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The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.
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Adenosina Trifosfatases , DNA Helicases , Proteínas Nucleares , Neoplasias da Próstata , Fatores de Transcrição , Adenosina Trifosfatases/metabolismo , Animais , Benzamidas , DNA Helicases/genética , Elementos Facilitadores Genéticos , Genes myc , Fator 3-alfa Nuclear de Hepatócito , Humanos , Masculino , Nitrilas , Proteínas Nucleares/genética , Oncogenes , Feniltioidantoína , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Receptores Androgênicos , Fatores de Transcrição/genética , Regulador Transcricional ERG , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The determination of molecular features that mediate clinically aggressive phenotypes in prostate cancer remains a major biological and clinical challenge1,2. Recent advances in interpretability of machine learning models as applied to biomedical problems may enable discovery and prediction in clinical cancer genomics3-5. Here we developed P-NET-a biologically informed deep learning model-to stratify patients with prostate cancer by treatment-resistance state and evaluate molecular drivers of treatment resistance for therapeutic targeting through complete model interpretability. We demonstrate that P-NET can predict cancer state using molecular data with a performance that is superior to other modelling approaches. Moreover, the biological interpretability within P-NET revealed established and novel molecularly altered candidates, such as MDM4 and FGFR1, which were implicated in predicting advanced disease and validated in vitro. Broadly, biologically informed fully interpretable neural networks enable preclinical discovery and clinical prediction in prostate cancer and may have general applicability across cancer types.
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Aprendizado Profundo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores Androgênicos/genética , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.
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We have measured the temperature dependence of the ClNO2 product yield in competition with hydrolysis following N2O5 uptake to aqueous NaCl solutions. For NaCl-D2O solutions spanning 0.0054-0.21 M, the ClNO2 product yield decreases on average by only 4 ± 3% from 5 to 25 °C. Less reproducible measurements at 0.54-2.4 M NaCl also fall within this range. The ratio of the rate constants for chlorination and hydrolysis of N2O5 in D2O is determined on average to be 1150 ± 90 at 25 °C up to 0.21 M NaCl, favoring chlorination. This ratio is observed to decrease significantly at the two highest concentrations. An Arrhenius analysis reveals that the activation energy for hydrolysis is just 3.0 ± 1.5 kJ/mol larger than for chlorination up to 0.21 M, indicating that Cl- and D2O attack on N2O5 has similar energetic barriers despite the differences in charge and complexity of these reactants. In combination with the measured preexponential ratio favoring chlorination of 300-200+400, we conclude that the strong preference of N2O5 to undergo chlorination over hydrolysis is driven by dynamic and entropic, rather than enthalpic, factors. Molecular dynamics simulations elucidate the distinct solvation between strongly hydrated Cl- and the hydrophobically solvated N2O5. Combining this molecular picture with the Arrhenius analysis implicates the role of water in mediating interactions between such distinctly solvated species and suggests a role for diffusion limitations on the chlorination reaction.
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Androgen receptor (AR) action is a hallmark of prostate cancer (PCa) with androgen deprivation being standard therapy. Yet, resistance arises and aberrant AR signaling promotes disease. We sought compounds that inhibited genes driving cancer but not normal growth and hypothesized that genes with consensus androgen response elements (cAREs) drive proliferation but genes with selective elements (sAREs) promote differentiation. In a high-throughput promoter-dependent drug screen, doxorubicin (dox) exhibited this ability, acting on DNA rather than AR. This dox effect was observed at low doses for multiple AR target genes in multiple PCa cell lines and also occurred in vivo. Transcriptomic analyses revealed that low dox downregulated cell cycle genes while high dox upregulated DNA damage response genes. In chromatin immunoprecipitation (ChIP) assays with low dox, AR binding to sARE-containing enhancers increased, whereas AR was lost from cAREs. Further, ChIP-seq analysis revealed a subset of genes for which AR binding in low dox increased at pre-existing sites that included sites for prostate-specific factors such as FOXA1. AR dependence on cofactors at sAREs may be the basis for differential modulation by dox that preserves expression of genes for survival but not cancer progression. Repurposing of dox may provide unique opportunities for PCa treatment.
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Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Elementos de Resposta , Animais , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Doxorrubicina/uso terapêutico , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , RNA-Seq , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We present the slow electron velocity map imaging spectroscopy of cryogenically cooled phenoxide, 1-naphthoxide, and 2-naphthoxide anions. The results allow us to examine the ground state and the lowest energy excited state in the corresponding neutral radicals. Care was taken to minimize autodetachment signals in the photoelectron spectra, allowing for more straightforward comparisons with Franck-Condon analyses. The ground states of these three aromatic oxide radicals all have the unpaired electron residing in a π orbital delocalized throughout the molecule. The electron affinity of 1-naphthoxy is measured to be 2.290(2) eV, while that of 2-naphthoxy is measured to be 2.404(2) eV, both of which are higher than that of the smaller phenoxy molecule at 2.253(1) eV. The first excited states have the unpaired electron residing in a more localized σ orbital, yielding measured term energies for the à state of 1.237(2) eV in 1-naphthoxy and 1.068(1) eV in 2-naphthoxy, while that of phenoxy is lower at 0.952(1) eV. The calculated Franck-Condon spectra generally showed good agreement with the experimental spectra, yielding assignments of the more active vibrations in each electronic state. Significant autodetachment signals arising from dipole bound states near the ground states of all three radicals were observed in our efforts to avoid them, and comparably less autodetachment signals were observed near the excited states. Besides this type of non-Franck-Condon intensities in the photoelectron spectra, we also observed minor features arising due to vibronic coupling in the ground states of all three radicals.
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We report the slow electron velocity map imaging spectroscopy of cryogenically cooled anthracene and fluoranthene radical anions, two similarly sized polycyclic aromatic hydrocarbon molecules. The results allow us to examine the lowest energy singlet and triplet states in the neutral molecules on equal footing from the anionic ground state. The analysis of the experimental spectra is aided by harmonic calculations and Franck-Condon simulations, which generally show good agreement with experimental values and spectra. The electron affinity of fluoranthene is measured to be 0.757(2) eV, which is larger than that of anthracene at 0.532(3) eV. The lowest energy triplet state in anthracene is observed at 1.872(3) eV above the singlet ground state, while that of fluoranthene is observed at 2.321(2) eV above its singlet ground state. Comparisons of experimental and calculated spectra show that in addition to the Franck-Condon active modes, there is a clear presence of vibrational modes that gain intensity via vibronic coupling in both the singlet and triplet states in both molecules. In addition, the triplet state generally exhibits increased vibronic coupling compared to the singlet state, with the fluoranthene triplet state exhibiting evidence of distortion from C2v symmetry.
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A velocity map imaging (VMI) setup consisting of multiple electrodes with three adjustable voltage parameters, designed for slow electron velocity map imaging applications, is presented. The motivations for this design are discussed in terms of parameters that influence the VMI resolution and functionality. Particularly, this VMI has two tunable potentials used to adjust for optimal focus, yielding good VMI focus across a relatively large energy range. It also allows for larger interaction volumes without significant sacrifice to the resolution via a smaller electric gradient at the interaction region. All the electrodes in this VMI have the same dimensions for practicality and flexibility, allowing for relatively easy modifications to suit different experimental needs. We have coupled this VMI to a cryogenic ion trap mass spectrometer that has a flexible source design. The performance is demonstrated with the photoelectron spectra of S- and CS2-. The latter has a long vibrational progression in the ground state, and the temperature dependence of the vibronic features is probed by changing the temperature of the ion trap.
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BACKGROUND: The Androgen Receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in all stages of prostate cancer progression, including progression to castration-resistance following androgen-deprivation therapy. Thus, identifying and targeting critical AR-regulated genes is one potential method to block castration-resistant cancer proliferation. Of particular importance are transcription factors that regulate stem cell pluripotency; many of these genes are emerging as critical oncogenes in numerous tumor cell types. Of these, Nanog has been previously shown to increase the self-renewal and stem-like properties of prostate cancer cells. Thus, we hypothesized that Nanog is a candidate AR target gene that may impart castration-resistance. METHODS: We modulated AR signaling in LNCaP prostate cancer cells and assayed for Nanog expression. Direct AR binding to the NANOG promoter was tested using AR Chromatin Immunoprecipation (ChIP) and analyses of publically available AR ChIP-sequencing data-sets. Nanog over-expressing cells were analyzed for cell growth and cytotoxicity in response to the AR antagonist enzalutamide and the microtubule stabilizing agent docetaxel. RESULTS: AR signaling upregulates Nanog mRNA and protein. AR binds directly to the NANOG promoter, and was not identified within 75 kb of the NANOGP8 pseudogene, suggesting the NANOG gene locus was preferentially activated. Nanog overexpression in LNCaP cells increases overall growth, but does not increase resistance to enzalutamide or docetaxel. CONCLUSIONS: Nanog is a novel oncogenic AR target gene in prostate cancer cells, and stable expression of Nanog increases proliferation and growth of prostate cancer cells, but not resistance to enzalutamide or docetaxel.
Assuntos
Antineoplásicos/farmacologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/fisiologia , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Humanos , Imunoprecipitação , Masculino , Proteína Homeobox Nanog , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Polimorfismo de Fragmento de Restrição , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Taxoides/farmacologia , Regulação para CimaRESUMO
There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate is anatomically and developmentally different from the human prostate and does not spontaneously form tumors. Genetically engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts are an alternative but must rely on an immunocompromised host. Therefore, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of androgen receptor-targeted and immunotherapies. These mice maintain multiple human immune cell lineages, including functional human T-cells and myeloid cells. Implications: To our knowledge, results illustrate the first model of human PCa that has an intact human immune system, metastasizes to clinically relevant locations, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.
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PURPOSE: Despite successful clinical management of castration-sensitive prostate cancer (CSPC), the 5-year survival rate for men with castration-resistant prostate cancer is only 32%. Combination treatment strategies to prevent disease recurrence are increasing, albeit in biomarker-unselected patients. Identifying a biomarker in CSPC to stratify patients who will progress on standard-of-care therapy could guide therapeutic strategies. EXPERIMENTAL DESIGN: Targeted deep sequencing was performed for the University of Illinois (UI) cohort (n = 30), and immunostaining was performed on a patient tissue microarray (n = 149). Bioinformatic analyses identified pathways associated with biomarker overexpression (OE) in the UI cohort, consolidated RNA sequencing samples accessed from Database of Genotypes and Phenotypes (n = 664), and GSE209954 (n = 68). Neutralizing antibody patritumab and ectopic HER3 OE were utilized for functional mechanistic experiments. RESULTS: We identified ERBB3 OE in diverse patient populations with CSPC, where it was associated with advanced disease at diagnosis. Bioinformatic analyses showed a positive correlation between ERBB3 expression and the androgen response pathway despite low dihydrotestosterone and stable expression of androgen receptor (AR) transcript in Black/African American men. At the protein level, HER3 expression was negatively correlated with intraprostatic androgen in Black/African American men. Mechanistically, HER3 promoted enzalutamide resistance in prostate cancer cell line models and HER3-targeted therapy resensitized therapy-resistant prostate cancer cell lines to enzalutamide. CONCLUSIONS: In diverse patient populations with CSPC, ERBB3 OE was associated with high AR signaling despite low intraprostatic androgen. Mechanistic studies demonstrated a direct link between HER3 and enzalutamide resistance. ERBB3 OE as a biomarker could thus stratify patients for intensification of therapy in castration-sensitive disease, including targeting HER3 directly to improve sensitivity to AR-targeted therapies.
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Benzamidas , Feniltioidantoína , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Androgênios/uso terapêutico , Recidiva Local de Neoplasia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Nitrilas/uso terapêutico , Biomarcadores , Castração , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Receptor ErbB-3/genéticaRESUMO
BACKGROUND: In the adult human prostate CD133 expression is thought to mark rare prostate epithelial stem cells and malignant tumor stem/initiating cells. Such putative stem cell populations are thought to proliferate slowly, but possess unlimited proliferative potential. Based on this, we hypothesized that CD133(pos) prostate cancer cells proliferate slower than CD133(neg) cells. METHODS: Human prostate cancer cell lines were analyzed for CD133 expression and DNA content using flow cytometry. Rates of cell division and DNA synthesis were determined using CFSE cell tracing and BrdU uptake, respectively. Changes in cell cycle distribution and the percentage of CD133(pos) cells were assayed under conditions of different cell density and AR-pathway modulation. Lastly, we over-expressed lentiviral CD133 to measure whether CD133 regulates the cell cycle. RESULTS: The cell cycle distribution differs between CD133(pos) and CD133(neg) cells in all three human prostate cancer cell lines studied. CD133(pos) cells have a greater proportion of cells in G2 and proliferate faster than CD133(neg) cells. High cell density increases the percentage of CD133(pos) cells without changing CD133(pos) cell cycle progression. Treatment with the AR agonist R1881, or the anti-androgen MDV3100, significantly changed the percentage and proliferation of CD133(pos) cells. Finally, ectopic over-expression of CD133 had no effect on cell cycle progression. CONCLUSIONS: Contrary to our hypothesis, we demonstrate that CD133(pos) cells proliferate faster than CD133(neg) cells. This association of CD133 expression with increased cell proliferation is not directly mediated by CD133, suggesting that surface CD133 is a downstream target gene of an undefined pathway controlling cell proliferation.
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Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Antígeno AC133 , Antagonistas de Androgênios/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Cinética , Masculino , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate does not spontaneously form tumors and is anatomically and developmentally different from the human prostate. Engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts represent an alternative but rely on an immunocompromised host. Accordingly, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of hormonal and immunotherapies. These mice maintain multiple human cell lineages, including functional human T-cells and myeloid cells. In 22Rv1 xenografts, subcutaneous tumor size was not significantly altered across conditions; however, metastasis to secondary sites differed in castrate huNOG vs background-matched immunocompromised mice treated with enzalutamide (enza). VCaP xenograft tumors showed decreases in growth with enza and anti-Programed-Death-1 treatments in huNOG mice, and no effect was seen with treatment in NOG mice. Enza responses in huNOG and NOG mice were distinct and associated with increased T-cells within tumors of enza treated huNOG mice, and increased T-cell activation. In huNOG-EXL mice, which support human myeloid development, there was a strong population of immunosuppressive regulatory T-cells and Myeloid-Derived-Suppressor-Cells (MDSCs), and enza treatment showed no difference in metastasis. Results illustrate, to our knowledge, the first model of human PCa that metastasizes to clinically relevant locations, has an intact human immune system, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.
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The development of androgen receptor signaling inhibitor (ARSI) drug resistance in prostate cancer (PC) remains therapeutically challenging. Our group has described the role of sex determining region Y-box 2 (SOX2) overexpression in ARSI-resistant PC. Continuing this work, we report that NR3C1, the gene encoding glucocorticoid receptor (GR), is a novel SOX2 target in PC, positively regulating its expression. Similar to ARSI treatment, SOX2-positive PC cells are insensitive to GR signaling inhibition using a GR modulating therapy. To understand SOX2-mediated nuclear hormone receptor signaling inhibitor (NHRSI) insensitivity, we performed RNA-seq in SOX2-positive and -negative PC cells following NHRSI treatment. RNA-seq prioritized differentially regulated genes mediating the cell cycle, including G2 checkpoint WEE1 Kinase (WEE1) and cyclin-dependent kinase 1 (CDK1). Additionally, WEE1 and CDK1 were differentially expressed in PC patient tumors dichotomized by high vs low SOX2 gene expression. Importantly, pharmacological targeting of WEE1 (WEE1i) in combination with an ARSI or GR modulator re-sensitizes SOX2-positive PC cells to nuclear hormone receptor signaling inhibition in vitro, and WEE1i combined with ARSI significantly slowed tumor growth in vivo. Collectively, our data suggest SOX2 predicts NHRSI resistance, and simultaneously indicates the addition of WEE1i to improve therapeutic efficacy of NHRSIs in SOX2-positive PC.
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Antineoplásicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Transdução de Sinais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Antagonistas de Receptores de Andrógenos/farmacologia , Receptores Citoplasmáticos e Nucleares , Linhagem Celular Tumoral , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição SOXB1/genéticaRESUMO
We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.
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BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current treatment strategies do not cure most children with recurrent or high-risk disease, underlying the need for novel therapeutic approaches. Retinoic acid has been shown to induce differentiation in a variety of cells including skeletal myoblasts and neuroblasts. In the setting of minimal residual disease, retinoic acid improves survival in neuroblastoma, another poorly differentiated childhood tumor. Whether such an approach is useful for rhabdomyosarcoma has not yet been investigated. Several in vitro studies have demonstrated an appreciable effect of retinoic acid on human RMS cellular proliferation and differentiation. PROCEDURE: We assessed the efficacy of ATRA on rhabdomyosarcoma, in vitro and in vivo, using cell lines and xenografts. RESULTS: ATRA slowed RMS cell proliferation, and promoted a more differentiated myogenic phenotype in both alveolar and embryonal RMS cell lines. Treatment of cultured murine myoblasts with retinoids increased Myogenin expression, but did not induce cell cycle arrest. Despite the favorable in vitro effects, ATRA failed to delay relapse of minimal residual disease using human RMS xenografts in immuno-suppressed NOD-SCID (NSG) mice. Interestingly, tumors that recurred after ATRA treatment showed evidence of enhanced muscle differentiation. CONCLUSION: Our results indicate that ATRA could increase the expression of some genes associated with muscle differentiation in rhabdomyosarcoma cells, but there was no benefit of single-agent therapy in an MRD model, likely because cell cycle arrest was uncoupled from the pro-differentiation effects of retinoids.
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Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Rabdomiossarcoma/patologia , Tretinoína/farmacologia , Animais , Western Blotting , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Mioblastos Esqueléticos/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
New strategies are needed to predict and overcome metastatic progression and therapy resistance in prostate cancer. One potential clinical target is the stem cell transcription factor SOX2, which has a critical role in prostate development and cancer. We thus investigated the impact of SOX2 expression on patient outcomes and its function within prostate cancer cells. Analyses of SOX2 expression among a case-control cohort of 1028 annotated tumor specimens demonstrated that SOX2 expression confers a more rapid time to metastasis and decreased patient survival after biochemical recurrence. SOX2 ChIP-Seq analyses revealed SOX2-binding sites within prostate cancer cells which differ significantly from canonical embryonic SOX2 gene targets, and prostate-specific SOX2 gene targets are associated with multiple oncogenic pathways. Interestingly, phenotypic and gene expression analyses after CRISPR-mediated deletion of SOX2 in castration-resistant prostate cancer cells, as well as ectopic SOX2 expression in androgen-sensitive prostate cancer cells, demonstrated that SOX2 promotes changes in multiple metabolic pathways and metabolites. SOX2 expression in prostate cancer cell lines confers increased glycolysis and glycolytic capacity, as well as increased basal and maximal oxidative respiration and increased spare respiratory capacity. Further, SOX2 expression was associated with increased quantities of mitochondria, and metabolomic analyses revealed SOX2-associated changes in the metabolism of purines, pyrimidines, amino acids and sugars, and the pentose phosphate pathway. Analyses of SOX2 gene targets with central functions metabolism (CERK, ECHS1, HS6SDT1, LPCAT4, PFKP, SLC16A3, SLC46A1, and TST) document significant expression correlation with SOX2 among RNA-Seq datasets derived from patient tumors and metastases. These data support a key role for SOX2 in metabolic reprogramming of prostate cancer cells and reveal new mechanisms to understand how SOX2 enables metastatic progression, lineage plasticity, and therapy resistance. Further, our data suggest clinical opportunities to exploit SOX2 as a biomarker for staging and imaging, as well as a potential pharmacologic target.
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Fatores de Transcrição SOXB1RESUMO
Metastatic castration-resistant prostate cancers (mCRPCs) are treated with therapies that antagonize the androgen receptor (AR). Nearly all patients develop resistance to AR-targeted therapies (ARTs). Our previous work identified CREB5 as an upregulated target gene in human mCRPC that promoted resistance to all clinically approved ART. The mechanisms by which CREB5 promotes progression of mCRPC or other cancers remains elusive. Integrating ChIP-seq and rapid immunoprecipitation and mass spectroscopy of endogenous proteins, we report that cells overexpressing CREB5 demonstrate extensive reprogramming of nuclear protein-protein interactions in response to the ART agent enzalutamide. Specifically, CREB5 physically interacts with AR, the pioneering actor FOXA1, and other known co-factors of AR and FOXA1 at transcription regulatory elements recently found to be active in mCRPC patients. We identified a subset of CREB5/FOXA1 co-interacting nuclear factors that have critical functions for AR transcription (GRHL2, HOXB13) while others (TBX3, NFIC) regulated cell viability and ART resistance and were amplified or overexpressed in mCRPC. Upon examining the nuclear protein interactions and the impact of CREB5 expression on the mCRPC patient transcriptome, we found that CREB5 was associated with Wnt signaling and epithelial to mesenchymal transitions, implicating these pathways in CREB5/FOXA1-mediated ART resistance. Overall, these observations define the molecular interactions among CREB5, FOXA1, and pathways that promote ART resistance.
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Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
In this work, we present the Wisconsin Oscillator, a small, inexpensive, low-power circuit for powering ion-guiding devices such as multipole ion guides, ion funnels, active ion-mobility devices, and non-mass-selective ion traps. The circuit can be constructed for under $30 and produces two antiphase RF waveforms of up to 250 Vp-p in the high kilohertz to low megahertz range while drawing less than 1 W of power. The output amplitude is determined by a 0-6.5 VDC drive voltage, and voltage amplification is achieved using a resonant LC circuit, negating the need for a large RF transformer. The Wisconsin Oscillator automatically oscillates with maximum amplitude at the resonant frequency defined by the onboard capacitors, inductors, and the capacitive load of the ion-guiding device. We show that our circuit can replace larger and more expensive RF power supplies without degradation of the ion signal and expect this circuit to be of use in miniature and portable mass spectrometers as well as in home-built systems utilizing ion-guiding devices.