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Background Taxane treatment may be a suitable therapeutic option for patients with castration‐resistant prostate cancer and high expression of constitutively active androgen receptor variants (AR‐Vs). The aim of the study was to compare the effects of cabazitaxel and androgen deprivation treatments in a prostate tumor xenograft model expressing high levels of constitutively active AR‐V7. Furthermore, mechanisms behind acquired cabazitaxel resistance were explored. Methods Mice were subcutaneously inoculated with 22Rv1 cells and treated with surgical castration (n = 7), abiraterone (n = 9), cabazitaxel (n = 6), castration plus abiraterone (n = 8), castration plus cabazitaxel (n = 11), or vehicle and/or sham operation (n = 23). Tumor growth was followed for about 2 months or to a volume of approximately 1000 mm3. Two cabazitaxel resistant cell lines; 22Rv1‐CabR1 and 22Rv1‐CabR2, were established from xenografts relapsing during cabazitaxel treatment. Differential gene expression between the cabazitaxel resistant and control 22Rv1 cells was examined by whole‐genome expression array analysis followed by immunoblotting, immunohistochemistry, and functional pathway analysis. Results Abiraterone treatment alone or in combination with surgical castration had no major effect on 22Rv1 tumor growth, while cabazitaxel significantly delayed and in some cases totally abolished 22Rv1 tumor growth on its own and in combination with surgical castration. The cabazitaxel resistant cell lines; 22Rv1‐CabR1 and 22Rv1‐CabR2, both showed upregulation of the ATP‐binding cassette sub‐family B member 1 (ABCB1) efflux pump. Treatment with ABCB1 inhibitor elacridar completely restored susceptibility to cabazitaxel, while treatment with AR‐antagonists bicalutamide and enzalutamide partly restored susceptibility to cabazitaxel in both cell lines. The cholesterol biosynthesis pathway was induced in the 22Rv1‐CabR2 cell line, which was confirmed by reduced sensitivity to simvastatin treatment. Conclusions Cabazitaxel efficiently inhibits prostate cancer growth despite the high expression of constitutively active AR‐V7. Acquired cabazitaxel resistance involving overexpression of efflux transporter ABCB1 can be reverted by bicalutamide or enzalutamide treatment, indicating the great clinical potential for combined treatment with cabazitaxel and anti‐androgens.

BACKGROUND: The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR va ...

Our results argue for a role for IDO mediated immune suppression in the early stages of prostate cancer progression. However, since the intra-tumor IDO expression in J(-/-) mice was indistinguishab ...

The epidermal growth factor receptor (EGFR) mediates regulatory signals in the normal prostate, but the functional importance of this is unclear.Adult male rats were castrated, or castrated + treated with gefitinib (Iressa, ZD1839), an EGFR tyrosine kinase inhibitor, for 3 days. Seven-day castrated rats were treated with testosterone, or testosterone + gefitinib, for 3 days.Both castration alone and testosterone treatment in castrated animals increased the mRNA and protein levels of EGFR and phospho-EGFR in the ventral prostate. Inhibition of EGFR during castration and during testosterone-stimulated prostate growth resulted in a decrease in total epithelial weight, epithelial cell proliferation, endothelial cell proliferation, and increased epithelial cell apoptosis.This study suggests that increased EGFR signaling during castration mediates stimulatory effects balancing castration-induced prostate regression, and that EGFR signaling is a necessary component in testosterone-stimulated prostate growth.

Castration-induced involution of the normal prostate is caused by primary effects in the prostate stroma and vasculature, but if this is the case also in tumors is unknown.Androgen-independent AT-1 prostate tumor cells were therefore injected into the ventral prostate (VP) in Copenhagen rats. Seven days later when the growing tumor was surrounded by normal VP tissue the rats were castrated and the effect examined 3 and 7 days later.Castration reduced vascular density in the surrounding VP tissue and this was accompanied by tumor cell hypoxia, apoptosis, and temporarily retarded tumor growth. Castration-induced VP tissue regression occurred more rapidly in the contra-lateral than in the tumor-bearing lobe.Androgen-independent tumor cell respond to castration when growing in an androgen-dependent environment. The presence of a tumor influences the castration response in the surrounding normal tissue. The microenvironment determines how prostate epithelial cells respond to castration.

Castration rapidly reduces stroma insulin-like growth factor (IGF)-1 synthesis and action in mouse prostate epithelium. We explore if similar changes are of importance for castration-induced prostate regression in humans.Epithelial and surrounding stroma cells were micro-dissected from patient biopsies obtained before and shortly after castration. IGF-1 mRNA levels were quantified by RT-PCR and related to epithelial apoptosis and IGF-1, IGF-1 receptor, and androgen receptor (AR) immunoreactivity.IGF-1 mRNA was principally produced in the stroma and IGF-R1 in the epithelium. Stroma IGF-1 mRNA levels were significantly decreased after castration in non-malignant but not malignant tissue. Lack of stroma IGF-1 reduction after castration was associated with low stroma AR expression before therapy. Reduction of IGF-1 mRNA levels in the tumor stroma and/or epithelium was associated with epithelial apoptosis after therapy.Low AR expression and maintained stroma IGF-1 synthesis may result in limited tumor cell death after castration therapy.

Angiopoietin 2 expression is related to histological grade, vascular density, metastases, and outcome in prostate cancer.

BACKGROUND: Androgen ablation is the standard treatment for advanced prostate cancer but the short-term cellular effects are largely unknown. METHODS: Sextant prostate biopsies were taken from 77 p ...

Characterization of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) as a model to study effects of castration therapy.

BACKGROUND Endoglin, a receptor for some of the members of the transforming growth factor-β (TGF-β) family, is expressed on proliferating endothelial cells and has been suggested as a marker of ongoing angiogenesis. In this study, endoglin was evaluated as a prognostic factor for prostate cancer progression. METHODS Immunohistochemical staining of endoglin was examined in 72 cases of prostate cancer and compared with immunohistochemical staining of the pan-endothelial marker von Willebrand factor (vWf), clinicopathological factors, and cancer-specific survival. Micro-vessels were measured in the most vascularized fields. Double staining with antibodies against smooth muscle actin and endoglin or vWf, respectively, was performed in order to evaluate vessel maturation. RESULTS Endoglin-stained tumor vessels were generally small and only 19% also stained with actin. Endoglin was a better prognostic marker than vWf. The median survival times were shorter for patients with tumor vascular count (vc) above median than for patients with vc below median (4 vs. 12 years, P = 0.0007, and 5 vs. 10 years, P = 0.018, for endoglin and vWf, respectively). Endoglin vc was associated with Gleason score (P = 0.001), local tumor stage (P = 0.0006), metastasis (P = 0.01), tumor cell immunoreactivity for TGF-β1 (P = 0.0003), and tumor cell proliferation index (rs = 0.319, P = 0.02). Endoglin, in contrast to vWf, vc was prognostic for survival in the subgroup of patients with Gleason score 5, 6, and 7 tumors. CONCLUSIONS Endoglin marks principally small, probably newly formed tumor vessels in zthe prostate, and is a promising prognostic marker for prostate cancer patients. Prostate 51: 268–275, 2002. © 2002 Wiley-Liss, Inc.

BACKGROUND Inhibitory effects of gonadotropin-releasing hormone (GnRH) analogs on prostate cancer cell proliferation, both in vivo and in vitro, indicate the presence of specific binding sites for GnRH on prostate cancer cells. To investigate this issue further, we examined the expression of GnRH receptor (GnRH-R) mRNA and protein in human prostate biopsies as well as in other extrapituitary tissues. METHODS The relative quantity of GnRH-R mRNA was determined by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in human prostate biopsies. Extrapituitary GnRH-R levels were determined by a semiquantitative PCR reaction. RESULTS Using PCR, a relatively high expression level of GnRH-R mRNA was found in prostate tumor tissue followed by normal prostate, thymus, and kidney expression levels. The levels showed by heart, brain, placenta, lung, liver, skeletal muscle, pancreas, colon, ovary, small intestine, spleen, and testis were low but detectable, whereas peripheral blood leukocyte showed no demonstrable product. GnRH-R immunoreactivity was localized in both luminal and basal epithelial cells in benign and malignant prostate tissue, and GnRH-R were also observed in intraprostatic lymphocytes. The relative GnRH-R mRNA levels in prostate biopsies from 16 patients showed a wide range of individual differences, but these differences were not related to histological grade. Castration therapy did not significantly influence GnRH-R mRNA expression in normal and malignant prostate tissue. CONCLUSIONS These results suggest that epithelial cells and infiltrating lymphocytes are targets for GnRH action in the human prostate. Comparative data show relatively high GnRH-R expression in human prostate tissue compared to other human tissues. Prostate 47:276–284, 2001. © 2001 Wiley-Liss, Inc.

BACKGROUND Continuous administration of gonadotropin-releasing hormone (GnRH) agonists in prostate cancer patients results in involution of the tumors due to suppression of androgen production. In addition to the effect of GnRH at the hypothalamic-pituitary level, experiments in vitro on breast, ovary, and prostatic cells have shown an inhibition of cell proliferation, indicating the presence of local GnRH receptors (GnRH-R). The aim of the present study was to investigate the expression of GnRH-R mRNA in the normal rat ventral prostate (VP) and Dunning R3327 PAP adenocarcinoma and to evaluate the effects of castration on receptor mRNA expression. METHODS RNA was prepared from ovaries, pituitaries, VP, and Dunning tumors from both intact and castrated animals. GnRH-R mRNA levels were quantified by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS GnRH-R mRNA was detected in normal VP and Dunning tumors. Normal VP showed lower amounts of GnRH-R mRNA compared to Dunning tumors. An elevation of mRNA expression was observed 7 days after castration in Dunning tumors. CONCLUSIONS GnRH-R mRNA was found in both VP and Dunning tumors, indicating the presence of a local GnRH system. Normal VP showed lower amounts of GnRH-R mRNA when compared to malignant tissues. GnRH-R mRNA levels were elevated in Dunning tumors following castration. Prostate 39:101–107, 1999. © 1999 Wiley-Liss, Inc.

BACKGROUND. The mechanism behind castration-induced apoptosis in prostate cells is unknown, but data from other species suggest that transforming growth factor β1 (TGF-β1) may be involved. METHODS. By using quantitative RT-PCR and immunohistochemistry, expression of TGF-β1 and its receptors type I and II (RI and RII) was studied in normal and tumor areas of core biopsies taken before and 2-11 days after castration therapy. The TGF-β responses were related to changes in apoptotic index and to changes in serum prostate-specific antigen (PSA). RESULTS. In normal prostate tissue, apoptosis was generally increased by castration, and apoptosis was accompanied by an increase in TGF-β1 and RII mRNA levels (P < 0.05). In tumors, apoptosis was seen only in 44% of the cases and in these, but not in the others, TGF-β1, RI, and RII mRNA levels were increased (P < 0.05). In the patients showing a prognostically favorable PSA response (nadir PSA

BACKGROUND Prostate tumors express high levels of transforming growth factor-β1 (TGF-β1) and seem to acquire resistance to its antiproliferative effects with tumor progression. Moreover, TGF-β1 could be involved in tumor-promoting processes such as angiogenesis, cell migration, and immunosuppression. METHODS Immunoreactivity for TGF-β1 and its receptors type I and type II (TGFβ-RI and TGFβ-RII), tumor vascular count, and cell proliferation were studied in 73 cases of prostate cancer, diagnosed between 1975–1983 and followed with surveillance. RESULTS Patients with tumor overproduction of TGF-β1 had shorter median cancer-specific survival than patients with normal TGF-β1 immunoreactivity (5.0 vs. 10 years, P = 0.006). Furthermore, increased TGF-β1 staining was associated with tumor grade, high vascular counts, and metastasis (P = 0.02, 0.02, and 0.01, respectively). Patients with loss of tumor TGFβ-RII expression in combination with TGF-β1 overproduction showed particularly short survival (2.6 vs. 10 years, P = 0.0000), when compared to patients with normal immunoreactivity. CONCLUSIONS Overproduction of TGF-β1 and loss of TGFβ-RII expression are associated with poor clinical outcome in prostate cancer, and TGF-β1 may promote tumor progression by stimulating angiogenesis and metastasis. Prostate 37:19–29, 1998. © 1998 Wiley-Liss, Inc.

Indoleamine 2,3-dioxygenase (IDO) activity has been shown to be expressed in local lymph nodes and induce immune suppression of tumor immunity. Here we analyze the effect of IDO expression on prostate tumor growth using the transgenic adenocarcinoma of mouse prostate (TRAMP) animal model.Mice deficient in IDO expression were crossed to TRAMP mice and the time to the appearance of palpable tumors were measured. Immune histology was used to analyze the IDO expressing cells in tumors and in local lymph nodes. The levels of the substrate for IDO (tryptophane) and its product (kynurenine) was measured by HPLC.We found that systemic IDO activity, determined as the kynurenine/tryptophan ratio in serum, correlated with the presence of palpable tumor. Immunohistological analysis showed increased numbers of IDO expressing cells in local lymph nodes. In tumors, IDO expression could be detected in the tumor stroma by both CD31+ and CD31(-) cells. Essentially no CD45+, IDO expressing cells could be detected in the tumors. The influence of IDO activity on tumor progression was analyzed by back-crossing TRAMP mice with IDO(-/-) animals and J-chain negative (J(-/-)) mice that have perturbed IDO activity. In both crosses a delayed tumor incidence was observed.Our results argue for a role for IDO mediated immune suppression in the early stages of prostate cancer progression. However, since the intra-tumor IDO expression in J(-/-) mice was indistinguishable from that of C57BL/6 animals the IDO expression in the tumor tissue appears to be irrelevant for TRAMP tumor incidence.

BACKGROUND: The role of androgen receptors (ARs) in the prostate tumor cell environment is largely unknown. METHODS: AR immunostaining was evaluated in relation to stroma morphology, expression of AR co-activator ARA55, tumor characteristics and clinical outcome in normal and prostate cancer (PCa) tissue obtained at transurethral resection in men treated with expectancy, and in diagnostic transrectal core biopsies in men treated with surgical castration. Stroma composition was studied by Masson-trichrome and desmin staining. Levels of AR and ARA55 mRNA were quantified by laser micro-dissection and RT-PCR. RESULTS: The percentage of cells with positive nuclear AR immunostaining in the tumor and normal stroma was inversely related to Gleason score, tumor size, tumor stage, metastasis, response to castration therapy, and cancer-specific survival. The AR staining in the normal stroma provided independent prognostic information in Cox multiple linear regression analysis. Loss of stroma AR staining was linked to low expression of ARA55 in stroma smooth muscle cells, and in tumors also to gradual disappearance of this cell type. CONCLUSIONS: PCa aggressiveness and efficacy of castration therapy are related to AR levels in the tumor stroma and importantly to AR levels in the surrounding normal prostate tissue stroma. .

As changed paracrine support from androgen receptor (AR)-positive cells in the prostate stroma contribute to castration-induced glandular involution, we examined if the subsequent relapse to androgen-independent epithelial cell growth could be related to reactivation of AR signaling in the stroma.Human prostate tissue taken before, within 14 days, and at suspected local tumor relapse after surgical castration therapy was immunostained for AR.Castration initially decreased nuclear AR staining in epithelial and stroma cells, in both tumor and non-malignant tissue, but after some months, it reappeared.Local tumor relapse was associated with reappearance of nuclear AR not only in tumor epithelial cells but also in the tumor stroma. Reappearance of nuclear AR in non-malignant prostate cells may be a physiological response to long-term systemic androgen ablation that could influence tumor growth.

The mechanisms by which castration induces prostate involution are largely unknown.Early responses to castration in mouse ventral prostate (VP) were explored by quantitative microscopy, cDNA array expression, quantitative RT-PCR, and Western blot analysis. As several changes occurred in the insulin-like growth factor (IGF) system this was studied in more detail. Laser micro-dissection was used to localize sites of IGF-1 and IGF-1 receptor (IGF-R1) production. IGF-1 protein levels and IGF-R1 mediated signaling via insulin regulated substrate 1 and 2 (IRS-1 and 2) were examined. IGF-1 was injected into the VP in intact, and castrated mice and effects studied 1 day later.IGF-1 and IGF binding protein 2 (IGFBP-2) mRNA were rapidly reduced whereas IGFBP-3 and IGF-R1 mRNA were increased after castration. IGF-1 was principally produced in the stromal compartment, while IGF-R1 was produced in both epithelial and stromal cells. IGF-1 and IRS-1 protein levels were decreased 1 and 3 days after castration, respectively, while IRS-2 was unchanged. Inactivating phosphorylation of IRS-1 at serine 307 was increased 1 day after castration, and activating phosphorylation at tyrosine 612 was decreased 2 days later. These changes were accompanied by decreased cell proliferation and increased cell death in the glandular and vascular compartment. Local injection of IGF-1 increased vascular density and epithelial cell proliferation in intact mice, but had no effect in castrated animals.Decreased IGF-1 levels and action may mediate some of the key features of castration-induced prostate involution.

In order to learn more about short- and long-term effects of castration therapy, relevant model systems for prostate cancer are required. In this study, we examined whether the transgenic adenocarcinoma of the mouse prostate (TRAMP) tumor response to castration in C57BL/6 mice mimics that seen in patients.Transgenic animals were examined before and 3 days after castration, at the ages of 17, 24, and 36 weeks. Moreover, 24-weeks old animals were castrated and followed for 6 months. Immunohistochemistry (IHC) and stereology were used to evaluate epithelial cell proliferation and death, blood vessel volume, androgen receptor (AR) expression, and transgenic expression of SV40 large T.Cancer developed preferentially in the dorso-lateral prostate lobe. Tumor burden and incidence of metastases increased with age. The majority of tumors were well differentiated, while poorly differentiated, large tumors and macroscopic metastases developed in 8% of the animals. Well and moderately differentiated tumors responded to castration with cessation of proliferation and induction of apoptosis. Poorly differentiated tumors and metastases did not respond. Castration prevented local tumor growth for at least 6 months in 82% of the cases. Although, 45% of the treated animals developed wide-spread metastatic disease suggesting that castration may enhance growth of distant metastases.The C57Bl/6 TRAMP tumor in several ways mimics how prostate cancer in patients responds to castration both in the short and long term, but some differences may also exist. This model can preferably be used to elucidate how this treatment works, and to test how it can be improved by additional therapies.

Androgen ablation is the standard treatment for advanced prostate cancer but the short-term cellular effects are largely unknown.Sextant prostate biopsies were taken from 77 prostate cancer patients before and 1-10 days after castration treatment. Apoptosis, cell proliferation, and morphology were studied in malignant and non-malignant tissue, using stereological and immunohistochemical methods.Epithelial cell proliferation was significantly decreased both in non-malignant and malignant epithelium already 1 day after therapy. It remained low until day 7, but increased thereafter in the remaining non-malignant epithelial cells and in some tumors. Epithelial cell apoptosis was significantly increased during the first week and then returned to basal levels. The maximal apoptotic indexes, seven- and two-times the intact levels in the non-malignant and malignant glands, respectively, were found at days 3-4 or even earlier in the tumors. Signs of tumor shrinkage such as glandular collapse and decreased tumor cell size were observed from day 3 in most tumors.The present study shows that the magnitude and kinetics of the response to castration in the normal human prostate is very similar to the response previously described in rodents. We also demonstrate that most human prostate tumors rapidly respond to castration indicating the need for further evaluation of when and how to best monitor the effects of hormone ablation therapy in prostate cancer patients.

In several malignant tissues, the angiopoietins are principal regulators of vascular growth and regression, but in normal prostate and prostate tumors the role of the angiopoietins is unknown.Angiopoietin (ang) 1 and 2 were immunolocalized in TUR-diagnosed prostate tumors with long follow-up and the expression was related to vascular density and clinicopathological variables.Ang 1 was strongly expressed in the basal epithelial cell layer in non-malignant tissue, whereas tumors had lower levels localized to the epithelial cells. A weak ang 2 immunoreaction was observed in non-malignant tissue and in low to intermediate Gleason score (GS) tumors, with a similar expression pattern. However, most high GS tumors showed an intense ang 2 staining. Ang 2 was significantly correlated to GS, density of endoglin stained blood vessels, metastases, and to cancer specific survival.We conclude that ang 2 probably is an important regulator of angiogenesis in prostate cancer.

Endoglin, a receptor for some of the members of the transforming growth factor-beta (TGF-beta) family, is expressed on proliferating endothelial cells and has been suggested as a marker of ongoing angiogenesis. In this study, endoglin was evaluated as a prognostic factor for prostate cancer progression.Immunohistochemical staining of endoglin was examined in 72 cases of prostate cancer and compared with immunohistochemical staining of the pan-endothelial marker von Willebrand factor (vWf), clinicopathological factors, and cancer-specific survival. Micro-vessels were measured in the most vascularized fields. Double staining with antibodies against smooth muscle actin and endoglin or vWf, respectively, was performed in order to evaluate vessel maturation.Endoglin-stained tumor vessels were generally small and only 19% also stained with actin. Endoglin was a better prognostic marker than vWf. The median survival times were shorter for patients with tumor vascular count (vc) above median than for patients with vc below median (4 vs. 12 years, P = 0.0007, and 5 vs. 10 years, P = 0.018, for endoglin and vWf, respectively). Endoglin vc was associated with Gleason score (P = 0.001), local tumor stage (P = 0.0006), metastasis (P = 0.01), tumor cell immunoreactivity for TGF-beta1 (P = 0.0003), and tumor cell proliferation index (r(s) = 0.319, P = 0.02). Endoglin, in contrast to vWf, vc was prognostic for survival in the subgroup of patients with Gleason score 5, 6, and 7 tumors.Endoglin marks principally small, probably newly formed tumor vessels in zthe prostate, and is a promising prognostic marker for prostate cancer patients.

BACKGROUND: Angiogenesis is important for prostate organogenesis and prostate cancer progression. It is not yet known whether androgens promote part of their control of prostate structure and function by influencing angiogenesis. The aim of this study was to explore the possible androgenic regulation of the angiogenic factor vascular endothelial growth factor (VEGF) and its receptors flt-1 and flk-1/KDR in the rat ventral prostate (VP) and Dunning R3327 PAP adenocarcinoma.METHODS: RNA was prepared from VP and tumors of intact and castrated rats. VEGF, flt-1, and flk-1/KDR mRNA levels were determined using competitive RT-PCR.RESULTS: VEGF121, VEGF165, and VEGF189 together with flt-1 and flk-1/KDR mRNA were detected. The VEGF, but not flt-1 mRNA levels were significantly decreased in the VP after castration. The Dunning tumor expressed high levels of mRNA for VEGF and its receptors compared to the VP. The flt-1 mRNA level in the tumor increased after castration, while the VEGF mRNA levels were unchanged.CONCLUSIONS: Decreased mRNA expression of VEGF, but not flt-1, was found in the rat VP after castration. However, in the Dunning tumor, castration did not alter the expression of VEGF mRNA. Moreover, elevated levels of both mRNA for VEGF and its receptors relative to the VP were observed, indicating that the VEGF system may be important for Dunning tumor development.