T877A Mutation Research Articles

Computational approaches for evaluation of isobavachin as potential inhibitor against t877a and w741l mutations in prostate cancer

Prostate cancer is the World’s second most common cancer, with the fifth-highest male mortality rate. Point mutations such as T877A and W741L are frequently seen in advanced prostate cancer patients, conferring drug-resistance and hence driving cancer growth. Such occurrence of drug resistance in prostate cancer necessitates designing of suitable ligands to ensure better interactions with the receptors which can block the progression of the disease. The present study focus on the modification of plant-derived flavonoids that might act as inhibitors against such point mutations namely, T877A and W741L. In T877A mutation threonine is substituted by alanine at the 877 codon and W741L mutation, tryptophan is substituted by lysine at the 741 codon in prostate cancer. The study revolved on the aspect of the evaluation of Isobavachin and its derivatives as a potential agent to tackle such point mutations by using the in silico approach. A total of 98 molecular dockings were performed to find the ligand-receptor complexes with the lowest binding energy employing Autodock Software to conduct the blind and site-specific docking. Additionally, ligands were screened for Drug-likeness and toxicity using several tools yielding eight possible drug candidates. Based on the results of Molecular Docking, Drug-likeness, and ADMET testing, ten structures, including six complexes and three receptors were subjected to molecular dynamics simulation of 100 ns covering RMSD, RMSF, Rg, and MM/PBSA. Based on the simulation results, Isobavachin, IsoMod4, and IsoMod7 were concluded to be stable and exhibited potential properties for developing a novel drug to combat prostate cancer and its associated drug-resistance. Communicated by Ramaswamy H. Sarma

Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR.

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

New Insights into the Binding Mechanism of Co-regulator BUD31 to AR AF2 Site: Structural Determination and Analysis of the Mutation Effect.

Introduction Androgen Receptor (AR) plays a pivotal role in the development of male sex and contributes to prostate cancer growth. Different from other nuclear receptors that bind to the co-regulator LxxLL motif in coregulator peptide interaction, the AR Ligand Binding Domain (LBD) prefers to bind to the FxxLF motif. BUD31, a novel co-regulator with FxxLF motif, has been demonstrated to suppress wild-type and mutated AR-mediated prostate cancer growth.Methods To find out the interaction mechanisms of BUD31 with WT/T877A/W741L AR complex, molecular dynamics simulations were employed to study the complex BUD31 and WT/mutant ARs. The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) results demonstrated that T877A and W741L point mutations can reduce the binding affinity between BUD31 and AR. The RMSF and dynamic cross-correlation analysis indicated that amino acid point mutations can affect the motions of loop residues in the AR structure.Results These results indicated that AR co-regulator binding site AF2 can serve as a target for drug discovery to solve the resistance problem.

Interaction energies between two antiandrogenic and one androgenic agonist receptor in the presence of a T877A mutation in prostate cancer: a quantum chemistry analysis

We provide a detailed picture of the interactions between the androgen receptor T877A mutation and three different compounds used in the treatment of prostate cancer.

Resveratrol, piceatannol and analogs inhibit activation of both wild-type and T877A mutant androgen receptor

Prostate cancer growth and progression are mainly dependent on androgens and many current prostate cancer treatment options target the synthesis or function of androgens. We have previously reported that resveratrol and synthetic analogs of resveratrol with a higher bioavailability inhibit the synthesis of androgens in human adrenocortical H295R cells. Now we have studied the antiandrogenic properties of resveratrol, piceatannol and analogs in two different prostate cell lines; LNCaP and RWPE. LNCaP carry a T877A mutation in the androgen receptor while RWPE has a wild-type androgen receptor. We found that resveratrol, piceatannol and all studied analogs were able to inhibit a dihydrotestosterone-induced activation of the androgen receptor, showing that they act as antiandrogens. In LNCaP cells, all studied compounds were able to statistically significantly decrease the androgenic signaling in concentrations ≥1μM and the synthetic analogs trimethylresveratrol (RSVTM) and tetramethylpiceatannol (PICTM) were the most potent compounds. RWPE cells were not as responsive to the studied compounds as the LNCaP cells. A statistically significant decrease in the androgenic signaling was observed at concentrations ≤5μM for most compounds and RSVTM was found to be the most potent compound. Further, we studied the effects of resveratrol, piceatannol and analogs on the levels of prostate-specific antigen (PSA) in LNCaP cells and found that all studied compounds decreased the level of PSA and that the synthetic analogs diacetylresveratrol (RSVDA), triacetylresveratrol (RSVTA) and RSVTM were the most potent compounds, decreasing the PSA level by approx. 50% at concentrations ≥10μM. In a cell-free receptor binding assay we were unable to show binding of resveratrol or analogs to the ligand binding domain of the androgen receptor, indicating that the observed effects are mediated via other mechanisms than direct ligand competition. We conclude that the resveratrol, piceatannol and analogs are highly interesting for chemoprevention of prostate cancer, since they have a high potency both as inhibitors of androgen synthesis and androgen receptor activation.

A Molecular Modeling Study of the Hydroxyflutamide Resistance Mechanism Induced by Androgen Receptor Mutations.

Hydroxyflutamide (HF), an active metabolite of the first generation antiandrogen flutamide, was used in clinic to treat prostate cancer targeting androgen receptor (AR). However, a drug resistance problem appears after about one year’s treatment. AR T877A is the first mutation that was found to cause a resistance problem. Then W741C_T877A and F876L_T877A mutations were also reported to cause resistance to HF, while W741C and F876L single mutations cannot. In this study, molecular dynamics (MD) simulations combined with the molecular mechanics generalized Born surface area (MM-GBSA) method have been carried out to analyze the interaction mechanism between HF and wild-type (WT)/mutant ARs. The obtained results indicate that AR helix 12 (H12) plays a pivotal role in the resistance of HF. It can affect the coactivator binding site at the activation function 2 domain (AF2, surrounded by H3, H4, and H12). When H12 closes to the AR ligand-binding domain (LBD) like a lid, the coactivator binding site can be formed to promote transcription. However, once H12 is opened to expose LBD, the coactivator binding site will be distorted, leading to invalid transcription. Moreover, per-residue free energy decomposition analyses indicate that N705, T877, and M895 are vital residues in the agonist/antagonist mechanism of HF.

Abstract 1720: Circulating tumor cell (CTC) enrichment and DNA mutation detection by sequencing

Abstract Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. To evaluate the potential of utilizing PREP100 in CTC molecular analysis, we first prepared mock samples using cultured prostate cancer cells PC3 and LnCAP and analyzed captured cells by PCR amplicon sequencing. Using Celsee PREP100, we were able to recover the average of 79% (ranging from 40% to 100%) of 110 - 1,100 PC3 cells and 60 - 1,500 LnCAP cells and detect p.K139fs*3 deletion in PC3 cells and T877A mutation in LnCAP cells. In addition, we also spiked in the two cell lines into normal donor blood samples and analyzed the captured cells by PCR amplicon sequencing. Preliminary experiments showed that 40% of 250 spiked PC3 cells and 74% of 50 spiked LnCAP cells were captured. Further, we were able to detect p.K139fs*3 deletion in the retrieved 50 - 1,000 spiked PC3 cells. Our results illustrate that Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs. Citation Format: Ruqin Kou, Eric Zhao, Conrad Leung, Priya Gogoi, Will Chow, Yixin Wang. Circulating tumor cell (CTC) enrichment and DNA mutation detection by sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1720. doi:10.1158/1538-7445.AM2017-1720

Molecular Dynamics Studies on the Enzalutamide Resistance Mechanisms Induced by Androgen Receptor Mutations.

The second-generation antiandrogen enzalutamide, targeting androgen receptor (AR), was approved to treat castration resistant prostate cancer (CRPC) in 2012. Its resistance was observed when it was in the clinical research stage. AR mutation is the main factor of enzalutamide resistance. AR F876L and F876L_T877A mutations were reported to switch enzalutamide from AR antagonist to agonist, but W741C cannot. There are various mutations in the ligand binding domain of AR LBD, such as L701H, W741L, H874Y, T877A, and M895T, if these mutations can lead to drug resistance problem or not is not known. In this work, molecular dynamics (MD) simulations and molecular mechanics Generalized Born (GB) surface area (MM-GBSA) calculations were employed to explore the interaction mechanisms between enzalutamide and wild-type (WT)/mutant ARs. The simulation results indicate that helix 12 (H12), which lies on the top of the AR LBD like a cover, plays a vital role for the function of enzalutamide. When C-ring of enzalutamide locates near to H12, the distance between enzalutamide and H12 is reduced, which prevents H12 from closing and distort the coactivator binding site, resulting in the inactivation of transcription. In this case, enzalutamide acts as an AR antagonist. However, when the C-ring of enzalutamide is near to helix H11 or the Loop 11-12, H12 tends to close to form a coactivator binding site to facilitate transcription, enzalutamide acts as an AR agonist. Moreover, per-residue free energy decomposition analysis indicates that M895 and I899 are key residues in the antagonist mechanism of enzalutamide. J. Cell. Biochem. 118: 2792-2801, 2017. © 2017 Wiley Periodicals, Inc.

Abstract A20: Use of cell-free plasma DNA for dynamic monitoring of response to cytotoxic chemotherapy

Abstract Background: There is great interest in utilizing blood-based circulating biomarkers to gain molecular insight into cancer. Most of the published data examines circulating free DNA (cfDNA) obtained at a single time-point. Here, we developed and utilized an methodology for monitoring cfDNA at multiple time-points within 24 hours of cytotoxic chemotherapy infusions over sequential cycles. Methods: To monitor the short- and long-term responses to treatment, we analyzed cfDNA from plasma drawn immediately before (baseline), 5 hours and 24 hours after administration of docetaxel chemotherapy. We quantified the extracted cfDNA via qPCR of Long interspersed nuclear elements (LINE1). We then amplified and performed targeted deep sequencing (Illumina Miseq) of androgen receptor (AR) exon 8. To reduce PCR variability, we pooled 10 technical replicate amplifications. Results: Detailed data and time courses are presented for a patient with castrate resistant prostate cancer metastatic to bone treated with docetaxel/everolimus/bevacizumab according to a recently published trial (J Clin Oncol 33, 2015 suppl; abstr 5066). LINE1 was measured at 34.4 pg/mcl at baseline prior to treatment. Targeted deep sequencing of AR exon 8 revealed two independent mutations at 11% (H874A) and 13% (T877A), respectively. 5 hours after start of docetaxel infusion, total DNA content measured by LINE1 decreased to 0.4 pg/mcl. However, H874A and T877A mutations were observed at frequencies similar to the baseline values. A similar pattern was observed for samples obtained at Cycle 2 (21 days later). Specifically, baseline values for LINE1 (67.4 pg/mcl), H874A (3%), and T877A (7%) were noted to change to LINE1 (8.5 pg/mcl), H874A (6%), and T877A (17%) 5 hours later. Conclusion: We conclude that cytotoxic chemotherapy can produce effects immediately observable on cfDNA markers. The generalizability and clinical relevance of these findings will require follow up studies involving more patients. Citation Format: Daniel Ruderman, Katherin Patsch, Naim Matasci, Anjana Soundararajan, Patricia Diaz, Mitchell E. Gross. Use of cell-free plasma DNA for dynamic monitoring of response to cytotoxic chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A20.

Molecular mechanism of R-bicalutamide switching from androgen receptor antagonist to agonist induced by amino acid mutations using molecular dynamics simulations and free energy calculation.

R-bicalutamide, a first generation antiandrogen, was used to treat prostate cancer for decades. Although it is very effective at the beginning, resistance appears after 2-3years of treatment. Mutation of androgen receptor (AR) is considered a main reason for drug resistance. It is reported that AR W741C, W741L, W741C_T877A, T877A, F876L, F876L_T877A and L701H mutations can convert R-bicalutamide from AR antagonist to agonist, but the switching mechanisms are not clear. In this study, molecular dynamics simulations and molecular mechanics generalized Born surface area (MM-GBSA) calculations were performed to analyze the interaction mechanisms between R-bicalutamide and wild type/mutant ARs. The results indicate that helix H12, which lies on the top of AR LBD like a cover, plays a vital role in R-bicalutamide binding. When interacting with AR, the B-ring of R-bicalutamide pushes H12 aside, distorting the coactivator binding site (AF2) resulting in the inactivation of transcription. Several residue mutations appear to enlarge the distance between the B-ring of R-bicalutamide and H12, reducing steric clash, which is conducive to a closed H12 conformation, leading to the formation of the coactivator binding site AF2 and increased transcription. Hydrogen bond and per-residue free energy decomposition analyses are also investigated to explore the interacting mechanisms, and M895 is found to be a key residue in the antagonist mechanism. The obtained molecular mechanisms will aid rational screening and design of novel AR antagonists, even to mutant AR.

Abstract CT134: Androgen receptor (AR) mutations in patients (pts) with castration-resistant prostate cancer (CRPC) with and without prior abiraterone acetate (AA) treatment

Abstract Background: ARN-509 is a second-generation antiandrogen with antitumor activity in CRPC. Activating mutations in the AR ligand-binding domain (LBD) have been associated with resistance to first- (T877A) and second- (F876L) generation antiandrogens. We evaluated the type and frequency of relevant AR LBD mutations in ARN-509-treated CRPC pts at baseline (BL) and disease progression (PD). Methods: ARN-509-001 was a phase I/II study that evaluated ARN-509 activity in nonmetastatic (M0), chemotherapy-naïve, and post-AA CRPC. Of the 97 pts enrolled in phase II at a dose of 240 mg/d, 92 were evaluable for the mutation analysis at BL and 82 at PD. Relevant mutations in circulating tumor DNA were detected using a digital PCR method called BEAMing (Beads, Emulsification, Amplification, and Magnetics) (Richardson AL. Clin Cancer Res. 2012). Results: Median duration of therapy was ∼16 months. One pt in the M0 cohort and one in the chemotherapy-naïve cohort had the F876L mutation at BL. Two pts in the chemotherapy-naïve cohort and one in the post-AA cohort acquired the AR F876L mutation during treatment. Pts with M0 CRPC did not acquire a mutation (Table 1). Three pts in the post-AA cohort had the T877A mutation at BL; the T877A mutation was not detected in any other cohort at BL. In the post-AA cohort, one pt acquired the T877A mutation during treatment while another lost the mutation (Table). The two pts with detectable F876L at BL developed prostate-specific antigen (PSA) progression at 4 and 6 months, respectively, compared with a median time to PSA progression of 16.4 months in the remainder of pts. Conclusions: Pts with metastatic CRPC who were treated with ARN-509 had a low rate of acquisition of the AR F876L (3/82 = 4%) and AR T877A (1/82 = 1%) mutations. These results suggest that ARN-509 may be continued in the setting of a rising PSA. Larger studies are needed to confirm the prevalence of F876L, T877A, and the conversion rate. AR F876LAR T877AEvaluableBLPDAcquiredBLPDAcquiredpatientsn/Nn/Nn/Nn/NTotal922/925/823/823/923/821/82M0491/491/470/470/500/470/47Chemotherapy-naïve241/243/202/200/200/200/20Post-AA190/191/151/153/193/151/15 Citation Format: Dana E. Rathkopf, Matthew R. Smith, Emmanuel S. Antonarakis, Charles J. Ryan, William R. Berry, Neal D. Shore, Glenn Liu, Celestia Higano, Joshi J. Alumkal, Ralph Hauke, Ronald Tutrone, Mansoor Saleh, Edna Chow Maneval, Shibu Thomas, Deborah Ricci, Margaret K. Yu, Carla J. de Boer, Angela Trinh, Thian Kheoh, Rajesh Bandekar, Howard I. Scher. Androgen receptor (AR) mutations in patients (pts) with castration-resistant prostate cancer (CRPC) with and without prior abiraterone acetate (AA) treatment. [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 CT134. doi:10.1158/1538-7445.AM2015-CT134

Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer.

Although novel agents targeting the androgen-androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients. Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR. On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ(2)). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ(2)) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression). AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC.

Direct effects of the selective CYP17 lyase (L) inhibitor, VT-464, on the androgen receptor (AR) and its oral activity in an F876L tumor mouse xenograft model.

263 Background: MDV3100 inhibits binding of androgens to AR and abiraterone is known to block androgen production through CYP17 inhibition; both are effective treatments for castration-resistant prostate cancer (CRPC, yet cross-resistance to both agents is problematic. The AR mutations F876L and T877A increase resistance to MDV3100 (M) or abiraterone (A), respectively. A regimen resistance is likely mediated through T877A activation by stimulatory progesterone (P4), pregnenolone (P5), or prednisone. The oral, non-steroidal L-inhibitor, VT-464, in phase 2 CRPC studies without prednisone coadministration, does not increase P4 or P5. Methods: The effects of VT-464, A, and M on AR transcription activity and cell proliferation were assessed and compared using reporter assays, and their oral effects in an M-resistant (F876L) human tumor mouse model were also assessed. Results: VT-464 competitively antagonized (Schild analysis) wild-type AR (IC50 =18 µM) and its potency was not decreased by AR overexpression (VCaP IC50=7.4μM). VT-464 fully antagonized AR T877A (IC50=0.52 μM) and inhibited LNCaP cell (AR T877A mutation) proliferation (IC50=0.70 μM) with potency similar to M. In contrast to M, VT-464 fully inhibited F876L (IC50=2.5 μM). VT-464 did not activate any AR forms up to the highest concentration tested (60 μM). In vivo, greater F876L tumor inhibition was observed with oral VT-464 compared to oral M or abiraterone acetate. Conclusions: VT-464 more potently antagonized mutated AR forms associated with A or M resistance than wild-type; AR antagonism contributed to xenograft activity and appears relevant to its clinical pharmacology. Due to its high L inhibition selectivity and potent mutant AR antagonist activity, VT-464 has a suitable profile for the treatment of early- or late-stage CRPC patients. VT-464 has advanced into phase 2 studies funded by Innocrin (NCT#02012920), the NCI (NCT02130700), and PCF in M- and A-resistant populations.

18 F-RB390: Innovative ligand for imaging the T877A androgen receptor mutant in prostate cancer via positron emission tomography (PET)

Detecting prostate cancer before spreading or predicting a favorable therapy are challenging issues for impacting patient's survival. Presently, 2-[(18) F]-fluoro-2-deoxy-D-glucose ((18) F-FDG) and/or (18) F-fluorocholine ((18) F-FCH) are the generally used PET-tracers in oncology yet do not emphasize the T877A androgen receptor (AR) mutation being exclusively present in cancerous tissue and escaping androgen deprivation treatment. We designed and synthesized fluorinated 5α-dihydrotestosterone (DHT) derivatives to target T877A-AR. We performed binding assays to select suitable candidates using COS-7 cells transfected with wild-type or T877A AR (WT-AR, T877A-AR) expressing plasmids and investigated cellular uptake of candidate (18) F-RB390. Stability, biodistribution analyses and PET-Imaging were assessed by injecting (18) F-RB390 (10MBq), with and without co-injection of an excess of unlabeled DHT in C4-2 and PC-3 tumor bearing male SCID mice (n = 12). RB390 presented a higher relative binding affinity (RBA) (28.1%, IC50 = 32 nM) for T877A-AR than for WT-AR (1.7%, IC50 = 357 nM) related to DHT (RBA = 100%). A small fraction of (18) F-RB390 was metabolized when incubated with murine liver homogenate or human blood for 3 hr. The metabolite of RB390, 3-hydroxysteroid RB448, presented similar binding characteristics as RB390. (18) F-RB390 but not (18) F-FDG or (18) F-FCH accumulated 2.5× more in COS-7 cells transfected with pSG5AR-T877A than with control plasmid. Accumulation was reduced with an excess of DHT. PET/CT imaging and biodistribution studies revealed a significantly higher uptake of (18) F-RB390 in T877A mutation positive xenografts compared to PC-3 control tumors. This effect was blunted with DHT. Given the differential binding capacity and the favorable radioactivity pattern, (18) F-RB390 represents the portrayal of the first imaging ligand with predictive potential for mutant T877A-AR in prostate cancer for guiding therapy. Prostate 75:348-359, 2015. © 2014 Wiley Periodicals, Inc.

Abstract 3930: Ligand-independent activity of the mutant LNCaP androgen receptor drives cell proliferation in hormone-independent LNCaP tumor cells

Abstract Androgen-dependent prostate tumor cells can adapt to androgen ablation by up-regulating expression of the androgen receptor (AR). Increased expression of AR may sensitize cells to residual androgens in the environment or to de novo-synthesized ligands. We have shown previously that hormone-independent LNCaP 104-R1 cells, which arise after long-term androgen deprivation of androgen-dependent 104-S cells, express much higher levels of AR than 104-S cells. LNCaP prostate tumor cells express a mutant T877A AR that can be activated by progesterone, raising the possibility that LNCaP cells progress to apparent hormone independence during androgen deprivation by initiating de novo synthesis of progesterone. In support of this, we have found that androgen-dependent LNCaP 104-S cells grow independently of exogenous androgen after lentivirus-mediated over-expression of T877A AR, but not wild-type (wt) AR. This difference was attributable to the T877A substitution and not to sequence differences in the N-terminus between wt and LNCaP cell-derived AR. However, this growth was insensitive to the anti-androgen bicalutamide, similar to the bicalutamide-insensitive growth of hormone-independent 104-R1 cells. Lentiviral sh-RNAi-mediated knockdown of AR resulted in growth inhibition in both 104-S and 104-R1 cells. Analysis of T877A AR transactivation in 104-S and 104-R1 cells by reporter gene assay revealed that T877A AR displayed significant constitutive and bicalutamide-insensitive activity. Wild-type AR, on the other hand, showed low basal activity in the absence of added ligand and modest induction by bicalutamide. Co-expression of CYP11A1 (cholesterol side chain cleavage enzyme) and HSD3B2 (3-beta hydroxysteroid dehydrogenase-2), which sequentially generate pregnenolone and progesterone, strongly activated T877A AR, but not wt AR in 104-S cells. This activation could be inhibited by bicalutamide or ketoconazole, an inhibitor of CYP11A1. Expression of CYP11A1 alone in 104-S cells increased cell proliferation in the absence of androgen that was bicalutamide-sensitive. This indicates that 104-S cells may express low level but functional HSD3B activity, because pregnenolone does not activate T877A AR. Secondly, the bicalutamide-sensitivity of progesterone-driven cell proliferation and bicalutamide-insensitivity of T877A AR-driven proliferation, together with the observations above, suggest that de novo synthesis of progesterone or another ligand in LNCaP cells is most likely not responsible for T877A AR-driven proliferation in cells in which T877A AR is over-expressed. Rather, the T877A mutation may confer upon the receptor constitutive ligand-independent activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3930. doi:1538-7445.AM2012-3930

Modulation of androgen receptor signaling in hormonal therapy-resistant prostate cancer cell lines.

BackgroundProstate epithelial cells depend on androgens for survival and function. In (early) prostate cancer (PCa) androgens also regulate tumor growth, which is exploited by hormonal therapies in metastatic disease. The aim of the present study was to characterize the androgen receptor (AR) response in hormonal therapy-resistant PC346 cells and identify potential disease markers.Methodology/Principal FindingsHuman 19K oligoarrays were used to establish the androgen-regulated expression profile of androgen-responsive PC346C cells and its derivative therapy-resistant sublines: PC346DCC (vestigial AR levels), PC346Flu1 (AR overexpression) and PC346Flu2 (T877A AR mutation). In total, 107 transcripts were differentially-expressed in PC346C and derivatives after R1881 or hydroxyflutamide stimulations. The AR-regulated expression profiles reflected the AR modifications of respective therapy-resistant sublines: AR overexpression resulted in stronger and broader transcriptional response to R1881 stimulation, AR down-regulation correlated with deficient response of AR-target genes and the T877A mutation resulted in transcriptional response to both R1881 and hydroxyflutamide. This AR-target signature was linked to multiple publicly available cell line and tumor derived PCa databases, revealing that distinct functional clusters were differentially modulated during PCa progression. Differentiation and secretory functions were up-regulated in primary PCa but repressed in metastasis, whereas proliferation, cytoskeletal remodeling and adhesion were overexpressed in metastasis. Finally, the androgen-regulated genes ENDOD1, MCCC2 and ACSL3 were selected as potential disease markers for RT-PCR quantification in a distinct set of human prostate specimens. ENDOD1 and ACSL3 showed down-regulation in high-grade and metastatic PCa, while MCCC2 was overexpressed in low-grade PCa.Conclusions/SignificanceAR modifications altered the transcriptional response to (anti)androgens in therapy-resistant cells. Furthermore, selective down-regulation of genes involved in differentiation and up-regulation of genes promoting proliferation and invasion suggest a disturbed balance between the growth and differentiation functions of the AR pathway during PCa progression. These findings may have implications in the current treatment and development of novel therapeutical approaches for metastatic PCa.

Androgen receptor W741C and T877A mutations in AIDL cells, an androgen-independent subline of prostate cancer LNCaP cells

The androgen-independent LNCaP (AIDL) cell line was generated by maintaining prostate cancer LNCaP cells in a hormone-deprived medium. Notably, synthetic androgen R1881-related gene response is attenuated in AIDL cells as compared to the parental LNCaP cells. The aim of this study was to clarify the mechanisms underlying androgen sensitivity in AIDL cells. We first examined the expression of androgen receptor (AR) and its co-regulators. However, no significant difference in mRNA expression was found between LNCaP and AIDL cells. Remarkably, AR protein levels were induced by R1881 and DHT in LNCaP cells, but not in AIDL cells. We next performed the cDNA sequencing to detect mutations in the AR gene. The T877A mutation was detected both in LNCaP and AIDL cells. Furthermore, AIDL cells harbored a missense substitution (TGG → TGT) in the AR gene, which caused a point mutation at codon 741 (W741C). Double T877A and W741C AR mutants have been previously reported to exhibit reduced androgen sensitivity. Hence, the low-androgen-sensitive responses of AIDL cells may be explained, at least in part, by AR gene mutations.

Abstract 1864: Differential effects of the isoflavone genistein on androgen receptor expression and cell proliferation comparing prostate cancer cells with mutant and wild type androgen receptor

Abstract In advanced prostate cancer, cells become androgen independent and therefore resistant to androgen ablation therapy. One mechanism by which prostate cancer bypasses androgen ablation therapy is acquisition of androgen receptor (AR) mutations some of which render it promiscuous enabling activation by a broad group of steroids such as estrogen, progesterone, and even anti-androgens. Genistein, an exogenous steroid (phytoestrogen) and isoflavone found in soy, has estrogenic activity. Most studies have shown that genistein has anti-proliferative effects on prostate cancer cells without regard for the status of the AR. It has been suggested that activation of the mutant AR (MT-AR) by genistein could lead to increased cellular proliferation. Previous studies examining genistein's effect on AR expression used prostate cell lines (i.e. LNCaP) with ARs carrying the promiscuous T877A mutation, without comparison to cells with wild type (WT) AR. Genistein does not bind WT-AR, and thus, may not have the same effect on AR expression as it does in the presence of MT-AR. We set out to compare the effects of genistein in the presence of WT-AR versus MT-AR, using human prostate cancer cell lines with WT-AR (LAPC-4) and MT-AR (LNCaP). Cells were treated with increasing concentrations (0, 0.5, 1, 10, 25, and 50 μM) of genistein. Real time PCR, Western blot analysis, PSA luciferase assay, cell counting by hemocytometer, and MTS proliferation assays were used to determine levels of AR mRNA, protein, transcriptional activity, and cell proliferation, respectively. Genistein caused an androgen-independent biphasic changes in AR mRNA, protein levels, and transcriptional activity, and in cell proliferation in LNCaP cells, which reached a maximum at 1μM of genistein (40-45% increase in AR protein and mRNA expression, 42% increase in PSA luciferase activity, and 2-fold increase in cell proliferation compared to controls). These effects reversed at a concentration of 10 μM. In contrast, in LAPC-4 cells AR mRNA and protein levels and transcriptional activity as well as cell proliferation decreased linearly with increasing dose of genistein, without showing the stimulatory bi-phasic trend observed in LNCaP cells. These decreases were significant, beginning at 1 μM genistein, at which concentration AR mRNA was reduced by 30%, protein expression by 22%, PSA luciferase activity by 40%, and cell proliferation by 56%. These results demonstrate that at physiological concentrations, genistein can exert a mitogenic effect in the presence of MT-AR. Our findings highlight the significance of promiscuous mutations such as T877A for the AR response to genistein treatment and indicate that men with advanced prostatic cancers which carry such AR mutations could be adversely affected by genistein. (Supported in part by Grant No. CA116195) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2011-1864

Noncanonical Wnt signaling mediates androgen-dependent tumor growth in a mouse model of prostate cancer

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.

Tumor Suppressor Function of Androgen Receptor Coactivator ARA70α in Prostate Cancer

Androgen receptor (AR), a member of the steroid receptor family, is a transcription factor that has an important role in the regulation of both prostate cell proliferation and growth suppression. AR coactivators may influence the transition between cell growth and growth suppression. We have shown previously that the internally spliced ARA70 isoform, ARA70beta, promotes prostate cancer cell growth and invasion. Here we report that the full length ARA70alpha, in contrast, represses prostate cancer cell proliferation and anchorage-independent growth in vitro and inhibits tumor growth in nude mice xenograft experiments in vivo. Further, the growth inhibition by ARA70alpha is AR-dependent and mediated through induction of apoptosis rather than cell cycle arrest. Interestingly, AR with T877A mutation in LNCaP cells decreased its physical and functional interaction with ARA70alpha, facilitating the growth of LNCaP cells. The tumor suppressor function of ARA70alpha is consistent with our previous findings that ARA70alpha expression is decreased in prostate cancer cells compared with benign prostate. ARA70alpha also reduced the invasion ability of LNCaP cells. Although growth inhibition by ARA70alpha is AR-dependent, the inhibition of cell invasion is an androgen-independent process. These results strongly suggest that ARA70alpha functions as a tumor suppressor gene.