New Therapeutic Approach to Suppress Castration-Resistant Prostate Cancer Using ASC-J9 via Targeting Androgen Receptor in Selective Prostate Cells

Using androgen receptor (AR) knockout mice to determine AR functions in selective prostate cancer (PCa) cells, we determined that AR might play differential roles in various cell types, either to promote or suppress PCa development/progression. These observations partially explain the failure of current androgen deprivation therapy (ADT) to reduce/prevent androgen binding to AR in every cell. Herein, we identified the AR degradation enhancer ASC-J9, which selectively degrades AR protein via interruption of the AR-AR selective coregulator interaction. Such selective interruption could, therefore, suppress AR-mediated PCa growth in the androgen-sensitive stage before ADT and in the castration-resistant stage after ADT. Mechanistic dissection suggested that ASC-J9 could activate the proteasome-dependent pathway to promote AR degradation through the enhanced association of AR-Mdm2 complex. The consequences of ASC-J9-promoted AR degradation included reduced androgen binding to AR, AR N-C terminal interaction, and AR nuclear translocation. Such inhibitory regulation could then result in suppression of AR transactivation and AR-mediated cell growth in eight different mouse models, including intact or castrated nude mice xenografted with androgen-sensitive LNCaP cells or androgen-insensitive C81 cells and castrated nude mice xenografted with castration-resistant C4-2 and CWR22Rv1 cells, and TRAMP and Pten^+/− mice. These results demonstrate that ASC-J9 could serve as an AR degradation enhancer that effectively suppresses PCa development/progression in the androgen-sensitive and castration-resistant stages.Androgen/androgen receptor (AR) signaling plays essential roles in prostate cancer (PCa) progression and results in castration resistance.^1–4 Currently, most, if not all, androgen deprivation therapy (ADT) targets androgens via surgical and/or medical castration to reduce/prevent androgen binding to AR.⁵ However, few, if any, of these ADTs with various antiandrogens, including the recently developed enzalutamide,⁶ have the capacity to eliminate all PCa cells in the later castration-resistant stage. Therefore, degradation of AR during/after ADT can be considered to have clinical benefits for patients with advanced PCa with substantial AR.⁶ These conclusions suggest that identifying a novel compound(s) that could degrade/diminish AR protein in the castration-resistant stage, unlike currently used antiandrogens, may yield better therapeutic efficacies to battle PCa in the castration-resistant stage.Early studies via isolation of three PCa primary cells (PCa1, PCa2, and PCa3) from the same patient found that androgen/AR signaling could function differentially to either suppress or promote PCa growth.⁷ Using the cre-loxP strategy in mice to selectively knockout AR in various PCa cells, Niu and colleagues^3,4,8 observed that the loss of AR in cytokeratin 5/cytokeratin 8–positive basal intermediate epithelial cells led to increased PCa metastasis, yet loss of AR in cytokeratin 8–positive luminal epithelial cells resulted in suppressed PCa progression with increased cell apoptosis. In contrast, loss of AR in stromal fibroblasts and smooth muscle cells resulted in suppression of prostate/PCa growth.^9,10 These results conclude that AR can either promote or suppress PCa progression in different types of PCa cells.Because only one AR gene has been identified,¹¹ we hypothesized that these differential AR roles in various PCa cells in the same patient could be due to the existence of different AR-AR coregulator complexes. This hypothesis led us to screen the AR degradation enhancer 5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one (ASC-J9) from natural products and their derivatives by selectively interrupting the interaction between AR and selective AR coregulators, such as AR–AR-associated protein (ARA) 70 and AR-ARA55, which are expressed mainly in luminal epithelial cells and stromal cells, respectively, in which AR may function with positive roles to either maintain cell survival or promote cell proliferation. Results from four different human PCa cell lines and eight different in vivo mouse models concluded that ASC-J9 could function as a promising AR degradation enhancer to suppress PCa progression before and after castration resistance with few adverse effects.     

Androgen depletion up‐regulates cadherin‐11 expression in prostate cancer

Men with castration‐resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin‐11 (also known as OB‐cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen‐deprivation therapy up‐regulates cadherin‐11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22/26 (85%) primary PCa tumours from men with castration‐resistant PCa expressed cadherin‐11. In contrast, only 7/50 (14%) androgen‐dependent PCa tumours expressed cadherin‐11. In the MDA–PCa‐2b xenograft animal model, cadherin‐11 was expressed in the recurrent tumours following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin‐11 and androgen receptor (AR), and cadherin‐11 is expressed in very low levels or not expressed in AR‐positive cell lines, including LNCaP, C4‐2B4 and VCaP cells. We showed that AR likely regulates cadherin‐11 expression in PCa through an indirect mechanism. Although re‐expression of AR in the AR‐negative PC3 cells led to the inhibition of cadherin‐11 expression, depletion of androgen from the culture medium or down‐regulation of AR by RNA interference in the C4‐2B4 cells or VCaP cells only produced a modest increase of cadherin‐11 expression. Promoter analysis indicated that cadherin‐11 promoter does not contain a typical AR‐binding element, and AR elicits a modest inhibition of cadherin‐11 promoter activity, suggesting that AR does not regulate cadherin‐11 expression directly. Together, these results suggest that androgen deprivation up‐regulates cadherin‐11 expression in prostate cancer, and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin‐11 expression or function may be considered when applying androgen‐ablation therapy. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Stromal Androgen Receptor Roles in the Development of Normal Prostate,Benign Prostate Hyperplasia,and Prostate Cancer

The prostate is an androgen-sensitive organ that needs proper androgen/androgen receptor (AR) signals for normal development. The progression of prostate diseases, including benign prostate hyperplasia (BPH) and prostate cancer (PCa), also needs proper androgen/AR signals. Tissue recombination studies report that stromal, but not epithelial, AR plays more critical roles via the mesenchymal-epithelial interactions to influence the early process of prostate development. However, in BPH and PCa, much more attention has been focused on epithelial AR roles. However, accumulating evidence indicates that stromal AR is also irreplaceable and plays critical roles in prostate disease progression. Herein, we summarize the roles of stromal AR in the development of normal prostate, BPH, and PCa, with evidence from the recent results of in vitro cell line studies, tissue recombination experiments, and AR knockout animal models. Current evidence suggests that stromal AR may play positive roles to promote BPH and PCa progression, and targeting stromal AR selectively with AR degradation enhancer, ASC-J9, may allow development of better therapies with fewer adverse effects to battle BPH and PCa.The prostate contains mainly the stromal cells and epithelial cells that are separated by base members and merged in extracellular matrix. Stromal cells include fibroblasts, smooth muscle cells (SMCs), and other minor inflammatory cells, nerve cells, and endothelial cells.The prostate is developed from the endodermal urogenital sinus¹ that contains an outer layer of embryonic connective tissue urogenital sinus mesenchyme (UGM) and an inner layer of urogenital sinus epithelium (UGE).¹ The initial step of prostate development in UGM involves the differentiation of fibroblasts and SMCs,¹ and in response to the UGM androgen/androgen receptor (AR) signals, UGE can grow into the surrounding stromal cells and develop into the prostate epithelial cells as part of the normal prostate development.The ability of the UGM to induce epithelial development and the developed epithelial cells, in return, to direct UGM to undergo differentiation, suggesting that the reciprocal developmental interactions between UGM and UGE might be governed by androgen/AR signals, which are essential for the development of normal prostate, benign prostate hyperplasia (BPH), and prostate cancer (PCa). Prostate development factors, including its proliferation, differentiation, morphogenesis, and functional maintenance, are all influenced by androgen/AR signals.² Androgen/AR signals also play vital roles in the initiation and progression of BPH and PCa,^3,4 which may require the proper interaction with various AR coregulators.²AR is a member of the nuclear receptor superfamily that can be activated and translocated from cytoplasm to nucleus after binding the testosterone or dihydrotestosterone.^5–7 In prostate, AR is expressed in both epithelial and stromal tissues. The transactivated AR in nucleus may then function through modulation of various downstream target genes to influence the development and maintenance of the prostate. In addition to influencing cell growth directly, epithelial AR and stromal AR can also function through epithelial-mesenchymal transition (EMT) to influence prostate development. EMT is a process by which epithelial cells lose their cell-cell adhesion and gain migratory properties to become mesenchymal-like and/or mesenchymal stem cells. These potent mesenchymal cells may then differentiate into different cell types to influence the progression of BHP⁸ and PCa.⁹This review will focus on the discussion of the roles of stromal AR in the development of normal prostate and prostate diseases.     

Evidence for steroidogenic potential in human prostate cell lines and tissues

Malignant prostate cancer (PCa) is usually treated with androgen deprivation therapies (ADTs). Recurrent PCa is resistant to ADT. This research investigated whether PCa can potentially produce androgens de novo, making them androgen self-sufficient. Steroidogenic enzymes required for androgen synthesis from cholesterol (CYP11A1, CYP17A1, HSD3β, HSD17β3) were investigated in human primary PCa (n = 90), lymph node metastases (LNMs; n = 8), and benign prostatic hyperplasia (BPH; n = 6) with the use of IHC. Six prostate cell lines were investigated for mRNA and protein for steroidogenic enzymes and for endogenous synthesis of testosterone and 5α-dihydrotestosterone. All enzymes were identified in PCa, LNMs, BPH, and cell lines. CYP11A1 (rate-limiting enzyme) was expressed in cancerous and noncancerous prostate glands. CYP11A1, CYP17A1, HSD3β, and HSD17β3 were identified, respectively, in 78%, 52%, 16%, and 82% of human BPH and PCa samples. Approximately 10% of primary PCa, LNMs, and BPH expressed all four enzymes simultaneously. CYP11A1 expression was stable, CYP17A1 increased, and HSD3β and HSD17β3 decreased with disease progression. CYP17A1 expression was significantly correlated with CYP11A1 (P = 0.0009), HSD3β (P = 0.0297), and HSD17β3 (P = 0.0090) in vivo, suggesting CYP17A1 has a key role in prostatic steroidogenesis similar to testis and adrenal roles. In vitro, all cell lines expressed mRNA for all enzymes. Protein was not always detectable; however, all cell lines synthesized androgen from cholesterol. The results indicate that monitoring steroidogenic metabolites in patients with PCa may provide useful information for therapy intervention.     

Somatic copy number alterations by whole‐exome sequencing implicates YWHAZ and PTK2 in castration‐resistant prostate cancer

Castration‐resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole‐exome sequencing on five CRPC/normal paired formalin‐fixed paraffin‐embedded (FFPE) samples, using the SOLiD4 next‐generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock‐down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock‐down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole‐exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next‐generation sequencing technologies. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Stromal Androgen Receptor in Prostate Development and Cancer

The androgen receptor (AR) in stromal cells contributes significantly to the development and growth of prostate during fetal stages as well as during prostate carcinogenesis and cancer progression. During prostate development, stromal AR induces and promotes epithelial cell growth, as observed from tissue recombinant and mouse knockout studies. During prostate carcinogenesis and progression, the stromal cells begin to lose AR expression as early as at the stage of high-grade prostatic intraepithelial neoplasia. The extent of loss of stromal AR is directly proportional to the degree of differentiation (Gleason grade) and progression of prostate cancer (PCa). Co-culture studies suggested that stromal AR inhibits the growth of malignant epithelial cells, possibly through expression of certain paracrine factors in the presence of androgens. This functional reversal of stromal AR, from growth promotion during fetal prostate development to mediating certain growth-inhibiting effects in cancer, explains to some extent the reason that loss of AR expression in stromal cells may be crucial for development of resistance to androgen ablation therapy for PCa. From a translational perspective, it generates the need to re-examine the current therapeutic options and opens a fundamental new direction for therapeutic interventions, especially in advanced PCa.CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Prostate cancer (PCa) is the most common non-skin malignancy in the male population within the United States and is the second most common cancer in men worldwide.¹ It is also one of the leading causes of cancer-related deaths in males in the United States.Normal human prostate is composed of an epithelial tissue and an adjacent stroma. The epithelium is composed of two principal cell types, the tall columnar secretory luminal cells that line the glandular ducts and the flattened basal cells surrounding them. In addition, some rare neuroendocrine cells are also present. Often, the terms mesenchyme and stroma are loosely used. Herein, mesenchyme refers to the mesodermal-derived fetal or newborn tissues with instructive induction potential. The word stroma describes the tissues surrounding the prostatic epithelium, later in development. In the adult human prostate, the stroma is composed mainly of smooth muscle cells. However, it also includes some fibroblasts, nerves, blood vessels, and various infiltrating immune and inflammatory cell types.Circulating androgens mediate the development and function of prostate by stimulating the androgen receptor (AR). Rat studies have shown that in stroma, AR is expressed in mesenchymal cells of the urogenital sinus (UGS), especially those adjacent to the epithelium, concurrent with the formation of prostatic buds.^2,3 As the prostate develops and the mesenchymal cells differentiate to form smooth muscle, AR expression is widespread, but not universal, throughout the muscle. In the past, investigators have mainly focused on studying epithelial AR function in prostate. Relatively limited data are available to describe the expression and function of stromal AR in prostate development^2–14 and cancer. Stromal AR is involved in both prostate development and prostate carcinogenesis, with distinct functions in these two processes. We examine the current knowledge and understanding of stromal AR function, including its translational significance.     

Prostate cancer derived prostatic acid phosphatase promotes an osteoblastic response in the bone microenvironment

Approximately 90 % of patients who die of prostate cancer (PCa) have bone metastases, often promoting osteoblastic lesions. We observed that 88 % of castration-resistant PCa (CRPC) bone metastases express prostatic acid phosphatase (PAP), a soluble secreted protein expressed by prostate epithelial cells in predominately osteoblastic (n = 18) or osteolytic (n = 15) lesions. Additionally, conditioned media (CM) of an osteoblastic PCa xenograft LuCaP 23.1 contained significant levels of PAP and promoted mineralization in mouse and human calvaria-derived cells (MC3T3-E1 and HCO). To demonstrate that PAP promotes mineralization, we stimulated MC3T3-E1 cells with PAP and observed increased mineralization, which could be blocked with the specific PAP inhibitor, phosphonic acid. Furthermore, the mineralization promoted by LuCaP 23.1 CM was also blocked by phosphonic acid, suggesting PAP is responsible for the mineralization promoting activity of LuCaP 23.1. In addition, gene expression arrays comparing osteoblastic to osteolytic CRPC (n = 14) identified betacellulin (BTC) as a gene upregulated during the osteoblastic response in osteoblasts during new bone formation. Moreover, BTC levels were increased in bone marrow stromal cells in response to LuCaP 23.1 CM in vitro. Because new bone formation does occur in osteoblastic and can occur in osteolytic CRPC bone metastases, we confirmed by immunohistochemistry (n = 36) that BTC was highly expressed in osteoblasts involved in new bone formation occurring in both osteoblastic and osteolytic sites. These studies suggest a role for PAP in promoting the osteoblastic reaction in CRPC bone metastases and identify BTC as a novel downstream protein expressed in osteoblasts during new bone formation.     

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing, CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, pre-existing SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SC-like BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (AR(low)) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The AR(low) seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.     

Androgen Receptor Influences on Body Defense System via Modulation of Innate and Adaptive Immune Systems: Lessons from Conditional AR Knockout Mice

Upon insult, such as infection or tissue injury, the innate and adaptive immune systems initiate a series of responses to defend the body. Recent studies from immune cell-specific androgen receptor (AR) knockout mice demonstrated that androgen and its receptor (androgen/AR) play significant roles in both immune regulations. In the innate immunity, androgen/AR is required for generation and proper function of neutrophils; androgen/AR also regulates wound healing processes through macrophage recruitment and proinflammatory cytokine production. In adaptive immunity, androgen/AR exerts suppressive effects on development and activation of T and B cells. Removal of such suppression causes thymic enlargement and excessive export of immature B cells. Altogether, androgen/AR plays distinct roles in individual immune cells, and targeting androgen/AR may help in treatment and management of immune-related diseases.The immune system includes both innate and adaptive immune responses. Once insult to the body (eg, infection) is initially encountered, the innate system acts within minutes, followed some hours later by early induced responses conducted by recruited effector cells, such as neutrophils and macrophages, in a nonspecific manner.^1,2 In most cases, infections are eliminated by the innate immune system. When pathogens breach the first line of immune defense, however, the adaptive immune system is activated with antigen-specific effectors (eg, T and B cells), and the generation of memory cells ensures a long-lasting and prompt response to recurrent infection with the same pathogens. On the other hand, the context of the activated innate immunity also shapes the outcome of adaptive immune responses.³Androgen and androgen receptor (AR) signals control the development and function of both male and female reproductive systems.^4–6 The AR gene is located on the X chromosome in both the human and the murine genome. AR is a prototypical nuclear receptor, containing an N-terminal domain, a ligand-binding domain, a DNA-binding domain, and a C-terminal domain.⁷ After binding of its ligands, testosterone or 5α-dihydrotestosterone, AR translocates into the nucleus and binds to androgen responsive elements on the promoters or enhancers of target genes, thereby turning on their expression.⁸ The expression of AR has been detected in various immune cell lineages, including neutrophils, mast cells, macrophages, B cells, and T cells,^9–11 implying the involvement of androgen and its receptor (androgen/AR) in regulating the development and function of the immune system.It has long been suspected that, in both animals and humans, the male sex hormones may modulate the development and function of the immune system. Males are at higher risk of developing sepsis, acute respiratory distress, and multiorgan failure after soft-tissue traumatic hemorrhagic shock and thermal injury, in part because of immune suppression and abnormal activation of neutrophils.¹² On the other hand, males are less prone to autoimmune diseases. Of more than 70 chronic disorders categorized as autoimmune diseases, many affect predominantly women.^13,14 Various studies have uncovered important immune regulatory functions of androgen/AR (as discussed below), and such nonclassical roles of androgen/AR outside the reproductive system are important for understanding the pathogenesis of these immunological conditions.Recent studies using conditional AR knockout (ARKO) mice, with knockout of AR in selective immune cells, opened a new era for investigating the nonclassical roles of androgen/AR involved in immune regulation; these studies also suggest potential for the development of new therapeutic strategies for treating immune-related diseases.^6,15 In this review, we summarize the roles of androgen/AR in both the innate and adaptive components of the immune system (specifically neutrophils, macrophages, T cells, and B cells), as well as new evidence from studies using conditional ARKO mice. We also address the potential influences of androgen/AR on immune-related diseases.     

Tissue Slice Grafts : An in Vivo Model of Human Prostate Androgen Signaling

We developed a tissue slice graft (TSG) model by implanting thin, precision-cut tissue slices derived from fresh primary prostatic adenocarcinomas under the renal capsule of immunodeficient mice. This new in vivo model not only allows analysis of approximately all of the cell types present in prostate cancer within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Both benign and cancer tissues displayed characteristic histology and expression of cell-type specific markers for up to 3 months. Moreover, androgen-regulated protein expression diminished in TSGs after androgen ablation of the host and was restored after androgen repletion. Finally, many normal secretory epithelial cells and cancer cells in TSGs remained viable 2 months after androgen ablation, consistent with similar observations in postprostatectomy specimens following neoadjuvant androgen ablation. Among these were putative Nkx3.1⁺ stem cells. Our novel TSG model has the appropriate characteristics to serve as a useful tool to model all stages of disease, including normal tissue, premalignant lesions, well-differentiated cancer, and poorly differentiated cancer.Over the years, significant efforts have been made to develop realistic model systems to investigate the biology of benign and malignant prostatic epithelial cells. These models are vital in prostate research and have enabled important discoveries. However, there are intrinsic limitations in these experimental models that limit their use.¹ For example, most of the immortal cell lines maintain little of the secretory differentiation that is characteristic of normal and cancerous epithelial cells of the prostate. Many do not express androgen receptor (AR) or prostate specific antigen (PSA), or express a mutated AR.^2,3 Similarly, primary cultures derived from normal or cancer tissues typically lose functional AR and androgen-sensitive growth and gene expression.⁴ Their limited life span also makes it difficult to generate sufficient cells for long-term studies.⁴ Moreover, the interactions between the epithelial, stromal, and vascular compartments of the prostate, which have been demonstrated to be essential for normal prostate development and function as well as in the development and progression of prostate cancer (PCa), are also missing in these in vitro model systems.¹Commonly used in vivo models of human PCa such as xenografts generated from cell lines suffer similar drawbacks of lack of expression of wild-type AR and/or the complex biochemical and physical interactions between the various cellular, tissue, and hormonal compartments that characterize human PCa. Animal models of PCa such as transgenic and knock-out mouse models maintain an intact prostate architecture; however, these systems do not model the progression of human PCa because the single molecule transgenic and knock-out mice either rarely develop a pathology beyond hyperplasia or prostatic intraepithelial neoplasia or rapidly progress to poorly differentiated cancer.⁵ In addition, differences in the anatomy and physiology between the rodent and human prostate also make it difficult to generalize conclusions obtained by using these models.⁶Xenografts derived from direct implantation of small pieces of tumors freshly taken from patients into mice, so-called “tumorgrafts,” are thought to be the most realistic experimental models of human cancer because they recapitulate the parent tumors microscopically as well as molecularly.⁷ In several malignancies, tumorgrafts have gained popularity recently because they are very predictive of drug response.⁸ The ability to generate prostate tumorgrafts has been demonstrated by several groups.^{9,10,11,12,13} The take rate of prostate tumorgrafts under the renal capsule of immunodeficient mice is >90% using a recently optimized protocol.¹⁰ This model has been used to compare angiogenesis in PCa versus benign prostate and to quantify apoptotic activity after castration in human prostate tissue.^11,13Androgen signaling plays a key role not only in the growth and function of normal prostate, but also in the development and progression of PCa.¹⁴ Androgen deprivation therapy is a common treatment for men with advanced PCa.¹⁵ Prostate tissue grafts have been used to determine the response of benign and cancerous tissues to androgen deprivation.^12,13 It was reported that benign glandular structures in postcastration grafts were populated by basal, secretory, and squamous cells, whereas cancer glands in the grafts resembled the original cancer tissue.¹² However, it is not clear whether androgen signaling is decreased in castrated grafts and whether the pathway activity can resume after androgen restoration.We have modified the “tumorgraft” model by the use of thin, precision-cut tissue slices. This protocol extends previous applications of such tissue slices in vitro.^16,17,18 This new in vivo model not only allows analysis of approximately all of the cell types present in PCa within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Using this model, we investigated the responses of prostate tissue to androgen ablation and restoration.     

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss‐driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70–hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP‐CR subclone) and a matched TMA of hormone‐naïve (HNPC) and castrate‐resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co‐expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR⁺ tumours was significantly higher than in AR^? tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP‐CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co‐expressed with Hsp70–hsp90 client proteins. Up‐regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.