Androgen receptor variants and prostate cancer in humanized AR mice

Androgen, acting via the androgen receptor (AR), is central to male development, differentiation and hormone-dependent diseases such as prostate cancer. AR is actively involved in the initiation of prostate cancer, the transition to androgen independence, and many mechanisms of resistance to therapy. To examine genetic variation of AR in cancer, we created mice by germ-line gene targeting in which human AR sequence replaces that of the mouse. Since shorter length of a polymorphic N-terminal glutamine (Q) tract has been linked to prostate cancer risk, we introduced alleles with 12, 21 or 48 Qs to test this association. The three “humanized” AR mouse strains (h/mAR) are normal physiologically, as well as by cellular and molecular criteria, although slight differences are detected in AR target gene expression, correlating inversely with Q tract length. However, distinct allele-dependent differences in tumorigenesis are evident when these mice are crossed to a transgenic prostate cancer model. Remarkably, Q tract variation also differentially impacts disease progression following androgen depletion. This finding emphasizes the importance of AR function in androgen-independent as well as androgen-dependent disease. These mice provide a novel genetic paradigm in which to dissect opposing functions of AR in tumor suppression versus oncogenesis.     

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine.     

Androgen receptor expression in primary prostate cancers of lobund-wistar rats and in tumor-derived cell lines

Summary Prostate tumors were induced in Lobund-Wistar rats by treatment with N-methyl-N-nitrosourea (MNU) and testosterone propionate (TP). Androgen receptor (AR) expression was confirmed in 16 (100%) of the primary prostate cancers, with strong uniform staining in well-differentiated tumors and more variable AR immunoreactivity in poorly differentiated tumors. Epithelial cell lines were established from nine of the tumors. At early passages, four of the tumor cell lines tested were strongly immunoreactive for AR; however, only two of the cell lines, E2(A) and F2, have remained AR-positive. These cell lines specifically bind ³H-DHT at 40 and 19 fmol/mg protein, respectively, and express a 110 kDa AR immunoreactive protein. Proliferation in in vitro culture of both E2(A) and F2 cells was increased in the presence of 5α-dihydrotestosterone (DHT). The antiandrogen, hydroxyflutamide was able to prevent the DHT-induced growth of E2(A) but not F2 cells. Furthermore, hydroxyflutamide alone increased proliferation of F2 cells, suggesting that the androgen signalling pathway in this cell line may be abnormal. Tumorigenicity of the AR-expressing and nonexpressing cell lines was confirmed by xenograft formation following subcutaneous inoculation into intact male nude mice. In summary, carcinogen-induced prostate tumors of Lobund-Wistar rats express AR and two of nine cell lines derived from the tumors express AR. Further evaluation of AR structure in primary prostate tumors forming spontaneously or following MNU and TP induction will determine whether, as in human prostate cancers, disease progression in Lobund-Wistar rats is associated with mutations in the AR gene.     

Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence

Despite earlier detection and recent advances in surgery and radiation, prostate cancer is second only to lung cancer in male cancer deaths in the United States. Hormone therapy in the form of medical or surgical castration remains the mainstay of systemic treatment in prostate cancer. Over the last 15 years with the clinical use of prostate specific antigen (PSA), there has been a shift to using hormone therapy earlier in the disease course and for longer duration. Despite initial favorable response to hormone therapy, over a period of time these tumors will develop androgen‐independence that results in death. The androgen receptor (AR) is central to the initiation and growth of prostate cancer and to its response to hormone therapy. Analyses have shown that AR continues to be expressed in androgen‐independent tumors and AR signaling remains intact as demonstrated by the expression of the AR regulated gene, PSA. Androgen‐independent prostate cancers have demonstrated a variety of AR alterations that are either not found in hormone naïve tumors or found at lower frequency. These changes include AR amplification, AR point mutation, and changes in expression of AR co‐regulatory proteins. These AR changes result in a “super AR” that can respond to lower concentrations of androgens or to a wider variety of agonistic ligands. There is also mounting evidence that AR can be activated in a ligand independent fashion by compounds such as growth factors or cytokines working independently or in combination. These growth factors working through receptor tyrosine kinase pathways may promote AR activation and growth in low androgen environments. The clinical significance of these AR alterations in the development and progression of androgen‐independent prostate cancer remains to be determined. Understanding the changes in AR signaling in the evolution of androgen‐independent prostate cancer will be key to the development of more effective hormone therapy. © 2003 Wiley‐Liss, Inc.     

Effects of the flavonoid baicalin and its metabolite baicalein on androgen receptor expression, cell cycle progression and apoptosis of prostate cancer cell lines

Recent studies on the Chinese herbal medicine PC SPES showed biological activities against prostate cancer in vitro, in vivo and in patients with advanced stages of the disease. In investigating its mode of action, we have isolated a few of the active compounds. Among them, baicalin was the most abundant (about 6%) in the ethanol extract of PC SPES, as determined by HPLC. Baicalin is known to be converted in vivo to baicalein by the cleavage of the glycoside moiety. Therefore, it is useful to compare their activities in vitro. The effects of baicalin and baicalein were studied in androgen-positive and -negative human prostate cancer lines LNCaP and JCA-1, respectively. Inhibition of cell growth by 50% (ED(50)) in LNCaP cells was seen at concentrations of 60.8 +/- 3.2 and 29.8 +/- 2.2 microM baicalin and baicalein, respectively. More potent growth inhibitory effects were observed in androgen-negative JCA-1 cells, for which the ED(50) values for baicalin and baicalein were 46.8 +/- 0.7 and 17.7 +/- 3.4, respectively. Thus, it appears that cell growth inhibition by these flavonoids is independent of androgen receptor status. Both agents (1) caused an apparent accumulation of cells in G(1) at the ED(50) concentration, (2) induced apoptosis at higher concentrations, and (3) decreased expression of the androgen receptor in LNCaP cells.     

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Background  

Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells.     

The role of the growth hormone/insulin-like growth factor axis in tumor growth and progression: Lessons from animal models

Over the past two decades it has become widely appreciated that a relationship exists between the insulin-like growth factors (IGFs) and cancer. Many cancers have been shown to overexpress the IGF-I receptor and produce the ligands (IGF-I or IGF-II) and some combinations of the six IGF-binding proteins. With the recent demonstration by epidemiological studies that an elevated serum IGF-I level is associated with an increased relative risk of developing a number of epithelial cancers, interest has been sparked in this area of research with the possibility of targeting the IGF-I receptor in cancer treatment protocols. This review highlights many of the most relevant studies in this exciting area of research, focusing in particular on lessons learned from animal models of cancer.     

Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression

High levels of peripheral-type benzodiazepine receptor (PBR), the alternative-binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non-tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon-rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

Androgen receptor expression and DNA content of paraffin-embedded archival human prostate tumors

BACKGROUND: Androgen receptors (AR) are expressed in human prostate cells and immunohistochemistry has been used for qualitative analysis of AR expression in prostate tumor cells. Quantitative and multiparametric analysis of receptor expression could be of diagnostic and prognostic value in the management of patients on antiandrogen therapy. Multiparametric flow cytometric methods have been developed for analysis of hormone receptor expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded human solid tumors. The present study was undertaken for analysis of AR expression and DNA content in archival human prostate tumors. METHODS: AR expression and DNA content were measured in nuclei isolated by enzyme digestion from thick sections cut from 51 paraffin-embedded human prostate tumors. AR expression in different subpopulations was studied by gated analysis. The relationship among AR activity, DNA content, and histopathological grade was analyzed. RESULTS: Distinct aneuploid populations were observed in 23% of tumors examined. AR activity was observed in all the specimens and the percentage of AR- positive nuclei in the 48 samples analyzed was <10% (n = 4), 11-50% (n = 39), and >51% (n = 5). Tumor subpopulations with aneuploid DNA content had higher AR expression (percent AR-positive cells and mean log fluorescence) than the diploid subpopulations. No strong correlation was seen between AR expression and histopathological grade of the tumors. CONCLUSIONS: Flow cytometric analysis of archival prostate tumor can be used for rapid determination of aneuploid DNA content and AR expression in subpopulations of nuclei isolated from formalin-fixed/paraffin-embedded prostate tumor blocks.     

Androgen receptor activation by G(s) signaling in prostate cancer cells

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.     

The androgen receptor (AR) in syndromes of androgen insensitivity and in prostate cancer

The actions of androgens, principally testosterone and 5alpha-dihydrotestosterone, are mediated by a specific receptor protein, the androgen receptor (AR), which is encoded by a single-copy gene located on the human X-chromosome. This receptor protein is a prototypical member of the nuclear receptor family and modulates a range of processes during embryogenesis and in the adult. During embryogenesis, normal AR function is critical to the development of the male phenotype and defects of the AR cause a range of phenotypic abnormalities of male sexual development. Complete loss of AR function has been traced to a number of distinct types of genetic events, including abnormalities of mRNA splicing, the introduction of premature termination codons, and amino acid substitution mutations. An interesting subset of mutations is that in which the AR is completely undetectable using sensitive immunoassays. In all instances, these functional abnormalities are associated with a phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, partial defects of AR function are almost invariably caused by amino acid substitutions within the DNA- and hormone-binding domains of the receptor protein. Such partial defects of receptor function may be caused by changes in either receptor function or receptor abundance.The alterations of AR function and expression that have been characterized in clinical prostatic cancers and in prostate cancer cell lines differ in several important respects. A number of studies have documented the emergence of considerable heterogeneity of AR expression at early stages in the development of prostate cancer. Despite these early changes of AR expression, a substantial body of information suggests that the AR is expressed in advanced forms of prostate cancer, in some cases as the result of amplification events. While infrequent in localized tumors, mutations of the AR have been identified in a number of advanced prostatic cancers and in some instances appear to alter the ligand specificity of the AR. Finally, it appears that other signaling pathways can act to influence AR function.     

Genistein increases epidermal growth factor receptor signaling and promotes tumor progression in advanced human prostate cancer

Genistein is an isoflavone found in soy, and its chemo-preventive and -therapeutic effects have been well established from in vitro studies. Recently, however, its therapeutic actions in vivo have been questioned due to contradictory reports from animal studies, which rely on rodent models or implantation of cell lines into animals. To clarify in vivo effects of genistein in advanced prostate cancer patients, we developed a patient-derived prostate cancer xenograft model, in which a clinical prostatectomy sample was grafted into immune deficient mice. Our results showed an increased lymph node (LN) and secondary organ metastases in genistein-treated mice compared to untreated controls. Interestingly, invasive malignant cells aggregated to form islands/micrometastasis only in the secondary organs of the genistein-treated groups, not in the untreated control group. To understand the underlying mechanism for metastatic progression, we examined cell proliferation and apoptosis on paraffin-sections. Immunohistological data show that tumors of genistein-treated groups have more proliferating and fewer apoptotic cancer cells than those of the untreated group. Our immunoblotting data suggest that increased proliferation and metastasis are linked to enhanced activities of tyrosine kinases, EGFR and its downstream Src, in genistein-treated groups. Despite the chemopreventive effects proposed by earlier in vitro studies, the cancer promoting effect of genistein observed here suggests the need for careful selection of patients and safer planning of clinical trials.     

Immunohistological detection of BRAF25 in human prostate tumor and cancer specimens

BRAF25 is an alternatively spliced protein of BRAF35 (see associated paper). We have mapped the BRAF25 gene to chromosome sub-band 19p13.3, a region where loss of chromosomal heterozygosity has been reported in about 50% of ovarian cancers. Because of the high incidence of genetic links of prostate cancer to breast and ovarian cancers, we investigated the BRAF25 expression in the prostate specimens. Immunohistochemical analysis using antibodies specific for BRAF25 revealed a strong immunostaining in sections of the benign prostatic hyperplasia (BPH). The staining was concentrated on the nuclei of cells facing the lumen of prostatic glands, even though the sporadic nuclei of cells in stromas were also stained. However, the expression of BRAF25 was dramatically reduced in intermediate prostate cancer and absent in advanced prostate cancer. Preincubation of the antibody with the immunizing peptide abolished immunostaining in BPH specimens. Therefore, the expression of BRAF25 was gradually lost in prostate cancer.     

Androgen inhibits the growth of carcinoma cell lines established from prostate cancer xenografts that escape androgen treatment

Most prostate cancers escape endocrine therapy by diverse mechanisms. One of them might be growth repression by androgen. We reported that androgen represses the growth in culture of MOP cells (a sub-line of LNCaP cells) and that of MOP cell xenografts, although tumor growth becomes androgen-independent (AI). Here we explore whether AI tumors contain androgen-responsive cells. ME carcinoma cells were established from AI tumors. The responses to androgen were examined by cell counting, DAPI labeling, flow cytometry, PSA immunoassay and tumor size follow-up. Androgen receptors (AR) were analyzed by western blotting and DNA sequencing. The pattern of responses of these cells to androgen was compared to that of MOP cells and that of JAC cells established from LNCaP-like MOP cells. R1881, a synthetic androgen: (1) repressed the growth of all the six ME cell lines obtained, MOP and JAC cells, (2) augmented the secretion of PSA, (3) induced spectacular cell bubbling/fragmentation and (4) blocked the cell cycle and induced a modest increase of apoptosis. All the androgen-repressed cells expressed the same level of mutated AR as LNCaP cells. In nude mice, the growth of ME-2 cell xenografts displayed transient androgen repression similar to that of MOP cells. In culture neither fibroblasts nor extra-cellular matrix altered the effects of R1881 on cell proliferation. These results demonstrate that androgen-independent tumors contain androgen-responsive cells. The apparent discrepancy between the responses to androgen of tumors and those of carcinoma cells in culture suggests that microenvironmental factors contribute to the androgen responsiveness of tumor cells in vivo. These modifications, albeit unspecified, could be suitable targets for restoring the androgen responsiveness of AI tumors.     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

The immunoglobulin superfamily of neuronal cell adhesion molecules: Lessons from animal models and correlation with human disease

Neuronal cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in the formation of neural circuits at different levels: cell migration, axonal and dendritic targeting as well as synapse formation. Furthermore, in perinatal and adult life, neuronal IgCAMs are required for the formation and maintenance of specialized axonal membrane domains, synaptic plasticity and neurogenesis. Mutations in the corresponding human genes have been correlated to several human neuronal disorders. Perturbing neuronal IgCAMs in animal models provides powerful means to understand the molecular and cellular basis of such human disorders. In this review, we concentrate on the NCAM, L1 and contactin subfamilies of neuronal IgCAMs summarizing recent functional studies from model systems and highlighting their links to disease pathogenesis.     

Alterations of expression and regulation of transforming growth factor beta in human cancer prostate cell lines

TGFβ can promote and/or suppress prostate tumor growth through multiple and opposing actions. Alterations of its expression, secretion, regulation or of the sensitivity of target cells can lead to a favorable environment for tumor development. To gain a better insight in TGFβ function during cancer progression, we have used different cultured human prostate cells: preneoplastic PNT2 cells, the androgen-dependent LNCaP and the androgen-independent PC3 and DU145 prostate cancer cell lines. We have studied by specific ELISA assays in conditioned media (CM), the secretion of TGFβ1 and TGFβ2 in basal conditions and after hormonal treatment (DHT or E2) and the expression of TGFβ1 mRNA by Northern blot. We have also compared the effect of fibroblast CM on TGFβ secretion by the different cell types. Compared to PNT2 cells, cancer cell lines secrete lower levels of active TGFβ which are not increased in the presence of fibroblast CM. LNCaP cells respond to androgen or estrogen treatment by a 10-fold increase of active TGFβ secretion while PC3 and DU145 are unresponsive. In conclusion, prostate cancer cell lines have lost part of their ability to secrete and activate TGFβ, and to regulate this secretion through stromal–epithelial interactions. Androgen-sensitive cancer cells may compensate this loss by hormonal regulation.