Castrated Male Mice Research Articles

Inhibition of estrogen biosynthesis enhances lymphoma growth in mice.

Most lymphomas show higher incidence and poorer prognosis in males compared to females. However, the endocrine contribution to this gender difference is not entirely known. Here we show that castration accelerates lymphoma growth in C57BL6 male mice grafted with murine EG7 T cell lymphoma cells. However, the androgen receptor antagonist Bicalutamide did not affect lymphoma growth, suggesting no impact of androgen receptor signaling on lymphoma progression. In contrast, inhibition of androgen-to-estrogen conversion by the aromatase inhibitor (AI) Letrozole induced faster lymphoma growth in mice, suggesting that androgens impact lymphoma growth through its conversion to estrogens. This was supported by the inability of dihydrotestosterone, which is not converted to estrogens by aromatase, to influence lymphoma growth in castrated male mice. Lymphoma growth was also stimulated in immunocompromised mice grafted with human B cell lymphoma (Granta-519) and treated with either reversible or irreversible AIs, showing that the blockage of estrogen synthesis caused enhanced growth of both murine T and human B cell lymphomas and with different AIs. Additionally, AI-treated EG7 lymphomas showed accelerated growth not only in male but also in intact female mice. Altogether, our results demonstrate that aromatase inhibition accelerates lymphoma growth but not androgens per se, highlighting a protective role of estrogens in lymphoma pathogenesis. These results also raise concern that the use of AIs in women with breast cancer might enhance lymphoma progression.

Dysfunction of tear film and ultrastructural changes of corneal epithelial cells in castrated male mice

Background Studies indicate that the decline of androgen level affects the secretion function of epithelial cells of meibomian glands and lacrimal glands, which results in the abnormality of tear quality and quantity. However, whether androgen level regulates the mucin adhesion to ocular surface is still not elucidated. Objective This study was to investigate the tear functional changes and corneal ultrastructural changes in castrated male mice. Methods Fifty-six SPF male BALB/c mice were devided into normal control group, sham group, and orchectomy 1-week group, orchectomy 2-week group, orchectomy 4-week group, orchectomy 6-week group, orchectomy 8-week group according to random number table. Orchectomy was performed in the mice of the orchectomy 1-, 2-, 4-, 6-and 8-week group, and only incision rather than enucleation of testis was carried out in the mice of the sham group. Peripheral venous blood samples were collected during eyeball enucleation in all the mice and serum androgen concentration was detected by radioimmunoassay. Tear secretion level was measured with phenol red thread. Break-up time of tear film (BUT) was tested, and corneal inflorescence staining was scored based on the criterion of Toshida. The corneas of all the mice were collected for ultrastructural examination under the transmission electron microscope. The study complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission. Results Mouse serum androgen concentration was (573.87±6.74)ng/μl in the normal control group and (579.74±8.24)ng/μl in the sham group, and there was no significant difference between these two groups (t=1.432, P=0.251), however, the serum androgen concentration was 0 ng/μl in all castrated mice after orchectomy. The tear secretion levels were (5.26±0.99), (4.89±0.56), (4.62±0.83), (4.28±0.63), (3.36±0.56), (2.60±0.27) and (2.08±0.35)mm/5 minutes in the normal control group, sham group and orchectomy 1-, 2-, 4-, 6-and 8-week groups, respectively, and the tear secretion levels were significantly lower in the orchectomy 2-, 4-, 6-and 8-week groups than those in the normal control group (all at P<0.05). The BUT were (68.33±12.86), (62.47±3.75), (58.67±10.03), (47.17±7.64), (39.51±3.39), (32.67±3.88) and (31.00±2.36) seconds in the normal control group, sham group and orchectomy 1-, 2-, 4-, 6-and 8-week groups, respectively, and the BUTs were significantly shorter in the orchectomy 2-, 4-, 6-and 8-week groups than those in the normal control group (all at P<0.05). The fluorescein stain score of corneal epithelium was gradually increased with the lapse of castrated time. The decrease of number, shortening and edema of microvilli as well as separation of desmosomes of corneal epithelial cells were found under the transmission electron microscope in castrated mice. Conclusions Decreasing of serum androgen level, declining of aqueous tears, instability of tear film and damage of corneal epithelial cell ultrastructures occur in castrated male mice, and these manifestations are similar to dry eyes. Key words: [Key words] Androgen; Epithelial cells, corneal; Dry eye; Tear; Ultrastructure; Orchectomy; Disease models, animal; Mice, BALB/c

Both Estrogen and Androgen Modify the Response to Activation of Neurokinin-3 and κ-Opioid Receptors in Arcuate Kisspeptin Neurons From Male Mice.

Gonadal steroids regulate the pattern of GnRH secretion. Arcuate kisspeptin (kisspeptin, neurokinin B, and dynorphin [KNDy]) neurons may convey steroid feedback to GnRH neurons. KNDy neurons increase action potential firing upon the activation of neurokinin B receptors (neurokinin-3 receptor [NK3R]) and decrease firing upon the activation of dynorphin receptors (κ-opioid receptor [KOR]). In KNDy neurons from intact vs castrated male mice, NK3R-mediated stimulation is attenuated and KOR-mediated inhibition enhanced, suggesting gonadal secretions are involved. Estradiol suppresses spontaneous GnRH neuron firing in male mice, but the mediators of the effects on firing in KNDy neurons are unknown. We hypothesized the same gonadal steroids affecting GnRH firing pattern would regulate KNDy neuron response to NK3R and KOR agonists. To test this possibility, extracellular recordings were made from KNDy neurons in brain slices from intact, untreated castrated or castrated adult male mice treated in vivo with steroid receptor agonists. As observed previously, the stimulation of KNDy neurons by the NK3R agonist senktide was attenuated in intact vs castrated mice and suppression by dynorphin was enhanced. In contrast to observations of steroid effects on the GnRH neuron firing pattern, both estradiol and DHT suppressed senktide-induced KNDy neuron firing and enhanced the inhibition caused by dynorphin. An estrogen receptor-α agonist but not an estrogen receptor-β agonist mimicked the effects of estradiol on NK3R activation. These observations suggest the steroid modulation of responses to activation of NK3R and KOR as mechanisms for negative feedback in KNDy neurons and support the contribution of these neurons to steroid-sensitive elements of a GnRH pulse generator.

Abstract 4073: Efficient conditional reprogramming of differentiated prostate cancer cells back to a stem cell-like state with increased aggressive properties

Abstract Androgen receptor- (AR-) expressing prostate cancer (PCa) cell lines can transdifferentiate to neuro-ectodermal cell lineages under androgen deprivation. We have proposed that this transdifferentiation is mediated by initial regression to an unstable cancer stem cell-like (CSCL) intermediate state and we have developed a serum- and androgen-free custom cell culture medium that now allows us to efficiently reprogram PCa cells to this CSCL state and expand them for characterization. Exposure of adherent cultures of parental LNCaP, VCaP, LAPC4 or CWR22rv1 cells to the reprogramming medium induced a rapid and efficient (&amp;gt;67%) morphological change of plated cells to smaller, rounded cells with large nuclei and prominent nucleoli that grow in 3-dimensions. Following transfer of the reprogrammed cells they, thereafter, grow as cell rosettes that will form larger spheroids. Analysis of changes in gene expression by microarray gene expression profiling or immunohistochemistry (IHC) showed a remarkable overexpression of stem cell genes in the reprogrammed cells and a change in cell surface CD protein expression that resembles neural stem cells. Reprogramming drastically reduced expression of AR RNA and protein in all cell lines. Once reprogrammed, the CSCLs have significantly higher tumor-initiating capacity based upon tumors formed from cell serial dilutions xenografted into intact or castrated male nude mice. In vitro, reprogrammed cells were significantly more resistant to the anti-androgen enzalutamide and to hypoxia compared to parentals. By placing CSCLs into specific differentiation mediums, we could differentiate them back into neural-, oligodendrocyte-, glia- or osteoblast-like cells based upon induced expression of lineage-specific RNAs/proteins or return them to prostate-like cells (that re-express AR) by supplementing them with serum and androgen. Reprogrammed cells have an EMT phenotype based on increased expression of EMT-related genes and significantly increased invasiveness in in vitro and in vivo models. Comparison of gene expression changes acquired by reprogrammed CSCLs amongst the different cell models revealed a shared set of 65 over-/under-expressed genes (≥1.8-fold) between them. Finally, we have found that growth of parental LNCaP cells in androgen-deprived or enzalutamide-supplemented (serum-containing) growth medium increased the population of cells bearing CSCL surface CD stem markers. In summary, our studies show that commonly used PCa cell lines have the ability to efficiently transition between a differentiated and a CSCL state in an appropriate microenvironment that is depleted of androgens. While in the CSCL state, these cells show increased invasiveness, increased resistance to hormonal therapeutics and a propensity to transdifferentiate to alternate cell lineages under appropriate stimulation. Citation Format: Josselin Caradec, Amy A. Lubik, Mannan Nouri, Na Li, Manuel Altimarano-Dimas, Jennifer Bishop, Mani Moniri, Down Cochrane, Martin Gleave, Ralph Buttyan. Efficient conditional reprogramming of differentiated prostate cancer cells back to a stem cell-like state with increased aggressive properties. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4073. doi:10.1158/1538-7445.AM2015-4073

A sexual dimorphism influences bicyclol-induced hepatic heat shock factor 1 activation and hepatoprotection.

Bicyclol [4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2-hydroxy-methyl-2'-methoxycarbonyl biphenyl] is a synthetic hepatoprotectant widely used in clinical practice, but resistance to this treatment is often observed. We found that the hepatoprotective effect of bicyclol was greatly compromised in female and castrated male mice. This study was to dissect the molecular basis behind the sex difference, which might underlie the clinical uncertainty. We compared bicyclol-induced hepatoprotection between male and female mice using acute liver damage models. Inducible knockout by the Cre/loxp system was used to decipher the role of heat shock transcription factor 1 (HSF1). Functional experiments, western blot, and histopathological analysis were used to determine the key causative factors which might antagonize bicyclol in female livers. HSF1 activation and heat shock protein 70 (Hsp70) expression, which were responsible for bicyclol-induced hepatoprotection, were compromised in female and castrated male livers. Compromised HSF1 activation was a result of HSF1 phosphorylation at serine 303, which was catalyzed by glycogen synthase kinase 3β (GSK3β). Testosterone was necessary for bicyclol to inhibit hepatic GSK3β activity. Administration of testosterone or GSK3β inhibitors restored bicyclol-induced protection in females. Bicyclol induces sex-specific hepatoprotection based on a sex-specific HSF1/Hsp70 response, in which testosterone and GSK3β play key roles. Because a lot of patients suffering from liver diseases have very low testosterone levels, our results give a possible explanation for the clinical variation in bicyclol-induced hepatoprotection, as well as practicable solutions to improve the effect of bicyclol.

Influence of gonadal hormones on the behavioral effects of intermittent hypoxia in mice.

Obstructive sleep apnea (OSA) is characterized by repetitive upper airway obstruction resulting in cyclic intermittent hypoxia (IH) during sleep in affected individuals. OSA occurs more frequently in postmenopausal than premenopausal women and the severity of OSA increases after menopause. Gonadal hormones can influence brain and behavior; testosterone and estrogens in particular can enhance spatial learning and memory. We hypothesized that estrogens may protect mice from IH-induced hippocampal morphological and behavioral changes. To test this hypothesis we exposed intact or gonadectomized male and female mice to room air or IH [15 cycles/h, 8 h/day, fraction of inspired oxygen (FiO 2) nadir of 5%] for a total of 30 days. During the final 4 days of IH, mice were tested for anxiety- and depressive-like behaviors. After cessation of IH exposure mice were tested on the Barnes maze and passive avoidance tests to assess learning and memory. Ovariectomy paired with IH treatment, impaired spatial learning and memory compared to all other female groups. Intact male mice receiving IH treatment also had impaired learning and memory compared with intact or castrated male mice exposed to room air. Learning and memory changes were mirrored by changes in basilar dendritic length of the CA1 region of the hippocampus. These data suggest that estrogens provide protection against IH-induced deficits, whereas androgens partially exacerbate IH-induced deficits on learning and memory.

Abstract 610: Preclinical evaluation of novel androgen receptor N-terminal domain inhibitor EPI-002 for the treatment of castration-resistant prostate cancer

Abstract Background: Androgen receptor (AR) is a ligand-activated transcription factor that plays critical roles in lethal and incurable castration-resistant prostate cancer (CRPC). EPI-002 is a novel small molecule that specifically targets the AR N-terminal domain (NTD), while conventional therapies target the ligand-binding domain (LBD). Our previous work suggests that EPI-002 may have clinical potential to treat CRPC. Here, the objective was to evaluate EPI-002 against several clinically relevant conditions such as elevated expression of AR co-activators, variable lengths of the poly-glutamine tract (CAG-repeats) within AR NTD, and constitutively active AR splice variants lacking the LBD. Methods: To evaluate the effect of EPI-002 on AR transcriptional activity against overexpressed co-activators such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells transfected with an AR-driven reporter and various amounts of the co-activators. AR-negative COS1 cells were employed for reporter assays to examine if the length of CAG-repeats (varying from 0 to 49) affects EPI-002 inhibition. The effect of EPI-002 on constitutively active AR splice variants was studied in LNCaP95 cells, which express endogenous AR-V7 variant. The expression of several genes regulated by AR-V7 was analyzed by QPCR in LNCaP95 cells treated with antiandrogen enzalutamide or EPI-002. The proliferation of LNCaP95 cells treated with EPI-002 was measured using BrdU incorporation. The efficacy of EPI-002 on LNCaP95 xenografts was studied in castrated male mice by oral delivery and compared to control and enzalutamide. Results: EPI-002 effectively inhibited AR transcriptional activity in spite of overexpressed co-activators SRC1, SRC2, SRC3, or p300. The variable lengths of CAG-repeats within the NTD had no effect on the inhibition of AR by EPI-002. In LNCaP95 cells, EPI-002 significantly blocked the transcriptional activity of AR-V7 indicated by decreased levels of mRNA of AR-V7 regulated genes such as UBE2C. In contrast, enzalutamide had no effect on expression of these genes. EPI-002 significantly inhibited the androgen-independent and antiandrogen-resistant proliferation of LNCaP95 cells. In castrated male mice, EPI-002 attenuated the growth of LNCaP95 xenografts, whereas enzalutamide had no significant effect. By the end of the 28-day study, animals treated with EPI-002 had xenografts that were half of the size of those from the control arm. Conclusions: AR NTD inhibitor EPI-002 was effective against several clinically relevant conditions, including overexpressed co-activators, variable lengths of CAG-repeats, and constitutively active AR-V7. These findings support EPI-002 as a promising therapeutic agent to treat CRPC, particularly against castration-resistant tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting therapies. Citation Format: Yu Chi (Kevin) Yang, Nasrin (Rina) Mawji, Jean Wang, Marianne Sadar. Preclinical evaluation of novel androgen receptor N-terminal domain inhibitor EPI-002 for the treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 610. doi:10.1158/1538-7445.AM2014-610

Abstract 4517: Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer

Abstract Prostate cancer is one of the most common malignancy diagnosed in men. Although androgen deprivation therapy is effective at the beginning of treatment, the majority of patients eventually progress to hormone refractory prostate cancer (HRPC), which leads to a poor outcome. HRPC mouse models have been developed to study transition from hormone dependent (HD) to hormone refractory (HR) disease; however, the exact mechanism behind this phenotypic conversion remains unclear. By using serial in vivo transplantation of LNCap xenograft tumors in castrated male SCID mice, we developed, validated and characterized a mouse model of HRPC, HR-LNCap. In vivo passaged tumor fragments form solid tumors in castrated male SCID mice. By comparison, HR-LNCap tumors grow more aggressively than parental LNCap tumors in SCID mice. A tumor cell line established from HR-LNCap xenograft tumors was able to grow in charcoal stripped serum containing medium, indicating it is androgen independent. HR-LNCap tumors lost sensitivity to androgen receptor antagonist bicalutamide, but remained sensitive to MDV-3100, a second generation AR antagonist approved for treating HRPC. Characterization of this model revealed that HR-LNCap tumors had unique AR signaling pathway profiles, including elevated AR expression and decreased expression of the AR-regulated genes, prostate specific antigen (PSA) and FKBP5, when compared with parental LNCap tumors. RNA sequencing was performed on HR-LNCap tumors to determine expression signature of the model. Interestingly, additional pathway analysis showed that HR-LNCap tumors have significantly higher levels of pAKT in comparison with parental LNCap tumors, indicating activation of AKT/mTOR pathway. Treatment of HR-LNCap tumor bearing mice with a small molecule TOR kinase inhibitor CC214-2 showed significant tumor growth inhibition. Further analysis demonstrated that treatment with CC214-2 inhibited TORC1 and TORC2 pathway markers, pAKT, pS6 and p4EBP1, in HR-LNCap tumors. Taken together, this study describes a mouse model of HRPC with a unique molecular profile, which can be used to evaluate novel therapeutics against HRPC. Citation Format: Wenqing Yang, Jing Jiu, Rama Krishna Narla, Heather K. Raymon. Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2014-4517

Abstract 3808: Preclinical efficacy of the novel, oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) on castration resistant prostate cancer

Abstract Exportin 1/ Chromosomal Maintenance Protein 1 (XPO1/CRM1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of tumor suppressor proteins such as p53, FOXO, PTEN, pRB and I-κB. Selinexor is an orally bioavailable XPO1 inhibitor that represents a novel class of small molecule compounds with potent activity against a wide variety of cancers. Here we report the activity of Selinexor against Castration resistant prostate cancer (CRPC). CRPC progression is mediated by activation of various adaptive Androgen Receptor (AR) signaling pathways. These pathways are currently being targeted by novel anti-androgen therapies such as abiraterone acetate and enzalutamide with favorable outcomes. However, resistance to anti-androgen therapy can be developed through deregulation of tumor suppressor pathways. XPO-1 is highly expressed in prostate cancer cells and therefore we tested the effects of Selinexor on CRPC cells and tumors models. Treatment of the C4-2B prostate cancer cell line with Selinexor in vitro significantly inhibited cell proliferation and resulted in nuclear accumulation of p53 and p21. In addition, Selinexor inhibited expression of the tumor-promoting gene Ubiquitin-Conjugating Enzyme E2C (UBE2C), which is activated by AR in these cells. Selinexor was also tested in tumor graft models of two patient tumors in castrated male mice. The MDA-PCa-133 tumor is an adenocarcinoma derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumors in castrated male mice express full-length AR and Prostate-Specific Antigen (PSA) but lack expression of p53. The MDA-PCa-144-13 tumor is a small cell carcinoma derived from a lethal variant of prostate cancer with anaplastic clinical phenotype and does not express AR or PSA but expresses a gain-of-function p53 mutant. Vehicle or Selinexor treatment (10 mg/kg p.o. QoDX3 M/W/F) of MDA-PCa-133 tumors for 34 days resulted in almost complete inhibition of tumor growth with a 30-fold reduction of PSA (p&amp;lt;0.001). Treatment of MDA-PCa-144-13 tumors for 22 days also resulted in significant reduction of tumor volume (&amp;gt;8 fold compared to vehicle only; p&amp;lt;0.0047). Immunohistochemical analysis of MDA-PCa-133 tumors showed that Selinexor induced nuclear accumulation of XPO1 cargos FOXO3a and p27 with concomitant reduction in the proliferation marker KI67. In conclusion, Selinexor demonstrated robust inhibition of CRPC tumor growth and activated multiple tumor suppressors in prostate cancer cells. Selinexor is now being evaluated in Phase 1 clinical studies in patients with advanced hematological and solid tumor cancers, and preliminary results show adequate tolerability with evidence of anti cancer activity. Future clinical studies in patients with CRPC are planned and will provide more information on the use of Selinexor as monotherapy and in combination with anti-androgen therapy of CRPC. Citation Format: Sankar Narayan Maity, Guanglin Wu, Jing-Fang Lu, Anh Hoang, Yosef Landesman, Dilara McCauley, Sharon Shacham, Michael G. Kauffman, Ana M. Aparicio, Eleni Efstathiou, John C. Araujo, Christopher J. Logothetis. Preclinical efficacy of the novel, oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) on castration resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3808. doi:10.1158/1538-7445.AM2014-3808

Sodium valproate enhances urethane tumorigenicity in lungs of male but not female mice.

In the study, the possible effect of sodium valproate (NaVP) on urethane-induced lung tumors in mice has been evaluated. BALB/c mice (n = 120; 4-6 weeks old, both sexes) were used in the following groups: 1) urethane-treated, 2) urethane-NaVP-treated, 3) only NaVP-treated, 4) control. In the same groups, castrated male mice (n = 48) were investigated. Urethane was given by intraperitoneal injections 10 mg/mouse, twice a week, the total dose 50 mg/mouse. In NaVP-treated mice, the 0.4 % NaVP aqueous solution was offered to mice ad libitum. The duration of the experiment was 6 months. The number of tumors per mouse in urethane-NaVP-treated males was significantly higher than in those treated with urethane only (13.82 ± 1.12 vs 6.77 ± 0.43, p < 0.0001). No significant difference in the number of tumors per mouse was revealed while comparing the female urethane- and urethane-NaVP-treated groups (6.50 ± 0.79 vs 8.15 ± 0.55, p = 0.105). No difference in the number of tumors per mouse was found in urethane-NaVP-treated castrated males as compared with urethane-treated castrated males. However, in the urethane-NaVP-treated castrated males the number of tumors per mouse was significantly lower than in analogous non-castrated males (7.8 ± 1.67 vs 13.82 ± 1.12, p < 0.01). NaVP combined with urethane potentiates urethane tumorigenicity in BALB/c non-castrated but not in female and castrated male mice. These data indicate an important role of testosterone in the urethane-NaVP induced lung tumorigenesis.

MP24-05 TARGETING THE VAV3 ONCOGENE ENHANCES DOCETAXEL-INDUCED APOPTOSIS THROUGH THE INHIBITION OF ANDROGEN RECEPTOR PHOSPHORYLATION IN LNCAP PROSTATE CANCER CELLS UNDER CHRONIC HYPOXIA IN VITRO AND IN VIVO

growth rates, protein, and gene expression were analyzed. Effects of combination AR-ASO and OGX-011 on ENZ-R LNCaP progression rates were assessed in castrated male athymic mice. RESULTS: ENZ-R LNCaP-derived sub-lines expressed high levels of CLU and AR compared to CRPC LNCaP cell lines. LNCaP cells overexpressing CLU acquire resistance to AR-ASO induced apoptosis as shown by decreased cell growth inhibition rates and apoptosis induction. AR-ASO induced timeand dose-dependent increases in CLU levels in parental and ENZ-R LNCaP cells. Co-targeted suppression of CLU and AR synergistically inhibited ENZ-R cell growth rates compared to monotherapy. Increased apoptosis (PARP and caspase-3 cleavage, sub G0/G1 fraction) together with reduced AKT phosphorylation was most enhanced with combined therapy. In vivo, combined OGX-011 + AR-ASO significantly delayed ENZ-R LNCaP tumor progression compared to scramble ASO + AR-ASO. CONCLUSIONS: Suppression of CLU combined with AR knockdown inhibited ENZ-R LNCaP cell growth, providing pre-clinical proof-ofprinciple as a promising AR co-targeting approach in ENZ-R CRPC.

AKR1C3 overexpression may serve as a promising biomarker for prostate cancer progression

BackgroundAldo-keto reductase family 1 member C3 (AKR1C3) is a key steroidogenic enzyme that is overexpressed in prostate cancer (PCa) and is associated with the development of castration-resistant prostate cancer (CRPC). The aim of this study was to investigate the correlation between the expression level of AKR1C3 and the progression of PCa.MethodsSixty human prostate needle biopsy tissue specimens and ten LNCaP xenografts from intact or castrated male mice were included in the study. The relationship between the level of AKR1C3 expression by immunohistochemistry and evaluation factors for PCa progression, including prostate-specific antigen (PSA), Gleason score (GS) and age, were analyzed.ResultsLow immunoreactivity of AKR1C3 was detected in normal prostate epithelium, benign prostatic hyperplasia (BPH) and prostatic intraepithelial neoplasia (PIN). Positive staining was gradually increased with an elevated GS in PCa epithelium and LNCaP xenografts in mice after castration. The Spearman’s r values (rs) of AKR1C3 to GS and PSA levels were 0.396 (P = 0.025) and -0.377 (P = 0.036), respectively, in PCa biopsies. The rs of AKR1C3 to age was 0.76 (P = 0.011). No statistically significant difference was found with other variables.ConclusionOur study suggests that the level of AKR.1C3 expression is positively correlated with an elevated GS, indicating that AKR1C3 can serve as a promising biomarker for the progression of PCa.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7748245591110149.

Gonadal hormones differently modulate cutaneous wound healing of chronically stressed mice

Gonadal hormones influence physiological responses to stress and cutaneous wound healing. The aim of this study was to investigate the role of gonadal hormones on cutaneous wound healing in chronically stressed mice. Male and female mice were gonadectomized, and after 25 days, they were spun daily at 115 rpm for 15 min every hour until euthanasia. Twenty-eight days after the gonadectomy, an excisional lesion was created. The animals were killed 7 or 14 days after wounding, and the lesions were collected. Myofibroblast density, macrophage number, catecholamine level, collagen deposition, and blood vessel number were evaluated. In the intact and gonadectomized groups, stress increased the plasma catecholamine levels in both genders. In intact groups, stress impaired wound contraction and re-epithelialization and increased the macrophage number in males but not in females. In addition, stress compromised myofibroblastic differentiation and blood vessel formation and decreased collagen deposition in males but not in females. In contrast to intact mice, wound healing in ovariectomized female mice was affected by stress, while wound healing in castrated male mice was not. In conclusion, gender differences contribute to the cutaneous wound healing of chronically stressed mice. In addition, androgens contribute to the stress-induced impairment of the healing of cutaneous wounds but estrogens inhibit it.

Long Noncoding RNA MALAT-1 is a New Potential Therapeutic Target for Castration Resistant Prostate Cancer

To understand the role of MALAT-1 in prostate cancer we evaluated its expression in prostate cancer tissues and cell lines. We also studied the therapeutic effects of MALAT-1 silencing on castration resistant prostate cancer cells invitro and invivo. Quantitative reverse transcriptase-polymerase chain reaction was used to detect MALAT-1 expression in prostate cancer tissues and cell lines. siRNA against MALAT-1 was designed and the silencing effect was examined by quantitative reverse transcriptase-polymerase chain reaction. The biological effects of MALAT-1 siRNA on cells were investigated by examining cell proliferation using a cell counting kit and cell colony assays as well as cell migration by invitro scratch assay, cell invasion by Transwell® invasion assay and cell cycle by flow cytometry. We further investigated the effect of therapeutic siRNA targeting MALAT-1 on castration resistant prostate cancer invivo. MALAT-1 was up-regulated in human prostate cancer tissues and cell lines. Higher MALAT-1 expression correlated with high Gleason score, prostate specific antigen, tumor stage and castration resistant prostate cancer. MALAT-1 down-regulation by siRNA inhibited prostate cancer cell growth, invasion and migration, and induced castration resistant prostate cancer cell cycle arrest in the G0/G1 phases. Importantly, intratumor delivery of therapeutic siRNA targeting MALAT-1 elicited delayed tumor growth and reduced metastasis of prostate cancer xenografts in castrated male nude mice, followed by the concomitant prolongation of survival of tumor bearing mice. MALAT-1 may be needed to maintain prostate tumorigenicity and it is involved in prostate cancer progression. Thus, MALAT-1 may serve as a potential therapeutic target for castration resistant prostate cancer.

Castration Induces Parkinson Disease Pathologies in Young Male Mice via Inducible Nitric-oxide Synthase

Although Parkinson disease (PD) is a progressive neurodegenerative disorder, available animal models do not exhibit irreversible neurodegeneration, and this is a major obstacle in finding out an effective drug against this disease. Here we delineate a new irreversible model to study PD pathogenesis. The model is based on simple castration of young male mice. Levels of inducible nitric-oxide synthase (iNOS), glial markers (glial fibrillary acidic protein and CD11b), and α-synuclein were higher in nigra of castrated male mice than normal male mice. On the other hand, after castration, the level of glial-derived neurotrophic factor (GDNF) markedly decreased in the nigra of male mice. Accordingly, castration also induced the loss of tyrosine hydroxylase-positive neurons in the nigra and decrease in tyrosine hydroxylase-positive fibers and neurotransmitters in the striatum. Reversal of nigrostriatal pathologies in castrated male mice by subcutaneous implantation of 5α-dihydrotestosterone pellets validates an important role of male sex hormone in castration-induced nigrostriatal pathology. Interestingly, castration was unable to cause glial activation, decrease nigral GDNF, augment the death of nigral dopaminergic neurons, induce the loss of striatal fibers, and impair neurotransmitters in iNOS(-/-) male mice. Furthermore, we demonstrate that iNOS-derived NO is responsible for decreased expression of GDNF in activated astrocytes. Together, our results suggest that castration induces nigrostriatal pathologies via iNOS-mediated decrease in GDNF. These results are important because castrated young male mice may be used as a simple, toxin-free, and nontransgenic animal model to study PD-related nigrostriatal pathologies, paving the way for easy drug screening against PD.

Abstract A21: Orphan nuclear receptor TLX recruits lysine-specific demethylase 1 to repress androgen receptor gene transcription and functions to promote hormone-resistant growth of prostate cancer cells

Abstract Prostate cancer (PCa) is the most frequently diagnosed type of cancer and the second leading cause of death from cancer in males in the United States. The initial growth of most localized PCa is slow and androgen-dependent. Thus, surgical treatment (prostatectomy) and hormone therapy (androgen deprivation) are effective initially. However, many patients relapse and progress to a fatal androgen-independent and bone-metastatic stage shortly when both primary and secondary hormone therapies fail. Unfortunately, the current options for the metastatic hormone-refractory PCa are still limited and mostly ineffective, resulting in high mortality of this disease. Although dysregulated androgen receptor (AR) signaling pathway has been proposed to contribute to the development of hormone-resistance, the involved mechanisms are still not fully defined. Our present findings revealed that the orphan nuclear receptor TLX (NR2E1) exhibited increased expression patterns in androgen-independent and antiandrogen-resistant PCa cells, and also the high-grade and hormone-refractory clinical PCa tissues. Stable overexpression of TLX in AR-positive LNCaP cells could promote both androgen-independent and antiandrogen-resistant cell growth in vitro, and enhance tumor growth capacity in castrated male SCID mice in vivo. Decreased expression of AR and AR target genes in LNCaP-TLX cells were identified, while results of AR-promoter driven luciferase reporter assay also revealed that TLX overexpression could suppress the AR transcription in non-prostatic HEK293 cells. Furthermore, we also identified the direct binding site of TLX on the AR promoter by chromatin immunoprecipitation analyses in TLX-transfected HEK293 cells. The molecular mechanism may involve the TLX-mediated recruitment of lysine-specific demethylase 1 (LSD1) as treatment with an inhibitor of LSD1 pargyline could reverse the transrepressive regulation of TLX on AR-promoter driven luciferase activity and restore AR gene expression levels in LNCaP cells. In summary, our results showed for the first time that overexpression of TLX might contribute to the hormone-resistant PCa cell growth and advanced progression of PCa. LSD1 might serve as a corepressor in TLX-mediated direct transrepression on AR gene expression. This work is partly supported by an RGC-General Research Fund CUHK461012. Citation Format: Lin Jia, Dinglan Wu, Shan Yu, Franky Leung Chan. Orphan nuclear receptor TLX recruits lysine-specific demethylase 1 to repress androgen receptor gene transcription and functions to promote hormone-resistant growth of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A21.

Of mice and men‐warning: Intact versus castrated adult male mice as xenograft hosts are equivalent to hypogonadal versus abiraterone treated aging human males, respectively

Immune deficient male mice bearing human prostate cancer xenografts are used to evaluate therapeutic response to novel androgen ablation approaches and the results compared to surgical castration based upon assumption that testosterone microenvironment in intact and castrated adult male mice mimics eugonadal and castrated aging adult human males. To test these assumptions, serum total testosterone (TT) and free testosterone (FT) were determined longitudinally in groups (n > 20) of intact versus castrated adult male nude, NOG, and immune competent C57BL/6 mice. In adult male mice, TT and FT varies by 30- to 100-fold within the same animal providing a microenvironment that is only equivalent to hypogonadal, not eugonadal, adult human males (TT is 1.7 ± 1.2 ng/ml [5.8 ± 4.1 nM] in nude and 2.5 ± 1.3 ng/ml [8.7 ± 4.4 nM] in NOG mice versus >4.2 ng/ml [14.7 nM] in eugonadal humans). This was confirmed based upon enhanced growth of androgen dependent human prostate cancer xenografts inoculated into mice supplemented with exogenous testosterone to elevate and chronically maintain serum TT at a level (5 ng/ml [18 nM]) equivalent to a 50-year-old eugonadal human male. In castrated mice, TT and FT range from 2 to 20 pg/ml (7-70 pM) and <0.8 pg/ml (<2.6 pM), respectively, which is equivalent to castrate resistant prostate cancer (CRPC) patients treated with abiraterone. This was confirmed based upon the inability of another CYP17A1 inhibitor, ketoconazole, to inhibit the growth of CRPC xenografts in castrated mice. Adult male mice supplemented with testosterone mimic eugonadal human males, while unsupplemented animals mimic standard androgen ablation and castrated animals mimic abiraterone treated patients. These studies confirm what is claimed in Robert Burns' poem "To a Mouse" that "The best laid schemes of mice and men/often go awry.".

Regulation of Arcuate Neurons Coexpressing Kisspeptin, Neurokinin B, and Dynorphin by Modulators of Neurokinin 3 and κ-Opioid Receptors in Adult Male Mice

Pulsatile GnRH release is essential to fertility and is modulated by gonadal steroids, most likely via steroid-sensitive afferents. Arcuate neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) are steroid-sensitive and have been postulated to both generate GnRH pulses and mediate steroid feedback on pulse frequency. KNDy neurons are proposed to interact with one another via NKB and dynorphin to activate and inhibit the KNDy network, respectively, and thus alter kisspeptin output to GnRH neurons. To test the roles of NKB and dynorphin on KNDy neurons and the steroid sensitivity of these actions, targeted extracellular recordings were made of Tac2(NKB)-GFP-identified neurons from castrate and intact male mice. Single-cell PCR confirmed most of these cells had a KNDy phenotype. The neurokinin 3 receptor (NK3R) agonist senktide increased action potential firing activity of KNDy neurons. Dynorphin reduced spontaneous KNDy neuron activity, but antagonism of κ-opioid receptors (KOR) failed to induce firing activity in quiescent KNDy neurons. Senktide-induced activation was greater in KNDy neurons from castrate mice, whereas dynorphin-induced suppression was greater in KNDy neurons from intact mice. Interactions of dynorphin with senktide-induced activity were more complex; dynorphin treatment after senktide had no consistent inhibitory effect, whereas pretreatment with dynorphin decreased senktide-induced activity only in KNDy neurons from intact but not castrate mice. These data suggest dynorphin-mediated inhibition of senktide-induced activity requires gonadal steroid feedback. Together, these observations support the hypotheses that activation of NK3R and KOR, respectively, excites and inhibits KNDy neurons and that gonadal steroids modulate these effects.

Diethylstilboestrol Exposure Does Not Reduce Testosterone Production in Human Fetal Testis Xenografts

In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15–19 weeks’ gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5–e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus.

Sex Bias in Experimental Immune-Mediated, Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs, Estrogen, and IL-6

Background and AimsImmune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.MethodsTo model DILI, we immunized BALB/c, BALB/cBy, IL-6–deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.ResultsBALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.Conclusions17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.