIN lesions are harbingers of more aggressive disease. To date, pathological, molecular and clinical parameters do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion PCA in subsequent biopsies.
PMCID: PMC3717517  PMID: 18519767
23.  Bicalutamide 50 mg monotherapy in patients with isolated high‐grade PIN: findings in repeat biopsies at 6 months 
Journal of Clinical Pathology  2006;60(4):443-446.
To evaluate morphological findings in repeat biopsies in patients with isolated high‐grade prostatic intraepithelial neoplasia (HGPIN) after a 6‐month course of bicalutamide (Casodex) 50 mg/day.
20 consecutive patients with isolated HGPIN in prostate biopsies were treated for 6 months with bicalutamide 50 mg/day. After treatment, the patients were resubmitted to prostate biopsy mapping. The control group included 22 untreated consecutive patients with isolated high‐grade PIN with repeat biopsies taken 6 months after the initial biopsies.
In the initial biopsies of the treated group, HGPIN was monofocal in 12 patients and plurifocal in 8. In the repeat biopsies HGPIN was present in 2 patients, monofocal in both, whereas prostate adenocarcinoma (PCa) was discovered in one. In the control group, HGPIN was monofocal in 15 and plurifocal in 7. In the repeat biopsies HGPIN was present in six patients, being monofocal in three and plurifocal in the other three. PCa was present in one.
There was a lower incidence of HGPIN (treated group vs control: 10% vs 27.2%) after 6 months of bicalutamide. Reduction in its extent was also observed (treated group vs control: monofocal 100% vs 50%). Treatment did not affect the incidence of cancer (treated vs control: 5% vs 4.5%).
PMCID: PMC2001123  PMID: 16822873
24.  HP1γ expression is elevated in prostate cancer and is superior to Gleason score as a predictor of biochemical recurrence after radical prostatectomy 
BMC Cancer  2013;13:148.
Aberrant chromatin structure in cancer cells results from altered proteins involved in its packaging. Heterochromatin protein 1 gamma (HP1γ) is a non-histone heterochromatic protein that functions to maintain chromatin stability and is important in embryonic development. Given an interest in the role developmental genes play in cancer, we investigated HP1γ expression in prostate cancer (PCa) and its prognostic associations.
Tissue microarrays consisting of benign (N = 96), localized cancer (N = 146), metastatic PCa (N = 44), and HGPIN (N = 50) were immunoflourescently stained for HP1γ and Ki-67. Using a novel, automated quantitative imaging system, VECTRA™, epithelial staining in both the nucleus and cytoplasm was quantified and compared against clinicopathologic variables.
HP1γ is significantly elevated in HGPIN (80%), localized PCa (76%), and metastatic PCa (98%) compared to benign tissues from both the nuclear and cytoplasmic compartments (P < 0.0001). Increased nuclear and total HP1γ expression was associated with Gleason score (P = 0.02 and P = 0.04 respectively). Given known binding to the C-terminus of Ki-67, a co-expression analysis was performed that revealed a correlation between nuclear and cytoplasmic HP1γ and Ki-67 (Pearson Coefficient 0.321 and 0.562 respectively, P < 0.0001). Cox survival analysis demonstrated that cytoplasmic HP1γ expression was an independent prognostic marker and out-performed pathological Gleason score for predicting PSA-recurrence after radical prostatectomy.
In this first detailed analysis of HP1γ expression in cancer, VECTRA™ demonstrates compartmentalized and total HP1γ protein expression is increased in PCa and that expression correlates with clinical outcomes better than Gleason score. Given the critical role HP1γ plays in chromatin organization and gene expression, it represents a novel prognostic and therapeutic target.
PMCID: PMC3618256  PMID: 23522301
Prostate cancer; HP1γ; Prognosis; PSA-Recurrence; Ki-67; Tissue microarray
25.  Subsequent prostate cancer detection in patients with prostatic intraepithelial neoplasia or atypical small acinar proliferation 
To evaluate the predictors of prostate cancer in follow-up of patients diagnosed on initial biopsy with high-grade prostatic intraepithelial neoplasia (HGPIN) or atypical small acinar proliferation (ASAP).
We studied 201 patients with HGPIN and 22 patients with ASAP on initial prostatic biopsy who had subsequent prostatic biopsies. The mean time of follow-up was 17.3 months (range 1–62). The mean number of biopsy sessions was 2.5 (range 2–6), and the median number of biopsy cores was 10 (range 6–14).
On subsequent biopsies, the rate of prostate cancer was 21.9% (44/201) in HGPIN patients. Of these, 32/201 patients (15.9%), 9/66 patients (13.6%) and 3/18 patients (16.6%) were found to have cancer on the first, second and third follow-up biopsy sessions, respectively. In ASAP patients, the cancer detection rate was 13/22 (59.1%), all of whom were found on the first follow-up biopsy. There was a statistically significant difference between the cancer detection rate in ASAP and HGPIN patients (p < 0.001). Multivariate analysis showed that the independent predictors of cancer were the number of cores in the initial biopsy, the number of cores (> 10) in the follow-up biopsy and a prostate specific antigen (PSA) density of ≥ 0.15 (odds ratio 0.77, 3.46 and 2.7,8 respectively; p < 0.04). Conversely, in ASAP patients none of these variables were found to be associated with cancer diagnosis.
ASAP is a strong predictive factor associated with cancer when compared with HGPIN. The factors predictive of cancer on follow-up biopsy of HGPIN are number of cores on initial biopsy, more than 10 cores in rebiopsy and elevated PSA density. As the cancer detection rate on repeated biopsy of HGPIN patients is the same as that of patients without HGPIN, perhaps the standard of repeat biopsy in all patients with HGPIN should be revisited.
PMCID: PMC2422970  PMID: 18542796

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