Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in human prostate cancer

Progression of prostate cancer from an androgen sensitive to androgen insensitive tumor has previously been shown to be accompanied by a change in alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in a rat model of prostate cancer. This change results in loss of the FGF-R2(IIIb) isoform and predominant expression of the FGF-R2(IIIc) isoform. We sought to determine whether this change in FGF-R2 splicing is also associated with androgen insensitivity in human prostate tumors. We analysed three well characterized human prostate cancer cell lines and three metastatic prostate tumors which have been maintained as xenografts in nude mice. One of the cell lines, LNCaP, and two of the xenografts, DUKAP-1 and DUKAP-2, have been characterized as androgen sensitive, whereas two of the cell lines, DU-145 and PC-3, and one of the xenografts, DU9479, display androgen independent growth. Using an RT-PCR based assay, we demonstrated that progressive loss of the FGF-R2(111b) isoform correlated with androgen insensitivity in these human prostate cancer models. These findings lend support to the hypothesis that that loss of FGF-R2(IIIb) may be one step in a series of events which lead to progression of human prostate cancer.     

Organic solvents identify specific ligand binding sites on protein surfaces

Over many years, a great deal of attention has focused on the growth regulatory effects of androgens in prostate cells. This, has also prompted widespread interest in the role of these steroid hormones in prostate cancer pathogenesis. Even so, no-one has so far been able to identify the exact relationship between androgenic hormone levels and the risk of these diseases though differences in hormonal patterns amongst racial ethnic groups has been reported to reflect diversities in prostate cancer incidence. One of the difficulties stems from the fact that serum hormone levels do not reflect the changes observed in prostate tissue androgen concentrations as the normal prostate progresses to a disease state. In this article efforts will be directed towards understanding some of the intra-prostate-specific mechanisms responsible for activating and/or repressing the androgen-dependent gene network associated with the gradual transition to a hormone refractive neoplastic state.     

Neuroendocrine differentiation in prostatic carcinoma during hormonal treatment

OBJECTIVES: Neuroendocrine differentiation (NED) is a common feature in adenocarcinoma of the prostate. Several studies suggest that NED may have a major impact on cancer progression as neuroendocrine (NE) secretory products have been shown to possess growth stimulatory effects. NED has also been proposed to constitute part of the mechanism by which a prostate cancer cell progresses toward androgen independence as NE tumor cells have been demonstrated to be devoid of androgen receptor immunoreactivity. In this retrospective study, we evaluated NED status in prostate cancer specimens from patients undergoing androgen ablation therapy. METHODS: The degree of NED in transurethral resection of the prostate (TURP) samples from 53 patients with prostate cancer was investigated by immunocytochemistry using polyclonal rabbit immunoglobin G (IgG) against chromogranin A (CgA). Changes in NED with time were determined by a manual semiquantitative cell counting method. RESULTS: During androgen withdrawal therapy, 21 tumors (40%) displayed increased NED concomitant with histopathologic tumor progression, whereas 29 carcinomas (55%) showed no change in NED status. However, a majority of the histopathologically unchanged tumors displayed marked NED at the first TURP and an increase in NED was by definition not possible. In only 3 cases (5%) was a decrease in NED observed with time. CONCLUSIONS: Androgen ablation therapy may be a contributing factor to the increase in NED of prostatic adenocarcinoma with time, and our findings imply that androgen withdrawal therapy enhances the selection and progression of NED, androgen-independent tumor cells.     

Suppression of caveolin expression induces androgen sensitivity in metastatic androgen-insensitive mouse prostate cancer cells

Although prostate cancer cells are often initially sensitive to androgen ablation, they eventually lose this response and continue to survive, grow and spread in the absence of androgenic steroids. The mechanism(s) that underlie resistance to androgen ablation therapy remain mostly unknown. We have demonstrated that elevated caveolin protein levels are associated with human prostate cancer progression in pathological specimens. Here we show that suppression of caveolin expression by a stably transfected antisense caveolin-1 cDNA vector converted androgen-insensitive metastatic mouse prostate cancer cells to an androgen-sensitive phenotype. Orthotopically grown tumors and low-density cell cultures derived from antisense caveolin clones had increased apoptosis in the absence of androgenic steroids, whereas similarly grown tumors and cells from vector (control) clones and parental cells were not sensitive to androgens. Studies using a representative antisense caveolin clone showed that selection for androgen resistance in vivo correlated with increased caveolin levels, and that adenovirus-mediated caveolin expression blocked androgen sensitivity. Our results identify a new candidate gene for hormone-resistant prostate cancer in man and indicate that androgen insensitivity can be an inherent property of metastatic prostate cancer.     

Regulation of prostate growth by fibroblast growth factors

Growth of the prostate is controlled by androgen. However, there is information indicating that androgen may not act directly, but may act indirectly through polypeptide growth factors, to control prostate growth. This review will focus on the involvement of members of the fibroblast growth factor (FGF) family in this process. The properties of FGFs and FGF-receptors are described that implicate these molecules in growth control. Information is provided that prostate stromal cells synthesize FGF2 and FGF7. FGF2 is a potent mitogen for stromal cells; whereas, FGF7 is exclusively a mitogen for epithelial cells. Transforming growth factor beta (TGF beta), also produced by prostate cells, inhibit cell growth. This suggests that prostate growth is controlled by autocrine and paracrine mechanisms. Evidence is presented that altered FGF expression accompanies benign prostatic hyperplasia and prostate cancer. A model is proposed whereby androgen regulates TGF beta, influencing FGF2 and FGF7 expression, and in turn regulating growth of the prostatic stroma and epithelium. An imbalance in the influence of these growth factors may contribute to prostate disease.     

Similar clinical outcomes in African-American and non-African-American males treated with suramin for metastatic prostate cancer

African-American males have a higher incidence of prostate cancer than non-African-American males and an overall poorer prognosis. Environmental factors such as socioeconomic status and biological factors such as an increased frequency of androgen receptor mutation have been identified as causal. As androgen ablation therapy is ubiquitous in the treatment of metastatic prostate cancer, little information is available on clinical outcome independent of hormone therapy. Our experience at the Warren G. Magnusson Clinical Center, National Institutes of Health with the anticancer agent, suramin, offers the opportunity to study clinical outcome in patients treated with an agent whose tumoricidal activity is not dependent on androgen receptor function. Clinical outcome was examined retrospectively in 43 patients treated on a single suramin-based protocol and evaluated as a function of ethnic background. No significant difference in time to disease progression or survival was observed between African Americans (n = 4) and the other 39 patients. These findings are consistent with the hypothesis that therapies that work through mechanisms independent of the androgen receptor may result in similar outcomes across ethnic groups.     

Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines

Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), for the functional activity of AR in LNCaP and PC-3 human prostate carcinoma cells. Our results are as follows. (i) Clone 33 LNCaP cells express AR, PAcP, and PSA, and cell growth is stimulated by 5alpha-dihydrotestosterone (DHT). Stimulation of cell growth correlates with decreased cellular PAcP activity. (ii) In clone 81 LNCaP cells, the expression of PAcP decreases with a concurrent decrease in the degree of androgen stimulation of cell growth, whereas the expression of PSA mRNA level is up-regulated by DHT, as in clone 33 cells. Conversely, in PAcP cDNA-transfected clone 81 cells, an additional expression of cellular PAcP correlates with an increased stimulation by androgen, higher than the corresponding control cells. (iii) PC-3 cells express a low level of functional AR with no detectable PAcP or PSA, and the growth of PC-3 cells is not affected by DHT treatment. Nevertheless, in two PAcP cDNA-transfected PC-3 sublines, the expression of exogenous cellular PAcP correlates with androgen stimulation. This androgen stimulation of cell growth concurs with an increased tyrosine phosphorylation of a phosphoprotein of 185 kDa. In summary, the data indicate that the expression of AR alone is not sufficient for androgen stimulation of cell growth. Furthermore, in AR-expressing prostate cancer cells, the expression of cellular PAcP correlates with androgen stimulation of cell proliferation.     

Inhibition of caspases by cytokine response modifier A blocks androgen ablation-mediated prostate cancer cell death in vivo

Androgen withdrawal is a major therapeutic modality in the treatment of prostate cancer. Although tumors initially respond, they subsequently relapse, and these recurring tumors are androgen independent. To examine possible mechanisms to explain the androgen independence of prostate cancer, we have expressed cytokine response modifier A (CrmA), a competitive inhibitor of caspases, interleukin 1beta-converting enzyme-like proteases, which mediate apoptotic cell death, in the human androgen-dependent prostate cancer cell line LNCaP. LNCaP cells require androgens for continuous growth in culture and to form tumors in nude mice. The expression of CrmA in LNCaP cells prevented the decreased growth rate induced by androgen withdrawal in tissue culture. When CrmA-expressing LNCaP (LNCaP-CrmA) cells were implanted s.c. in nude mice, the tumors grew six times faster than parental cells. Androgen ablation by castration before tumor implantation suppressed the ability of control LNCaP cells expressing nonfunctional CrmA mutant (R291T) to form tumors, but LNCaP-CrmA cells formed tumors similar in size to those formed in normal mice. When orchiectomy was performed 10 days after tumor implantation, control LNCaP cells expressing a nonfunctional CrmA mutant (R291T) regressed, but LNCaP-CrmA tumors continued to grow. Thus, inhibition of caspases prevents androgen withdrawal-induced prostate cancer cell death, suggesting that caspase activation is normally an important part of this process.     

Studies on the expression of fibroblast growth factors and fibroblast growth factor receptors in human prostate cancer

PURPOSE: We tried to clarify the role of fibroblast growth factors (FGFs) and those receptors (FGF-Rs) in cell proliferation of human prostate cancer. METHODS: The mRNA expression of FGF1, FGF2, FGF7, FGF-R1, FGF-R2 (IIIb), and FGF-R2 (IIIc) was investigated by RT-PCR in androgen sensitive cells (LNCaP), androgen-independent cells (PC3) and primary cultured stromal (PS) and epithelial cells (PE) from benign prostatic hyperplasia (BPH). Expression of the mRNA of FGF-R1, FGF-R2 (IIIb) and FGF-R2 (IIIc) in human prostate cancer tissue was similarly analyzed. Furthermore, the level of FGF-R1 expression in human prostate cancer was measured by semi-quantitative RT-PCR. RESULTS: FGF-R1 mRNA was detected in LNCaP, PC3 and the primary cultured stromal cells of BPH. FGF-R2 (IIIb) was seen in LNCaP cells and the primary cultured epithelial cells of BPH, while FGF-R2 (IIIc) was only observed in PC3. FGF1 mRNA was expressed in LNCaP and PC3, while FGF2 mRNA was in PC3 alone. The expression of FGF7 mRNA was detected only in the primary cultured stromal cells. Of 17 patients with human prostate cancer, FGF-R2 (IIIb) was detected in 2 and FGF-R2 (IIIc) in 15. Histological type of two cases having FGF-R2 (IIIb) were well differentiated adenocarcinoma. The mRNA levels of FGF-R1 in poorly and moderately differentiated types were significantly higher than those in well differentiated ones (p < 0.05). CONCLUSION: These findings suggest that several changes of expression in FGFs and FGF-Rs may correlate with malignant progression of human prostate cancer.     

Fourier-transform infrared conformational study of bovine insulin in surfactant solutions

BACKGROUND: bcl-2 protein is an oncoprotein encoded by a protooncogene bcl-2 which is thought to be a key gene for the regulation of cell life and death. bcl-2 protein has been reported to be expressed in a subset of prostate cancers, and its biological role in this disease has not yet been elucidated. This present study focused on the cell cycle analysis of bcl-2 immunoreactivity in prostate tumor samples and correlated findings with the prognosis of the patients. METHODS: Archival tissues from 37 prostate cancers were submitted for immunohistochemistry and DNA analysis via flow cytometry. All tissues were obtained before the patients received any treatment. Immunohistochemistry was performed using streptavidin biotin procedures, and DNA flow cytometry was done by a combination of two methods. RESULTS: Fourteen of 37 prostate cancers (38%) exhibited bcl-2 immunoreactivity. bcl-2 positive prostate cancers significantly correlated with DNA aneuploidy and higher proliferation indices, but not with histological differentiation. Clinically, there was a poorer prognosis for patients with bcl-2 positive prostate cancer. CONCLUSIONS: Our data indicates that bcl-2 protein expression in prostate cancer is associated with cell proliferation and may serve as a predictive factor for the prognosis of prostate cancer.     

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.     

Androgen metabolism and prostate cancer: establishing a model of genetic susceptibility

The prostate is an androgen-regulated organ, which has led to long-standing interest in the role of androgens in prostate carcinogenesis. Although evidence of a hormonal etiology for prostate cancer is strong, it is almost entirely circumstantial. Much of the problem in proving a causal relationship relates to the continued difficulties in reliably measuring human tissue-specific exposure to endogenous steroid hormones. The international and racial-ethnic variations in prostate cancer incidence, combined with the effects of migration on risk patterns, have suggested that whereas environmental factors are likely to be important, genetic factors might also play a central role in determining prostate cancer risk. We are developing a polygenic model of prostate carcinogenesis focused around a series of genes involved in androgen biosynthesis and androgen activation, transport, and metabolism in the prostate. In this developing model, we have initially targeted four genes based on three main criteria: (a) all encode products that play important roles in inducing androgen stimulation in the prostate; (b) all are polymorphic; and (c) all show substantial allelic variation in the polymorphic marker among the racial-ethnic groups of greatest interest in terms of prostate cancer risk. In addition to studying how the polymorphic markers of interest are related to prostate cancer development within and between racial-ethnic groups, we are concurrently evaluating whether genotypic variations correlate in the anticipated direction with biochemical parameters in vitro and in vivo. We summarize the development of this model and the state of knowledge related to each of the genes comprising the current model. We discuss the extent to which the current model can explain demographic variation in prostate cancer risk as well as the potential for future expansion of the model to incorporate environmental risk factors as well as additional genes. The model, when fully developed, can potentially provide a basis for targeting populations for screening interventions and/or preventive strategies aimed at the multigene products or at the genes themselves.     

Development of seven new human prostate tumor xenograft models and their histopathological characterization

Seven human prostate tumor models were established by transplanting tumor fragments in NMRI athymic nude mice. Once established, the tumors were serially transplantable in both NMRI and BALB/c nude mice. The xenografts originated from primary prostatic carcinomas (prostatectomy specimens), transurethral resection material, and metastatic lesions (pelvic lymph nodes and scrotal skin). Histological examination revealed that, in the course of several mouse passages (8 to 23), tumors retained their resemblance to the original patient material. The PC-295, PC-310, PC-329, and PC-346 tumors are dependent on androgens for their growth. The PC-324, PC-339, and PC-374 tumors are androgen independent, although growth of PC-374 tumors still seemed androgen sensitive. All tumors are diploid, except for the PC-374, which is tetraploid. The diploid PC-295 tumor has an additional small population of tetraploid cells. All xenografts displayed a heterogeneous expression pattern of the androgen receptor except for the PC-324 and PC-339 tumors in which the androgen receptor could not be detected. Prostatic acid phosphatase and prostate-specific antigen were retained during serial transplantation in all tumors but the PC-324 and PC-339. This panel of permanent human prostate tumor models comprises tumors representing both the androgen-dependent and -independent stages of human prostate cancer with various degrees of differentiation and, therefore, is of great value for the study of many aspects of growth and progression of human prostate cancer.