Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions. Androgen receptor (AR) remains a critical therapeutic target in the treatment of metastatic castration-resistant prostate cancer (CRPC), due to overactive AR signaling . Next-geAmong more than 20 alternatively spliced AR variants identified to date, AR-V7, which arises from cryptic exon inclusion, is the most prevalent variant in CRPC followed by the exon-skipping AR-v567. Expression of AR-V7 has been clinically associated with adverse patient outcomes including increase rates of metastases and inferior survival rates, and resistance to current standard of care treatment with abiraterone, enzalutamide, and taxane chemotherapy . TogethePreviously we showed that AR-fl binds microtubules (MTs) via the hinge domain and uses them as tracks for fast nuclear import . TaxanesIn this study, we set out to investigate the mechanisms mediating AR-V7 nuclear import and its subnuclear biophysical properties in association with chromatin to identify unique, targetable biological features.Nuclear translocation is a pre-requisite for the transcriptional activity of AR-fl and all other nuclear hormone receptors. Nuclear import is mediated by a conserved bipartite nuclear localization signal (NLS) motif, which in AR-fl and AR-v567 is comprised of parts of exons 3 and 4. AR-V7 lacks exon 4, which is the second half of the canonical bipartite NLS, and although its cryptic exon 3 has been implicated in NLS reconstitution , the canTo quantify nuclear translocation kinetics of AR proteins in response to treatment, we performed fluorescence recovery after photobleaching (FRAP) analysis in the M12 human metastatic prostate cancer cells stably expressing each GFP-tagged AR protein . The halTreatment with R1881 accelerated AR-fl nuclear import by decreasing the T½ from 23 to 11 s while addition of DTX or IPZ significantly attenuated T½ to 23 and 19 s, respectively . The nucMost nuclear import pathways involve the small GTPase Ran, which catalyzes the release of cargo protein from importin in the nucleus. As AR-V7 does not use importin-α/β pathway for nuclear import, we set out to determine whether it requires active transport via interaction with the nucleoporins, NPC components that mediate transport of proteins larger than 40 kDa. Thus, we incubated cells with wheat germ agglutinin (WGA), a well-established inhibitor of nucleoporin-mediated nuclear transport  and idenAndrogen-regulated gene expression requires AR-fl receptor dimerization in the nucleus mediated by the zinc finger (D-box) domain, prior to DNA binding. AR-V7 transcriptional activity has been shown to depend on the D-box domain which mediates AR-V7 homodimerization in the nucleus . To deteIn CRPC, AR-fl is often co-expressed with AR variants (AR-V) in patient tumors and it has been suggested that active AR signaling in castrate conditions is partially due to AR-V heterodimerization with AR-fl resulting in its nuclear translocation in the absence of ligand . To deteMechanistically, we sought to determine whether AR-V7 might form a heterodimer with unliganded AR-fl in the cytoplasm driving the latter into the nucleus. Treatment with IPZ showed that when AR-V7 and AR-fl were co-expressed in the same cell, IPZ inhibited the nuclear localization of AR-fl only; while it had no effect on AR-V7 , consistTo investigate whether AR-V7 transcriptional output mediates the fractional AR-fl nuclear translocation, we introduced the DNA-binding domain (DBD) mutation (A573D) known to abrogate the transcriptional activity of canonical AR, into AR-V7 (AR-V7-A573D). We found that the A573D mutation abrogated AR-V7 transcriptional activity, but not its nuclear localization, compared to unaltered AR-V7 . This isIt is well established that agonist-bound AR-fl is transcriptionally active and relatively immobile in the nucleus; while antagonist-bound AR-fl is highly mobile and transcriptionally inactive . AR-V7, To better understand the kinetics of subnuclear mobility of each protein, we generated photo-convertible mEos4b-AR-fl or mEos4b-AR-V7 and followed them by live-cell imaging . Photo-cTo resolve the conundrum between the high nuclear mobility of AR-V7 and its high transcriptional activity, we introduced the A573D DBD mutation into AR-V7 or AR-fl expression plasmids and introduced them into PC3 cells. We detected a significant increase in the mobility of the mutant AR-fl-A573D compared to AR-fl, in the presence of ligand, with T ½ of 4 s versus 17 s, respectively, p<0.0001 . No diffTo investigate whether the high intranuclear mobility of AR-V7 is due to its reduced occupancy rate on target AREs on chromatin, we performed ChIP for AR-V7 or AR-fl in 22Rv1 cells with endogenous expression of both proteins. ChIP-qPCR data showed that R1881 increased the occupancy of AR-fl on the enhancer regions of PSA and FKBP5 . In contInhibition of androgen receptor signaling remains the cornerstone of contemporary therapeutic strategies for patients with metastatic CRPC. Reactivation of AR signaling is a hallmark of CRPC, largely mediated by the nuclear activity of the AR splice variant AR-V7 .In this study, we identify that AR-V7 nuclear import does not use the canonical NLS-dependent importin-α/β pathway, in contrast to AR-fl and AR-v567. Earlier findings suggested that the C-terminal cryptic exon 3 (CE3) domain of AR-V7 might mediate its nuclear import due to it similarity with the second bipartite NLS of AR-fl . The samSurprisingly, we found that the same inactivating mutations introduced in the D-box domain (A596T and S597T), identified in patients with androgen insensitivity syndrome , had a dAnalysis of single PC3 cells co-expressing tagged AR-FL and AR-V7, showed significant increase in AR-fl nuclear localization, in the absence of ligand  in agreeThis study also revealed unique nuclear biology of AR-V7, distinct from that of ligand-bound AR-fl, suggesting a distinct mode of transcriptional action. The chromatin binding dynamics of nuclear hormone receptors  have been closely correlated with their respective transcriptional output and affinity to their ligand . It is wPC3, LNCaP, C4-2, HEK-293T, and 22Rv1 cell lines were obtained from the ATCC. We generated the C4-2 cell line (tet-on GFP-AR-V7 or GFP-AR-V7-D-Box mutant), which stably expresses tetracycline-inducible GFP-AR-V7 or GFP-AR-V7-DBox mutant by infecting the lentiviral construct (detailed information in Plasmid Constructions section). The stable M12 cell lines expressing GFP-tagged AR-fl, AR-v567, or AR-V7 were described previously . MycoplaPrimary antibodies were used: rabbit polyclonal anti-AR-N-terminal (AR-N-21), rabbit monoclonal anti-AR-V7, rabbit monoclonal anti-AR-C-terminal, rabbit polyclonal anti-actin, rabbit polyclonal anti-beta tubulin, rabbit polyclonal anti-Histone H3, rabbit polyclonal anti-GFP, mouse monoclonal anti-mCherry (1C51). DTX (Taxotere), IPZ, and cytochalasin D were obtained from Sigma-Aldrich. WGA Alexa Fluor 594 Conjugate (W11262) were purchased from molecular probes. See Key resources table for antibodies used in this study.The following plasmids pmCherry-AR-fl, pEGFP-C1-AR-fl, pEGFP-C1-AR-v567, and pEGFP-AR-V7  were useThe photoconvertible mEos4b-C1 backbone (Addgene plasmid #54812) was a gift from Dr. Michael Davidson . We subc90,000 cells were plated on coverslips and transfected with or without plasmid (refer to Plasmid constructions section) using FuGENE HD (Promega) or AMAXA Nucleofector R Kit (Lonza), according to the supplier’s instructions. Transfected cells were fixed or imaged live within 24–48 hr after transfection and 4–8 hr of doxycycline or R1881 treatment. Cells were analyzed using Hoechst nuclear stain, confocal microscopy, and ImageJ.Live-cell imaging was carried out on cells either microinjected or transfected with the plasmids described above. Cells were grown on No. 1.5 coverglass mounted on 35 mm MatTek dish and cultured in RPMI 1640 supplemented with 5% charcoal-stripped FBS, 25 mM HEPES, 2 mM sodium pyruvate, and 2 ml L-glutamine. Live-cell imaging and FRAP were carried out on a Zeiss LSM 700 confocal microscope equipped with an on-stage live-cell chamber . Photo bleaching in the region of interest was carried out with the 405 nm laser at maximum power for three iterations. A single z-section was imaged before and at time intervals. The normalized intensity of region of interests and the half-time of recovery required for the fluorescence intensity to reach 50% of its pre-bleach intensity (T1/2) were obtained using Zeiss Zen software. Photo-conversion imaging analysis was performed in PC3 cells plated on 35 mm MatTek dish after transient transfection with photoconvertible AR constructs described above. PC3 cells transfected with mEos4b-AR-fl was further incubated with 10 nM R1881 for 1 hr before the photo-conversion. Briefly, region of interest at nuclei of cells was photo-converted by applying 405 nm laser (60% power for 400 ms) using the Mosaic system  equipped with spinning disk confocal microscope (Zeiss/Perkin-Elmer) at Bio-imaging resource center at the Rockefeller University. The time-lapse images were captured in two different channels  before and after photo-conversion and images acquisition was performed with MetaMorph software.PC3 cells stably expressing the ARE-reporter vector with mCherry fluorescent reporter signal was plated on 35 mm MatTek dish. GFP-tagged AR-fl or AR-V7 was microinjected in the nuclei of cells. The synthetic androgen R1881 was added to the cells microinjected with GFP-AR-fl construct. After overnight incubation, GFP-AR and ARE-reporter mCherry signal were imaged using Zeiss scanning confocal microscope.ACGCTGGACAGGGGGCAAAAG, R: GGGCAGGGCACATGGTTCACT), FKBP5 , and ACTIN . Relative quantitation was used to determine fold change in expression levels by the comparative Ct method.For relative quantitation of AR target genes, quantitative real-time PCR was performed on 100 ng input RNA using Power SYBR green RNA-to-Ct 1 step kit (Applied Biosystems) and primers specific for PSA . Equal volumes of sheared chromatin were immunoprecipitated with rabbit AR-V7 antibody (RevMab 31-1109-00), rabbit AR antibody (Abcam 52615), or rabbit IgG control (Santa Cruz Biotechnology). After extensive washing, crosslinking was reversed, and DNA fragments were purified using Macherey-Nagel kit (740609). Q-PCR amplification was performed using the ABI 7500 fast system (Fast SYBR Green 4385612 Applied Biosystems) and the relative standard curve method in a 96-well format. Primers used: FKBP5e: F-GGT TCC TGG GCA GGA GTA AG; R-AAC GTG GAT CCC ACA CTC TC; PSAe: F-TGG GAC AAC TTG CAA ACC TG; R-GAT CCA GGC TTG CTT ACT GT; AREneg: F-GCT GAT TCA ATT ACC TCC CAG AA; R-AGT TTG GGA CAG ACG GGA AA. The input chromatin for each sample was analyzed at four concentrations  to generate a standard curve per primer pair and per 96-well plate. The sheared chromatin was diluted 1/6 before being used for Q-PCR. All reactions were run in triplicate.C4.2 cell line expressing tet-inducible GFP-AR-V7 was treated with either 10 nM R1881 for AR nuclear translocation or 1 µg/ml doxycycline for GFP-AR-V7 induction. Subcellular fractionation of cells was performed using the subcellular protein fractionation kit (Thermo Fisher Scientific) and each extract was subjected to immunoblotting with indicated antibodies.The Student’s two-tailed t-test was used to determine the mean differences between two groups. p<0.05 is considered significant. Data were presented as mean ± SEM. This work performs a careful study of AR-V7, a splice variant of androgen receptor (AR) that lacks the androgen-binding domain, is constitutively active, and is typically expressed as prostate cancers become resistant to anti-androgen therapies. Clinically, there is intense interest in overcoming anti-androgen resistance, and part of this includes understanding differences between AR-V7 and AR, to be able to therapeutically target AR-V7. This manuscript provides a robust analysis of the regulation of nuclear import and the chromatin-binding features of AR-V7 versus AR. The work reveals that AR-V7 exhibits fast nuclear import kinetics in an NLS- and importin-α/β- independent manner, dependent on the dimerization (D-box) domain mediates AR-V7 nuclear import, revealing a new function for this domain versus its role in the full-length AR. The work also shows that AR-V7 employs an unconventional mode of transcription characterized by high intranuclear mobility, with transient and unstable chromatin interactions, likely reflecting a "Hit-and-Run" mechanism. This greatly enlarges the mechanistic understanding of AR-V7 function, and may help with developing new therapeutic agents. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Erica Golemis as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Prof. Dr. John T. Isaacs (Reviewer #2).Thank you for submitting your article "AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in Castrate-Resistant Prostate Cancer" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Overall, the reviewers agree that the experiments are carefully performed and the data are convincing, and the study is important. The reviewers concur that there are some experiments that are essential to solidify the findings.1. The authors show that neither DTX or IPZ affect AR-V7 nuclear localization, suggesting that nuclear import of AR-V7 is independent of microtubules and importin-B. However, the authors do show that WGA, an inhibitor of nucleoporin-mediated transport, results in cytoplasmic sequestration of AR-V7. They suggest in the discussion that a β-like importin family member with the capacity to mediate import for proteins without a classical NLS might be a candidate importer. This should be investigated more thoroughly and some attempt made to nominate the nuclear transporter for AR-V7, as this would further delineate how the regulation of AR-V7 and AR-FL differ, and is directly related to the main thrust of the authors' manuscript.2. One major issue throughout is that most studies are done by ectopic expression of tagged plasmids in AR-null cells (PC3). It is unclear if these results would hold in situations where AR-V7 and AR-FL are co-expressed. Some of the key experiments – for example Figure 1F, Figure 2, Figure 3A, etc. should be performed in cell lines co-expressing AR-V7 and AR-FL , with and without isoform-specific knockdown of AR-FL/AR-V7. Additionally, C-terminal endogenous tagging of AR-FL and AR-V7 by CRISPR-mediated knockin, followed by a repeating of some of these same experiments, should be considered.3. The results in Figure 3A suggest that co-expression of AR-V7 and AR-FL increases AR-FL expression in the nucleus under basal conditions. This is intriguing and may be consistent with the findings of Watson et al., PNAS 2010, suggesting that AR-V7 activity requires AR-FL expression. Increased AR-FL nuclear localization under low androgen conditions may also drive castration resistance, and perhaps AR-V7 drives this indirectly by promoting AR-FL nuclear import, rather than directly. Can the authors determine whether the increased nuclear AR-FL upon AR-V7 co-expression is active (by reporter gene activity or the expression of downstream targets)? Does co-expression or AR-V7 and AR-FL in an AR-dependent line drive increased AR-FL nuclear localization and enzalutamide resistance? Although IPZ treatment indicated that FL-AR import still requires importin, it does not rule out the possibility that FL-AR and AR-V7 may dimerize within the nucleus and lead to increased nuclear retention of FL-AR. This can be tested using a D-box mutant of FL-AR co-expressed with AR-V7.Optional Revisions1. Figure 1D – is there a specific reason why this experiment was done in M12 cells while most other experiments seem to be done in PC3?2. Can the authors more clearly label what is the difference between the "AR-V7 with AR-fl" and "AR-fl with AR-V7" conditions in Figure 3B?3. Figure 4C – to eye, all the replicate dots look identical (but perhaps there is just very little variability between them?). Please double check this. Statistics also needed for this panel. Also would check some additional AR-V7 target genes as delineated in Cato et al., Cancer Cell 2019 . Particularly given that study and authors' data suggesting that AR-V7 is a repressor, would make sense to check the impact on genes that are repressed, rather than activated by AR-V7.4. The authors could expand the discussion to emphasis their thoughts as to the implication of the differences between AR FL vs. ARv7 driven transcriptional regulation.5. The authors have determined that RanGTPase activity and the nucleoporin complex are partially required for nuclear import of AR-V7. It might benefit the readers if the authors can briefly describe what other mechanisms exist which can explain these findings.6. In Figure 4D, the DNA binding mutant (A573D) does not seem to affect AR-V7 nuclear localization, but it was not quantified and not mentioned in the text. It would be helpful to include the quantification data.7. In figure 5, the authors showed that co-expression of AR-V7 with FL-AR does not affect AR-V7 mobility when the FL-AR is activated by R1881. It would be interesting to see whether AR-V7 mobility is changed in the presence of Apo-AR as a fraction of Apo-AR may dimerize with AR-V7 and activate a subset of genes using mechanisms which are different from FL-AR and AR-V7 alone.8. Although it has been suggested that fast nuclear mobility of AR appears to be associated with antagonist bound AR, however, it has also been shown recently that ER antagonist/SERDs immobilized ER on chromatin and which may account for the antagonist activities of these compounds . Furthermore, with respect to GR, it has been shown that the intranuclear mobility of this receptor correlated with the affinity of the ligand with which this receptor binds. Therefore, these other previous findings should be discussed in the context of the current finding.9. Line 200, please correct this sentence "FRAP analysis of AR-fl identified slow the fluorescence…. Essential revisions:Overall, the reviewers agree that the experiments are carefully performed and the data are convincing, and the study is important. The reviewers concur that there are some experiments that are essential to solidify the findings.1. The authors show that neither DTX or IPZ affect AR-V7 nuclear localization, suggesting that nuclear import of AR-V7 is independent of microtubules and importin-B. However, the authors do show that WGA, an inhibitor of nucleoporin-mediated transport, results in cytoplasmic sequestration of AR-V7. They suggest in the discussion that a β-like importin family member with the capacity to mediate import for proteins without a classical NLS might be a candidate importer. This should be investigated more thoroughly and some attempt made to nominate the nuclear transporter for AR-V7, as this would further delineate how the regulation of AR-V7 and AR-FL differ, and is directly related to the main thrust of the authors' manuscript.We took the reviewer’s suggestion to nominate a putative nuclear import receptor for AR-V7.As the reviewers suggested to “make an attempt to nominate the nuclear transporter of AR-V7” these are the experiments we performed. Given that AR-V7 does not contain a canonical NLS and ubiquitination of the tumor suppressor PTEN, also without canonical NLS, facilitates its nuclear import via importin-11 (IPO11), we tested the involvement of IPO11 in AR-V7 nuclear import. Using a dominant-negative mutant IPO11, which abrogates its nuclear import function, we show no effect on AR-V7 nuclear localization. This data is now shown in Figure 1- supplement 2. In ongoing studies beyond the scope of this manuscript, we plan to systematically test the involvement of individual nuclear import receptors, including non-canonical importins and transportins with knockdown experiments. The discussion is updated to reflect these new findings.2. One major issue throughout is that most studies are done by ectopic expression of tagged plasmids in AR-null cells (PC3). It is unclear if these results would hold in situations where AR-V7 and AR-FL are co-expressed. Some of the key experiments – for example Figure 1F, Figure 2, Figure 3A, etc. should be performed in cell lines co-expressing AR-V7 and AR-FL , with and without isoform-specific knockdown of AR-FL/AR-V7. Additionally, C-terminal endogenous tagging of AR-FL and AR-V7 by CRISPR-mediated knockin, followed by a repeating of some of these same experiments, should be considered.To address this point, we have repeated a few key experiments in cell lines co-expressing endogenous AR-V7 and AR-fl. Specifically, we have confirmed the results shown in Figure 2 on the effect of the catalytic mutant RanQ69L on the impaired nuclear localization of AR-V7, in C4-2 cells with endogenous AR-fl stably expressing inducible GFP-AR-V7. In addition, transient transfection of cells endogenously co-expressing AR-fl and AR-V7, such as 22RV1 cells, corroborated the above findings. These data are now shown in Figure 2-supplment 1 and discussed in results and discussion. We have expanded and clarified the methods to reflect this.To confirm the partial cytoplasmic accumulation of AR-V7-D-Box, observed following microinjection in PC3 cells , we generated C4-2 cells with endogenous AR-fl to stably express doxycycline inducible GFP-AR-V7 D-Box mutant. Quantitation of the subcellular localization of the AR-V7 D-Box mutant revealed statistically significant increase in AR-V7’s cytoplasmic localization in the presence of the D-Box mutations, further suggesting that the D-Box domain may be involved in AR-V7 nuclear localization. These data are now displayed in new Figure 2E-2F and discussed in the text.Finally, we considered the CRISPR knock-in tagging suggestion, however, it is unfortunately not feasible as AR-V7 is an RNA splice variant and not present in the genome.3. The results in Figure 3A suggest that co-expression of AR-V7 and AR-FL increases AR-FL expression in the nucleus under basal conditions. This is intriguing and may be consistent with the findings of Watson et al., PNAS 2010, suggesting that AR-V7 activity requires AR-FL expression. Increased AR-FL nuclear localization under low androgen conditions may also drive castration resistance, and perhaps AR-V7 drives this indirectly by promoting AR-FL nuclear import, rather than directly. Can the authors determine whether the increased nuclear AR-FL upon AR-V7 co-expression is active (by reporter gene activity or the expression of downstream targets)? Does co-expression or AR-V7 and AR-FL in an AR-dependent line drive increased AR-FL nuclear localization and enzalutamide resistance? Although IPZ treatment indicated that FL-AR import still requires importin, it does not rule out the possibility that FL-AR and AR-V7 may dimerize within the nucleus and lead to increased nuclear retention of FL-AR. This can be tested using a D-box mutant of FL-AR co-expressed with AR-V7.As the reviewer suggested, we tried very hard to determine whether nuclear AR-fl in response to AR-V7 co-expression is transcriptionally active. However, due to the very small number of cells co-expressing both constructs, we could not reliably use reporter gene activity which reflects population averaging. To answer this important question, we are currently developing a single-cell sensitive AR-fl-specific fluorescent reporter assay so that we can image single cells that co-express both proteins and get a fluorescent readout, indicative of active AR-fl.To answer the question of whether co-expression or AR-V7 and AR-FL in an AR-dependent line drive increased AR-FL nuclear localization, we have co-transfected an AR-dependent line (LNCaP) with AR-fl and AR-V7 and observed increased nuclear localization of AR-fl in the presence of AR-V7. In addition, a similar experiment performed in C4-2 cells with inducible AR-V7 confirmed the effect of AR-V7 on enhanced AR-fl nuclear localization. These data are now shown in figure 3C and 3F, respectively. As the number of LNCaP cells co-expressing both AR-Fl and AR-V7 was less than 10% of the transfected population, enzalutamide resistance could not be assessed using population-averaging classical cytotoxicity assays.Optional Revisions1. Figure 1D – is there a specific reason why this experiment was done in M12 cells while most other experiments seem to be done in PC3?Yes, the AR-independent M12 cells were stably expressing each construct but they were challenging to culture or microinject/transfect. Therefore, we switched to PC3 cells, which are easily cultured and are a more relevant model.2. Can the authors more clearly label what is the difference between the "AR-V7 with AR-fl" and "AR-fl with AR-V7" conditions in Figure 3B?We have changed labeling and legend in the main figure and supplement to highlight which proteins are microinjected in each condition.3. Figure 4C – to eye, all the replicate dots look identical (but perhaps there is just very little variability between them?). Please double check this. Statistics also needed for this panel. Also would check some additional AR-V7 target genes as delineated in Cato et al., Cancer Cell 2019 . Particularly given that study and authors’ data suggesting that AR-V7 is a repressor, would make sense to check the impact on genes that are repressed, rather than activated by AR-V7.The reviewer is right that the variation in the values was too small to show in the graph.4. The authors could expand the discussion to emphasis their thoughts as to the implication of the differences between AR FL vs. aRv7 driven transcriptional regulation.Yes, we have added elaborating ideas to the Discussion section. We highlight those alternate modes of transcriptional action for AR-V7 may be promoted by differences in protein structure and post-translational modifications relative to AR-fl. Cato et al. suggest that AR-V7 binds preferentially to transcriptional co-repressors, likely due to differences in H3K27 acetylation. How structural differences may impact co-regulator binding with AR-V7 relative to AR-fl remain to be investigated.5. The authors have determined that RanGTPase activity and the nucleoporin complex are partially required for nuclear import of AR-V7. It might benefit the readers if the authors can briefly describe what other mechanisms exist which can explain these findings.Our work revealed the partial requirement for RanGTPase activity in AR-V7 nuclear import by utilizing a RanQ96L mutant, which has lowered intrinsic GTPase activity. This suggests that RanGTPase is not the only factor that regulates the nucleo-cytoplasmic transport of AR-V7, yet it plays a role. Functionally, this may suggest that AR-V7 can enter the nucleus even if its nuclear importer is not re-exported into the cytosol. To clarify, our work also showed that AR-V7 import depends completely on its passage through the nuclear pores. We have edited the discussion accordingly.6. In Figure 4D, the DNA binding mutant (A573D) does not seem to affect AR-V7 nuclear localization, but it was not quantified and not mentioned in the text. It would be helpful to include the quantification data.The reviewer is correct, we did not observe that the A573D mutation affected AR-V7 nuclear localization based on the quantification and did not include it in the manuscript.7. In figure 5, the authors showed that co-expression of AR-V7 with FL-AR does not affect AR-V7 mobility when the FL-AR is activated by R1881. It would be interesting to see whether AR-V7 mobility is changed in the presence of Apo-AR as a fraction of Apo-AR may dimerize with AR-V7 and activate a subset of genes using mechanisms which are different from FL-AR and AR-V7 alone.The reviewers are correct, and that FRAP studies in cells co-expressing of AR-V7 and Apo-AR could potentially suggest a heterodimerization in the nucleus. However, this would be challenging to measure since the fraction of Apo-AR present in the nucleus is likely to be low relative to the amount of AR-V7, and we would not see a meaningful difference in FRAP studies.8. Although it has been suggested that fast nuclear mobility of AR appears to be associated with antagonist bound AR, however, it has also been shown recently that ER antagonist/SERDs immobilized ER on chromatin and which may account for the antagonist activities of these compounds . Furthermore, with respect to GR, it has been shown that the intranuclear mobility of this receptor correlated with the affinity of the ligand with which this receptor binds. Therefore, these other previous findings should be discussed in the context of the current finding.We have modified the discussion to address this point.9. Line 200, please correct this sentence "FRAP analysis of AR-fl identified slow the fluorescence….We have corrected this sentence in the manuscript.


Clinicians awarded overall high scores in NR and SSR reports. One rater acknowledged a significantly higher level of clarity and time saving when comparing SSR to NR. Our findings highlight that the standardized structured reporting of radical prostatectomy specimens, qualifying as level 5 reports, significantly increases objectively measured content quality and the level of completeness. The implementation of nationwide SSR in Germany, particularly in oncologic pathology, can serve pathologists, clinicians, and patients.Prostate cancer represents one of the most common malignant tumors in male patients in Germany. The pathological reporting of radical prostatectomy specimens following a structured process constitutes an excellent prototype for the introduction of software-based standardized structured reporting in pathology. This can lead to reports of higher quality and could create a fundamental improvement for future AI applications. A software-based reporting template was used to generate standardized structured pathological reports of radical prostatectomy specimens of patients treated at the University Hospital Klinikum rechts der Isar of Technische Universität München, Germany. Narrative reports (NR) and standardized structured reports (SSR) were analyzed with regard to completeness, and clinicians’ satisfaction with each report type was evaluated. SSR show considerably higher completeness than NR. A total of 10 categories out of 32 were significantly more complete in SSR than in NR ( Prostate cancer is one of the most common malignant tumors and accounts for 25% of all new cancer cases in male patients in Germany . PatholoAnnually, more than 400 radical prostatectomy specimens reach the Institute of Pathology at Technische Universität München. About 20 residents and senior pathologists generate different versions of reports on radical prostatectomy specimens. The lack of standardized structure yields discrepancies between reports of both inter- and intrapersonal nature. Prostate cancer surgery and radical prostatectomy follow a structured process  and therIn this single-center study, we aim to investigate the impact of introducing an electronic reporting tool on overall reporting quality and the clinicians’ perception by comparing traditional narrative and innovative standardized structured pathology reports. In addition, the potential use of generated datasets for future AI applications is discussed.The study was approved by the ethics committee of the TUM school of medicine, Technische Universität München, Munich, Germany . This pilot study is based on a combined retrospective and prospective analysis on histopathological reports of patients treated via radical prostatectomy at the University Hospital Klinikum rechts der Isar of the Technische Universität München (TUM), Germany. The first retrospective cohort contained 101 consecutive patients treated between January and March 2019. Histopathological reports were generated at the Institute of Pathology at TUM by 12 individual board-certified pathologists. All reports were composed in a narrative style (referred to as “narrative reports/NR”) in accordance to the WHO Classification of Tumours of the Urinary System and Male Genital Organs  [n = 33) whose pathology reports were created by a single board- certified pathologist (AMS) using a software-based structured reporting tool based on the recently published ICCR template for radical prostatectomy specimens [The second prospective cohort consisted of randomly selected patients treated between February and July 2021 (n = 2) and one patient without histological evidence for malignant tumor in the specimen were excluded from the study leading to a total number of n = 131.Patients with neoadjuvant therapy . Due to different pathological tumor stages, scores between 23 and 32 points were possible. The individual reports were evaluated by awarding 0 , 0.5 (answered incompletely or indirectly), or 1 point (answered explicitly and completely) for each category resulting in a total quality score for every report expressed in percentage of the maximum score.In detail, a total of 23 required and recommended categories and 9 subcategories (17 considered as “required” and 15 as “recommended” by ICCR) were defined and scored in all NR and SSR (The different report (sub-) categories were assessed individually by comparing NR and SSR on a feature level. One point was awarded only if the required information was clearly stated (complete as well as explicit). Otherwise, this category yielded 0 points. Each category received a total score, expressed as percentage for better comparability.The diagnostic quality of the reports was not addressed in the current study.To investigate clinician’s perception, 62 reports  were evaluated by two board-certified urologists using a questionnaire , SPSS , and GraphPad .U test. The two-sided t-test was performed to compare individual questionnaire outcomes. p-values lower than 0.05 were considered statistically significant.Basic descriptive statistics were used for specification of patients and tumor characteristics. The two-sided Fisher’s exact test was performed to evaluate the significance of NR vs. SSR in each category. Overall mean of NR against SSR was analyzed utilizing a Mann–Whitney n = 99) by 12 different board-certified pathologists and 32 reports were prospectively generated by a single board-certified pathologist using a structured reporting software (SSR n = 32). Clinicopathological details are given in 134 patients who underwent radical prostatectomy for prostate cancer at the Urology Department, University Hospital Klinikum rechts der Isar of the TUM between 2019 and 2021 were selected. Three patients had to be excluded: two patients received neoadjuvant therapy and one showed no evidence of a malignant tumor in the specimen. Out of the 131 analyzed pathology reports 99 were written in a traditional narrative reporting style (NR p < 0.0001) was observed. Comparable results were found when exclusively comparing the categories defined as mandatory by the ICCR (p < 0.0001).High total scores of the overall content quality expressed as percentage of the maximum achievable score were observed in both NR and SSR  were complete in all of the narrative and structured reports. However, the above-mentioned categories were provided as a free-text format in NR, and therefore more difficult to find than in the synoptic format of SSR.p < 0.0001, p = 0.0144 and p < 0.0001, respectively). Moreover, a clear statement on extraprostatic extension, extent (if available), urinary bladder neck invasion and presence/absence of intraductal carcinoma was significantly more common in SSR , the percentage of Gleason pattern 4/5 and presence/absence of lymphovascular invasion  were compared with a comparable, randomly selected NR sub-cohort (n = 30) employing a random number generator. All 62 reports were evaluated by two different board-certified urologists (rater 1 and 2) using a questionnaire addressing completeness, time saving, quality, comprehensibility, and clarity for each individual report. The results of the analysis are shown in All SSR (p < 0.0001). In detail, clear differences were observed for time saving aspects  and clarity of the report . In contrast, no differences between NR and SSR were observed in the evaluation of first rater’s perception.Each report could achieve a total score between 5 being the best and 28 being the worst score. Overall, both NR and SSR received favorable grades by the two raters . However, relevant differences were observed. SSR received significantly higher total scores by rater 2 compared to NR , thus, reflecting the individual reporting style of the pathologist. These narrative reports without defined content and single text fields qualify as level 2 of 6 according to the Ontario scale . The Ontp < 0.0001)  see . Likewis001) see . On the For referring physicians, it is important to receive a clear and precise statement from pathologists. This was addressed in our questionnaire which was answered by two board-certified urologists. Whereas one of the raters noticed no differences in all five categories addressed in the above-mentioned questionnaire , the other rater observed significantly higher clarity and time saving in SSR compared to NR reports. The time saving aspect might be related to the different formatting of non-structured NR and SSR using a synoptic format and reaches beyond the completeness of ICCR categories. n = 131), differences were noticed between NR and SSR, the latter revealing the information at first glance. SSR display a synoptic format with bullet points and are therefore quick and easy to review. In NR, there is a considerable variation depending on the author. Some pathologists use a text-only format, others a combination of complete sentences and a synoptic summary of the most important information at the end of the report. The exact location of clinically relevant information differs considerably between SSR and NR as well as between NR from different pathologists. In SSR, all information crucial for the planning of further treatment is clearly arranged at the end of the report. This can save time in extracting the relevant data for the respective clinician. The lack of synoptic formatting of data goes beyond time saving aspects. On top of that, the introduction and further implementation of artificial intelligence in the field of pathology and other fields of medicine depends on both complete and uniformly structured data [During the analysis of all reports (red data ,16. Due red data ,27.In addition to advantages for clinical studies and translational research, the implementation of a software-based reporting system serves the pathologist during the decision-making process. The software offers a clear structure with text-fields, drop-down menus, and boxes, thus serving as a checklist during the specimen evaluation. This is of particular interest for young residents and can also serve as a self-monitoring tool for experienced pathologists.Moreover, new versions and updates of reporting guidelines can be easily incorporated into a software-based reporting program and, therefore, made available for all pathologists using the software. Thus, constant report quality in accordance with the latest standards can be maintained. In the long run, it can be assumed that a structured and software-supported method of reporting will save time in everyday medical practice, benefitting both pathologists and urologists. In conclusion, our pilot study on radical prostatectomy specimen reporting shows that SSR significantly increases objectively measured content quality and level of completeness. Given the currently shown correlation between the use of SSR and improved patient care with higher survival rates in patients with colorectal cancer from the Netherlands , the impThe rating urologists were not given any precise information in advance and no expectations were formulated, to not influence the evaluation process. However, due to different conditions in both groups, limitations may arise when comparing the results of this pilot study. Cohort 1 includes more than three times as many subjects as cohort 2. However, the distribution of pT stages shows no relevant difference between the two groups.The Influence of the examiner-dependent factor could be greater in the SR group, as only one pathologist was responsible for all findings. In addition, the selection of reports evaluated by the two urologists could cause errors. The urologists worked with all of the SR but only one randomly selected third of NR. Additionally, the number of urologists rating the reports is small, so no general statement can be made but a tendency can be shown. Therefore, larger studies are required to confirm our results.


HSPB2 mRNA expression levels in breast cancer, which represents the most frequent malignancy in females and one of the three most common cancer types worldwide. To this end, malignant breast tumors along with paired non-cancerous breast tissue specimens were used. HSPB2 expression levels were quantified in these two cohorts using a sensitive and accurate SYBR green-based quantitative real-time polymerase chain reaction (q-RT-PCR). Extensive biostatistical analyses were performed including Kaplan–Meier and Cox regression survival analyses for the assessment of the results. The significant downregulation of HSPB2 gene expression was revealed in breast tumors compared to their adjacent non-cancerous breast tissues. Notably, high HSPB2 mRNA expression predicts poor disease-free survival and overall survival of breast cancer patients. Multivariate Cox regression analysis revealed that HSPB2 mRNA overexpression is a significant predictor of poor prognosis in breast cancer, independent of other clinicopathological factors. In conclusion, high HSPB2 mRNA expression levels are associated with breast cancer patients’ relapse and poor survival.Small heat shock proteins (sHSPs) are ubiquitous ATP-independent chaperones that contribute to the maintenance of proteome integrity and functionality. Recent evidence suggests that sHSPs are ubiquitously expressed in numerous types of tumors and have been proposed to be implicated in oncogenesis and malignant progression. Heat shock protein family B member 2 (HSPB2) is a member of the sHSPs, which is found to be expressed, among others, in human breast cancer cell lines and constitutes an inhibitor of apical caspase activation in the extrinsic apoptotic pathway. In this study, we investigated the potential prognostic significance of  Breast cancer (BrCa) is the most frequent malignancy in females and one of the three most common cancers worldwide, followed by lung and colorectal cancer. In 2020, 2.3 million women were diagnosed with BrCa, and 685,000 deaths occurred globally ,2. EarlyBrCa is a complex heterogeneous disease classified into three major subtypes according to estrogen receptor (ER) or progesterone receptor (PR) expression and erb-b2 receptor tyrosine kinase 2  gene amplification. Notably, tumors expressing ER and/or PR are termed “hormone receptor-positive”, tumors expressing HER2 are called “HER2-positive”, and tumors lacking these three markers are considered “triple negative” ,7. TheraMolecular chaperones, also known as heat shock proteins (HSPs) constitute a large family of molecular machines involved in the proper folding, unfolding, and assembly of polypeptides in order to maintain their structure and function ,10. In pSeveral pieces of evidence suggest that HSPs are abnormally expressed in different types of cancer, including, among others, breast, colorectal, lung, prostate, pancreatic, bladder, and ovarian malignancies ,16,17,18HSPB2 gene was also reported to be expressed in human breast cancer cell lines and constitutes an inhibitor of apical caspase activation in the extrinsic apoptotic pathway [HSPB2 expression levels in BrCa tumors and matched adjacent normal tissue, and we also evaluated its potential association with patients’ relapse and overall survival.Heat shock protein family B member 2 (HSPB2) is a member of the small HSPs (sHSPs), which consist of ubiquitous ATP-independent chaperones with low molecular weights ,22,23. H pathway . HoweverHSPB2 gene are affected during oncogenic transformation. To this end, we used a genetically defined model of stepwise carcinogenesis, in which human primary mammary epithelial cells (HMEC) were immortalized by the expression of the human telomerase catalytic subunit (hTERT) (HME cells) followed by p53/pRb inactivation due to concomitant simian virus 40 large T antigen (LT) expression (HMLE cells); HMLE cells were then transformed by the co-expression of the oncogene H-RasV12, resulting in the generation of breast metastatic malignant cells (HMLER cells) [HSPB2 mRNA levels are significantly decreased in the HMLE and HMLER cell lines during oncogenesis as compared to normal HMEC cells  . Our anaEC cells ; notablyIn the current study, two groups of samples were used, namely one group of 150 cancerous tissue specimens and another group of 16 paired non-cancerous tissue specimens from patients with primary BrCa. The cohort of patients consisted of 150 women, with a total median age of 60 years, ranging from 31 to 90 years, at the time of diagnosis. Moreover, concerning the histological grade and according to the World Health Organization (WHO) classification system, 8 patients were diagnosed with grade I (well-differentiated), 97 with grade II (moderately differentiated), and 45 with grade III malignant tumors (poorly differentiated). Furthermore, according to the TNM staging system, 43 malignant lesions were characterized as stage I (28.7%), 89 as stage II (59.3%), and 18 as stage III (12.0%). The clinical and biological traits of patients with BrCa included in the current study are shown in HSPB2 mRNA levels among 16 pairs of malignant breast tumors and their adjacent non-cancerous breast tissues revealed the downregulation of the HSPB2 mRNA expression in the majority of malignant breast tumors (p < 0.001)  . Specifi 3094.54 . These fHSPB2 mRNA expression status in each tissue specimen as positive or negative, an optimal cut-off value was set, as described in the “Materials and Methods” section. We found that 75 (50%) samples were classified as HSPB2 mRNA-positive, when the relative HSPB2 mRNA expression was equal to or higher than 656.5 RQU and 75 (50%) samples as HSPB2 mRNA-negative (relative HSPB2 mRNA expression lower than 656.5 RQU). We then investigated the potential association of HSPB2 mRNA expression status with patients’ clinicopathological parameters including molecular subtype, anatomic stage, mitotic rate, and HER2, ER, and PR status. We found no significant association between HSPB2 gene expression levels and the mentioned clinicopathological parameters (not shown).To classify the HSPB2 mRNA expression regarding patients’ relapse was revealed by the univariate Cox regression analysis . Kaplan–Meier curves revealed that HSPB2-positive BrCa patients have a significantly lower DFS compared to those who are HSPB2-negative (p = 0.011) . Consequently, high HSPB2 mRNA expression levels represent an independent prognostic indicator for tumor recurrence in BrCa patients.The independent significance of analysis . Specifi= 0.011) A. The siHSPB2 mRNA expression status for patients’ overall survival (OS). To this end, a univariable Cox regression analysis was performed and revealed that high HSPB2 mRNA expression predicts poor OS for BrCa patients. Specifically, BrCa patients bearing HSPB2-positive tumors had a significantly shorter OS time interval than those with lower HSPB2 mRNA levels . Furthermore, the molecular subtype and prognostic stage were found to be significant prognosticators of OS  . Therefore, the HSPB2-positive mRNA expression status in BrCa could be considered a novel independent indicator of poor OS.The prognostic value of = 0.009) B. AdditiHSPB2 mRNA expression status. The stratification according to molecular subtype revealed that patients with triple-negative or HER2-positive tumors had significantly lower DFS (p < 0.001) and OS (p < 0.001) rates .Determination of tumor characteristics such as molecular subtype and tumor grade is crucial in the prognosis of patients diagnosed with BrCa . Thus, p1) rates  comparedp < 0.001) and OS (p < 0.001), compared to those with tumors of anatomic stage I or II, regardless of the HSPB2 expression status (HSPB2-positive tumors had remarkably lower DFS (p = 0.006) and OS (p = 0.011) rates, in comparison with those with HSPB2-negative tumors.Additionally, after stratification, according to the anatomic stage, patients with tumors of anatomic stage III were found to have a remarkably shorter DFS (n status . Furtherp < 0.001) and OS (p < 0.001) intervals, as compared to patients of stage I or II tumors, regardless of the HSPB2 expression status (HSPB2-positive tumors showed an increased probability of a poorer DFS (p = 0.003) and OS (p = 0.024), as compared to those with HSPB2-negative tumors.The stratification of patients according to prognostic stage revealed that patients of prognostic stage III had significantly shorter DFS , and is of the utmost importance for cell homeodynamics and survival ,30. HSPsBrCa is a highly heterogeneous disease characterized by not only various phenotypes and molecular subgroups, but also by different responses to treatment . DependiOver the last decades, growing evidence has shown that sHSPs expression is frequently deregulated in diverse cancer types and is proposed to profoundly impact malignant progression. In particular, sHSPs have been associated with several hallmarks of cancer, including tumorigenesis, cell growth, the evasion of apoptosis, immune surveillance, angiogenesis, metastasis, and chemoresistance ,42,43,44HSPB2 mRNA expression levels in BrCa patients.HSPB2 expression is particularly elevated (despite ubiquitous expression) in cardiac and skeletal muscle ,25 and wHSPB2 gene transcription is predominantly downregulated in malignant breast tumors compared to their adjacent non-cancerous breast tissues. In line with our findings, a recent comprehensive transcriptomic study of the HSP gene family in BrCa patients from both The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohorts revealed that HSPB2 gene expression levels are profoundly downregulated in all BrCa molecular subtypes as compared to normal breast tissues [HSPB2 in BrCa samples, due to HSPB2 gene deletion [HSPB2 in cancer, as well; specifically, HSPB2 mRNA was lower in esophageal squamous cell carcinoma cell lines, due to hypermethylation of the promoter of the HSPB2 gene [HSPB2 mRNA was also found to be barely expressed in pancreatic cancer [HSPB2, whereas most studies stating the overexpression of HSPs have examined their protein levels, a possible explanation is that HSPB2 protein expression levels do not perfectly correlate with HSPB2 mRNA levels. Indeed, as shown by transcriptomics and proteomics data of HSPB2 expression in various BrCa cell lines, deposited to the Expression Atlas database [Despite the fact that many sHSPs are overexpressed in a wide range of solid tumors , we obse tissues . Salhia deletion . Other sPB2 gene , while Hc cancer . Since adatabase , the lowHSPB2 mRNA overexpression is a significant predictor of poor prognosis in BrCa, independent of other clinicopathological factors, including molecular subtype and prognostic stage. Additionally, Kaplan–Meier survival curves revealed that patients with HSPB2-positive tumors were more likely to have poor outcomes, such as relapse and death. Consistently, the elevated levels of diverse HSPs expression in specific malignancies have been associated with a poor prognosis and an increased resistance to therapies [Multivariate Cox regression analysis revealed that herapies ,51,52,53Heat shock factor 1 (HSF1) constitutes the most robust regulator of HSPs expression to maintain (among others) proteome stability . A growiRPRM, ADGRB1, and STEAP3 genes [Furthermore, although the role of HSPB2 has not been investigated so widely as the role of other HSPs, it was found to confer resistance to apoptosis in human BrCa cell lines, as it inhibited the apical caspase activation in the extrinsic apoptotic pathway , as wellP3 genes . In addiP3 genes . The conP3 genes ,60. TherP3 genes ,60,61,62The major implication of HSPs in cell transformation and tumor progression largely supports the notion that HSPs-targeting drugs could constitute a promising approach in cancer therapy ,60,63,64HSPB2 mRNA expression status could be combined with other well-validated clinical biomarkers, which could tailor the therapeutic options aiming to improve the outcome of cancer therapy and a patient’s overall survival, while minimizing the associated risk.Molecular biomarkers represent biological molecules used to infer disease risk, diagnosis, prognosis, and therapeutic response . It is e2, 95% humidity, and 37 °C.Human mammary epithelial cells HMEC were obtained from Lonza Group AG , while the human mammary adenocarcinoma cell line MCF-7 was obtained from the American Tissue Culture Collection . The HMEC-derived cell lines HME, HMLE, and HMLER were a kind gift by Prof. Robert A. Weinberg . HMEC, HME, HMLE, and HMLER cells were grown in MEGM™ Mammary Epithelial Cell Growth Medium BulletKit™ . MCF-7 cells were cultured in DMEM  supplemented with 2 g/L glucose, 10% FBS, and 1% penicillin/streptomycin. All cell lines were maintained in a humidified incubator at 5% COOne hundred and fifty (150) BrCa samples and 16 paired, non-cancerous tissue samples were collected from patients with primary BrCa, subjected to surgery at the “Saint Savvas” Cancer Hospital of Athens, Athens, Greece. The biological and clinicopathological data collected included the age of patients, the dimensions of the resected tumor, the infiltration of regional lymph nodes, the presence of distant metastasis, the histological and intrinsic (molecular) subtypes, as well as the histological grade of the tumor, the expression status of PR, ER, HER2, and the mitotic rate based on the Ki-67 index. All tumors were independently characterized by two pathologists. The anatomic (TNM) and prognostic stages were determined, based on these data. All breast tissue specimens were stored in liquid nitrogen immediately after surgery. Survival data were available for 145 out of 150 patients, and the median follow-up time was 97 months.The study was conducted in compliance with the 1964 Declaration of Helsinki and its later amendments and was approved by the institutional Ethics Committee of the “Saint Savvas” Cancer Hospital of Athens, Athens, Greece. Written informed consent was obtained from all patients.® , following the manufacturer’s protocol instructions. Spectrophotometric evaluation of the concentration and purity of the isolated RNA samples was conducted using a BioSpec-nano Micro-volume UV–Vis Spectrophotometer . Two micrograms μg of total RNA were subjected to reverse transcription, using M-MLV reverse transcriptase  and oligo-dT 5 μM, according to the manufacturer’s instructions, to obtain first-strand cDNA.Total RNA was extracted from each sample using the TRI reagentHSPB2; moreover, specific primer pairs were designed for housekeeping genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hydroxymethylbilane synthase (HMBS), which were chosen among others as reference genes, according to supporting literature data [HSPB2 primers were 5′-CCGAGTACGAATTTGCCAACC-3′ and 5′-AGGCCGGACATAGTAGCCAT-3′. Those of GAPDH were 5′-GTCAAGGCTGAGAACGGGAA-3′ and 5′-TCGCCCCACTTGATTTTGGA-3′, and those of HMBS were 5′-AAGAGACCATGCAGGCTACCA-3′ and 5′-ACAAGTTGGCCAGGCTGATG-3′.A real-time qPCR assay was developed and carried out, using the SYBR Green Chemistry, in an ABI 7500 Real-Time PCR System . Specific primers were designed for ure data  and our 2O, and 0.5 μL of 10-fold diluted cDNA. We optimized the qPCR assays by standardizing the primer concentration and the thermal protocol, so as to observe a unique melting curve for each assay. The uniqueness and specificity of each amplicon were also assessed by agarose gel electrophoresis. The cDNA derived from the MCF-7 cells was used as a calibrator to render “−∆Ct” values calculated in distinct qPCR runs comparable. The comparative Ct method (2−∆∆Ct) was applied for relative quantification [HSPB2 expression of each sample was defined as the ratio of HSPB2 molecules to GAPDH and HMBS molecules, divided by the same ratio that was calculated for the calibrator (MCF-7 cells) and was determined in relative quantification units (RQU).The qPCR mixture contained KAPA SYBR FAST qPCR Master Mix Universal, supplemented with ROX as a passive reference dye, forward and reverse primers at a final concentration of 200 nM, 2.5 μL DEPC-treated Hfication ,73. The HSPB2 mRNA expression between paired samples. Next, the BrCa patients were categorized into two distinct groups, namely HSPB2-positive and HSPB2-negative patients; this categorization was done based on the median value of HSPB2 mRNA expression (656.5 RQU). More specifically, patients with an HSPB2 mRNA expression value higher than 656.5 RQU were characterized as HSPB2-positive, whereas those with an HSPB2 mRNA expression value lower than 656.5 RQU were characterized as HSPB2-negative. Potential associations between the expression of HSPB2 mRNA and other categorical clinicopathological variables were investigated, using a two-tailed χ2-test.The distribution in the patients’ cohort was not normal; thus, non-parametric tests were used. Firstly, descriptive statistics were performed. The Wilcoxon signed-rank test was carried out to assess the difference in p-value < 0.050.Kaplan–Meier survival analysis was then applied, with regard to disease-free survival (DFS) and overall survival (OS). Differences between Kaplan–Meier curves were assessed using the log-rank (Mantel–Cox) test. Bootstrap (1000 random samples) univariate and multivariable Cox regression analyses were carried out, concerning also DFS and OS; further, the bias-corrected and accelerated (BCa) 95% confidence interval (CI) of each hazard ratio (HR) was estimated. Next, Kaplan–Meier survival analysis was performed in subgroups of the cohort, in which patients were stratified according to specific clinicopathological characteristics. Each outcome was considered statistically significant if the HSPB2 molecular chaperone gene transcription is predominantly downregulated in BrCa, whereas increased HSPB2 gene expression levels are associated with patients’ relapse and poor survival. Consequently, HSPB2 mRNA expression status could be potentially used to assess the clinical prognosis of patients with this frequent malignancy, independent of other clinicopathological factors.Our findings suggest that the


Prostate cancer (PCa) is today the second most common cancer in the world, with almost 400,000 deaths annually. Multiple factors are involved in the etiology of PCa, such as older age, genetic mutations, ethnicity, diet, or inflammation. Modern treatment of PCa involves radical surgical treatment or radiation therapy in the stages when the tumor is limited to the prostate. When metastases develop, the standard procedure is androgen deprivation therapy, which aims to reduce the level of circulating testosterone, which is achieved by surgical or medical castration. However, when the level of testosterone decreases to the castration level, the tumor cells adapt to the new conditions through different mechanisms, which enable their unhindered growth and survival, despite the therapy. New knowledge about the biology of the so-called of castration-resistant PCa and the way it adapts to therapy will enable the development of new drugs, whose goal is to prolong the survival of patients with this stage of the disease, which will be discussed in this review. Homo sapiens, which happened relatively recently with the development of animal husbandry—animal fat, especially saturated, can contribute to PCa progression, while unsaturated fatty acids (FA) abundant in fish and vegetable oils reduce PCa risk .,4,5.3,4,The prostate secretes numerous non-peptide  and peptide substances  ,7,8,9. TAccording to 2020 GLOBOCAN data, PCa is the second most common cancer among men and the fifth leading cause of cancer death, with approximately 1.4 million new cases and approximately 375,000 deaths worldwide . The incPCa is diagnosed in only 2% of cases in people under the age of 50, and over 60% of patients are diagnosed after the age of 65 . In 2018BRCA1 and BRCA2. African Americans in the USA have the highest incidence rate globally, which is explained by African ancestry and multiple environmental factors, such as hypovitaminosis D. About 15% of PCas are thought to be hereditary. Thus, the risk of developing PCa is 2× higher if the father had PCa, 3× if the brother had PCa, and if both father and brother had PCa, as much as 5 times ..180].Finally, abiraterone is an androgen biosynthesis inhibitor in testicular, adrenal, and prostatic tumor tissue targeting CYP17. It is also an AR antagonist and 3β-hydroxysteroid dehydrogenase inhibitor with affinity for CYP11B1 and a panel of hepatic CYP enzymes. A newer drug, galeterone, also blocks CYP17, AR, and the transcriptional activity of this receptor in the nucleus of tumor cells .Other mechanisms of therapeutic targeting of the AR pathway involve pharmacological antagonism at the receptor level and inhibition of the conversion of testosterone into a more active metabolite—DHEA—which is achieved by blocking the enzyme 5α-reductase. Examples of AR antagonists include: flutamide, bicalutamide, nilutamide, apalutamide and enzalutamide .These antiandrogens bind to the ligand binding site on the AR and thus prevent androgen binding. Unlike bicalutamide, apalutamide is an antiandrogen with a high affinity for the AR that does not have agonistic activity ; it also blocks the binding of AR to DNA in the nucleus of PCa cells. Darolutamide has an advantage over enzalutamide and apalutamide, as it blocks both overexpressed and mutated AR, which is the main mechanism of resistance to the other two mentioned antagonists. Additionally, darolutamide blocks the transcriptional activity of AR with point mutations F877L, H875Y/T878A, F877L/T878A, and T878G. The clinical efficacy of AR antagonists is lower when they are used as monotherapy, since AR blockade leads to the termination of the negative feedback loop and increased secretion of LH and thus of testosterone. Better results in the treatment of metastatic PCa are achieved if AR antagonists are combined with GnRH analogues; then AR antagonists block the effects of adrenal androgens that GnRH analogues do not. A newer approach in antagonizing the AR is the blockade of the AF-1 region (activation function-1) on the N-terminal part of the AR and this is the mechanism of action of the new substance, EPI-506, which is the precursor of the active molecule EPI-002; said antagonist is in phase I/II clinical trials for patients with mCRPCa resistant to abiraterone and/or enzalutamide. Spironolactone is also a weak antagonist of AR and testosterone biosynthesis, and cyproterone acetate, a progestin, can block AR to some extent. On the other hand, finasteride and dutasteride, as 5α-reductase inhibitors, prevent testosterone from converting into DHEA, especially in the male external genitalia. Both drugs are used in the treatment of benign prostatic hypertrophy.It is estimated that 20–40% of PCa patients have a recurrence after initial radiation therapy or surgical removal of the prostate. Then, drugs that suppress endogenous androgens  are introduced into the treatment. However, 12–18 months after initiation of ADT, CRPCa develops with disease progression. AR gene amplification has been identified in 30–50% of CRPCa cases and is associated with poor prognosis and shorter overall survival, as well as shorter progression-free survival .One of the important causes of resistance to antiandrogens in PCa therapy is changes at the gene level. Muller et al. (2012) showed ten years ago that in some tumor tissues there is a co-deletion of genes that suppress tumor development and they are located in the same part of the chromosome . Such isPoint mutations of the AR gene;Amplification of AR and CYP17;Changes in androgen biosynthesis;Changes in cofactors for AR;Creation of different variants of AR.Overall, antiandrogen therapy suppresses most PCas for a certain period of time, but resistance to ADT is virtually inevitable. Nearly all cancers will progress to CRPCA if given ADT for an adequate period of time. The resistance mechanisms leading to CRPCA can be divided into five groups:These mutations are rare in primary hormone-dependent tumours but occur more frequently in CRPCa (>10%), especially in cases where the tumor has progressed despite systemic hormone therapy and the use of new antiandrogens. Not only that, but AR gene mutation is among the nine most significant gene mutations in CRPCa . The resThis resistance mechanism often occurs with ADT and plays an important role in the loss of efficacy of the new antiandrogens, enzalutamide and abiraterone. It has been identified in as many as 80% of CRPCa cases . At the Tumor cells can escape inhibition of androgen synthesis by goserelin or abiraterone by increasing, intratumorally, androgen synthesis and/or AR, but also via glucocorticoid receptors and glucocorticoids . EvadingThe EGF/IGF signaling pathway that is mediated by phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt/PKB) and depends on the level of phosphatidylinositol 3,4,5-triphosphate (PIP3) and PTEN in the cell;The Ras signaling pathway (Ras family of proto-oncogenes) that occurs via Cdc42-linked kinase (Ack), Ras/Raf mitogen-activated protein kinase/ERK kinase (MEK), and the proto-oncogene tyrosine protein kinase Src;IL6 cytokine signaling pathway that activates AR through Janus kinase signal transducer and activator of transcription (JAK1), signal transducer and activator of transcription 3 (STAT3), and intermediate histone acetyltransferasep300.In conditions of AR blockade due to ADT, rescue pathways are activated in the form of at least three alternative signaling pathways that involve growth factors and stimulate AR signaling pathways independently of androgens, i.e., steroids :The EGF/In a tumor consisting of several million cells, there are already those that have activated drug resistance mechanisms and will contribute to the development of CRPCA. Induced resistance is also possible. However, tumor size does not necessarily increase the likelihood of developing CRPCA. There are many other factors . Shore et al. (2021) emphasized predictive factors for progression to CRPCA: Gleason score, extent of metastatic spread, hereditary genetic factors/mutations, and hormonal kinetics . For exaSignal transduction of all nuclear receptors, including AR, begins with cofactor recruitment. Previously, the ligands bound to the receptors. They subsequently move into the nucleus and form dimers with a conformational change into an active form that can interact with promoter regions. The dimer–cofactor complex (coactivators or corepressors) activates or inhibits transcription. The ligand nature determines the receptor conformation and cofactor affinity ,192.Since 1995, when the first cofactor, SRC-1, was isolated, their number has increased to over 200 . They caLi et al. (2002) showed that changes in the expression of AR cofactors could play a vital role in the development of prostate tumors. They confirmed by in situ hybridization that in PCa epithelial samples, there is increased expression of Ran/ARA24 and PIAS1 and slightly less of SRC1, TMF1/ARA160, TRAP220, ARA54, and ELE1/ARA70 . The autFujita et al. (2019) showed that the expression of proteins from the SRC family (steroid receptor coactivator), SRC1, -2, and -3, is correlated with the aggressiveness of PCa . In miceAnother AR coactivator, CBP/p300, also has an oncogenic effect. It increases during androgen deprivation, and blocking its bromodomain reduces tumor growth . SimilarWild-type full-length AR (AR-FL) consists of an N-terminal domain, a DNA-binding domain, and a ligand-binding domain , encoded by exon 1, exons 2–3, and exons 4–8, respectively. Along with AR-FL, in CRPCA, splice variants are also particularly prevalent: AR-V1, AR-V7, and AR-567. Splice variants have a low abundant LBD and can be constitutively active in androgen-responsive genes even without ligands. At the same time, AR-FL interacts with both coactivators and corepressors, while AR-V7 is mainly linked to corepressors. This is partly due to isoform-specific variations in H3K27 acetylation . The role of the HOXB13 gene and its product, the transcription factor, is vital for AR-V7 signaling as well.CRPCA resistance to enzalutamide results from the absence of the target , whereas abiraterone increases the expression of ARf1, AR-V7, and AR-567 ,198. AR-From Charles Huggins and androgen deprivation in the treatment of mPCa  to the nUntil just a few years ago, the mCSPCa treatment was exclusively via androgen deprivation with either luteinizing hormone-releasing hormone (LHRH) analogues, LHRH antagonists, or subcapsular orchiectomy. Attempts to add peripheral anti-androgens of the first generation did not lead to a prolongation of patient survival, so their administration was not recommended by experts . Today, p < 0.001) [The first study to add a specific therapy to ADT was the CHAARTED study, which compared docetaxel with placebo in mCSPCa patients. Patients were stratified into a group with “high volume” disease, which is defined by the presence of visceral metastases and/or at least four bone metastases, at least one of which is in the peduncle of the skeleton; and another group that does not have these characteristics called “low volume”. Although the study demonstrated longer survival in the intent-to-treat (ITT) population, a sub analysis found that only those patients with high-volume disease benefited  . Given tp < 0.001) and rPFS . Grade 3 side effects of hypertension and hypokalemia were more common in the abiraterone group [LATITUDE is a study that examined the addition of abiraterone to ADT in patients with initially metastatic castration-sensitive “high-risk” PCa, defined by the presence of at least two of three features: visceral metastases, at least three bony metastases, and a Gleason score ≥8. In this study, 1199 patients were randomized to either abiraterone and prednisolone or placebo and prednisolone. All patients were on ADT. Co-primary endpoints were overall survival and radiological time to disease progression. Abiraterone prolonged OS compared to placebo . The most common side effect of apalutamide was rash [p < 0.0001) despite crossover that was allowed in the study [Apalutamide is a newer generation antiandrogen. In the study, TITAN was compared with placebo in patients with mCSPCa. The study included all patients regardless of volume or risk of disease and all were on ADT. Primary endpoints were rPFS and OS. In the primary analysis, after a median follow-up of 22.7 months, the proportion of patients who were free of radiological progression in the apalutamide group at two years was 68.2% versus 47.5% in the placebo group  [Similar to apalutamide, enzalutamide has also demonstrated efficacy in mCSPCa. The ARCHES study compared enzalutamide with placebo in addition to standard ADT. Patients were stratified by disease volume and prior docetaxel use. In all subgroups, enzalutamide prolonged the time to radiological disease progression. Adverse effects of Grade 3 and greater were equally common in both groups . In an u< 0.001) . The ENZ< 0.001) .p < 0.0001) and OS . The addition of abiraterone to ADT and docetaxel did not increase the rates of neutropenia, febrile neutropenia, fatigue, or neuropathy. Triple therapy achieved the greatest benefit in high-volume disease according to CHAARTED criteria [The most recent treatment concept for mCSPCa is the so-called triple therapy, which includes simultaneous administration of ADT, antiandrogens, and docetaxel chemotherapy. The PEACE-1 study is a 2 × 2 factorial design study in which a control group of patients received standard of care (SOC) ADT plus docetaxel, one study group received SOC plus radiotherapy, one group received SOC plus abiraterone, and the last group received SOC plus abiraterone and radiotherapy. A total of 1173 patients were randomized in the study. Patients receiving abiraterone had longer rPFS . In addition, all key secondary objectives were achieved in this study: time to PSA progression, radiological time to disease progression (rPFS), as well as PSA response rate. Fluid retention, hypertension, and hypokalemia were the most common side effects in the AA group [p < 0.001). Similar to AA, all secondary objectives were met in the study with enzalutamide. The most common side effects in the enzalutamide group were hot flashes, fatigue, and diarrhea, while 0.5% of patients had seizures [As already mentioned, for a long time ADT was the only option that prolonged the survival of mCRPCa. The TAX-327 study examined three chemotherapy options in mCRPCa, three-weekly docetaxel, weekly docetaxel, and mitoxantrone. Symptomatic patients with mCRPCa, as well as those who had visceral metastases, were included in the study. The primary target was OS. Both docetaxel groups had longer OS compared to mitoxantrone. Since 2004, three-week docetaxel has become the standard treatment for symptomatic patients with mCRPCa . A littlAA group . Enzalutseizures .p < 0.001), as was median OS  [p < 0.001) and longer OS at the time of publication. In the enzalutamide group, 72% of patients were alive, while in the placebo group, that percentage was 63%  [p < 0.0001) [A large number of patients enter the mCRPCa stage of the disease due to a jump in PSA or the appearance of new asymptomatic metastases. Considering this fact, studies were developed for mCRPCa in asymptomatic or minimally symptomatic patients who had not previously received docetaxel. Study COU-AA-302 compared AA with placebo in combination with prednisolone. The study enrolled 1088 patients and the co-primary endpoints were rPFS and OS. Median rPFS was longer in the AA group  . In a st< 0.001) . In addi 0.0001) . The saf 0.0001) ,216.p < 0.001). Median OS was longer in the cabazitaxel group . The most common side effect of Gr3 cabazitaxel was neutropenia in 44.7% of patients.Cabazitaxel is a taxane cytostatic. In the TROPIC study, it demonstrated longer PFS and OS compared to mitoxantrone when discontinued after progression to docetaxel therapy . After tp < 0.001) and overall survival . Grade 3 side effects were more frequent in the 177Lu-PSMA617 group, but quality of life was preserved [Radiopharmaceuticals are an interesting field of mCRPCa treatment. The first registered radiopharmaceutical for the treatment of this disease was the alpha emitter Radium-223, which demonstrated longer OS compared to placebo in docetaxel-naïve patients with mCRPCa. This type of treatment did not show significant side effects but was reserved only for patients with bone metastases . The VISBRCA1, BRCA2, and ATM mutations, while cohort B included patients with 12 other prespecified genes. Cohort A included 245 patients, while cohort B included 142. Olaparib prolonged PFS in cohort A . In addition, benefit was shown in objective response (ORR) and time to PSA progression. Additionally, rPFS was prolonged with olaparib in the entire study population (cohorts A and B). The most common side effects of olaparib were anemia and nausea. Statistical significance for overall survival has not yet been proven in the aforementioned analysis [Comprehensive genomic profiling (CGP) and personalized therapy of malignant diseases is the focus of interest of the oncology public. ESMO recommends CGP in the treatment of mCRPCa to select the most adequate therapy . Severalanalysis . Howeveranalysis . In the analysis . We are Immunotherapy with checkpoint inhibitors (ICI) plays a significant role in the treatment of various urological tumors ,229. In The definition of castration resistance in addition to radiological progression includes an increase in PSA with the patient’s castrate testosterone level. Sometimes PSA progression occurs without visible metastases by conventional imaging methods including skeletal scintigraphy and CT scans. What is also the fact that in this situation, patients with a shorter PSA doubling have a shorter time to the appearance of metastases and a worse prognosis . In 2015Autophagy is a survival mechanism that cancer cells use to deal with metabolic stress caused by various treatments, such as castration therapy. Additionally, autophagy has been shown to promote castration resistance. Evidence suggests that the expression of CAMKK2, AMPK, and ULK1 in males with PCa is related to the disease’s prognosis and progression. When AMPK-ULK1 signaling is inhibited, autophagy, cell proliferation, and colony formation are also inhibited in Lymph Node Carcinoma of the Prostate (LNCaP) cells. These findings add to the growing evidence that the CAMKK2-AMPK-ULK1 signaling cascade promotes autophagy and, thus, PCa and castration resistance .Despite their distinct action methods, abiraterone and enzalutamide are currently used in treating CRPCa. Enzalutamide is a drug that exerts multiple effects on androgen signaling, including: (i) the inhibition of testosterone binding to AR, (ii) the suppression of AR nuclear translocation, and (iii) the inhibition of AR binding to DNA. It has been demonstrated that the administration of enzalutamide induces an autophagy cascade in the C4-2B subline of LNCaP cells, which is mediated by the activation of AMPK and the inhibition of mTOR. This subpopulation of cells can replicate the behavior of CRPCa in humans by growing in castrated hosts. Moreover, it has been demonstrated that inhibiting AMPK in cells exposed to enzalutamide inhibits autophagy and promotes cell death. These results indicate that autophagy is a crucial survival strategy in CRPC . HoweverThe same evidence has been presented in the study of apalutamide, another AR antagonist used in CRPCa. Apalutamide treatment of LNCaP cells induces autophagy, which has been shown to have a strong anti-tumor effect when combined with autophagy inhibitors such as 3-methyladenine or siRNAs . LNCaP cStandard treatment for men with metastatic PCa or CRPCa includes the microtubule-stabilizing drug docetaxel. A number of studies have demonstrated that autophagic activity during taxane-based therapy varies considerably. While some research suggests that taxane therapy may stimulate autophagy, others have found the opposite to be true ,247. HowClinical responses after receiving prostate-specific membrane antigen (PSMA)-targeted radioligand treatment (RLT) with 177Lu or 225Ac radionuclides have been seen in between 57 and 76% of PCa patients ,250. HowADT remains the mainstay of primary PCa pharmacotherapy, but not as a monotherapy in most metastatic patients. Remission after ADT usually lasts 1–4 years, although in some cases it is longer (10 years). New strategies in the treatment of this malignancy include the development of inhibitors of androgen signals, the use of a combination of antiandrogens as a gold standard, as well as intermittent regimens of these drugs. Survival is prolonged, but still the cancer returns; that is, there is an exacerbation. It is a fact that even in CRPCa, cancer cells are dependent on androgens, but the mechanism of resistance to antiandrogens often remains unknown ,253,254.Not all targets listed in Another novel approach in PCa treatment could be modulation of autophagy. There have been a number of attempts to restore sensitivity to docetaxel or ADT by inhibiting autophagy. In preclinical models, the results have been encouraging because blocking autophagy has been shown to increase cell death in response to anti-cancer treatment. Currently, the focus of research is on conducting clinical trials to verify the findings of preclinical reports in patients with refractory malignancies . Small iRecently, the efficacy of chloroquine in combination with metal-based chemotherapeutic drugs such as palladium has been investigated . The palOther inhibitors, such as metformin or fenofibrate, have also been studied. Metformin is an oral biguanide frequently used to treat type 2 diabetes, well known by mTOR inhibition. While it is expected that this should increase autophagy, metformin actually inhibits autophagy by blocking Beclin-1 . A recenUnfortunately, the clinical use of these drugs could be challenging. For example, a high dose of pantoprazole, a drug that has been considered as a possible chemotherapy adjuvant, was shown to be beneficial in blocking autophagy and preventing taxanes resistance in a variety of solid tumors . However


Glucocorticoids (GC) are steroids hormones that drive circulating glucose availability through gluconeogenesis in the liver. However, alternative splicing of the GR mRNA produces two isoforms, termed GRα and GRβ. GRα is the classic receptor that binds to GCs and mediates the most described actions of GCs. GRβ does not bind GCs and acts as a dominant-negative inhibitor of GRα. Moreover, GRβ has intrinsic and GRα-independent transcriptional activity. To date, it remains unknown if GRβ modulates glucose handling in hepatocytes. Therefore, the study aims to characterize the impact of GRβ overexpression on glucose uptake and storage using an in vitro hepatocyte model. Here we show that GRβ overexpression inhibits the induction of gluconeogenic genes by dexamethasone. Moreover, GRβ activates the Akt pathway, increases glucose transports mRNA, increasing glucose uptake and glycogen storage as an insulin-mimetic. Our results suggest that GRβ has agonist-independent insulin-mimetic actions in HepG2 cells. Glucocorticoids (GCs) are steroid hormones that tune systemic energy metabolism to the circadian rhythm and psychological stress . PsycholGC signaling deploys a transcriptional program mediated by ubiquitous intracellular proteins, the Glucocorticoid receptors (GRs), which act as master transcription factors. Alternative splicing of the GR mRNA produces two isoforms, termed GRα and GRβ . GRα is Consistent with their diverging mechanisms of action, GRβ signaling closely recapitulates insulin. In cultured murine embryonic fibroblasts, GRβ mRNA reportedly increases after insulin stimulation, occurring in mice liver after fasting-refeeding. In both cases, GRα mRNA remained unchanged . AccordiThis evidence strongly implies a regulatory role of GRβ on metabolic signaling. However, it remains unknown if GRβ regulates glucose uptake and storage and if it enhances the insulin effects in hepatocytes. Thus, this study aims to characterize the impact of GRβ on glucose uptake and storage, using the cell line HepG2 as an in vitro hepatocyte model.To assess the role of GRβ in hepatocyte glucose homeostasis, we used the hepatocarcinoma-derived cell line HepG2, which has the presence of both GR isoforms . We over473) and p70S6K (Thr389), an effect that was emulated by GRβ overexpression. Most notably, GRβ alone induced Akt and p70S6K phosphorylation in the same magnitude as insulin  . We founWe found that GRβ increased glucose uptake to the same extent that insulin, and when we measured glycogen levels on the cells, we found an increase in the glycogen accumulation with GRβ associated with an increase in the glycogen synthase proteins levels, suggesting a regulation of anabolic processes, which is contrary to the GRα actions. Thus, a direct relationship between the glucocorticoid receptor and the mitochondria has been described ,18. HoweIn conclusion, GRβ overexpression activates the central canonical regulators of the insulin signaling cascade (Akt and P70S6K), increases glucose uptake, and glycogen storage in the HepG2 cell line, showing an additive effect with insulin in some markers of glucose handling, suggesting a converge in the response, ultimately GRβ acting as insulin-mimetic. v/v) fetal bovine serum (FBS), 1% (v/v) penicillin-streptomycin  solution in a humidified atmosphere with 5% CO2 at 37 °C. When cells reached 80% confluence, they were transfected with a plasmid-encoded the human GRβ kindly donated by John Cidlowski or an empty vector using lipofectamine 2000  in optiMEM  medium overnight. Then, the optiMEM medium was removed and replaced with fresh DMEN + 10% FBS medium for 1 h. After, cells were stimulated with dexamethasone  1 μM for 24 h or insulin  100 nM for 30 min or 3 h. HepG2 cells (ATCC HB-8065) were cultured in DMEM high glucose  supplemented with 10%  and phosphatase inhibitor . Proteins were resolved in SDS-polyacrylamide gels and then subjected to immunoblotting using antibodies specific for phospho-p70S6K , p70S6K , phospho-Akt , Akt , glycogen synthase  and β-actin . After, membranes were incubated with secondary antibodies anti-mouse  or anti-rabbit  conjugated to horseradish peroxidase and the ECL detection kit .Total RNA was obtained from HepG2 cells using TRIzol reagent . According to manufacturer protocol, cDNA was prepared from 1 μg of RNA using SuperScript II enzyme . Real-time PCR was performed using Eco Real-Time PCR System , using Fast SYBR Green Master Mix . The primers sequences are presented in For glucose uptake, HepG2 cells were serum-starved for 3 h; then cells were stimulated with insulin 100 nM for 30 min and incubated with 2-NBDG 300 μmol/L for 15 min at 37 °C as previously described . Imagingt-test was performed to compare two groups. Multiple groups were analyzed using two-way ANOVAs followed by Sidak’s multiple comparisons test. All analyses were performed with GraphPad software . p-value < 0.05 was considered statistically significant.Data are presented as mean ± SEM of at least 3 independent experiments. The student’s


Platelet-activating factor (PAF) has been suggested to be a potent inflammatory mediator in Allergic rhinitis (AR) pathogenesis. Vitamin E, an essential nutrient that comprises tocopherol and tocotrienol, is known as a potential therapeutic agent for airway allergic inflammation. This study aimed to investigate the beneficial effects of intranasal Tocotrienol-rich fraction (TRF) on PAF-induced AR in a rat model.Sprague Dawley rats were randomly assigned into 3 groups: Control, PAF-induced AR and PAF-induced AR with TRF treatment. To induce AR, 50 μl of 16 μg/ml PAF was nasally instilled into each nostril. From day 1 to 7 after AR induction, 10 μl of 16 μg/μl TRF was delivered intranasally to the TRF treatment group. Complete upper skulls were collected for histopathological evaluation on day 8.The average severity scores of AR were significantly higher in the PAF-induced AR rats compared to both control and PAF-induced AR with TRF treatment. The histologic examination of the nasal structures showed moderate degree of inflammation and polymorphonuclear cells infiltration in the lamina propria, mucosa damage and vascular congestion in the PAF-induced AR rats. TRF was able to ameliorate the AR symptoms by restoring the nasal structures back to normal. H&E staining demonstrated a statistically significant benefit upon TRF treatment, where minimal degree of inflammation, and a reduction in the infiltration of polymorphonuclear cells, mucosa damage and vascular congestion were observed.TRF exhibited symptomatic relief action in AR potentially due to its antioxidant, anti-inflammatory and anti-allergic properties. Allergic rhinitis (AR) is a common inflammatory disorder of the nasal mucosa that is clinically symptomized by sneezing, nasal itching, rhinorrhoea, and nasal congestion . It has The pathophysiology of AR involves a complex Immunoglobulin E (IgE)-mediated hypersensitivity reaction triggered by inhaled allergens. Upon exposure, immune cells process the allergens and stimulate B-cell mediated immune response. The cross-linking of IgE antibody on the surface of the activated inflammatory cells triggers mast cell degranulation in the early phase of allergic response, releasing preformed and newly synthesized mediators such as platelet-activating factor (PAF), histamine and proinflammatory cytokines that promote vascular permeability, rhinorrhoea and vasodilation , 6. ThesVitamin E is a robust lipid soluble antioxidant that presents peroxyl radical-scavenging activity. It comprises of 2 homologs, Tocopherol (TP) and Tocotrienol (T3), with each homolog further classified into α, β, γ and δ isoforms. TP and T3 have the same basic chemical structure characterized by a long chain hydrocarbon at the 2-position of a chromanol ring. TP consists of a side chain made of a phytylin group while T3 consists of an isoprenyl group with 3 unsaturated double bonds at positions 3’,7’ and 11’ , 15. BotCurrently, therapeutic interventions of AR include pharmacotherapy and immunotherapy . AccordiTocotrienol-rich fraction (TRF) was obtained from Davos Life Science Sdn. Bhd. . Platelet-activating factor (PAF), bovine serum albumin (BSA), glycerine, tween-80, ketamine chloride, xylazine hydrochloride and pentobarbital were obtained from Sigma-Aldrich .This study was conducted with male Sprague Dawley rats aged 8 to 9 weeks (weighing 250 to 300 g), that were without any evidence of upper airway infection. The rats were purchased from Invivos Pte Ltd and housed at the Biological Resource Centre (BRC), A*Star Singapore under standard laboratory conditions (12 h light/12 h dark schedule at 24 °C ± 2 °C). The rats were fed with standard pellet diet and water ad libitum. This study was approved by the BRC Institutional Animal Care and Use Committee (IACUC).The rats were acclimatized to laboratory conditions for 72 h prior to experiment and randomly assigned to 3 groups. Group 1 (n = 5) is the control group where the healthy rats were not exposed to the AR. Group 2 (n = 7) represents the PAF-induced AR group where the rats were intranasally induced with 50 µl of 16 µg/ml PAF per nostril. Group 3 (n = 7) is the PAF-induced AR with TRF treatment group where the AR rats additionally received 10 µl of 16 µg/µl TRF per nostril.The experimental design for the AR induction and treatment was shown in Fig. From day 1 to 7 post-induction, the AR-induced rats were administered with 10 µl of excipient solution containing 5% saline, 5% glycerine, 5% tween-80 and 85% water into each nostril. While the TRF treatment group received additional 16 µg/µl of TRF in the same excipient solution at 10 µl per nostril. No treatment was given to the control group.All the rats were euthanized with 120 mg/kg of pentobarbital on day 8. The rats were decapitated and the eyes, skin, fur, muscle tissues, and the mandibles were excised. The complete upper skull was then fixed in 10% buffered formalin. The samples were dehydrated in ascending grades of alcohol and xylene and embedded in paraffin for histological examination by Advanced Molecular Pathology Laboratory (AMPL), A*STAR. Tissue sections were taken from paraffin blocks at 5 µm thickness and stained with hematoxylin and eosin (H&E). The stained samples were evaluated with a light microscope by a certified veterinary pathologist blinded to the protocol.The significance of nonneoplastic lesions in nasal tissues was evaluated semi quantitatively by applying grading scheme of 0 to 5 severity grades with reference to previous reports , 31. SevThe data were analysed using Graphpad Prism version 9.1.0 . Comparison of the histological evaluation graded by the veterinary pathologist in all groups were performed with one-way ANOVA and subsequently post-hoc Tukey test. Statistical significance value was set at p < 0.05.Figure The histological evaluation of the nasal cavity, nasal septum and nasal turbinate including degree of inflammation, inflammatory cells infiltrate and mucosa damage in all groups are presented in Fig. In the control group, none to minimal degree of inflammation, polymorphonuclear cells infiltration and mucosa damage were observed Fig. A. H&E stCompared to the control group, there was a greater degree of inflammation and significant submucosal infiltration of polymorphonuclear cells in the PAF-induced AR group, with p < 0.05. An extensive damage in the mucosa was significantly observed as compared to the control and TRF treated group, with p < 0.0001 Fig. A. H&E stThe overall histological parameters in the PAF-induced AR group receiving TRF treatment were restored to the regular nasal structures close to the healthy control, particularly in mucosa damage with p < 0.0001 Fig. A. There Oxidative stress (OS) and inflammation are intricately linked where OS is intensified upon triggering inflammation . The imb2- release in PAF-induced human eosinophils [Current evidence suggests the involvement of PAF in the generation of ROS and acts as a powerful stimulator of acute inflammatory processes in allergic reactions , 38. A rinophils . All thiinophils . Unlike inophils .There is now increasing evidence for the immunological roles of platelets in inflammation, particularly in the development of allergic responses like asthma. Allergic asthma is a chronic inflammatory disease leading to mucus hypersecretion, as well as the hyperresponsiveness, obstruction and remodelling in the airways. Upon allergen exposure, platelets entail in the IgE-FcεRI-dependent pathway, resulted in the infiltration of central effector cells in allergic inflammation, and the release of platelets-derived factors such as type 2 cytokines and various biochemical mediators including PAF , 47. ROSIn the present study, we demonstrated the protective actions of TRF in ameliorating AR symptoms in a PAF-induced AR rat model. The dosage of TRF in the range of 5 to 16 μg/μl delivered at 10 μl was examined, with reference to the gold standard treatment of using nasal corticosteroids. Mometasone furoate (MF) as an intranasal aqueous spray is among the most common prescribed corticosteroids for AR. Its efficacy has been evaluated in several randomized, double-blind, placebo-controlled clinical trials involving adults and children with AR. Overall clinical data show MF aqueous spray, administered once daily at 100 μg per nostril for 2–12 weeks, is not only effective in treating AR, but also prevents the onset of symptoms in AR patients , 52. In T3 possesses both antioxidant and anti-inflammatory properties. It not only can neutralize free radicals, but also has an anti-inflammatory action in allergic disorders , 54. In Nutritional epigenetics has established a new framework for gene-diet interactions by studying heritable modifications in gene expression without alterations in the underlying DNA sequence. The best-known epigenetic mechanism, DNA methylation, has been proposed to drive immune programming associated with pro-allergic in the epigenetic regulation of asthma and allergic diseases at the Th2 locus control region . VitaminAt the moment, there is a lack of intervention human trial into the management of AR using T3. Nevertheless, the relationship between dietary vitamin E and allergic diseases in humans has been previously reported. Vitamin E supplementation was shown to protect young children from the development of atopy and wheezing . FurtherThere may be some limitations to this study. More molecular research into signaling pathways such as phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and nuclear factor erythroid 2–related factor 2/heme oxygenase-1 (Nrf2/HO-1) would be beneficial in validating the potential mechanism of T3's antioxidant and anti-allergic effects. Despite significant anatomical similarities between humans and animals, the physiological and genetic variations may preclude dosage used in animal studies from being fully replicated in human trials. The inter-species dose extrapolation by using allometric scaling should be explored to determine a safer dose of PAF in humans. Finally, further randomized controlled clinical trials are needed to determine the therapeutic effects of TRF in the treatment of AR.TRF exhibited symptomatic relief actions in AR potentially due to its antioxidant, anti-inflammatory and anti-allergic properties. This is the first study to demonstrate the significance of intranasally administered TRF, which may offer an alternative therapeutic option in the prevention and reversal of AR.