Selectivity indices were even higher than those reported for the reference drug, 5-fluorouracil (SI = 0.96), the starting compound resveratrol (SI = 0.45) and the equimolar mixture of curcumin plus resveratrol (SI = 0.77). The previous hybrids showed good antiproliferative activity.We synthesized twelve hybrids based on curcumin and resveratrol, and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, along with the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids  Colorectal cancer (CRC) is the third most diagnosed malignancy and the fourth leading cause of cancer-related death worldwide. Despite the progress in diagnosis and treatment, incidence and mortality of CRC cancer is unacceptably high . CurrentCurcuma longa Linn. Several studies with this compound have revealed important functions in cancer control + calcd. for C24H21O4 [M + H]+ 373,1361Yield 20%; m.p. 119–120 °C; IR (cm3b)(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-((E)-4-methoxystyryl)phenyl)prop-2-en-1-one (−1): νmax 3248 (OH), 1641 (C = O), 1568 (C = C), 1452 (C = CAr), 1263 (C-O-C); 1H-NMR  δ 8.15 , 7.78 , 7.74 , 7.69 , 7.61 , 7.52 , 7.41 , 7.30 , 7.22 , 6.98 , 6.84 , 3.88 , 3.80 . 13C-NMR  δ 188.42 (C = O), 159.90 (Ar-O), 150.45 (Ar-O), 148.50 (Ar-O), 145.16 (C = C), 142.35 (Ar), 136.78 (Ar), 131.31 (C = C), 129.76 (C = C), 129.47 (2C Ar), 129.05 (Ar), 128.75 (2C Ar), 126.73 (Ar), 126.62 (Ar), 125.58 (Ar), 124.70 (C = C), 118.90 (Ar), 116.07 (Ar), 114.73 (2C Ar), 114.36 (Ar), 112.12 (Ar), 56.28 (OCH3), 55.66 (OCH3). HRMS-ESI (m/z): 387,1589 [M+H]+ calcd. for C25H23O4 [M + H]+ 387.1518Yield 50%; m.p. 187–190 °C; IR (cm3c)(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-((E)-4-nitrostyryl)phenyl)prop-2-en-1-one (−1): νmax 3232 (OH), 1643 (C = O), 1556 (C = C), 1452 (C = CAr), 1303 (NO2), 1276 (C-O-C); 1H-NMR  δ 8.13 , 7.81 , 7.73–7.63 , 7.53 , 7.49 , 7.34 , 7.30 , 7.08 , 6.85 , 6.73 , 3.88 . 13C-NMR  δ 188.30 (C = O), 150.81 (Ar-O), 150.13 (Ar-O), 148.45 (Ar-NO2), 144.93 (C = C), 145.02 (Ar), 142.99 (Ar), 136.17 (Ar), 132.13 (Ar), 129.50 (C = C), 128.55 (2C Ar), 126.83 (C = C), 126.42 (Ar), 126.31 (Ar), 124.84 (Ar), 124.58 (C = C), 122.81 (Ar), 119.07 (Ar), 116.04 (Ar), 112.58 (2C Ar), 112.11 (Ar), 56.29 (OCH3). HRMS-ESI (m/z): 402,1977 [M+H]+ calcd. for C24H20NO5 [M + H]+ 402.1263Yield 35%; m.p. 227–230 °C; IR (cm3d)(E)-1-(4-((E)-2,3-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3359 (OH), 1651 (C = O), 1575 (C = C), 1510 (C = CAr), 1267 (C-O-C); 1H-NMR  δ 8.03 , 7.77 , 7.66 , 7.59 , 7.40 , 7.27 , 7.23 , 7.18 , 7.14 , 7.08 , 6.96 , 6.88 , 3.97 (OCH3), 3.90 (OCH3), 3.88 (OCH3). 13C-NMR  δ 189.79 (C = O), 153.17 (Ar-O), 148.36 (Ar-O), 147.27 (Ar-O), 146.87 (Ar-O), 145.07 (C = C), 142.00 (Ar), 137.32 (Ar), 131.03 (Ar), 129.06 (2C Ar), 128.84 (C = C), 127.59 (Ar), 126.75 (2C Ar), 125.61 (C = C), 124.28 (C = C), 123.45 (Ar), 119.71 (Ar), 117.99 (Ar), 114.95 (Ar), 111.99 (Ar), 110.07 (Ar), 61.29 (OCH3), 56.07 (OCH3), 55.89 (OCH3). HRMS-ESI (m/z): 417,1696 [M+H]+ calcd. for C26H25O5 [M + H]+ 417.1623Yield 55%; m.p. 63–65 °C; IR (cm3e)(E)-1-(4-((E)-2,4-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3349 (OH), 1649 (C = O), 1575 (C = C), 1508 (C = CAr), 1259 (C-O-C); 1H-NMR  δ 7.99 , 7.76 , 7.60 , 7,53 , 7,53 , 7.40 , 7.22 , 7.13 , 7.06 , 6.95 , 6.53 , 6.48 , 4.60 (OH), 3.95 (OCH3), 3.88 (OCH3), 3.84 (OCH3). 13C-NMR  δ 189.77 (C = O), 161.13 (Ar-O), 158.42 (Ar-O), 148.31 (Ar-O), 146.88 (Ar-O), 144.85 (C = C), 142.85 (Ar), 136.63 (Ar), 129.04 (2C Ar), 127.69 (C = C), 127.64 (Ar), 126.31 (2C Ar), 126.09 (C = C), 125.80 (C = C), 123.37 (Ar), 119.74 (Ar), 118.98 (Ar), 114.96 (Ar), 110.12 (Ar), 105.17 (Ar), 98.51 (Ar), 56.06 (OCH3), 55.59 (OCH3), 55.50 (OCH3). HRMS-ESI (m/z): 417,1696 [M+H]+ calcd. for C26H25O5 [M + H]+ 417.1623Yield 40%; m.p. 78–82 °C; IR (cm3f)(E)-1-(4-((E)-2,5-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3379 (OH), 1651 (C = O), 1577 (C = C), 1508 (C = CAr), 1265 (C-O-C); 1H-NMR  δ 8.01 , 7.77 , 7.63 , 7.56 , 7.40 , 7.22 , 7.18–7.08 , 6.96 , 6.84 , 6.18 , 3.94 (OCH3), 3.85 (OCH3), 3.82 (OCH3). 13C-NMR  δ 189.78 (C = O), 153.74 (Ar-O), 151.73 (Ar-O), 148.38 (Ar-O), 146.91 (Ar-O), 145.05 (C = C), 142.18 (Ar), 137.10 (Ar), 129.03 (2C Ar), 128.19 (C = C), 127.57 (Ar), 126.67 (2C Ar), 126.58 (C = C), 125.99 (C = C), 123.41 (Ar), 119.62 (Ar), 114.98 (Ar), 114.54 (Ar), 112.31 (Ar), 111.81 (Ar), 110.16 (Ar), 56.25 (OCH3), 56.04 (OCH3), 55.85 (OCH3). HRMS-ESI (m/z): 417,1696 [M+H]+ calcd. for C26H25O5 [M + H]+ 417.1623Yield 38%; m.p. 47–51 °C; IR (cm3g)(E)-1-(4-((E)-2,6-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3365 (OH), 1649 (C = O), 1573 (C = C), 1510 (C = CAr), 1249 (C-O-C); 1H-NMR  δ 8.01 , 7.76 , 7.60–7.66 , 7.41 , 7.25–7.17 , 7.14 , 6.96 , 6.60 , 6.12 , 3.95 , 3.91 . 13C-NMR  δ 189.86 (C = O), 158.91 (2 Ar-O), 148.20 (Ar-O), 146.82 (Ar-O), 144.73 (C = C), 143.92 (Ar), 136.64 (Ar), 131.13 (C = C), 128.94 (2 Ar), 128.06 (Ar), 127.69 (Ar), 126.44 (2 Ar), 123.33 (C = C), 122.74 (Ar), 119.87 (C = C), 114.89 (Ar), 114.28 (Ar), 110.06 (Ar), 103.97 (2 Ar), 56.06 (OCH3), 55.88 (2 OCH3). HRMS-ESI (m/z): 417,1696 [M+H]+ calcd. for C26H25O5 [M+H]+ 417.1623Yield 35%; m.p. 87–90 °C; IR (cm3h)(E)-1-(4-((E)-3,5-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3388 (OH), 1651 (C = O), 1577 (C = C), 1508 (C = CAr), 1267 (C-O-C); 1H-NMR  δ 8.00 , 7.76 , 7.58 , 7.39 , 7.21 , 7.16–7.08 , 6.96 , 6.69 , 6.42 , 3.93 (OCH3), 3.82 (2 OCH3). 13C-NMR  δ 189.69 (C = O), 161.04 (2 Ar-O), 148.49 (Ar-O), 146.97 (Ar-O), 145.18 (C = C), 141.43 (Ar), 138.77 (Ar), 137.34 (Ar), 131.26, 129.07 (2C Ar), 128.09 (C = C), 127.49 (C = C), 126.66 (2C Ar), 123.43 (C = C), 119.46 (Ar), 115.04 (Ar), 110.24 (Ar), 104.91 (2C Ar), 100.59 (Ar), 56.04 (OCH3), 55.44 (2 OCH3). HRMS-ESI (m/z): 417,1696 [M+H]+ calcd. for C26H25O5 [M + H]+ 417.1623Yield 35%; m.p. 49–51 °C; IR (cm3i)(E)-1-(4-((E)-3,4-dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3390 (OH), 1649 (C = O), 1577 (C = C), 1508 (C = CAr), 1265 (C-O-C); 1H-NMR  δ 8.01 , 7.77 , 7.60 , 7.40 , 7.23 , 7.19 , 7.13 , 7.11–7.07 , 7.02 , 6.96 , 6.88 , 3.96 (OCH3), 3.96 (OCH3), 3.91 (OCH3). 13C-NMR  δ 189.68 (C = O), 149.51 (Ar-O), 149.22 (Ar-O), 148.32 (Ar-O), 146.86 (Ar-O), 145.00 (C = C), 141.95 (Ar), 136.98 (Ar), 131.17 (Ar), 129.89 (C = C), 129.10 (2C Ar), 127.60 (C = C), 126.33 (2C Ar), 125.67 (Ar), 123.37 (C = C), 120.56 (Ar), 119.64, 114.94 (Ar), 111.22 (Ar), 110.16 (Ar), 108.85 (Ar), 56.07 (OCH3), 56.01 (OCH3), 55.95 (OCH3). HRMS-ESI (m/z): 417,1696 [M+H]+ calcd. for C26H25O5 [M + H]+ 417.1623Yield 55%; m.p. 67–70 °C; IR (cm3j)(E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-((E)-4-hydroxy-3-methoxystyryl)phenyl)prop-2-en-1-one (−1): νmax 3232 (OH), 1641 (C = O), 1589 (C = C), 1508 (C = CAr), 1269 (C-O-C); 1H-NMR  δ 8.01 , 7.77 , 7.58 , 7.40 , 7.22 , 7.15–7.10 , 7.08–7.03 , 6.99 , 6.93 , 6.10 , 5.88 , 3.95 . 13C-NMR  δ 189.70 (C = O), 148.34 (Ar-O), 146.88 (Ar-O), 146.86 (Ar-O), 146.25 (Ar-O), 145.03 (C = C), 142.01 (Ar), 136.87 (Ar), 131.32 (Ar), 129.45 (C = C), 129.10 (2C Ar), 127.58 (C = C), 126.27 (2C Ar), 125.32 (C = C), 123.36 (Ar), 121.08 (Ar), 119.57 (Ar), 114.96 (Ar), 114.75 (Ar), 110.19 (Ar), 108.48 (Ar), 56.06 (OCH3), 55.98 (OCH3). HRMS-ESI (m/z): 403,1540 [M+H]+ calcd. for C25H23O5 [M + H]+ 403.1467Yield 21%; m.p. 142–146 °C; IR (cm3k)(E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-((E)-3,4,5-trimethoxystyryl)phenyl)prop-2-en-1-one (−1): νmax 3390 (OH), 1651 (C = O), 1579 (C = C), 1504 (C = CAr), 1265 (C-O-C); 1H-NMR  δ 8.03 , 7.77 , 7.62 , 7.41 , 7.24 , 7.17 , 7.14 , 7.06 , 6.97 , 6.78 , 3.97 (OCH3), 3.93 (2 OCH3), 3.89 (OCH3). 13C-NMR  δ 189.69 (C = O), 153.51 (2 Ar-O), 148.35 (Ar-O), 146.87 (Ar-O), 145.12 (C = C), 141.56 (Ar), 137.28 (Ar-O), 132.54 (Ar), 131.28 (Ar), 129.11 (2C Ar), 127.57 (C = C), 127.09 (C = C), 126.51 (2C Ar), 123.37 (C = C), 119.61 (Ar), 114.95 (Ar), 110.18 (Ar), 103.90 (2C Ar), 61.06 (OCH3), 56.20 (OCH3), 56.08 (OCH3). HRMS-ESI (m/z): 447,1803 [M+H]+ calcd. for C27H27O6: 447.1729Yield 55%; m.p. 74–77 °C; IR (cm3l)(E)-1-(4-((E)-4-Hydroxy-3,5-dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (−1): νmax 3332 (OH), 1641 (C = O), 1589 (C = C), 1508 (C = CAr), 1269 (C-O-C); 1H-NMR  δ 8.02 , 7.77 , 7.59 , 7.40 , 7.19–7.12 , 7.16 , 7.14–7.11 , 7.00 , 6.95 , 6.79 , 6.02 , 5.69 , 3.96 (OCH3), 3.95 (2 OCH3). 13C-NMR  δ 189.64 (C = O), 148.31 (Ar-O), 147.29 (2 Ar-O), 146.85 (Ar-O), 145.02 (C = C), 141.82 (Ar-O), 136.99 (Ar), 135.41 (Ar), 131.49 (Ar), 129.10 (2C Ar), 128.42 (C = C), 127.59 (C = C), 126.30 (2C Ar), 125.69 (Ar), 123.34 (C = C), 119.59 (Ar), 114.93 (Ar), 110.17 (Ar), 103.69 (2C Ar), 56.38 (2 OCH3), 56.07 (OCH3). HRMS-ESI (m/z): 433,1646 [M+H]+ calcd. for C26H25O6: 433.1572.Yield 40%; m.p. 86–88 °C; IR , its metastatic derivative (SW620) and non-malignant cells (CHO-K1). These were obtained from The European Collection of Authenticated Cell Cultures  and maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% heat-inactivated (56 °C) horse serum, 1% penicillin/streptomycin and 1% non-essential amino acids . For all experiments, horse serum was reduced to 3%, and the medium was supplemented with 5 mg/mL transferrin, 5 ng/mL selenium and 10 mg/mL insulin  .2. All cultures were allowed to grow for 24 h, and afterward they were treated with DMSO (vehicle control 1%) or increasing concentrations (0.01–200 µM) of the synthesized hybrids, as well as curcumin and resveratrol (lead compounds), the equimolar mixture of these and 5-fluorouracil . After treatment, cells were fixed with trichloroacetic acid (50% v/v) (MERCK) for a period of 1 h at 4 °C. Cell proteins were determined by staining with 0.4% (w/v) SRB , and then they were washed with 1% acetic acid for the removal of unbound SRB and left for air-drying. Protein bound SRB was solubilized in 10 mM Tris-base and the absorbance was measured at 492 nm in a microplate reader (Mindray MR-96A) [Cytotoxicity of the synthesized hybrids, lead and reference compounds was evaluated through Sulforhodamine B (SRB) assay, a colorimetric test that is based on staining of total cellular protein of adherent cells. Cells were seeded to a final density of 20,000 cells/well in 96-well tissue culture plates and incubated at 37 °C in a humidified atmosphere at 5% CO MR-96A) . All exp50 values) or DMSO , for 0, 2, 4, 6 and 8 days. Culture media was replaced every 48 h. After each incubation time, cells were fixed, stained and read as previously described for this technique [Antiproliferative effect of the most active compounds was also tested through Sulforhodamine B (SRB) assay. Briefly, cells were seeded to a final density of 2500 cells/well in 96-well tissue culture plates and incubated in the same conditions described for cytotoxicity. Cultures were allowed to grow for 24 h and then were treated with increasing concentrations of the selected hybrids .All experiments were performed at least three times. Data are reported as mean ± SE (standard error). Statistical differences between control group (non-treated) and treated cells were evaluated by one-way ANOVA followed by the Dunnett’s test. Values with


Amino acid sequences of mugwort were determined by mass spectrometry. Afterward, cross-reactivity between mugwort tropomyosin and Dermatophagoides pteronyssinus 10 (Der p10) was analysed by ELISA inhibition and basophil activation experiments. To compare immunologic responses eliciting by two different tropomyosins, peripheral blood mononuclear cells (PBMCs) of HDM-monosensitized patients were stimulated by mugwort, HDM, Der p10 and synthetic peptides representing mugwort tropomyosin respectively.+AR patients were mainly sensitized to cat and mugwort, and the positive rate of monosensitized HDM+AR out-clinic patients was increased during the mugwort pollen season. Tropomyosin protein was able to find in mugwort. Synthetic tropomyosin peptide of mugwort activated basophils which were primed by HDM-specific IgE; ELISA inhibition experiment showed synthetic tropomyosin peptide of mugwort inhibited IgE binding to HDM tropomyosin, Der p10. Unlike HDM and Derp 10, mugwort and mugwort tropomyosin mainly induced IFN-γ and IL-17 release in PBMCs of monosensitized HDM+AR patients, but not IL-5.Polysensitized HDM+ patients to reduce mugwort exposure in mugwort pollen season in virtue of the tropomyosin induced mild inflammation.Pan-allergen tropomyosin accounts for the cross-reactivity between mugwort and HDM, which reminds HDMThe online version contains supplementary material available at 10.1186/s12948-021-00142-z. Allergic rhinitis (AR), an upper airway allergic inflammatory disease, causes symptoms of sneeze, runny nose, nasal obstruction and itchy nose, which is predominantly mediated by type 2 helper (Th2) cells and immunoglobulin E (IgE) , 2. AmonAllergen specific immunotherapy (AIT) is an effective therapeutic method for monosensitized AR patients . However+AR patients. In view of this, the present study has specifically investigated cross-reactivity between HDM and mugwort in HDM+AR patients.HDM and mugwort have been regarded as the two most common and clinically relevant sensitizing allergens in AR patients in Asia . HDM croSubjects with AR based on criteria of the AR and its Impact on Asthma (ARIA) consensus statement  were recPenicillium notatum, Cladosporium herbarum, Aspergillus fumigatus, Alternaria alternata) and hops], and one with a mix of food allergens . Furthermore, concentrations of Der f2 specific IgE, Der p1 specific IgE, and total IgE were also measured using the ImmunoCAP system . Allergen-specific IgE > 0.35 kU/L was considered as positive.The presence of IgE antibodies in blood was determined using a EUROLINE Atopy Screen , which comprised two sets of allergens; one with a mix of aeroallergens .+AR patients and 1 mugwort+ AR patient using Ficoll-Hypaque density gradient centrifugation according to the standard protocol . Cells were plated at a density of 1 × 106 cells/well in a 24-well plate in 0.5 mL RPMI 1640  culture medium containing either HDM , mugwort , or synthetic peptides of mugwort tropomyosin, and then incubated at 37 °C in 5% CO2 for 48 h. Cells incubated with RPMI 1640 medium alone were used as controls. After incubation, the cell suspensions were collected and the supernatants were assessed for IL-5, IL-17, and IFN-γ using Luminex xMAP suspension array technology in a Bio-Plex 200 system . All cytokine kits were purchased from R&D Company and the results were expressed as pg/mL.PBMCs were isolated from the blood of 6 healthy donors, 16 HDM+AR patients. The influence of polysensitization on asthma development was assessed by the Chi squared test. The prevalence of different allergens in HDM+AR patients was estimated using Fisher’s exact test and logistic regression. The Wilcoxon test was used for paired comparisons of the effect of specific antigen stimulation on the release of cytokines from PBMCs, and the effect of synthetic mugwort peptides on blocking HDM specific IgEs between groups. P values of less than 0.05 were regarded as statistically significant.Statistical analysis was performed using the SPSS version 22.0 software package . Categorical variables were described using frequencies and/or percentages and continuous variables were presented as mean ± standard deviation (SD). Multiple logistic regression was used to analyse the possible risk factors for polysensitized HDM+AR patients were recruited into the study. Overall, 64.6% of the HDM+AR patients were monosensitized, and 35.4% polysensitized (Table p = 0.001). Type of sensitizing allergens was further analysed in polysensitized HDM+AR patients in parallel with the HDM-specific IgE level. Regardless of the level of HDM-specific IgE detected, sensitization was greatest to inhalant allergens in the polysensitized HDM+AR patients , mugwort , house dust , cockroach  and hops   to produce high levels of IL-5 (0.59–61.56 pg/mL) and IL-17 (0.91–5.27 pg/mL), but not IFN-γ  to produce IL-17 (1.36–2.24 pg/mL) and IFN-γ (3.7–4.79 pg/mL), but not IL-5   Fig. e.We demonstrated for the first time that cross-reactive protein tropomyosin in mugwort and HDM is responsible for the cross-reactivity between HDM and mugwort. However, unlike HDM and Derp 10, mugwort and synthetic tropomyosin peptide induced Th1 and Th17 in PBMCs of HDM monosensitized AR patients, but not Th2 response.+AR patients who are sensitized to mugwort. Furthermore, the number of monosensitized HDM+AR patients is increased from July to August. Mite densities indeed vary with seasons and areas. Reportedly, three peaks for the domestic mites density in Beijing appear in September to October, January and May [+AR patients in July and August might likely be affected by mugwort. Although amino acid sequences of mugwort (A. sieversian) tropomyosin proteins are different from that of HDM tropomyosin Der p10. Considering that the sequence of the same protein varies in different species and cross-reactivity is thought to occur when a protein of similar sequence, structure or family binds to T and B cell receptors [Tropomyosin, a pan-allergen, belongs to a family of phylogenetically conserved proteins with multiple isoforms present in muscle and non-muscle cells of vertebrates and invertebrates . It has  and May . Thus, teceptors . Therefo+AR patients with mugwort induced synthesis of IL-17 and IFN-γ, whereas stimulation with HDM induced synthesis of IL-5 and IL-17. Our group has previously demonstrated that single-nucleotide polymorphisms (SNPs) in IL-17A and IL-17F gene regions are potentially associated with the development of AR and comorbid asthma in Chinese subjects [+AR patients may be associated with clinical symptoms of patients. In this study, the finding for Der p10-induced synthesis of IL-5 in monosensitized HDM+AR patients was in accordance with the findings of stimulation by HDM. In the meanwhile, stimulation synthesized tropomyosin peptide of mugwort could induce IL-17 and IFN-γ by PBMCs of HDM+AR subjects, suggesting that tropomyosin is responsible for the cross-reactivity between HDM and mugwort, eliciting non-Th2 response.The present study has indicated that stimulation of PBMCs of monosensitized HDMsubjects . Similarsubjects . As ment+AR patients in comparison to HDM. Since mugwort stimulation may be related to clinical symptoms of HDM sensitized patients in autumn pollen season, avoiding to expose to mugwort should be recommended to HDM+AR patients who cross-react to mugwort. Furthermore, therapeutic agents for targeting non-Th2 response may potentially alleviate symptoms of patients. Whether HDM+AR patients are benefit for mugwort AIT due to the present of cross-reactive protein tropomyosin still needs to be further investigated.In conclusion, we have for the first time demonstrated that mugwort tropomyosin cross-react to Der p10, and therefore might play a role in eliciting a non-Th2 response in polysensitized HDMAdditional file 1: Table S1. Identified corresponding proteins in Artemisia sieversian.


In the last decade, there have been more than 40 randomized trials evaluating early mobilization and rehabilitation in intensive care units (ICU) . Such trDespite supporting evidence and guidelines, implementation of ICU mobilization and rehabilitation is highly variable. Hence, Early mobilization and rehabilitation is more successful in ICUs with a culture that prioritizes and values this intervention . MobilitA structured QI approach can greatly enhance successful implementation of early mobilization and rehabilitation . One appA systematic review identified 28 unique barriers to early mobilization and rehabilitation, including patient-related barriers , structural barriers , procedural barriers , and cultural barriers  . There aThe multi-professional team effort required for early mobilization and rehabilitation program depends on optimal communication. We recommend that interprofessional communication is facilitated using a structure adapted to the individual ICU that allows  mobilization goals, including an opportunity for all team members to raise concerns and ensure flow of information regarding mobility goals and achievement across staff and over time .ICU patients’ experience with early mobilization and rehabilitation is variable. It may be tiring, uncomfortable and difficult, while at other times be motivating and rewarding for patients . With imMeta-analyses have demonstrated the safety of in-bed and out-of-bed ICU mobilization, with rare occurrence of serious events . One metPatients’ sedation and delirium status is a common barrier to early mobilization and rehabilitation . More brBarriers to early mobilization and rehabilitation may include ICUAW, impaired physical functioning, traumatic injuries, and obesity . EquipmeImportant knowledge gaps exist regarding exercise, including the timing, type, and dose of interventions. There is some evidence suggesting that starting rehabilitation within 2 or 3 days of ICU admission may be superior to later initiation . AdditioMobility and rehabilitation-related measures, appropriate to the ICU setting and integrated into clinical care, are needed to set patient goals and track their progress, allocate scarce rehabilitation resources to those patients who may benefit the most, and conduct evaluations of structured quality improvement programs . UnderstEvidence is still evolving about early mobilization in ICU with ongoing large, multi-center trials. Further research is needed to understand the optimal timing, type and dose of interventions, and their effect on long-term patient outcomes. These 10 strategies provide guidance for implementing early mobilization and rehabilitation in the ICU with the goal of optimizing safety and effectiveness to improve patients’ experiences and outcomes.


AR) mRNA level is upregulated in a subset of UCC cases. In an AR‐positive UCC‐derived cell line model, UM‐UC‐3‐AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell‐like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen‐stimulated UM‐UC‐3‐AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor . Here, we explored the role of androgen receptor (AR) signalling in UCC progression. We detected AR expression in a subset of patients with UCC. Androgen treatment increased clonogenicity and migration of a UCC‐derived cell line, while AR blockade reverted these effects. Finally, androgens regulated the expression of nearly 300 genes, some of which correlated with  ADTandrogen deprivation therapyARandrogen receptorCISin situ carcinomaCpcrossing pointCSScharcoal‐stripped serumCYPcytochrome P450FDRfalse discovery rateGSEAgene set enrichment analysisICCimmunocytochemistryIHCimmunohistochemistryMIBCmuscle‐invasive bladder cancerNESnormalized enrichment scoresNMIBCnonmuscle invasive bladder cancerPCprincipal componentPCAprincipal component analysisPCaprostate cancerPSAprostate‐specific antigensiRNAssmall interfering RNAsTCGAThe Cancer Genome AtlasUCCbladder urothelial cell carcinoma1Bladder urothelial cell carcinoma (UCC) is the 12th most commonly diagnosed malignancy worldwide and the 14th leading cause of cancer death among men and women . IdentifAlthough UCC is not considered a hormone‐related type of cancer, there are indications of the involvement of hormonal factors in the aetiology of bladder cancer. The incidence of UCC in men is approximately threefold higher than in women , 5, 6. FAR gene completely prevented bladder carcinogenesis [Steroids are key mediators of sex‐specific differences. Androgens, the male sex steroids, exert their function through binding to the androgen receptor (AR). The AR is a transcription factor that plays a key role in the development and maintenance of male sexual organs. The androgen and AR signalling pathway is well known for its contribution to the progression of prostate cancer (PCa), and androgen deprivation therapy (ADT) is a first‐line treatment for advanced PCa . Surgicaogenesis . Interesogenesis , 11. AltAR in various nonmalignant bladder and UCC specimens to correlate AR levels with gender and tumour stage. Next, we investigated the role of AR in several cancer‐related pathways using a new AR‐positive bladder cancer cell line model, UM‐UC‐3‐AR. Finally, we have defined the transcriptome signature accompanying phenotypical changes driven by androgen/AR signalling and used expression data from UCC patients to pin point those genes of potential clinical relevance. Our results provide new insights into the role of the AR in UCC proposing novel therapeutic routes for the treatment of this disease.Here, we profiled the expression of 22.1n = 12), NMIBC in situ carcinoma , Ta (n = 37) and T1 (n = 11), and MIBC T2 (n = 27), T3 (n = 14) and T4 (n = 13) specimens were used. For qPCR validation, independent samples were used, including NB (n = 5), CIS (n = 1), Ta (n = 14), T1 (n = 4), T2 (n = 7), T3 (n = 6) and T4 (n = 4). Normal bladder urothelium was obtained by macrodissection of benign urothelium from NMIBC bladder cancer specimens. Specimens were processed by step sectioning and selected for purity of benign (99–100%) or cancer cells (at least 70%). Patient and tumour characteristics can be found in Tables The experiments were undertaken with the understanding and written consent of each subject. The use of patient materials and the study methodologies were approved by the local ethics committee of the Radboud University Medical Center (CMO Arnhem‐Nijmegen) and conformed to the standards set by the Declaration of Helsinki. Upon transurethral resection of tumour tissue or cystectomy, specimens were snap‐frozen in liquid nitrogen. For microarray analysis, normal bladder . Concentration and purity of the RNA were determined on a NanoDrop 1000 Spectrophotometer.2.32 values were used to determine AR gene expression in those specimens.Microarray gene expression analysis on these tissue samples was performed and described previously . Normali2.4HP1BP3 or HPRT1 were used for normalization. Relative gene expression levels were calculated according to Pfaffl et al. [For RT‐qPCR analysis, 2 µg of DNase I‐treated total RNA was used to generate cDNA, using random hexamer primers  and SuperScript II Reverse Transcriptase . Real‐time PCR analysis was performed using LightCycler 480 SYBR Green I Master Mix and a LightCycler 480 Machine (Roche). Crossing‐point (Cp) values were determined using the LightCycler 480 SW 1.5 software (Roche). RNA not subjected to reverse transcriptase was used as a negative control. The average Cp values from two different experimental runs were used for the analysis. Expression levels of l et al. . Primers2.5m R1881  or EtOH (0.1%) as vehicle control. Cells were fixed with 4% paraformaldehyde and left to dry overnight. Slides with fixed tissue (IHC) or cells (ICC) were exposed to boiling 10 mm sodium citrate (pH 6.0) for 10 min. The rabbit polyclonal AR antibody N20  was diluted 1 : 5000 in PBS/1% BSA and incubated for 1 h at room temperature, followed by exposure to Poly‐HRP goat anti‐Ms/Rb/Rt IgG  solution diluted 1 : 1 in PBS/1% BSA for 30 min. Slides were incubated with Bright‐DAB (Immunologic) for 10 min, counterstained with haematoxylin and mounted in Permount Mounting Medium (Thermo Fisher Scientific).For immunohistochemistry (IHC), 4‐µm‐thick UCC tissue sections containing > 50% epithelial cells were cut. For immunocytochemistry (ICC), 5637, T24, 253J, UM‐UC‐3 and UM‐UC‐3‐AR cells (8000/well) were seeded in Lab‐Tek II 8‐Well Chamber Slides (Thermo Fisher). For AR nuclear translocation experiments, cells were seeded in androgen‐deprived medium. After 24 h, cells were treated for 24 h with 10 n2.6−1d‐glucose, 4 mml‐glutamine and 1 mm pyruvate, supplemented with 10% FCS (Sigma) and 1x nonessential amino acids . The 5637 (HTB‐9), T24 (HTB‐4), 253J [ml‐glutamine and 10% FCS. All cell cultures were maintained in a humidified atmosphere at 37 °C and 5% CO2. Cells were frequently tested for Mycoplasma infection, using a Mycoplasma‐specific PCR. All cell lines were authenticated using the PowerPlex 21 System  by Eurofins Genomics (Germany) and propagated for no more than 6 months or 30 passages after resuscitation from the authenticated stocks.The UM‐UC‐3 cell line was purchased from the ATCC . The fully AR‐positive UM‐UC‐3‐AR line was obtained from the UM‐UC‐3 parental line by clonal selection. UM‐UC‐3 and UM‐UC‐3‐AR cells were cultured in Dulbecco’s modified Eagle medium with 1 g·L4), 253J  and LNCa2.76 of UM‐UC‐3, UM‐UC‐3‐AR, LNCaP and 5637 cells were seeded per well in 6‐well plates. Twenty four hours later, cells were lysed using Laemmli lysis buffer  supplemented with 300 mm β‐mercaptoethanol. Lysates were homogenized by sheering them through a 0.5 × 25‐mm syringe needle. The protein concentration was measured after staining with Coomassie Brilliant Blue  using BSA as a standard. A total of 50 µg of protein was used for SDS/PAGE using 7.5% polyacrylamide gels. Proteins were electrotransferred onto PVDF membranes . Membranes were blocked for 1 h in PBS‐T/5% nonfat dry milk and incubated overnight with the primary antibody. The rabbit polyclonal AR antibody N20  and the mouse monoclonal antibody anti‐β‐actin  were diluted 1 : 50 000 and 1 : 5000 in PBS‐T/5% nonfat dry milk, respectively. The PO‐conjugated donkey anti‐rabbit antibody  or sheep anti‐mouse antibody  diluted 1 :50 000 in PBS‐T was used as secondary antibodies. Protein bands were detected using the ECL Detection Kit and Hyperfilm (Amersham Biosciences).A total of 1.2 × 102.8−1 of KaryoMAX  for 30 min. From each cell line, five metaphases were fully analysed by GTG banding at ~ 450 band level to exclude gross chromosomal aberrations. Count and analysis of chromosome marks were manually executed using reference marks described for UM‐UC‐3 by the ATCC: m1 = der(1)t , m2 = ?t(1p5p), m3 = i(3q), m4 = t(7q14q) and m5 = ?t(2p3p).UM‐UC‐3 and UM‐UC‐3‐AR were seeded in T25 culture flasks until reaching 40% confluency. Cells were arrested in metaphase by exposing them to 0.2 µg·mL2.9AR gene glycine repeat region, Phusion High‐Fidelity Polymerase and the Phusion High‐Fidelity buffer specialized for GC‐rich regions (Thermo Fisher) and primers listed in Table AR, SPIN4, PCA3 and GAPDH intronic regions. PCA3 and GAPDH were used for normalization. Normalized AR and SPIN4 copy numbers were adjusted to those from healthy female genomic DNA. Copy number of both genes from a benign prostate hyperplasia specimen was used as a control.Genomic DNA from UM‐UC‐3 and UM‐UC‐3‐AR was isolated using the QIAaMP DNA Mini Kit  following the manufacturer’s instructions. For amplification of the 2.10Renilla (Promega) control vector, and X‐tremeGENE™ 9 Transfection Reagent (Roche) was added per well. One day after transfection, 0.1 or 0.5 nm of R1881 (Organon) was added to the medium in combination with 1 or 5 µm enzalutamide , respectively. DMSO and EtOH (< 1%) were used as vehicle controls. The luciferase activities were measured 16 h later using the Dual‐Luciferase Reporter Assay System (Promega). Luciferase signals were measured on a VVICTOR3 Multilabel Reader .A total of 10 000 cells/well were seeded in FCS‐containing medium in 96‐well plates in triplicate. Transfection mix containing 90 ng of the pCMV‐PSA85‐Luc Reporter Construct , 10 ng of the pRL‐TK 2.11AR and a negative control No. 1 siRNA were used .Silencer Select small interfering RNAs (siRNAs) directed against the 2.12m R1881 (Organon) in combination with 1 µm enzalutamide . DMSO and EtOH (0.2%) were used as vehicle controls. For siRNA experiments, 200 000 cells were seeded and transfected as described above. One day post‐transfection, cells were harvested and reseeded in 6‐well plates in the presence of 0.1 nm R1881. Medium was refreshed after 1 week. After 14 days, colonies were washed with PBS, fixed with 3% paraformaldehyde, and stained with 0.01% crystal violet (Merck) for 60 min. The number and size of colonies were analysed using the fiji software [UM‐UC‐3 and UM‐UC‐3‐AR cells were seeded in 6‐well plates at a density of 250 cells/well and cultured in medium containing 0.1 nsoftware . Morphol2.13m of synthetic androgen R1881 (Organon) was added to the medium in combination with 1 µm enzalutamide . DMSO and EtOH (0.2%) were used as vehicle controls. For siRNA experiments, 10 000 cells/well were seeded and transfected as described above. One day after transfection, cells were treated with R1881 and enzalutamide at concentrations described before. Cell viability was measured at regular time intervals using 3‐‐2,5‐diphenyltetrazolium bromide (1 mg·mL−1) assays. Doubling time was calculated using the Doubling Time tool at www.doubling‐time.com.A total of 10 000 cells/well were seeded in 96‐well plates in triplicate, in charcoal‐stripped serum (CSS)‐containing medium to wash away traces of androgens previously reported to be present in FCS . One day2.14m R1881 (Organon) in combination with 1 µm enzalutamide . DMSO and EtOH (0.2%) were used as vehicle controls. For siRNA‐mediated AR knockdown, siRNA transfection was performed 24 h after seeding. Treatment with 0.1 nm R1881 started 24 h after transfection. When cells reached 100% confluency , a wound was made by mechanically scratching the cell monolayer with a 1‐mL pipette tip. Medium was replaced, and cells were allowed to migrate into the cell‐free area. Wound size was measured at regular time intervals, and cell migration was calculated using the difference in wound size between each time point.A total of 70 000 UM‐UC‐3 or UM‐UC‐3‐AR cells were seeded per well in a 24‐well plate CSS‐containing medium. One day after seeding, cells were treated with 0.1 n2.15For RNA sequencing analysis, samples were purified using an RNeasy RNA Extraction Kit (Qiagen) with DNase I treatment. Total RNA (1 µg per sample) was used for library preparation using the RNA HyperPrep Kit with RiboErase . A fragmentation step was carried out for 6.5 min, and NEXTFLEX DNA barcodes  were used for adapter ligation. Libraries were amplified using 10 amplification cycles. Library concentration was measured using the dsDNA Fluorescence Quantification Assays , and library size was determined using the BioAnalyzer High Sensitivity DNA Kit . Sequencing was performed using an Illumina NextSeq 500, and 50‐bp paired‐end reads were generated.2.16Complex Heatmap package [Paired‐end reads were aligned to the human GRCh38 genome (Release 95—January 2019) using the STAR RNA‐seq aligner version 2.4.0 , with –s2.17Hallmarks of Cancer gene set database was used with permutations set to 1000 to determine the FDR. Normalized enrichment scores (NESs) were obtained after analysis.Gene set enrichment analysis (GSEA) was done using the GSEA software supplied by the Broad Institute . The gen2.18AR and of androgen‐responsive genes identified in UM‐UC‐3‐AR are available from the public The Cancer Genome Atlas (TCGA) database, containing 408 MIBC cases  of s Tables . The cli2.19t‐tests were performed using graphpad prism . A P‐value of < 0.05 was considered statistically significant, and P < 0.05 is represented by one star (*), P < 0.01 is represented by two stars (**), P < 0.001 is represented by three stars (***) and P < 0.0001 is represented by four stars (****).The data of bar and dot plots are presented as means ± SEM from at least three independent experiments. Two‐tailed paired and unpaired 2‐transformed . Spearman’s correlation of log2‐transformed expression values (TCGA or microarray) was calculated using the R package psych [prcomp from the package stats [ggplot2 from the package tydiverse [For correlations of TCGA data, FPKM values were logge psych . A correge psych . The buige stats  was usedydiverse  was used33.1AR mRNA in urothelial bladder tissue, we performed a microarray analysis on NMIBC , MIBC  and nonmalignant bladder urothelial specimens. A subset of UCC tissue samples (27/108) expressed AR mRNA at levels above those from nonmalignant tissue  and a glycine (GGC) tract. More than 16 glycine (GGC) copies have been demonstrated to form a hairpin structure that inhibits translation [AR gene , indicating that it should encode for a functional protein. As a transcription factor, the main role of AR is to promote transcriptional activation of AR‐targeted genes. The KLK3 gene, encoding the prostate‐specific antigen (PSA), is a well‐established AR‐responsive gene [KLK3 luciferase reporter construct, we were able to monitor AR‐mediated transcription in UM‐UC‐3, UM‐UC‐3‐AR and LNCaP cells. In a dose‐dependent manner, androgen stimulation significantly increased AR‐mediated transcriptional activity observed in FCS‐containing media in UM‐UC‐3‐AR and LNCaP, but not in UM‐UC‐3 cells , paraclones (para = beyond) or meroclones  [AR knockdown did not achieve a significant effect in the overall colony formation  , 35. Morion Fig. E,F, it dion Fig. . These d3.4AR expression   Fig. . A total05) Fig. , with 6905) Fig. . Among tAR and 265 out of 284 genes were present in our microarray data set  and low‐AR , using median AR expression as a cut‐off. We then compared the median expression levels of the androgen‐responsive genes between these two groups. A total of 128/265 genes from our UCC cohort displayed an expression pattern we defined as consistent with the regulation observed in UM‐UC‐3‐AR. In brief, from the genes downregulated by enzalutamide treatment and/or upon siRNA‐mediated AR knockdown in UM‐UC‐3‐AR 75/175 showed similar expression dynamics to AR in the high‐AR and low‐AR groups. From the genes upregulated after enzalutamide treatment and/or AR knockdown in our cell line model, the expression of 53/90 genes was higher in the low‐AR group compared with the high‐AR group  and one of genes that positively correlated with AR (cluster 2). From the genes, which expression significantly (FDR < 0.05) correlated with AR levels, 15 genes had a correlation coefficient > 0.3 or < −0.3 .An expression heat map of ort Fig. . To furtion Fig. . InteresCGA Fig. . Lastly,ers Fig. . Despiteets Fig. . No othe4AR mRNA levels, UM‐UC‐3 and UM‐UC‐3‐AR represent the UCC cases with low‐ and high‐AR expression levels, respectively. Both lines UM‐UC‐3 and UM‐UC‐3‐AR contain the same number of glycine repeats within exon 1, the same number of X chromosomes and the same AR gene copy number. Thus, additional levels of gene regulation should underlie the different AR mRNA expression levels in these two isogenic cell lines, which also could explain the differences in AR levels found in bladder tumours.AR signalling is one of the main drivers of sex differences and is suggested to be involved in bladder carcinogenesis , 37, 38.in vitro that can be classified as holoclones, meroclones and paraclones. These morphologies are suggested to represent stem cells, early progenitor cells and late progenitor cells, respectively [Although AR levels in UM‐UC‐3‐AR were significantly lower than those in LNCaP, reflecting difference between UCC and PCa patients, these levels were sufficient to mediate phenotypic changes. Ligand‐bound AR regulated the clonogenic capacity of UM‐UC‐3‐AR cells. Androgen stimulation resulted in the formation of big‐sized holoclones. Addition of AR inhibitor enzalutamide significantly reduced colony formation, and similarly to siRNA‐mediated knockdown, it resulted in the generation of small paraclones. PCa‐derived cells, seeded at low densities, have been shown to form three different colony morphologies ectively , 35. Theectively . TogetheFKBP5, LDH‐A, GREB1, PDLIM1, TNK2, VASP, ARHGAP31 and ZHX3 [TFCP2L1 and SH3RF3 [With these phenotypic changes, several genes were significantly upregulated upon hormone stimulation, among which some previously described for their role in promoting migration and colony formation of diverse type of cancer models such as and ZHX3 , 46, 47,d SH3RF3 , 49. TheNNMT and LDHA, genes previously reported to be upregulated in UCC patients [BCL6 and H19, whose high expression in UCC is associated with an adverse prognosis [SOCS6 and SEMA3F, genes downregulated upon androgen treatment in our model, is associated with a malignant phenotype in UCC [Androgen treatment of UM‐UC‐3‐AR cells also induced expression of patients , 51, andrognosis , 53. Cone in UCC , 55. TheLDH‐A was also part of the hypoxia signature in our enrichment analysis, the pathway with highest NES in response to androgen stimulation. LDH‐A relates to stem cell function and is associated with poor postradiotherapy outcome in UCC [GREB1 was first reported to be oestrogen‐responsive [GSEA demonstrated that transcriptome dynamics observed in UM‐UC‐3‐AR indeed correspond to an androgen response signature, which included genes encoding for the AR co‐regulators PIAS1 and CCND3 , 57, ande in UCC . The oese in UCC . Indeed,sponsive  and latesponsive . Other ssponsive  and elevsponsive  have beeCYP4F12, PPP1R3C, RERE and PLAU defined as androgen‐responsive in UM‐UC‐3‐AR significantly correlated with AR in two independent UCC data sets. The CYP4F12 gene encodes a member of the cytochrome P450 (or CYP) superfamily of enzymes, which are involved in the synthesis of cholesterol and steroids (e.g. androgens), and in drug metabolism. The latter function being particularly important in tissues regularly exposed to disposal products  such as the bladder. Indeed, deregulation of some CYP proteins has been proposed as a risk factor for the development of bladder cancer [CYP4F12 mRNA expression was shown to be downregulated upon treatment with AR inhibitors enzalutamide or ARN‐509 in PCa‐derived cells [PPP1R3C, a gene that encodes a protein involved in glycogen synthesis and part of the ‘hypoxia’ and the ‘myogenesis’ signatures from our GSEA, were also shown before to be androgen‐responsive [PLAU, a gene that is known to be downregulated by androgens in PCa models [RERE mRNA as a consequence of miR‐22 inhibition resulted in increased migration and cell proliferation in colon cancer [AR and these four genes, the direct or indirect role of AR protein in the regulation of CYP4F12, PPP1R3C, RERE and PLAU transcription and function in UCC is yet to be elucidated.Four genes r cancer , 68, buted cells , in linesponsive , as oppoa models . Both ren cancer . Curiousn cancer . AlthougAR knockdown that were not affected by enzalutamide treatment. Enzalutamide promotes the accumulation of AR in the cytosol [Our transcriptome data also revealed a cluster of androgen‐responsive genes downregulated upon  cytosol , which c cytosol . The traAR mRNA levels were significantly upregulated in NMIBC compared with MIBC. The lower frequency of AR‐positive cases in MIBC also suggests that AR‐independent but androgen‐dependent mechanisms support high stage disease. Certainly, a recent publication has shown that the SLC39A9 membrane protein acts as an AR that, when activated by androgens, increases migration and invasion of AR‐negative MIBC cells in vitro and in vivo [A negative association between AR expression and tumour stage has been reported before, both at the mRNA and protein levels . Indeed, in vivo . Our traAR mRNA levels, both in the microarray analysis and in the RT‐qPCR validation. A subset of UCC tumours, both NMIBC and MIBC, expressed AR mRNA levels above those detected in nonmalignant bladder urothelium specimens. Our in vitro data indicate that androgen/AR signalling is involved in the acquisition of a more aggressive phenotype of bladder tumour cells and that it can be reverted with blockage of AR signalling, advocating the potential benefits of AR‐targeted therapy for UCC. In low‐grade NMIBC, AR‐targeted therapy may prevent the recurrence of disease, and in high‐grade NMIBC and MIBC, it may prevent progression. Indeed, in PCa patients with concomitant NMIBC, progression of NMIBC to MIBC was only reported in patients who did not receive ADT as a treatment for their prostate tumour [AR mRNA levels. Together, our data provide new understandings in the role of androgens and AR signalling in UCC and suggest that ADT is a realistic option for the treatment of this disease.Several studies, including ours, reported that the incidence of UCC is three to four times higher in men than in women , 6. Howee tumour . Further5Our study provides biological evidence of AR signalling in UCC with several pathways being differentially regulated in response to androgen treatment. Ligand‐bound AR activates canonical and noncanonical AR target genes and potentiates migration and colony formation. The contribution of AR to the development of an aggressive phenotype in UCC highlights its potential clinical relevance as a novel therapeutic target.The authors declare no conflict of interest.MVLV, GWV and JAS contributed to the experimental design. MVLV, JJD, MA‐H, FS and GvZ performed experiments and data analysis. MVLV prepared the manuscript. GWV, JJD, JPMS, MA‐H, FS, GvZ, DS, MV and JAS revised the manuscript.https://publons.com/publon/10.1002/1878‐0261.12957.The peer review history for this article is available at Fig. S1. AR expression in bladder urothelial cell carcinoma tissue samples.Click here for additional data file.Fig. S2. Characterization of UM‐UC‐3 and UM‐UC‐3‐AR.Click here for additional data file.Fig. S3. Colony formation assay.Click here for additional data file.Fig. S4. Migration capacity of UM‐UC‐3 cells.Click here for additional data file.Fig. S5. Expression of androgen‐responsive genes and AR in UCC patients.Click here for additional data file.Table S1. Clinical information of bladder urothelial cell carcinoma patients from the Nijmegen cohort used in microarray analysis.Table S2. Clinical information from bladder urothelial cell carcinoma patients used in RT‐qPCR analysis.Table S3. Primer sequences for real‐time semi‐quantitative (RT)‐qPCR analysis.Table S4. Primer sequences for glycine repeat and gene copy number analysis.Table S5. siRNA sequences.Table S6. Transcript expression data from muscle‐invasive bladder cancer cases from the TCGA database.Table S7. Clinical information of muscle invasive bladder cancer patients, from publically available TCGA used for in silico analysis.Table S8. RT‐qPCR Cp values from bladder urothelial cell carcinoma and non‐malignant urothelium tissue samples.Table S9. Transcriptome profile after Enzalutamide treatment or siRNA‐mediated AR knockdown of androgen‐treated UM‐UC‐3‐AR cells.Table S10. Microarray expression values of AR and 265 androgen‐AR responsive genes (identified in UM‐UC‐3‐AR) in 97 bladder urothelial cell carcinoma patients from the Nijmegen cohort.Table S11. Median expression (log2) of AR and 266 androgen‐responsive genes (identified in UM‐UC‐3‐AR) in 97 bladder urothelial cell carcinoma (UCC) patients from the Nijmegen cohort (microarray data set).Table S12. Spearman correlation coefficient from subset of androgen‐responsive genes correlating with AR in bladder urothelial cell carcinoma patients from the Nijmegen cohort (microarray data set).Table S13. Median expression (log2) of AR and 254 androgen‐responsive genes (identified in UM‐UC‐3‐AR) in 408 muscle invasive bladder cancer patients, from the TCGA cohort.Table S14. Spearman correlation coefficient from subset of androgen‐responsive genes correlating with AR in MIBC patients from TCGA.Click here for additional data file.


The skin’s barrier ability is an essential function for terrestrial survival, which is controlled by intercellular lipids within the stratum corneum (SC) layer. In this barrier, free fatty acids (FFAs) are an important lipid class. As seen in inflammatory skin diseases, when the lipid chain length is reduced, a reduction in the barrier’s performance is observed. In this study, we have investigated the contributing effects of various FFA chain lengths on the lamellar phase, lateral packing. The repeat distance of the lamellar phase increased with FFA chain length (C20–C28), while shorter FFAs (C16 to C18) had the opposite behaviour. While the lateral packing was affected, the orthorhombic to hexagonal to fluid phase transitions were not affected by the FFA chain length. Porcine SC lipid composition mimicking model was then used to investigate the proportional effect of shorter FFA C16, up to 50% content of the total FFA mixture. At this level, no difference in the overall lamellar phases and lateral packing was observed, while a significant increase in the water permeability was detected. Our results demonstrate a FFA C16 threshold that must be exceeded before the structure and barrier function of the long periodicity phase (LPP) is affected. These results are important to understand the lipid behaviour in this unique LPP structure as well as for the understanding, treatment, and development of inflammatory skin conditions. The skin has the task of protecting the body from external influences and assisting in maintaining a homeostatic environment within the body . It is tThe lipid matrix is comprised of three major lipid classes, ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs), at approximately equimolar ratios. These extracellular lipids form two distinct co-existing crystalline lamellar structures with repeat distances of approximately 13 and 6 nm, which are referred to as the long and short periodicity phases  . Within One of the common symptoms of many inflammatory skin diseases is a reduced skin barrier function. With these diseases, changes to the lipid matrix’s composition and arrangement are observed, which may explain the reduced barrier capability ,13. ThesTherefore in this work, we have studied the effect of FFA chain length between the lengths of C16 and C28. We have studied how each of the major FFA chain lengths, observed in both healthy and diseased skin, affect the lipid organization and barrier function with the use of LPP lipid models. The LPP and its mainly orthorhombic lateral packing are crucial aspects for the skin barrier, and as a result, is the focus of this study. Lipid matrix models are models that are capable of closely resembling the structural and barrier behaviour as found in native SC. We show that the alterations in the LPP behaviour are not linear with chain length and that shorter FFA chain length models exhibit a different behaviour compared to their longer chained counterparts. Similar behaviour is also observed with the lateral packing, only models containing FFAs C20 and longer have the orthorhombic phase transition temperature measured to increase with increasing chain length, which may be explained by the phase separation of the shorter FFAs. We compare the phase separation behaviour between the LPP models with different lipid compositions. We also show that the LPP is a robust structure, and can withstand a certain threshold of change before its barrier function is affected. The significance of our results in inflammatory skin diseases is discussed.Esterified omega-hydroxyacyl-sphingosine (CER EOS), as well as shorter CERs including: N-(tetracosanoyl)-sphingosine (CER NS), N-(tetracosanoyl)-phytosphingosine (CER NP); N-(2R-hydroxy-tetracosanoyl)-sphingosine (CER AS), and N-(2R-hydroxy-tetracosanoyl)-phytosphingosine (CER AP), were all kindly donated by Evonik, Essen, Germany. CER nomenclature was used based on the definitions from Motta et al. . The sphTwo different models were prepared with synthetic lipids, with the ratio among CERs, CHOL, and FFAs remain in an equimolar ratio; however, the composition of the lipid subclasses was changed. In the simplistic model, the lipid composition was comprised of CER EOS and NS in a 40:60 molar ratio, CHOL, and a single-chain length FFA C16–C24) to monitor the behavioural changes induced by the various FFA chain lengths. In addition, a porcine SC lipid composition mimicking model was used to determine the adaptability of the LPP. The CERs subclasses consisted of CER EOS (C30): CER NS: CER NP: CER AS: CER NP (C16): CER AP in a molar ratio of 40:36:11:3:6:4, a similar composition found in porcine SC . The FFA to monit2. Once sprayed, the models were heated until melted (85 °C) and equilibrated for 30 min. Once cooled, samples were hydrated for ≥15 h in a humid environment (pH 5.0). Before the measurement, a 2 × 5 mm2 strip of the sample was loaded into the beam. At the ESRF the SAXD images were generated using a Pilatus 1M detector with 981 × 1043 pixels of 172 μm2 spatial resolution. The calibration of the detectors was performed using silver behenate. For the diffraction measurements, the X-ray wavelength was set at 1.033 Å and the detector-to-sample distance was 2.1 m. The samples were measured for 120 s, at 25 °C. The number of repeats (n) for each condition was ≥2. The two-dimensional (2D) scattering was integrated over a 40° segment perpendicular to the orientation of the sample, of which the segment starting point was located at the beam centre.The SAXD measurements were performed at the European Synchrotron Radiation Facility  at station BM26B, and the NCD-SWEET beamline at the ALBA synchrotron . For measurements at the ESRF, 0.75 mg of lipid at the desired composition was prepared in 2:1 hexane/ethanol solvent at a concentration of 4.5 mg/mL. The mixture was sprayed using a y-axis adapted Camag Linomat IV sample applicator  on a nuclepore polycarbonate filter, under a steady flow of nitrogen, over an area of 1 cmn = 2.Samples for measuring at ALBA were prepared using a similar method; however, 1 mg of lipids was sprayed over an area of 1 × 3 mm, and before measuring, samples were hydrated in an oxygen-free, 84% relative humidity environment for 5 days at room temperature. At ALBA, the wavelength was set at 0.999 Å, and the Pilatus 1M detector was set at a distance of 2.148 m from the sample. The experimental set up was calibrated with silver behenate. The samples were measured for 20 s at 23 °C. The one-dimensional intensity profiles were calculated by integrating starting from the beam centre, over a 90° segment. Samples were measured d) was calculated using least square fitting and the equation: d = 2hπ/q, in which h is the order number of the diffraction peak at a certain position (q).The 1D intensity profiles were calculated using the Bragg equation. Peak position was fitted with Fityk , and the3/MeOH), at a concentration of 5 mg/mL. The mixture was sprayed on an AgBr window over an area of 1 cm2. Once sprayed, the model was equilibrated to 85 °C for 30 min and cooled. The lipids were then hydrated in deuterated acetate buffer (pH 5.0) at 37 °C for ≥ 15 h, to ensure the sample had sufficient time to fully hydrate.The sample preparation method for the FTIR measurements was similar as described for the SAXD measurements. 1.0 mg of lipid at the desired compositions was dissolved in 2:1 chloroform/methanol (CHCl−1 resolution. The data were collected in steps of 4 min in absorbance mode. The spectra of the temperature run were collected using the software Spectrum TimeBase (PerkinElmer). Measurements were performed at n ≥ 2.FTIR spectroscopy measurements were performed using the Frontier FTIR spectrometer , equipped with a broad-band mercury cadmium telluride detector. ≥10 min before and during measuring, the sample compartment was purged with dry air. The lipid models were measured between 0 °C and 90 °C, at a rate of 0.25 °C/min, at a 1 cm2 and CD2 symmetric stretching vibration  was determined by the centre of gravity (CoG) method. The peak height for CoG was set between 80 and 90%. The lateral phase transition temperatures were determined by the linear regression fit of the peak shift of the νsCH2 and νsCD2, and by the CH2 and CD2 scissoring vibrational peak splitting distance . The spectra were deconvoluted using the Spectrum (PerkinElmer) software and plotted with in-house Enthought Canopy scripts. The ratio between orthorhombic and hexagonal lateral packing was calculated by first fitting the δCH2 peaks with a Pearson7 function in Fityk, then by dividing the height of the second orthorhombic phase peak (1475 cm−1) from the hexagonal peak (1472 cm−1).The peak position of the CH2. Samples were equilibrated at 85 °C for 30 min before cooling back to room temperature. Once equilibrated the sample was loaded into a PermeGear in-line diffusion cell , which was filled with Milli-Q water and left to hydrate for 30 min before measuring.The samples were prepared in a similar way as described for the X-ray samples. A total of 0.9 mg of lipids were dissolved in hexane/ethanol (2:1) solution, to a concentration of 4.5 mg/mL. The lipids were sprayed on the porous filter (Whatman Nuclepore Track-Etch Membrane), under a steady flow of nitrogen, over an area of 1 cmn ≥ 4. One-way ANOVA with Dunnett multiple comparison test was performed to analyse the TEWL data. The significance level for rejection of the null hypothesis was set at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).TEWL measurements were performed using an AquaFlux  and the software BioFlux, recording for a total of 30 min with 10 sec intervals. The average steady-state flux (J) for each sample was determined as the average flux value measured during the final 3 min of the measurement, The effect of the FFA chain length on the LPP repeat distance was identified with SAXD measurements. Simple models forming the LPP were prepared with a single FFA chain length varying between C16 and C28. The full 1D diffraction profiles at each FFA chain length can be found in sCH2, and the splitting behaviour of δCH2. The highly ordered orthorhombic phase is associated with a high conformational ordering that is when the νsCH2 peak is located at ~≤2850 cm−1. As the lipid lateral packing changes from an orthorhombic to hexagonal phase the peak position shifts by ~1 cm−1, and further increases by ~2.5 cm−1 when the lipids change from a hexagonal to a fluid phase  containing C24 model, the fluid mid transition temperature was similar for the protiated and deuterated lipids, occurring at 71 and 70 ± 0.0 °C, respectively, implying the dFFAs are mixed with the protiated lipids. In contrast, the mid hexagonal to fluid phase transition of the νsCH2 and νsCD2 in the dFFA C16 model, has a difference of ~10 °C (78.9 ± 0.6 and 69.0 ± 3.1 °C respectively), thus implying that a portion of the FFA C16 has phase separated from the LPP. Deuterated FFA C16 has a melting temperature of 61–64 °C; however, the phase-separated lipids in the C16 model have a higher melting temperature of 68–71 °C; this shift in the melting temperature implies it is not purely FFA C16 that has separated. The SAXD curves do show an additional phase in the shorter C16 model; thus, a proportion of C16 FFAs that had phase-separated may have incorporated themselves into the structure responsible for the additional peak. However, it is unlikely to be pure FFA C16 since it has a repeat distance of 3.5 nm  or shorter (C16–C18). The difference in behaviour between shorter and longer FFAs may be explained due to the hydrophobic mismatching between the FFA and CER chains. Previously it has been shown that FFA C24 is located throughout the length of the LPP  or short. In our Throughout our lifecycle, the lipid composition within the SC changes due to factors such as age and seasonal influences ,32; thusTo determine how relevant the changes in the LPP and its lateral packing are to the barrier function, the compositional adaptability before a structural change or reduction in barrier performance was determined. A lipid model that mimicked the composition of porcine SC was used. This composition is composed of readily available synthetic lipids, which can mimic the barrier’s ability, and structural properties of human SC ,23. The sCH2 thermotropic curves were similar, with the orthorhombic to hexagonal mid-transition temperature for the 1.8, 20, 30, 40, and 50% being 34.6 ± 0.1, 33.0 ± 0.4, 30.6 ± 0.5, 33.5 ± 1.6, and 31.6 ± 1.5, and for hexagonal to fluid mid-phase transitions being 71.8 ± 0.8, 69.6 ± 2.8, 74.5 ± 0.5, 70.2 ± 2.0, and 68.8 ± 34 °C, respectively; an example of each of the individual curves is seen in 2 in −1 regardless of the FFA C16 content, indicating the mean orthorhombic domain size did not change. However more noticeable is the increase in the intensity of the central hexagonal peak as the concentration of FFA C16 increases in the models. The increase in the height ratio of the hexagonal peak 1467 cm−1 compared to the orthorhombic peak at 1475 cm−1 from 0.29 in the 1.8% model to 0.67 in the 50% model, suggests a significant increase in the proportion of lipids packed in the hexagonal phase.To further probe the phase separation behaviour occurring at higher FFA C16 concentrations, lateral packing was monitored, at the same FFA C16 proportion. At all concentrations measured between 1.8 and 50% the ν2 and δCD2 peaks in 2 curve shows a disruptive orthorhombic splitting at 24 °C. 2 vibration in To try and identify the phase-separated lipid composition, the mixing behaviour of FFA C16 was monitored. The phase separation was most prominent when FFA C16 concentration was at 50% , and thi−1 (d = 3.67 nm), a similar length found in our curves [Based on the short repeat distance of the phase separated structure, another lipid with a similar length is CER NP (C16). It is not possible to investigate if this lipid had phase separated with FTIR since deuterated CER NP (C16) is currently not commercially available. Alternatively, the phase separation may be due to a small proportion of CER NP (C24). Phase separated crystalline NP has been previously reported to locate at q = 1.71 nmr curves . CER NP r curves . It is a2/h. A similar flux value is measured at 1.1 ± 0.4 g/m2/h when the FFAC16 content was increased to 20%. As the FFA C16 content increased to 30 and 40%, a higher steady-state flux value was measured at 1.8 ± 0.2  and 2.8 ± 0.6 g/m2/h, respectively .Regardless of the physical alterations, knowledge of barrier properties is also important to understand due to increasing concentrations of FFA C16; thus, the effect on the inside-outside barrier function was measured with TEWL. The same modified porcine lipid models were measured as described previously. The average steady-state fluxes are presented in sCH2 and νsCD2 wavenumber values represent the packing density of the lipid chains, with the higher the wavenumber, the lower the lipid chain packing density. This could be the result of an increasing proportion of the lipids shifting from the orthorhombic to hexagonal phase. In our FFA C16 50% models the lipids had a νsCH2 and νsCD2 wavenumber of 2850.1 and 2090.7 cm−1, respectively; these values are higher compared to the 1.8% SC lipid model with wavenumbers around 2846 and 2089.4 cm−1, respectively [The poorer barrier ability found in models with an increasing proportion of shorter FFA may be explained by the increase in overall conformational disordering, arising from these shorter FFAs. The νectively . This inectively . A similectively . To mimiectively , the FFAectively . At thisThe results of this study imply that FFA chain length does have an effect on the LPP structure and barrier function at inflammatory skin disease relevant concentrations. However, it is important to identify how significant these are in relation to other changes that are observed simultaneously in diseased skin. Aside from shortening the FFA chain length, other alterations commonly seen in inflammatory skin diseased SC include CER chain length shortening , CER heaIn summary, our results show a non-uniform effect that the FFAs chain length has on the LPP in SC lipid models. The behavioural change observed depends on whether a mismatch with the remaining lipid chain is present. Modulations of the FFA C16 concentration in porcine mimicking lipid models showed a threshold concentration of 30% of total FFA content before the barrier performance and lateral packing density were reduced and phase separation occurred. This study expands on the current knowledge of the FFA chain length effect, and the stability the LPP structure in response to changes in FFA chain length before a decrease in its barrier performance is observed. These results are highly relevant for understanding the mechanisms governing the change in inflammatory skin diseases, as well as for the development of treatments for such alterations in the skin.


In recent years, many therapeutic advances have been made in the management of castration-resistant prostate cancer, with the development and approval of many new drugs. The androgen receptor (AR) is the main driver in prostate cancer growth and progression and the most effective therapeutic agents are still directed against this pathway. Among these, new generation hormonal agents (NHA) including enzalutamide, abiraterone acetate, apalutamide, and darolutamide have shown to improve overall survival and quality of life of prostate cancer patients. Unfortunately, despite the demonstrated benefit, not all patients respond to treatment and almost all are destined to develop a resistant phenotype. Although the resistance mechanisms are not fully understood, the most studied ones include the activation of both dependent and independent AR signalling pathways. Recent findings about multiple growth-promoting and survival pathways in advanced prostate cancer suggest the presence of alternative mechanisms involved in disease progression, and an interplay between these pathways and AR signalling. In this review we discuss the possible mechanisms of primary and acquired resistance to NHA with a focus on AR independent pathways. At the time of diagnosis, most patients have a localized disease treatable with surgery or radiotherapy with curative intent. About 5%-10% of PC is diagnosed at an advanced stage. While 5-year survival is close to 100% for localized and locally advanced disease, it is nearly 30% in metastatic patients. Androgen deprivation therapy (ADT), both surgical and biochemical, is the standard of care in metastatic castration-sensitive prostate cancer (mCSPC), owing to the driving role of androgen receptor (AR) in the growth of this tumour. Recent findings demonstrated that, in patients with high volume de novo mCSPC, the addition of docetaxel to ADT results in significantly improved outcomes when compared with ADT alone,4. Similar results have been recently presented for the combination of new generation hormonal treatments and ADT in high risk mCSPC-7. Despite the brilliant response rates (approximately 80%-90%), almost all patients experience disease progression within 18-36 months from the start of ADT, moving to the last phase of disease, castration-resistant prostate cancer (CRPC). Recently, many therapeutic advances have been made in metastatic CRPC (mCRPC), with the development and approval of new generation orally available hormonal agents: abiraterone, a strong CYP17A1 inhibitor blocking androgens biosynthesis, and enzalutamide, a potent AR antagonist. The phase III trials COU-AA-301 and AFFIRM demonstrated an improvement in overall survival (OS) in patients treated respectively with abiraterone and enzalutamide in post-docetaxel setting . In September 2019, the Food and Drug Administration (FDA) approved apalutamide for mCSPC patients. Darolutamide is a next generation AR antagonist, with higher affinity for AR than enzalutamide. Approval of darolutamide for the treatment of non-metastatic CRPC was based on the benefit in MFS reported in the phase III ARAMIS study. Recent studies on growth-promoting and survival pathways in advanced PC suggest the crucial role of alternative AR-independent mechanisms in disease progression and an interplay between these pathways and AR signaling. In this review we discuss the possible mechanisms of primary and acquired resistance to NHA with a specific focus on AR independent pathways.Prostate cancer (PC) is the most frequent cancer in men and accounts for almost 1 in 5 new diagnoses. Although in recent years there has been a decrease in mortality due to earlier detection and advances in treatment, PC is still the second leading cause of cancer death among men in the USAAndrogens are sex hormones required for the development of the male reproductive system and secondary sexual characteristics. Testosterone (T) and 5-a-dihydrotestosterone (DHT) are mostly produced in the testicles in the adult male. No more than 10% of their synthesis occurs in the adrenal glands.. The transcriptional activity and the interaction with other pathways involved in cellular signalling is modulated by several coactivator and suppressor proteins. The androgen/AR complex can also act through non-DNA binding-dependent pathways. Activation of second messenger signalling pathways have been identified in several cell lines. There is evidence suggesting that some of the non-DNA binding-dependent actions of androgens are mediated via the activation of membrane-bound protein receptors, starting intracellular signalling cascades even in the presence of low levels of androgens. The AR promotes physiological epithelial differentiation, but, in PC, abnormal AR activation can dysregulate the expression of genes involved in the control of proliferation and survival of tumour cells.Testosterone and DHT act via the androgen receptor (AR), a ligand-dependent nuclear transcription factor, encoded by a gene located on chromosome Xq11-12. The receptor structure consists of three main functional domains: the N-terminal transcriptional regulation domain, the DNA binding domain and the ligand binding domain (LBD). In the absence of ligand, the AR is cytoplasmic, linked to heat-shock and other chaperone proteins. When androgens bind AR, they induce a conformational change resulting in dissociation of chaperone proteins and exposure of the nuclear localization signal, responsible for AR import into the nucleus. The androgen/AR complex translocates to the nucleus where it dimerizes and binds the androgen response elements to modulate gene transcription; PC cells are able to synthesize androgens and modify AR, enabling it to activate even in the presence of low levels of androgens. The mechanisms underlying this phenomenon are associated with the selective pressure of ADT and include AR gene overexpression, AR gene mutation, AR splice variants expression, and upregulation of transcriptional coactivators.In recent years there has been a significant increase in the understanding of prostate cancer biology. It is currently known that the shift from CSPC to CRPC is not necessarily due to androgen deprivation therapy (ADT) resistance, but it is caused by cell adaptation to a microenvironment with low androgens levels. In fact, despite the significant T levels decrease induced by ADT, CRPC remains driven by AR signaling. According to this accepted definition of primary resistance, acquired resistance is considered as treatment failure occurring later during treatment. A significant proportion of patients treated with NHA develop primary resistance, and consequently an understanding of the mechanisms of resistance is essential in order to develop new therapeutic strategies.Despite the significant benefits of NHA, some CRPC patients do not respond to therapy because of primary resistance. In addition, all patients treated will acquire resistance after a certain period from the beginning of treatment. Primary resistance is commonly defined as treatment failure within the first 3 months after commencing treatment, due to clinical progression, with or without radiological confirmation. In a recent preclinical study, apalutamide and enzalutamide exhibited agonist activity in resistant PC cell lines due to a missense mutation (F876L) in the AR LBD. The AR F876L mutation was detected in plasma samples collected from patients with progressive CRPC after apalutamide treatment. Naïve samples lacked the mutation, suggesting that F876L is involved in the development of resistance to second generation antiandrogens. No data are currently available about the mechanisms of resistance to darolutamide.There are few data regarding the mechanisms of resistance that occur during treatment with apalutamide. In the pivotal SPARTAN trial, the frequency of AR signalling anomalies detected in the apalutamide group at the end of treatment was low, and AR aberrations were associated with a shorter median progression-free survival only in the placebo group,29.Adaptive resistance of PC to abiraterone and enzalutamide treatment is much better understood, and it can be due to the activation of both dependent and independent AR signalling pathways. This type of adaptation is more common in patients who progressed during new generation hormonal therapy than in treatment-naïve patients, so it has been considered as a potential resistance mechanism.AR amplification/overexpression is the most common genomic aberration in CRPC patients; up to 80% of these patients show AR overexpression.AR mutations can be found in up to 30% of CRPC patients treated with ADT. Their incidence may increase during treatment with abiraterone and enzalutamide because, when AR signalling is more effective suppressed, clonal selection of tumour cells can enhance AR somatic mutations and the consequent aberrant transcription. Most AR point mutations are clustered in the LBD, altering the steroid-binding pocket and enabling its activation by alternative ligands including progesterone, hydrocortisone, oestradiol, and some AR antagonists. ARV expression is significantly increased during ADT and is related to PC progression. The most common AR splicing variant, AR-V7, is associated with resistance to both abiraterone and enzalutamide and with short survival.Transcriptionally activated AR splice variants (ARVs) play a critical role in the development and progression of CRPC. ARVs lack the LBD, remaining constitutively active even without androgen binding. Studies in CRPC xenografts have shown that several genes involved in the androgen synthesis pathway, including CYP17A1, are upregulated during hormonal therapy. The raised intra-tumoural level of androgens stimulates both paracrine and autocrine activation of AR, irrespective of serum androgens levels.Intra-prostatic synthesis can be an important source of androgens under the selective pressure of hormonal treatment. The intra-tumour synthesis of steroid hormones, usually limited in naïve primary PC, significantly increases in CRPC patientset al. examined GR expression and function in cell lines and human tissues of PC patients. They observed that the GR is expressed minimally in primary PC tissue and that GR expression notably increases during long-term treatment with enzalutamide. These findings confirm previous results of a study by Arora et al. that showed how GR overexpression confers clinical resistance to enzalutamide. In particular, AR inhibition resulted in GR upregulation due to the lack of AR-mediated feedback repression on GR expression. The GR substituted for the AR to activate AR target genes crucial for the maintenance of a resistant state. In the same study, the GR agonist dexamethasone was shown to induce in vitro resistance to enzalutamide while a GR antagonist restored sensitivity. The frequency and the clinical relevance of GR-driven CRPC are currently not known.The glucocorticoid receptor (GR) is a component of the steroid nuclear receptor family. Similarly to the AR, it is composed of three functional modules: the N-terminal binding, the DNA binding and the ligand binding domain. Acquired resistance to NHA can occur due to an increased expression of the GR, that shares response elements with the AR in various target genes. Puhr . The PR can be involved in resistance to NHA through continued progesterone production. These findings prove a mechanism of escape from AR blockade via alternative nuclear receptors during drug exposure. Therapeutic inhibition of GR activity by mifepristone, a steroidal GR and PR antagonist, prevented CRPC growth and delayed progression in preclinical models. A phase II study evaluating enzalutamide in addition to mifepristone in patients with mCRPC is ongoing to determine if the combination of the two drugs delays time to PSA progression  also belongs to the steroid nuclear receptor family and has been shown to be increased in CRPC. PR isoforms A and B are expressed in prostate stromal fibroblasts and smooth muscle cells, but not in epithelial cells. Reciprocal interactions between epithelium and stroma are crucial for the maintenance of prostate homeostasis and functioning. Both PR isoforms are known to regulate cell proliferation. While the epithelial phenotype is characterized by apical–basal cell polarity and strong intercellular adhesions, mesenchymal cells are totally different, being neither adherent nor polarized. EMT can be induced by ADT in metastatic PC and it confers invasive potential to tumour epithelial cells. Snail and Twist are transcription factors able to induce the repression of E-cadherin expression, a key event in EMT. The TGF-β superfamily members induce Snail transcriptional factor. Aberrant TGF-β pathway and the protein kinase C (PKC)/Twist1 signalling activation are putative mechanisms for the occurrence of resistance. There are currently no direct Twist inhibitors available, but Shiota et al. investigated a PKC inhibitor to switch back resistance in PC cell lines with promising results. A phase II trial  process is crucial for physiological embryonic development and differentiation of the urinary genital system, but it is also activated in pathological conditions, such as fibrosis or cancer progressionet al. demonstrated that metformin, an oral hypoglycaemic agent, is able to revert enzalutamide resistance restoring sensitivity to the drug in mice xenografts by the inhibition of EMT. Based on the effect of metformin on the activation of STAT3 and expression of TGF-β1, the authors proposed that metformin exerts its anticancer effect by targeting the TGF-β1/STAT3 axis, involved in EMT activation. Currently, there are two ongoing clinical trials evaluating the anticancer role of metformin in combination with enzalutamide in mCRPC  levels. These patients are usually treated with cisplatin or carboplatin combined with etoposide, obtaining high response rates but short duration of disease control. A systematic review of published clinical cases revealed a median time to development of NEPC of 20 months and a median OS after NEPC diagnosis of 7 months.A subset of patients with advanced PC may eventually develop a neuroendocrine (NE) phenotype as an adaptive response to intense AR signalling inhibition. An aggressive NE phenotype can also appear de novo, but it more commonly arises during hormonal therapy. The prevalence of neuroendocrine prostate cancer (NEPC) is approximately 1% in primary PC and up to 25%-30% in mCRPC. AUR-KA is best known for its role in mitosis, but its interactions with the oncogene N-myc are not completely understood. In neuroblastoma, a positive feedback loop has been described in which AUR-KA induces and stabilizes N-myc. Alisertib was also studied in combination with abiraterone in patients with mCRPC progressing during abiraterone treatment. This phase I/II trial was stopped early because of intolerable toxicity and no clear signs of therapeutic activity.NEPC is histologically different from PC, and is characterized by small round blue cells, which do not express AR or secrete PSA, but express NE markers such as chromogranin A and neuron specific enolase. Recent genomic profiling studies have shown a significant overexpression and gene amplification of Aurora Kinase A (AUR-KA) and N-myc in 40% of NEPC and 5% of advanced PC, and a loss of AR target gene expression. The demonstration that REST plays a role in the prostate epithelium differentiation, and that its expression is negatively related to the recurrence of disease, suggests a possible role of REST as a prognostic biomarker.Recent studies have shown that enzalutamide treatment may cause a lower expression of the repressor element 1 silencing transcription factor (REST), a mediator of AR activity that plays a leading role in restricting neuronal differentiation of stem cells. Preclinical tests revealed that in LNCaP cells subjected to prolonged incubation with androgen-depleted media, the reduced expression of REST coincided with an increased expression of chromogranin A. Gene expression profiling demonstrated that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including AUR-KA. The retinoblastoma tumour suppressor gene 1 (RB1) is a key factor in cell cycle control. RB1 is more commonly mutated in metastatic- and recurrent-PC than in primary tumours, particularly in NEPC variants. This indicates that there is a selective pressure for RB1 loss which facilitates lineage plasticity and progression of PC cells initiated by PTEN mutation. The additional loss of the tumour suppressor gene Tp53 causes resistance to antiandrogen therapy . Cycline-D kinase inhibitors, such as palbociclib, ribociclib, and abemaciclib, may prevent RB1 phosphorylation and inactivation, restoring sensitivity to hormonal treatment. Gene expression profiling studies revealed that both mouse and human NEPC exhibit increased expression of epigenetic reprogramming factors such as the enhancer of zeste homolog 2 (Ezh2). Ezh2 inhibitors, including GSK-126/343/503, have recently been developed and they demonstrated restoration of AR expression and sensitivity to antiandrogen therapy in PTEN and RB1 double-knockout mice.Preclinical studies have shown that aggressive variants of PC are characterized by combined alterations in RB1, Tp53 and/or PTEN. The immune checkpoints CTLA4 and PD-1/PD-L1 are the main targets of immunotherapy. They have been shown to negatively modulate cytotoxic T lymphocyte activity in tumours, enabling immunological escape. In contrast to several other tumours, advanced PC is characterized by minimal immune cell infiltrate and a relatively low mutational burden, suggesting a possible lower sensitivity to treatment with immune checkpoint inhibitors (ICIs). However, mCRPC showed higher mutational load compared to primary tumors.Immune escape strategies protect PC from detection and destruction by the immune system. In physiological conditions, immune checkpoint pathways are essential for self-tolerance preservation and modulation of normal immune responses. Increased expression of immune checkpoint molecules by tumour cells deeply affects specific T-cell immunity in the tumour microenvironment. This mechanism inhibits the elimination of cancer cells, therefore facilitating tumour progressionet al. demonstrated that patients progressing during enzalutamide treatment had significantly increased PD-L1/2+ dendritic cells and a high frequency of PD-1+ T cells in blood, compared to those naïve or responding to treatment.Bishop . In a phase II study, pembrolizumab in monotherapy was tested in 258 docetaxel-refractory patients already treated with at least one new generation hormonal therapy. A complete or partial response was observed in about 5% of patients, irrespective of their PD-L1 status. The median duration of response was not reached in PD-L1 positive disease.Pembrolizumab, an anti-PD-1 antibody, showed preliminary antitumor activity in PD-L1 positive mCRPC patients in the phase Ib KEYNOTE-028 trialThe use of PD-1 and PD-L1 as predictive biomarkers of response to immunotherapy is still undefined..A phase II single-arm study reported ICIs anti-tumour activity in mCRPC patients progressing during enzalutamide. Ten patients were treated with pembrolizumab in addition to standard dose of enzalutamide. Three subjects experienced a rapid PSA reduction and two of them achieved a partial response, including a patient with microsatellite instability, which has proven to be a predictive factor for this therapeutic approachThe therapeutic association of pembrolizumab with enzalutamide in mCRPC is currently being studied in the phase I KEYNOTE-365 trial . Synergistic inhibition of PI3K and AR signalling induced PC regression, indicating that both pathways coordinate to support cell survival. Thomas et al. confirmed these results both in vitro and in vivo, examining the association of the PI3K/AKT inhibitor capivasertib with the antiandrogen bicalutamide. The combined targeting of PI3K/AKT pathway and AR axis significantly delayed CRPC progression. Recently, results of a phase I dose-escalation study of enzalutamide in combination with capivasertib were published, showing anti-tumour activity of the combination therapy in the subgroup of patients harbouring aberrations in the PI3K/AKT/mTOR pathway. The PI3K isoform p110β regulates cell mitosis and survival. BL140, a newly developed isoform β-specific inhibitor, has been shown to effectively suppress PC growth in all PC lines tested.Carver . Two phase I trials are testing a combination strategy of enzalutamide with the mTOR inhibitors everolimus  inhibitor in mCRPC, due to dose reductions secondary to toxicity. These findings support PTEN loss as a predictive factor for treatment response. A phase III trial is testing the addition of ipatasertib to abiraterone in men with asymptomatic or mildly symptomatic, previously untreated mCRPC  and suppression of mTOR. In addition, the authors demonstrated that small interfering RNA targeting AMPK significantly repressed autophagy and increased cell death in PC cells exposed to enzalutamide or conventional ADT, suggesting that autophagy represents a crucial survival mechanism in CRPC. Lastly, modulators of autophagy such as metformin and clomipramine significantly enhanced enzalutamide activity and reduced cancer growth in mouse models implanted with enzalutamide-resistant cells.Autophagy is a catabolic process of self-digestion in which cellular components are isolated from the rest of the cell within autolysosomes for degradation and recycling, in order to maintain cellular homeostasis. The physiological balance between autophagy and apoptosis can be lost in cancer development because of a dysfunction in apoptosis. The subsequent upregulation of autophagy increases nourishment for high-proliferative tumour cells.Resistance to hormonal therapy is often associated with stress-induced activation of molecular chaperones involved in the autophagy process. Clusterin (CLU) is a heat-shock chaperone-like protein induced by antiandrogens such as enzalutamide. CLU inhibition by OGX-011 repressed enzalutamide-induced activation of AKT and MAPK pathways. The inhibition of both AR  and CLU (with OGX-011) synergistically increased apoptotic rates and delayed the appearance of CRPC in LNCaP tumour and PSA progression in vivoIn vivo, BET inhibitors were more effective than direct AR antagonists in blocking cell proliferation in mouse models with CRPC xenografts. In enzalutamide-resistant PC models, enzalutamide and apalutamide showed stronger tumour growth inhibition when associated with BET inhibitors, JQ1 and OTX015, respectively.The oncogene C-myc promotes cells growth and proliferation by increasing ribosome biosynthesis. Bromodomain and extra-terminal enhancer (BET) proteins are involved in regulating chromatin remodelling by C-myc .MET is a tyrosine kinase receptor activated by a unique ligand, known as hepatocyte growth factor (HGF). The HGF/MET signalling system plays a fundamental role in tumour growth, invasion, and in metastasis development in many types of malignancies. MET overexpression has been described in primary and advanced PC and it correlates with poor prognosiset al. showed that the AR represses c-MET expression in a ligand-dependent manner in PC xenografts. Although ADT can restrain the expression of growth-promoting genes activated by the AR, it may also reduce the repressive role of androgen signalling on c-MET expression. Accordingly, there is a strong rationale supporting the combination of inhibition of the HGF/MET pathway with ADT.Verras . The phase II trial testing the same combination was stopped early after the results of the phase III study COMET1, in which cabozantinib did not significantly improve OS compared to prednisone in heavily treated mCRPC patients.Cabozantinib is an orally available receptor tyrosine kinase inhibitor with strong activity against MET and VEGFR2. The safety profile of cabozantinib in combination with abiraterone was defined in a phase I studyPreclinical work has revealed that crizotinib, a multi-kinase inhibitor including MET, can repress PC growth in cell lines and mouse models. A phase I trial is ongoing to study the toxicity and pharmacokinetic profile of crizotinib in association with enzalutamide in mCRPC, before or after treatment with docetaxel  is highly expressed in CRPC and it regulates the transcriptional activity of the AR. Experimental drugs targeting IL-6 showed unsuccessful results, however.The activation of the NF-κB/p52 pathway plays a role in the development of resistance to enzalutamide. Nadiminty . The identification of defects in the DNA repair mechanism provides a strong rationale for the use of PARP inhibitors (PARPi) in PC treatment. The phase II TOPARP-A study demonstrated that olaparib treatment in patients who did not benefit from standard therapies, and who had defects in DNA-repair genes, was associated with high response rates. In 2016 the FDA designated olaparib as an innovative therapy for mCRPC patients with BRCA1/2 or ATM gene mutation who have received prior chemotherapy with a taxane and at least one new generation hormonal therapy. PARP1 is also implicated in the regulation of AR transcriptional activity. The AR requires active PARP1 to modulate its association with chromatin. There is accumulating evidence that second-generation AR signalling inhibitors might induce “BRCAness”-102 and there is potential synergy between AR inhibitors and PARP inhibitors in the treatment of mCRPC. Proof of the synergy between PARPi and hormonal agents derives from a randomized phase II trial testing the combination of olaparib with abiraterone in docetaxel-pretreated mCRPC patients. A benefit in rPFS as primary endpoint was shown in the experimental group, regardless of DNA repair genes mutation status. Combination therapy with NHA and PARPi is also being studied in the first-line setting of mCRPC. Pre-clinical studies suggested that the combination of enzalutamide and PARPi can lead to BRCAness because of the ability of enzalutamide to down-regulate the expression of genes involved in the DNA repair system. The rationale of testing this co-treatment as first-line therapy is to induce the BRCAness phenotype and to expand the use of PARPi in the treatment landscape of mCRPC. Given the cross-talk between the AR and the DNA repair system, several studies are ongoing to test the activity of PARPi in combination with new generation hormonal therapies in mCRPC.Human DNA is constantly exposed to damage by endogenous and exogenous agents, and a complex repair system is active to protect cells’ genome stability. Poly-ADP-ribose polymerase (PARP) enzymes are primarily involved in detecting single strand damage and activating the repair process In vitro and in vivo studies are ongoing with the aim of detecting predictive biomarkers of response, and to investigate new approaches to bypassing resistance.The advent of new generation hormonal therapies has given way to a new era in the treatment of CRPC. Second generation antiandrogens have increased specificity and higher affinity for the AR than previous hormonal agents, resulting in more effective suppression of AR signalling. These drugs significantly improve patients’ quality of life and prolonged overall and metastasis-free survival. Despite these advances, a proportion of patients do not benefit at all from the treatment and, among those who respond, resistance will undoubtedly occur after a variable time. Primary and acquired resistance is a multifactorial phenomenon that is caused not only by AR-dependent mechanisms, but also by the activation of other pathways bypassing AR signalling. Metastatic PC is currently understood to be a heterogeneous disease, characterized by a coexistence of AR-driven and AR-independent neoplastic cells. This heterogeneity is the prime driver for distinct biological behaviours observed and for the different responses to NHA. In recent years, many efforts have been made to investigate the genomic landscape of PC. The aim is to change the future of healthcare by moving towards precision medicine and finding the best therapeutic approach for individual patients. Genomic sequencing of PC patient samples led to the discovery of potentially targetable gene mutations, such as PI3K/AKT, MET, BRCA2, ATM, and others. Several novel agents targeting the identified non-AR-driven pathways involved in the pathogenesis and progression of PC are currently under investigation to prevent or overcome these mechanisms of resistance, some with very promising results. PARP inhibitors are the most advanced molecules in clinical trials for CRPC, both as single agents or in combination with hormonal therapy. The future challenge for clinicians will be to better understand the biological heterogeneity of PC and to select patients with a high chance of response to different treatments.