A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family... more A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family history-positive prostate cancer in patients of European descent, occurring in up to 5% of prostate cancer families. To date, the molecular features of prostate tumors occurring in HOXB13 G84E carriers have not been studied in a large cohort of patients. We identified 101 heterozygous carriers of G84E who underwent radical prostatectomy for prostate cancer between 1985 and 2011 and matched these men by race, age and tumor grade to 99 HOXB13 wild-type controls. Immunostaining for HOXB13, PTEN, ERG, p53 and SPINK1 as well as RNA in situ hybridization for ETV1/4/5 were performed using genetically validated assays. Tumors from G84E carriers generally expressed HOXB13 protein at a level comparable to benign and wild-type glands. ETS gene expression (either ERG or ETV1/4/5) was seen in 36% (36/101) of tumors from G84E carriers compared to 68% (65/96) of the controls (p < 0.0001). PTEN was ...
Further understanding of the molecular mechanisms responsible for prostate cancer (CaP) developme... more Further understanding of the molecular mechanisms responsible for prostate cancer (CaP) development and progression is paramount for overcoming the current diagnostic and therapeutic hurdles presented by this urologic disease. The β-catenin nuclear signaling molecule has been widely implicated as an oncogene in human cancer, including CaP. Pooling together knowledge gathered on the contributions of β-catenin and other factors to human neoplasia may assist in the development of better strategies for management and treatment of prostate tumors of all stages (early, advanced/ androgen-dependent, advanced/androgen-independent). Although there is considerable lack of comprehension regarding the function of β-catenin transcriptional activity in prostate tumors in vivo, recent evidence indicates the probability that β-catenin contributes to multiple signaling pathways for which a causative role in CaP is already known. In this review, we will approach such pathway interactions, perhaps the...
A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant ... more A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components β-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasi...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 26, 2017
PURPOSE:TP53 missense mutations may help to identify prostate cancer (PCa) with lethal potential.... more PURPOSE:TP53 missense mutations may help to identify prostate cancer (PCa) with lethal potential. Here, we pre-analytically, analytically and clinically validated a robust immunohistochemistry (IHC) assay to detect subclonal and focal TP53 missense mutations in PCa. <p>EXPERIMENTAL DESIGN: The p53 IHC assay was performed in a CLIA-accredited laboratory on the Ventana Benchmark immunostaining system. p53 protein nuclear accumulation was defined as any p53 nuclear labeling in >10% of tumor cells. 54 formalin fixed paraffin embedded (FFPE) cell lines from the NCI-60 panel and 103 FFPE PCa tissues (88 primary adenocarcinomas, 15 metastases) with known TP53 mutation status were studied. DU145 and VCaP xenografts were subjected to varying fixation conditions to investigate the effects of pre-analytic variables. Clinical validation was performed in two partially overlapping radical prostatectomy (RP) cohorts.</p> <p>RESULTS: p53 nuclear accumulation by IHC was 100% sen...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 7, 2015
Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asp... more Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asporin (ASPN) as a potential mediator of metastatic progression found within the tumor microenvironment. ASPN contains an aspartic acid (D)-repeat domain and germline polymorphisms in D-repeat-length have been associated with degenerative diseases. Associations of germline ASPN D polymorphisms with risk of prostate cancer progression to metastatic disease have not been assessed. Germline ASPN D-repeat-length was retrospectively analyzed in 1600 men who underwent radical prostatectomy for clinically localized prostate cancer and in 548 non-cancer controls. Multivariable Cox proportional hazards models were used to test the associations of ASPN variations with risk of subsequent oncologic outcomes including metastasis. Orthotopic xenografts were used to establish allele- and stroma-specific roles for ASPN D variants in metastatic prostate cancer. Variation at the ASPN D locus was differentia...
Chronic inflammation and obesity may contribute to the genesis or progression of BPH and BPH-asso... more Chronic inflammation and obesity may contribute to the genesis or progression of BPH and BPH-associated lower urinary tract symptoms (LUTS). The influence of variants in genes related to these states on BPH has not been studied extensively. Thus, we evaluated the association of 17 single-nucleotide polymorphisms (SNPs) in immune response genes (IL1B, IL6, IL8, IL10, TNF, CRP, TLR4 and RNASEL) and genes involved in obesity, including insulin regulation (LEP, ADIPOQ, PPARG and TCF7L2), with BPH. BPH cases (N = 568) and age-frequency matched controls (N=568) were selected from among adult male CLUE II cohort participants who responded in 2000 to a mailed questionnaire. BPH was defined as BPH surgery, use of BPH medications or symptomatic BPH (American Urological Association Symptom Index Score ⩾ 15). Controls were men who had not had BPH surgery, did not use BPH medications and whose symptom score was ⩽ 7. Age-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated...
Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in beta-oxidation of branched-chain f... more Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in beta-oxidation of branched-chain fatty acids and bile acid intermediates. Recent work has identified AMACR as a new diagnostic marker for prostate cancer (PCa). The data from the present study suggest that AMACR is also functionally important for the growth of PCa cells. Overexpressed AMACR from both clinical tissues and PCa cell lines is wild type by sequence analysis and functionally active by enzymatic assay. Correspondingly, enzyme activity of AMACR increases approximately 4-fold in PCa in comparison with adjacent normal prostate. Small interference RNA (siRNA) against AMACR, but not the control inverted siRNA, reduced the expression of AMACR and significantly impaired proliferation of the androgen-responsive PCa cell line LAPC-4. No effect was observed in HeLaS3 cells, which express AMACR at a low level. Cell cycle analyses revealed a G(2)-M cell cycle arrest in LAPC-4 cells treated with siRNA compared with mock tre...
Prostate cancer is among the most common malignancies. It is estimated that 1 in 6 men in the Uni... more Prostate cancer is among the most common malignancies. It is estimated that 1 in 6 men in the United States will be diagnosed with this disease. Despite the high prevalence and importance of prostate cancer, the molecular mechanisms underlying its development and progression remain poorly understood. This article reviews new information about the roles of oxidants and electrophiles in prostate cancer; the potential importance of chronic inflammation and atrophy in prostate carcinogenesis, and implications for chemoprevention; evidence supporting telomere shortening and genetic instability in the etiology of prostate cancer; and ␣-methylacyl-coenzyme A racemase (AMACR) as a potential marker for prostate carcinogenesis. These new results show that at least some high-grade prostatic intraepithelial neoplasias (PIN) and early adenocarcinomas appear to arise from proliferative inflammatory atrophy (PIA). Inflammation and other environmental factors may lead to the destruction of prostate epithelial cells, and increased proliferation may occur as a response to this cell death. Such proliferation may be mechanistically related to decreased p27 Kip1 observed in PIA. The decreased apoptosis associated with these events may also be related to increased expression of Bcl-2. Increased oxidant and electrophile stress in the setting of increased proliferation associated with these events may lead to elevated glutathione S-transferase P1 (GSTP1) expression as a genomic-protective measure. However, aberrant methylation of the CpG island of the GSTP1 gene promoter silences GSTP1 gene expression and protein levels, setting the stage for additional genetic damage and accelerated progression toward PIN and carcinoma. Additional results show that AMACR may be an important new marker of prostate cancer, and its use in combination with p63 staining may provide the basis for an improved method for identification of prostate cancer.
Overexpression of alpha-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fat... more Overexpression of alpha-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fatty acid beta-oxidation, in prostate cancer has been reported. Here, we report that an enzyme downstream from AMACR in the peroxisomal branched chain fatty acid beta-oxidation pathway-D-bifunctional protein (DBP)-is also upregulated in prostate cancer at both mRNA and protein levels, accompanied by increased enzymatic activity. Furthermore, our data suggest that pristanoyl-CoA oxidase (ACOX3), which is expressed at extremely low level in other human organs studied including the liver, might contribute significantly to peroxisomal branched chain fatty acid beta-oxidation in human prostate tissue and some prostate cancer cell lines. In contrast to these results for peroxisomal enzymes, no significant expression changes of mitochondrial fatty acid beta-oxidation enzymes were observed in prostate cancer tissues through comprehensive quantitative RT-PCR screening. These data for the first time provide evidence for the selective over-activation of peroxisomal branched chain fatty acid beta-oxidation in prostate cancer, emphasizing a new metabolic change during prostate oncogenesis.
BACKGROUND-Because of the dramatically different clinical course of aggressive and indolent prost... more BACKGROUND-Because of the dramatically different clinical course of aggressive and indolent prostate carcinoma (PCa), markers that distinguish between these phenotypes are of critical importance. Apoptosis is an important protective mechanism for unrestrained cellular growth and metastasis. Therefore, dysfunction in this pathway is a key step in cancer progression. As such, genetic variants in apoptosis genes are potential markers of aggressive PCa. Recent work in breast carcinoma has implicated the histidine variant of CASP8 D302H (rs1045485) as a protective risk allele. METHODS-We tested the hypothesis that the Hvariant was protective for aggressive PCa in a pooled analysis of 796 aggressive cases and 2,060 controls. RESULTS. The H allele was associated with a reduced risk of aggressive PCa (ORper allele = 0.67, 95% CI: 0.54-0.83, Ptrend = 0.0003). The results were similar for European-Americans (ORper allele = 0.68; 95% CI: 0.54-0.86) and African-Americans (ORper allele = 0.61; 95% CI: 0.34-1.10). We further determined from the full series of 1,160 cases and 1,166 controls in the Prostate, Lung, Colorectal, Ovarian (PLCO) population that the protective effect of the H allele tended to be limited to high-grade and advanced PCa (all cases ORper allele = 0.94; 95% CI: 0.79-1.11; localized, low-grade disease ORper allele = 0.98; 95% CI: 0.79-1.23; and aggressive disease ORper allele = 0.73; 95% CI: 0.50-1.07). CONCLUSION-These results suggest that histidine variant of CASP8 D302H is a protective allele for aggressive PCa with potential utility for identification of patients at differential risk for this clinically significant phenotype.
BACKGROUND-Malignant transformation is often accompanied by morphological and functional alterati... more BACKGROUND-Malignant transformation is often accompanied by morphological and functional alterations in subcellular organelles. The Golgi apparatus is a subcellular structure primarily involved in modification and sorting of macromolecules for secretion and transport to other cellular destinations. Molecular alterations associated with the Golgi apparatus may take place during prostate carcinogenesis but such alterations have not been documented. METHODS-To demonstrate that the Golgi apparatus undergoes alterations during prostate carcinogenesis, we examined the expression and localization of two candidate molecules, Golgi phosphoprotein 2 (GOLPH2) and myosin VI (MYO6), both overexpressed in prostate cancer as initially identified by expression microarray analysis. RESULTS-Elevated GOLPH2 expression in prostate cancers was validated through real-time RT-PCR, Western blot, and tissue microarray analysis, and its Golgi localization in surgical prostate cancer tissues confirmed using two-color immunofluorescence. In addition, distinctive juxtanuclear MYO6 staining pattern consistent with Golgi localization was observed in surgical prostate cancer tissues. Two-color immunofluorescence revealed intensive Golgi-specific staining for both GOLPH2 and myosin VI in prostate cancer cells but not in the adjacent normal prostate epithelium. CONCLUSIONS-We show that the Golgi apparatus in prostate cancer cells differs from the normal Golgi by elevated levels of two molecules, GOLPH2 and MYO6. These results for the first time demonstrated consistent cancer cell-specific alterations in the molecular composition of the Golgi apparatus. Such alterations can be explored for discovery of novel prostate cancer biomarkers through targeted organellar approaches.
BACKGROUND-TNFRSF10C, is located on 8p21.3, one of the most frequently deleted loci in the genome... more BACKGROUND-TNFRSF10C, is located on 8p21.3, one of the most frequently deleted loci in the genome of prostate cancer (PCa). Hypermethylation of TNFRSF10C promoter CpG island (CGI) had been reported in many tumors including PCa. However, the interplay between somatic deletion and promoter hypermethylation of TNFRSF10C on PCa development has not been investigated. METHODS-Methylation status of promoter CGI and deletion status of the TNFRSF10C locus was investigated by bisulfite sequencing and Affymetrix SNP array, respectively in 59 pairs of PCa tumor and matched normal samples with 3 PCa cell lines. TNFRSF10C gene expression changes in relation to cancer-associated genetic/epigenetic changes in clinical specimens, and change of TNFRSF10C expression before and after 5-aza-2'-deoxycytidine treatment in the PC3 PCa cell line were assessed by real-time RT-PCR. RESULTS-We found that TNFRSF10C promoter CGI was differentially methylated in 46 of 59 primary cancers (78.0%). Hemizygous deletion at TNFRSF10C was found in 44 of the 59 prostate tumors (74.5%). Interestingly, in 94.9% of the tumors (56 out of 59), TNFRSF10C was either hemizygously deleted or its promoter CGI hypermethylated. Deletion and/or methylation of the TNFRSF10C gene was correlated with decreased mRNA expression of the gene in clinical specimens. Demethylation of the TNFRSF10C promoter CGI was accompanied by transcriptional re-activation of TNFRSF10C in the prostate cancer cell line PC3. CONCLUSION-We found a notably high frequency of promoter CGI methylation and deletion of TNFRSF10C in PCa tissues. Our results indicated that inactivation of TNFRSF10C by chromosomal deletion and promoter methylation may play an important role in PCa development.
Recent identifications of associations between novel variants in inflammation-related genes and s... more Recent identifications of associations between novel variants in inflammation-related genes and several common diseases emphasize the need for systematic evaluations of these genes in disease susceptibility. Considering that many genes are involved in the complex inflammation responses and many genetic variants in these genes have the potential to alter the functions and expression of these genes, we assembled a list of key inflammation-related genes to facilitate the identification of genetic associations of diseases with an inflammation-related etiology. We first reviewed various phases of inflammation responses, including the development of immune cells, sensing of danger, influx of cells to sites of insult, activation and functional responses of immune and non-immune cells, and resolution of the immune response. Assisted by the Ingenuity Pathway Analysis, we then identified 17 functional sub-pathways that are involved in one or multiple phases. This organization would greatly increase the chance of detecting gene-gene interactions by hierarchical clustering of genes with their functional closeness in a pathway. Finally, as an example application, we have developed tagging single nucleotide polymorphism (tSNP) arrays for populations of European and African descent to capture all the common variants of these key inflammation-related genes. Assays of these tSNPs have been designed and assembled into two Affymetrix ParAllele customized chips, one each for European (12,011 SNPs) and African (21,542 SNPs) populations. These tSNPs have greater coverage for these inflammationrelated genes compared to the existing genome-wide arrays, particularly in the African population. These tSNP arrays can facilitate systematic evaluation of inflammation pathways in disease susceptibility. For additional applications, other genotyping platforms could also be employed. For existing genome-wide association data, this list of key inflammation-related genes and associated subpathways can facilitate comprehensive inflammation pathway-focused association analyses.
b-catenin signaling may contribute to prostate cancer (CaP) progression. Although b-catenin is kn... more b-catenin signaling may contribute to prostate cancer (CaP) progression. Although b-catenin is known to upregulate T cell factor (TCF) target gene expression in CaP cells, recent evidence demonstrates its capacity to enhance ligand-dependent androgen receptor (AR) function. Thus, we wished to further understand the interaction between these two pathways. We find in both CaP cells (CWR22-Rv1, LAPC-4, DU145) and non-CaP cells (HEK-293, TSU, SW480, HCT-116) that bcatenin/TCF-related transcription (CRT), as measured by activation of a synthetic promoter and that of cyclin D1, is inhibited by androgen treatment. This inhibition is AR-dependent, as it only occurs in cells expressing AR endogenously or transiently, and is abrogated by AR antagonists. Additional analyses convey that the liganddependent nature of CRT suppression depends on transactivation-competent AR in the nucleus, but not on indirect effects stemming from AR target gene expression. Given the recent work identifying an AR/bcatenin interaction, and from our finding that liganded AR does not prompt gross changes in the constitutive nuclear localization of TCF4 or mutant b-catenin, we hypothesized that transcription factor (i.e. AR and TCF) competition for b-catenin recruitment may explain, in part, androgen-induced suppression of CRT. To address this idea, we expressed an AR mutant lacking its DNAbinding domain (DBD). This receptor could not orchestrate ligand-dependent CRT repression, thereby providing support for those recent data implicating the AR DBD/LBD as necessary for b-catenin interaction. Further supporting this hypothesis, TCF/LEF overexpression counteracts androgen-induced suppression of CRT, and requires b-catenin binding activity to do so. Interestingly, TCF4 over-expression potently antagonizes AR function; however, this inhibition may occur independently of b-catenin/TCF4 interaction. These results from TCF4 over-expression analyses, taken together, provide further evidence that AR-mediated suppression of CRT is a consequence of limiting amounts of b-catenin, and not AR target gene expression. Our analyses point to a reciprocal balance between AR and CRT function that may shape critical processes during normal prostate development and tumor progression.
Understanding the molecular etiology of prostate cancer (CaP) progression is paramount for broade... more Understanding the molecular etiology of prostate cancer (CaP) progression is paramount for broadening current diagnostic and therapeutic modalities. Current interest in the role of wnt pathway signaling in prostate tumorigenesis was generated with the ®nding of b-catenin mutation and corresponding nuclear localization in primary lesions. The recent ®nding of b-catenin-induced enhancement of androgen receptor (AR) function potentially ties b-catenin to key regulatory steps of prostate cell growth, dierentiation, and transformation. By immunohistological analysis of metastatic tumors, we detected nuclear bcatenin in 20% of lethal CaP cases, suggesting a more common role for b-catenin in advanced disease than would be predicted by its mutation rate. Interestingly, bcatenin nuclear localization was found to occur concomitantly with androgen-induced regrowth of normal rat prostate. These in vivo observations likely implicate b-catenin involvement in both normal and neoplastic prostate physiology, thus prompting our interest in further characterizing modes of b-catenin signaling in prostate cells. Extending our previous ®ndings, we demonstrate that transient b-catenin over-expression stimulates T cell factor (TCF) signaling in most CaP cell lines. Further, this activity is not subject to crossregulation by phosphoinositide-3-kinase (PI3-K)/Akt signaling, a stimulatory pathway often upregulated in CaP upon PTEN inactivation. Consistent with a previous report, we observed that transient b-catenin overexpression enhances AR-mediated transcription o two natural target gene promoters. However, we were unable to recapitulate b-catenin-induced stimulation of ectopically expressed AR in AR-negative cells, suggesting that other AR-associated factors are required for this activity. Although LNCaP cells are capable of this mode of AR co-stimulation, stable expression of mutant b-catenin did not alter their proliferative response to androgen. In total, our characterization of b-catenin signaling in CaP reveals the complex nature of its activity in prostate tissue, indicating that b-catenin potentially contributes to multiple stimulatory inputs required for disease progression.
The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, pro... more The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.
Purpose: We assessed the feasibility of a novel urinary test for prostate cancer based on the pre... more Purpose: We assessed the feasibility of a novel urinary test for prostate cancer based on the presence of ␣ methylacyl coenzyme A racemase (AMACR) protein in voided urine specimens obtained after prostate biopsy. Materials and Methods: Clean catch voided urine specimens were prospectively collected from 26 consecutive men immediately after transrectal ultrasound guided prostate biopsy for suspected malignancy. The presence of AMACR was evaluated in a blinded manner by Western blot analysis and correlated with biopsy results and patient clinical information. Results: AMACR was detected in the urine in 18 of 26 patients (69%). AMACR was detected in all 12 patients with biopsy confirmed adenocarcinoma of the prostate (100% sensitivity, 95% CI 75 to 100), in 5 of 12 with no evidence of cancer on biopsy (58% specificity, 95% CI 29 to 78) and in 1 of 2 (50%, 95% CI 3 to 80) with atypia on biopsy. Overall AMACR detection was associated with cancer status by prostate biopsy in 21 of 26 patients (86%). Conclusions: We report the feasibility of a novel, noninvasive, nonprostate specific antigen based molecular approach to detect prostate cancer in voided urine. To our knowledge this is the first report of AMACR protein detection in the urine of patients with prostate cancer. A screening test based on urinary AMACR may develop into a useful adjunct to serum prostate specific antigen and digital rectal examination for identifying men at increased risk for harboring prostate cancer despite negative biopsy. Such a test has potential application for stratifying patients into low and high risk groups for surveillance vs repeat biopsy.
Lipoprotein lipase (LPL) is in chromosome 8p22, site of one of the most common somatic deletions ... more Lipoprotein lipase (LPL) is in chromosome 8p22, site of one of the most common somatic deletions in prostate tumors. Additionally, a CpG island (CGI) was identified in the LPL promoter region. To test the hypothesis that LPL is a tumor suppressor gene, which is inactivated by somatic deletion and hypermethylation in prostate cancer, we evaluated somatic DNA deletion and methylation status at LPL in 56 pairs of DNA samples isolated from prostate cancer tissues and matching normal controls and 11 prostate cell lines. We found that the DNA in 21 of 56 primary cancers (38%) was methylated in the LPL promoter CGI, whereas no methylation was detected in any normal samples. In addition, we found a hemizygous deletion at LPL in 38 of the 56 tumors (68%). When the results of deletion and methylation were considered together, we found LPL promoter CGI methylation occurred in 45% of LPL deleted tumors and in 22% of LPL retained tumors. Within several clinical characteristics tested, the preoperative PSA levels were found to be significantly higher in subjects with LPL promoter CGI methylation compared with subjects without LPL promoter methylation (p = 0.0012). Additionally, demethylation of the LPL promoter CGI was accompanied by transcriptional reactivation of LPL in the prostate cancer cell lines DU145 and PC3. In summary, we report a novel finding that the LPL gene is commonly methylated in prostate tumors, and our results suggest that biallelic inactivation of LPL by chromosomal deletion and promoter hypermethylation may play a role in human prostate cancer.
Purpose: Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly com... more Purpose: Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide the diagnosis and therapy of this aggressive tumor. Experimental Design: We examined the status of RB1, TP53, and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy-number alteration analysis, and sequencing of formalin-fixed paraffin-embedded specimens. Results: We found retinoblastoma (Rb) protein loss in 90% of small cell carcinoma cases (26 of 29) with RB1 allelic loss in 85% of cases (11 of 13). Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3 of 7) showed Rb protein loss. In contrast, only 7% of primary high-grade acinar carcinomas (10 of 150), 11% of primary acinar carcinomas with neuroendocrine differentiation (4 of 35), and 15% of metas...
Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are cr... more Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. Experimental Design: PTEN IHC was validated by employing formalin fixed and paraffin-embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymorphism microarray analysis was done on a subset of these cases. Results: PTEN prote...
A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family... more A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family history-positive prostate cancer in patients of European descent, occurring in up to 5% of prostate cancer families. To date, the molecular features of prostate tumors occurring in HOXB13 G84E carriers have not been studied in a large cohort of patients. We identified 101 heterozygous carriers of G84E who underwent radical prostatectomy for prostate cancer between 1985 and 2011 and matched these men by race, age and tumor grade to 99 HOXB13 wild-type controls. Immunostaining for HOXB13, PTEN, ERG, p53 and SPINK1 as well as RNA in situ hybridization for ETV1/4/5 were performed using genetically validated assays. Tumors from G84E carriers generally expressed HOXB13 protein at a level comparable to benign and wild-type glands. ETS gene expression (either ERG or ETV1/4/5) was seen in 36% (36/101) of tumors from G84E carriers compared to 68% (65/96) of the controls (p < 0.0001). PTEN was ...
Further understanding of the molecular mechanisms responsible for prostate cancer (CaP) developme... more Further understanding of the molecular mechanisms responsible for prostate cancer (CaP) development and progression is paramount for overcoming the current diagnostic and therapeutic hurdles presented by this urologic disease. The β-catenin nuclear signaling molecule has been widely implicated as an oncogene in human cancer, including CaP. Pooling together knowledge gathered on the contributions of β-catenin and other factors to human neoplasia may assist in the development of better strategies for management and treatment of prostate tumors of all stages (early, advanced/ androgen-dependent, advanced/androgen-independent). Although there is considerable lack of comprehension regarding the function of β-catenin transcriptional activity in prostate tumors in vivo, recent evidence indicates the probability that β-catenin contributes to multiple signaling pathways for which a causative role in CaP is already known. In this review, we will approach such pathway interactions, perhaps the...
A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant ... more A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components β-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasi...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 26, 2017
PURPOSE:TP53 missense mutations may help to identify prostate cancer (PCa) with lethal potential.... more PURPOSE:TP53 missense mutations may help to identify prostate cancer (PCa) with lethal potential. Here, we pre-analytically, analytically and clinically validated a robust immunohistochemistry (IHC) assay to detect subclonal and focal TP53 missense mutations in PCa. <p>EXPERIMENTAL DESIGN: The p53 IHC assay was performed in a CLIA-accredited laboratory on the Ventana Benchmark immunostaining system. p53 protein nuclear accumulation was defined as any p53 nuclear labeling in >10% of tumor cells. 54 formalin fixed paraffin embedded (FFPE) cell lines from the NCI-60 panel and 103 FFPE PCa tissues (88 primary adenocarcinomas, 15 metastases) with known TP53 mutation status were studied. DU145 and VCaP xenografts were subjected to varying fixation conditions to investigate the effects of pre-analytic variables. Clinical validation was performed in two partially overlapping radical prostatectomy (RP) cohorts.</p> <p>RESULTS: p53 nuclear accumulation by IHC was 100% sen...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 7, 2015
Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asp... more Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asporin (ASPN) as a potential mediator of metastatic progression found within the tumor microenvironment. ASPN contains an aspartic acid (D)-repeat domain and germline polymorphisms in D-repeat-length have been associated with degenerative diseases. Associations of germline ASPN D polymorphisms with risk of prostate cancer progression to metastatic disease have not been assessed. Germline ASPN D-repeat-length was retrospectively analyzed in 1600 men who underwent radical prostatectomy for clinically localized prostate cancer and in 548 non-cancer controls. Multivariable Cox proportional hazards models were used to test the associations of ASPN variations with risk of subsequent oncologic outcomes including metastasis. Orthotopic xenografts were used to establish allele- and stroma-specific roles for ASPN D variants in metastatic prostate cancer. Variation at the ASPN D locus was differentia...
Chronic inflammation and obesity may contribute to the genesis or progression of BPH and BPH-asso... more Chronic inflammation and obesity may contribute to the genesis or progression of BPH and BPH-associated lower urinary tract symptoms (LUTS). The influence of variants in genes related to these states on BPH has not been studied extensively. Thus, we evaluated the association of 17 single-nucleotide polymorphisms (SNPs) in immune response genes (IL1B, IL6, IL8, IL10, TNF, CRP, TLR4 and RNASEL) and genes involved in obesity, including insulin regulation (LEP, ADIPOQ, PPARG and TCF7L2), with BPH. BPH cases (N = 568) and age-frequency matched controls (N=568) were selected from among adult male CLUE II cohort participants who responded in 2000 to a mailed questionnaire. BPH was defined as BPH surgery, use of BPH medications or symptomatic BPH (American Urological Association Symptom Index Score ⩾ 15). Controls were men who had not had BPH surgery, did not use BPH medications and whose symptom score was ⩽ 7. Age-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated...
Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in beta-oxidation of branched-chain f... more Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in beta-oxidation of branched-chain fatty acids and bile acid intermediates. Recent work has identified AMACR as a new diagnostic marker for prostate cancer (PCa). The data from the present study suggest that AMACR is also functionally important for the growth of PCa cells. Overexpressed AMACR from both clinical tissues and PCa cell lines is wild type by sequence analysis and functionally active by enzymatic assay. Correspondingly, enzyme activity of AMACR increases approximately 4-fold in PCa in comparison with adjacent normal prostate. Small interference RNA (siRNA) against AMACR, but not the control inverted siRNA, reduced the expression of AMACR and significantly impaired proliferation of the androgen-responsive PCa cell line LAPC-4. No effect was observed in HeLaS3 cells, which express AMACR at a low level. Cell cycle analyses revealed a G(2)-M cell cycle arrest in LAPC-4 cells treated with siRNA compared with mock tre...
Prostate cancer is among the most common malignancies. It is estimated that 1 in 6 men in the Uni... more Prostate cancer is among the most common malignancies. It is estimated that 1 in 6 men in the United States will be diagnosed with this disease. Despite the high prevalence and importance of prostate cancer, the molecular mechanisms underlying its development and progression remain poorly understood. This article reviews new information about the roles of oxidants and electrophiles in prostate cancer; the potential importance of chronic inflammation and atrophy in prostate carcinogenesis, and implications for chemoprevention; evidence supporting telomere shortening and genetic instability in the etiology of prostate cancer; and ␣-methylacyl-coenzyme A racemase (AMACR) as a potential marker for prostate carcinogenesis. These new results show that at least some high-grade prostatic intraepithelial neoplasias (PIN) and early adenocarcinomas appear to arise from proliferative inflammatory atrophy (PIA). Inflammation and other environmental factors may lead to the destruction of prostate epithelial cells, and increased proliferation may occur as a response to this cell death. Such proliferation may be mechanistically related to decreased p27 Kip1 observed in PIA. The decreased apoptosis associated with these events may also be related to increased expression of Bcl-2. Increased oxidant and electrophile stress in the setting of increased proliferation associated with these events may lead to elevated glutathione S-transferase P1 (GSTP1) expression as a genomic-protective measure. However, aberrant methylation of the CpG island of the GSTP1 gene promoter silences GSTP1 gene expression and protein levels, setting the stage for additional genetic damage and accelerated progression toward PIN and carcinoma. Additional results show that AMACR may be an important new marker of prostate cancer, and its use in combination with p63 staining may provide the basis for an improved method for identification of prostate cancer.
Overexpression of alpha-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fat... more Overexpression of alpha-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fatty acid beta-oxidation, in prostate cancer has been reported. Here, we report that an enzyme downstream from AMACR in the peroxisomal branched chain fatty acid beta-oxidation pathway-D-bifunctional protein (DBP)-is also upregulated in prostate cancer at both mRNA and protein levels, accompanied by increased enzymatic activity. Furthermore, our data suggest that pristanoyl-CoA oxidase (ACOX3), which is expressed at extremely low level in other human organs studied including the liver, might contribute significantly to peroxisomal branched chain fatty acid beta-oxidation in human prostate tissue and some prostate cancer cell lines. In contrast to these results for peroxisomal enzymes, no significant expression changes of mitochondrial fatty acid beta-oxidation enzymes were observed in prostate cancer tissues through comprehensive quantitative RT-PCR screening. These data for the first time provide evidence for the selective over-activation of peroxisomal branched chain fatty acid beta-oxidation in prostate cancer, emphasizing a new metabolic change during prostate oncogenesis.
BACKGROUND-Because of the dramatically different clinical course of aggressive and indolent prost... more BACKGROUND-Because of the dramatically different clinical course of aggressive and indolent prostate carcinoma (PCa), markers that distinguish between these phenotypes are of critical importance. Apoptosis is an important protective mechanism for unrestrained cellular growth and metastasis. Therefore, dysfunction in this pathway is a key step in cancer progression. As such, genetic variants in apoptosis genes are potential markers of aggressive PCa. Recent work in breast carcinoma has implicated the histidine variant of CASP8 D302H (rs1045485) as a protective risk allele. METHODS-We tested the hypothesis that the Hvariant was protective for aggressive PCa in a pooled analysis of 796 aggressive cases and 2,060 controls. RESULTS. The H allele was associated with a reduced risk of aggressive PCa (ORper allele = 0.67, 95% CI: 0.54-0.83, Ptrend = 0.0003). The results were similar for European-Americans (ORper allele = 0.68; 95% CI: 0.54-0.86) and African-Americans (ORper allele = 0.61; 95% CI: 0.34-1.10). We further determined from the full series of 1,160 cases and 1,166 controls in the Prostate, Lung, Colorectal, Ovarian (PLCO) population that the protective effect of the H allele tended to be limited to high-grade and advanced PCa (all cases ORper allele = 0.94; 95% CI: 0.79-1.11; localized, low-grade disease ORper allele = 0.98; 95% CI: 0.79-1.23; and aggressive disease ORper allele = 0.73; 95% CI: 0.50-1.07). CONCLUSION-These results suggest that histidine variant of CASP8 D302H is a protective allele for aggressive PCa with potential utility for identification of patients at differential risk for this clinically significant phenotype.
BACKGROUND-Malignant transformation is often accompanied by morphological and functional alterati... more BACKGROUND-Malignant transformation is often accompanied by morphological and functional alterations in subcellular organelles. The Golgi apparatus is a subcellular structure primarily involved in modification and sorting of macromolecules for secretion and transport to other cellular destinations. Molecular alterations associated with the Golgi apparatus may take place during prostate carcinogenesis but such alterations have not been documented. METHODS-To demonstrate that the Golgi apparatus undergoes alterations during prostate carcinogenesis, we examined the expression and localization of two candidate molecules, Golgi phosphoprotein 2 (GOLPH2) and myosin VI (MYO6), both overexpressed in prostate cancer as initially identified by expression microarray analysis. RESULTS-Elevated GOLPH2 expression in prostate cancers was validated through real-time RT-PCR, Western blot, and tissue microarray analysis, and its Golgi localization in surgical prostate cancer tissues confirmed using two-color immunofluorescence. In addition, distinctive juxtanuclear MYO6 staining pattern consistent with Golgi localization was observed in surgical prostate cancer tissues. Two-color immunofluorescence revealed intensive Golgi-specific staining for both GOLPH2 and myosin VI in prostate cancer cells but not in the adjacent normal prostate epithelium. CONCLUSIONS-We show that the Golgi apparatus in prostate cancer cells differs from the normal Golgi by elevated levels of two molecules, GOLPH2 and MYO6. These results for the first time demonstrated consistent cancer cell-specific alterations in the molecular composition of the Golgi apparatus. Such alterations can be explored for discovery of novel prostate cancer biomarkers through targeted organellar approaches.
BACKGROUND-TNFRSF10C, is located on 8p21.3, one of the most frequently deleted loci in the genome... more BACKGROUND-TNFRSF10C, is located on 8p21.3, one of the most frequently deleted loci in the genome of prostate cancer (PCa). Hypermethylation of TNFRSF10C promoter CpG island (CGI) had been reported in many tumors including PCa. However, the interplay between somatic deletion and promoter hypermethylation of TNFRSF10C on PCa development has not been investigated. METHODS-Methylation status of promoter CGI and deletion status of the TNFRSF10C locus was investigated by bisulfite sequencing and Affymetrix SNP array, respectively in 59 pairs of PCa tumor and matched normal samples with 3 PCa cell lines. TNFRSF10C gene expression changes in relation to cancer-associated genetic/epigenetic changes in clinical specimens, and change of TNFRSF10C expression before and after 5-aza-2'-deoxycytidine treatment in the PC3 PCa cell line were assessed by real-time RT-PCR. RESULTS-We found that TNFRSF10C promoter CGI was differentially methylated in 46 of 59 primary cancers (78.0%). Hemizygous deletion at TNFRSF10C was found in 44 of the 59 prostate tumors (74.5%). Interestingly, in 94.9% of the tumors (56 out of 59), TNFRSF10C was either hemizygously deleted or its promoter CGI hypermethylated. Deletion and/or methylation of the TNFRSF10C gene was correlated with decreased mRNA expression of the gene in clinical specimens. Demethylation of the TNFRSF10C promoter CGI was accompanied by transcriptional re-activation of TNFRSF10C in the prostate cancer cell line PC3. CONCLUSION-We found a notably high frequency of promoter CGI methylation and deletion of TNFRSF10C in PCa tissues. Our results indicated that inactivation of TNFRSF10C by chromosomal deletion and promoter methylation may play an important role in PCa development.
Recent identifications of associations between novel variants in inflammation-related genes and s... more Recent identifications of associations between novel variants in inflammation-related genes and several common diseases emphasize the need for systematic evaluations of these genes in disease susceptibility. Considering that many genes are involved in the complex inflammation responses and many genetic variants in these genes have the potential to alter the functions and expression of these genes, we assembled a list of key inflammation-related genes to facilitate the identification of genetic associations of diseases with an inflammation-related etiology. We first reviewed various phases of inflammation responses, including the development of immune cells, sensing of danger, influx of cells to sites of insult, activation and functional responses of immune and non-immune cells, and resolution of the immune response. Assisted by the Ingenuity Pathway Analysis, we then identified 17 functional sub-pathways that are involved in one or multiple phases. This organization would greatly increase the chance of detecting gene-gene interactions by hierarchical clustering of genes with their functional closeness in a pathway. Finally, as an example application, we have developed tagging single nucleotide polymorphism (tSNP) arrays for populations of European and African descent to capture all the common variants of these key inflammation-related genes. Assays of these tSNPs have been designed and assembled into two Affymetrix ParAllele customized chips, one each for European (12,011 SNPs) and African (21,542 SNPs) populations. These tSNPs have greater coverage for these inflammationrelated genes compared to the existing genome-wide arrays, particularly in the African population. These tSNP arrays can facilitate systematic evaluation of inflammation pathways in disease susceptibility. For additional applications, other genotyping platforms could also be employed. For existing genome-wide association data, this list of key inflammation-related genes and associated subpathways can facilitate comprehensive inflammation pathway-focused association analyses.
b-catenin signaling may contribute to prostate cancer (CaP) progression. Although b-catenin is kn... more b-catenin signaling may contribute to prostate cancer (CaP) progression. Although b-catenin is known to upregulate T cell factor (TCF) target gene expression in CaP cells, recent evidence demonstrates its capacity to enhance ligand-dependent androgen receptor (AR) function. Thus, we wished to further understand the interaction between these two pathways. We find in both CaP cells (CWR22-Rv1, LAPC-4, DU145) and non-CaP cells (HEK-293, TSU, SW480, HCT-116) that bcatenin/TCF-related transcription (CRT), as measured by activation of a synthetic promoter and that of cyclin D1, is inhibited by androgen treatment. This inhibition is AR-dependent, as it only occurs in cells expressing AR endogenously or transiently, and is abrogated by AR antagonists. Additional analyses convey that the liganddependent nature of CRT suppression depends on transactivation-competent AR in the nucleus, but not on indirect effects stemming from AR target gene expression. Given the recent work identifying an AR/bcatenin interaction, and from our finding that liganded AR does not prompt gross changes in the constitutive nuclear localization of TCF4 or mutant b-catenin, we hypothesized that transcription factor (i.e. AR and TCF) competition for b-catenin recruitment may explain, in part, androgen-induced suppression of CRT. To address this idea, we expressed an AR mutant lacking its DNAbinding domain (DBD). This receptor could not orchestrate ligand-dependent CRT repression, thereby providing support for those recent data implicating the AR DBD/LBD as necessary for b-catenin interaction. Further supporting this hypothesis, TCF/LEF overexpression counteracts androgen-induced suppression of CRT, and requires b-catenin binding activity to do so. Interestingly, TCF4 over-expression potently antagonizes AR function; however, this inhibition may occur independently of b-catenin/TCF4 interaction. These results from TCF4 over-expression analyses, taken together, provide further evidence that AR-mediated suppression of CRT is a consequence of limiting amounts of b-catenin, and not AR target gene expression. Our analyses point to a reciprocal balance between AR and CRT function that may shape critical processes during normal prostate development and tumor progression.
Understanding the molecular etiology of prostate cancer (CaP) progression is paramount for broade... more Understanding the molecular etiology of prostate cancer (CaP) progression is paramount for broadening current diagnostic and therapeutic modalities. Current interest in the role of wnt pathway signaling in prostate tumorigenesis was generated with the ®nding of b-catenin mutation and corresponding nuclear localization in primary lesions. The recent ®nding of b-catenin-induced enhancement of androgen receptor (AR) function potentially ties b-catenin to key regulatory steps of prostate cell growth, dierentiation, and transformation. By immunohistological analysis of metastatic tumors, we detected nuclear bcatenin in 20% of lethal CaP cases, suggesting a more common role for b-catenin in advanced disease than would be predicted by its mutation rate. Interestingly, bcatenin nuclear localization was found to occur concomitantly with androgen-induced regrowth of normal rat prostate. These in vivo observations likely implicate b-catenin involvement in both normal and neoplastic prostate physiology, thus prompting our interest in further characterizing modes of b-catenin signaling in prostate cells. Extending our previous ®ndings, we demonstrate that transient b-catenin over-expression stimulates T cell factor (TCF) signaling in most CaP cell lines. Further, this activity is not subject to crossregulation by phosphoinositide-3-kinase (PI3-K)/Akt signaling, a stimulatory pathway often upregulated in CaP upon PTEN inactivation. Consistent with a previous report, we observed that transient b-catenin overexpression enhances AR-mediated transcription o two natural target gene promoters. However, we were unable to recapitulate b-catenin-induced stimulation of ectopically expressed AR in AR-negative cells, suggesting that other AR-associated factors are required for this activity. Although LNCaP cells are capable of this mode of AR co-stimulation, stable expression of mutant b-catenin did not alter their proliferative response to androgen. In total, our characterization of b-catenin signaling in CaP reveals the complex nature of its activity in prostate tissue, indicating that b-catenin potentially contributes to multiple stimulatory inputs required for disease progression.
The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, pro... more The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.
Purpose: We assessed the feasibility of a novel urinary test for prostate cancer based on the pre... more Purpose: We assessed the feasibility of a novel urinary test for prostate cancer based on the presence of ␣ methylacyl coenzyme A racemase (AMACR) protein in voided urine specimens obtained after prostate biopsy. Materials and Methods: Clean catch voided urine specimens were prospectively collected from 26 consecutive men immediately after transrectal ultrasound guided prostate biopsy for suspected malignancy. The presence of AMACR was evaluated in a blinded manner by Western blot analysis and correlated with biopsy results and patient clinical information. Results: AMACR was detected in the urine in 18 of 26 patients (69%). AMACR was detected in all 12 patients with biopsy confirmed adenocarcinoma of the prostate (100% sensitivity, 95% CI 75 to 100), in 5 of 12 with no evidence of cancer on biopsy (58% specificity, 95% CI 29 to 78) and in 1 of 2 (50%, 95% CI 3 to 80) with atypia on biopsy. Overall AMACR detection was associated with cancer status by prostate biopsy in 21 of 26 patients (86%). Conclusions: We report the feasibility of a novel, noninvasive, nonprostate specific antigen based molecular approach to detect prostate cancer in voided urine. To our knowledge this is the first report of AMACR protein detection in the urine of patients with prostate cancer. A screening test based on urinary AMACR may develop into a useful adjunct to serum prostate specific antigen and digital rectal examination for identifying men at increased risk for harboring prostate cancer despite negative biopsy. Such a test has potential application for stratifying patients into low and high risk groups for surveillance vs repeat biopsy.
Lipoprotein lipase (LPL) is in chromosome 8p22, site of one of the most common somatic deletions ... more Lipoprotein lipase (LPL) is in chromosome 8p22, site of one of the most common somatic deletions in prostate tumors. Additionally, a CpG island (CGI) was identified in the LPL promoter region. To test the hypothesis that LPL is a tumor suppressor gene, which is inactivated by somatic deletion and hypermethylation in prostate cancer, we evaluated somatic DNA deletion and methylation status at LPL in 56 pairs of DNA samples isolated from prostate cancer tissues and matching normal controls and 11 prostate cell lines. We found that the DNA in 21 of 56 primary cancers (38%) was methylated in the LPL promoter CGI, whereas no methylation was detected in any normal samples. In addition, we found a hemizygous deletion at LPL in 38 of the 56 tumors (68%). When the results of deletion and methylation were considered together, we found LPL promoter CGI methylation occurred in 45% of LPL deleted tumors and in 22% of LPL retained tumors. Within several clinical characteristics tested, the preoperative PSA levels were found to be significantly higher in subjects with LPL promoter CGI methylation compared with subjects without LPL promoter methylation (p = 0.0012). Additionally, demethylation of the LPL promoter CGI was accompanied by transcriptional reactivation of LPL in the prostate cancer cell lines DU145 and PC3. In summary, we report a novel finding that the LPL gene is commonly methylated in prostate tumors, and our results suggest that biallelic inactivation of LPL by chromosomal deletion and promoter hypermethylation may play a role in human prostate cancer.
Purpose: Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly com... more Purpose: Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide the diagnosis and therapy of this aggressive tumor. Experimental Design: We examined the status of RB1, TP53, and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy-number alteration analysis, and sequencing of formalin-fixed paraffin-embedded specimens. Results: We found retinoblastoma (Rb) protein loss in 90% of small cell carcinoma cases (26 of 29) with RB1 allelic loss in 85% of cases (11 of 13). Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3 of 7) showed Rb protein loss. In contrast, only 7% of primary high-grade acinar carcinomas (10 of 150), 11% of primary acinar carcinomas with neuroendocrine differentiation (4 of 35), and 15% of metas...
Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are cr... more Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. Experimental Design: PTEN IHC was validated by employing formalin fixed and paraffin-embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymorphism microarray analysis was done on a subset of these cases. Results: PTEN prote...
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Papers by William Isaacs