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BACKGROUND Metastatic prostate cancer is a common and lethal disease for which there are no therapies that produce cures or long-term durable remissions. Clinically relevant preclinical models are needed to increase our understanding of biology of this malignancy and to evaluate new agents that might provide effective treatment. Our objective was to establish and characterize patient-derived xenografts (PDXs) from advanced prostate cancer (PC) for investigation of biology and evaluation of new treatment modalities. METHODS Samples of advanced PC obtained from primary prostate cancer obtained at surgery or from metastases collected at time of death were implanted into immunocompromised mice to establish PDXs. Established PDXs were propagated in vivo. Genomic, transcriptomic, and STR profiles were generated. Responses to androgen deprivation and docetaxel in vivo were characterized. RESULTS We established multiple PDXs (LuCaP series), which represent the major genomic and phenotypic features of the disease in humans, including amplification of androgen receptor, PTEN deletion, TP53 deletion and mutation, RB1 loss, TMPRSS2-ERG rearrangements, SPOP mutation, hypermutation due to MSH2/MSH6 genomic aberrations, and BRCA2 loss. The PDX models also exhibit variation in intra-tumoral androgen levels. Our in vivo results show heterogeneity of response to androgen deprivation and docetaxel, standard therapies for advanced PC, similar to the responses of patients to these treatments. CONCLUSIONS The LuCaP PDX series reflects the diverse molecular composition of human castration-resistant PC and allows for hypothesis-driven cause-and-effect studies of mechanisms underlying treatment response and resistance. Prostate. © 2017 Wiley Periodicals, Inc.

Background Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. Methods One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. Results Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. Conclusion This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the practicality of maintaining PDX models. Cryopreservation may also increase the accessibility of these important resources and provide new opportunities for preclinical studies on a broader spectrum of prostate tumors.

BACKGROUND The TMPRSS2-ERG gene fusion is detected in approximately half of primary prostate cancers (PCa) yet the prognostic significance remains unclear. We hypothesized that ERG promotes the expression of common genes in primary PCa and metastatic castration-resistant PCa (CRPC), with the objective of identifying ERG-associated pathways, which may promote the transition from primary PCa to CRPC. METHODS We constructed tissue microarrays (TMA) from 127 radical prostatectomy specimens, 20 LuCaP patient-derived xenografts (PDX), and 152 CRPC metastases obtained immediately at time of death. Nuclear ERG was assessed by immunohistochemistry (IHC). To characterize the molecular features of ERG-expressing PCa, a subset of IHC confirmed ERG+ or ERG− specimens including 11 radical prostatectomies, 20 LuCaP PDXs, and 45 CRPC metastases underwent gene expression analysis. Genes were ranked based on expression in primary PCa and CRPC. Common genes of interest were targeted for IHC analysis and expression compared with biochemical recurrence (BCR) status. RESULTS IHC revealed that 43% of primary PCa, 35% of the LuCaP PDXs, and 18% of the CRPC metastases were ERG+ (12 of 48 patients [25%] had at least one ERG+ metastasis). Based on gene expression data and previous literature, two proteins involved in calcium signaling (NCALD, CACNA1D), a protein involved in inflammation (HLA-DMB), CD3 positive immune cells, and a novel ERG-associated protein, DCLK1 were evaluated in primary PCa and CRPC metastases. In ERG+ primary PCa, a weak association was seen with NCALD and CACNA1D protein expression. HLA-DMB association with ERG was decreased and CD3 cell number association with ERG was changed from positive to negative in CRPC metastases compared to primary PCa. DCLK1 was upregulated at the protein level in unpaired ERG+ primary PCa and CRPC metastases (P = 0.0013 and P

BACKGROUND Despite multiple new therapies available to patients with advanced castration-resistant prostate cancer (CRPC), the overall survival benefit still remains relatively short. Therefore, it is important to investigate additional treatment options that could achieve greater efficacy. Because of tumor heterogeneity and the development of resistance to treatment with single agents, combination therapies using existing drugs with new agents can potentially broaden individual therapeutic windows and achieve improved efficacy and safety profiles. The objective of the current studies was to evaluate the efficacy of combination of enzalutamide (ENZ) with prostate specific membrane antigen antibody drug conjugate (PSMA ADC) to inhibit CRPC patient-derived xenografts (PDX) in a preclinical setting. METHODS Subcutaneous LuCaP 96CR prostate cancer PDX bearing mice were treated with a single dose of PSMA ADC (2.0 mg/kg) or 5 days a week ENZ (50 mg/kg) as monotherapy or with a combination of these two agents. The effects of the PSMA ADC+ENZ combination were compared to PSMA ADC alone, ENZ alone, and placebo control. IHC analyses were performed to determine PSMA, AR, ARV7, and GR expression and effects on proliferation. RESULTS All treatments inhibited tumor progression but with different efficacy. At 6 weeks, in the control and ENZ groups all tumors were progressing, while in the PSMA ADC group only 5/11 were progressing, two remained unchanged and four tumors had decreased tumor volume. Moreover, all animals in the PSMA ADC+ENZ group had smaller tumors at week 6 when compared to their size at enrollment (week 0). A 14-week followup showed that all three treatments resulted in significant survival benefits but the combination effects were the most pronounced resulting in PSMA ADC+ENZ versus ENZ HR = 0.093 (P = 0.0045) and PSMA ADC+ENZ versus PSMA ADC HR = 0.051 (P =

BACKGROUND It is timely and important to develop new treatment modalities for advanced prostate cancer, because even the newly FDA approved treatments, despite providing significant survival benefits, do not constitute cure of this disease. Antibody drug conjugates (ADCs) represent a promising approach to cancer therapy. Prostate-specific membrane antigen (PSMA) is expressed in advanced prostate cancer and targeting this protein is used for imaging of advanced prostate cancer as well as development of targeting strategies. The objective of our studies was to evaluate the efficacy of PSMA ADC against a series of patient-derived prostate cancer xenografts (LuCaP 58, LuCaP 77, LuCaP 96CR, and LuCaP 105) with different characteristics, including varying levels of PSMA expression and responses to androgen suppression. METHODS Mice bearing subcutaneous LuCaP prostate cancer-derived xenografts received PSMA antibody monomethyl auristatin E (MMAE) drug conjugate (PSMA ADC) in which the antibody and MMAE are linked via a protease-cleavable linker. PSMA ADC dose ranged from 1 to 6 mg/kg. Unmodified PSMA mAb + free MMAE at the amount equivalent to those contained in 6 mg/kg PSMA ADC was used as control. All treatments were administered once a week via tail-vein injections and repeated four times once a week and tumor responses were monitored for 10 weeks. IHC analyses were performed to determine PSMA and AR expression and effects on proliferation. RESULTS Treatment responses varied widely across the tumor models, from complete tumor regressions in LuCaP 96CR to largely unimpeded tumor progression of LuCaP 58, which had the lowest baseline level of PSMA expression. Intermediate antitumor effects were seen for LuCaP 77 and LuCaP 105 tumors, despite their having similar basal expression of PSMA as LuCaP 96CR. Interestingly, we detected substantial differences in responses even within the same model, indicating that PSMA expression is not the only factor involved in treatment outcomes. CONCLUSIONS Our results show high efficacy of PSMA ADC in advanced prostate cancer but also considerable variability in effects despite PSMA expression. Further studies to identify tumor characteristics that are predictive of treatment response are ongoing. Prostate 75:303–313, 2015. © 2014 Wiley Periodicals, Inc.

BACKGROUND Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have been described and marketed in the past decades. Unfortunately, some of these mAbs are poorly characterized, which might lead to their inappropriate use and misinterpretation of the acquired results. METHODS We collected the 13 most frequently used mAbs against GCPII and quantitatively characterized their binding to GCPII by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Using a peptide library, we mapped epitopes recognized by a given mAb. Finally, we assessed the applicability of these mAbs to routine experimental setups, including Western blotting, immunohistochemistry, and flow cytometry. RESULTS ELISA and SPR analyses revealed that mAbs J591, J415, D2B, 107-1A4, GCP-05, and 2G7 bind preferentially to GCPII in native form, while mAbs YPSMA-1, YPSMA-2, GCP-02, GCP-04, and 3E6 bind solely to denatured GCPII. mAbs 24.4E6 and 7E11-C5.3 recognize both forms of GCPII. Additionally, we determined that GCP-02 and 3E6 cross-react with mouse GCPII, while GCP-04 recognizes GCPII and GCPIII proteins from both human and mouse. CONCLUSION This comparative analysis provides the first detailed quantitative characterization of the most commonly used mAbs against GCPII and can serve as a guideline for the scientific community to use them in a proper and efficient way. Prostate 74: 1674–1690, 2014. © 2014 Wiley Periodicals, Inc.

BACKGROUND Approximately 90% of patients who die of Prostate Cancer (PCa) have bone metastases, which promote a spectrum of osteoblastic, osteolytic or mixed bone responses. Numerous secreted proteins have been reported to promote osteoblastic or osteolytic bone responses. We determined whether previously identified and/or novel proteins were associated with the osteoblastic or osteolytic response in clinical specimens of PCa bone metastases. METHODS Gene expression was analyzed on 14 PCa metastases from 11 patients by microarray profiling and qRT-PCR, and protein expression was analyzed on 33 PCa metastases from 30 patients by immunohistochemistry on highly osteoblastic and highly osteolytic bone specimens. RESULTS Transcript and protein levels of BMP-2, BMP-7, DKK-1, ET-1, and Sclerostin were not significantly different between osteoblastic and osteolytic metastases. However, levels of OPG, PGK1, and Substance P proteins were increased in osteoblastic samples. In addition, Emu1, MMP-12, and sFRP-1 were proteins identified with a novel role of being associated with either the osteoblastic or osteolytic bone response. CONCLUSIONS This is the first detailed analysis of bone remodeling proteins in human specimens of PCa bone metastases. Three proteins not previously shown to be involved may have a role in the PCa bone response. Furthermore, our data suggests that the relative expression of numerous, rather than a single, bone remodeling proteins determine the bone response in PCa bone metastases. Prostate 73: 932–940, 2013. © 2013 Wiley Periodicals, Inc.

BACKGROUND A number of minimally invasive technologies exist for the treatment of prostate cancer (CaP), each with their associated morbidities. We sought to test the efficacy of low dose alternating electric current (LDAEC) to inhibit CaP growth in a preclinical setting and determine its effect on normal tissue. METHODS In the first study, two power settings, 15 or 25 mA of current, and two treatment times, 15 or 60 min, were evaluated in C4-2B CaP xenografts. In the second study, power was regulated to maintain an intra-tumoral temperature of ≤45°C in C4-2B and LuCaP 35 tumors. In both studies, tumor volume, serum PSA levels, survival and histology were analyzed. In a third study, LDAEC was applied to mice hamstrings with evaluation of gait and histology. RESULTS The most effective tumor volume reduction in the first study was seen with tumors treated with 25 mA for 15 min (62 ± 9.4% decrease, P = 0.001). Longer treatment time did not enhance treatment effect. Using 45°C to govern delivery of LDAEC resulted in a near 100% reduction in tumor volume in 8/10 mice with C4-2B tumors (P

Background Prostate cancer (PCa) has a propensity to metastasize to bone. Tumor cells replace bone marrow and can elicit an osteoblastic, osteolytic, or mixed bone response. Our objective was to elucidate the mechanisms and key factors involved in promoting osteoclastogenesis in PCa bone metastasis.

The TMPRSS2-ERG gene fusion is detected in approximately half of primary prostate cancers (PCa) yet the prognostic significance remains unclear. We hypothesized that ERG promotes the expression of common genes in primary PCa and metastatic castration-resistant PCa (CRPC), with the objective of identifying ERG-associated pathways, which may promote the transition from primary PCa to CRPC.We constructed tissue microarrays (TMA) from 127 radical prostatectomy specimens, 20 LuCaP patient-derived xenografts (PDX), and 152 CRPC metastases obtained immediately at time of death. Nuclear ERG was assessed by immunohistochemistry (IHC). To characterize the molecular features of ERG-expressing PCa, a subset of IHC confirmed ERG+ or ERG- specimens including 11 radical prostatectomies, 20 LuCaP PDXs, and 45 CRPC metastases underwent gene expression analysis. Genes were ranked based on expression in primary PCa and CRPC. Common genes of interest were targeted for IHC analysis and expression compared with biochemical recurrence (BCR) status.IHC revealed that 43% of primary PCa, 35% of the LuCaP PDXs, and 18% of the CRPC metastases were ERG+ (12 of 48 patients [25%] had at least one ERG+ metastasis). Based on gene expression data and previous literature, two proteins involved in calcium signaling (NCALD, CACNA1D), a protein involved in inflammation (HLA-DMB), CD3 positive immune cells, and a novel ERG-associated protein, DCLK1 were evaluated in primary PCa and CRPC metastases. In ERG+ primary PCa, a weak association was seen with NCALD and CACNA1D protein expression. HLA-DMB association with ERG was decreased and CD3 cell number association with ERG was changed from positive to negative in CRPC metastases compared to primary PCa. DCLK1 was upregulated at the protein level in unpaired ERG+ primary PCa and CRPC metastases (P = 0.0013 and P 0.0001, respectively). In primary PCa, ERG status or expression of targeted proteins was not associated with BCR-free survival. However, for primary PCa, ERG+DCLK1+ patients exhibited shorter time to BCR (P = 0.06) compared with ERG+DCLK1- patients.This study examined ERG expression in primary PCa and CRPC. We have identified altered levels of inflammatory mediators associated with ERG expression. We determined expression of DCLK1 correlates with ERG expression and may play a role in primary PCa progression to metastatic CPRC. Prostate 76:810-822, 2016. © 2016 Wiley Periodicals, Inc.

Despite multiple new therapies available to patients with advanced castration-resistant prostate cancer (CRPC), the overall survival benefit still remains relatively short. Therefore, it is important to investigate additional treatment options that could achieve greater efficacy. Because of tumor heterogeneity and the development of resistance to treatment with single agents, combination therapies using existing drugs with new agents can potentially broaden individual therapeutic windows and achieve improved efficacy and safety profiles. The objective of the current studies was to evaluate the efficacy of combination of enzalutamide (ENZ) with prostate specific membrane antigen antibody drug conjugate (PSMA ADC) to inhibit CRPC patient-derived xenografts (PDX) in a preclinical setting.Subcutaneous LuCaP 96CR prostate cancer PDX bearing mice were treated with a single dose of PSMA ADC (2.0 mg/kg) or 5 days a week ENZ (50 mg/kg) as monotherapy or with a combination of these two agents. The effects of the PSMA ADC+ENZ combination were compared to PSMA ADC alone, ENZ alone, and placebo control. IHC analyses were performed to determine PSMA, AR, ARV7, and GR expression and effects on proliferation.All treatments inhibited tumor progression but with different efficacy. At 6 weeks, in the control and ENZ groups all tumors were progressing, while in the PSMA ADC group only 5/11 were progressing, two remained unchanged and four tumors had decreased tumor volume. Moreover, all animals in the PSMA ADC+ENZ group had smaller tumors at week 6 when compared to their size at enrollment (week 0). A 14-week followup showed that all three treatments resulted in significant survival benefits but the combination effects were the most pronounced resulting in PSMA ADC+ENZ versus ENZ HR = 0.093 (P = 0.0045) and PSMA ADC+ENZ versus PSMA ADC HR = 0.051 (P = 0.0001) with no deaths observed in the combination group.Our results clearly indicate that the combination of PSMA ADC+ENZ possesses strong antitumor activity and significantly improves survival over ENZ monotherapy using the LuCaP 96CR PDX model. These results provide a strong rationale for clinical testing of PSMA ADC in combination with ENZ and/or other androgen-directed treatment strategies.

BACKGROUND Spontaneous metastasis following implantation at the orthotopic site is a highly desired feature in prostate cancer (CaP) models, since it would enable studies of mechanisms associated with tumor cell dissemination. METHODS LuCaP 23.8 and LuCaP 35, hormone-sensitive CaP xenografts that express PSA and the wild-type androgen receptor, were grown orthotopically in SCID mice. When tumor volumes reached ∼250–500 mg, primary tumors were removed to allow micrometastases to grow. RESULTS Using this procedure we generated macroscopic metastases (>20 mg) in 71% (LuCaP 23.8) and 100% (LuCaP 35) of animals, respectively. CONCLUSIONS These models may be used to evaluate new treatment modalities and study mechanisms associated with development of metastases. Prostate 56: 110–114, 2003. © 2003 Wiley-Liss, Inc.

Background Generation of suitable in vivo models is critical for understanding of processes associated with development and progression of prostate cancer (CaP). Methods Lymph nodes containing metastatic androgen-independent CaP were implanted into athymic mice. A xenograft designated LuCaP 35 and its hormone-independent variant LuCaP 35V were established and characterized. Results LuCaP 35 is an androgen-sensitive, prostate-specific antigen (PSA)-producing xenograft. It expresses the wild-type androgen receptor and exhibits deletions in chromosome 8p, but not in chromosome 10. The response of LuCaP 35 to androgen ablation is similar to that observed in man. Using recurring LuCaP 35 tumors we have also established an androgen-insensitive variant of LuCaP 35. Conclusions The availability of hormone-dependent and -independent variants of LuCaP 35, which exhibit many properties analogous to those of CaP in man, provides an excellent model system to study the processes associated with development of androgen independence and to evaluate new treatment modalities. Prostate 55: 239–246, 2003. © 2003 Wiley-Liss, Inc.

BACKGROUND To improve the therapy of advanced prostate cancer (CaP), it is critical to develop animal models that mimic CaP bone metastases. Unlike the human disease, CaP xenograft models rarely metastasize spontaneously to bone from the orthotopic site of primary tumor growth. METHODS Single-cell suspensions of LNCaP, PC-3, LuCaP 35, and LuCaP 23.1 CaP cells were injected directly into tibia of SCID mice. Immunohistochemistry and bone histomorphometrical analyses were performed to characterize these osseous-CaP models. RESULTS LuCaP 23.1 yields an osteoblastic response, LNCaP yields mixed lesions, and LuCaP 35 and PC-3 result in osteolytic responses. We have detected osteoprotegerin, RANK ligand, parathyroid hormone-related protein, and endothelin-1, proteins associated with bone growth and remodeling, in the CaP cells grown in the bone. CONCLUSIONS These animal models can be used to study biological interactions, pathways, and potential therapeutic targets, and also to evaluate new agents for treatment and prevention of CaP bone metastasis. Prostate 52: 20–33, 2002. © 2002 Wiley-Liss, Inc.

It is timely and important to develop new treatment modalities for advanced prostate cancer, because even the newly FDA approved treatments, despite providing significant survival benefits, do not constitute cure of this disease. Antibody drug conjugates (ADCs) represent a promising approach to cancer therapy. Prostate-specific membrane antigen (PSMA) is expressed in advanced prostate cancer and targeting this protein is used for imaging of advanced prostate cancer as well as development of targeting strategies. The objective of our studies was to evaluate the efficacy of PSMA ADC against a series of patient-derived prostate cancer xenografts (LuCaP 58, LuCaP 77, LuCaP 96CR, and LuCaP 105) with different characteristics, including varying levels of PSMA expression and responses to androgen suppression.Mice bearing subcutaneous LuCaP prostate cancer-derived xenografts received PSMA antibody monomethyl auristatin E (MMAE) drug conjugate (PSMA ADC) in which the antibody and MMAE are linked via a protease-cleavable linker. PSMA ADC dose ranged from 1 to 6 mg/kg. Unmodified PSMA mAb + free MMAE at the amount equivalent to those contained in 6 mg/kg PSMA ADC was used as control. All treatments were administered once a week via tail-vein injections and repeated four times once a week and tumor responses were monitored for 10 weeks. IHC analyses were performed to determine PSMA and AR expression and effects on proliferation.Treatment responses varied widely across the tumor models, from complete tumor regressions in LuCaP 96CR to largely unimpeded tumor progression of LuCaP 58, which had the lowest baseline level of PSMA expression. Intermediate antitumor effects were seen for LuCaP 77 and LuCaP 105 tumors, despite their having similar basal expression of PSMA as LuCaP 96CR. Interestingly, we detected substantial differences in responses even within the same model, indicating that PSMA expression is not the only factor involved in treatment outcomes.Our results show high efficacy of PSMA ADC in advanced prostate cancer but also considerable variability in effects despite PSMA expression. Further studies to identify tumor characteristics that are predictive of treatment response are ongoing.

Background Our objective was to evaluate five preclinical prostate cancer (CaP) xenograft models to determine whether (1) prostate-specific antigen (PSA) formed complexes in murine serum, (2) the percentage of free PSA (f-PSA) was characteristic of a given xenograft line, and (3) the percentage of f-PSA was similar to that in the patient at time of tumor harvest. Our fourth objective was to identify which murine serpin(s) bind(s) to PSA in vivo. Methods Xenografts were established from metastatic foci. The percentage of f-PSA, and total PSA (t-PSA) in serum of animals bearing CaP xenografts was determined by immunoassay. Size exclusion high-performance liquid chromatography and Western blots were used to evaluate the presence of PSA complexes in murine serum. Edman degradation was used to determine the N-terminal sequence of complexed proteins. Results PSA was detected as both free and complexed forms in murine serum from all mice bearing the CaP xenografts. Three xenografts (related sublines) produced PSA that resulted in low mean percentages of f-PSA (1.9-6.4%). In sera from the other two xenografts, the mean percentages of f-PSA were high (>25%); patient sera, where available at time of tumor acquisition, were in agreement. Western blots showed that murine protease inhibitors formed complexes with PSA. Edman degradation yielded a sequence with 80% homology over 15 amino acids with that of murine alpha1-protease inhibitor (alpha1-PI). Conclusions Our data have shown that the majority of PSA secreted by these CaP xenografts complexes in murine serum with a protease inhibitor with high homology to murine alpha1-PI and that the percentage of f-PSA is a characteristic of each xenograft line tested, which is in agreement with patient values at time of tumor harvest. These CaP xenografts offer opportunities for study of human PSA biology and phenomenology.

BACKGROUND Our objective was to investigate the presence of prostate specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) mRNA and protein in prostate cancer cell lines, and the complexing characteristics of expressed PSA. METHODS RT-PCR, immunohistochemistry, and Western blots were employed. Trypsin treatment of PSA was performed to establish the possible presence of an activatable form of PSA. RESULTS ACT mRNA and protein were detected in LNCaP, PC-3, and DU 145 by RT-PCR and by immunohistochemistry, respectively. Only LNCaP cells were positive for PSA mRNA and protein. LNCaP expressed approximately 30% active PSA, approximately 40% putative zymogen form of PSA, and approximately 30% stably inactive PSA. CONCLUSIONS We have shown that the majority of PSA expressed by LNCaP cells is present in free, noncomplexed forms in the conditioned media. A portion (40%) can be activated by trypsin, while the rest is stably inactive PSA. LNCaP cells may serve as a source of the "unreactive" PSA present in prostate cancer patients' serum.

BACKGROUND Little is known about the genetic events in the malignant transformation of prostatic cells. This is due in large measure to the cellular heterogeneity of the prostate. METHODS An amplification method was devised to synthesize cDNA from small samples of cancer and benign tissues of the same resected glands. Differential gene expression of candidate informative markers between cancer and benign was screened by the polymerase chain reaction with gene-specific oligonucleotide primers. RESULTS The expression of a transcription factor, ETS-2, was shown to be elevated in some cancer specimens. Elevated expression was also noted for neuron-specific enolase (NSE) and another transcription factor, SEF2. CONCLUSIONS Our method can be used to identify quickly genes that are differentially expressed between benign and cancerous prostate cells. Transcription factors, such as ETS-2, may play a significant role in cancer progression. Prostate 30:145–153, 1997. © 1997 Wiley-Liss, Inc.

Approximately 90% of patients who die of Prostate Cancer (PCa) have bone metastases, which promote a spectrum of osteoblastic, osteolytic or mixed bone responses. Numerous secreted proteins have been reported to promote osteoblastic or osteolytic bone responses. We determined whether previously identified and/or novel proteins were associated with the osteoblastic or osteolytic response in clinical specimens of PCa bone metastases.Gene expression was analyzed on 14 PCa metastases from 11 patients by microarray profiling and qRT-PCR, and protein expression was analyzed on 33 PCa metastases from 30 patients by immunohistochemistry on highly osteoblastic and highly osteolytic bone specimens.Transcript and protein levels of BMP-2, BMP-7, DKK-1, ET-1, and Sclerostin were not significantly different between osteoblastic and osteolytic metastases. However, levels of OPG, PGK1, and Substance P proteins were increased in osteoblastic samples. In addition, Emu1, MMP-12, and sFRP-1 were proteins identified with a novel role of being associated with either the osteoblastic or osteolytic bone response.This is the first detailed analysis of bone remodeling proteins in human specimens of PCa bone metastases. Three proteins not previously shown to be involved may have a role in the PCa bone response. Furthermore, our data suggests that the relative expression of numerous, rather than a single, bone remodeling proteins determine the bone response in PCa bone metastases.

Background—Angiopoietin-2 is expressed in prostate cancer (PCa) bone, liver, and lymph node metastases, whereas, its competitor angiopoietin-1 has limited expression in these tissues. Therefore, we hypothesized that the inhibition of angiopoietin-2 activity in PCa metastasis (removed) will impede angiogenesis, tumor growth and alter bone response in vivo. Methods—To test our hypothesis we used L1–10, a peptide-Fc fusion that inhibits interactions between angiopoietin-2 and its receptor tie2. We blocked angiopoietin-2 activity using L1–10 in established subcutaneous and intra-tibial LuCaP 23.1 xenografts. We then determined the effect of L1–10 on survival, tumor growth, serum PSA, proliferation, microvessel density, and angiogenesis-associated gene expression in subcutaneous tumors. We also determined serum PSA, tumor area and bone response in intra-tibial tumors. Results—The administration of L1–10 decreased tumor volume and serum PSA, and increased survival in SCID mice bearing subcutaneous LuCaP 23.1 tumors. Histomorphometric analysis, showed a further significant decrease in tumor epithelial area within the L1–10-treated LuCaP 23.1 subcutaneous tumors (p=0.0063). There was also a significant decrease in cell proliferation (p=0.012), microvessel density (p=0.012), and a significant increase in ANGPT-2 and HIF-1α mRNA expression (p≤0.05) associated with L1–10 treatment. Alternatively, in LuCaP 23.1 intratibial tumors L1–10 treatment did not significantly change serum PSA, tumor area or bone response. Conclusions—Our results demonstrate that inhibiting angiopoietin-2 activity impedes angiogenesis and growth of LuCaP 23.1 PCa xenografts. Based on these data, we hypothesize that angiopoietin-2 inhibition in combination with other therapies may represent a potential therapy for patients with metastatic disease.

A number of minimally invasive technologies exist for the treatment of prostate cancer (CaP), each with their associated morbidities. We sought to test the efficacy of low dose alternating electric current (LDAEC) to inhibit CaP growth in a preclinical setting and determine its effect on normal tissue.In the first study, two power settings, 15 or 25 mA of current, and two treatment times, 15 or 60 min, were evaluated in C4-2B CaP xenografts. In the second study, power was regulated to maintain an intra-tumoral temperature ofor=45 degrees C in C4-2B and LuCaP 35 tumors. In both studies, tumor volume, serum PSA levels, survival and histology were analyzed. In a third study, LDAEC was applied to mice hamstrings with evaluation of gait and histology.The most effective tumor volume reduction in the first study was seen with tumors treated with 25 mA for 15 min (62 +/- 9.4% decrease, P = 0.001). Longer treatment time did not enhance treatment effect. Using 45 degrees C to govern delivery of LDAEC resulted in a near 100% reduction in tumor volume in 8/10 mice with C4-2B tumors (P0.001) with similar inhibition of LuCaP 35 tumors (P = 0.01). This treatment, although resulting in skeletal muscle necrosis, did not affect nerves, smooth muscle and blood vessels.LDAEC demonstrates efficacy against C4-2B and LuCaP 35 CaP xenografts while causing no harm to nerves and blood vessels. These results warrant further investigations into the use of LDAEC as a treatment for CaP.

Prostate cancer (PCa) has a propensity to metastasize to bone. Tumor cells replace bone marrow and can elicit an osteoblastic, osteolytic, or mixed bone response. Our objective was to elucidate the mechanisms and key factors involved in promoting osteoclastogenesis in PCa bone metastasis.We cultured osteoblast-like MC3T3-E1 cells with conditioned medium (CM) from PC-3 and C4-2B cells. MC3T3-E1 mineralization decreased in the presence of PC-3 CM, whereas C4-2B CM had no effect on mineralization. Using oligo arrays and validating by real-time PCR, we observed a decrease in the expression of mineralization-associated genes in MC3T3-E1 cells grown in the presence of PC-3 CM. In addition, PC-3 CM induced the expression of osteoclastogenesis-associated genes IGFBP-5, IL-6, MCP-1, and RANKL while decreasing OPG expression in MC3T3-E1 cells. Furthermore, CM from MC3T3-E1 cells cultured in the presence of PC-3 CM, in association with soluble RANKL, increased osteoclastogenesis in RAW 264.7 cells. Investigation of PCa metastases and xenografts by immunohistochemistry revealed that the osteoclastic factor IL-6 was expressed in the majority of PCa bone metastases and to a lesser extent in PCa soft tissue metastases. In vitro it was determined that soluble IL-6R (sIL-6R) was necessary for IL-6 to inhibit mineralization in MC3T3-E1 cells.PC-3 cells inhibit osteoblast activity and induce osteoblasts to produce osteoclastic factors that promote osteoclastogenesis, and one of these factors, IL-6, is highly expressed in PCa bone metastases.IL-6 may have an important role in promoting osteoclastogenesis in PCa bone metastasis through its' interaction with sIL-6R.

BACKGROUND The mitogen-activated protein kinases (MAPKs) regulate cell growth, differentiation, and stress responses, and many critical signaling pathways are subject to cross-regulation by MAPK signaling. Previous studies have yielded evidence of cross-talk between the MAPK pathways and androgen receptor (AR) signaling, which plays a critical role in growth control of both normal prostate and prostate cancer (PCa). Objective of this study was to evaluate the expression of MAPK-like protein nemo-like kinase (NLK) in PCa and its effects on AR-mediated transcription. METHODS Real-time PCR and IHC were used to evaluate levels of NLK in prostatic samples. Effects of over-expression of NLK on apoptosis and proliferation were determined using Western blot and flow cytometry. Effects on AR signaling were evaluated using over-expression and knockdown of NLK in PCa cells in combination with PCR, Western blotting and reporter assays. RESULTS Our results show that the expression of NLK is decreased in PCa metastases in comparison to normal prostate epithelium and primary PCa. Our results also show that over-expression of NLK resulted in induction of apoptosis, which was more pronounced in AR-expressing LNCaP versus AR-negative PC-3 cells. Higher levels of NLK decreased levels of AR mRNA and protein as well as inhibited AR-mediated transcription. CONCLUSIONS NLK expression is altered during PCa progression and it is involved in regulation of AR signaling in these cells. A deeper understanding of the roles of NLK in regulation of AR-mediated transcription and control of PCa progression may point the way to new modes of therapeutic intervention in this disease. Prostate 69: 1481–1492, 2009. © 2009 Wiley-Liss, Inc.

Introduction mTOR activity is increased in advanced prostate cancer (CaP) as a result of a high rate of PTEN mutations. RAD001 (Everolimus) is a new orally available mTOR inhibitor. The objective of our study was to evaluate the effects of RAD001 on the growth of CaP in the bone, both alone and in combination with docetaxel and zoledronic acid. Methods C4-2 CaP cells were injected into tibiae of mice and the animals were treated with RAD001, docetaxel, and zoledronic acid alone or in combination. Histomorphometrical analysis, serum PSA measurements, bone mineral density (BMD), and µCT were used to determine the effects of treatment on tumor and bone. Results All three agents alone decreased tumor volume, and RAD001 and docetaxel also decreased levels of serum PSA by 68% and 65%, respectively (both P

BACKGROUND. Prostate cancer (CaP) bone metastases express numerous proteins associated with bone cells. Specific transcription factors, including Runx2, regulate the expressionofmanybone-relatedfactorsinosteoblasts.Expressionofthesetranscriptionfactors in CaP may be linked to the ability of CaP bone metastases to influence bone remodeling. METHODS. CaP tissues and cell lines were analyzed for expression of Runx2 mRNA by RT-PCR and in situ hybridization, and protein by immunohistochemistry, Western blotting, and electrophoretic mobility shift assays (EMSA). RESULTS. Runx2 mRNA and protein were detected in CaP tissues and cell lines. A specific Runx2: OSE2 complex could be formed with PC-3 nuclear extracts. CONCLUSIONS. Expression of Runx2 in CaP may be the molecular switch that is associated with expression of various bone-specific factors in CaP. In turn, expression of these factors can influence bone remodeling and possibly play a role in the growth and survival of CaP in bone. Prostate 56: 13–22, 2003. # 2003 Wiley-Liss, Inc.