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It is now widely recognized that carcinoma-associated fibroblasts which are believed to be myofibroblasts, promote the transformation of prostate epithelial cells to cancer cells, enhance their proliferation and invasiveness, and induce the acquisition of resistance to cancer therapy and immune evasiveness. Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein that is remarkably prostate-specific. PAGE4 is also a stress-response protein that functions as a transcriptional regulator and is upregulated in early-stage prostate cancer (PCa) and its precursor lesions. However, PAGE4 is downregulated in high-grade PCa and metastatic disease. Here, we show that PAGE4 is highly expressed in the stromal cells surrounding the cancer-adjacent "normal" glands and low-grade PCa lesions but not in lesions proximal to high-grade PCa. Overexpression of PAGE4 in a stromal cell line inhibits the migration and invasion of PCa epithelial cells in multiple coculture systems. PAGE4 overexpression also inhibits the downregulation of E-cadherin in PCa epithelial cells when cocultured with stromal cells. Furthermore, signaling via tumor necrosis factor-α and transforming growth factor-β pathways is decreased in the stromal cells overexpressing PAGE4 suggesting that PAGE4 appears to play a protective role against disease progression by perturbing interactions between epithelial cells and stromal cells in PCa. Taken together, these findings support previous observations that upregulation of PAGE4 in PCa correlates with a better prognosis and highlight PAGE4 as a novel therapeutic target for early-stage "low-risk" disease.

A causal link between benign prostatic hyperplasia (BPH) and prostate cancer has long been suspected but not widely accepted. A new model is proposed that supports such a connection. In contrast to the prevailing wisdom, our model, that draws on dynamical systems theory, suggests that in response to stress, epithelial cells in the unstable gland can give rise to both types of diseases via a phenotypic switching mechanism. The central idea is that phenotypic switching is a stochastic process which exploits the plasticity of the epithelial cell. It is driven by 'noise' contributed by the conformational dynamics of proteins that are intrinsically disordered. In a system that is noisy when stressed, disorder promotes promiscuity, unmasks latent information, and rewires the network to cause phenotypic switching. Cells with newly acquired phenotypes can transcend the traditional zonal boundaries to give rise to BPH or prostate cancer depending on the microenvironment. Establishing causality between the two diseases may provide us with an opportunity to better understand their etiology and guide prevention and treatment strategies. J. Cell. Biochem. 117: 2235-2240, 2016. © 2016 Wiley Periodicals, Inc.

Resistance is a significant limitation to the effectiveness of cancer therapies. The PI3K/Akt and MAP kinase pathways play important roles in a variety of normal cellular processes and tumorigenesis. This study is designed to explore the relationship of these signaling pathways with multidrug resistance in prostate cancer (PCa). The PI3K/Akt and MAP kinase pathways were investigated utilizing paclitaxel resistant DU145-TxR PCa cells and their parental non-resistant DU145 cells to determine their relationship with resistance to paclitaxel and other anticancer drugs. Our results demonstrate that the PI3K/Akt and MAP kinase pathways are upregulated in DU145-TxR cells compared to the DU145 cells. Inactivating these pathways using the PI3K/Akt pathway inhibitor LY294002 or the MAP kinase pathway inhibitor PD98059 renders the DU145-TxR cells more sensitive to paclitaxel. We investigated the effects of these inhibitors on other anticancer drugs including docetaxel, vinblastine, doxorubicin, 10-Hydroxycamptothecin (10-HCPT) and cisplatin and find that both inhibitors induces DU145-TxR cells to be more sensitive only to the microtubule-targeting drugs (paclitaxel, docetaxel and vinblastine). Furthermore, the treatment with these inhibitors induces cleaved-PARP production in DU145-TxR cells, suggesting that apoptosis induction might be one of the mechanisms for the reversal of drug resistance. In conclusion, the PI3K/Akt and MAP kinase pathways are associated with resistance to multiple chemotherapeutic drugs. Inactivating these pathways renders these PCa cells more sensitive to microtubule-targeting drugs such as paclitaxel, docetaxel and vinblastine. Combination therapies with novel inhibitors of these two signaling pathways potentially represents a more effective treatment for drug resistant PCa.

Background A need remains for new therapeutic approaches for men with advanced prostate cancer, particularly earlier in the disease course. Objective To assess the ability of an oral selective estrogen receptor α agonist (GTx-758) to lower testosterone concentrations compared with leuprolide while minimizing estrogen deficiency–related side effects of androgen-deprivation therapy. Design, setting, and participants Hormone-naive advanced prostate cancer patients were randomized to oral GTx-758 1000mg/d, 2000mg/d, or leuprolide depot. Intervention GTx-758 and leuprolide. Outcome measurements and statistical analysis The primary end point was the proportion of patients achieving total testosterone ≤50 ng/dl by day 60. Secondary end points included serum free testosterone, prostate-specific antigen (PSA), sex hormone-binding globulin, hot flashes, bone turnover markers, and insulin-like growth factor (IGF)-1 levels. Results and limitations Of 159 randomized patients, leuprolide reduced total testosterone to ≤50 ng/dl in a greater proportion of patients than GTx-758 by day 60 (43.4%, 63.6%, and 88.2% of subjects receiving GTx-758 1000mg [ p p =0.004], and leuprolide, respectively). GTx-758 reduced free testosterone and PSA earlier and to a greater degree than leuprolide. GTx-758 led to fewer hot flashes, decreases in bone turnover markers, and alterations in IGF-1 compared with leuprolide. A higher incidence of venous thromboembolic events (VTEs) was seen with GTx-758 (4.1%) compared with leuprolide (0.0%). Conclusions Although leuprolide reduced total testosterone to ≤50 ng/dl in a greater proportion of patients compared with GTx-758, GTx-758 was superior in lowering free testosterone and PSA. GTx-758 reduced estrogen deficiency side effects of hot flashes, bone loss, and insulin resistance but with a higher incidence of VTEs. Patient summary This paper reports findings that leuprolide lowered total testosterone more than GTx-758 but that GTx-758 lowered free testosterone and prostate-specific antigen more than leuprolide. GTx-758 also reduced estrogen deficiency side effects, albeit at a higher rate of vascular events. Trial registration Clinicaltrials.gov identifier NCT01615120.

Aim: To develop and apply a heat-responsive and secreted reporter assay for comparing cellular response to nanoparticle (NP)- and macroscopic-mediated sublethal hyperthermia. Materials & methods: Reporter cells were heated by water bath (macroscopic heating) or iron oxide NPs activated by alternating magnetic fields (nanoscopic heating). Cellular responses to these thermal stresses were measured in the conditioned media by secreted luciferase assay. Results & conclusion: Reporter activity was responsive to macroscopic and nanoparticle heating and activity correlated with measured macroscopic thermal dose. Significant cellular responses were observed with NP heating under doses that were insufficient to measurably change the temperature of the system. Under these conditions, the reporter response correlated with proximity to cells loaded with heated nanoparticles. These results suggest that NP and macroscopic hyperthermia may be distinctive under conditions of mild hyperthermia. Original submitted 10 May 2013; Revised submitted 31 October 2013

Prostate cancer is a heterogeneous disease and thus, it is important to understand whether among the heterogeneous collection of cell types, androgen-deprivation insensitive cells exist prior to hormonal manipulation. We established several LNCaP subclones with distinct insensitivities to androgen deprivation from a parental LNCaP cell line. In the resulting clones, the sensitivity to androgen-deprivation negatively correlated with their PSA expression levels. In two of these clones, an androgen insensitive clone, LNCaP-cl1, and an androgen sensitive clone, LNCaP-cl5, the DNA copy number differed significantly, indicating that these clones contain genetically distinct cells. LNCaP-cl1 had higher PSA expression but lower invasiveness and tumor growth potential than LNCaP-cl5. The expression levels of two genes that are known to be regulated by miR-21, an androgen-regulated microRNA, Sprouty1 (SPRY1) and Jagged1 (JAG1) were significantly lower in LNCaP-cl1 than in LNCaP-cl5. Knocking down SPRY1 in LNCaP cells enhanced PSA expression and cell proliferation. JAG1 administration in LNCaP cells enhanced cell invasion and JAG1 knockdown in PC3 cells suppressed cell invasion and tumor formation. These results indicated that the expression differences in SPRY1 and JAG1 may contribute to the phenotypic differences between the LNCaP-cl1 and LNCaP-cl5 clones. In tissue samples, SPRY1 expression levels were significantly lower in prostate cancer patients with PSA recurrence after surgical treatment (P = 0.0076) and JAG1 expression levels were significantly higher in Gleason sum (GS) 8–9 disease than in GS 5–6 (P = 0.0121). In summary a random population of LNCaP cells comprises a heterogeneous group of cells with different androgen-deprivation sensitivities and potential for invasiveness.

A causal link between benign prostatic hyperplasia (BPH) and prostate cancer has long been suspected but not widely accepted. A new model is proposed that supports such a connection. In contrast to the prevailing wisdom, our model, that draws on dynamical systems theory, suggests that in response to stress, epithelial cells in the unstable gland can give rise to both types of diseases via a phenotypic switching mechanism. The central idea is that phenotypic switching is a stochastic process which exploits the plasticity of the epithelial cell. It is driven by 'noise' contributed by the conformational dynamics of proteins that are intrinsically disordered. In a system that is noisy when stressed, disorder promotes promiscuity, unmasks latent information, and rewires the network to cause phenotypic switching. Cells with newly acquired phenotypes can transcend the traditional zonal boundaries to give rise to BPH or prostate cancer depending on the microenvironment. Establishing causality between the two diseases may provide us with an opportunity to better understand their etiology and guide prevention and treatment strategies. J. Cell. Biochem. 117: 2235-2240, 2016. © 2016 Wiley Periodicals, Inc.

Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling that is frequently altered in expression in several types of cancers. In prostate cancer (PCa), Cyr61 is highly expressed in organ-confined disease. Further, Cyr61 expression levels are associated with a lower risk of disease recurrence, and can be quantitatively measured in the serum. Considered together, these results indicate that Cyr61 is a potential and clinically useful tissue, as well as serum-based biomarker for differentiating lethal and non-lethal PCa.

The cancer/testis antigens (CTAs) are a group of heterogeneous proteins that are typically expressed in the testis but aberrantly expressed in several types of cancer. Although overexpression of CTAs is frequently associated with advanced disease and poorer prognosis, the significance of this correlation is unclear since the functions of the CTAs in the disease process remain poorly understood. Here, employing a bioinformatics approach, we show that a majority of CTAs are intrinsically disordered proteins (IDPs). IDPs are proteins that, under physiological conditions in vitro, lack rigid 3D structures either along their entire length or in localized regions. Despite the lack of structure, most IDPs can transition from disorder to order upon binding to biological targets and often promote highly promiscuous interactions. IDPs play important roles in transcriptional regulation and signaling via regulatory protein networks and are often associated with dosage sensitivity. Consistent with these observations, we find that several CTAs can bind DNA, and their forced expression appears to increase cell growth implying a potential dosage-sensitive function. Furthermore, the CTAs appear to occupy "hub" positions in protein regulatory networks that typically adopt a "scale-free" power law distribution. Taken together, our data provide a novel perspective on the CTAs implicating them in processing and transducing information in altered physiological states in a dosage-sensitive manner. Identifying the CTAs that occupy hub positions in protein regulatory networks would allow a better understanding of their functions as well as the development of novel therapeutics to treat cancer.

The microenvironment is central to many aspects of cancer pathobiology and has been proposed to play a role in the development of cancer cell resistant to therapy. To examine the response to microenvironmental conditions, two paclitaxel resistant prostate cancer cell lines (stable and reversible) and one reversible heat resistant cell line were studied. In comparison to their parental cell lines, both paclitaxel resistant cell lines (stable and reversible) were more sensitive to microenvironmental heat, potentially yielding a synergistic therapeutic opportunity. In the two phenotypic cells repopulated after acute heat or paclitaxel treatments, there was an inverse correlation between paclitaxel and heat resistance: resistance to paclitaxel imparted sensitivity to heat; resistance to heat imparted sensitivity to paclitaxel. These studies indicate that as cancer cells evolve resistance to single microenvironmental stress they may be more sensitive to others, perhaps allowing us to design new approaches for prostate cancer therapy.

Most proteins encoded by the nuclear genome are synthesized in the cytoplasm and fold into precise 3D structures. During synthesis, the nascent polypeptide begins to fold as it traverses the large subunit of the ribosome and is assisted by molecular chaperones in attaining its precise folded/highly ordered state efficiently and in a biologically relevant timescale. Proteins that are misfolded are culled, re-routed, and marked by mechanisms such as ubiquitinylation for degradation ensuring strict quality control (QC). In addition to the highly ordered “globular” proteins, emerging evidence indicates that a large fraction of the proteome also comprises the so-called “Intrinsically Disordered Proteins” (IDPs). IDPs are proteins that lack rigid 3D structures and instead, exist as dynamic ensembles. The dynamic structures in the IDPs have many similarities with “normal” globular proteins such as the native (ordered), and non-native (molten globule, pre-molten globule, and coil-like) states seen during folding of “normal” globular proteins. However, unlike the case of the nascent globular proteins, IDPs evade being detected as “misfolded” and degraded by the cell’s QC system. We refer to this paradox as the order/disorder paradox and postulate that the IDPs capitalize on their intrinsic promiscuity and ability to undergo disorder-to-order transitions upon binding to biological targets (coupled folding and binding) to escape the cell’s surveillance machinery. Understanding the mechanism by which the IDPs evade the quality check has wide implications from protein folding to disease biology since the aggregation of misfolded proteins underlies several debilitating illnesses such as many neurodegenerative diseases and cancer.

The metastatic invasion of cancer cells from primary tumors to distant ecological niches, rather than the primary tumors, is the cause of much cancer mortality [Zhang QB, et al. (2010) Int J Cancer 126:2534–2541; Chambers AF, Goss PE (2008) Breast Cancer Res 10:114]. Metastasis is a three-dimensional invasion process where cells spread from their site of origin and colonize distant microenvironmental niches. It is critical to be able to assess quantitatively the metastatic potential of cancer cells [Harma V, et al. (2010) PLoS ONE 5:e10431]. We have constructed a microfabricated chip with a three-dimensional topology consisting of lowlands and isolated square highlands (Tepuis), which stand hundreds of microns above the lowlands, in order to assess cancer cell metastatic potential as they invade the highlands. As a test case, the invasive ascents of the Tepui by highly metastatic PC-3 and noninvasive LNCaP prostate cancer cells were used. The vertical ascent by prostate cancer cells from the lowlands to the tops of the Tepui was imaged using confocal microscopy and used as a measure of the relative invasiveness. The less-metastatic cells (LNCaP) never populated all available tops, leaving about 15% of them unoccupied, whereas the more metastatic PC-3 cells occupied all available Tepuis. We argue that this distinct difference in invasiveness is due to contact inhibition.

Background The cancer/testis antigens (CTAs) are a unique group of proteins normally expressed in germ cells but aberrantly expressed in several types of cancers including prostate cancer (PCa). However, their role in PCa has not been fully explored.

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. J.

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.

The microenvironment of the cancer cell is pivotal to its phenotypic regulation. One of the central components of the microenvironment is temperature. An elevation in environmental temperature has been shown to increase the cancer cell's susceptibility to chemo- and radiation therapy. The goal of the studies described here was to identify some of the pathways that are modified by a mild increase in temperature in cancer cells. Using prostate cancer cells as a model system we found that in addition to the well described and anticipated up-regulation of the heat shock family of proteins, there is a significant down-regulation of certain members of the "cold shock" family of proteins such as, RNA binding motif protein 3 (RBM3) and cold inducible RNA binding protein (CIRBP). siRNA-mediated down-regulation of the cold shock protein (CSP) encoding mRNAs dramatically attenuates cell survival in the absence of any heat application. Furthermore, we also demonstrate that knocking down the CSPs can enhance the therapeutic response of prostate cancer cells to chemotherapy. Our findings suggest that down-regulating CSPs in cancer cells may "mimic" the stress response the cells experience when exposed to heat treatment rendering them more susceptible to therapy. Thus, the pharmacological modulation of RBM3 and CIRBP may represent novel therapeutic approaches for prostate cancer.

(Z)-1-1-Dichloro-2,3-diphenylcyclopropane (A(II)) and (Z)-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane [2-(4-methoxyphenyl)-A(II)] inhibit tubulin polymerization, PSA production, and the proliferation of human prostate cancer cells. The actions of the agents were studied in three transgenic adenocarcinomas of the mouse prostate (TRAMP) cell lines. Antiproliferative potencies were determined and cells treated with the more potent 2-(4-methoxyphenyl)-A(II) were examined for induction of apoptosis. Microarray analyses were conducted to determine the apoptosis-related genes up- and down-regulated by the agent. 2-(4-Methoxyphenyl)-A(II) concentration-dependently inhibited growth of all three cell lines. Fifty percent and 100% growth inhibitory and 50% lethal concentrations were determined to be 0.3, 1.5, and 5 muM, respectively. Minimum detectable apoptosis-inducing concentrations by ELISA were 0.10 to 0.14 muM. PARP cleavage and two-color flow cytometry assays verified apoptosis induction. Microarray analyses showed Bok and Siva-pending to be up-regulated and that Birc, Dad1, and Atf5 were down-regulated. 2-(4-methoxyphenyl)-A(II) inhibits proliferation and induces apoptosis in the in vivo-adaptable TRAMP cells, suggesting the compound should be further examined in preclinical models.

OBJECTIVES To compare results of urinary cytology, quantitative detection of human complement factor H-related protein (BTA TRAK), and urinary fragments of cytokeratins 8 and 18 (UBC IRMA) with the recurrence status in patients with pTapT1 bladder cancer and to define the possible role of these methods in a surveillance protocol. METHODS We collected urine from 88 consecutive patients with primary pTapT1 tumors before the first transurethral resection (TURB) and before each follow-up cystoscopy. In all samples urinary cytology and quantitative BTA and UBC tests were performed. We compared results with recurrence status and with tumor characteristics in the case of recurrence. We constructed receiver operator characteristic (ROC) curves for quantitative methods. In addition, we evaluated individual cutoffs based on pretreatment levels. RESULTS During the mean follow-up of 16.96 months, we performed 313 cystoscopies, 93 of which were positive in 51 patients. The sensitivity and specificity of cytology, BTA, and UBC were 19.8% and 99%, 53.8% and 83.9%, and 12.1% and 97.2%, respectively. The sensitivity of pTis detection was 66.6%, 0%, and 100%, respectively. With cutoffs set to a sensitivity of 90%, the specificity of BTA and UBC dropped to 24.8% and 20.4%, respectively. Individually calculated cutoffs did not provide a significant benefit. CONCLUSIONS Because of high specificity and sensitivity in pTis detection, urinary cytology fulfills requirements for an adjunctive method to cystoscopy. Quantitative BTA and UBC tests have a low sensitivity in the detection of bladder cancer recurrence and cannot be used routinely to reduce the number of cystoscopies during follow-up.

As the average age of the US population increases, the number of men with benign prostatic hyperplasia will also rise. Lower urinary tract symptoms are found in almost all men as they age, yet we do not have a dependable means to detect, classify, and predict potential response to therapy and disease progression. A molecular biomarker, JM-27, may address some of these needs. Serum levels of JM-27 detect disease and differentiate among disease states as classified by the associated symptoms. Although larger validation studies to more extensively characterize this test are under way, current data suggest that JM-27 differentiates men with highly symptomatic disease from those with asymptomatic disease.

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)α 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, PURα 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 PURα in PC3 and DU145 cells negatively regulates cell proliferation, resulting in decreases in PCNA expression. CONCLUSION Further dissection of the role of PURα in cell growth regulation may reveal a novel target for HRPC. Prostate 68:1048–1056, 2008. © 2008 Wiley-Liss, Inc.

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.

OBJECTIVES Nuclear matrix proteins (NMPs), which constitute the nuclear skeleton, play a role in regulating gene expression and are thought to play an important role in carcinogenesis. Five NMPs specific to renal cell carcinoma (RCC) have been previously identified, RCCA 1 to 5. Our aim was to identify the sequence of these novel NMPs that are associated with RCC. METHODS NMPs were extracted from five conventional clear cell RCC specimens from patients undergoing surgical excision for presumed RCC. The RCCA proteins identified were analyzed by matrix-assisted light desorption ionization mass spectrometry to determine their peptide mass fingerprint. This fingerprint was then analyzed by searching the Mascot and National Institutes of Health BLAST (basic local alignment search tool) databases to determine protein identification. RESULTS Only RCCA-1 and RCCA-2 were able to be consistently identified from each tumor specimen by the reverse staining method necessary to perform mass spectrometry. Mass spectrometry peptide mass fingerprinting revealed that RCCA-1 appears to be a differentially spliced form of nucleoporin p54 that has been identified in endometrial carcinoma. RCCA-2 was identified as an albumin-like protein. CONCLUSIONS This is the first report of nucleoporin p54 and albumin alterations in RCC. The identification of an abnormal nucleoporin in RCC suggests that alterations in nuclear transport might play a role in the development of this disease. The alterations in these NMPs suggest future areas for investigation in identifying diagnostic markers of, or therapeutic targets for, RCC.

Contains fulltext : 47839schalken.pdf (Publisher’s version ) (Closed access) This is the first of 2 articles that summarize the findings of the International Consensus Panel on cytology and bladder tumor markers. The objectives of our panel were to reach a consensus on the areas where markers are needed, to define the attributes of an ideal tumor marker, and to identify which marker(s) would be suitable for diagnosis and/or surveillance of bladder cancer. Our panel consisted of urologists and researchers from Europe, Asia, and the United States who reviewed original articles, reviews, and book chapters on individual bladder tumor markers published in the English language mainly using the PubMed search engine. Panel members also met during 3 international meetings to write recommendations regarding bladder tumor markers. The panel found that the most practical use of noninvasive tests is to monitor bladder cancer recurrence, thereby reducing the number of surveillance cystoscopies performed each year. Markers also may be useful in the screening of high-risk individuals for early detection of bladder cancer. However, more prospective studies are needed to strengthen this argument. Case-control and cohort studies show that several markers have a higher sensitivity to detect bladder cancer. However, cytology is the superior marker in terms of specificity, although some markers in limited numbers of studies have shown specificity equivalent to that of cytology. Our panel believes that several bladder tumor markers are more accurate in detecting bladder cancer than prostate-specific antigen (PSA) is in detecting prostate cancer. However, bladder tumor markers are held to a higher standard than PSA. Therefore, use of bladder tumor markers in the management of patients with bladder cancer will require the willingness of both urologists and clinicians to accept them.

Previously, we demonstrated using a rat model of spinal cord injury (SCI) that bladder wall tissue compliance significantly increased within the first 2 weeks following injury. In order to explore the potential molecular-level mechanisms of this event, the present study quantified molecules pertinent to bladder tissue remodeling and changes in mechanical properties. An initial gene array analysis followed by real-time qPCR revealed that the message levels for tropoelastin and lysyl oxidase were as high as 8-fold in SCI rats compared to normal. Furthermore, both the message and protein levels of TGF-beta1 and IGF-1, known stimulators of elastin synthesis, in SCI rat bladders were significantly higher compared to those of normal rats. Taken together, it can be speculated that functional changes of the bladder associated with SCI induce release of select growth factors, which, in turn, stimulate elastogenesis that lead to alteration of biomechanical properties of the wall tissue.

Purpose: Benign prostatic hyperplasia (BPH) is among the most common diseases affecting quality of life in older men. Despise intense research efforts to our knowledge the genetic mechanisms underlying the BPH disease process and therapeutic markers needed to determine patient responsiveness to a particular form of pharmacological therapy are still not identified. This has slowed the development of new therapeutic agents for the treatment of BPH. Materials and Methods: We investigated the cellular effects of certain drugs on BPH. Besides doxazosin, which is the prevailing drug to treat BPH, the effect of ibuprofen was examined as a new therapeutic approach due to strong evidence of a correlation between BPH and inflammation. Results: This study showed that doxazosin as well as ibuprofen significantly decreases cell viability and induced apoptosis in 267 B1 cells as well as in BPH-1 cells. In addition, we observed that the administration of doxazosin and ibuprofen to BPH-1 cells decreased the expression of JM-27, a protein particularly expressed in prostate that is highly up-regulated in symptomatic BPH. Conclusions: Our findings suggest that ibuprofen may be a new therapeutic target for the treatment of BPH. Further research must be done to investigate the potential use of ibuprofen in patients with BPH and examine if JM-27 expression in patients with BPH may stratify individuals who may be most responsive to pharmacological treatment.

Purpose: Bladder cancer specific nuclear structural alterations have been identified. We examined the expression pattern of one of these proteins, BLCA-1, in tissue and urine samples from individuals with bladder cancer as well as in samples from normal controls. Materials and Methods: BLCA-1 sequence data were used to produce antibodies to this protein, which were used in immunoblot and enzyme-linked immunosorbent assays. Results: BLCA-1 was detectable in tissue from patients with bladder cancer but not in normal adjacent areas of the bladder or in normal donor bladder tissue. This protein was also detectable in the urine of patients with bladder cancer by immunoblot and immunoassay. Using a cutoff of 0.025 optical density units (absorbance value) BLCA-1 was detected in 20 of 25 urine samples from patients with bladder cancer but in only 6 of 46 normal, high risk, prostate or renal cancer samples tested, resulting in a test with 80% sensitivity and 87% specificity. Expression of this protein did not appear to correlate with tumor grade. Conclusions: This research indicates that BLCA-1 is a urine based marker of bladder cancer which may be useful for the detection of this disease.

The use of prostate specific antigen (PSA) to screen individuals for prostate cancer has changed the management of the disease, permitting early detection in men. Repeat biopsies for persistently increased PSA are not uncommon with prostate cancer never being detected in many of the men. The novel prostate cancer marker, EPCA, is expressed throughout the prostate of individuals with prostate cancer but not in those without the disease.The expression of ECPA was studied to identify those men with initially negative biopsies who were ultimately found to have prostate cancer. Negative biopsies, subsequent biopsies and prostatectomy specimens were evaluated by quantitative immunohistochemistry.The EPCA staining intensity in the samples analyzed from the patient cohort with prostate carcinoma were significantly different compared to controls (p0.001) with almost no overlap in staining results. Sensitivity of the EPCA immunohistochemistry is 84% and specificity is 85%.An immunohistochemical test for EPCA could serve as an adjunct to the current diagnostic approach to a patient who undergoes prostate needle biopsy, and could identify individuals with prostate cancer as much as 5 or more years earlier than the current diagnostic approach and algorithms. In addition, this test might also help limit the numbers of biopsies performed as part of subsequent routine evaluation for increased PSA.

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors and has potential tumor-suppressive functions in prostate and other cancer types. Vitamin D3 (VD3) exerts its biological actions by binding within cells to VDR. The VDR then interacts with specific regions of the DNA in cells, and triggers changes in the activity of genes involved in cell division, cell survival, and cellular function. Using human primary cultures and the prostate cancer (PCa) cell line, ALVA-31, we examined the effects of VD3 under different culture conditions. Complete G0/G1 arrest of ALVA-31 cells and approximately 50% inhibition of tumor stromal cell growth was observed. To determine changes in gene expression patterns related to VD3 activity, microarray analysis was performed. More than approximately 20,000 genes were evaluated for twofold relative increases and decreases in expression levels. A number of the gene targets that were up- and down-regulated are related to potential mechanisms of prostatic growth regulation. These include estrogen receptor (ER), heat shock proteins: 70 and 90, Apaf1, Her-2/neu, and paxillin. Utilizing antibodies generated against these targets, we were able to confirm the changes at the protein level. These newly reported gene expression patterns provide novel information not only potential markers, but also on the genes involved in VD3 induced apoptosis in PCa.

Resistance is a significant limitation to the effectiveness of cancer therapies. The PI3K/Akt and MAP kinase pathways play important roles in a variety of normal cellular processes and tumorigenesis. This study is designed to explore the relationship of these signaling pathways with multidrug resistance in prostate cancer (PCa). The PI3K/Akt and MAP kinase pathways were investigated utilizing paclitaxel resistant DU145-TxR PCa cells and their parental non-resistant DU145 cells to determine their relationship with resistance to paclitaxel and other anticancer drugs. Our results demonstrate that the PI3K/Akt and MAP kinase pathways are upregulated in DU145-TxR cells compared to the DU145 cells. Inactivating these pathways using the PI3K/Akt pathway inhibitor LY294002 or the MAP kinase pathway inhibitor PD98059 renders the DU145-TxR cells more sensitive to paclitaxel. We investigated the effects of these inhibitors on other anticancer drugs including docetaxel, vinblastine, doxorubicin, 10-Hydroxycamptothecin (10-HCPT) and cisplatin and find that both inhibitors induces DU145-TxR cells to be more sensitive only to the microtubule-targeting drugs (paclitaxel, docetaxel and vinblastine). Furthermore, the treatment with these inhibitors induces cleaved-PARP production in DU145-TxR cells, suggesting that apoptosis induction might be one of the mechanisms for the reversal of drug resistance. In conclusion, the PI3K/Akt and MAP kinase pathways are associated with resistance to multiple chemotherapeutic drugs. Inactivating these pathways renders these PCa cells more sensitive to microtubule-targeting drugs such as paclitaxel, docetaxel and vinblastine. Combination therapies with novel inhibitors of these two signaling pathways potentially represents a more effective treatment for drug resistant PCa.

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. J. Cell. Biochem. 86: 203–212, 2002. © 2002 Wiley-Liss, Inc.

UV exposure and serum levels of vitamin D have been linked in several studies with prostate cancer risk. At the cellular level, the principal action of vitamin D is mediated though vitamin D receptors (VDR). Since prostate cancer is a disease strongly associated with age, we examined the presence of VDR in normal prostate from donors of various ages to determine if the VDR expression pattern changed with age. We also compared the VDR expression in the peripheral and central zones of the prostate to determine if the expression pattern varied by location. Immunohistochemical studies were performed on paraffin-embedded tissue from cases selected by the following age decades; 10-19, 20-29, 30-39, 40-49, 50-59, and 60-69. Both the central and peripheral zones were examined for VDR expression. The intensity of VDR expression in prostate was compared with expression in different types of human tissues. Mean VDR expression was lowest in the 10-19 years of age group. The intensity of the nuclear VDR was higher though the fifth decade, and then declined in cases of ages 60-70. When multiple sections of the same donor prostate were compared, VDR expression was greater in the peripheral zone compared to the central zone.

BACKGROUND We previously found that in the absence of testosterone (T), calcitriol promotes proliferation of normal prostatic stroma, while in the presence of T, it has a differentiating effect on prostatic epithelium. The present study was conducted to determine the effect of calcitriol exposure in utero on the postnatal development of the normal prostate. METHODS Pregnant rats were injected subcutaneously with either 1.25 μg of calcitriol or vehicle alone on alternate days till delivery. Calcitriol-exposed and control pups were sacrificed at age 25 days (prepuberty), 63 days (postpuberty), or 102 days (adults), and their prostates and seminal vesicles were harvested and weighed. RESULTS Pups prenatally exposed to calcitriol and sacrificed before puberty (25 days) had a 35% greater mean prostatic weight than controls (0.0314 vs. 0.0422 g, P < 0.007), and calcitriol-exposed adult rats (102 days) had a 68% greater mean prostatic weight than controls (0.1365 vs. 0.2304 g, P < 0.005). No differences were observed in seminal vesicle weights, and in serum calcium and testosterone levels. A disproportionately high mortality rate from sudden death (71%) was observed at puberty in uncastrated male rats prenatally exposed to calcitriol. CONCLUSIONS These findings suggest that high-dose calcitriol exposure in utero may uniquely influence subsequent prostatic growth. Nonandrogenic steroids such as calcitriol may also be involved in genetic imprinting of the prostate. Prostate 41:181–189, 1999. © 1999 Wiley-Liss, Inc.

The regulation of cell processes is integrally connected to cellular and extracellular structure. Studies over the past three decades have demonstrated the complex interactions of cell structure and function. The relationship of cellular structure and function has perhaps been most studied in the transformed cell. The hallmark of transformation is alterations in the shape of the cell and the nucleus. Many of the cellular alterations observed in the cancer process are structural, including changes in extracellular matrix-cytoskeletal interactions, cytoskeletal elements, as well as nuclear structure. This review focuses on the structural components of the nucleus, the nuclear matrix, and their role in the cancer process and the use of these structural components of the nucleus, the nuclear matrix, and their role in the cancer process and the use of these structural components as cancer specific biomarkers. J. Cell. Biochem. Suppls. 32/33:183-191, 1999.

Objectives Matrix metalloproteinases (MMPs) are proteins that degrade the extracellular matrix and have been shown to be elevated in the urine of patients with cancer. One action of MMPs is the degradation of collagen IV that plays a role in tumor invasion and metastasis. This degradation can be measured by a fluorescent microplate activity assay that has been suggested to identify patients with renal cell carcinoma (RCC). Our aim was to confirm the utility of urinary MMP activity as a diagnostic test for RCC. Methods Urine samples from 21 patients undergoing nephrectomy for renal masses, as well as from 2 patients undergoing retroperitoneal mass excision for presumed local recurrence, were collected. Urine samples from 47 healthy volunteers were also collected. After concentration, the urine samples were incubated with fluorescein-labeled collagen IV. The fluorescence activity in each sample was measured using a conventional fluorescent microplate reader to determine the degree of collagen IV degradation in each specimen. Results Of the 21 patients undergoing nephrectomy, 15 had RCC, and both patients undergoing retroperitoneal mass excision had pathologically confirmed RCC recurrence. The mean number of fluorescence units emitted from the urine of patients with RCC was 48,924 units (range 0 to 275,879). The mean number of fluorescence units emitted from the urine of healthy patients was 29,834 units (range 0 to 400,086). This difference was not statistically significant (P = 0.34). Conclusions Despite previous evidence, urinary MMP activity was not an adequate test to identify RCC. Most normal urine samples had significant MMP activity.

The nuclear matrix has been linked to several important cellular functions within cells, such as DNA organization and replication, as well as regulation of gene expression. It has been reported that the nuclear matrix protein composition is altered in cells grown on different extracellular matrices in vitro. This study examined the nuclear matrix protein composition of tumors produced by MAT-LyLu (MLL) rat prostate tumor cells implanted at different organ sites within the rat. When high resolution two-dimensional gels were utilized to compare nuclear matrix protein composition to the prostate orthotopic tumor, it was found that there were distinct protein differences depending upon where the tumor grew. In particular, there were 14 proteins found in the lung, six proteins found in intramuscular, 17 proteins is the heart, and five proteins in the tail vein tumor tissue that were not present in the prostate orthotopic tumor tissue. Therefore, this study adds evidence to support that the nuclear matrix composition of a cell is dependent, at least in part, by the extracellular matrix and/or different cellular environments and may have a role in site-specific differences in tumor properties.

The nuclear matrix is the framework scaffolding of the nucleus and has been demonstrated to be an important component in a number of nuclear processes including transcription, replication, and RNA splicing and transport. In the interphase nucleus, DNA is specifically organized in a three-dimensional fashion. An example of this fact is that actively transcribed genes have been demonstrated to associate with the nuclear matrix. In this study, nuclear matrix proteins from various rat tissues, including two androgen-regulated tissues, the seminal vesicle and ventral prostate, were examined to determine if they contained proteins that associate with consensus binding sequences for several proteins involved in the regulation of transcription. Specific interactions were identified between proteins of the nuclear matrix and these transcriptional activator binding sequences. In addition, the sizes of the complexes binding to the DNA sequences appeared to vary in some of the tissues. These data support the concept that the nuclear matrix may serve as a support structure to bring together specific DNA sequences with factors involved in the regulation of gene expression.

Background The Cancer/Testis Antigens (CTAs) are an important group of proteins that are typically restricted to the testis in the normal adult but are aberrantly expressed in several types of cancers. As a result of their restricted expression patterns, the CTAs could serve as unique biomarkers for cancer diagnosis/prognosis. The aim of this study was to identify promising CTAs that are associated with prostate cancer (PCa) recurrence following radical prostatectomy (RP). Methods The expression of 5 CTAs was measured by quantitative multiplex real-time PCR using prostate tissue samples obtained from 72 patients with apparently clinically localized PCa with a median of two years follow-up (range, 1 to 14 years). Results The expression of CTAs namely, CEP55, NUF2, PBK and TTK were significantly higher while PAGE4 was significantly lower in patients with recurrent disease. All CTAs with the exception of TTK were significantly correlated with the prostatectomy Gleason score, but none were correlated with age, stage, or preoperative PSA levels. In univariate proportional hazards models, CEP55 (HR = 3.59, 95% CI: 1.50-8.60), p = 0.004; NUF2 (HR = 2.28, 95% CI: 1.11-4.67), p = 0.024; and PAGE4 (HR = 0.44, 95% CI: 0.21-0.93), p = 0.031 were significantly associated with the risk of PCa recurrence. However, the results were no longer significant after adjustment for prostatectomy Gleason score. Conclusions To our knowledge, this is the first study to identify CTAs as biomarkers that can differentiate patients with recurrent and non-recurrent disease following RP and underscores its potential impact on PCa prognosis and treatment.

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.

Among the urologic cancers, prostate cancer is by far the most common, and it appears to have the potential to affect almost all men throughout the world as they age. A number of studies have shown that many men with prostate cancer will not die from their disease, but rather with the disease but from other causes. These men have a form of prostate cancer that is described as “very low risk” and has often been called indolent. There are however a group of men that have a form of prostate cancer that is much more aggressive and life threatening. Unlike other cancer types, we have few tools to provide for the molecular classification of prostate cancer. This limits the potential to personalize treatment approaches for the disease. Several recent studies have focused on attempting to provide a better classification of prostate cancer in order to differentiate the types of the disease as well as to provide novel treatment targets. These approaches have utilized the latest technologies and sequencing capabilities to accomplish this goal. Two recent studies by Taylor et al. 1 and Yu et al. 2 are good examples of these approaches. Understanding the significance of these studies and their potential applications to prostate cancer is critical to reaching a better definition of the disease which impacts so many men. Taylor et al. 1 examined global DNA copy numbers (i.e. the numbers of copies of each gene) along with expression profiles (mRNAs that are expressed) to examine 218 prostate cancer tumors, both from individuals with localized disease as well as those with metastases. In other tumor types including lung cancer, breast cancer and glioblastoma, genomic alterations have been relatively easily identified. However, from this study by Taylor et al. 1 it appears that point mutations (single changes in the DNA sequence) may occur only rarely in prostate cancers. In fact, as the authors themselves conclude in their paper, there are no single genes that are commonly mutated in prostate cancer. Genes that are known to be altered in prostate cancer—for example, p53 and the tumor suppressor PTEN—are altered not by mutation, but by copy number changes. However, this comprehensive study did reveal several interesting modifications of the androgen pathway that seem to be important in prostate cancer. A co-activator of the androgen receptor (AR) known as NCOA2 was shown to be altered in a number of the prostate cancers studied. Functional studies demonstrated that altering NCOA2 levels may provide additional outputs to the androgen pathway and, therefore, has the potential to drive the disease. Interestingly, rather than finding actual changes to the AR, they observed changes in co-factors like NCOA2 and NCOR2 in localized primary tumors. Another interesting change observed by the authors in their study was that an area on chromosome 3 (3p14) appears to be deleted in a number of prostate cancers. Three interesting genes FOXP1, RYBP, and SHQ1 lie within this region. These have the potential to serve as tumor suppressors or other kinds of regulators that the absence of which appears to be important in the development of prostate cancer. Overall, it is interesting to note the lack of consistent and robust genetic changes that are found within prostate cancer. This seems to represent the diverse nature of the disease and may further demonstrate the importance of developing strategies to subclassify prostate cancers into distinct entities. Overall, the strengths of the study by Taylor et al. 1, include the comprehensive nature of the genomic analysis that is performed. This is supported by strong clinical data that provides meaning to the studies being performed. Finally, the population size examined is reasonable. Some of the limitations to the study outlined include the fact that the authors included, did not look at truly normal individuals. They therefore do not know which genetic alterations may indeed be associated with prostate cancer versus those truly without the disease. They also focused on relatively large tumor sizes. In doing so, they may have missed some important smaller lesions that could contain signatures with the potential to drive prostate cancer to a more aggressive state. With the known multi-focality of the disease, examining the various tumors within the prostate may be important in gaining an understanding of the true genetic basis of the disease. For example, another study by Miller and colleagues 3 demonstrated that many of the smaller lesions within the prostate have the same genetic signature as the resulting metastases unlike some of the larger lesions that are typically studied. Despite these limitations, however, the study is important as it reveals that prostate cancer is a complex disease that will not be as easy to decipher as a number of other cancers have been, at least at the genomic level. In an earlier issue of Cancer Cell, investigators from the University of Michigan utilized used a different approach to try to better decipher the genetic pathways associated with the progression of prostate cancer. In this case, Yu et al. 2 have utilized a technique known as chromatin immunoprecipitation (ChIP) together with extensive sequencing to decipher new aspects of not only the identified gene fusion of TMPRSS2 to several members of the ETS transcription factor family. These gene fusions have been observed in a large percentage of prostate cancers. The researchers studied prostate cancer cell lines and a single prostate cancer specimen. Chromatin immunoprecipitation utilizes antibodies to pull out specific proteins and their associated DNA sequences. The sequences are then analyzed to determine the specific genes that might be associated with individual proteins. For example, here the researchers looked at the AR to determine the associated sequences. They also looked at several other genes that are known to be important in prostate cancer: ERG, FoxA1 and RNA POLII, along with the sequences that are associated with the genes and which may be specific for prostate cancer. They identified a number of genes that have previously been shown to be associated with the AR. In addition to the canonical ARE sequence, the next most abundant set of sequences associated with the AR were those belonging to the ETS transcription factor family. This finding coincides nicely with previously identified gene fusions to demonstrate the androgen regulation of this family of factors in prostate cancer. Finally, the authors showed that ERG is associated with more than 90% of the AR genes. These findings illustrate the relationship between the pathways of ERG and the AR. The investigators proposed that the TMPRSS2-ERG gene fusion alters the androgen pathway and connects the androgen pathway to the Polycomb group of proteins. They went on to suggest that therapies targeting the AR may not be effective, that the true driver of prostate cancer may be the fusion of TMPRSS2-ERG, and that higher doses of testosterone may be more therapeutically effective than hormonal withdrawal. This study extensively catalogues the transcription factors and system modifications that are associated with prostate cancer, a data-set that will be extremely valuable for future studies. The major strength of the work by Yu et al. 2 is the comprehensive nature by which they studied the association between the AR, the Polycomb network and the TMPRSS2-ERG gene fusion. They used a number of prostate cancer cell lines to conduct their investigations and their results highlight the importance of TMPRSS2-ERG fusion in prostate cancer, suggesting a direct role in the regulation of castrate-resistant prostate cancer. One of the limitations of this work is that it focuses almost entirely on prostate cancer cell lines as models and does not involve interrogation of human samples beyond a single prostate cancer specimen. Another limitation is that TMPRSS2-ERG gene fusions have generally been found to be associated with earlier stage prostate cancer rather than the later stage disease that would be more typically associated with castrate-resistant prostate cancer. Therefore, the clinical significance of these findings still needs to be demonstrated. These two papers represent comprehensive and unique analyses of the genomic alterations and pathways that are associated with prostate cancer. Using state-of-the-art approaches these investigators have delved deeply into prostate cancer to begin to dissect out some of the critical pathways and components that are associated with the disease. Their conclusions support the idea that prostate cancer is a complex disease for which simple answers will not be forthcoming. At the same time, we are beginning to get clues about the critical regulators that may be involved in the development of the disease as well as its progression to a more malignant phenotype. Future studies will certainly build upon these fundamental studies as we continue to understand more about a disease that affects almost all men as they age.