Conclusions: Our approach proved effective for efficient lead finding, activity enhancement and preliminary control of selectivity over other receptors. The phenanthridinone skeleton appears to be a promising steroid surrogate.Background: Nuclear receptors (NRs) are considered as potential drug targets because they control diverse biological functions. However, steroidal ligands for NRs have the potential to cross-react with other nuclear receptors, so development of non-steroidal NR ligands is desirable to obtain safer agents for clinical use. We anticipated that efficient lead finding and enhancement of activity toward nuclear receptors recognizing endogenous steroidal ligands might be achieved by exhaustive evaluation of a steroid surrogate library coupled with examination of structure-activity relationships (SAR). Method: We evaluated our library of RORs (retinoic acid receptor-related orphan receptors) inverse agonists and/or PR (progesterone receptor) antagonists based on the phenanthridinone skeleton for antagonistic activities toward liver X receptors (LXRs), androgen receptor (AR) and glucocorticoid receptor (GR) and examined their SAR. Results: Potent LXRβ, AR, and GR antagonists were identified. SAR studies led to a potent AR antagonist (IC Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate DNA transcription by binding small molecular agonists such as hormones. Forty-eight structurally conserved kinds of NRs have been identified, and many of them recognize endogenous steroidal ligands . BecauseThe number of fold structures of human proteins is at least 50 times smaller than the number of human proteins . This fa1 [2 [3 [4 and 5 for Niemann-Pick disease type C1 (NPC1) [H)-one skeleton as a cyclized carba-analog of the skeleton of LXRs pan agonist T0901317 (6). Because endogenous ligands for the above proteins  all have a steroid skeleton, we hypothesized that if the phenanthridin-6(5H)-one scaffold acts as a steroid surrogate, phenanthridinone derivatives would also bind to other NRs that recognize endogenous steroidal ligands. The approach described here is expected to be useful in the development of novel steroid surrogates, such as the phenanthridinone skeleton, enabling efficient lead finding of ligands for target proteins that recognize endogenous steroidal ligands, and the generation of more selective ligands by further molecular modifications of new and convenient scaffolds  at any position, etc.), compared to the steroid skeleton. Here, we show that application of this approach to our small library of phenanthridinone-based RORs inverse agonists and PR antagonists led to several selective LXRβ, AR and glucocorticoid receptor (GR) antagonists.We have previously developed LXRs antagonist 1 , retinoi1 [2 , progest1 [2 [3 , and pha1 (NPC1)  and Niem1 (NPC1) . These cAR and GR, members of NRs, recognize endogenous steroidal ligands as shown in 1–3, 7–42 and 44 were prepared as described previously [50 values. Reproducibility of our assay systems is shown in 6), RU486, and OHF, are shown in 8–10) or a hydroxymethyl group (11) at the 2-position of 7 resulted in retention or decrease of the PR-antagonistic activity [6 and human LXRs LBD, the hydroxyl group of 6 forms a hydrogen bond with a histidine residue (His421 for LXRα and His435 for LXRβ) in helix 11 [11) resulted in retention of the activity, whereas introduction of hexafluoropropanol (12) resulted in 6-fold and 9-fold increases of PR and AR activity, respectively. On the other hand, for LXRs and RORs, 11 showed weaker activity than 7 whereas 12 showed stronger activity than 7, suggesting that the two trifluoromethyl groups might be associated with more potent activity and the hydroxyl group might not form a strong hydrogen bond with histidine in these receptors, in contrast to the other receptors. This idea is consistent with the reported co-crystal structure of T0901317 (6) complexed with RORγ [12 showed not only PR-antagonistic activity and RORs inverse agonistic activity, but also LXRα, LXRβ, AR and GR antagonistic activity (H)-one scaffold acts as a steroid surrogate.For this work, compounds eviously ,10,15. Peviously  while ROeviously ,10. Concactivity . Introduhelix 11 ,18. The ith RORγ . Overallactivity . These r12 and 15) resulted in enhancement of the GR antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent AR antagonistic activity with an IC50 value of 0.10 μM, and more than 200-fold selectivity for AR over RORs and GR, and about 7.8- and 50-fold selectivity over PR and LXRs, respectively. The longest alkyl analog 17 showed decreased activity toward all NRs, suggesting that the binding pocket hosting the N-alkylated derivatives might not be able to accommodate a group larger than a hexyl group.Next, SAR at the nitrogen atom is shown in 12 and 15) resulted in enhancement of the GR antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent AR antagonistic activity with an IC50 value of 0.10 μM, and more than 200-fold selectivity for AR over RORs and GR, and about 7.8- and 50-fold selectivity over PR and LXRs, respectively. The longest alkyl analog 17 showed decreased activity towards all NRs, suggesting that the binding pocket hosting the N-alkylated derivatives might not be able to accommodate a group larger than a hexyl group.Next, SAR at the nitrogen atom is shown in 18–24; 19 and 20 showed more than 3-fold and 2-fold enhanced PR activity compared with the unsubstituted analog 12, respectively [23 showed 5-fold weaker PR activity than 12. In the case of RORs, we have reported that introduction of a methoxy group at the 9-position (23) enhanced ROR activities [18 showed decreased activity for all receptors tested. Enhanced activity was observed when a methoxy group was introduced at the 4-, 9- or 10-position for LXRα, 9- or 10-position for LXRβ, 3- and 8-position for AR, and 3- or 4-position for GR. Especially, 3-methoxy analog 19 showed 5.7-fold improved GR activity compared with 12. Overall, introduction of a methoxy group resulted in increased activity in almost all cases except RORβ, but the most suitable position depended on the NR . The introduction of a substituent at every position is useful to investigate substituent effects. In this context, it is important to note that phenanthridin-6(5H)-one analogs bearing substituent(s) at any position can be quite easily synthesized, and this represents a considerable advantage compared to the steroid skeleton.Next, the effect of introduction of a methoxy group at every position (methoxy scanning) was investigated 18–24;  because ectively . In cont25–3125 exhibited similar or weaker activity to the 3-methoxy and 4-methoxy analogs 19 and 20. A possible explanation might be the direction of the two methoxy groups, that is, one or both might occupy a space not suitable for receptor interaction due to steric hindrance. This idea is supported by the fact that 3,4-dioxolane analog 26 showed stronger activity than 25 toward PR, AR and GR. Compound 26 was the most potent GR antagonist with an IC50 value of 0.76 μM, although this compound also showed PR and AR antagonistic activities with IC50 values of 0.23 and 0.54 μM, respectively. When two methoxy groups were introduced at distal positions, the activity was improved. Thus, 3,8-dimethoxy analog 27 and 4,8-dimethoxy analog 28 showed more potent PR-antagonistic activity than monomethoxy analogs 19, 20, and 22, and 3,8-dimethoxy analog 27 showed more potent AR antagonistic activity than monomethoxy analogs 19 and 22.The results of methoxy scanning prompted us to investigate the effect of introduction of two methoxy groups 25–31. In the 32–35  enhanced inverse agonistic activity toward RORγ [2 also exhibited potent LXRβ antagonistic activity with the IC50 value of 0.88 μM. Replacement of fluorine at the 9-position (35) with chlorine (2) caused enhanced activity toward RORs and LXRβ, but decreased activity toward PR, AR, and GR, suggesting that molecular modification at the 9-position would be important to improve selectivity for metabolic NRs over steroid receptors.We then examined various substituents at the 9-position . We have20 showed enhanced RORα, LXRα and PR activity, and 4-fluoro analog 32 showed enhanced LXRα, PR and AR activity. Compounds 39–44 and 2 showed weak activity toward RORs and GR, indicating that the alkyl group on the nitrogen atom is important for activity toward these receptors. We have reported that 4-alkyl analogs 40–42 showed greater PR activity than the unsubstituted analog 1, and 4-methyl analog 40 had an IC50 value of 0.15 µM [40–42 showed decreased activity compared with the unsubstituted analog 1, whereas 4-fluoro analog 32 showed greater activity than 1. Based on the reported SARs of non-steroidal AR antagonists, an electron-withdrawing substituent neighboring the amide bond is expected to be important for potent AR antagonism [43 was designed and synthesized as shown in 43 showed greater AR activity and weaker RORs, LXRs, PR and GR activity than 4-fluoro analog 32. Compound 43 showed the IC50 value of 0.059 μM, being 2.9-fold more potent than clinically used OHF under our assay conditions. Compound 43 showed more than 340-fold selectivity over RORs and LXRβ, and 90-fold and 6-fold selectivity over LXRα and PR, respectively. This result indicates that molecular modification at the 4-position can increase selectivity for AR over the other NRs.Next, we focused on the 4-position , because 0.15 µM . As for tagonism . Therefo43 did not show antiandrogenic activity in LNCaP, whereas several other AR antagonists including 33 decreased the PSA level with an IC50 of 190 nM  spectrometer . Chemical shifts (δ) are reported in parts per million. Flash column chromatography was performed on silica gel 60N (40–50 mm) . HPLC analyses to check purity were performed on an analytical column  Inertsil ODS-4 reversed-phase column, 5 μm, 4.6 mm × 150 mm) eluted with a mobile phase consisting of CH3CN/water at a flow rate of 1.0 mL/min, with UV (ultraviolet) monitoring at 254 nm, at 37 °C.45)  in toluene (11 mL) were added hexafluoroacetone trihydrate  and p-TsOH . The mixture was stirred for 8.5 h at 110 °C, and then cooled to room temperature. Water was added, and the resulting mixture was extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/AcOEt = 10:1-3:1) to give 46 as a pink solid (9%). 1H NMR  δ: 7.58 , 7.35 , .To a solution of 2-chloroaniline  was added to it, and stirring was continued at 100 °C for 4 h. The resulting mixture was cooled, diluted with water, and extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/AcOEt = 4:1) to give 47  as a brown solid. 1H NMR  δ: 7.94 , 7.89 , 7.76 , 7.69 , 7.52 , 7.24 .To a solution of EDC  and DMAP  in DMF (4.0 mL) was added 2-iodobenzoic acid  under an Ar atmosphere. The mixture was stirred for 30 min at room temperature, then 47  in DMF (1.5 mL) at 0 °C. The mixture was stirred for 1 h at 0 °C, then SEMCl  was added at 0 °C, and stirring was continued for 6 h at room temperature. The resulting mixture was diluted with AcOEt, quenched with water, and extracted with AcOEt. The combined organic layer was washed with water, dried over Na2SO4, and concentrated. The residue was purified by column chromatography (n-hexane/AcOEt = 20:1 to 10:1) to give 48  as a colorless oil. The compound was used for the next reaction without further purification.Sodium hydride  was added to a solution of 48  in DMA (1.0 mL) were added PCy3·HBF4 , Cs2CO3  and Pd(OAc)2  under an Ar atmosphere. The mixture was stirred for 3 h at 130 °C, then cooled to room temperature, and water was added. The resulting mixture was extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/AcOEt = 20:1) to give 49 as a brown oil . 1H NMR  δ: 8.56 , 8.53 , 8.26 , 7.62 , 7.47 , 5.85 , 4.95 , 3.88 , 3.74 , 1.02 , 0.98 , 0.02 , −0.05 .To a solution of 49  in THF (0.8 mL) at room temperature. The reaction mixture was stirred under reflux for 9 h, then diluted with water, and extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl3/MeOH = 40:1) to give 43 as a pale yellow solid (18%).TBAF  was added to a solution of 1H NMR  δ: 8.35 , 8.27 , 7.86–7.83 , 7.68 , 7.63 , 7.41 . HLPC purity 97.0% (area %).T-47D alkaline phosphatase assays were performed as described in the literature . Brieflyv/v) fetal bovine serum, and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were seeded in clear-bottomed white 96-well plates at a density of 1 × 104 cells/well and incubated for 6 h prior to transfection. Cells were co-transfected with 30 ng of a NR expression plasmid, 50 ng of a luciferase reporter and 10 ng of CMX-β-galactosidase expression vector per well, using the calcium phosphate co-precipitation method. After 24 h, transfected cells were treated with dimethyl sulfoxide (DMSO) or DMSO solution of test compounds for 24 h. T0901317 (0.3 μM), T0901317 (0.1 μM), dihydrotestosterone (0.3 nM), and dexamethasone (1 nM) were used as agonists for LXRα, LXRβ, AR and GR, respectively. After addition of a luciferase substrate, luminescence was detected with a luminometer. The luciferase activity of each sample was normalized by β-galactosidase activity: 2-nitrophenyl-β-d-galactopyranoside was added, and the absorbance was measured at 405 nm. Each sample was carried out in triplicate. A six-point sigmoidal dose-response curve was generated for each compound. The IC50 value for each compound was calculated by linear approximation of two points adjacent to 50% inhibition with logarithmic scale. In 50 ± SEM.Human embryonic kidney (HEK) 293 cells  were maintained in DMEM (Dulbecco’s Modified Eagle’s medium) containing 5% (Nuclear receptor plasmids: GR (CMX-hGR), AR (CMX-hAR), ROR (pcDNA3.1(-)-hRORα1, pcDNA3.1(-)-hRORβ1, pcDNA3.1(-)-hRORγ1), LXR .Luciferase reporter plasmids: GR (MTV-Luc), AR (ARE-Luc), ROR (RORE-TK-Luc), LXR (TK-MH100x4-Luc).Plasmids for AR, GR and LXRs were provided by Prof. Dr. Makishima, and plasmids for RORs were provided by Itsuu Institute .v/v) fetal bovine serum, and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were cultured in RPMI1640 without phenol red containing 10% (v/v) charcoal-stripped fetal bovine serum, and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air for 3 days. Then cells were plated in 96-well plates at 1 × 104 cells/well and incubated overnight. On the next day, cells were treated with fresh media containing test compound plus dihydrotestosterone , and incubated for 48 h. Then, PSA levels were measured in the culture supernatants, after 100-fold dilution with the medium, using ELISA  according to the manufacturer’s instructions. All data points were carried out twice in triplicate.The human prostate cancer cell line LNCaP  was routinely maintained in RPMI1640 containing 10% (50: 0.88 μM), AR (IC50: 0.10 μM), GR (IC50: 0.76 μM) were efficiently identified by exhaustive evaluation of a small steroid surrogate library. In addition, a potent AR antagonist 43 (IC50: 0.059 μM) belonging to a new chemical class was developed. Further, AR antagonist 33 showed antiandrogenic activity toward AR antagonist-resistant cell line LNCaP with the IC50 value of 190 nM. Structure-activity relationship studies of phenanthridinone analogs revealed that (1) hydrogen at the nitrogen is important for AR and PR activity, and a longer alkyl group is favorable for RORs and GR activity, (2) molecular modification at the 9-position can increase selectivity for metabolic NRs over steroid receptors, and (3) an electron-withdrawing substituent on the phenyl group is important for AR antagonistic activity, while modification at the 4-position can increase selectivity for AR over other NRs. The phenanthridinone skeleton is more readily modifiable than the steroid skeleton. Thus, our strategy of efficient lead finding, activity enhancement and preliminary control of selectivity over other receptors should expand the utility of the multi-template approach, even though the compounds obtained here still require further optimization.Development of non-steroidal NR ligands is desirable to obtain safer, more selective agents for clinical use. Here, non-steroidal antagonists for multiple target proteins including LXRβ (IC


Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death. Because CRPC maintains a dependency on AR and androgens3, the development of new agents that antagonize AR signaling has resulted in increased overall survival. For example, enzalutamide (Enz) is a specific AR antagonist that increases overall PCa survival4. However, initial insensitivity or acquired resistance to Enz is a common occurrence, indicating that new therapies are required for CRPC5.Prostate cancer (PCa) is a leading cause of cancer-related death in men, especially when metastasis occurs. Although initially responsive to androgen deprivation therapy (ADT), PCa cells can adapt to grow in low androgen levels by inducing androgen receptor (AR) expression and signaling, which leads to the progression of castration-resistant PCa (CRPC)8. Since resistance to Enz is a common occurrence in the clinic5, we hypothesize that adding a multi-DUB inhibitor such as BA to ADT may provide a powerful approach against CRPC by decreasing AR expression, increasing cell death, and possibly overcome resistance to Enz with minimal toxicity to normal cells.The strategy of discovering small molecule drugs to enhance protein degradation including AR has not been fully exploited as a therapeutic option in CRPC. We previously reported that the PCa-specific ability of betulinic acid (BA), a plant-derived small molecule, to decrease several pro-survival proteins including AR and increase cell death may be due to inhibition of multiple deubiquitinases (DUBs) in cancer but not in non-cancer cells11. Recent findings indicate that DUBs have critical regulatory roles in most pathways involving Ub. There are approximately 100 human DUBs, the best characterized being Ub specific proteases (USP) and Ub C-terminal hydrolases (UCHL). DUBs increase the stability of key proteins by negatively regulating UPS-mediated degradation. Removal of poly-Ub from key proliferation and pro-survival proteins renders them less susceptible to degradation by the UPS and increases their levels. In fact, several DUBs are reported to be overexpressed in cancer and are characterized as oncogenes11.Reversible ubiquitination (Ub) is a crucial mechanism in the regulation of the ubiquitin proteasome system (UPS) and the concentrations of many pro-survival proteins15. There is evidence that specific DUBs regulate AR protein stability and downstream signaling. For example, USP10 is an AR cofactor important for activation of AR regulated genes18 and USP26 can also influence AR activity and stability19. More recently, USP12, 22, 7, and 14 have been shown to regulate AR accumulation, signaling, and binding to the chromatin23. Because DUBs appear to have a role in oncogenic transformation11, recent attention has focused on the identification of small molecule inhibitors of DUBs26. The idea is that inhibiting DUBs will elevate poly-Ub on proliferation/pro-survival proteins, increase their recognition and degradation by the UPS, result in greater apoptosis, and improve drug efficacy. Several small molecule DUB inhibitors increase accumulation of poly-Ub proteins and result in greater apoptosis in cancer cells32. Currently, DUB inhibitors are in the preclinical stage of research and no results from clinical trials are yet known.Several studies suggest that DUBs are valid targets for treatment of PCa35. We identified several DUBs that are inhibited by BA using PCa cells or recombinant proteins. Another multi-DUB inhibitor, WP113027, also reduced AR protein, increased poly-Ub/cell death, and decreased DUB activity in PCa but not in non-cancer prostate epithelial cells. Finally, we showed that the AR antagonist Enz combined with BA enhanced AR degradation and cell death in CRPC cells. Overall, our results suggest that small molecule multi-DUB inhibitors such as BA can be combined with Enz to efficiently reduce AR expression and increase apoptotic cell death of CRPC cells with less toxic side effects.In this report, we focused on the ability of BA to reduce AR expression in PCa cells, which makes it an attractive anti-CRPC agent. Our results showed that BA decreased AR protein stability, which is dependent on an active UPS but not on AKT, ERK, or JNK signaling. BA also decreased AR mRNA, possibly due to increased Ub-histone 2A (Ub-H2A), a known epigenetic transcriptional repressor7. BA is also reported to increase necrotic cell death via the mitochondrial permeability transport pore (MPTP) in a manner independent of pro-apoptotic Bax/Bak36. We determined in human PCa cell lines LNCaP, DU145, and PC3 the extent of apoptosis and necrosis dependent cell death after treatment with BA. Results indicated that the pan-caspase apoptosis inhibitor QVD reduced BA increase of cell death and cleaved (cl)-PARP (marker of apoptosis) in LNCaP and DU145. In contrast, QVD decreased cl-PARP but had no effect on cell death by BA in PC3  chase experiment. 22Rv1 cells were simultaneously treated with the protein synthesis inhibitor Chx + DMSO control (Chx/C) or BA (Chx/BA) and cells harvested at 0, 4, 8, 16, and 24 h. Western blot analysis showed that at 8, 16, and 24, Chx + BA reduced AR and AR-V7 protein greater than Chx + control Fig. . Similar44, we investigated if inhibition of AKT, ERK, and JNK signaling pathways alters AR protein and apoptotic cell death. Treatment of LNCaP cells with BA increased phospho (P)-AKT, P-ERK, and P-JNK compared to control treated cells, suggesting that BA stimulated these signaling pathways . Results showed that Btz antagonized the BA decrease in AR and increase in cell death and cl- PARP Fig. . Since Bays Fig. . Howeverays Fig. . We suggays Fig. . Overall38, the effect of BA on AR mRNA expression is not known. LNCaP cells were treated with BA for 0, 2, 4, 8, 14, and 24 h and AR mRNA measured by real-time quantitative polymerase chain reaction (qPCR). The results demonstrated that BA reduced AR mRNA (and protein) starting at 8 h  BA increased Ub-H2A (23 kDa contains one Ub not poly-Ub) by 14 h, a time when AR mRNA is substantially decreased  H2A Fig. . ImmunofH2A Fig. . Finallyors Fig. . Our pre46. Since HA-UbVS only binds to active DUBs, the HA tag allows for the labeling of active DUBs present in PCa cells after BA treatment and analysis by western blot with anti-HA8. To begin to identify the specific DUBs inhibited by BA, we labeled LNCaP and 22Rv1 lysates with HA-UbVS after BA treatment and analyzed by western blot with a DUB-specific antibody . Standard LNCaP and 22Rv1 protein lysates without HA-UbVS labeling was also analyzed by western blot and the ratio of active USP/HA over total USP determined. Our results showed that BA treatment (1) decreased USP10 activity in both LNCaP and 22Rv1; (2) decreased USP9X activity in 22Rv1; and (3) had little effect on USP7 activity , a potent, irreversible, and specific inhibitor of most DUBs18. Stable knockdown of USP10 in LNCaP and 22Rv1 cells with 3 different shRNAs resulted in a 1.5 to 5-fold decrease in AR and AR-V7 protein compared to shGFP control cells  of normal prostate Fig. . In PCa 47. Using multiple recDUBs, the results demonstrated that 10 μM BA specifically inhibited recUSP21 activity by 80%, with an IC50 of 6.1 μM DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry μM Fig. . BA alsoDUB labeling with HA-UbVS showed that BA treatment of LNCaP and 22Rv1 cells inhibited active USP21 Fig. . Since U8. However, it is not clear if BA’s PCa-specific effect is a unique feature of its anti-cancer capability independent of DUB inhibition or if other DUB inhibitors have a similar effect. WP1130 (WP) is a small molecule inhibitor of multiple DUBs  that increases cl-PARP and poly-Ub accumulation in lymphoma cells and decreases the oncogenic transcription factor ERG, a key driver of PCa48. Our results showed that like BA, treatment of LNCaP cells with WP  potently induced cell death , decreased AR protein, and increased cl-PARP/poly-Ub  were sensitive but 22Rv1 and LN-AI/CSS (CRPC) were resistant to Enz (0.5–20 μM)  Fig. . TreatmeμM) Fig. . SimilarμM) Fig. . Overall8. It appears that the DUBs  that regulate AR expression are also important in regulating AR-V7 variant. In addition to reducing AR protein, our data indicated that BA also decreased AR mRNA, making it a very attractive agent for the treatment of PCa and CRPC  will replicate in animal models of CRPC is worth investigation. To begin to address the concern that BA and WP’s low solubility will have limitations for clinical use, the therapeutic value of a new orally bioavailable BA formulation and a more soluble analog of WP have been investigated in preclinical mouse models60.There is substantial evidence that resistance to Enz is due to changes in AR and increased expression of AR variantsion Fig. , there iion Fig. . The WP 62. However, like Enz, it is probable that these new AR-specific agents will not be effective in a subset of CRPC patients or rapidly develop resistance. A major advantage of using multi-DUB inhibitors such as BA or WP is that neither agent requires AR for efficacy and they can target multiple pro-survival and proliferation genes. Since AR expression is heterogeneous especially in metastatic CRPC57, inclusion of agents such as BA or WP that also kill AR negative CRPC cells will likely have a positive role in preventing disease recurrence with less toxicity to normal cells.There is a great emphasis on discovering newer AR antagonists or new agents that can specifically degrade AR and its variants with the expectation that Enz resistant CRPC will respondBA and WP were obtained from Biovision ; QVD from R & D Systems ; CsA and SP600125 from Enzo Life Sciences ; Chx from Sigma-Aldrich ; Btz and U0126 from LC Laboratories ; LY294002 from EMD Millipore ; HA-UbVS from Boston Biochem ; Enz from Selleckchem ; Coomassie blue from EMD Biosciences ; and trypan blue (0.4%) from Invitrogen . All other reagents were purchased from Sigma-Aldrich.63 were obtained from the American Type Culture Collection (ATCC) and used within 6 months of resuscitation of original cultures. Unlike LNCaP, LN-AI cells can grow for long-term in RPMI 1640 with 5% charcoal-stripped fetal bovine serum  and are referred to as LN-AI/CSS64. All other PCa cells were maintained in RPMI 1640 medium  with 5% fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin (Invitrogen). Media for viral transduced LNCaP and 22Rv1 cells also contained puromycin . RWPE-1 normal prostate epithelial cells were obtained from ATCC and maintained in Keratinocyte-SFM media (Invitrogen)65.Human PCa cell lines LNCaP, DU145, PC3, and 22Rv1PCa cells were cultured in media containing BA (10 μM) +/− CsA (10 μM), QVD (10 μM), Chx (25 μg/ml), Btz (5 nM), LY294002 (20 μM), U0126 (10 μM), SP600125 (20 μM), Enz (10 μM), or DMSO (0.1–0.25%) control for varying times (24–72 h). WP was used at 1, 2.5, and 5 μM or WP (2.5 μM) + Enz (10 μM) for 16 h. In all the experiments, floating and trypsinized attached cells were pooled for further analysis.Treated and control cells were harvested, resuspended in PBS, diluted 1:1 in 0.4% trypan blue, dead blue and live non-blue cells immediately counted using a hemocytometer, and the % dead blue cells determined from at least three independent experiments done in duplicate.64. The following antibodies were used: cl-PARP (9541), P-AKT , AKT (9272), P-ERK1/2 , ERK1/2 (9102), P-JNK , Ub-H2A (Lys119) (D27C4) from Cell Signaling Technology ; AR , Ub (P4D1), JNK1 (FL), actin (C-11), horseradish peroxidase-conjugated secondary antibody from Santa Cruz Biotechnology ; USP7 (A300-033A), USP9X (A301-350A), USP10 (A300-900A) from Bethyl Laboratories ; and USP21 (HPA028397) from Sigma-Aldrich). Precision Plus Protein Dual Color Standards  was used to estimate molecular weights in kDa. After immunodetection, our preference for loading controls was staining of total proteins transferred to the membrane with Coomassie blue because drug treatments often affect the levels of typical housekeeping proteins such as actin or tubulin. Blot images were cropped for clarity of the presentation. Full uncropped blot images are available upon request.Preparation of total protein lysates and western blot analysis was done as previously described64. AR  or Ub (1 μg) antibody was added to 400 μg of total protein and incubated 4 °C overnight with rotation. Negative control IP was addition of an equal amount of non-specific mouse Ig (Santa Cruz). Protein A/G agarose  was added for 2 h, 4 °C with rotation followed by centrifugation, washing pellets 3x with PBS, and final pellet resuspended in SDS loading buffer for analysis by western blot . Total protein lysates were run on a separate gel.LNCaP and 22Rv1 cells were treated with BA (10 μM) and control (0.25% DMSO) for 18–20 h and cell pellets immediately lysed in NP40 lysis buffer (same used for western blot)49. The following DNA oligonucleotides from Eurofins MWG Operon  were used for qPCR: AR sense 5′-GACGACCAGATGGCTGTCATT-3′ and antisense 5′-GGGCGAAGTAGAGCATCCT-3′ (106 amplicon) (Primer Bank)66. Values from two independent experiments done in duplicate were normalized to the housekeeping reference genes ribosomal protein, large, P0 (RPL0) sense 5′-GCAATGTTGCCAGTGTCTG-3′ and antisense 5′-GCCTTGACCTTTTCAGCAA-3′ (141 amplicon) and β-2 micoglobulin sense 5′-ATGAGTATGCCTGCCGTGTGA-3′ and antisense 5-GGCATCTTCAAACCTCCATG-3′ (101 amplicon)67.The procedure for qPCR was done as previously describedAdherent and non-adherent LNCaP cells were harvested after a 24 h treatment with BA (10 μM) or control, applied to slides by smearing, air dried, fixed in formalin for 10 min, permeabilized with 0.1% Triton X-100/PBS for 10 min, rinsed with water, and blocked with goat serum  for 30 min. Ub-H2A was immunostained using Ub-H2A (Lys119) (D27C4)  at 1:50 dilution for 1 h followed by secondary antibody Alexa Fluor 488 (green) goat anti-mouse IgG  for 1 h. Mounting medium with DAPI was from Vector Laboratories. Color images were acquired using a Nikon Eclipse 90i fluorescence microscope and merged using Adobe Photoshop 7.8. Immunostaining for Ub-H2A (D27C4)  was performed using a 1/200 dilution of mouse monoclonal and the MOM Peroxidase Kit (Vector Laboratories) following the manufacturer’s instructions and as we previously described68.We obtained paraffin sections from TRAMP mice with PCa treated with BA (10 mg/kg body weight) or vehicle control from our previous study68. The slide contains unstained paraffin sections of 64 cases of PCa categorized as Gleason grade 4–6 (n = 12), 7 (n = 23), and 8–10 (n = 29), and normal prostate tissue (n = 4).Human PCa tissue microarray PR803a was purchased from US Biomax, Inc.  and utilized for immunostaining of USP10  using the methods previously described2, 250 mM sucrose, 1 mM DTT, 1 mM PMSF), centrifuged, 25 µg of protein incubated with 0.5 μg HA-UbVS for 1.5 h at room temperature, and samples analyzed by western blot using anti-USP10, 21, 9X, and 7 to detect DUB activity (USP/HA). Lysates without HA-UbVS were also analyzed by western blot to detect effect on total USP10, 21, 9X, and 7. Quantitative differences for USP/HA and total USP were determined by measuring pixel intensity from images , normalization to Coomassie blue stained total protein, and the ratio of USP/HA/total USP determined (control 0 h = 1). A similar analysis was done with LNCaP and 22Rv1 cells treated with WP (2.5 μM) or control . DUB-Glo Protease Assay  was used to determine the effect of WP (5 μM) on DUB activity in confluent RWPE-1 and PC3 cells treated with WP or control for 24 h as previously described8. Light units (control treatment = 1) from 4 samples were determined from two independent experiments.LNCaP and 22Rv1 cells treated with BA (10 μM) or control  were harvested, lysed in DUB buffer  targeting USP10 were cloned into pLKO.1 lentivirus vector: shUSP10-1 sense: 5′-CCGGGCCTCTCTTTAGTGGCTCTTTCTCGAGAAAGAGCCACT AAAGAGAGGCTTTTTG-3′; shUSP10-1 antisense: 5′-AATTCAAAAAGCCTCTCTTTAGTGGC TCTTTCTCGAGAAAGAGCCACTAAAGAGAGGC-3′; shUSP10-2 sense: 5′-CCGGCCTATGTG GAAACTAAGTATTCTCGAGAATACTTAGTTTCCACATAGGTTTTTG-3′; shUSP10-2 anti- sense: 5′-AATTCAAAAACCTATGTGGAAACTAAGTATTCTCGAGAATACTTAGTTTCCACATA GG-3′; shUSP10-3 sense: 5′-CCGGCCCATGATAGACAGCTTTGTTCTCGAGAACAAAGCTG TCTATCATGGGTTTTTG-3′; shUSP10-3 antisense: 5′-AATTCAAAAACCCATGATAGACAGC TTTGTTCTCGAGAACAAAGCTGTCTATCATGGG-3′. The following DNA oligonucleotides (Eurofins MWG Operon) targeting USP21 were cloned into pLKO.1 lentivirus vector: shUSP21-1 sense: 5′-CCGGGCCTTTCTACTCTGATGACAACTCGAGTTGTCATCAGAGTAGAAAGGCTT TTTG-3′; shUSP21-1 antisense: 5′-AATTCAAAAAGCCTTTCTACTCTGATGACAACTCGAGTT GTCATCAGAGTAGAAAGGC-3′; shUSP21-2 sense: 5′-CCGGGACCCTCTGCAATATCACTTT CTCGAGAAAGTGATATTGCAGAGGGTCTTTTTG-3′; shUSP21-2 antisense: 5′-AATTCAAAA AGACCCTCTGCAATATCACTTTCTCGAGAAAGTGATATTGCAGAGGGTC-3′; shUSP21-3 sense: 5′-CCGGCCACTTTGAGACGTAGCACTTCTCGAGAAGTGCTACGTCTCAAAGTGGTT TTTG-3′; shUSP21-3 antisense: 5′-AATTCAAAAACCACTTTGAGACGTAGCACTTCTCGAGA AGTGCTACGTCTCAAAGTGG-3′. All DNA sequences were previously validated and obtained from Mission shRNA library (Sigma-Aldrich). The control shRNA was targeted against green fluorescent protein (GFP). Flag-HA-USP10 (22543) plasmid was purchased from Addgene 71. Retrovirus production and infection were done as previously described72. We used LNCaP/EV (empty vector) as the negative control cells72. Differences in AR protein between shUSP10-1, -2, -3 and shUSP21-1, -2, -3 were compared to shGFP (LNCaP and 22Rv1) and between LNCaP/USP10 and LNCaP/EV were determined by western blot as previously described73.The shRNA design, lentivirus production, and infection were done as previously describedThe CellTiter Aqueous colorimetric method from Promega was used to determine cell viability of LNCaP, 22Rv1, and LN-AI/CSS PCa cells in media containing Enz (0.5–20 µM) or control (0.1% DMSO). Cell viability was normalized against the DMSO control and the data expressed as a percentage of control from three independent experiments done in triplicate.t-test  from 2–3 independent experiments done in duplicate or triplicate with P < 0.05 considered significant.Statistical differences between drug-treated and control PCa cells were determined by two-tailed Student’s Supplementary Figures 1-7 and Table 1


A high-throughput screening assay (HTS) was set up to detect α1-AR subtype-selective agonists by a dual-luciferase reporter assay in HEK293 cells. Using the HTS assay, two novel compounds, CHE3 and CHK3, were discovered as α1-ARs agonists in α1-ARs expressed in HEK293 cells. These compounds also showed moderate/weak anti-proliferative activities against tested cancer cell lines. The HTS assay proposed in this study represents a potential method for discovering more α1-AR subtype-selective ligands.α Human ADRA1A/1B/1D-expression vector (NCBI: NM_000680.1/NM_000679.2/NM_000678.2) and negative control vector (NEG) were purchased from GeneCopoeia, Inc. Rockville, MD, USA; Catalog No.: EX-A0967-M29, EX-Y3321-M29, EX-Y2008-M29 and EX-NEG-M29). CHE3 and CHK3  (included as a control for transfection efficiency) using Lipofectamine 2000 transfection reagent . The medium was removed after 4 h and replaced with high-glucose DMEM for 18 h. Cells were then treated with agonists (at concentrations of 0.01–20 μM) and incubated for 8 h.HEK293 cells were plated at a density of 1050/IC50 values in μM, and the ED50 of phenylephrine (μM) was calculated by plotting the data using nonlinear regression analysis in GraphPad Prism 5 software. The pKB of the antagonist was calculated by plotting log (dose ratio–1) against the antagonist dose (μM) and subjecting the data to Schild regression, where the intercept of the x-axis represents the pKB.Firefly and Renilla luciferase activities, which are indicated as RLUs, were determined using Dual-Glo luciferase assay kits (Promega) according to the manufacturer’s instructions. RLUs were measured using a luminometer  and are reported as the mean ± SEM of three individual experiments. For agonists, fold of induction = RLUinduced/RLUuninduced. For antagonists, % of control = 100 × RLU (agonist + antagonist)/RLU. All RLUs were normalized against firefly RLUs/Renilla RLUs. Data are expressed as EC1-ARs in HEK293 cells were detected by fluorescence microscopy. Human ADRA1-expression vector: ADRA1A, ADRA1B and ADRA1D were tagged by eGFP tag. The cells were treated as described above for α1-ARs HTS assay and observed using a confocal microscope The expression of α1-ARs according to the protocol as described above. Total RNA was extracted and quantified followed the references using a spectrophotometer at 260/280 nm. Then cDNA was synthesized from total RNA by using oligo-dT primer. Each cDNA specimen and cloned human α1-AR subtypes SYBR Green PCR for each α1-AR subtype was performed by an Applied Biosystems 7500 Real Time PCR System. The primers and probes for α1A-, α1B-, and α1D-AR were designed as follows [1A-AR forward, 5'-ATCATCTCCATCGACCGCTACA-3'; a1a-AR reverse, 5'-TCACTTGCTCCGAGTCCGACTT-3'; a1B-AR forward, 5'-GCTCCTTCTACATCCCTCTGG-3'; a1B-AR reverse, 5'-AGGGTAGCCAGCACAAGATGA-3'; a1D-AR forward, 5'-ACCACGCGCAGCCTCGAGGCAGGC-3'; a1D-AR reverse, 5′-GAGCGAGCTGCGGAAGGTGTGGCC-3′. The PCR mix contained 2 μL cDNA template, 10 μL of 2× SYBR®Premix Ex Taq II , 0.4 μL of 50× ROX Reference Dye II , 0.8 μL each of primers for α1A-, α1B-, or α1D-AR, in a total volume of 20 μL. GAPDH  was used as internal control to standardize the quantitation of the three target genes. All experiments were carried out in triplicate.HEK293 cells were grown in 6-well plates and transiently transfected with α follows : α1A-AR 5 cells/mL, and cultured at 37 °C in a humidified incubator (5% CO2) for 24 h, followed by exposure to various concentrations of compounds tested for 24 h. Subsequently 10 μL of CCK-8 (Dojindo) was added to each well, the cells were then incubated for an additional 1 h at 37 °C to convert WST-8 into formazan. Cell growth inhibition was determined by measuring the absorbance (Abs) at λ = 450 nm using amicroplate reader. Three independent experiments were performed. Cell growth inhibition was calculated according to the following equation:Cell proliferation was measured with the Cell Counting Kit-8 (CCK-8) assay kit . Cells were harvested during logarithmic growth phase and seeded in 96-well plates at a density of 1 × 1050) were obtained from liner regression analysis of the concentration-response curves plotted for each tested compound.The half maximal inhibitory concentrations (ICt test. Values of p < 0.05 were considered to indicate statistical significance.Data are expressed as means ± standard errors. Differences among the mean values were analyzed by an unpaired Student’s 1-AR subtype-selective agonists/antagonists. Two novel compounds, CHE3 and CHK3, were discovered as α1-AR agonists using this assay. These compounds displayed moderate cytotoxic activities against tested cancer cell lines. The HTS assay proposed in this study represents a potential method for discovering more α1-AR subtype-selective ligands.The present study reported a rapid and sensitive assay for detecting α


E-isomers. To extend the understanding of conformational and thermodynamic preferences of Z-isomeric super-statin analogues, this study focused on analyzing pitavastatin and its lactonized derivatives via NMR spectroscopy and ab initio calculations. Z-isomeric pitavastatin analogues exist in solution as a pair of interconverting rotamers, where the Gibbs free energies between the major and minor rotamers are within 0.12 and 0.25 kcal mol−1 and the rotational energy barriers are between 15.0 and 15.9 kcal mol−1. The analysis of long-range coupling constants and ab initio calculations revealed that rotation across the C5'–C7 single bond is essential for generating a pair of atropisomers. The overall comparison of the results between Z-isomeric pitavastatin and rosuvastatin analogues demonstrated that the former are to some extent more flexible to attain numerous conformations. Demonstrating how structural differences between super-statin analogues induce distinctive conformational preferences provides important insight into the super-statins’ conformational variability and may well improve future drug design.Super-statins are synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the rate-limiting enzyme responsible for the biosynthesis of cholesterol. All of the super-statins with a C=C double bond spacer between the heterocyclic and the dihydroxycarboxylic moiety that are currently on the market exist as  E-oriented geometric isomers. In the early discovery stage of super-statins it was shown that some of their Z-isomeric analogues showed only weak  of 0° ± 30° and 180° ± 30°  and θ and –1) and global minimum at –1). Conformations exhibiting  minima at torsion angle θ = 90°, 150° and 270° all minimized to 164° ; an additional local minimum was found at θ = 321° . Frequency calculations verified that the optimized geometries at  minima were stable points on the potential energy surface.Initial structures were generated by Chem3D Pro 10.0 software and energy minimization at B3LYP/6-311+G level was performed using Gaussian 09 . Torsion-6-((Z)-2-(2-Cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)vinyl)-4-hydroxytetrahydro-2H-pyran-2-one (P-2). A solution of Bu4NF∙3H2O  and AcOH  in THF (2.5 mL) was cooled in ice bath. Then a solution of P-1  in THF (2.5 mL) was added. The solution was left to stir for 24 h at room temperature. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (5 mL). The organic layer was washed with water (5 mL), saturated solution of NaHCO3 (5 mL), brine (5 mL) and water (5 mL) respectively. Organic layer was dried over Na2SO4 and evaporated under reduced pressure. The residue was purified by column chromatography  to give 170 mg (73% yield) of P-2. 1H-NMR : 7.91 , 7.67 , 7.54–7.24 , 6.61 , 5.80 , 5.16 , 4.24 , 2.61 , 2.50 2), 2.41 , 1.98–1.48 , 1.29 2), 1.06 2). 13C-NMR : 169.8 (C-1), 163.3 , 161.4 (Ar), 148.2 (Ar), 145.8 (Ar), 134.0 , 133.4 (C-6), 132.7 , 130.1 (Ar), 129.9 (Ar), 129.7 (C-7), 128.7 (Ar), 126.53 (Ar), 126.49 (Ar), 126.4 (Ar), 116.3 , 116.1 , 73.4 (C-5), 63.0 (C-3), 39.2 (C-2), 35.9 (C-4), 15.9 (CH(CH2)2), 11.7 2). IR (KBr): 3424, 3063, 3007, 2954, 2922, 1708, 1513, 1488, 1255, 1222, 1159, 1034, 764, 668, cm−1. Mp: 190.8 °C (onset), 192.7 °C (peak). HRMS (ESI): MH+, found: 404.1656. C25H23FNO3 requires 404.1656. c 0.90, CH2Cl2]. -7-(2-Cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)-3,5-dihydroxyhept-6-enoate calcium (P-3). To a solution of P-2  in THF–water, 4:1 (5 mL) at 35 °C was added 8M aq NaOH . After 16 h the reaction was finished and THF was evaporated under reduced pressure. Sodium salt of Z-isomeric pitavastatin spontaneously precipitated from the aqueous solution. The suspension was cooled in an ice bath and THF (1 mL approx) was added drop by drop till all of the precipitate has dissolved again. To a concentrated solution of sodium salt of Z-isomeric pitavastatin was added a solution of CaCl2  in water (0.5 mL). Immediately occurred white precipitate was left to stir for 1 h. Then it was filtered off, washed with water (5 × 2 mL) and dried at 90 °C under vacuum to give 340 mg (77% yield) of P-3. 1H-NMR : 7.90 , 7.60 , 7.49–7.12 , 6.42 , 5.62 , 4.17 , 3.94 , 2.52 2), 2.28–1.94 , 1.80–0.72 2, 2∙OH). 13C-NMR : 181.8 (C-1), 163.9 , 162.3 (Ar), 148.5 (Ar), 146.4 , 137.3 , 134.4 , 133.1 , 130.1 (Ar), 129.8 (Ar), 129.4 (Ar), 127.7 (C-7), 127.0 (Ar), 126.8 (Ar), 126.6 (Ar), 116.4 , 116.3 , 69.0 (C-3), 68.7 (C-5), 44.9 (C-4), 43.8 (C-2), 16.3 (CH(CH2)2), 11.6 2). IR (KBr): 3397, 3067, 3008, 2919, 1561, 1513, 1489, 1417, 1222, 1158, 844, 765, 560 cm−1. Mp:/(amorphous). HRMS (ESI): MH+, found: 422.1764. C25H25FNO4 requires 422.1762. c 0.98, MeOH] for sodium salt of pitavastatin P-3. c 0.25, DMSO] for calcium salt of pitavastatin P-3.Z-isomeric pitavastatin analogues P-1, P-2, and P-3 were synthesized and characterized using various NMR spectroscopy techniques. Acquiring NMR spectra at lower temperature revealed that the resonance line broadening observed at room temperature originates from the dynamic exchange between two rotamers. Firstly, according to the 4JH5-H7 long-range coupling constants, the preferred orientation along the C5–C6 bond for pitavastatin analogues is anti. This orientation is in addition supported by ab initio calculations. Secondly, the determination of mole fractions of minor conformers for pitavastatin analogues, which were between 1.4 and 2.7 fold greater than for rosuvastatin analogues, enabled calculations of thermodynamic preferences. The values of Gibbs free energies for the equilibrium between the major and minor conformers of pitavastatin analogues at room temperature are consequently smaller than for rosuvastatin analogues. Finally, the experimentally established values of rotational energy barriers ΔG‡ for pitavastatin and rosuvastatin analogues are very comparable with each other and in good agreement with their calculated values. The presented results confirm that for Z-isomeric pitavastatin analogues the rotation across the C5'–C7 bond is crucial for generating a pair of atropisomers. Nevertheless, the orientation across the C=C double bond is obviously essential for generating a pair of conformers, for which atropisomerism was not detected in E-isomeric pitavastatin and rosuvastatin analogues.The


Telomere stability is important for cell viability, as cells with telomere DNA damage that is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice variant AR-V7, but full-length AR was found to be the predominant form of AR associated with telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this combined treatment approach to induce telomere DNA damage and inhibit the DDR was effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express AR splice variant. The critical role of the androgen receptor (AR) in prostate cancer cell proliferation and survival is the enduring basis for treating advanced prostate cancer with drugs that block AR function or androgen biosynthesis , 2. HoweTelomeres are the DNA-protein structures that cap the ends of linear chromosomes, which are double-stranded DNA with a single-stranded overhang . Telomer1 DNA content , presumably a result of cells entering mitosis with a level of telomere DNA damage that is incompatible with survival , and the antibody-bound material was then precipitated with 30 μl protein-G Sepharose beads (Invitrogen) that had been pre-equilibrated with 30 μg bovine serum albumin (BSA) and 5 μg sheared Escherichia coli DNA for 30 min at 4°C. In order to isolate DNA from the ChIP pellet (ChIP DNA), cross-linking was reversed at 65°C for 4 h, treated with RNAase A and proteinase K at 37°C, and extracted with 0.5 ml phenol/chloroform/isoamylalcohol. In order to probe ChIP DNA for AR binding sites in the PSA gene, NDR G1 gene and telomeres, ChIP DNA was subjected to PCR using primers for PSA ARE III: 5’- cttctagggtgaccagagcag (forward) and 5'- gcaggcatccttgcaagatg (reverse), for NDR-G1 ARE 5’-gccacctgggtagctttgta (forward) and 5’-agaggagccgccaaattaaa (reverse) and for chromosome 17p telomeres (Chr. 17p-Tel): 5’-gaatccacggattgctttgtgtactt (sub-telomeric forward) and 5’-tgctccgtgcatctggcatc(ccctaa)5 (telomeric reverse). Cycle parameters for ARE III were 94°C for 2 min, 94°C for 30 sec, 55°C for 30 sec and 68°C for 1 min for 30 cycles. Cycle parameters for telomere DNA were 95°C for 3 min 94°C for 30 sec, 60°C for 30 sec, 68°C for 1 min for 35 cycles. The number of cycles for each sequence was chosen from the linear range of amplification [ChIP analysis was performed as described elsewhere . Brieflyfication .5 cells/well of a six-well plate) were transfected with 200 pmol siRNA targeting AR exon 1 , AR exond AR-V7 ), AR crynd AR-V7 ), or a cnd AR-V7  is also nd AR-V7 . Cells w4 cells/well of a six-well plate) were treated as described in figures for 24 hr, then washed to remove drugs and allowed to grow for 10–14 days, then fixed and stained with 0.01% crystal violet [This procedure is essentially as described , 27. Cell violet , 28. The6 22Rv1 cells as described by Wu et al.[3, tumor-bearing mice were randomly assigned to the following 4 treatment groups: vehicle , ENZ  alone (7 mice); ATMi KU59403 alone (7 mice); ENZ + KU59403 (6 mice). All treatments were carried out 5 days/week for 4 weeks. KU59403 (25 mg/kg) was administered twice daily by i.p. injection [3 when mice were sacrificed, and tumors were harvested.All experiments were performed in accordance with protocols (IACUC #1555) approved by the Institutional Animal Care and Use Committee of Henry Ford Health System. Mice were anesthetized by carbon dioxide inhalation in an approved chamber prior to cervical dislocation. In order to test the effect of AR antagonist ENZ and ATM inhibitor KU59403  on 22Rv1 tumors, athymic nude mice (Charles River) were inoculated subcutaneously with 4 X 10Wu et al., and whenjection . ENZ  staining of pATM using anti-pATM antibodies (Santa Cruz) and the IHC analysis Kit (Vector Laboratories), and apoptotic cell death was assessed by using a TUNEL assay Kit (InVitrogen), following the manufacturer’s suggested protocols.Data are presented as mean +/-SD of three or more independent experiments. Statistical significance was calculated using the two-tailed Student t-test, using GraphPad Prism Software. A p-value <0.05 was considered significant.Despite resistance to the growth inhibitory effect of ENZ, this AR antagonist nevertheless induces telomere DNA damage in 22Rv1 CRPC cells , as wellENZ also induces activation of ATM  in 22Rv1Our observation that AR antagonist induces telomere damage in AR-positive prostate cancer cells indicates a role of AR in telomere stability in these cells –8. Our sOne approach to identifying AR association with telomeres is dual-label immunofluorescence of AR that colocalizes with TIN2 in prostate cancer cells . The vasWe previously used actinomycin D to demonstrate that the role of AR in telomere stability is independent of AR transcriptional activity, as inhibiting the expression of AR-target genes in LNCaP cells with actinomycin D does not cause telomere DNA damage . This leNotably, actinomycin D inhibits transcription as a result of its ability to intercalate into DNA; however, owing to differences in histone modifications and compactness of nucleosomes, actinomycin D intercalates and disrupts DNA-protein interactions at least three times more efficiently in euchromatin than in heterochromatin –39. TeloThus, we hypothesized that actinomycin D might disrupt euchromatin-associated AR more efficiently than telomere-associated AR. We first tested this hypothesis using LNCaP cells that express only full-length AR. We treated cells with actinomycin D and then prepared them for immunolabeling of AR and TIN2. Actinomycin D treatment indeed decreased the amount of nuclear AR, but had no effect on TIN2 protein . Most noWe further validated the association of actinomycin D-resistant AR with telomeres by analyzing AR-chromatin immunoprecipitate (AR-ChIP) for the presence of telomere DNA, using PCR primers that amplify sequence extending from the sub-telomere region of chromosome 17p into its telomere (17p-Tel) . As expeBy contrast, AR-ChIP prepared from actinomycin D-treated cells , affirmiWe next used actinomycin D to address the question whether the AR splice variant AR-V7 in 22Rv1 cells is associated with telomeres and whether AR-V7 plays a role in telomere stability. Dual immunofluorescence labeling of 22Rv1 cells was carried out using TIN2 antibody and either antibody AR-441 that recognizes both full-length AR and variant AR-V7, or antibody specific to AR-V7 . ColocalThe apparent presence of a small subset of AR-V7 at telomeres in 22Rv1 cells  suggestsWe next investigated the role of full-length AR and AR splice variant AR-V7 in the cell death response to ATMi. Knockdown of full-length AR alone did not affect 22Rv1 cell survival, presumably because AR-V7 was still expressed; however, knockdown of full-length AR plus treatment with ATMi KU60019 significantly decreased cell survival (p = 0.001) , similarWe treated established CRPC 22Rv1 tumors with vehicle, ENZ alone, ATMi alone, or ENZ in combination with ATMi . We usedin vivo as it had no detrimental effect on body weight when administered alone or in combination with ENZ, compared to controls  in vivo , as it dThe expression of constitutively active AR splice variants such as AR-V7, which lacks the ligand binding domain, is considered an important mechanism of AR antagonist-resistant growth of metastatic CRPC , 17. HowThus, our data suggest that the use of ENZ in combination with a DDR inhibitor, such as an ATMi, may be effective in prolonging disease-free survival of patients with AR-positive mCRPC, even the 19–59% that co-express an AR splice variant , 45.The AR is a well-characterized transcription factor that regulates the expression of many genes , includiWhen applied to the treatment of CRPC 22Rv1 cells and tumors, combined treatment with AR antagonist plus ATMi significantly inhibited growth, whereas each drug alone was ineffective. This feature fits the criterion for synthetic lethality , althougThere is growing interest in combined targeting of AR and DNA repair in CRPC, using ENZ or androgen ablation plus an inhibitor of poly (ADP-ribose) polymerase (PARP) , 51 or aMany cell types, including prostate cancer, use AR to modulate specific gene expression, but prostate cancer cells uniquely require AR to regulate cell proliferation and cell survival. Similarly, most cells do not need AR for telomere stability, but our studies have shown a critical role of AR in telomere stability in prostate cancer cells . Our datIn summary, we have demonstrated that CRPC 22Rv1 tumor growth is resistant to AR antagonist or ATMi as monotherapies, but is significantly inhibited by combined treatment. CRPC cells that express both full-length AR and splice variant AR-V7 and are not growth inhibited by AR antagonist are inferred to depend on, and are said to be driven by AR-V7; but, ironically, it is their expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi.S1 Fig(A) The concentration of AR antagonist enzalutamide (ENZ) that induces telomere DNA damage in prostate cancer cells is lower in charcoal-stripped serum (CSS) than in untreated serum (FCS).Dose-response effect of enzalutamide (ENZ) on telomere DNA damage (A), lack of effect of ATM inhibitor on AR-target gene expression (B), and effect of ENZ + ATM inhibitor KU60019 on cell survival (C-F) in prostate cancer cells.  Exponentially growing LNCaP cells were treated as indicated for 24 hr, in either FCS-containing medium (hormone-replete) or CSS-containing medium (hormone-depleted). To prepare cells for treatment with AR antagonist ENZ in CSS medium, exponentially growing LNCaP cells in FCS medium were washed twice with phenol red-free RPMI medium (Thermo Fisher Scientific) for 1 hr, and incubated in phenol red-free RPMI medium supplemented with 10% CSS (InVitrogen) for 26 hr prior to treatment with AR antagonists. After 24-hr treatment with 1–10 μM AR antagonist, cells were labeled with antibodies to γ-H2AX (marker of DNA damage) and TIN2 (telomere specific protein), and cells with a TIF response  were counted. Data are expressed as mean ± SD of 3 independent experiments. The concentration of ENZ that induces telomere DNA damage in LNCaP cells was lower in hormone-depleted CSS medium (1 μM) than in hormone-replete FCS medium (10 μM). (B) ATMi (KU60019) has no effect on expression of the AR target gene PSA. 22Rv1 cells were treated without or with 10 μM KU60019 for 24 hr. PSA and GAPDH mRNA levels were assayed by RT‐PCR. (C-F) Dose-response effect of ENZ in the absence vs. presence of 10 μM ATMi on survival of androgen-sensitive LNCaP and CRPC 22Rv1, C4-2B, and LNCaP/AR cells. Cells were treated for 24 hr as indicated, then washed to remove drugs and allowed to grow for 14 days (colony formation assay). The survival fraction is plotted relative to vehicle-treated controls; mean ± SD of 3 independent experiments.(TIF)Click here for additional data file.S2 Fig(A)ENZ induces telomere DNA damage (A) and activates ATM at telomeres (B) in CRPC cells.  22Rv1 cells were treated without  or with 5 μM ENZ in FCS-containing medium for 6 hr, then labeled with antibodies to DNA damage marker γ-H2AX (red) and the telomere marker TIN2 (green). Dual-labeled foci (indicated by yellow) are shown in the ‘merge’ panel, indicating DNA damage at telomeres of ENZ-treated 22Rv1 cells. (B) 22Rv1 cells were treated with or without 5 μM ENZ for 6 hr, then labeled with antibodies to phosphorylated ATM  and TIN2 (green). Colocalization of pATM (activated ATM) and TIN2 is shown in the ‘merge’ panels, indicating the presence of activated ATM at telomeres of ENZ-treated 22Rv1 cells. Higher magnification inserts of representative cells in the merge images in A and B facilitate the visualization of the presence or absence of colocalization.(TIF)Click here for additional data file.S3 FigA) Data for each group are shown as mean ± SEM. *, p<0.05; **, p<0.001; ***, p<0.0001. B) Growth curves are shown for each tumor.These data supplement the data shown in (TIF)Click here for additional data file.S4 Fig3. Time to sacrifice was not adjusted for differences in tumor size at the start of treatment.Survival was defined as the number of days until sacrifice, when tumor size was ~2,000 mm(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Tmax), mean (Tmean) and minimum (Tmin) air temperatures and the diurnal temperature range (DTR) during the Chinese New Year holiday are evaluated with two methods using daily meteorological observations collected at 2200 stations in China from 1961 to 2015. These two methods yield nearly equivalent results that reflect strong variations in the defined holiday effects. During the period from 1961 to 1980, Tmean, Tmax, Tmin and the DTR all exhibit cooling holiday effects, this effect as measured by the DTR disappears during the period from 1981 to 2000. However, during the period from 2001 to 2015 warming holiday effects are observed for Tmax and the DTR. The evaluation shows that the holiday effect is neither unique nor statistically significant. These results indicate that the holiday effect is primarily caused by natural atmospheric oscillations, because ΔT oscillates noticeably with periods of approximately 7.1 days, 8.5 days and 16.2 days, and these oscillations can account for approximately 75.6% of the variance in ΔT. The oscillation identified here is consistent with the fundamental theory of Rossby wave in the atmosphere.Anthropogenic emissions are generally lower during holidays than they are on workdays, this pattern is expected to result in temperature variations. Variations in the daily maximum ( The effects of holidays and weekends on meteorological and environmental variables  represent an important aspect of the detection and attribution of climate change. These effects have been analyzed in several studies using observed meteorological and satellite datasets5.Whether regional and global climate changes can be attributed to natural and anthropogenic causes is a central issue6. Thus, researchers have generally focused on anthropogenic impacts while assuming that the weekly cycles of consumption and emissions resulting from human activity could help explain the effects of weekends on the climate7.The effects of holidays and weekends on meteorological and environmental variables have been attributed to anthropogenic causes for decades. Human activities, such as commercial transportation and industrial activities, occur with reduced intensities on weekends relative to weekdays in most countrieset al.8, who argued that weekly cycles of smoke and hot gas emissions from factory chimneys led to reductions in rainfall on Sundays. Choi et al.9 found an relationship between weekly anthropogenic air pollutant emissions and meteorological conditions over China at a regional scale and suggested a possible link between the formation of clouds and the weekly variations in the mass concentrations of atmospheric aerosols10. On a larger scale, Gordon11 analyzed the weekly cycles of meteorological variables in the Northern Hemisphere and observed that the average daily temperature was lowest on Sunday and highest on Wednesday from the period of 1979 to 1992. Ohashi et al.12 calculated the weekend effect on the air temperatures over the city of Osaka in Japan from 2013-2014 and found that the daytime temperatures were more than 1 °C higher on weekdays than on weekends because of the anthropogenic electricity use.The first investigation of the potential relationships between human activities and weekends was conducted by Ashworth 13 studied mesospheric temperatures in Scandinavia from June to August 1992 and identified a significant quasi-16-day temperature oscillation pattern. Jiang et al.14 found that zonal mean temperatures oscillate with periods of approximately 9 and 13.5 days. Such oscillations have been attributed to multiple natural processes, such as the North Atlantic Oscillation (NAO) and Rossby waves. The NAO is a large-scale meridional oscillation of atmospheric mass that occurs between the subtropical anticyclone near the Azores and the subpolar low pressure system, which is related to a wide range of climate variation over the Northern Hemisphere across a variety of time scales16. Rossby waves are long-period turbulent waves that propagate both within the atmosphere and over the oceans, and display considerable variations in their length and time scales19.In addition to weekly cycles, other short-term oscillations of atmospheric phenomena with different periods have been widely reported. Espy and Witt21. The NAO has been regarded as a dominant pattern that can significantly impact both temperature and precipitation with a time scale of greater than 10 days23. Hurrell24 concluded that the temperature variabilities over Scandinavia and central Asia are strongly related to NAO. Delworth and Zeng25 argued that NAO fluctuations can modulate hemispheric temperature by several tenths of a degree.The impacts of natural factors  on multiple meteorological elements, including temperature and precipitation, have been extensively investigated26. Morioka et al.27 found that the temperature variabilities in the South Atlantic and Indian Oceans may be related to quasi-stationary oceanic Rossby waves. Kikuchi and Wang28 argued that Rossby wave trains play an essential role in the initiation, development, and propagation of quasi-biweekly oscillation in air temperature over the globe.Studies have also examined the relationship between Rossby waves and meteorological elements6. Kim et al.29 attempted to compare the influences of natural and anthropogenic factors on the weekend effect using diurnal temperature data from 1950 to 2000 and concluded that the natural weekend effect induced by Rossby waves was much stronger than the anthropogenic weekend effect. Godfrey et al.30 suggested that meteorological patterns occurring frequently on or near a specific calendar date are more likely to be statistical phantoms than genuine climatic phenomena.However, whether natural or anthropogenic factors cause the weekend effect is still debatedThe reductions in anthropogenic emissions that occur during major holidays, such as the Chinese New Year holiday, provide good opportunities to study the impacts of holidays on observed air temperatures. The Chinese New Year holiday is a national week-long holiday that has been celebrated for centuries. It provides a good opportunity to investigate the holiday effect on observed air temperatures. Based on daily air temperature observations at 2 meters above the surface collected at 2200 weather stations in China from 1961 to 2015 , which is the area of the grid cell multiplied by the total area of all of the grid cells.31, the holiday effect values obtained for three separate sub-periods, namely, 1961–1980  becomes weaker. Additionally, the cooling holiday effect nearly disappears when the DTR (0.01 °C) is considered. A warming holiday effect is observed over the Tibetan Plateau  and along the northern border of Xinjiang Province .During the period from 1981 to 2000, the cooling holiday effect becomes stronger in terms of Tmean and Tmin . More importantly, the holiday effects of Tmax  and the DTR become opposite from each other. Similarly, warming holiday effects are observed with regards to Tmean, Tmax, Tmin and the DTR over the Tibetan Plateau; however, in Xinjiang Province, the warming holiday effect is only obvious in the DTR.During the period from 2001 to 2015, the cooling holiday effect is once again weaker with regard to Tmean, Tmax, Tmin and the DTR are similar to those using Method 1  are significant whether at p = 0.01 or p = 0.05 level [see supplementary Table Method 1 allows us to investigate the inter-annual variabilities in the holiday effect. A two-tailed p-value. The FDR approach at p = 0.01 or p = 0.05 level is also applied to these p-values to obtain the significant results. When the test statistic used for the significance test is the coefficient of variation , most of the stations , the holiday effect values obtained for all of the stations are insignificant. These differences in the significant results may be related to the different test statistics employed for the significance testing.Method 2 allows us to investigate the characteristics of the holiday effect at an intra-annual scale. The percentage of samples whose test statistic are larger than that of the 7-day holiday ΔT series, is regarded as the see Fig. . When tht-tests, most stations .T . IMF1 through IMF7 contain information on the different signal sources of the ΔTmin value, including their frequencies. IMF8 denotes the trend in the data series. The contribution from the variance in each IMF is calculated for each station by dividing the variance in each IMF by the sum of all of the variances from all of the IMFs. Figure Tmean, Tmax, Tmin and the DTR.Eight primary IMFs are obtained for each station after decomposing the daily series of ΔTmean, ΔTmax and ΔTmin range from 21.1% to 29.8%  vary only slightly relative to those over the whole period (1961–2015). The standard deviations in the variance contributions among all of the stations vary from 4.2% to 7.6%.IMF1 through IMF3 are regarded as the dominant IMFs because they account for 72.9–82.2% of the oscillations in ΔT. During the period from 1961 to 2015, the variance contributions of each dominant IMF in ΔTmean, ΔTmax, ΔTmin and the ΔDTR according to the lunar calendar. For IMF2, the periods of ΔTmean, ΔTmax, ΔTmin and the ΔDTR are 9.1 days, 8.7 days, 8.4 days and 7.9 days, respectively. The periods of both ΔTmin and the ΔDTR are approximately 8 days. IMF3 has a period of 17.7 days in ΔTmean and periods of 16.6 days, 16.0 days and 14.5 days in ΔTmax, ΔTmin and the ΔDTR, respectively. The standard deviations of the periods of IMF1 and IMF2 are 0.4–0.7 days, and the standard deviation is 1.5–2.0 days for the period of IMF3. The periodic features during the three separate periods  are similar to those over the whole period (1961–2015).Figure To evaluate the validities of the method used in Method 2 see Fig. , the firTo further uncover the primary cause of the holiday effect on the air temperatures, the data processing scheme is repeated using only the data collected on dry days. The relevant figures are shown in Figs Tmean, Tmax and Tmin all exhibit cooling holiday effects throughout most of the regions of China when only data on dry days are used. The corresponding simple spatial averages are approximately −0.41 °C to −0.49 °C  were detected, both of which demonstrate highly variable length and time scales. This result demonstrates that the so-called holiday effect is likely related to natural atmospheric oscillations. The spatial pattern of the holiday effect may be regarded as an extension of such oscillations. The central region and most of the southern regions of China are located in the troughs of these oscillations, whereas other areas, including the Tibetan Plateau, are located on the oscillation peaksves Fig. 19.Therefore, the so-called holiday effect is mainly attributed to the superposition of natural atmospheric oscillations with different periods . Although a positive correlation is found between the holiday effect and the NAO , current methods still cannot quantitatively distinguish the specific contributions from natural processes and human activities to the air temperatures during the holiday week. Further research is thus required.31.Located on the western shore of the Pacific Ocean, China experiences a typical monsoon climate with cold, dry winters and hot, rainy summers. The terrain in China descends from the west toward the east, with elevations that reach up to 8336 m in the southwest see Fig. . China iThe Chinese New Year, which is the most important festival in China, has been celebrated for thousands of years. During the holiday season, almost all factories are shut down and people are off work. The official holiday typically lasts 7 days, and it begins on the first day of lunar January (the Chinese New Year’s Day) according to the Chinese lunar calendar. On the solar calendar , this holiday falls in either January or February. To reconcile the differences between these two calendars, the Chinese lunar calendar has 12 lunar months in a typical year and 13 lunar months in a leap year. There are generally three leap years and two typical years in a five-year period.Tmean, Tmax, Tmin and the DTR collected at 2200 stations in China from 1961 to 2015  adjustments with reference series and metadata for each station44, The detailed process of this method was described in previous studies45. This method has also been demonstrated to efficiently adjust the shifts in time series caused by changes of location of stations, instruments and observation surroundings47. Moreover, only stations with observational records that are at least 82% complete are used in the individual data analysis. 82% is used as the threshold to obtain enough stations in each part of the data analysis and increase the reliability of the results. Approximate 2200 stations satisfy this requirement.Apart from the basic data quality control, the inhomogeneity of the datasets has also been consideredTmean, Tmax, Tmin and the DTR of the holiday week (a 7-day period) with those of the work week immediately preceding and following the holiday week  during each year, and it has been used in similar studies48. This method is used to allow direct comparison with the previous studies.In this study, two methods are used to evaluate the holiday effect on air temperatures during the week of the Chinese New Year holiday. Method 1 is a traditional method that directly compares the temporally-averaged values of Tmean, Tmax and Tmin are calculated for each day  for each station, as shown in Fig. th polynomial order is used to improve the regression results. The regression sufficiently capture the seasonal cycles, with a correlation coefficient of 0.98, and a standard error less than 0.3 °C. The daily deviations in air temperatures  during the Chinese New Year Holiday.In Method 2, 9ΔT, Fig.  are obtaΔT, Fig.  from theΔT, Fig. . The aveΔT, Fig. . Apart ft-test. A holiday effect value is obtained at each station for every year. The null hypothesis is that the inter-annual variations in the holiday effect at each station resemble a normal distribution with a mean of zero.Two different tests were used to assess the statistical significance of the holiday effect on air temperatures. Method 1 allows us to test the significance of the holiday effect at an inter-annual scale for each station using a single two-tailed 55 is applied to the ΔT series for each station to examine the significance of the holiday effect  at an intra-annual scale see Fig. . The coosee Fig. . The 7-dp-values are calculated as the proportion of test statistics that exceed those obtained during the 7-day holiday week during the 5,000 iterations. Following Earl et al.54, two types of test statistics are calculated, namely, the coefficient of variation  and the range of ΔT .The test statistics obtained from the Monte-Carlo-produced 7-day ΔT series are then compared with those of the 7-day holiday ΔT series. This procedure is repeated 5,000 times for each station. The 56. In order to evaluate this issue, the false discovery rate (FDR) approach, which controls the expected proportion of null hypotheses that are mistakenly rejected , is applied to the p-values obtained from multiple hypothesis tests57. It has been demonstrated that the FDR is directly applicable to the field significance problem56, and has been applied to a variety of climate studies58. The FDR approach at p = 0.01 or p = 0.05 level is applied to the p-values to increase the chances that significant results in this study are not an evaluation of “field significance”.Studies have pointed out that strong spatial correlations exist in air temperature data, which may lead to an evaluation of field significance  in multiple hypothesis tests59 and is widely employed in geophysical research60. The EEMD is a refinement of the original empirical mode decomposition (EMD) method, which decomposes an original signal into a finite number of intrinsic mode functions (IMFs). Each IMF contains the local features of the original signal at different time scales, and the number of IMFs is positively correlated with the temporal scale of the data series61. The instantaneous frequency of each IMF can be obtained by performing a Hilbert transform, and the average period of each IMF can be easily calculated from the frequency.The method used here combines ensemble empirical mode decomposition (EEMD) with a Hilbert transform to further analyze the air temperature oscillations. This method is highly effective for decompositionTmax and increase Tmin62, which may impact the inferred magnitude of the holiday effect on the air temperatures. Therefore, the holiday effect is also examined using only data from dry days.After analyzing the holiday effect using data on all available days, the data processing was repeated using only data collected on dry days. In other words, the data collected during all precipitation days  were excluded. Precipitation and clouds can decrease Supplementary Information