Increased transcription and high translation efficiency lead to accumulation of androgen receptor splice variant after androgen deprivation therapy.

Upregulation of androgen receptor splice variants (AR-Vs), especially AR-V7, is associated with castration resistance of prostate cancer. At the RNA level, AR-V7 upregulation is generally coupled with increased full-length AR (AR-FL); consequently, AR-V7 and AR-Vs collectively constitute a minority of the AR population. However, Western blotting showed that the relative abundance of AR-V proteins is much higher in many castration-resistant prostate cancers (CRPCs). To address the mechanism underlying this discrepancy, we analyzed RNA-seq data from ~350 CRPC samples and found a positive correlation between all canonical and alternative AR splicing. This indicates that increased alternative splicing is not at the expense of canonical splicing. Instead, androgen deprivation releases AR-FL from repressing the transcription of the AR gene to induce coordinated increase of AR-FL and AR-V mRNAs. At the protein level, however, androgen deprivation induces AR-FL, but not AR-V, degradation. Moreover, AR-V7 is translated much faster than AR-FL. Thus, androgen-deprivation-induced AR-gene transcription and AR-FL protein decay, together with efficient AR-V7 translation, explain the discrepancy between the relative AR-V mRNA and protein abundances in many CRPCs, highlighting the inevitability of AR-V induction after endocrine therapy.

P-cresol degradation through Fe(III)-EDDS/H2O2 Fenton-like reaction enhanced by manganese ion: Effect of pH and reaction mechanism.

P-cresol is a highly toxic phenolic pollutant in coal chemical wastewater. The effective removal of p-cresol is of great significance to the ecological environment. In this study, the degradation of p-cresol by the Fe(III)-EDDS/H2O2 Fenton-like reaction modified by Mn2+ was investigated. The results showed that the removal rate of p-cresol could be significantly increased by the addition of Mn2+ under neutral and weakly alkaline conditions (pH 6.5-8.5). Acidic conditions (pH 3.5) were not conducive to the Fenton-like reaction. This is because a neutral or weakly alkaline environment is conducive to Mn2+-EDDS complex formation, which can produce O2·- to accelerate the reduction of Fe(III), and the efficiency of p-cresol degradation through a Fenton-like reaction catalyzed by the Fe(III)-EDDS complex is significantly improved. In addition, the degradation of EDDS through ·OH was reduced by O2·-, which maintained and stabilized the Mn2+-EDDS complex and Fe(III)-EDDS complex. Under neutral conditions, the optimal dosage of Fe(III) is 0.7 mM, and the optimal molar ratios are EDDS/Fe(III) = 1: 1, Mn2+/Fe(III) = 1: 1, and H2O2/Fe(III) = 15: 1. The addition of free radical clearance isopropanol and CHCl3 proved that ·OH was the main active substance in the p-cresol degradation process.

Effect of hydroxypropyl-ß-cyclodextrin on the cometabolism of phenol and phenanthrene by a novel Chryseobacterium sp.

Cometabolic degradation is an effective method to remove the polycyclic aromatic hydrocarbons (PAHs) with phenol as growth substrate from coal chemical wastewater (CCW). Unfortunately, the toxicity and low solubility of PAHs always restrict their degradation. In this study, Chryseobacterium sp. H202 was firstly isolated from the aerobic segment of CCW. Then, to improve the cometabolic degradation of PAHs, the effects of hydroxypropyl-ß-cyclodextrin (HPCD) were investigated. Phenanthrene removal was accelerated in the presence of phenol; however, the degradation of phenol was inhibited because of the toxicity of phenanthrene. Addition of 50 mg/L HPCD accelerated the degradation of phenol and effectively improved the phenanthrene removal rate by about 55%. Inclusion of HPCD appeared to increase the apparent solubility and reduce the toxicity of phenanthrene, thereby improving the cometabolic degradation of phenol and phenanthrene. Therefore, HPCD can enhance the degradation of phenanthrene with phenol as the growth substrate during CCW treatment.

Interplay between Cytoplasmic and Nuclear Androgen Receptor Splice Variants Mediates Castration Resistance.

Androgen receptor splice variants (AR-V) are implicated in resistance of prostate cancer to androgen-directed therapies. When expressed alone in cells, some AR-Vs (e.g., AR-V7) localize primarily to the nucleus, whereas others (e.g., AR-V1, AR-V4, and AR-V6) localize mainly to the cytoplasm. Significantly, the latter are often coexpressed with the nucleus-predominant AR-Vs and the full-length AR (AR-FL). An important question to be addressed is whether the cytoplasmic-localized AR-Vs play a role in castration-resistant prostate cancer (CRPC) through interaction with the nucleus-predominant AR-Vs and AR-FL. Here, it is demonstrated that AR-V1, -V4, and -V6 can dimerize with both AR-V7 and AR-FL. Consequently, AR-V7 and androgen-bound AR-FL induced nuclear localization of AR-V1, -V4, and -V6, and these variants, in turn, mitigated the ability of the antiandrogen enzalutamide to inhibit androgen-induced AR-FL nuclear localization. Interestingly, the impact of nuclear localization of AR-V4 and -V6 on AR transactivation differs from that of AR-V1. Nuclear localization leads to an increased ability of AR-V4 and -V6 to transactivate both canonical AR targets and AR-V-specific targets and to confer castration-resistant cell growth. However, although AR-V1, which lacks inherent transcriptional activity, appears to activate AR-FL in an androgen-independent manner, it significantly antagonizes AR-V7 transactivation. Together, these data demonstrate that the complex interactions among different AR-Vs and AR-FL play a significant role in castration-resistant disease. IMPLICATIONS: This study suggests important consequences for clinical castration resistance due to simultaneous expression of AR-FL and AR-Vs in patient tumors and suggests that dissecting these interactions should help develop effective strategies to disrupt AR-V signaling. Mol Cancer Res; 15(1); 59-68. ©2016 AACR.

A Whole Blood Assay for AR-V7 and ARv567es in Patients with Prostate Cancer.

PURPOSE: Most prostate cancer mortality can be attributed to metastatic castration resistant prostate cancer, an advanced stage that remains incurable despite recent advances. The AR (androgen receptor) signaling axis remains active in castration resistant prostate cancer. Recent studies suggest that expression of the AR-V (AR splice variant) AR-V7 may underlie resistance to abiraterone and enzalutamide. However, controversy exists over the optimal assay. Our objective was to develop a fast and sensitive assay for AR-Vs in patients. MATERIALS AND METHODS: Two approaches were assessed in this study. The first approach was based on depletion of leukocytes and the second one used RNA purified directly from whole blood preserved in PAXgene® tubes. Transcript expression was analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: Through a side-by-side comparison we found that the whole blood approach was suitable to detect AR-Vs. The specificity of the assay was corroborated in a cancer-free cohort. Using the PAXgene assay samples from a cohort of 46 patients with castration resistant prostate cancer were analyzed. Overall, AR-V7 and ARv567es were detected in 67.53% and 29.87% of samples, respectively. Statistical analysis revealed a strong association of AR-V positivity with a history of second line hormonal therapies. CONCLUSIONS: To our knowledge this is the first study to demonstrate that PAXgene preserved whole blood can be used to obtain clinically relevant information regarding the expression of 2 AR-Vs. These data on a castration resistant prostate cancer cohort support a role for AR-Vs in resistance to therapies targeting the AR ligand-binding domain.

Androgen receptor splice variants circumvent AR blockade by microtubule-targeting agents.

Docetaxel-based chemotherapy is established as a first-line treatment and standard of care for patients with metastatic castration-resistant prostate cancer. However, half of the patients do not respond to treatment and those do respond eventually become refractory. A better understanding of the resistance mechanisms to taxane chemotherapy is both urgent and clinical significant, as taxanes (docetaxel and cabazitaxel) are being used in various clinical settings. Sustained signaling through the androgen receptor (AR) has been established as a hallmark of CRPC. Recently, splicing variants of AR (AR-Vs) that lack the ligand-binding domain (LBD) have been identified. These variants are constitutively active and drive prostate cancer growth in a castration-resistant manner. In taxane-resistant cell lines, we found the expression of a major variant, AR-V7, was upregulated. Furthermore, ectopic expression of two clinically relevant AR-Vs (AR-V7 and ARV567es), but not the full-length AR (AR-FL), reduced the sensitivities to taxanes in LNCaP cells. Treatment with taxanes inhibited the transcriptional activity of AR-FL, but not those of AR-Vs. This could be explained, at least in part, due to the inability of taxanes to block the nuclear translocation of AR-Vs. Through a series of deletion constructs, the microtubule-binding activity was mapped to the LBD of AR. Finally, taxane-induced cytoplasm sequestration of AR-FL was alleviated when AR-Vs were present. These findings provide evidence that constitutively active AR-Vs maintain the AR signaling axis by evading the inhibitory effects of microtubule-targeting agents, suggesting that these AR-Vs play a role in resistance to taxane chemotherapy.

Androgen Receptor Splice Variants Dimerize to Transactivate Target Genes.

Constitutively active androgen receptor splice variants (AR-V) lacking the ligand-binding domain have been implicated in the pathogenesis of castration-resistant prostate cancer and in mediating resistance to newer drugs that target the androgen axis. AR-V regulates expression of both canonical AR targets and a unique set of cancer-specific targets that are enriched for cell-cycle functions. However, little is known about how AR-V controls gene expression. Here, we report that two major AR-Vs, termed AR-V7 and AR(v567es), not only homodimerize and heterodimerize with each other but also heterodimerize with full-length androgen receptor (AR-FL) in an androgen-independent manner. We found that heterodimerization of AR-V and AR-FL was mediated by N- and C-terminal interactions and by the DNA-binding domain of each molecule, whereas AR-V homodimerization was mediated only by DNA-binding domain interactions. Notably, AR-V dimerization was required to transactivate target genes and to confer castration-resistant cell growth. Our results clarify the mechanism by which AR-Vs mediate gene regulation and provide a pivotal pathway for rational drug design to disrupt AR-V signaling as a rational strategy for the effective treatment of advanced prostate cancer.

20(S)-protopanaxadiol inhibition of progression and growth of castration-resistant prostate cancer.

Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy.

Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy.

Upregulation of constitutively-active androgen receptor splice variants (AR-Vs) has been implicated in AR-driven tumor progression in castration-resistant prostate cancer. To date, functional studies of AR-Vs have been focused mainly on their ability to regulate gene expression independent of the full-length AR (AR-FL). Here, we showed that AR-V7 and ARv567es, two major AR-Vs, both facilitated AR-FL nuclear localization in the absence of androgen and mitigated the ability of the antiandrogen enzalutamide to inhibit AR-FL nuclear trafficking. AR-V bound to the promoter of its specific target without AR-FL, but co-occupied the promoter of canonical AR target with AR-FL in a mutually-dependent manner. AR-V expression attenuated both androgen and enzalutamide modulation of AR-FL activity/cell growth, and mitigated the in vivo antitumor efficacy of enzalutamide. Furthermore, ARv567es levels were upregulated in xenograft tumors that had acquired enzalutamide resistance. Collectively, this study highlights a dual function of AR-Vs in mediating castration resistance. In addition to trans-activating target genes independent of AR-FL, AR-Vs can serve as a "rheostat" to control the degree of response of AR-FL to androgen-directed therapy via activating AR-FL in an androgen-independent manner. The findings shed new insights into the mechanisms of AR-V-mediated castration resistance and have significant therapeutic implications.

Castration-resistant prostate cancer: adaptive responses in the androgen axis.

The androgen signaling axis in prostate cancer is associated with multiple adaptive mechanisms in response to castration. Herein we review these adaptations with an emphasis on recent molecular insights into the growth and development of castration resistant prostate cancer (CRPC). Alterations include both conventional and novel intracrine androgen synthesis pathways and androgen transport as well as androgen receptor (AR) overexpression, mutation, and splice variation. Each of these underlying mechanisms are potentially linked to post-castration growth, especially after treatment with newer hormonal agents such as abiraterone and enzalutamide. Post-translational AR modifications are well documented and these can affect receptor activity, stability, localization, and interaction with other proteins. Changes in recruitment of androgen receptor associated co-activators/repressors and a distinct AR-induced transcriptional program can dramatically alter proliferation, invasion, and metastasis in a ligand and context-dependent manner. Numerous previously uncharacterized non-coding RNAs, some of which are androgen regulated, may also have important biological function in this disease. Taken together, the view of CRPC has changed dramatically in the last several years. This has occurred not only within the setting of multiple treatment paradigm changes, but also as a multiplicity of potential molecular mechanisms underlying this disease state have been explored and discovered.

Methylselenol prodrug enhances MDV3100 efficacy for treatment of castration-resistant prostate cancer.

The next-generation antiandrogen MDV3100 prolongs overall survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patient responses are variable, and survival benefit remains relatively small. Developing effective modality to improve MDV3100 efficacy is urgently needed. Recent evidence suggests that constitutively active androgen receptor splice variants (AR-Vs) drive resistance to MDV3100. In our study, we show that methylselenol prodrug downregulates the expression and activity of both the full-length AR (AR-FL) and AR-Vs. The downregulation is independent of androgen and could be attributable to repressed transcription of the AR gene. Cotreatment with methylselenol prodrug and MDV3100 suppresses AR signaling more dramatically than either agent alone, and synergistically inhibits the growth of CRPC cells in vitro. The combinatorial efficacy is observed in not only AR-V-expressing cells but also cells expressing predominantly AR-FL, likely owing to the ability of the two drugs to block the AR signaling cascade at distinct steps. Ectopic expression of AR-FL or AR-V7 attenuates the combinatorial efficacy, indicating that downregulating AR-FL and AR-V7 is importantly involved in mediating the combinatorial efficacy. Significantly, methylselenol prodrug also downregulates AR-FL and AR-Vs in vivo and substantially improves the antitumor efficacy of MDV3100. These findings support a potential combination therapy for improving MDV3100 efficacy, and provide a rationale for evaluating the clinical application of combining methylselenol prodrug with MDV3100 for the treatment of CRPC.

20(S)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen receptor.

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

Splicing variants of androgen receptor in prostate cancer.

Significant advances in our understanding of continued androgen receptor (AR) signaling in castration-resistant prostate cancer have led to the development and FDA approval of two next-generation androgen-directed therapies, abiraterone and enzalutamide. These new therapies heralded a new era of prostate cancer therapy. However, disease progression during androgen-directed therapies remains the most critical challenge in the clinical management of prostate cancer. Accumulating evidence points to an important contribution of constitutively-active AR splice variants to AR-driven tumor progression during androgen-directed therapies. In this review, we will focus on the structure, activity, detection, clinical relevance, and mechanisms of production of AR splice variants.

[Progress on the epidemiology of echinococcosis].

Echinococcosis is a zoonotic parasitic disease caused by the larval stages belonging to the genus Echinococcus. Echinococcosis is a major public health problem in many countries and regions. The epidemiological study of echinococcosis would contribute to the control and elimination of this disease. This paper summarizes the research status and progress on epidemiology of echinococcosis.

Methylseleninic acid enhances paclitaxel efficacy for the treatment of triple-negative breast cancer.

A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.

Telomerase as an important target of androgen signaling blockade for prostate cancer treatment.

As the mainstay treatment for advanced prostate cancer, androgen deprivation therapy (ADT) targets the action of androgen receptor (AR) by reducing androgen level and/or by using anti-androgen to compete with androgens for binding to AR. Albeit effective in extending survival, ADT is associated with dose-limiting toxicity and the development of castration-resistant prostate cancer (CRPC) after prolonged use. Because CRPC is lethal and incurable, developing effective strategies to enhance the efficacy of ADT and circumvent resistance becomes an urgent task. Continuous AR signaling constitutes one major mechanism underlying the development of CRPC. The present study showed that methylseleninic acid (MSA), an agent that effectively reduces AR abundance, could enhance the cancer-killing efficacy of the anti-androgen bicalutamide in androgen-dependent and CRPC cells. We found that the combination of MSA and bicalutamide produced a robust downregulation of prostate-specific antigen and a recently identified AR target, telomerase, and its catalytic subunit, human telomerase reverse transcriptase. The downregulation of hTERT occurs mainly at the transcriptional level, and reduced AR occupancy of the promoter contributes to downregulation. Furthermore, apoptosis induction by the two agents is significantly mitigated by the restoration of hTERT. Our findings thus indicate that MSA in combination with anti-androgen could represent a viable approach to improve the therapeutic outcome of ADT. Given the critical role of hTERT/telomerase downregulation in mediating the combination effect and the fact that hTERT/telomerase could be measured in blood and urine, hTERT/telomerase could serve as an ideal tumor-specific biomarker to monitor the efficacy of the combination therapy noninvasively.

[Accumulation of phenolic acids in continuously cropped cucumber soil and their effects on soil microbes and enzyme activities].

The study showed that in solar greenhouse continuously cropped cucumber soil, phenolic acids p-hydroxybenzoic acid, ferulic acid and benzoic acid had an obvious accumulation with increasing cropping year, and their contents were significantly higher after continuously cropped for 5 approximately 9 years than for 1 approximately 3 years. With the increasing concentration of treated exogenous phenolic acids, the amounts of bacteria, actinomycetes, total microbes, ammonibacteria, and nitrifying bacteria in cucumber root area increased first, but decreased then. Soil bacteria and actinomycetes had the largest amount at the concentration of 80 microg phenolic acids x g(-1) soil, while soil fungi (including Fusarium and Phytophthora) increased rapidly when the concentration of phenolic acids was lower than 120 microg x g(-1) soil. With increasing phenolic acids concentration, soil enzyme activities also increased first but decreased then, with the peak values differed in different treatments.