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.

The androgen receptor (AR) is critical in the normal development and function of the prostate, as well as in prostate carcinogenesis. Androgen deprivation therapy is the mainstay in the treatment of advanced prostate cancer, however, after an initial response, the disease inevitably progresses to castration-resistant prostate cancer (CRPC). Recent evidence suggests that continued AR activation, sometimes in a ligand-independent manner, is commonly associated with the development of CRPC. Thus, novel agents targeting the AR are urgently needed as a strategic step in developing new therapies for this disease state. In this study, we investigated the effect of berberine on AR signaling in prostate cancer. We report that berberine decreased the transcriptional activity of AR. Berberine did not affect AR mRNA expression, but induced AR protein degradation. Several ligand-binding domain truncated AR splice variants have been identified and these variants are believed to promote the development of CRPC in patients. Interestingly, we found that these variants were more susceptible to berberine-induced degradation than the full-length AR. Furthermore, the growth of LNCaP xenografts in nude mice was inhibited by berberine and AR expression was reduced in the tumors, whereas the morphology and AR expression in normal prostates were not affected. This report is the first to show that berberine suppresses AR signaling and suggests that berberine or its derivatives is a promising agent for the prevention and/or treatment of prostate cancer.

The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)-and 2-oxoglutarate-dependent dioxygenases that efficiently repair N 1 -methyladenine and N 3 -methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N 1 -methyladenine and N 3 -methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo. In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.The nucleic acids in living cells are subject to modification by both endogenous and environmental agents (1). Direct-acting chemicals constantly damage nucleic acids and generate various methyl lesions with mutagenic and/or cytotoxic consequences (2, 3). O 6 -Methylguanine (O 6 mG) 2 and N 3 -methyladenine lesions have the highest potential for methylating damage by an SN1 agent such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), which block replication and are thought to be toxic (4, 5). For the most part, the SN2 agent such as methyl methane sulfonate (MMS) produces N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) lesions in single-stranded DNA (ssDNA). Accumulation of these adducts can lead to cell death (6, 7). Organisms have evolved several mechanisms to efficiently remove various methyl lesions, including suicidal methyltransferases, DNA glycosylases, and the AlkB family dioxygenases (see Fig. 1A) (8, 9). To date, AlkB repair appears to be the major natural defense mechanism with the power to restore the canonical base structure in vivo. Escherichia coli AlkB and its human homologues, ALKBH2 and ALKBH3, utilize iron(II) and 2-oxoglutarate (2OG) to achieve oxidative demethylation of m 1 A and m 3 C (see Fig. 1B) (10 -12). The lack of AlkB repair results in increased sensitivity to MMS, elevated level of mutations, and reduced cell proliferation (13-16). Furthermore, the accumulation of m 1 A and m 3 C lesions could also occur on RNA. Research suggests that the oxidative demethylation in mRNA and tRNA acts as a part of AlkB or ALKBH3 repair to protect cells against MMS (17, 18). The scope of substrates for AlkB repair has been largely extended to all simple N-alkyl lesions at the WatsonCrick base-pairing interface on the four bases (19), thus indicating the importance of oxidative demethylation for cell survival. In addition, human enzymes have been broadly linked to cancer. The housekeeping ...

Purpose The majority of prostate cancer mortality can be attributed to metastatic castration-resistant prostate cancer, an advanced stage which remains incurable despite recent advances. The androgen receptor (AR) signaling axis remains active in CRPC. Recent studies suggest that the expression of an AR splice variant, AR-V7, may underlie resistance to abiraterone and enzalutamide. However, controversy exists over the optimal assay. The objective of this study is to develop a fast and sensitive assay for AR splice variants (AR-Vs) in patients. Materials and Methods Two approaches were assessed in this study. The first was based on depletion of leukocytes and the second used RNA purified directly from whole blood preserved in PAXgene tubes. Transcript expression was analyzed by quantitative RT-PCR. Results Through side-by-side comparison, we concluded that the whole-blood approach was suitable for the detection of AR-Vs. The specificity of the assay was corroborated in a cancer-free cohort. Using the PAXgene assay, samples from a cohort of 46 CRPC patients were analyzed. Overall, AR-V7 and ARv567es were detected in 67.53% and 29.87% of the 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 study is the first to demonstrate PAXgene-preserved whole blood can be used to obtain clinically relevant information regarding the expression of two AR-Vs. The data from a CRPC cohort support a role of AR-Vs in resistance to therapies targeting the AR ligand-binding domain.