Prognostic features and markers for testicular cancer management.

Testicular neoplasm accounts for about 1% of all cancers in men. Over the last 40 years, the incidence of testicular cancer has increased in northern European male populations for unknown reasons. When diagnosed at early stage, testicular cancer is usually curable with a high survival rate. In the past three decades, successful multidisciplinary approaches for the management of testicular cancer have significantly increased patient survival rates. Utilization of tumor markers and accurate prognostic classification has also contributed to successful therapy. In this article, we highlight the most commonly used tumor markers and several potential "novel" markers for testicular cancer as part of the ongoing effort in biomarker research and discovery. In addition, this article also identifies several key prognostic features that have been demonstrated to play a role in predicting relapse. These features include tumor size, rete testis invasion, lymphovascular invasion, and tumor histology. Together with tumor markers, these prognostic factors should be taken into account for risk-adapted management of testicular cancer.

Identification of nuclear structural protein alterations associated with seminomas.

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high-resolution two-dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR-related lipid transfer protein 7 (StARD7). In the non-seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease.

Biomarkers for prostate cancer.

The detection of prostate cancer using a blood test has by many standards changed the face of the disease. Despite this tremendous success, there are limitations attributed to the use of prostate specific antigen (PSA) as a means to screen and detect prostate cancer. PSA, as its name implies, is not specific for prostate cancer and as such is often found elevated in other prostatic diseases/symptoms associated with the aging male. Clearly, more specific marker(s) that could identify which individuals actually have prostate cancer and differentiate them from those without the disease would be of tremendous value. The search for more accurate and clinically useful biomarkers of prostate cancer has been extensive. This has focused on individual markers, as well as groups of markers. Included among these are PSA isoforms, pathological indicators and stains, nucleic acids and others. This article highlights the discovery of PSA as a first blood-based biomarker for prostate cancer detection, as well as other molecular biomarkers and their potential application in detection of the disease.

Analysis of a serum test for prostate cancer that detects a second epitope of EPCA-2.

BACKGROUND: We have previously shown that EPCA-2 can serve as a highly specific and sensitive serum marker for prostate cancer. As a component of our validation of this marker, we have performed an initial evaluation of an assay that detects a distinct epitope of the same protein: EPCA-2.19. The goals of this study are to characterize the sensitivity and specificity of the EPCA-2.19 assay, in a non-screening population, and to demonstrate that such test based has similar characteristics as the initial assay produced. METHODS: Three hundred twenty-eight serum samples from men with PSA values < and >2.5 ng/ml who had negative biopsies, men with BPH, men with organ-confined and non-organ-confined prostate cancer, as well as control populations were evaluated using the EPCA-2.19 assay. RESULTS: At a cut-off of 0.5 ng/ml and above, EPCA-2.19 has a specificity of 94% and a sensitivity of 91% in separating normal men with PSA < and >2.5 ng/ml, and men with BPH from those with prostate cancer. Receiver Operator Curve analyses of the EPCA-2.19 assay demonstrate an area under the curve of 0.982 (95% CI 0.952-0.996, P < 0.0001). CONCLUSIONS: This study confirms our earlier findings that the assay that detects against a second epitope of EPCA-2 yields almost identical results to those obtained for the first published assay (EPCA-2.22). While this provides some validation of our earlier studies, larger multi-institutional studies still need to be performed.

Purine-rich element binding protein (PUR) alpha induces endoplasmic reticulum stress response, and cell differentiation pathways in prostate cancer cells.

BACKGROUND: Following androgen ablation treatment for advanced prostate cancer, almost all men relapse after a period of initial response to therapy, which eventually is life threatening. We have previously found that purine-rich element binding protein, PURalpha, was significantly repressed in androgen-independent prostate cancer cell lines in comparison to an androgen-dependent line. Moreover, over-expressing PURalpha in androgen-independent prostate cancer cells attenuated their cell proliferation. The aim of the studies described here was to uncover some of the mechanisms by which over-expression of PURalpha attenuates cell proliferation. METHODS: A set of common genes induced by over-expressing PURalpha both in PC3 and LNCaP cells was analyzed by DNA microarray. The results were then validated utilizing quantitative reverse transcription-PCR. Using a 5.3-kb region of the PSA promoter containing androgen response elements, the participation of PURalpha in androgen regulated gene expression was determined. RESULTS: Genes involved in stress response and cell differentiation were induced in cells over-expressing PURalpha. Some of the genes that are targets of androgen regulation are also induced. Most strikingly, ectopic expression of PURalpha induced transcriptional activity of the 5.3-kb PSA promoter containing androgen response elements, without androgen stimulation. CONCLUSION: Based upon the consideration that some of the genes involved in cell stress and differentiation are also regulated by androgens our data suggest that PURalpha shares some common pathways regulated by the androgen receptor. These findings suggest that regulation of PURalpha expression in prostate cancer cells may serve as a therapeutic target for hormone refractory prostate cancer.

The potential role of purine-rich element binding protein (PUR) alpha as a novel treatment target for hormone-refractory prostate cancer.

BACKGROUND: Hormonal therapy for advanced prostate cancer is typically effective at first, but almost all men suffer refractory disease which often is life threatening. The nuclear matrix comprises not only of the structural elements of the nucleus, but is associated with many components of the molecular machinery. Our aim is to find novel targets for the treatment of hormone-refractory prostate cancer (HRPC) by focusing on the composition of the nuclear matrix proteins (NMPs). METHODS: LN96 cells were established at our Institution after long-term culturing of LNCaP cells under androgen deprived conditions. The composition of NMPs of LNCaP cells and LN96 cells were analyzed by two-dimensional (2D) electrophoresis and spots differentially expressed were investigated by mass spectrometry for identification. Among the spots identified, we analyzed the potential functional role of the identified proteins in prostate cancer cells by establishing stable overexpressed cells. RESULTS: We found that purine-rich element binding protein (PUR)alpha was significantly repressed not only in NMPs but also in total protein and mRNA levels of LN96 cells in comparison to LNCaP cells under the same steroid deprived conditions. Moreover, PURalpha was decreased in its expression both at the protein and mRNA levels in the androgen-independent prostate cancer cell lines, PC3 and DU145 in comparison to LNCaP cells. Stably overexpressing PURalpha in PC3 and DU145 cells negatively regulates cell proliferation, resulting in decreases in PCNA expression. CONCLUSION: Further dissection of the role of PURalpha in cell growth regulation may reveal a novel target for HRPC.

Evaluation of colon cancer-specific antigen 2 as a potential serum marker for colorectal cancer.

PURPOSE: A blood test to detect colon cancer at a preventable stage would represent a major advancement. We have previously identified colon cancer-specific markers using focused proteomics analysis of nuclear structural proteins. Two of these markers, colon cancer-specific antigen (CCSA)-3 and CCSA-4, have been developed into blood-based markers that are able to distinguish individuals with colorectal cancer from those without. CCSA-2 is a distinct novel colon cancer marker identified using focused proteomics. EXPERIMENTAL DESIGN: Using an indirect ELISA on serum samples obtained from two institutions, we evaluated CCSA-2 as a serum-based colon cancer marker. A total of 111 serum samples from individuals who underwent colonoscopy and were subsequently diagnosed as either being normal or having hyperplastic polyps, nonadvanced adenomas, advanced adenomas, and colorectal cancer were evaluated. A diverse control population that consisted of 125 serum samples was also included in this study. RESULTS: Receiver operating characteristic analyses were used to measure the sensitivity and specificity of CCSA-2. CCSA-2 at a cutoff of 10.8 mug/mL has overall specificity of 78.4% [95% confidence interval (95% CI), 67.3-87.1%] and sensitivity of 97.3% (95% CI, 85.8-99.5%) in separating individuals with advanced adenomas and colorectal cancer from normal, hyperplastic, and nonadvanced adenoma populations. The receiver operating characteristic curve for CCSA-2 has an area under the curve of 0.90 (95% CI, 0.83-0.95). CONCLUSION: Our initial study shows that CCSA-2 is a potential serum-based marker for colon cancer detection with high sensitivity and specificity.

The use of a colon cancer associated nuclear antigen CCSA-2 for the blood based detection of colon cancer.

The early diagnosis of colorectal cancer (CRC) is central for effective treatment, as prognosis is directly related to the stage of the disease. Development of tumor markers found in the blood from patients, which can detect CRC at an early stage, should have a major impact in morbidity and mortality of this disease. The nuclear matrix is the structural scaffolding of the nucleus and specific nuclear matrix proteins (NMPs) have been identified as an "fingerprint" for various cancer types. Previous studies from our laboratory have identified four colon cancer associated NMPs termed colon cancer-specific antigen (CCSA)-2 to (CCSA)-5. The objective of the present study was to analyze the expression of one of these proteins, CCSA-2 in serum from various patient populations and to determine whether CCSA-2 antibodies could be used in a clinically applicable serum-based immunoassay specifically to detect colon cancer. Using an indirect ELISA, which detects CCSA-2, the protein was measured in the serum from 174 individuals, including healthy individuals, patients with colon cancer, patients with diverticulosis, colon polyps, inflammatory bowel disease (IBD) as well as other cancer types. With a predetermined cutoff absorbance of 0.6 OD we have successfully utilized this approach to develop an immunoassay that detected colon cancer. The immunoassay showed a sensitivity of 88.8% (24/27) and an overall specificity of 84.2% (106/127). This initial study showed the potential of CCSA-2 to serve as a highly specific blood based marker for colon cancer. Although potentially promising, the results of this study must be confirmed in larger independent validation studies.

Nuclear structure as a source of cancer specific biomarkers.

There are few biomarkers that have been developed which have proven clinical utility for the detection and prognosis of cancer. Cancer is diagnosed today, in large part, by examining cells under the microscope and determining the shape and texture of the nucleus. The molecular underpinnings of this hallmark of cancer are the components of the nuclear matrix. Utilizing proteomics focused on this subset of proteins, biomarkers have been identified that are specific for cancer types including prostate, colon and bladder cancer. These cancer biomarkers now serve as the basis of assays which can specifically identify individuals with cancer by sampling their blood and/or urine. In addition, these may serve as potential therapeutic targeting or imaging approaches.

Initial analyses of colon cancer-specific antigen (CCSA)-3 and CCSA-4 as colorectal cancer-associated serum markers.

Colon cancer-specific antigen (CCSA)-3 and CCSA-4 are novel colon cancer markers identified by focused proteomic analysis of nuclear structural proteins. The goal of these studies was to evaluate serum-based CCSA-3 and CCSA-4 in the detection of individuals with preneoplastic and neoplastic lesions using ELISAs. Serum samples from 107 subjects undergoing colonoscopy, 28 subjects with colorectal cancer, and 125 subjects with benign disease or other types of cancer were evaluated. Individuals who underwent colonoscopy were classified into mutually exclusive categories, including normal colon, hyperplastic polyp, nonadvanced adenoma, and advanced adenoma. Sensitivity and specificity for both CCSA-3 and CCSA-4 were evaluated using receiver operating characteristic (ROC) curves. At a cutoff of 2 microg/mL for CCSA-3 and 0.3 microg/mL for CCSA-4, each marker detected all 28 colorectal cancers, for a sensitivity of 100% (lower 95% confidence bound, 89.8%). The sensitivity for detection of the combined end point of colorectal cancer and advanced adenoma for CCSA-3 was 89.1% [95% confidence interval (95% CI), 76.4-96.4%] and for CCSA-4 was 84.8% (95% CI, 71.1-93.7%) and 91.3% (95% CI, 79.2-97.6%) for either marker positive. The specificity in individuals with normal, hyperplastic polyps, or nonadvanced adenomas was 82.0% (95% CI, 72.4-89.4%) and 91.0% (95% CI, 83.0-96.0%) for CCSA-3 and CCSA-4, respectively. ROC curves for CCSA-3 and CCSA-4 reveal an area under the curve of 0.94 (95% CI, 0.90-0.98%). In these initial analyses, CCSA-3 and CCSA-4 show promise as potential serum markers for detection of colorectal cancer and advanced adenomas.

EPCA-2: a highly specific serum marker for prostate cancer.

OBJECTIVES: To describe the initial assessment of early prostate cancer antigen (EPCA)-2 as a serum marker for the detection of prostate cancer and to examine its sensitivity and specificity. METHODS: Serum samples were obtained from 385 men: those with prostate-specific antigen (PSA) levels less than 2.5 ng/mL, PSA levels of 2.5 ng/mL or greater with negative biopsy findings, benign prostatic hyperplasia, organ-confined prostate cancer, non-organ-confined disease, and prostate cancer with PSA levels less than 2.5 ng/mL. In addition, a diverse group of controls was assessed with an enzyme-linked immunosorbent assay to detect an epitope of the EPCA-2 protein, EPCA-2.22. RESULTS: Using a cutoff of 30 ng/mL, the EPCA-2.22 assay had a 92% specificity (95% confidence interval 85% to 96%) for healthy men and men with benign prostatic hyperplasia and 94% sensitivity (95% confidence interval [CI] 93% to 99%) for overall prostate cancer. The specificity for PSA in these selected groups of patients was 65% (95% CI 55% to 75%). Additionally, EPCA-2.22 was highly accurate in differentiating between localized and extracapsular disease (area under the curve 0.89, 95% CI 0.82 to 0.97, P <0.0001) in contrast to PSA (area under the curve 0.62, 95% CI 0.50 to 0.75, P = 0.05). CONCLUSIONS: The results of our study have shown that EPCA-2 is a novel biomarker associated with prostate cancer that has high sensitivity and specificity and accurately differentiates between men with organ-confined and non-organ-confined disease.

The Group 3 LIM domain protein paxillin potentiates androgen receptor transactivation in prostate cancer cell lines.

Paxillin, a member of the group 3 subfamily of LIM domain proteins, is localized within focal adhesions and participates in a number of signal transduction pathways mobilized upon activation of cell surface receptors. In recent years, a number of group 3 LIM domain proteins have been found to also localize within the nucleus and exert direct effects on transcription. We show here that paxillin is present within nuclei and can target the nuclear matrix of CV-1 cells, cultured prostate cancer cell lines, and human prostate tissue. The increased targeting of androgen receptor to the nuclear matrix upon overexpression of paxillin may be brought about by direct interactions between paxillin and the receptor, which were detected in vitro. Paxillin functions as a coactivator for androgen receptor and glucocorticoid receptor, but not estrogen receptor alpha, similar to its close relative Hic-5/ARA55. Both paxillin and Hic-5/ARA55 use their COOH-terminal LIM domain to interact with steroid receptors. However, paxillin is distinguished from Hic-5/ARA55 by both the location of its receptor coactivation domain (i.e., COOH-terminal LIM domain) and by the dominant-negative activity of its NH(2)-terminal domain. Thus, highly related group 3 LIM domain proteins may use distinct mechanisms to modulate steroid hormone receptor transactivation.

Vitamin D and androgen regulation of prostatic growth.

Vitamin D has been reported to inhibit the growth of prostate cancer cells and model systems. In this study, we examined the interaction between 1,25-dihydroxyvitamin D(3) (1,25 D) in the presence or absence of endogenous testosterone on the growth and development of the adult rat prostate. Male Sprague-Dawley rats (165 days old) were either kept intact or castrated. Seven days after castration, the rats were treated with vehicle (control) or 1,25 D for 3 weeks and then sacrificed. Both ventral and dorsal lateral prostates were harvested; whole tissue lysates were collected and AR and VDR protein levels were analyzed by immunoblot analyses. Administration of 1,25 D in the intact animals decreased the prostatic size by 40%, compared to control animals, whereas 1,25 D did not influence the size of the prostate in castrated rats. 1,25 D administration in intact groups also increased both the AR and VDR protein levels by approximately twofold, whereas in castrated groups, 1,25 D only increased the AR protein level by 1.5-2.5-fold. 1,25 D in the presence of endogenous testosterone inhibits prostatic growth, whereas 1,25 D in the absence of endogenous testosterone does not affect prostatic growth. The growth inhibitory activity of 1,25 D in the presence of testosterone may be mediated through the ligand activated AR and VDR pathways. These studies may reveal important information about the potential efficacy of 1,25 D as well as hormonal status in understanding the development of prostate diseases.

Regulation of androgen and vitamin d receptors by 1,25-dihydroxyvitamin D3 in human prostate epithelial and stromal cells.

PURPOSE: The mechanisms of the interaction between 1,25-dihydroxyvitamin D(3) (1,25 D) and androgens, and their respective receptors in their action on the prostate are not completely understood. We examined the interplay of 1,25 D and androgens on the epithelial and stromal cells of the prostate. MATERIALS AND METHODS: The human neonatal prostatic epithelial cell line 267B-1 (BRFF, Inc., Ijamsville, Maryland) and primary cultures of human prostate stromal cells were treated with medium containing 5 or 10 microM 1,25 D or ethanol (control) in the presence or absence of 10 nM dihydrotestosterone (DHT) (Sigma Chemical Co., St. Louis, Missouri). Protein levels of androgen receptor (AR) and vitamin D receptor (VDR) were determined by immunoblot analysis of whole cell extracts. Electrophoresis mobility shift assays were used to determine AR and VDR DNA binding activities. RESULTS: The VDR protein level of 267B-1 cells was increased in the presence of 1,25 D (with the maximum effects seen at 24 hours) regardless of the presence or absence of DHT. In addition, exogenous DHT increased the AR and VDR DNA binding activities of 267B-1 and stromal cells in the presence of 1,25 D. CONCLUSIONS: ARs in the normal prostate are regulated by androgens, whereas VDRs in the normal prostate can be regulated by 1,25 D as well as by other androgens such as testosterone. This finding further supports the concept that 1,25 D as a steroid hormone, in addition to other androgens such as DHT, may have a role in the growth and differentiation of normal prostate.

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

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

Effects of 1,25-dihydroxyvitamin D3 on the distribution of androgen and vitamin D receptors in human prostate neonatal epithelial cells.

Although many studies have examined the mechanisms of 1,25-dihydroxyvitamin D(3) (calcitriol or 1,25 D) action in different prostate cancer cell lines, little is known regarding the influence of this steroid on the normal prostate. The presence of both VDR and AR in normal prostatic tissues raises the distinct possibility of an important role for this hormone in the normal gland. In order to ascertain the possible role of 1,25 D on both AR and VDR in the normal prostate, the effects of calcitriol and dihydrotestosterone (DHT) on the normal human neonatal prostatic epithelial cell line, 267B-1, were examined. These studies were approached by focusing on how 1,25 D in the presence or absence of DHT affects the distribution of AR and VDR in the cytoplasmic and nuclear compartments of the cells in terms of their protein levels and DNA binding activities. Immunoblot analyses show that 1,25 D increases the AR protein level in both the cytoplasmic and nuclear fractions but not the VDR protein level. On the other hand, the gel shift assays demonstrate that 1,25 D increases both the AR- and VDR-DNA binding activities in the nuclear fraction, whereas there is no increase in DNA binding activities in the cytoplasmic fraction. Addition of DHT along with 1,25 D does not affect the DNA binding activities of both AR and VDR. Overall, these studies suggest that 1,25 D actions on the normal prostate cells may be mediated independently through AR and VDR, respectively.

Characterization of the nuclear matrix proteins in a transgenic mouse model for prostate cancer.

The nuclear matrix (NM) contains a number of proteins that have been found to be associated with transformation. We have previously identified changes in the NM associated with prostate cancer. In this study, we examine the molecular changes that are associated with prostate cancer development in transgenic adenocarcinoma of mouse prostate (TRAMP) model by studying the differences in the NM proteins (NMPs). We collected prostates from the TRAMP males at six critical time points: 6 weeks (puberty), 11 and 19 weeks (development of mild hyperplasia), 25 weeks (development of severe hyperplasia), 31 and 37 weeks (development of neoplasia). The nuclear matrices from the prostates collected at these time points were then isolated and the NMPs were characterized by high-resolution two-dimensional gel electrophoresis. We found three NMPs (E1A, E1B, and E1C) that were present in the 6-week-old prostate and two NMPs (E2A and E2B) that were present in the 11-week-old prostate. These NMPs were absent in the 31- and 37-week-old prostate. We also found five NMPs (E3A-E3E) that were present in the 31-week-old prostate, but absent in the earlier time points. In addition, three NMPs (Le1, Le2, Le3) were present at higher expression in the 6-, 11-, 19-, and 25-weeks old TRAMP prostates, but they were expressed lower during the development of neoplasia at 31- and 37-weeks old. Identification of these NMPs permits the development of novel markers that can characterize various stages of prostate cancer development as well as potentially therapeutic targets.

Nuclear matrix proteins as biomarkers in prostate cancer.

The nuclear matrix (NM) is the structural framework of the nucleus that consists of the peripheral lamins and pore complexes, an internal ribonucleic protein network, and residual nucleoli. The NM contains proteins that contribute to the preservation of nuclear shape and its organization. These protein components better known as the NM proteins have been demonstrated to be tissue specific, and are altered in many cancers, including prostate cancer. Alterations in nuclear morphology are hallmarks of cancer and are believed to be associated with changes in NM protein composition. Prostate cancer is the most frequently diagnosed cancer in American men and many investigators have identified unique NM proteins that appear to be specific for this disease. These NM protein changes are associated with the development of prostate cancer, as well as in some cases being indicative of cancer stage. Identification of these NM proteins specific for prostate cancer provides an insight to understanding the molecular changes associated with this disease. This article reviews the role of NM proteins as tumor biomarkers in prostate cancer and the potential application of these proteins as therapeutic targets in the treatment of this disease.